oral and poster presenting authors index

Transcription

International Conference of the Thai Veterinary Medical Association
under the Royal Patronage (37
th
: Nonthaburi, Thailand)
Proceeding : The 37th International Conference on
Veterinary Science IICAB , APHIS, FAO Joint Symposiums
“ASIA WEB FOR WORLD FOOD SECURITY”
February 29th - March 2nd, 2012 / organized by ICVS organizing committee, TVMA;
Edited by Aranya Ponpornpisit and Kanjana Imsilp. ISBN 978-974-7870-47-3
Nonthaburi : Infinity Color Printing, 2012. CD: http://copydvdcd.com
1.
IVCS
2.
IICAB
3.
APHIS
4.
FAO
5.
TVMA
I.
Thai Veterinary Medical Association under the Royal Patronage
II. Aranya Ponpornpisit
III. Kanjana Imsilp
Proceeding Production
Aranya
Ponpornpisit
Navakanit
Sachanonta
Kwanhatai
Thongpalad
Yutthapong Chungpitakudom
Pajaree
Charuvichitratana
All oral and poster presentation abstracts published in The 37th International Conference on Veterinary Science
IICAB, APHIS, FAO Joint Symposiums: “ASIA WEB FOR WORLD FOOD SECURITY” proceeding pass a peer review process by the scientific committee of the conference. Proceedings did not require all authors of a research
paper to sign the letter of submission, nor do they impose an order on the list of authors. Submission to the
conference is obtained by the scientific committee meaning that all the listed authors have agreed all of the
contents. The corresponding (submitting) author is responsible for having ensured that this agreement has
been reached, and for managing all communication between the committee and all co-authors, before and after
publication. Each author is responsible for the content and accuracy of the entire manuscript.
© The Thai Veterinary Medical Association under the Royal Patronage
CONTENTS
Welcome Messages from TVMA President
5
Welcome Messages from Organizing Committee Chairperson
6
Welcome Messages from Scientific Committee Chairperson
7
TVMA Board Committee 2010-2011
9
Organizing Committee
14
Organizing Subcommittee
16
Conference Venue
21
Scientific Program
23
Scientific Program February 29th 2012
24
Scientific Program March 1st 2012
25
Scientific Program March 2nd 2012
27
ICVS-USDA-APHIS Joint Symposium
29
ICVS-FAO Joint Symposium
29
Oral Presentation
30
ICVS KUGENENG Symposium
31
WSPA Symposium
32
TAVLD and TSVA Symposium
32
Invited Speakers
34
Oral and Poster Presentation Contents
59
Oral Presentation Abstracts
68
Poster Presentation Abstracts
91
Oral and Poster Presentation Full Papers
154
Oral Presentation Full Papers
155
Poster Presentation Full Papers
191
Oral and Poster Presenting Authors Index
275
Invited Speaker Full Paper Contents
285
Sponsored
401
Welcome Message
On behalf of the Thai Veterinary Medical
Association under Royal Patronage (TVMA), we are pleased to warmly
welcome you to the 37th International Conference on Veterinary Science,37th ICVS on a new schedule during 29th
February – 2nd March 2012 at IMPACT Forum MuangthongThani, Nonthaburi, Thailand
The 37th ICVS Organizing Committee have been working hard over the past months to put together a memorable
program, both in the official sessions and the social functions. During the conference, there will be numerous opportunities for TVMA members and participants to collaborate and update themselves over our specialized field
of endeavor. We do apology for unexpected flood disaster that happened in the previous schedule. However ,
TVMA is pleased to assure that we are going to be sharing with each and everyone, under the theme of “ASIA WEB
FOR WORLD FOOD SECURITY”, and aims to emphasize the impact of veterinary related aspects of trade, global
warming, food safety and food security, emerging and re-emerging diseases, veterinary education and so on.
Hopefully, the conference will give valuable opportunities for the veterinarians and researchers to share their experiences and informations for the improvement of the Veterinary Sciences not only in the region but also globally.
We wish all participants a most rewarding conference and a memorable stay in Thailand, recognized as the land of
smiles. Let’s celebrate the 100 anniversary of the Veterinary Education in Thailand in 2012
Achariya Sailasuta D.V.M., F.R.V.C.S, Ph.D.
President of the Thai Veterinary Medical Association under Royal Patronage
Dear Colleagues,
It is our great pleasure to welcome you to The 37thICVS TVMA Annual Conference, at the IMPACT Forum,
Muang Thong Thani, Thailand during Feb 29th—March 2nd , 2012.
Last year was a wonderful experience to both TVMA and non-TVMA members. The 37thICVS scientific committee, thus, has been working harder to make this year program successful. The scientific program in
37thICVS have been arranged to meet the theme “Asia Web for World Food Security”.
It consists of out-
standing invited speakers whose expertise's in their fields are internationally well-recognized. The symposiums lining up such as ICVS-APHIS, ICVS-FAO joint symposiums and those from numerous associations including WSPA, TSVA, KUGENENG, Cancer Center and TAVLD are also intriguing.
In the 37th ICVS, eighty-three participants have submitted manuscripts for presentations both in oral and
poster sessions. Many of those can make your eyes wide open. We, thus, hope that this year overall program
would amaze you since it is closely related to our real lives.
On behalf of the 37thICVS Scientific Committee, I would like to thank all wonderful participants and hope that
your 37thICVS experiences will be valuable. We are so sure that your participations in this 37thICVS are
strengthening the ICVS as well as our professions. Last but not least we will be looking forward to seeing you
again in the 38thICVS.
All the best!
Kanjana Imsilp, D.V.M., M.S., Ph.D.
Chair, the 37th ICVS Scientific Committee
Dear Colleagues,
Welcome to the 37th International Conference on Veterinary Science & IICAB, APHIS, FAO Joint Symposiums.
On behalf of the organizing committee, I am delighted and pleased to welcome all participants to the 37 th International Conference on Veterinary Science & IICAB, APHIS, FAO Joint Symposiums. In addition, there are also
guest organizations; CUVA, KUVA, TPVA, TSVA, TAVLD, AAVST, WSPA and KUGENENG, to join this scientific conference.
The 37th ICVS organizing committee and all guest organizations have put so many efforts to arrange you a
noteworthy program. The Scientific program prepared for this event is absolute strength and sound. Therefore,
you are ensured to enjoy the best of scientific content and the modest knowledge from prestigious speakers and
having opportunity to share experiences and information together.
Under the 37th ICVS theme: “ASIA WEB FOR WORLD FOOD SECURITY”, IICAB symposium on “Advances in
Immunology and Vaccinology for Livestock Diseases”, APHIS symposium and FAO symposium on “ONE
HEALTH in MOTION”, these would bring you an up to date in veterinary knowledge, expertise, and technology
which will drive bonding, networking, trading and fostering education among veterinary community in Thailand
and the region.
I would take this opportunity to express my deepest appreciation to the organizing committee and all the guest
organizations, particularly the IICAB, USDA-APHIS and FAO, in arranging this memorable program for our delegates. This challenging task has been accomplished as results of kind assistance from all of you. Congratulations!
I look forward to meeting all invited speakers, delegates and colleagues, who will provide oral and poster
presentation, from Thailand, the region, Asia, and the world in this 37th ICVS event. I also wish you have prolific
scientific discussions and enjoy your stay in Thailand, the land of smiles and cultural rich.
Siriwan Prapong, D.V.M., Ph.D.
Chairperson, the 37th ICVS Organizing Committee
TVMA BOARD Committee
-2011
age
omittee 2010
oyal Patron
R
e
th
r
e
d
n
ociation u
Medical Ass
ry
a
n
ri
te
e
V
The Thai
C
TVMA Board
Dr. ACHARIYA SAILASUTA
President
Dr.NOPPORN VAYUCHOTE
Consultant
Dr.PRATUANG SUDSAKORN
Consultant
Dr.VANDA SUJARIT
Consultant
-2011
omittee 2010
C
rd
a
l Patronage
o
B
A
TVM
er the Roya
d
n
u
n
o
ti
a
ical Associ
rinary Med
te
e
V
i
a
h
T
e
Th
DR. TRITSADEE
CHAOSUAN-CHAROEN
Vice - President
DR. THANIDA
HARINTHARANON
Secretary-General
DR. KANUENGNIT
KORTHAMMARIT
Treasurer
DR. SITHIPORN
ANANJINDA
Register
DR. SIRIWAN
PRAPONG
Committee
DR. RUTJAWATE
TAHARN -KLAEW
Committee
DR. KITTI
SUPCHUKUN
Vice President
DR. BUNIKAR
CHULLABODHI
Assistant Secretary - General
DR. SAKCHAI
SRIBOONSUE
Committee
DR. AMNART
POAPOLATHEP
Editor in chief
DR. NANTARIKA
CHANSUE
Committee
DR. TUANGTHONG
PATCHIMASIRI
Committee
-2011
age
omittee 2010
oyal Patron
R
e
th
r
e
d
n
ociation u
Medical Ass
ry
a
n
ri
te
e
V
The Thai
C
TVMA Board
DR. RUNGROTE
OSATHANON
Public - relation
DR. TANIN
SHEEWAPALABOON
Committee
DR. ARANYA
PONPORNPISIT
Committee
DR. KAMONCHAI
THERASETTAMRONG
Committee
DR. CHANNARONG
RODKHUM
Committee
DR. ALONGKORN
MAHANNOP
Committee
DR. NARIT
PUTTEKULANGKURA
Committee
DR. ARIYA
TAECHABISAT
Committee
DR. BODIN
SUWATTANA
Committee
DR. PHORNCHAI
SUWANNAPHIROM
Committee
The 37th Organizing Committee
The 37th ICVS Organizing Committee
Dr. Aranya Ponpornpisit
Comittee
The 37th Organizing Subcommittee
The 37
th
ICVS Organizing Subcommittee
ICVS Secretariat Subcommittee
ACHARIYA SAILASUTA
Consultant
SIRIWAN PRAPONG
Consultant
SAYAMON SRISUWATANASAKUL
Consultant
CHANNARONG RODKHUM
Subcommittee Chairperson
NOPADON PIRARAT
Subcommittee
DOLLADA SRISAI
Subcommittee
ANTHICHA KUNJANTARACHOT
Subcommittee
PATTANAPON KAYANSAMRUAJ
Subcommittee
KANRAWEE THAWORANAN
Subcommittee
SUKHONTHA THONGBORISUT
Subcommittee
WORAPONG WILAIRAT
Subcommittee & Secretary
ICVS Trade Exhibition Co-ordinator Subcommittee
PRATUANG SUDSAKORN
Consultant
NOPPORN VAYUCHOTE
Consultant
SITTIPORN PRANEENIJ
Consultant
NIRANDOWN AUNGTRAKULSUK
Consultant
SORAWIS TANEETO
Consultant
APAI SUTTISUNK
Consultant
SAKCHAI SRIBOONSUE
BODIN SUWATTANA
Subcommittee
PAKORN PANUPAISAL
Subcommittee
SUMATE SUPCHUKHUN
Subcommittee
SOPAT CHAWANKUN
Subcommittee
WUTIKORN KANMUD
Subcommittee
TANIN SHEEWAPALABOON
ICVS Profit Management Subcommittee
ARIYA TECHAWISET
THANIN SHEWAPALABOON
Subcommittee
NARIT PUTHIKULANGKUL
Subcommittee
WACHIRA LIMTRAJIT
Subcommittee
The 37
th
ICVS Scientific Subcommittee
KANJANA IMSILP
PORNTIPPA LEKCHAROENSUK
Subcommittee
SUNEE KUNAKORNSAWAT
Subcommittee
SUWICHA KASEMSUWAN
Subcommittee
PIPAT ARUNVIPAS
Subcommittee
WORAKIJ CHEDCHOOTHUM
Subcommittee
PATCHARA PHUEKTES
Subcommittee
NARIN UPARAGARIN
Subcommittee
VISANU BOONYAWIWAT
Subcommittee
SOONTAREE PETCHDEE
Subcommittee
KANITTHA PETCH-UDOMSINSUK
Subcommittee
WIN SURACHETPONG
Subcommittee
PHANWIMOL TANHAN
Subcommittee
CHANTIMA PORKSAKORN
Subcommittee
NUVEE PRAPASARAKUL
Subcommittee
SARANYA POAPOLATHEP
ICVS Treasurer Subcommittee
MONAYA EKGATAT
PANUN TANACHAROENWATCH
Subcommittee
LADDA TRONGWONGSA
Subcommittee
SUREE THAMMASART
Subcommittee
SURAPONG WONGKASEMJIT
Subcommittee
REKA KANITPUN
Subcommittee
AMPUN YONGPISANPOB
Subcommittee
KANUENGNIJ KOTHAMRIT
Subcommittee
PATTARIN OPASCHAITAT
Subcommittee
SUPAPORN YONGPISANPOB
Subcommittee
The 37
th
ICVS Scientific Subcommittee
KANJANA IMSILP
PORNTIPPA LEKCHAROENSUK
Subcommittee
SUNEE KUNAKORNSAWAT
Subcommittee
SUWICHA KASEMSUWAN
Subcommittee
PIPAT ARUNVIPAS
Subcommittee
WORAKIJ CHEDCHOOTHUM
Subcommittee
PATCHARA PHUEKTES
Subcommittee
NARIN UPARAGARIN
Subcommittee
VISANU BOONYAWIWAT
Subcommittee
SOONTAREE PETCHDEE
Subcommittee
KANITTHA PETCH-UDOMSINSUK
Subcommittee
WIN SURACHETPONG
Subcommittee
PHANWIMOL TANHAN
Subcommittee
CHANTIMA PORKSAKORN
Subcommittee
NUVEE PRAPASARAKUL
Subcommittee
SARANYA POAPOLATHEP
ICVS Treasurer Subcommittee
MONAYA EKGATAT
PANUN TANACHAROENWATCH
Subcommittee
LADDA TRONGWONGSA
Subcommittee
SUREE THAMMASART
Subcommittee
SURAPONG WONGKASEMJIT
Subcommittee
REKA KANITPUN
Subcommittee
AMPUN YONGPISANPOB
Subcommittee
KANUENGNIJ KOTHAMRIT
Subcommittee
PATTARIN OPASCHAITAT
Subcommittee
SUPAPORN YONGPISANPOB
Subcommittee
The 37
th
ICVS Registration and Evaluation Subcommittee
SIRIWAN PRAPONG
Consultant
KANJANA IMSILP
Consultant
SITTIPORN ANANTAJINDA
Consultant
CHAYAKRIT SINTHUSING
WANDEE THIANGTUM
Subcommittee
WARAPORN PIMPRAPAI
Subcommittee
SANGCHAI YINGSAKMONGKOL
Subcommittee
NATTAPHONG AKRIMAJIRACHOTE
Subcommittee
MANEENOOCH KHIAO-IN
Subcommittee
NIYADA LANSABSAKUL
Subcommittee
NARUMOL KLANGKAEW
Subcommittee
NUCH CHOTECHUANG
Subcommittee
ICVS Steering Subcommittee
PIYANAN TAWEETHAVONSAWAT
SUPAWIWAT PONGLAOHAPUN
Subcommittee
NUTTHEE AM-IN
Subcommittee
SUKANYA LEETHONGDEE
Subcommittee
ANONGNART ASSAVACHEEP
Subcommittee
ARAYA RADTANAKATIKANON
Subcommittee
TREENATE JIRANANTASAK
Subcommittee
GRISNARONG WONGBANDUE
Subcommittee
SROISUDA CHOTIMANUKUL
Subcommittee
PATARAKRIT CHONGPAIBULPATANA
Subcommittee
NATTAWAN TANGMAHAKUL
Subcommittee
METINEE RODPHOL
Subcommittee
KRIENGKRAI CHATTRAKULCHAI
Subcommittee
NICOLE SIRISOPITH MEHL
Subcommittee
PATARAPOL PIAMSOMBOON
Subcommittee
PATARARAT KOHMANEE
Subcommittee
JUTHATIP KEAWCHAROEN
Subcommittee
The 37
th
ICVS Hospitality Subcommittee
TUANGTHONG PATCHIMASIRI
NAREE KETUSING
Subcommittee
NANTAPORN WANDEE
Subcommittee
PEERAWIT BUNPANGBAN
Subcommittee
ISARAYOS SIRIKANOKE
Subcommittee
UTTRA JAMIKORN
Subcommittee
ARAYA SUEBKAMPHET
Subcommittee
APIRADEE INTARAPAK
Subcommittee
MATURAWAN TUNHIKORN
Subcommittee
KITTICHAI EUNJIT
Subcommittee
Conference Venue
21
Scientific Program
30 
31
Invited Speaker Profile
33
Keynote Speaker
Tritsadee Chaosuancharoen, DVM
น.สพ. ทฤษดี ชาวสวนเจริญ
Director, Department of Livestock Development
อธิบดีกรมปศุสัตว์
Keynote Speaker
Sakchai Sriboonsue, DVM
น.สพ. ศักดิ์ชัย ศรีบุญซื่อ
Director, National Bureau of Agricultural Commodity and Food Standards (ACFS)
ผู้อำนวยกำรสำนักงำนมำตรฐำนสินค้ำเกษตรและอำหำรแห่งชำติ (มกอช.)
Speaker: Asia Web for World Food Security Symposium
Mr. Apichart Chongsakul
นาย อภิชาติ จงสกุล
Secretary General of the Office of Agricultural Economics
เลขำธิกำรสำนักงำนเศรษฐกิจกำรเกษตร
Mr. Pornsilp Patcharintanakul
นาย พรศิลป์ พัชรินทร์ตนะกุล
Vice Secretary General, Board of Trade of Thailand
รองเลขำธิกำรสภำหอกำรค้ำแห่งประเทศไทย
Associate Professor Dr.Sompop Manarungsan
รองศาสตราจารย์ ดร.สมภพ มานะรังสรรค์
President, Panyapiwat Institute of Management
อธิกำรบดี สถำบันกำรจัดกำรปัญญำภิวัฒน์
34 
Invited speaker
Dr. James Allen Roth, Distinguished Professor
Executive Director, IICAB.
College of Veterinary Medicine, Iowa State University, USA
Speaker: IICAB Symposium
Dr. Darunee Tantasuvan
สพ.ญ.ดร. ดรุณี ทันตสุวรรณ
Animal and Plant Health Inspection Service (APHIS), USDA, US Embassy
Speaker: APHIS Symposium
Dr. Darunee Tuntasuvan got the first degree on Animal Science from Kasetsart
University, Thailand in 1989. In 1991 she graduated from the same university on Veterinary
Medicine. Her last degree is on Immunoparasitology from the University of Berne,
Switzerland in 1992. She started her professional as a researcher in Parasitology at the
Regional lab in Khonkaen, Thailand. Then she worked at the National Institute of Animal
Health, Department of Livestock Development for 18 years. In 2002 she joined the
National Bureau of Agricultural Commodity and Food Standards.
Her current position is the Agricultural Scientist, USDA APHIS at the US Embassy
in Bangkok. Dr Darunee’s responsibility is to provide technical support for all APHIS
programs, especially HPAI control and trade issues related to animal health and plant
health.
Dr. Robert T.Tanaka
Agricultural Attaché, USDA-APHIS, US Embassy of Thailand
35
Invited speaker
Mr. Orestes Vasquez
Agricultural Attaché, USDA FAS, US Embassy of Thailand
Mr. Orestes Vasquez is the Agricultural Attaché at the US Embassy in Bangkok. In his current
position, Mr. Vasquez is responsible for creating economic opportunity for American agriculture. Prior
to his appointment to Bangkok, Mr. Vasquez worked in different capacities for FAS in Washington DC
including as an Agricultural Advisor to the Government of Afghanistan. Among his duties in Washington, DC monitor countries’ compliance with the World Trade Organization’s (WTO) Sanitary and Phytosanitary Agreement. In Afghanistan, he worked closely with the Government of Afghanistan in agricultural infrastructure rehabilitation and reconstruction projects
Mr. Vasquez was born and raised in Honduras where he attended the Pan-American School
of Agriculture graduating with a degree in Agricultural Engineering. Working for 7 years in the agribusiness sector in Central America, he attended the University of Idaho where he earned a Master’s in
Agricultural Economics.
Assistant Professor Dr. Narin Uparagarin
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. นรินทร์ อุประกรินทร์
Faculty of Veterinary Medicine, Kasetsart University
คณะสัตวแพทยศำสตร์ มหำวิทยำลัยเกษตรศำสตร์
Speaker: Poultry Enteric Diseases
Dr. Narin Upragarin graduated in Doctor of Veterinary Medicine (1992) from Kasetsart University. He received MS.c. (2003) in Animal Pathology program and Ph.D. (2005) at the Faculty of Veterinary Medicine, Utrecht University, The Netherlands. From 1992 until present time, he has been employed by the Department of Farm Resources and Production Medicine of the Faculty of Veterinary
Medicine, Kasetsart University, Kamphaengsan campus, Nakhonpathom, as a full-time staff member
lecturing on avian medicine. Since 1992, he has served extensively as an avian diagnostician/ technical service veterinarian for Thai poultry farmers and local veterinarians.
Main area of expertise : Diseases and Immunity in Chicken.
36 
Assistant Professor Dr. Visanu Boonyawiwat
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. วิศณุ บุญญาวิวัฒน์
Speaker: Veterinary Drug Products in Aquaculture: Practice/Malpractice
Speaker: Aquaculture Food Sustainable and Threat
Dr. Visanu Boonyawiwat is an assistant professor at the Department of Farm Animal Resources
and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University. He received his D.V.M. and
Ph.D. degress from Kasetsart University in 1994 and 2009, respectively. He is responsible for both teaching and doing researches mainly focus on aquatic medicine. He also act as an coordinator for student
exchange programs in aquatic animals among universities. He is outstanding speakers at several national
and international conferences in his field of expertise. He currently is the head of the Aquatic Diseases
Diagnostic Laboratory, the Diagnostic Center, Faculty of Veterinary Medicine, Kasetsart University.
Associate Professor Dr. Worawidh Wajjwalku
รองศาสตราจารย์ น.สพ.ดร. วรวิทย์ วัชชวัลคุ
Speaker: ICVS-KUGENENG Symposium
Chaiyakorn Thitiyanaporn, DVM
อาจารย์ น.สพ. ชัยกร ฐิติญาณพร
Speaker: Osteoarthritis treatment by bone bead surgery
Dr. Waraporn Aumarm
อาจารย์ สพ.ญ.ดร. วราภรณ์ อ่วมอ่าม
Speaker: Paracorticol Technique for Canine Surgery
37
Professor Dr. Roongroje Thanawongnuwech
ศาสตราจารย์ น.สพ.ดร. รุ่งโรจน์ ธนาวงษ์นุเวช
Faculty of Veterinary Science, Chulalongkorn University
คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Speaker: Complexity of PRRS I: Genetic and Antigenic Variation
Speaker: Complexity of PRRS II: Pathogenesis
Speaker: Initiative Forums for Preparedness of Swine EIDs
Dr. Roongroje Thanawongnuwech is a professor and the head of the Deparment of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University (CU), THAILAND. He received his D.V.M. from CU, and M.Sc. and Ph.D. degree from Iowa State University (ISU), respectively.
After working at CU for 2 years, he left to work as a post doctoral scholar at ISU on the interaction of
porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae in 2000 as
well a visiting scientist in Dr.Michael Johnson’s Lab, CSIRO, Australia in 2002. He has wide experience
in both small and large animal pathology, receiving several research awards in swine diseases, especially emerging and re-emerging animal diseases.
Dr.Roongroje Thanawongnuwech has been the Head of Veterinary Diagnostic Laboratory since
2000, Currently, he is the Head of Department of Veterinary Pathology, Chulalongkorn University. In
this function he manages the department research strategies on emerging and re-emerging animal
diseases. Prior to his current position, Roongroje Thanawongnuwech headed Veterinary Pathology
Unit between 2007-2009.
Dr. Win Surachetpong
อาจารย์ น.สพ.ดร. วิน สุรเชษฐพงษ์
Speaker: PRRSV-Host Interaction I: Innate Immune Response
38 
Associate Professor Dr. Sanipa Suradhat
รองศาสตราจารย์ สพ.ญ.ดร. สันนิภา สุรทัตต์
Speaker: PRRSV-Host Interaction II: Adaptive Immune Response
Dr.Sanipa Suradhat is an associate professor at the Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University (CU) in Bangkok, Thailand. She received a D.V.M.
(First class honor) degree from Chulalongkorn University in 1991, and a Ph.D. (Veterinary Microbiology)
from the Western College of Veterinary Medicine (WCVM), University of Saskatchewan, Saskatoon, Canada
in 1999. She is currently responsible for teaching and coordinating immunology courses of the D.V.M.
curriculum and graduate studies at the Faculty of Veterinary Science and The Interdisciplinary Program in
Medical Microbiology, Graduate School, CU. Currently, she is a member of an executive board of the
Faculty of Veterinary Science, a graduate program in Medical Microbiology, and several administrative
boards in both academic and research affairs. Her research interests include viral immunology and veterinary vaccinology. She has published several international research articles related to classical swine
fever vaccine, immunology of PRRSV, and the emerging influenza viruses in Thailand. Dr.Suradhat’s current research activities include development of a novel PRRSV vaccine and characterization of immunomodularoty proteins of PRRSV. She has also helped coordinating and supervising the research activities
related to influenza viruses at the Faculty of Veterinary Science, CU. She has a major role in establishment of the Chulalongkorn University Center of Emerging and Re-emerging Diseases in Animals (CU-EIDAs)
at the faculty of Veterinary Science, and currently serves as a project manager of CU-EIDAs.
Dr.Siripattra Netramai
สพ.ญ.สิริภัทรา เนตรมัย
Kasetsart University Veterinary Teaching Hospital Bangkhen, Kasetsart University
โรงพยำบำลสัตว์บำงเขน มหำวิทยำลัยเกษตรศำสตร์
Speaker: How to Deal with Lymphoma and Mast Cell Tumor in Dogs
39
Assistant Professor Dr. Pariwat Poolperm
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. ปริวรรต พูลเพิ่ม
Speaker: PRRS: Clinical Presentation and Management
Dr.Pariwat Poolperm currently is an assistant professor at the Department of Farm Resources
and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Kamphangsaen, Nakornpathom, THAILAND. He finished his D.V.M. and Ph.D. from Kasetsart University and North Carolina
State University, U.S.A., accordingly. His researches focus on swine herd health management. He has
participated and presented in several international conferences. He also published numerous publications both nationally and internationally.
Dr. Saranya Poapolathep
อาจารย์ สพ.ญ.ดร. ศรัญญา พัวพลเทพ
Speaker: How to Take Care of Dogs Receiving Chemotherapy and Irradiation
Dr.Saranya is a lecturer at the Department of Veterinary Pharmacology, Faculty of Veterinary
Medicine, Kasetsart University (KU) in Bangkok, Thailand. She received a D.V.M. degree from Kasetsart
University in 2001, and a Ph.D. (Veterinary Medical Science) from Graduate School of Agricultural and
Life Sciences, the University of Tokyo, Japan in 2010. She is currently responsible for teaching pharmacology and toxicology courses of the D.V.M. curriculum and graduates studies at Faculty of Veterinary Medicine, KU. Her research interests focus on mycotoxins, pharmacokinetics and drug residues.
Associate Professor Dr.Sunee Kunakornsawat
รองศาสตราจารย์ สพ.ญ.ดร.สุณี คุณากรสวัสดิ์
Speaker: Epistaxis: Nasal Tumors in Dogs
40 
Associate Professor Dr. Preeyaphan Udomprasert
รองศาสตราจารย์ น.สพ.ดร. ปรียพันธุ์ อุดมประเสริฐ
Moderator: PRRS, PED and FMD Disasters: The Real Monsters
Associate Professor Kitcha Urairong
รองศาสตราจารย์ น.สพ. กิจจา อุไรรงค์
Speaker: PRRS, PED and FMD Disasters: The Real Monsters
Assistant Professor Dr. Athipoo Nuntaprasert
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. อธิภู นันทประเสริฐ
Preecha Wongwijarn, DVM
น.สพ. ปรีชา วงศ์วิจารณ์
Department of Livestock Development
กรมปศุสตั ว์
Winai Thongmak, DVM
น.สพ.วินัย ทองมาก
Swine Production Specialist, Live Informatics Co.,Ltd.
ผู้จัดกำรแผนกวิชำกำรและที่ปรึกษำผลผลิตสุกร บริษัท ไลฟ์ อินโฟร์เมติกส์ จำกัด
Thanakorn Silamahakul, DVM
น.สพ.ธนากร ศิลามหากุล
ฟำร์มสุกรสิงห์บรุ ีอำหำรสัตว์
41
Selapoom Pairor, DVM
นายสัตว์แพทย์ เสลภูมิ ไพเราะ
Speaker: Blood Transfusion Technique in Dog and Cat
Assistant Professor Chayakit Sinthusing
ผู้ช่วยศาสตราจารย์ น.สพ. ชยกฤต สินธุสิงห์
Speaker: Hemoglobin Vesicle Transfusion in Canine Hypolvolaemic Shock
Pakthorn Lewchalermwong, DVM
น.สพ. ภัคธร ลิ่วเฉลิมวงศ์
Speaker: Brain emergency management
Assistant Professor Dr. Piyanan Taweethavonsawat
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. ปิยนันท์ ทวีถาวรสวัสดิ์
Speaker: Parasite in Wildlife
Angkana Sommanustweechai, DVM
สพ.ญ. อังคณา สมนัสทวีชัย
The Zoological Park Organization Under the Royal Patronage
องค์กำรสวนสัตว์ในพระบรมรำชูปถัมภ์
Speaker: Parasite in Wildlife
42 
Dr. Jeff M. Hammond
Pirbright Laboratory, Institute for Animal Health, World Reference Laboratory (WRL)
Speaker: Update on Infectivity, Transmission and Pathogenicity of FMDV and Research
Needs
Speaker: FMD Vaccine: Recommendations and Prospects of Improved Vaccine
Dr. Anan Jongkaewwattana
ดร.อนันต์ จงแก้ววัฒนา
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park
ศูนย์พันธุวิศวกรรมและเทคโนโลยีชีวภำพแห่งชำติ, อุทยำนวิทยำศำสตร์แห่งประเทศไทย
Speaker: Improvement of Porcine Epidemic Diarrhea Virus Culturing System using Verocell
Expressing Porcine Aminopeptidase-N and TMPRRS-2
Assistant Professor Dr. Pongrama Ramasuta
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. พงศ์ราม รามสูต
Deputy Dean for Research and Innovation, Faculty of Tropical Medicine, Mahidol University
รองคณบดีฝ่ำยวิจัย คณะเวชศำสตร์เขตร้อน มหำวิทยำลัยมหิดล
Speaker: Application of Phage Display Technology for the Production of Specific Fab to
Non-Structural Protein 3ABC of FMDV
Wilai Linjongsubongkoch, DVM
สพ.ญ. วิไล ลินจงสุบงกช
Director, Regional Reference Laboratory for Foot and Mouth Disease in South East Asia,
National Institute of Animal Health (NIAH)
ผู้อำนวยกำรศูนย์อ้ำงอิงโรคปำกและเท้ำเปื่อยภูมภิ ำคเอเชียตะวันออกเฉียงใต้
สถำบันสุขภำพสัตว์แห่งชำติ
Speaker: Update on Antigenic Variation of FMDV in South-East Asia
Komsilp Sahatrakul, DVM
น.สพ. คมศิลป์ สหตระกูล
Siam Ocean World Bangkok Co.Ltd.
สยำมโอเชียนเวิรล์ ด์ ศูนย์กำรค้ำสยำมพำรำกอน กรุงเทพ
Speaker: Role of Aquatic Veterinatian in Aquarium
43
Associate Professor Dr. Jirasak Tangtrongpiros
รองศาสตราจารย์ น.สพ.ดร. จิรศักดิ์ ตั้งตรงไพโรจน์
Speaker: White Stool Disease in Shrimp
Assistant Professor Dr. Channarong Rodkhum
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. ชาญณรงค์ รอดคา
Department of Veterinary Microbiology,
ภำควิชำจุลชีววิทยำ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Speaker: Clinical Examination and Treatment of Common Diseases in Ornamental Fish
Thanida Haetrakul, DVM
สพ.ญ. ฐนิดา เหตระกูล
Veterinary Medical Aquatic Animal Research Center,
ศูนย์วิจัยโรคสัตว์นำ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Speaker: Chulalongkorn University Ornamental Fish Hospital: Past to the Future
Dr. Orn-Anong Ratchtrachenchai
ดร. อรอนงค์ รัชตราเชนชัย
National Institute of Health of Thailand, Ministry of Public Health
สถำบันวิจัยวิทยำศำสตร์สำธำรณสุข กระทรวงสำธำรณสุข
Speaker: German E.coli outbreak: lesson learnt
44 
Professor Dr. Jiroj Sasipreeyajan
ศาสตราจารย์ น.สพ.ดร. จิโรจ ศศิปรียจันทร์
Speaker: Disease Situations of Chickens
Associate Professor Dr. Thongchai Chalermchaikit
รองศาสตราจารย์ น.สพ.ดร. ธงชัย เฉลิมชัยกิจ
Director of Center for Antimicrobial Resistance Monitoring in Food-borne Pathogen,
ผู้อำนวยกำรศูนย์ติดตำมกำรดือยำฯ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Speaker: Antimicrobial Usages and Food-borne Antimicrobial Resistance in Animals
Dr. Nipa Chokesajjawatee
ดร. นิภา โชคสัจจะวาที
Food Biotechnology laboratory, National Center for Genetic Engineering and Biotechnology
(BIOTEC), Thailand Science Park
หน่วยปฏิบัตเิ ทคโนโลยีชีวภำพทำงอำหำร, หน่วยปฏิบตั ิกำรเทคโนโลยีชีวภำพ,
Speaker: Microbial Risk Assessment and its Impacts on Food Safety
Mr. Kiddivong Sombuntham
นาย กิดดิวงค์ สมบุญธรรม
Secretary General, The Swine Raisers Association of Thailand
เลขำธิกำร สมำคมผูเ้ ลียงสุกรแห่งชำติ
45
Angsana Horcharoen, DVM
สพ.ญ. อังศนา ฮ้อเจริญ
Thai Swine Veterinary Association
สมำคมสัตวแพทย์ผู้ควบคุมฟำร์มสุกรไทย
Speaker: Initiative Forum for Preparedness of Swine EIDs
Rakthai Ngampak, DVM
น.สพ. รักไทย งามภักดิ์
Department of Livestock Development
กรมปศุสตั ว์
Speaker: Initiative Forum for Preparedness of Swine EIDs
Anan Sirimongkolkasem, MD
นพ. อนันต์ ศิริมงคลเกษม
Chairman, Thai Broiler Processing Exporters Association,
Managing Director, GFPT Public Company Limited
นำยกสมำคมผูผ้ ลิตไก่เพื่อส่งออกไทย, ประธำนกรรมกำรบริหำร บริษัท จีเอฟพีที จำกัด
Speaker: Poultry Production in Thailand: Outlook 2012
Ms. Chaweewan Kampa
นาง ฉวีวรรณ คาพา
Chairman, Thai Poultry Promotion Association of Thailand
under the patronage of His Majesty the King
นำยกสมำคมส่งเสริมกำรเลียงไก่แห่งประเทศไทยในพระบรมรำชูปถัมภ์
Nirandorn Auengtrakulsuk, DVM
น.สพ. นิรันดร เอื้องตระกูลสุข
Solicitor General, Department of Livestock Development
รองอธิบดีกรมปศุสัตว์
46 
Sumeth Sapchukun, DVM
น.สพ. สุเมธ ทรัพย์ชูกุล
Managing Director, Intervet (Thailand) Co.ltd.
กรรมการผู้จัดการบริษัท อินเตอร์เวท (ประเทศไทย) จากัด
Moderator: Poultry Production in Thailand: Outlook 2012
Associate Professor Dr. Janenuj Wongtavatchai
รองศาสตราจารย์ สพ.ญ.ดร. เจนนุช ว่องธวัชชัย
Dr. Thinnarat Srisuwan
น.สพ. ทินรัตน์ ศรีสุวรรณ
National Institute of Animal Health (NIAH)
สถำบันสุขภำพสัตว์แห่งชำติ
Dr. Jumroensri Thawonsuwan
ดร. จาเริญศรี ถาวรสุวรรณ
Coastal Aquatic Animal Health Research Institute
สถำบันวิจัยสุขภำพสัตว์นำชำยฝั่งสงขลำ (สสช.)
Speaker: Alternative Medicine in Aquaculture
47
Assistant Professor Dr. Siriwan Prapong
ผู้ช่วยศาสตราจารย์ สพ.ญ.ดร. ศิริวรรณ พราพงษ์
Chairman, Interdisciplinary Program in Genetic Engineering, Kasetsart University,
Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University
ประธำนคณะกรรมกำรบัณฑิตศึกษำ โครงกำรสหวิทยำกำรระดับบัณฑิตศึกษำ สำขำพันธุวิศวกรรม
มหำวิทยำลัยเกษตรศำสตร์, ภำควิชำสรีรวิทยำ คณะสัตวแพทยศำสตร์ มหำวิทยำลัยเกษตรศำสตร์
Speaker: Enhancing Food Security by Genetic Engineering
Assistant Professor Dr. Suttipun Kaewsompong
ผู้ช่วยศาสตราจารย์ ดร. สุทธิพันธุ์ แก้วสมพงษ์
Interdisciplinary Program in Genetic Engineering, Kasetsart University,
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University
โครงกำรสหวิทยำกำรระดับบัณฑิตศึกษำ สำขำพันธุวิศวกรรม มหำวิทยำลัยเกษตรศำสตร์,
ภำควิชำเทคโนโลยีชีวภำพ คณะอุตสำหกรรมเกษตร มหำวิทยำลัยเกษตรศำสตร์
Dr. Parichart Burns
ดร. ปาริชาติ เบิร์นส์
Interdisciplinary Program in Genetic Engineering, Kasetsart University,
National Center for Genetic Engineering and Biotechnology (BIOTEC)
โครงกำรสหวิทยำกำรระดับบัณฑิตศึกษำ สำขำพันธุวิศวกรรม มหำวิทยำลัยเกษตรศำสตร์,
ศูนย์พันธุวิศวกรรมและเทคโนโลยีชีวภำพแห่งชำติ
Dr. Chalinee Kongsawat
ดร. ชาลินี คงสวัสดิ์
Policy Study and Biosafety Unit,
National Center for Genetic Engineering and Biotechnology (BIOTEC),
Thailand Science Park
หน่วยศึกษำนโยบำยและควำมปลอดภัยทำงชีวภำพ
48 
Associate Professor Dr. Rod Straw
Surgical Oncology Specialist, Brisbane Veterinary Specialist Centre, Australia
Speaker: Becoming a Veterinary Oncologist: The Important Things to Know
Speaker: Clinical Small Animal Oncology Cases
Dr. Rod Straw is a registered surgical oncology specialist and has been working in
the area of veterinary oncology since 1986. He trained in the United States and was an
associate professor of oncology at Colorado State University before returning to Australia where he has been treating referred small animal cancer patients since 1995. His
expertises are in the areas of surgical, medical and radiation oncology. He has done
extensive clinical researches in chemotherapy delivery systems, bone cancer, bone
allografts, and bone morphogenesis. Dr.Straw has continuously published high quality
publications in a large number of international journals and contributed to numerous
textbooks. He is also an outstanding speaker at many iternational conferences. He
received the Smith Kline Beecham Research Award in 1991 and ASAVA Scientific Contibution Award in 2003. He also was the founder of Australia’s comprehensive cancer
centre with the Southern Hemisphere’s only Linear Accelerator dedicated to treating
animals with cancer using high energy external beam radiation. Currently, he has been
working as an oncology specialist at the Brisbane Veterinary Specialist Centre, Queensland, Australia.
Dr. Chaiyos Tanrattana
อาจารย์ น.สพ.ดร. ชัยยศ ธารรัตนะ
Moderator: Acupuncture as an Alternative for Animal Practice
Dr. Krongthong Oraveerakul
สพ.ญ. กรองทอง อรวีระกุล
Thonglor Pet Hospital
โรงพยำบำลสัตว์ทองหล่อ
Speaker: Acupuncture as an Alternative for Animal Practice
49
Associate Professor Dr. Phitaya Charupoonphol
รองศาสตราจารย์ นพ. พิทยา จารุพูนผล
Dean, Faculty of Public Health, Mahidol University
คณบดี คณะสำธำรณสุขศำสตร์ มหำวิทยำลัยมหิดล
Speaker: Acupuncture as an Alternative for Animal Practice
Associate Professor Dr. Kittisak Ajariyakhajorn
รองศาสตราจารย์ น.สพ.ดร. กิตติศักดิ์ อัจฉริยะขจร
Deputy Dean, Faculty of Veterinary Science, Chulalongkorn University
รองคณบดี คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Speaker: Current Situation of Bovine Infectious Diseases
Associate Professor Dr. Theera Rukkwamsuk
รองศาสตราจารย์ น.สพ.ดร. ธีระ รักความสุข
Dr. Aran Chanlun
อาจารย์ น.สพ.ดร. อรัญ จันทร์ลุน
Faculty of Veterinary Medicine, Khonkaen University
คณะสัตวแพทยศำสตร์ มหำวิทยำลัยขอนแก่น
Dr. Terdsak Yano
อาจารย์ น.สพ. เทิดศักดิ์ ญาโน
Faculty of Veterinary Medicine, Chiangmai University
คณะสัตวแพทยศำสตร์ มหำวิทยำลัยเชียงใหม่
50 
Associate Professor Dr. Sirintorn Yibchok-anun
รองศาสตราจารย์ สพ.ญ.ดร. ศิรินทร หยิบโชคอนันต์
Speaker: Antimicrobials Used in Small Animal Practice
Dr.Sirintorn Yibchok-anun is an associate professor at the Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, THAILAND. She
finished her D.V.M. from Chulalongkorn University. She received her Ph.D. in Veterinary
Physiology and Pharmacology from Iowa State University, Iowa, U.S.A. and also was a postdoctoral research associate there. Her researches focus on both clinical pharmacology
and pharmacokinetics. Dr.Sirintorn has continuously published high quality publications in
a large number of international journals. She is also an outstanding speaker at many conferences. Currently, she is the director of Chulalongkorn University Small Animal Hospital.
Assistant Professor Dr. Nuvee Prapasarakul
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. ณุวีร์ ประภัสระกูล
Speaker: Use of Antimicrobials by Thai Veterinarians and Bacterial Resistance Situations
Speaker: Bacterial Resistance: Problems and Solutions
Speaker: Significance of microbial sensitivity test and interpretation
Assistant Professor Dr. Nuvee Prapasarakul received his graduate degree from
Faculty of Veterinary Science, Chulalongkorn University, in 1997 and his Ph.D from Tokyo
University of Agriculture and Technology in 2003. He was granted the fellowship from
Crowford foundation, Murdoch University, Western Australia in 2006. At present, he has
been in charge for Head Department of Veterinary Microbilogy in his graduated university
and for editor of Thai Journal of Veterinary Practitioner since 2009. Assistant Professor
Dr.Nuvee Prapasarakul’s research on microbiological theories, especially diagnosis, pathogenesis and microbial controls for bacteria and fungi, are internationally recognized with
his publications, continuously.
51
Assistant Professor Dr. Kanjana Imsilp
ผู้ช่วยศาสตราจารย์ สพ.ญ.ดร. กาญจนา อิ่มศิลป์
Head, Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University
หัวหน้ำภำควิชำเภสัชวิทยำ คณะสัตวแพทยศำสตร์ มหำวิทยำลัยเกษตรศำสตร์
Moderator: Bacterial Resistance: Problems and Solutions
Dr. Kanjana currently is an assistant professor at the Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, THAILAND. She finished her D.V.M. and Ph.D. from Kasetsart University and University of Illinois at Urbana
Champaign, U.S.A., respectively. Her researches focus on pharmacokinetics and toxicokinetics, especially residues of antimicorbials and environmental toxicants in animals
and environments. She has participated and presented in several international conferences. She has also published numerous publications both nationally and internationally.
Dr. Nipattra Debavalya
อาจารย์ สพ.ญ.ดร. นิภัทรา เทพวัลย์
Speaker: Significance of Microbial Sensitivity Test and Interpretation
Speaker: Bacterial Resistance: Problems and Solutions
Associate Professor Dr. Thaweesak Songserm
รองศาสตราจารย์ น.สพ.ดร. ทวีศักดิ์ ส่งเสริม
Speaker: Overview: Duck Health Problems
Speaker: Overview: Quail Health Problems
Napaporn Senarat, DVM
สพ.ญ. นภาพร เสนารัตน์
Speaker: Rehabilitation
52 
Associate Professor Dr. Achariya Sailasuta
รองศาสตราจารย์ สพ.ญ.ดร. อัจฉริยา ไศละสูต
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University
ภำควิชำพยำธิวิทยำ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Speaker: Symposium on Canine Mast Cell Tumor
Associate Professor Dr. Anutep Rungsipipat
รองศาสตราจารย์ น.สพ.ดร. อนุเทพ รังสีพิพัฒน์
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University
ภำควิชำพยำธิวิทยำ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Sirin Theerawatanasirikul, DVM
สพ.ญ. ศิรินทร์ ธีระวัฒนศิริกุล
STAR:Molecular Biology Research on Animal Oncology
กลุ่มงำนวิจัยอณูชีววิทยำโรคมะเร็งในสัตว์ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Detachai Ketphun, DVM
น.สพ. เดชธชัย เกตุพันธุ์
STAR:Molecular Biology Research on Animal Oncology
กลุ่มงำนวิจัยอณูชีววิทยำโรคมะเร็งในสัตว์ คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Patarakrit Theewasutrakul, DVM
น.สพ. ภัทรกฤช ธีวะสุตระกูล
Animal Oncology Clinic, Small Animal Teaching Hospital,
คณะทำงำนคลินิกโรคมะเร็ง โรงพยำบำลสัตว์เล็ก คณะสัตวแพทยศำสตร์ จุฬำลงกรณ์มหำวิทยำลัย
Kasemsri Satjawiriyakul, DVM
สพ.ญ. เกษมศรี สัจจะวิริยะกุล
53
Dr. Somporn Techangamsuwan
อาจารย์ สพ.ญ.ดร. สมพร เตชะงามสุวรรณ
Assistant Professor Dr. Sompoch Weerakul
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. สมโภชน์ วีระกุล
Faculty of Veterinary Medicine, Khonkaen University
คณะสัตวแพทยศำสตร์ มหำวิทยำลัยขอนแก่น
Speaker: Answers to Problem(s) of Exotic Pets
Benchapol Lorsunyaluck, DVM
น.สพ. เบญจพล หล่อสัญญาลักษณ์
Wildlife and Exotic Pet Department,
Kasetsart University Veterinary Teaching Hospital Kampaengsan
แผนกสัตว์ปำ่ และคลีนิกสัตว์เลียงพิเศษ
โรงพยำบำลสัตว์ มหำวิทยำลัยเกษตรศำสตร์ วิทยำเขตกำแพงแสน
Speaker: Extra-label Drug Use (ELDU) in Exotic Pets
Dr.Chaowaphan Yinharnmingmongkol
น.สพ.ดร. เชาวพันธ์ ยินหาญมิ่งมงคล
Exotic Pet Clinic, Veterinary Teaching Hospital, Mahidol University
คลินิกสัตว์เลียงพิเศษ โรงพยำบำลสัตว์ประศุอำทร มหำวิทยำลัยมหิดล
Tossaporn Anuntakulnatee, DVM
น.สพ. ทศพร อนันตกุลนธี
Veterinary Teaching Hospital, Chulalongkorn University
โรงพยำบำลสัตว์เล็ก จุฬำลงกรณ์มหำวิทยำลัย
54 
Associate Professor Dr. Niwat Chansiripornchai
รองศาสตราจารย์ น.สพ.ดร. นิวัตร จันทร์ศิริพรชัย
Speaker: Infectious Coryza and Fown Cholera
Assoc.Prof.Dr.Niwat Chansiripronchai got DVM from Chulalongkorn University in
1993 and later he got MSc and PhD from SLU and Utrecht University in 2000 and 2004,
respectively. He has 18 years experience in Avian Health and Epidemiology. He published
more than 90 local and international publications and has 5 issues of textbooks. Nowadays, he is a chairperson of Graduate program in Veterinary Medicine, Chulalongkorn University.
Assistant Professor Dr. Pornchai Sanyathitiseree
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. พรชัย สัญฐิติเสรี
Speaker: Do and Don’t in Exotic Practice
Speaker: FAQs in Pet Birds and Avian
Dr.Kaset Sutacha
น.สพ. เกษตร สุเตชะ
Dr. Sirund Tuekum
น.สพ. สิรันดร์ ถึกอ่า
55
Assistant Professor Dr. Somchai Sajapitak
ผู้ช่วยศาสตราจารย์ น.สพ.ดร. สมชัย สัจจาพิทักษ์
Director, Kasetsart University Veterinary Teaching Hospital Nong Po
ผู้อำนวยกำรโรงพยำบำลสัตว์ มหำวิทยำลัยเกษตรศำสตร์ หนองโพ
Speaker: Update: Bovine Hormone
Dr. Somchai Sajapitak is an assistant professor in the Department of Large Animal
and Wildlife Medicine, Faculty of Veterinary Medicine, Kasetsart university. He received his
DVM from Kasetsart University. He later furthered his study and finished both MSc and
PhD from Nagoya University, Japan. His research focuses on large animal medicine. He
also is in an administrative position as the Director of Kasetsart University Veterinary
Teaching Hospital, Nongpho, Faculty of Veterinary Medicine, Kasetsart University.
Assistant Professor Adisorn Yawongsa
ผู้ช่วยศาสตราจารย์ น.สพ. อดิศร ยะวงศา
Director, Kasetsart University Veterinary Teaching Hospital Kampaengsan
ผู้อำนวยกำรโรงพยำบำลสัตว์ มหำวิทยำลัยเกษตรศำสตร์ วิทยำเขตกำแพงแสน
Speaker: Evidence-based Veterinary Medicine
Dr. Veerasak Punyapornwithaya
อาจารย์ น.สพ.ดร. วีระศักดิ์ ปัญญาพรวิทยา
Speaker: Epidemiology - Mastitis
Dr. Sukolrat Boonyayatra
อาจารย์ สพ.ญ.ดร. ศุกลรัตน์ บุญยยาตรา
Speaker: Update: Mastitis
Oral and Poster Presentation
Abstract
59
TABLE OF CONTENTS
ORAL AND POSTER PRESENTATION ABSTRACT .......................................................................... 69
ORAL PRESENTATION ABSTRACTS .............................................................................................. 70
1.TURMERIC THERAPY IN AFLATOXICOSIS OF BROILERS PATHOLOGIC STUDY............................ 71
2.CLONING AND CHARACTERIZATION OF SWAMP BUFFALO INTERFERON3.PRRSV CONTROL THROUGH MANAGEMENT OF PIGFLOW (PIGIMUNOFLOW) ........................ 73
4.INVESTIGATION OF BOVINE LEUKOCYTE ADHESION DEFICIENCY (BLAD) IN THAI
HOLSTEIN CATTLE ......................................................................................................................... 74
5.PREVALENCE SURVEY OF INTESTINAL AND BLOOD PARASITES IN DAIRY CATTLE IN
KAENG KA JAN DISTRICT, PETCHABURI PROVINCE, THAILAND .................................................... 75
6.INTESTINAL PARASITISM IN MONKEYS AT WILD LIFE FRIEND FOUNDATION,
MT. LOUKCHANG, PETCHABURI, THAILAND ................................................................................ 76
7.A COMPARISON OF FOOD-POISONING BACTERIAL CONTAMINATION ON FOOD
BETWEEN EUROPEAN COUNTRIES AND JAPAN ........................................................................... 77
8.COMPARISON OF MLST AND REP-PCR SYSTEM FOR DIFFERENTIATION OF
CAMPYLOBACTER JEJUNI ISOLATED FROM BROILER IN CHIANG MAI, THAILAND ……….............. 78
9.ROLE OF DOMESTIC ANIMALS IN CHIKUNGUNYA VIRUS ECOLOGY IN AN
ENDEMIC AREA IN THAILAND ..................................................................................................... 79
10.ESTIMATION OF CRITICAL PROPORTION FOR VACCINATION TO PREVENT
MAJOR OUTBREAKS OF HPAI H5N1 ............................................................................................ 80
11.ANIMAL-ASSISTED THERAPY FOR PERSONS WITH DISABILITIES VIA
BRAIN-COMPUTER INTERFACE SYSTEM ...................................................................................... 81
12.ESTABLISHMENT OF IN VITRO CULTURE OF SPERMATOGONIAL STEM CELL-LIKE
COLONY IN PUBERTAL DOMESTIC CAT (FELIS CATUS) ................................................................. 82
13.COMPARISON OF LOW-COST LONG-TERM FREEZING MEDIA FOR MICE
PANCREATIC ISLET CELLS ............................................................................................................. 83
60 
14.ORF5 GENETIC MODIFICATION OF PRRSV IN A MODIFIED LIVE PRRSV
VACCINATED SWINE FARM FOLLOWING AN OUTBREAK ........................................................... 84
15.VIRULENCE GENES PROFILING OF STREPTOCOCCUS AGALACTIAE STRAINS
ISOLATED FROM TILAPIAS (OREOCHROMIS SP.) AND THEIR CULTURING
ENVIRONMENTS IN THALAND .................................................................................................... 85
16.CONTRIBUTION OF THE MULTIDRUG EFFLUX SYSTEM MEXXY-OPRM
IN AMINOGLYCOSIDES RESISTANCE IN PSEUDOMONAS AERUGINOSA
CLINICAL ISOLATES FROM DOGS AND CATS ……………………………………………………………………....... 86
17.ANTIBIOTIC RESISTANCE AND VIRULENCE GENES OF ESCHERICHIA COLI
ISOLATED FROM SWINE ............................................................................................................ 87
18.CHARACTERIZATION OF CAMPYLOBACTER SPP. ISOLATED FROM
BROILER FLOCKS IN CHIANG MAI, THAILAND ........................................................................... 88
19.PARASITIC FOOD-BORNE ZOONOSES: AS THREAT FOR PUBLIC HEALTH
IN SOUTHEAST ASIA................................................................................................................... 89
20.CLONING AND SEQUENCING OF SIALOADHESIN AND CD163 CDNA FROM
THE FIELD PRRSV INFECTED PORCINE ALVEOLAR MACROPHAGES .......................................... 90
POSTER PRESENTATION ABSTRACTS ....................................................................................... 91
1.PEGYLATED LIPOSOMAL DOXORUBICIN-INDUCED PALMAR-PLANTAR
ERYTHRODYSTHESIA (HAND-FOOT SYNDROME) IN A NASAL CARCINOMA DOG .................... 92
2.EFFECT OF SOLID SURFACE VITRIFICATION ON MODIFICATION OF
LECTIN-BINDING CARBOHYDRATE CHAINS IN ZONA PELLUCIDA OF
CANINE CUMULUS-OOCYTE-COMPLEX ………………………………………………………………………………. 93
3.FOUR AFLATOXIN ANALOGUES IN DOG FOOD: A PRELIMINARY MONITORING .................. 94
4.MORPHOLOGY OF CAT TESTICULAR TISSUE AFTER CRYOPRESERVATION USING
DIFFERENT PROTOCOLS .......................................................................................................... 95
5.DIAGNOSIS OF ENCEPHALITOZOON INFECTION IN 2 LIVING RABBITS
AND SUCCESSFUL SURGICAL TREATMENT: A CASE REPORT .................................................. 96
6.NEW ADVANCE IN THE TREATMENT OF ACUTE PRIMARY GLAUCOMA
IN DOGS IN THAILAND: DIODE LASER TRANSSCLERAL CYCLOPHOTOCOAGULATION............. 97
61
7.CHARACTERISTICS OF GENTAMICIN-COATED HIGH POROUS CALCIUM SULFATE
BASED DELIVERY SYSTEM ........................................................................................................ 98
8.REFERENCE RANGE OF PROSTATIC SIZE AND VOLUME IN NORMAL HEALTHY
DOGS RELATED TO THE BODYWEIGHT .................................................................................... 99
9.INFLUENCE OF AGE ON CARDIOVASCULAR EFFECT OF PROPOFOL IN HEALTHY DOGS….....100
10.DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS PSEUDINTERMEDIUS
SCC MEC TYPEIII FROM CAT URINE ........................................................................................ 101
11.BLOOD COAGULATION FACTORS DEFECT IN CANINE EHRLICHIOSIS ……........................... 102
12.NITRIC OXIDE LEVELS IN CANINE GRADE II MAST CELL TUMORS ...................................... 103
13.GLUTATHIONE LEVELS IN CANINE MAMMARY CANCER ................................................... 104
14.BLOOD TRANSFUSION IN THAI DOMESTIC CATS WITH BLOOD FROM
DIFFERENT SPECIES ................................................................................................................ 105
15.DETERMINATION OF BLOOD TYPING POPULATION AND INCIDENCES OF
FELINE IMMUNODEFICIENCY VIRUS (FIV) AND FELINE LEUKEMIA VIRUS (FELV)
IN 5 BREEDS OF THAI DOMESTIC CATS................................................................................... 106
16.COMPARISON BETWEEN HEMODIALYSIS AND PERITONEAL DIALYSIS MANAGEMENT
IN RENAL INSUFFICIENCY DOGS. ........................................................................................... 107
17.COMMON MALIGNANT TUMORS IN DOGS IN BANGKOK: RETROSPECTIVE
STUDY DURING JUNE 2010 TO MAY 2011 ............................................................................ 108
18.CASE REPORT: THE CANINE SYSTEMIC ATHROSCLEROSIS ............................................... 109
19.INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) AND EPIDERMAL GROWTH
FACTOR (EGF) IMPROVE THE DEVELOPMENTAL COMPETENCE OF FELINE EMBRYOS
CULTURED SINGLY …………………………………………………………………………………………………………...110
20.A RELEVANCE OF OCT 4-IMMUNOHISTOCHEMICAL STAINING PATTERNS TO
THEIR RESPONSIBLE ON CANINE MAST CELL TUMORS GRADES: A PRELIMINARY STUDY ....111
21.EXTERNAL AUDITORY CANAL IN POSTMORTEM INTERVAL (PMI) ESTIMATION
USING HIGH PRECISION THERMOCOUPLE: A PILOT STUDY ................................................. 112
62 
22.SUBCUTANEOUS TISSUE GAS FORMATION; A GOOD INDICATOR OF ELAPSED
TIME SINCE DEATH. ..........................................................................................................113
23.POSTMORTEM RADIOGRAPHIC DIAGNOSIS OF RADIO-OPAQUE FOREIGN BODY
IN THE PROXIMAL DUODENUM OF A CANINE CARCASS ................................................. 114
24.RIBONUCLEIC ACIDS CONCENTRATION IN POSTMORTEM INTERVAL (PMI)
ESTIMATION IN DOGS IN THE TROPICS ........................................................................... 115
25.OPTIMIZED ELUTION OF CEFAZOLIN FROM CALCIUM SULFATE BEADS ..................... 116
26.RAPID FROZEN OOCYSTS AGAINST CAECAL COCCIDIOSIS IN BROILERS ..................... 117
27.EFFECTS OF PELLETING METHODS ON FERTILITY RATE OF SEMEN FROM
THAI NATIVE COCKS ......................................................................................................... 118
28.SELECTION OF LACTIC ACID BACTERIA ISOLATED FROM DUCK FOR
PROBIOTICS ADJUNCTS .................................................................................................... 119
29.ANTIBODY RESPONSE TO ACTINOBACILLUS PLEUROPNEUMONIAE IN
CHRONICALLY INFECTED PIG FARMS IN THAILAND .......................................................... 120
30.EFFICACY OF THE GLUTARALDEHYDE-QUATERNARY AMMONIUM COMPOUND,
DUALGUARD 20®, ON DISINFECTING PIG BUILDING .......................................................... 121
31.SURVIVAL OF ACTINOBACILLUS PLEUROPNEUMONIAE UNDER CONTROLLED
LABORATORY CONDITIONS. .............................................................................................. 122
32.INTERPRETATION THE RESULTS OF THE COMMERCIAL ELISA H1N1 AND H3N2
KITS AND HEMAGGLUTINATION INHIBITION ASSAY USING LOCAL THAI SIV
H1 AND H3 VIRUS ANTIGENS ............................................................................................. 123
33.DETECTION OF MYCOPLASMA HYORHINIS FROM LUNG SAMPLES OF
SEROSITIS NURSERY PIGS ................................................................................................... 124
34.EFFECTS OF DIETARY CHITO-OLIGOSACCHARIDE ADDITIVES ON GROWTH
PERFORMANCE AND ILEAL NUTRIENT DIGESTIBILITY IN WEANING PIGS ...........................125
35.EVIDENCE OF HP-PRRS VIRUS WATER TRANSMISSION IN THAI SWINE FARM .............. 126
63
36.DETECTION OF APXIV GENE IN A. PLEUROPNEUMONIAE ISOLATES FROM
THAI FATTENING PIGS IN 2011 ........................................................................................... 127
37.CURRENT INFORMATION ON PREVALENCE AND ANTIMICROBIAL
SUSCEPTIBILITY OF AEROMONAS SP. AND EDWARDSIELLA TARDA
ISOLATED FROM DISEASED GOLDFISH (CARASSIUS AURATUS) ........................................... 128
38.DETERMINATION OF SERUM CALCIUM CONCENTRATION IN
PERIPARTURIENT DAIRY COWS ............................................................................................ 129
39.PATHOGENICITY TO MEKONG GIANT CATFISH EGGS IN LABORATORY OF
WATER MOULDS ISOLATED FROM MEKONG GIANT CATFISH EGGS AND
THE REARING WATER ……………………………………………………………………………………………………...130
40.LAPAROSCOPIC OVARIECTOMY IN NATIVE-SAANEN GOATS ...........................................131
41.RISK FACTORS CONTRIBUTING TO ALCOHOL TEST POSITIVE IN SMALL
DAIRY HERDS IN SUPHANBURI PROVINCE, THAILAND ......................................................... 132
42.SOMATIC CELL COUNT AND MILK COMPOSIITON OF DAIRY COWS
KEPT BY SMALL-SCALE HOLDERS IN CENTRAL THAILAND .................................................... 133
43.INTRAMAMMARY INFECTION AT DRY OFF IN TWO LARGE DAIRY HERDS ...................... 134
44.EFFECT OF COLOSTRAL QUALITY ON SERUM PROTEIN IN DAIRY CALVES
RAISED IN SMALL HOLDER FARMS ...................................................................................... 135
45.THE SURVIVAL OF BACTERIAL MASTITIS PATHOGEN IN MILK SAMPLES ........................ 136
46.POLYMERASE CHAIN REACTION BASE-PREVALENCE OF
ENCEPHALITOZOON CUNICULI IN MEAT RABBITS ............................................................... 137
47.COMPARATIVE MORPHOMETRY AND ULTRASTRUCTURE OF HEPATOZOON
SPECIES INFECTED IN MANGROVE SNAKE (BOIGA DENDROPHILA MELANOTA)
AND KING COBRA (OPHIOPHAGUS HANNAH) ..................................................................... 138
48.RECENT TREND OF FOOD BORNE DISEASES IN JAPAN ................................................... 139
49.MOUSE STRAIN DIFFERENCES IN SUSCEPTIBILITY TO DIARRHETIC
SHELLFISH POISONING TOXINS, IN MOUSE BIOASSAY ........................................................ 140
64 
50.MICROBIOLOGICAL QUALITY AND ANTIBIOTIC RESIDUE OF BOILED
MILK IN BANGKOK AND METROPOLITAN REGION ...........................................................141
51.CULTIVATION OF JAVAN PORCUPINE (HYSTRIX JAVANICA) WHICH IS AN
ORIGIN SPECIES EXPECTED ANIMAL OF INDONESIA TO REINFORCE NATIONAL
FOOD SECURITY AND SOLVE THE PROBLEM OF ORGANIC WASTE....................................142
52.THE EVALUATION OF PERFORMANCE OF VETERINARY SERVICES (OIE PVS)
OF THAILAND: WHAT IS THE OIE PVS TOOL? .....................................................................143
53.SEROLOGIC SURVEY ON BARTONELLA SPP. AMONG VETERINARY PROFESSIONALS.....144
54.ANTIMICROBIAL RESISTANT BACTERIA FROM DOGS ISOLATED AT THE
VETERINARY DIAGNOSTIC UNIT, KASETSART UNIVERSITY ................................................. 145
55.THE PREVALENCE AND GENOTYPES OF CRYPTOSPORIDIUM SPP.
FROM WATER BUFFALOES IN KHON KAEN PROVINCE ....................................................... 146
56.MOLECULAR DETECTION OF TRYPANOSOMA EVANSI FROM SLAUGHTERED
COWS IN KHON KAEN PROVINCE ....................................................................................... 147
57.A NEW GENOTYPE OF BARTONELLA ISOLATES FROM DEER (RUSA TIMORENSIS)
IN THAILAND ...................................................................................................................... 148
58.THE EVALUATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
FOR EHRLICHIA CANIS DETECTION ..................................................................................... 149
59.VILLI AND CRYPT RATIO OF PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV)
INFECTED PIGLETS .............................................................................................................. 150
60.EPIDEMIOLOGY OF BOVINE VIRAL DIARRHEA VIRUS INFECTION IN
DAIRY FARMS IN NORTHEASTERN THAILAND .................................................................... 151
61.RELEVANT EVIDENCES FOR THE DIAGNOSIS OF ATYPICAL PESTIVIRUS
INFECTION IN A CALF ......................................................................................................... 152
62.IDENTIFICATION OF A PUTATIVE LRR CODING GENE IN L. INTERROGANS ................... 153
65
ORAL AND POSTER PRESENTATION FULL PAPER ................................................................154
ORAL PRESENTATION FULL PAPER ..................................................................................... 155
CLONING AND CHARACTERIZATION OF SWAMP BUFFALO INTERFERONINVESTIGATION OF BOVINE LEUKOCYTE ADHESION DEFICIENCY (BLAD) IN
THAI HOLSTEIN CATTLE ....................................................................................................... 158
PREVALENCE SURVEY OF INTESTINAL AND BLOOD PARASITES IN DAIRY CATTLE IN
KAENG KA JAN DISTRICT, PETCHABURI PROVINCE, THAILAND ........................................... 162
INTESTINAL PARASITISM IN MONKEYS AT WILD LIFE FRIEND FOUNDATION,
MT. LOUKCHANG, PETCHABURI, THAILAND ....................................................................... 164
A COMPARISON OF FOOD-POISONING BACTERIAL CONTAMINATION ON
FOOD BETWEEN EUROPEAN COUNTRIES AND JAPAN ........................................................ 166
COMPARISON OF MLST AND REP-PCR SYSTEM FOR DIFFERENTIATION OF
CAMPYLOBACTER JEJUNI ISOLATED FROM BROILER IN CHIANG MAI, THAILAND............... 168
ROLE OF DOMESTIC ANIMALS IN CHIKUNGUNYA VIRUS ECOLOGY IN AN
ENDEMIC AREA IN THAILAND .............................................................................................. 170
ANIMAL-ASSISTED THERAPY FOR PERSONS WITH DISABILITIES VIA BRAIN-COMPUTER
INTERFACE SYSTEM .............................................................................................................. 172
ESTABLISHMENT OF IN VITRO CULTURE OF SPERMATOGONIAL STEM CELL-LIKE
COLONY IN PUBERTAL DOMESTIC CAT (FELIS CATUS) ......................................................... 174
COMPARISON OF LOW-COST LONG-TERM FREEZING MEDIA FOR
MICE PANCREATIC ISLET CELLS ............................................................................................ 176
VIRULENCE GENES PROFILE OF STREPTOCOCCUS AGALACTIAE STRAINS
ISOLATED FROM TILAPIAS (OREOCHROMIS SP.) AND THEIR CULTURING
ENVIRONMENTS IN THAILAND ............................................................................................ 178
ANTIBIOTIC RESISTANCE AND VIRULENCE GENES OF ESCHERICHIA COLI
ISOLATED FROM SWINE ...................................................................................................... 183
66 
CHARACTERIZATION OF CAMPYLOBACTER SPP. ISOLATED FROM BROILER FLOCKS
IN CHIANG MAI, THAILAND ................................................................................................. 185
CLONING AND SEQUENCING OF SIALOADHESIN AND CD163 CDNA FROM
THE FIELD PRRSV INFECTED PORCINE ALVEOLAR MACROPHAGES ..................................... 189
POSTER PRESENTATION FULL PAPER ................................................................................. 191
PEGYLATED LIPOSOMAL DOXORUBICIN-INDUCED PALMAR-PLANTAR
ERYTHRODYSTHESIA (HAND-FOOT SYNDROME) IN A NASAL CARCINOMA DOG ................ 192
REFERENCE RANGE OF PROSTATIC SIZE AND VOLUME IN NORMAL HEALTHY DOGS
RELATED TO THE BODYWEIGHT .......................................................................................... 194
DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS PSEUDINTERMEDIUS
SCC MEC TYPEIII FROM CAT URINE ..................................................................................... 196
BLOOD COAGULATION FACTOR DEFECT IN CANINE EHRLICHIOSIS .................................... 198
NITRIC OXIDE LEVELS IN CANINE GRADE II MAST CELL TUMORS ........................................ 201
GLUTATHIONE LEVELS IN CANINE MAMMARY CANCER ..................................................... 204
DETERMINATION OF BLOOD TYPING POPULATION AND INCIDENCES OF FELINE
IMMUNODEFICIENCY VIRUS (FIV) AND FELINE LEUKEMIA VIRUS (FELV)
IN 5 BREEDS OF THAI DOMESTIC CATS................................................................................. 206
COMPARISON BETWEEN HEMODIALYSIS AND PERITONEAL DIALYSIS MANAGEMENT
IN RENAL INSUFFICIENCY DOGS. ......................................................................................... 208
COMMON MALIGNANT TUMORS IN DOGS IN BANGKOK: RETROSPECTIVE STUDY
DURING JUNE 2010 TO MAY 2011 ...................................................................................... 210
CASE REPORT: THE CANINE SYSTEMIC ATHEROSCLEROSIS IN A DOG ................................. 212
INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) AND EPIDERMAL GROWTH FACTOR (EGF) IMPROVE
THE DEVELOPMENTAL COMPETENCE OF FELINE EMBRYOS CULTURED SINGLY ……………….214
EXTERNAL AUDITORY CANAL IN POSTMORTEM INTERVAL (PMI) ESTIMATION
USING HIGH PRECISION THERMOCOUPLE: A PILOT STUDY ................................................ 216
67
SUBCUTANEOUS TISSUE GAS FORMATION, AN INDICATOR OF ELAPSED TIME
SINCE DEATH. ...................................................................................................................... 218
POSTMORTEM RADIOGRAPHIC DIAGNOSIS OF RADIO-OPAQUE FOREIGN BODY IN
THE PROXIMAL DUODENUM OF A CANINE CARCASS ......................................................... 220
RAPID FROZEN OOCYSTS AGAINST CAECAL COCCIDIOSIS IN BROILERS .............................. 222
EFFECTS OF PELLETING METHODS ON FERTILITY RATE OF SEMEN FROM THAI
NATIVE COCKS ..................................................................................................................... 224
SELECTION OF LACTIC ACID BACTERIA ISOLATED FROM DUCK FOR
PROBIOTICS ADJUNCTS ....................................................................................................... 226
ANTIBODY RESPONSE TO ACTINOBACILLUS PLEUROPNEUMONIAE IN
CHRONICALLY INFECTED PIG FARMS IN THAILAND ............................................................. 228
SURVIVAL OF ACTINOBACILLUS PLEUROPNEUMONIAE UNDER CONTROLLED
LABORATORY CONDITIONS……………………………………………………………………………………………...230
INTERPRETATION THE RESULTS OF THE COMMERCIAL ELISA H1N1 AND H3N2 KITS
AND HEMAGGLUTINATION INHIBITION ASSAY USING LOCAL THAI SIV H1 AND H3
VIRUS ANTIGENS ................................................................................................................. 232
DETECTION OF MYCOPLASMA HYORHINIS FROM LUNG SAMPLES OF SEROSITIS
NURSERY PIGS ..................................................................................................................... 234
EFFECTS OF DIETARY CHITO-OLIGOSACCHARIDE ADDITIVES ON GROWTH
PERFORMANCE AND ILEAL NUTRIENT DIGESTIBILITY IN WEANING PIGS………………………….236
EVIDENCE OF HP-PRRS VIRUS WATER TRANSMISSION IN THAI SWINE FARM ................... 238
DETECTION OF APXIV GENE IN A. PLEUROPNEUMONIAE ISOLATES FROM THAI
FATTENING PIGS IN 2011 .................................................................................................... 240
DETERMINATION OF SERUM CALCIUM CONCENTRATION IN PERIPARTURIENT
DAIRY COWS ....................................................................................................................... 242
68 
PATHOGENICITY TO MEKONG GIANT CATFISH EGGS IN LABORATORY OF
WATER MOULDS ISOLATED FROM MEKONG GIANT CATFISH EGGS AND
THE REARING WATER ………………………………………………………………………………….................... 244
LAPAROSCOPIC OVARIECTOMY IN NATIVE-SAANEN GOATS .............................................. 251
INFLUENCE OF COLOSTRAL QUALITY ON SERUM PROTEIN IN DAIRY CALVES RAISED
IN SMALL HOLDER FARMS……………………………………………………………………………………………….253
POLYMERASE CHAIN REACTION BASE-PREVALENCE OF ENCEPHALITOZOON CUNICULI
IN MEAT RABBITS ............................................................................................................... 255
COMPARATIVE MORPHOMETRY AND ULTRASTRUCTURE OF HEPATOZOON
SPECIES INFECTED IN MANGROVE SNAKE (BOIGA DENDROPHILA MELANOTA) AND
KING COBRA (OPHIOPHAGUS HANNAH) ............................................................................. 257
RECENT TREND OF FOOD BORNE DISEASES IN JAPAN ........................................................ 260
MOUSE STRAIN DIFFERENCES IN SUSCEPTIBILITY TO DIARRHETIC SHELLFISH
POISONING TOXINS, IN MOUSE BIOASSAY ......................................................................... 262
SEROLOGIC SURVEY ON BARTONELLA SPP. AMONG VETERINARY PROFESSIONALS .......... 264
ANTIMICROBIAL RESISTANT BACTERIA FROM DOGS ISOLATED AT THE VETERINARY
DIAGNOSTIC UNIT, KASETSART UNIVERSITY ....................................................................... 266
A NEW GENOTYPE OF BARTONELLA ISOLATES FROM DEER (RUSA TIMORENSIS) IN
THAILAND ............................................................................................................................ 269
THE EVALUATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR
EHRLICHIA CANIS DETECTION .............................................................................................. 271
IDENTIFICATION OF A PUTATIVE LRR CODING GENE IN L. INTERROGANS .......................... 273
ORAL AND POSTER PRESENTING AUTHORS INDEX .............................................................. 274
Oral Presentation Abstracts
71
TURMERIC THERAPY IN AFLATOXICOSIS OF BROILERS, PATHOLOGIC STUDY
N. Rangsaz1* , M. Gholamia Ahangaran2
1
Student of Veterinary Medicine Faculty, Member of Young Researchers Club,
Islamic Azad University, Shahrekord Branch, Shahrekord, Iran,
2
Assisstant Professor of Poultry Diseases, Faculty of Veterinary Medicine,
Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
*Corresponding author: Email: [email protected]
Aflatoxin is a secondary metabolite of many toxic fungi such as Aspergillus flavus,Aspergillus fumigatus and
A.parasiticus. This toxic metabolite can be considered as a loss agent of the poultry industry.In present study ,90
broilers divided into 4 group with three replicate equally including group (A), as negative control were used basal
diet, group (B), were used basal diet plus 3ppm productive aflatoxin, group (C), were consumed basal diet plus
0.05% ETE and group (D), received basal diet with ETE at concentration of 0.05% plus 3ppm productive aflatoxin.
Aflatoxin production by Aspergillus parasiticus (PTTC NO:1850) in maize was according to the shotwell method.
Many inhibitors and toxin binders to be exist that turmeric extract is applied as a natural substance in reducing the
lesions of aflatoxin in different organs of the broilers body. Results showed that aflatoxin in liver can induce lesions
such as hyperemia, necrosis and atrophy of hepatocytes, focal and disseminated haemorrage and chemotaxis of
macrophages in surrounding of portal vein.Using Ethanolic extract of turmeric can be useful with therapeutic
effects for alleviating the lesion in liver, Kidney and other organs histopathologicaly
Keywords : Turmeric, Aflatoxicosis, Broilers, Pathology
72 
CLONING AND CHARACTERIZATION OF SWAMP BUFFALO
INTERFERON-
*
Ukadej Boonyaprakob and Sommai Homsavart
Department of Physilogy, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
* Corresponding author : Email : [email protected]
Interferon-gamma (IFN-) cDNAs were cloned from buffy coat fractions of Thai swamp
buffaloes (Bubalus bubalis carabanesis) by RT-PCR and 3' Rapid Amplification of cDNA Ends
(RACE) PCR technique. The full-length cDNAs was 1,141 bp in length, with a 31 bp 5′UTR and a
609 bp 3′UTR. The open reading frame was 501 bp that translates into a 166 residues putative
protein with a 23 amino acid signal peptide and a mature protein with 143 amino acids.
Sequence analysis showed that the Thai swamp buffalo IFN- shared 99.4–100.0% identity at the
amino acid level to four previously published buffalo, 91.6-97.6% to other ruminants and less
than 65% to human IFN- sequences. Phylogenetic analysis based on the predicted IFN- peptide
sequences showed very close relationships among all buffalo breeds and a relatively close
relationship to other large ruminants. The signature motif of IFN- is also conserved in swamp
buffaloes IFN-. In order to study the transcriptional regulation of the swamp buffaloes IFN-, a
608 bp promoter was cloned and characterized. Results showed that the major transcription
factor binding sites were highly conserved among the IFN- promoter sequences of Thai Swamp
buffalo, Indian River buffalo, cattle, sheep, and human, indicating that regulation of IFN-
expression across species may be similar.
Keywords : Buffalo, IFN-gamma, polymorphism
73
PRRSV CONTROL THROUGH MANAGEMENT OF PIGFLOW (PIGIMUNOFLOW)
D. Nilubol*
Department of Veterinary Microbiology, Faculty of Veterinary Science,
Chulalongkorn University, THAILAND.
* Corresponding author Email : [email protected]
Previous attempt to eradicate PRRSV from PRRSV positive herd gained high degree of accomplishment
resulting in the production of PRRSV negative stock to replace old breeding stock. Several questions including duration of the herd staying free of PRRSV following the successful attempt and cost effectiveness of the protocol, however, have remained unanswerable. One attempt to keep the herd negative would be strict biosecurity preventing
the introduction of PRRSV into the herd, but this phenomenon become difficulty and unrealitic in some pig producing areas, especially in the highest pig proximity in western and eastern provinces of Thailand. PigImmunoFlow, a
concept of control PRRSV through management of pigflow according to immunity and infectious statuses of pigs,
was developed to facilitate PRRSV control in herds in area such that. PigImmunoFlow was a management protocol
implemented in the herd to reduce severity of PRRSV outbreak and control PRRSV in positive herds. PigImmunoFlow was developed based on scientific knowledge of PRRSV gained from research and field observations including
homologous and heterologous infection and immune response against such infection, transplacental infection and
dynamic of infection resulting herd characteristic based on PRRSV status. Practices in PigImmunoFlow include acclimatization and cool-down period of replacement gilts and proportion of replacement gilts or multiparous sows
introduced to breeding herd and management of pigflow to reach the concept of one-source, one-site and one
week system. Details and others managerial strategies are further discussed.
Keywords : PRRSV control
74 
INVESTIGATION OF BOVINE LEUKOCYTE ADHESION DEFICIENCY (BLAD)
IN THAI HOLSTEIN CATTLE
Kalaya Kengvikkum1*, Nattanant Sirivattanatanyakul1, Buh-nga Chindawanichsakul1
1
Bureau of Biotechnology for Animal production, Department of Livestock Development
Bovine leukocyte adhesion deficiency (BLAD) is an autosomal recessive genetic disease and mainly found in
Holstein cattle. It is caused by a point mutation (adenine to quinine at the position 383) in the gene coding subunit
CD18 for β2 integrin adhesion molecule. Affected cattle with homozygous form of gene have severe symptoms
such as ulcers of oral membranes, gingivitis, chronic pneumonia, impaired wound healing, anorexia and chronic
diarrhea. It causes economic losses to milk producers and breeders. In this study, blood and semen samples were
obtained from 220 cattle of Thai Holstein breed and isolated for DNA. The DNA materials were multiplied in PCR
using specific primer. In order to determine the area of mutation, the PCR products were digested with TaqI endonuclease enzyme. The fragments were analyzed on 2% agarose gel for the absence of a TaqI restriction site. Any
suspected samples with one TaqI restriction site must be followed by DNA sequencing technique to investigate the
incidence of the carriers. The aim of the study was to optimize PCR (Polymerase Chain Reaction), RFLP (Restriction
Fragment Length Polymorphism) and DNA sequencing analysis as a diagnostic test to identify BLAD carrier cattle.
Keywords : Bovine Leukocyte Adhesion Deficiency (BLAD), Thai Holstein cattle
75
KAENG KA JAN DISTRICT, PETCHABURI PROVINCE, THAILAND
A. Sommanustaweechai1, B. Sangkarak1, D. K. Nugroho1, F. Wu1, H. Sinel1, L. L. Bo1, N. Kiry1, P. Boosom1, P.
Wongnark1, S. I. Jayme1, S. Sinthasak1, S. Urbenjapol1, S. Khuhapan1, T. Lamaisri1, T. Kedkhuntod1, T. Srisuvan1, V. T.
Le1, W. Posuya1, S. Theraverapanya2, K. Unjit3, M. Pattiyapong3, N. Ketusing3, K. Taweeseneepitch4, K. Wongsathapornchai4, K. Chanachai4*
1
Participants of Veterinary Field Epidemiology in Action, Field Epidemiology Training Program for Veterinarian.
2
Petchaburi Provincial Livestock Office.
3
National Institute of Animal Health.
4
Field Epidemiology Training Program for Veterinarian, DEPARTMENT OF LIVESTOCK DEVELOPMENT, THAILAND.
*Corresponding author : Email : [email protected]
Intestinal and blood parasite cause a lot of losses to dairy cattle due to parasitic effect and carrier status. In
Petchaburi Province, middle part of Thailand, there are approximately ten thousand dairy cattle in 350 small-scale
dairy farms and half of them are in Kaeng Ka Jan District. Situation of intestinal and blood parasite in this area have
never been investigated. This study aimed to determine prevalence of intestinal, blood parasites and their risk factors in dairy cattle farm in Pa Deng Sub-district, Kaeng Ka Jan District. February 2011, 68 out of 154 dairy cattle
farms were selected for sample collection. Five to six cattle were selected from each farm for blood and feces collection. Intestinal parasites were determined by floatation and sedimentation techniques, while blood parasites
were identified by thin blood smear and haematocrit centrifuge techniques. Data from farms were collected using
questionnaires. Totally, 358 fecal and 382 blood samples were collected. Intestinal parasites that were found included nematodes (32%, 114/358), monensia (9%, 9/358), rumen flukes (7%, 25/358), liver flukes (4%, 14/358), and
coccidia (3%, 12/358). Theileria is only blood parasite that was identified in blood sample (19%, 71/382). Majority
of farms (96%, 65/68) routinely practiced deworming. Herd size (>20 cattle) was significantly associated with intestinal infestation (1.36, 95% CI 1.01-1.83), while the presence of Theileria significantly associated with low body condition score (1.61, 95% CI 1.01-2.56). The consequence of infestation to animal health and production performance
as well as more effective control measures should be further investigated.
Keywords : Petchaburi, intestinal parasite, blood parasite, dairy cattle
76 
MT. LOUKCHANG, PETCHABURI, THAILAND
Fanan Suksawat1*, Sineenart Chantarachart1, Pimchanok Thonglon1,
Pittaya Papirom2, Soawalak Sripakdee3, Anusak Kerdsin3, Surang Dejsirilerk3
1
Department of Veterinary Medicine, Department of Pathobiology,
Faculty of Veterinary Medicine, Khon Kaen University ,Khon Kaen, Thailand,
3
Department of Medical Sciences, Ministry of Public Health, Nontaburi,THAILAND..
2
Negligible wildlife raising remains a major problem in Thailand. One of popular animals that have been improperly reared, are monkeys. The objective of the current study was to microscopically identify intestinal parasites and blood parasites in monkeys at Wildlife Friends Foundation Thailand, Mt. Loukchang, Petchaburi Province.
Fecal and blood samples were randomly collected from 30 monkeys at the average age of 10 years. There were
7 pig-tailed macaques, 15 stump-tailed macaques, and 8 long-tailed macaques. Simple direct smear and simple
floatation methods were employed for intestinal parasitic detection, and thin blood smear was for blood parasitic
infection. Complete blood count was done to support if any obvious clinical signs. Of 30 monkeys, Strongyle type
were identified from 20 monkeys (66.66%) (0, 14, 6 from pig-tailed macaques, stump-tailed macaques, and longtailed macaques, respectively), Trichuris spp was identified from only one stump-tailed macaque (3.33%, 1/30). No
evidence of blood parasites was seen. The result suggests that Strongyle represent major intestinal parasite among
the studied monkeys. In the near future, identification of round worm species and the risk of parasitic infection
should be performed for possible zoonotic disease indication.
Keywords : Monkeys, intestinal parasite, blood parasite, Strongyle type, Trichuris spp
77
A COMPARISON OF FOOD-POISONING BACTERIAL CONTAMINATION ON FOOD
BETWEEN EUROPEAN COUNTRIES AND JAPAN
Hodaka Suzuki 1*, Shigeki Yamamoto 1
1
Division of Biomedical Food Research, National Institute of Health Sciences, JAPAN.
“Shokuhin no shokuchudokukin osenjittai chosa (The national survey of food-poisoning bacterial contamination on food)” has been performed annually in Japan since 1998. This surveillance is thought to be the useful
baseline study of bacterial contamination on food in Japan. On the other hand, “The community summary report
on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union” published
by the European Food Safety Authority (EFSA) is the annual surveillance report about zoonoses, zoonotic agents
and food-borne outbreaks in the EU based on the Directive 2003/99/EC. The results of these annual surveillances
were summarized for comparing the baseline of bacterial contamination on food between European countries and
Japan. Only Salmonella, Campylobacer and Escherichia coli O157 contamination, which are examined in the both
annual surveillances, were compared for 4 years (from 2005 to 2008). The contamination levels of these foodpoisoning bacteria on food were almost comparable between European countries and Japan, except for Salmonella
in chicken meat and Salmonella in sprouts. The percentage of Salmonella contamination in chicken was higher in
Japan compared to Europe. For example, in chicken meat, the percentage of Salmonella contamination was 6.4% in
average in Europe but 46.7% in Japan and, in minced chicken meat, the percentage of contamination was 6.6% in
average in Europe but 36.6% in Japan. In contrast, the percentage of Salmonella contamination in sprouts was
higher in Europe (2.1% in average) compared to Japan (0.1%).
Keywords : Food-poisoning bacteria
78 
CAMPYLOBACTER JEJUNI ISOLATED FROM BROILER IN CHIANG MAI, THAILAND
Nipa Chokesajjawatee1 Sarinya Pornaem1 Karl-Hans Zessin2 Thomas Alter3
Chomporn Chokboonmongkol4 Prapas Patchanee5*
1
National Center for Genetic Engineering and Biotechnology,
National Science and Technology Development Agency, Pathumthani, THAILAND,
2
Department Panel, Veterinary Public Health,
3
Institute of Food Hygiene, Free University Berlin, GERMANY,
4
Animal Health and Technical Service Office,
Bangkok Agro-Industrial Products Public CO., LTD, Bangkok, THAILAND,
5
Veterinary Public Health Center for Asia Pacific (VPHCAP),
Faculty of Veterinary Medicine, Chiang Mai University, THAILAND
* Corresponding author: E-mail: [email protected]
Campylobacter (C) jejuni is one of the most common foodborne pathogen caused of human enteritis
worldwide. Consumption of poultry product is an epidemiological factor for human campylobacteriosis which is
also a major concern in veterinary public health. Many molecular methods identifying a clonal differentiation
between C. jejuni have been reported. In addition to the discriminatory power of the method, other aspects such
as speed, simplicity and cost effectiveness are also important criteria to consider for epidemiological investigation
of Campylobacter infection. Multi Locus Sequence Typing (MLST) is an excellent subtyping method in the examination of global epidemiological study for foodborne pathogens. However, the cost and labor associated with the
MLST technique hinder its use in limited budget situation or in large-scale epidemiological survey. The MLST work
included amplification of seven house-keeping genes from the bacterial isolate, PCR product checking and purification, sequencing of each gene, and identification of the sequence type. Repetitive elements-based PCR (rep-PCR)
fingerprinting, with the use of simple PCR amplification and agarose gel electrophoresis procedure was adopted as
an alternative subtyping technique for C. jejuni in this study. This method employs Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence and a tri-nucleotide repeats GTG as the amplification targets. After amplification,
the PCR products were subjected to a simple agarose gel electrophoresis to generate DNA fingerprint patterns.
With a high throughput capability of the PCR and electrophoresis techniques, hundreds of sample could be
analyzed and the result could be obtained in the following day by a single investigator. In this study, verification
and validation of rep-PCR for C. jejuni subtyping was assessed. To understand a molecular epidemiology and to
compare molecular strain typing method, representing 16 C. jejuni isolated from broiler were analyzed using MLST
and rep-PCR. MLST was able to differentiate up to 9 sequence types (STs), while rep-PCR also provided 9 clonal
clusters with a similarity index cut-off value at 0.98. Clonal identification of all 16 C. jejuni isolates was identical for
both techniques. The rep-PCR typing method as shown in this study also demonstrated an equal discriminatory
power as that of MLST (0.8917). In conclusion, rep-PCR can be used to differentiate among different strains of
C. jejuni and provide sufficient epidemiological determination of both phylogenetic inference and clonal
differentiation of Campylobacter strains.
Keywords : Campylobacter, Broiler, Rep-PCR, MLST
79
ROLE OF DOMESTIC ANIMALS IN CHIKUNGUNYA VIRUS ECOLOGY
IN AN ENDEMIC AREA IN THAILAND
Sonthaya Tiawsirisup1*, Woraporn Sukhumavasi1, Theerayudh Sukmee2, Mathirut Mungthin3, Saovanee Leelayoova3, Tawee Naaglor3, Sakultip Amsakul4, Roongroj Thanawongnuwech1
1
Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, THAILAND,
2
Department of Microbiology, Phramongkutklao College of Medicine, THAILAND,
3
Department of Parasitology, Phramongkutklao College of Medicine, THAILAND,
4
Vector-borne Disease Control Center 11.3, Surat Thani,
Office of Disease Prevention and Control 11, Nakhon Si Thammarat, Ministry of Public Health, Thailand.
Chikungunya virus (CHIKV) is an emerging or re-emerging mosquito borne virus that can be found in several
countries in Asia and Africa. CHIKV belongs to Alphavirus genus of the Togaviridae family, and it is an enveloped,
single-stranded, positive-sense RNA virus. This virus was first discovered in Tanzania, east Africa in 1952 and was
identified in Thailand in 1958. This study was conducted to investigate the role of domestic animals in the
epidemiology of this virus in the epidemic areas. Evidence of CHIKV infection in dogs, cats, and monkeys were studied. One hundred and six serum samples were collected from Phangnga and Nakhon Si Thammarat province, southern Thailand during the epidemic of CHIKV. Fifteen monkey sera, 38 dog sera, and 20 cat sera were collected from
Phangnga province. Nineteen dog sera and 14 cat sera were collected from Nakhon Si Thammarat province. Viral
nucleic acid were extracted from individual serum sample and tested for CHIKV by using reverse transcription
polymerase chain reaction; however, no CHIKV nucleic acid was detected from all tested serum samples.
Keywords : Chikungunya virus, Domestic animals, Infection
80 
ESTIMATION OF CRITICAL PROPORTION FOR VACCINATION TO PREVENT
MAJOR OUTBREAKS OF HPAI H5N1
Thanawat Tiensin1*, Wandee Kongkaew2, Karoon Chanachai1, Thaweesak Songserm3,
Mirjam Nielen4, and Arjan Stegeman4
1
Bureau of Disease Control and Veterinary Services, Department of Livestock Development, THAILAND,
Regional Veterinary Research and Development Center, Department of Livestock Development, THAILAND,
3
Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND, and
4
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, THAILAND.
2
Transmission dynamics of H5N1 avian influenza virus within a poultry population have not been
determined during the recent epidemic in Asia. This study aimed at quantifying the transmission of H5N1 virus
within flocks during the epidemic in Thailand to estimate the transmission rate parameter (ß) and the basic reproduction number (R0). A comparison of transmission dynamic using experimental and field observations was
conducted. Although vaccination against HPAI H5N1 is applied widely, the number of regions where it successfully
eliminated virus infections is not satisfied. We derived the critical fraction of chickens needed to be immunized to
stop within-flock virus transmission using experimental and observational data.
The within-flock transmission of HPAI H5N1 virus in infected chicken flocks during the outbreaks in 2004 in
Thailand was quantified using a Generalized Linear Model. Assuming a 1-day and 4-day infectious period, the
accompanying R0 was 2.26 and 2.64, respectively (95% CI’s from 1.92-5.00). In addition, a transmission experiment
was estimated using a Maximum Likelihood estimator and R0 was quantified at 2.4 (95% CI 0.61-9.94). Using an
effective vaccine the critical proportion of a flock needed to be immunized is 1–1/R0. Taking the R0 point estimates,
this indicates that at least 60% of the flock has to be immunized successfully. However, to be on the safe side the
upper limits of the CI’s indicate that at least 90% (experimental data) or 80% (observational data) vaccination
coverage is needed. This technique can be applied to estimate the vaccine coverage in other infection diseases.
Keywords : Transmission dynamic, HPAI H5N1, R0
81
ANIMAL-ASSISTED THERAPY FOR PERSONS WITH DISABILITIES
VIA BRAIN-COMPUTER INTERFACE SYSTEM
Warangkhana Phanwanich1,2*, Yodchanan Wongsawat1
1
Department of Biomedical Engineering, Faculty of Engineering, Mahidol University, Thailand.
2
Animal Supplement & Pharmaceutical Co., Ltd., Vet Products Group, Thailand
Animal-assisted therapy (AAT) is the science that employs the merit of human-animal interaction to
alleviate the mental and physical problems of persons with disabilities. However, to achieve this goal of the AAT for
persons with severe disabilities (e.g. spinal cord injury, stroke, or amyotrophic lateral sclerosis), brain-computer
interface (BCI) system needs to be proposed. In this study, we propose the use of the steady-state visual evoked
potential (SSVEP) signals that yield natural responses to the visual stimulation at specific frequencies to interface
the disable person’s need to the animal without moving the body. Therefore, the disable person can command the
well-trained animals (e.g. dogs) via the proposed four designed commands, i.e., calling the dog’s name, playing
with the dog via greeting sound, giving the reward to the dog via the automatic feeding system, and telling the dog
to call for assistance. Since the severe persons with disabilities cannot move at all, the proposed BCI system also
integrates our previously developed real-time animal language interpretation system to enhance the two way
communication between the dog and the disable person. The accuracy of the proposed BCI system for AAT is
approximately 80% and the average emotional recognition rate in real dog is 100%.
Keywords : Animal-assisted therapy, persons with disabilities, brain-computer interface
82 
ESTABLISHMENT OF IN VITRO CULTURE OF SPERMATOGONIAL
STEM CELL-LIKE COLONY IN PUBERTAL DOMESTIC CAT (FELIS CATUS)
Narong Tiptanavattana, Mongkol Techakumphu and Theerawat Tharasanit*
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science,
Chulalongkorn University, Bangkok 10330, Thailand.
Spermatogonial stem cells (SSCs) play a central role to maintain the spermatogenesis and also to transmit
the male genetic profiles throughout post-pubertal male animals. These SSCs have been successfully isolated and
cultured in vitro from a number of domestic species such as mouse and rat. In cat, optimization of in vitro culture
system for SSCs is prerequisite for in-depth study of male germ cells. Until recently, successful isolation and culture
of these SSCs in domestic cat have yet to be reported. This study aimed to isolate and culture of SSCs from pubertal
cat testes. The testicular cells were enzymatically digested from testicular parenchyma and subsequently cultured
in vitro using a modified SSC culture medium previously described in mouse. The SSCs were identified by immunolabelling with GFRa-1 monoclonal antibody, and passaged onto mitomycin-treated CF-1 and sertoli cells. The
formation of SSC colony was daily observed under a phase contrast microscope.
Our culture technique could isolate the putative SSCs that demonstrated an intercellular bridge and germ
cell-like chain clusters by 7-14 days of culture. Although we could maintain the SSC colony for at least 54 days, the
culture system used in this study was not able to further support the proliferation of cat SSCs. In conclusion, we
firstly reported that SSCs can be isolated from pubertal cat testis and cultured in vitro, while other factors regulating the proliferation and senescence of SSCs in domestic cat remain to be elucidated.
Keywords : SSC, domestic cat, culture
83
COMPARISON OF LOW-COST LONG-TERM FREEZING MEDIA
FOR MICE PANCREATIC ISLET CELLS
Hathairat Chanphao1*, Narudee Kashemsant2, Siriruk Chantrakru3, Monchanok Vijarnsorn4
1
Center for Agricultural Biotechnology (CAB) - Kamphaeng Saen campus,
Department of Physiology, 3Department of Anatomy, and 4Department of Companion Animals Clinical Sciences,
Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
2
Pancreatic islet cells banks are essential for the modern treatment of diabetes mellitus by pancreatic transplant. To sustain the islet banks, a major challenge is the constant availability of donors. This can be partially compensated if using high quality freezing media (FM) to preserve the islets longer. Several studies in mice have investigated FM individually. However, this study attempts a comparative evaluation of three low-cost FM. The results of
this study in mice may also apply to the protocol of companion animals. Three FM were used to freeze isolated
pancreatic islets for one month, after which the insulin and ATP quantities were evaluated. This was an indicator of
the pancreatic cell functions. Insulin was evaluated before freezing and after thawing by glucose stimulated insulin
secretion (GSIS). The total insulin of the freeze-thaw islets remains the same as in the fresh islets. However, the
GSIS was impaired. In addition, islets frozen in 3M glycerol in DMEM tend to have the highest intracellular insulin,
which suggests a high functionality. Among the three FM, the highest intracellular ATP was found in islets preserved using 2M dimethylsulfoxide (DMSO) in DMEM. This indicates a high survival rate in this group. In conclusion,
this study suggests that the two above-mentioned FM are the most promising candidates for cryopreservation.
However, further experiments are required to explain the cause of impaired insulin release of the islets, such as
measuring intracellular Ca2+ and glucose transporter 2 proteins, thus clarifying the functional structure of the frozen pancreatic islets.
Keywords : freezing media, low-cost, long-term, islets
84 
ORF5 GENETIC MODIFICATION OF PRRSV IN A MODIFIED LIVE
PRRSV VACCINATED SWINE FARM FOLLOWING AN OUTBREAK
T. Hoonsuwan, T. Tripipat, D. Nilubol*
Department of Veterinary Microbiology, Faculty of Veterinary Science,
The objectives were to investigate dynamic and evolution of porcine reproductive and respiratory
syndrome virus (PRRSV) ORF5 following a modified live PRRSV vaccine (MLV) used in an outbreak herd. A PRRSV
positive herd with co-existence of European (EU) and North American (NA) genotypes and no history of MLV use
was recruited into the study. Following an outbreak, vaccination with a NA MLV (Boehlinger Ingelheim, USA) was
implemented. All sows were mass-vaccinated at monthly interval for 2 consecutive months followed by quarterly
vaccination regimen. All piglets were vaccinated at 7 days of age. Serum samples were collected monthly for twelve
consecutive months from 4 population groups of 5 samples each including replacement gilts, suckling, nursery and
finishing pigs. Two hundred and twenty three complete ORF5 genes consisting of 118 EU and 105 NA isolates were
obtained from the herd following a year of collection. Prior to and following vaccination, both EU and NA genotypes were independently evolved and co-existed in an individual pig. Following the vaccination, MLV vaccination
had no influence on increased heterogenicity of either EU or NA isolates, but it resulted in the emergence of two
novel PRRSV clusters of NA isolates in the herd including MLV-like and MLV-related clusters. MLV-like isolates
emerged and then disappeared within two months following a mass vaccination. However, there was an emergence of PRRSV cluster which was genetically related to the MLV vaccine use during the outbreak. The difference
between these 2 clusters was increased in the number of N-linked glycosylation positions. The results of the study
suggested that MLV had no influence on the development of previously existing isolates, but resulted in the introduction of novel isolates into the herd.
Keywords : PRRSV, ORF5, modified live vaccine, evolution
85
VIRULENCE GENES PROFILING OF STREPTOCOCCUS AGALACTIAE STRAINS
ISOLATED FROM TILAPIAS (OREOCHROMIS SP.)
AND THEIR CULTURING ENVIRONMENTS IN THAILAND
P. Kayansamruaj1, P. Nopadon2, C. Rodkhum1*
1
Department of Veterinary Microbiology 2Department of Veterinary Pathology, Faculty of Veterinary Science,
Chulalongkorn university, THAILAND.
* Corresponding author : Email : [email protected], [email protected]
Streptococcus agalactiae is a wide-host range pathogen which can produce disease in many species of
mammal and fish. The virulence characteristics of S. agalactiae have a variety depend on their host tropism. In this
study, we investigate some important virulence genes possessed among S. agalactiae strains isolated from tilapias
(Oreochromis sp.) and their culturing environments from several locations of Thailand. The purpose of this study is
to increase the understanding about virulence determinants of the bacterial strains. Forty-four (44) isolates of S.
agalactiae were collected and classified as molecular serotype Ia. The virulence genes contained in each bacterial
strain were identified using individual PCR with newly designed primers. The virulence genes target included in this
study are C5a peptidase (scpB), serine protease (cspA), fibrinogen-binding protein A and B (fbsA and fbsB), laminin
binding protein (lmb), D-alanylation ligase (dltA), immunogenic adhesion (bibA), GAPDH (gapC), pili-1 and pili-2
backbone (PI-1, PI-2a and PI-2b) encoding genes. The results showed that all virulence genes are contained in all
isolated S. agalactiae strains except for scpB and lmb which presence only in 2% of the S. agalactiae population and
PI-1a which not presence in any S. agalactiae isolates using in this study. The information from this study may indicated that S. agalactiae serotype Ia isolated from tilapias and their culturing environments in Thailand have a common virulence genes patterns.
Keywords: Streptococcus agalactiae; Tilapia; Virulence gene; Pathogenicity
86 
CONTRIBUTION OF THE MULTIDRUG EFFLUX SYSTEM MEXXY-OPRM
IN AMINOGLYCOSIDES RESISTANCE IN PSEUDOMONAS AERUGINOSA
CLINICAL ISOLATES FROM DOGS AND CATS
Kanchana Poonsuk1, Sirawit Pagdepanichkit1, Rungtip Chuanchuen*
1
Department of Veterinary Public Health, Faculty of Veterinary Science,
*
Corresponding author : Email : [email protected]
Aminoglycosides (AMG) is one of the drugs of choice for treatment of Pseudomonas aeruginosa (PA) infections. PA exhibit resistance to AMG via several mechanisms including the multidrug efflux system, of which MexXYOprM is the only multidrug efflux pump involved in AMG resistance. Fourteen PA clinical isolates from dogs and
cats were examined for expression of MexXY-OprM and the MexXY expression was detected in all isolates. Contribution of MexXY-OprM in AMG efflux was investigated by deletion of mexXY operon in 8 isolates generating Δ
(mexXY)::FRT and role of MexXY in AMG resistance was confirmed by complementation with plasmid containing
mexXY. Lack of mexXY resulted in 2-4 fold reduction of MIC value of 6 AMG drugs i.e. gentamycin, amikacin, neomycin, kanamycin, streptomycin and spectinomycin in comparison to corresponding patent strains. Complementation with mexXY restored the AMG MICs in all Δ(mexXY)::FRT mutants. The mexZ gene, the mexXY regulatory gene
was found to contain mutations i.e. Ala98-Gly and Thr94-Ala. Others AMG resistance mechanisms were also tested.
Mutation in rplY was found in two isolates. The expression of nuoG was identified in all 14 isolates and no mutation
was detected in galU. The results indicated that MexXY-OprM plays a role in AMG resistance in PA clinical isolates
from dogs and cats and the PA strains use combination mechanisms for being resistant to AMG.
Keywords : aminoglycoside resistance, MexXY-OprM, Pseudomonas aeruginosa
87
ANTIBIOTIC RESISTANCE AND VIRULENCE GENES OF
ESCHERICHIA COLI ISOLATED FROM SWINE
Khin Khin Lay1, Chailai Koowatananukul1, Rungtip Chuanchuen 1*
1
Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, THAILAND.
A total of 344 Escherichia coli isolated from fecal samples of swine were included in this study. All isolates
were tested phenotypically and genotypically for antimicrobial resistance to different classes of antimicrobial
agents. The occurrence of integrase 1 (intI1) gene was investigated by PCR and transfer of integrons with resistance
genes was tested by conjugation experiment. E.coli isolates were examined for the presence of 14 virulence genes
by multiplex PCR. All isolates were resistant to at least one antimicrobial agent and 98.3% were multidrug resistant.
Forty-two resistance patterns were observed. The common phenotypic resistance pattern was AMP-CHP-CIP-ERYGEN-STR-SUL-TET-TRI. Most of the resistant isolates possessed antimicrobial resistance genes corresponding to
their resistance phenotypes. The intI1 gene was present in 73%, of which 22.3% carried gene cassettes with size
ranging from 650-2,700 bp. Sequence analysis showed 5 distinct integrons profiles, in which partial sat, aadA1,
aadA22, dfrA12, aadA2, sat and psp were present in variable regions. The gene cassette aadA1 (26.8%) was most
frequently found among the isolates. Class 1 integrons were detected on conjugative plasmid in eight E. coli isolates. Ten virulence genes including astA (34.9%), eaeA (13.1%), elt (57.9%), estA (0.6%), estB (25%), faeG (4.7%),
fedA (16.9%), fasA (98.3%), paa, (22.7%) and sepA (14.5%) were identified in the E. coli isolates. This study demonstrated that E. coli from swine could serve as reservoirs for transferable antimicrobial resistance determinants and
virulence genes.
Keywords : E.coli, Class 1 integrons, antimcrobial resistance genes, virulence genes
88 
CHARACTERIZATION OF CAMPYLOBACTER SPP. ISOLATED FROM
BROILER FLOCKS IN CHIANG MAI, THAILAND
Chomporn Chokboonmongkol1 Karl-Hans Zessin 2 Thomas Alter3 Prapas Patchanee4*
1
Animal Health and Technical Service Office,
Bangkok Agro-Industrial Products Public CO., LTD, Bangkok, Thailand ,
2
Department Panel, Veterinary Public Health,
3
Institute of Food Hygiene, Free University Berlin, GERMANY,
4
Veterinary Public Health Center for Asia Pacific (VPHCAP),
Faculty of Veterinary Medicine, Chiang Mai University, THAILAND
* Corresponding author: Email : [email protected]
Ninety-eight broiler flocks raised in Chiang Mai area were included in this study. Samples collection was
followed the EU COMMISION DECISION protocol (2007/516/EC) including ten intact caeca and one whole carcass
per slaughter batch in order to determine Campylobacter spp. at broiler flocks and broiler carcasses, respectively.
This study was aimed to determine the prevalence of Campylobacter spp. in broiler flocks at the end of rearing
period and on broiler carcasses by using conventional microbiological and PCR method, and to phenotypic
characterize of Campylobacter isolates from broiler samples. Representing 16 C.jejuni was genetically analyzed
using Multi Locus Sequence Typing (MLST). C. jejuni was detected as a major Campylobacter spp. both in broiler
flocks and broiler carcasses, and prevalence of Campylobacter spp. were 11.2% (11/98) and 51% (50/98), respectively. Ciprofloxacin (81.3%) exhibited in a greatest proportion for antimicrobial drug resistance of 32 Campylobacter isolates followed by tetracycline (40.6%), ampicillin (31.3%) and erythromycin (9.38%). Eight different antimicrobial resistance patterns were demonstrated with nine different sequence types (STs) of C. jejuni which were
categorized into five clonal complexes. Three new STs were firstly reported in Thailand by this study. MLST analysis
provided similar pattern of allelic profile for C. jejuni isolated from the carcasses and caeca of broilers from the
same farm. The contamination of the chicken carcasses in the slaughter house, thus, as a result of C. jejuni
colonized in the chicken intestine. Different STs of C. jejuni isolated from chicken carcasses reflect routes of horizontal transmission during the steps of the slaughtering process. These findings provide solid scientific evidence for
cross contamination during the slaughtering process. Control measures in the slaughterhouse should be applied
strictly and in order to reduce Campylobacter prevalence on carcasses, modern slaughtering technologies need to
be implemented in the near future.
Keywords : Campylobacter, Broiler, Chiang Mai, MLST
89
PARASITIC FOOD-BORNE ZOONOSES: AS THREAT
FOR PUBLIC HEALTH IN SOUTHEAST ASIA
Rissar Siringo Ringo 1*, Sheila 1, Denny Widaya Lukman2
1
Undergraduate students of faculty of veterinary medicine, Bogor Agricultural University
2
Department of animal diseases science and veterinary public health,
faculty of veterinary medicine, Bogor Agricultural University
This day foodborne parasitic zoonoses becomes more common in worldwide. The extent of foodborne
parasitic zoonoses in Southeast Asia is considered a "tip of the iceberg" problem. Southeast Asia countries have a
large biodiversity as well as parasites species because of all of Southeast Asia countries have a tropical climate.
Until now, parasitic diseases in animal are still neglected and the reporting for parasitic disease hasn’t been applied
properly. Although a number of zoonotic parasites are often found and do cause serious illnesses in Southeast
Asia, some are more important as they cause serious public health problems, such as toxoplasmosis, trichinellosis,
cryptosporidiosis, anisakiasis, and cysticercosis. Southeast Asia countries play an important role in the world
livestock market, which can bring risks of foodborne zoonotic diseases through the trade. The occurrence of parasitic foodborne diseases is triggered by some factors, such as eating uncooked meat and fish. Furthermore, the
illegal trade of bush meat in Southeast Asia contributes to the emergence of zoonotic diseases since they may be
source of some zoonotic diseases. The recent food safety system is still focusing in microbial foodborne diseases
instead of parasitic ones. It is suggested to strengthen the control of parasitic foodborne zoonoses in food of
animal origin, specifically meat. The veterinary authority should play an important role in this control. The
veterinary curricula should involve the food safety which focuses on foodborne zoonotic diseases in order to
increase the competence of veterinary graduates on food safety system.
Keywords: foodborne zoonoses, parasites, public health
90 
CLONING AND SEQUENCING OF SIALOADHESIN AND CD163 CDNA
FROM THE FIELD PRRSV INFECTED PORCINE ALVEOLAR MACROPHAGES
Vo Phong Vu Anh Tuan1 and Athipoo Nuntaprasert 1*,
1
Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330 Thailand
*Corresponding author : E-mail: [email protected]
Sialoadhesin (Sn) and CD163 receptors were determined as two essential receptors on porcine alveolar
macrophages (PAMs) for PRRSV infection. Recently, Sn and CD163 genes were reported to be related to the
immunity of pigs after PRRS infection and may be used to improve PRRS immunity of pigs. The objective of present
study was cloned the Sn and CD163 cDNA from three strains (EU, US and China) of PRRSV infected farms and was
compared their nucleotide sequences and deduced amino acids with available databases. In this study, the six
positive EU, US and HP-PRRSV infected farms (two farms per each strain) were selected. The positive PRRSV
nursery pigs were confirmed the strains from sera using reverse transcriptase-PCR technique. Then, PAMs from
broncho-alveolar lavage of those PRRSV infected nursery pigs were obtained and isolated the total RNA. The Sn and
CD163 cDNA were further to amplify using reverse transcriptase-PCR technique and were cloned into pCR-XLTOPO® vector. The nucleotide sequences and the deduced amino acids of Sn and CD163 cDNA from different PRRSV
strains were analyzed and compared. In our study, Sn and CD163 cDNA from six PRRSV infected nursery pigs were
successfully amplified and cloned into the pCR-XL-TOPO® vectors. The transformants of those pCR-XL-TOPO-Sn and
pCR-XL-TOPO-CD163 were selected and sequenced. The analysis of nucleotide sequences and deduced amino acid
of both receptors are on the progress. This study might be helpful for supporting the idea for prevention of PRRSV
in the future.
Keywords : PRRSV, cDNA, Sialoadhesin, CD163, clone and sequence
Poster Presentation Abstract
92 
PEGYLATED LIPOSOMAL DOXORUBICIN-INDUCED PALMAR-PLANTAR
ERYTHRODYSTHESIA (HAND-FOOT SYNDROME) IN A NASAL CARCINOMA DOG
S.Kunakornsawat1*, K. Imsilp2, S Jeamprapai1, S Poapolathep2, S Netramai3
1
Department of Companion Animals Clinical Sciences,
Department of Pharmacology, and 3Veterinary Teaching Hospital, Bangkaen campus,
2
The aim of this report is increase clinical awareness of dermatological toxicity associated with administration of pegylated liposomal doxorubicin (Doxil®) induced palmar-plantar erythrodysthesia (PPES) (also known as
hand-foot syndrome in humans). Clinicians should be acquainted with this spectrum of mucocutaneous toxic
effects, as this product is becoming better recognized and more widely precribed.
A four-year-old, male crossbreed dog was diagnosed with stage I nasal carcinoma and received Doxil ® (0.5
mg/kg) intravenously, every 3 weeks for four total treatments. The dog responded well to the first chemotherapy
without development of any significant side effects (including no skin toxicity). The second cycle of therapy of
Doxil® (0.75 mg/kg) was administered intravenously. During this second peroid, cutaneous reaction (PPES) was
observed. A painful desquamating dermatitis characterized by skin changes, ranging from mild erythema,
hyperemia, and alopecia to severe crusting, ulceration, and epidermal necrosis was noted. Lameness associated
with apparent paw discomfort while bearing weight has been found. In conclusion, the use of Doxil ® at dose of
0.75 mg/kg developed cutaneous reaction in this dog. Early detection of PPES is crucial and can be achieved by the
recognition of sings and symptoms. Moreover, it may be necessary to delay treatment or stop cytotoxic
chemotherapy because PPES can have devastating consequences.
Keywords : palmar-plantar erythrodysthesia, doxorubicin, nasal carcinoma, dog
93
EFFECT OF SOLID SURFACE VITRIFICATION ON MODIFICATION OF LECTIN-BINDING
CARBOHYDRATE CHAINS IN ZONA PELLUCIDA OF CANINE CUMULUS-OOCYTE-COMPLEX
Chonticha Srirungruang1*, Urai Pongchairerk1, Kaitkanok Sirinarumitr2 and Sirirak Chantakru1
1
Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, THAILAND
2
Department of Small Animal Clinical Medicine, Faculty of Veterinary Medicine, Kasetsart University,
Bangkok 10900, THAILAND
Corresponding author: E-mail: [email protected]
The main purpose of cryopreservation of the oocytes is to preserve oocyte quality. The zona pellucida (ZP) is
glycoprotein whose carbohydrate residues initiate sperm binding and attachment to the oocytes which is a crucial
step for fertilization. Therefore, any structural changes in these molecules which might be caused by vitrification
will have great effects on fertilization. The objective of the study was to determine effect of solid surface vitrification (SSV) on carbohydrates in the ZP of canine cumulus-oocyte-complex (COCs) based on their lectin-binding properties COCs from each dog were randomly divided into five groups. The groups 2-4 except the control (group 1)
were equilibrated and separately introduced into different recipes of vitrification media consisting TCM 199
medium, supplemented with 0.5 M trehalose, 1.5 M ethylene glycol (EG) and 20% fetal bovine serum (FBS) was
used for COCs of the groups 2 and 4 and TCM 199 medium, supplemented with 0.5 M trehalose, 3 M EG and 20%
FBS for the groups 3 and 5. Only the COCs from groups 4 and 5 were vitrified. In lectin histochemistry, six
biotinylated lectins including PSA, s-WGA, LEL, GSL-II, VVA and ECL were incubated with paraffin-embedded
sections. Fluorescein-conjugated Avidin D and Rhodamine-conjugated Avidin D were then applied. The result
showed that cryoprotectants or/and SSV reduced PSA binding to the α-mannose on the ZP. SSV alone caused no
binding to N-acetyl glucosamine and α-and β-α-DN-acetyl glucosamine on the ZP. Thus, exposure of the
cryopreservation and SSV may modify structural changes of ZP sugars.
94 
FOUR AFLATOXIN ANALOGUES IN DOG FOOD: A PRELIMINARY MONITORING
Natthasit Tansakul1,*, Kamolchai Trongvanichnam1, Pareeya Udomkusonsri1 and Sasithorn Limsuwan2
1
Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND, .
Molecular Phytopathology and Mycotoxin Research, Department of Crop Sciences, Göttingen University,
Göttingen, GERMANY. Corresponding author
2
Aflatoxin (AF) is a carcinogenic and mutagenic metabolites produced by the fungal strains of Appergillusspp.
Four major aflatoxin analogs are aflatoxin B1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1) and aflatoxinG2
(AFG2). AFB1 is classified as a carcinogen class 1A. In tropical zone, fungal is commonly present in agricultural
commodities and thus could be a contaminant in food and feed. Risk of aflatoxin contaminated dog food has been
concerned. Therefore, aim of this study was set to evaluate mycotoxin, in particular aflatoxin, contamination in dog
food marketed in Thailand. Thirty samples collected from Pet shop’s shelf in central region of Thailand. Aflatoxin
analogues were detected by the HPLC-fluorescence with post-column bromination. Of thirty samples, there was no
sample free from aflatoxin contamination. An average of total aflatoxin concentration was 3.17 ng/g. As detected,
an average level of AFG1, AFB2 and AFB1 were 0.05, 0.70 and 2.49 ng/g, respectively, whereas AFG2 was not
detected in any sample. However, the contaminant level was far lower than the limit stipulated by Thai
Department of Livestock Development. Thus, dog food marketed in Thailand is safe. However, regarding the fact
that aflatoxin is naturally contamination in agricultural products including pet food, the effectiveness of its control
depends on a rigorous monitoring program.
95
MORPHOLOGY OF CAT TESTICULAR TISSUE AFTER CRYOPRESERVATION USING
DIFFERENT PROTOCOLS
Kattika Saisuwan1*, Kanjana Lapsongphon1 ,Siriwan Sakarin1, Kaywalee Chatdarong2,
Sayamon Srisuwatanasagul3, Paweena Thuwanut2
1
Sixth year students, Academic year 2009, Faculty of Veterinary Science, Chulalongkorn University,
Department of Obstretrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn Univ.,
3
Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, THAILAND, .
*corresponding author: E-mail: [email protected]
2
Testicular tissue cryopreservation is an alternative tool for gamete rescue in post-mortem and in animal
species that are at risk of becoming extinct. The objective of this study was to compare the morphology of cat
testicular tissue after cryopreservation by using different protocols. Testicular tissues were collected from domestic
cats divided into five groups ;(I) fresh controls (II) fresh tissue stored at 4oC for 24 h. (III) testicular tissue directly
frozen in -80oC(IV) testicular tissue directly frozen in -196oCand (V) testicular tissue stored above liquid nitrogen
vapour for 10 minutes then plunged into liquid nitrogen (196oC). Thereafter, they are processed for histological
evaluation under light microscope. Two major criteria were used to evaluated the morphology which were ;(I)
diameter of seminiferous tubules (II) the integrity and the structural changes (nuclei of intratubular cells and
epithelium) by giving a score of alteration from 0-5. The result demonstrated that there is no difference in the
diameter between fresh controls and fresh testis stored at 4 oC for 24 h. Among freezing groups, the diameter of
group V is significantly smaller than the others. Regarding the integrity and structure, the highest score of
alteration was observed in group V as well. In conclusion, after cryopreservation, the testicular tissue changes
depending on the different protocols used. Testicular tissues frozen without stored above liquid nitrogen vapour
show better morphology compared to those stored above liquid nitrogen vapour before freezing, and therefore,
may be the superior alternative method for testicular cryopreservation.
Keyword : Cat, Testicular tissue, Cryopreservation
Acknowledgements This work was a part of the 6th year senior project and supported by Academic Affairs, Faculty
of Veterinary Science, Chulalongkorn University
96 
DIAGNOSIS OF ENCEPHALITOZOON INFECTION IN 2 LIVING RABBITS
AND SUCCESSFUL SURGICAL TREATMENT: A CASE REPORT
Aree Thayananuphat1*, Tipawadee Seidkhuntod2, Natthanet Sritrakoon2,
Taksaon Duangurai2, and Preeda Lertwatcharasarakul3
1
Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University,
2
Kasetsart University Veterinary Teaching Hospital, Kasetsart University,
3
Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
Two 10 months old male mixed bred rabbits were presented with an abnormal mass within their right eyes.
Ocular examination revealed anterior uveitis, abscess-like masss at the iris and incipient cataract in the right eyes
whereas the left eyes were normal. Albendazole and anti-inflammatory medication were administered because
phacoclastic uveitis caused by encephalitizoon infection was suspected. Phacoemulsification was performed to
resolved eye problems. PCR of phacoemulsification fluid revealed encephalitozoon infection within the lens,
whereas aqueous humor revealed negative result. The surgical eye was functioned well with mild complications at
10 months postoperative follow up in the first rabbit. The second rabbit was lost to follow up after 2 weeks of
surgery. However, he navigated well with the normal eye covered at 2 weeks follow up. This is the first report of
encephalitozoon infection diagnosed in living rabbits and successful surgical treatment of phacoclastic uveitis due
to encephalitozoon infection in Thailand.
Key words: phacoclastic uveitis, phacoemulsification, Encephalitozoon, cataract, rabbit
97
NEW ADVANCE IN THE TREATMENT OF ACUTE PRIMARY GLAUCOMA IN DOGS
IN THAILAND: DIODE LASER TRANSSCLERAL CYCLOPHOTOCOAGULATION.
Natthanet Sritrakoon1, Aree Thayananuphat 2*
1
Ophthalmology Center, Kasetsart University Veterinary Teaching Hospital, Bankhean,
Department of Small Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
2
Glaucoma is a major cause of vision loss in dogs. Intraocular pressure (IOP) is possible controlled by
medical or surgical options. Diode laser transscleral cyclophotocoagulation (TSCP) is one of the cyclodestructive
techniques in surgical options that is less invasive and takes short surgical times. The purpose of this study is to
evaluate the result of diode laser TSCP for treatment of primary glaucoma in dogs. Records of 6 acute primary
glaucoma dogs in the Ophthalmology Center, Kasetsart University Veterinary Teaching Hospital, Bangkhean during
March to August 2011 were reviewed. Diode laser TSCP was underwent in all 6 dogs (6 eyes) after topical antiglaucoma medications were failed to reduce the IOP to normal limits. Dog 1-5 were under one diode laser TSCP
whereas diode laser TSCP was repeated in dog 6 at day 7 after the first surgery. The average IOP prior to surgery
was 47.2+18.30 (31-80) mmHg. Every eye received topical anti-glaucoma medications pre- and post-operatively.
The average IOP measured in 5 eyes at day 56 was 17.8+6.14 (12-27) mmHg and the IOP measured in 2 eyes at day
84 was 17 and 22 mmHg. All 6 glaucoma eyes were sighted at all times of the examination. Combination of diode
laser TSCP and topical anti-glaucoma medication was a new hope to control IOP in normal limits and maintain
vision in short term follow up in Thailand.
Key Words : diode laser transscleral cyclophotocoagulation, dog, glaucoma, intraocular pressure
98 
CHARACTERISTICS OF GENTAMICIN-COATED HIGH POROUS
CALCIUM SULFATE BASED DELIVERY SYSTEM
Chaiyakorn Thitiyanaporn1,2, Naris Thengchaisri3, Pareeya Udomkusonsri4*
1
Center for Agricultural Biotechnology (CAB),
Center for Agricultural Biotechnology (AG-BIOPERDO-CHE), Kasetsart University, THAILAND,
3
Department of Companion Animal Clinical Sciences, and
4
Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND
2
Calcium sulfate (CS) has been used as a bone substitute material and antibiotic delivery systems. Increased
porosity of CS bead may improve drug delivery capacity, elution property and osteogenic property. This study was
compare effects of bead materials including high porous calcium sulfate (HPCS), native calcium sulfate (NCS) and
polymethylmethacrylate (PMMA) beads on gentamicin elution and osteogenic properties using in vitro model.
Gentamicin beads were created by coating of HPCS, NCS and PMMA with 40 mg/ml of gentamicin or impregnating
gentamicin (3.8%, w/w) in PMMA. Physical properties, microstructure analysis and elution of gentamicin from
gentamicin-coated HPCS (G-HPCS), gentamicin-coated NCS (G-NCS), gentamicin-coated PMMA (G-PMMA) and
gentamicin-impregnated PMMA (GI-PMMA) were compared. The osteogenic properties of PMMA, NCS and HPCS
were compared using human osteoblast (h-OBs). Crystal size and porosity level in G-NCS were lower than G-HPCS
while both GI-PMMA and G-PMMA groups did not have. All type of beads could elute gentamicin for 10 days
except G-PMMA, which released gentamicin for 4 days. The highest gentamicin elution on the first day was found
in G-HPCS compared to other groups. Total gentamicin elution for 10 days from G-HPCS and G-NCS were not different but higher than G-PMMA and GI-PMMA. The PMMA, HPCS and NCS beads used in this study were not toxic to
h-OBs. The positive effect on h-OBs proliferation was found highest in HPCS beads compared to NCS and PMMA.
These results suggest that HPCS beads improve local antibiotic delivery as well as provide a positive effect on bone
cell proliferation.
Key words: Porosity, Calcium sulfate, Polymethylmethacrylate, Gentamicin sulfate, Elution
99
REFERENCE RANGE OF PROSTATIC SIZE AND VOLUME
IN NORMAL HEALTHY DOGS RELATED TO THE BODYWEIGHT
S. Ponglowhapan*, N. Sannamwong, N. Saengklub, P. Sriphutthachot
Department of Obstetrics Gynaecology and Reproduction,
Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330 THAILAND.
The objective of this study was to establish a reference range of prostatic size (width, length and depth)
and volume of healthy adult intact dogs. The study included 101 healthy intact dogs free from any prostatic
disorders as determined by clinical signs and ultrasonographic examination. Dogs, aged 1.5-4 years old, were
divided into 7 groups depending on their body weight (group I; >1-5, group II; >5-10, group III; >10-15, group IV;
>15-20, group V; >20-25, group VI; >25-30, group VII; >30 kg). Prostatic width, length and depth were measured
ultrasonographically and the volume was then calculated. Statistical analysis showed no differences in animal’s age
among groups (I-VII) and there were strong positive correlations between body weight and prostatic size as well as
body weight and prostatic volume. Group VII had higher prostatic volume compared to any groups (P < 0.05), and
no difference in the volume was detected between group I and II. The regression equation was expressed as
Prostatic Volume (cm3) = 0.33 x body weight (kg) + 3.28.
Keywords : Bodyweight, Canine, Prostate
100 
INFLUENCE OF AGE ON CARDIOVASCULAR EFFECT OF PROPOFOL IN HEALTHY DOGS.
THANAKORN SRIRAT3, ISSARAPORN PHROMNAO3, WANPITAK PONGKAN3,
TRASIDA PLOYNGAM1*, PREENUN JITASOMBUTI2, THANIKUL SRITHUNYARAT2
1
6th year student, Faculty of veterinary medicine, Khon Kaen university,
2
Department of Veterinary Medicine, Faculty of Veterinary Medicine, Khon Kaen University,
3
Department of surgery and theriogenology, Faculty of Veterinary Medicine, Khon Kaen University
Propofol has several cardiovascular effects, including hypotension, bradycardia, and negative inotropism.
We hypothesized that aging may increase cardiovascular risk of propofol in healthy dogs. In this study, dogs about
to be anesthesized for routine dental scaling were divided into two groups (group 1, < 5 years of age (n = 6); group
2, > 5 years of age (n = 6)). All dogs were anesthetic premedicated with acepromazine maleate (0.02 mg/kg) and
tramadol hydrochloride (3 mg/kg), induced with propofol (3-4 mg/kg), and maintained with isoflurane (1.5-2 %).
Blood pressure (BP), heart rate (HR), respiratory rate, electrocardiography, and body temperature were measured
at baseline, 5 min after premedication, and every 5 min after propofol induction. The results show that propofol
reduced BP significantly in both groups (group 1, 38.4±2.6 mmHg; group 2, 33.1±4.9 mmHg) at 5 min after
injection. While there were no significant different of BP between the two groups, HR in group 2 appeared to be
higher. HR at 5 min after induction was 106.67±5.7 beats/min in group 1 and 115.83±4.64 beats/min in group 2.
Although, BP was maintained to be equal in the two groups; this is in expense of a higher increase in HR in the
older dogs. Since BP is a function of peripheral resistance, cardiac output, as well as stroke volume and HR, the
results of this study suggest that aging dogs tend to have defects in either vascular-baroreflex counter- responses
to hypotensive effects of propofol or display exaggerated responses to its negative-inotropism effects.
Keywords : cardiovascular, propofol, dogs, age
101
DETECTION OF METHICILLIN RESISTANT
STAPHYLOCOCCUS PSEUDINTERMEDIUS SCC MEC TYPEIII FROM CAT URINE
W. Tonpitak1* and C. Sornklein1
1
Department of Microbiology, Faculty of Veterinary Medicine,
Mahanakorn University of Technology, THAILAND.
Staphylococcus (S.) pseudintermedius is the major causative agent of the pyoderma in dog and the various
infections in other animals. The methicillin resistant of Staphylococcus in the past few years is increasing in
humans, veterinary medicine and also zoonotic infection between pets and human are suspected. Furthermore,
the methicillin resistant Staphylococcus sp. have multi-drug resistant in many antibiotic groups. However, reports
of the methicillin resistant Staphylococcus pseudintermedius (MRSP) in cat are not many; especially those in
Thailand are not reported. The aim of this study is report the detection of the methicillin resistant Staphylococcus
pseudintermedius SCCmec typeIII from urine cat. The isolate was identified as Staphylococcus sp. by the conventional method, and then PCR-REA of the pta gene restricted by MboI are used to identify S. pseudintermedius. The
methicillin resistant was confirmed by the mecA gene detection by PCR. The isolate was identified as MRSP SCCmec
typeIII based on the identification of the mec gene complexes classA and the cassette chromosome recombinaseA3
and B3 gene by the multiplex PCRs. This finding is useful baseline of the prevalence of the MRSP in animals in
Thailand and also to concern the veterinarians to aware the protection of MRSP widespread zoonotic transmission.
Key words: Staphylococcus pseudintermedius, methicillin resistant, SCCmec type, urine, cat
102 
BLOOD COAGULATION FACTORS DEFECT IN CANINE EHRLICHIOSIS
Arayaporn Macotpet 1*, Kawintra Aiyaranoi1, Jutanun Laoumanotham1
Alongkorn Kulilung1, Ekkachai Patarapanwichien2, Eakkasit Barameechaithanan3
1
Department of Medicine, 2Department of Pathobiology and 3Veterinary Teaching Hospital,
Faculty of Veterinary Medicine, Khon Kaen University, THAILAND.
Ehrlichia canis is obligate intracellular bacteria with tropism for mononuclear cells. Haemostatic alterations
in dogs naturally infected by Ehrlichia canis were studied. Activated partial thromboplastin time (APTT),
prothrombin time (PT) and platelet count were measured for Ehrlichia canis-infected dogs (n = 19) and healthy
dogs (n = 16). Significant increased in APTT and reduction in platelet count (P < 0.05) for ehrlichiotic dogs were
observed. Furthermore by substitution test (APTT) 2 of 19 cases (10.53%) were possible the Factor IX defect and 1
of 19 cases (5.26%) was possible Factor XI defect. Therefore, defective of these coagulation factors may involve in
the pathophysiology of canine monocytic ehrlichiosis.
Key words: coagulation factors, ehrlichiosis, dog
103
NITRIC OXIDE LEVELS IN CANINE GRADE II MAST CELL TUMORS
Ekkachai Patarapanwichien1*, Arpapan Kanbanjong1, Karn Yongwahish1
Chonlada Prabwongsa1, Arayaporn Macotpet 2, Piyasak Wipoosak3
1
Department of Pathobiology, 2Department of Medicine and
3
Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Khon Kaen University, THAILAND.
Nitric oxide is a small, unstable, potentially toxic gas and is produced by nitric oxide synthase. Nitric oxide
has been invoked in nearly every normal and pathological conditions including both positive and negative affects in
tumor biology. Therefore, factor determination as indicative of increased nitric oxide radical formation is of
interest. The purpose of this study was to compare the mean nitric oxide levels between clinically healthy dogs (n =
26) and dogs suffering from spontaneous grade II mast cell tumors (n = 18). Mean nitric oxide levels were
4.35 + 3.25 μmol/L in the tumor group and 2.31+0.79 μmol/L in the healthy group. Mean nitric oxide was significantly higher in the tumor group compared to the healthy group (P < 0·05). The results are postulated that this may
lead to enhanced action of nitrogen radicals. Therefore plasma nitric oxide may be used as a indicator for nitric
oxide radical formation.
Key words: nitric oxide, mast cell tumors, dog
104 
GLUTATHIONE LEVELS IN CANINE MAMMARY CANCER
Ekkachai Patarapanwichien1*, Attapol Kanna1, Nillawan Tammasiri1
Nattapol Manojai1, Arayaporn Macotpet 2, Pongrat Jaisil3
1
Department of Pathobiology, 2Department of Medicine and 3Veterinary Teaching Hospital,
Faculty of Veterinary Medicine, Khon Kaen University, THAILAND.
Glutathione is a tripeptide with a structure of γ-L-glutamyl-L-cysteinyl-glycine, which is characterized by the
γ-glutamyl peptide and the reactive thiol group. Glutathione scavenges free radicals, detoxifies heavy metals, helps
ferry amino acids into the cells, helps in bile production, and much more. Cellular GSH plays a central role in the
body of defense against infection, free radicals and carcinogens. The purpose of this study was to compare the
mean glutathione levels between clinically healthy dogs (n = 21) and dogs suffering from spontaneous mammary
cancer (n = 18). The mean and standard deviation in each group were 10.89 + 2.39, and 6.76 + 1.99
mg% respectively. Serum glutathione level in mammary cancer group were significantly lower than those in healthy
group (P < 0·01). The results may support a role of antioxidation in mammary cancer.
Key words: glutathione, mammary cancer, dog
105
BLOOD TRANSFUSION IN THAI DOMESTIC CATS WITH BLOOD FROM DIFFERENT SPECIES
Panpicha Sattasathuchana1, Naris Thengchaisri1 , Monchai Lekjarernwong 2, Chayakit Sinthusing 1*.
1
Department of Small Animal Clinical Practice,
Veterinary Teaching Animal Hospital Kamphaeng Saen campus,
2
Five Thai Domestic Cats had presenting signs with depression, weakness, anorexia, and pale mucous
membrane. The result of blood examination showed regenerative anemia and packed cell volume (PCV) less than
12%. Blood transfusion would be made ready for the patients therefore blood donor had been important. The Royal tiger (Panthera tigris corbetti) is a Felidae as same as domestic cats that should be had the same blood groups
and could be transfused between species. The owner veterinary checked whole blood characteristic and cross
matching between royal tiger and patients. The result showed that tiger’s blood had been Osmolality between 345
+ 17 osm/kg more than blood osmolality of domestic cats 315 + 15 oms/kg (0.05 < p) significantly. Cross matching
resulted between tiger and patients had neither nor both hemagglutination and hemolytic reaction. Therefore
Royal tiger’s whole blood would be transfused to the patients and critical care monitoring at 24 hours, day 3, and
day 7 for determined blood transfusion reaction after transfused. All patients could be improved clinical signs and
PCV to higher than 22 % at 24 hours after transfusion.
Key word : blood transfusion, Thai domestic cat.
106 
DETERMINATION OF BLOOD TYPING POPULATION AND INCIDENCES
OF FELINE IMMUNODEFICIENCY VIRUS (FIV) AND FELINE LEUKEMIA VIRUS (FELV)
IN 5 BREEDS OF THAI DOMESTIC CATS
Chayakrit Sinthusingha1*, Monchai Lekjarernwong2, Wanchart Yippaditr3, Thunyakan Nithisakdiyanond4,
Lapapatr Phutdhikarnt4, Wanvisa Prasong4, and Atthariya Jiranaparat4
1
Department of Companion Animal Clinical Sciences,
3
Veterinary Teaching Animal Hospital Unit Hua-Hin,
4 th
6 -year veterinary student, Faculty of Veterinary Medicine, Kasetsart University,
Kampeansan Campus, 73140 THAILAND.
2
This study was investigated for determination of blood typing population and incidences of common infectious diseases in five Thai domestic cats such as Khao manee, Black Bombay, Korat, Burmese cat, Royal Siamese cat.
The blood sample was collected from 90 cats (each breed 18 cats) at 4 Thai domestic cats conservative institute ;
Samutsongkram, Phranakhon Sri Ayutthaya, Nakhonpathom and Singburi provinces determined blood typing.
Determination of Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) diseases by test kit. The
result showed majority of blood typing is A group and incidence of FIV was 33.34 % and FeLV diseases was 28 %
positive of all. Although FIV and FeLV positive cats is lower but the diseases are important and need to control and
prevent transmittion.
Key words : Thai domestic cats, Blood typing, Feline infectious disease
107
COMPARISON BETWEEN HEMODIALYSIS AND PERITONEAL
DIALYSIS MANAGEMENT IN RENAL INSUFFICIENCY DOGS.
Chayakit Sinthusing1*, Nakrob Pattanapon2, Chamaiporn Sodawichit 2, Chinnawat Kasemmongkolchai 2,
Ketkaew Wasanasuk 3, Teerapat Rungnirundorn 3.
1
Department of Small Animal Clinical Practice,
3
Veterinary Teaching Animal Hospital Bangkean campus,
Faculty of Veterinary Medicine Kasetsart University, THAILAND.
2
Twelve dogs had increased serum creatinine levels more than 7 mg/dl indicating renal insufficiency. They
also had signs of depresssion, anorexia, vomiting, and oliguria. These dogs were divided into two groups (n = 6) to
compare effectiveness of treatments using either hemodialysis or peritoneal dialysis. The peritoneal dialysis group
was treated 6 times per day whereas the hemodialysis group was treated 3 hours per day twice a week for 1 month
Parameters used for determination were serum creatinine level, blood electrolytes, blood gas analysis, and
complete blood count. The result showed that serum creatinine levels of dogs in hemodialysis group were
significantly reduced more than dogs in peritoneal dialysis group (p < 0.05) after the first day of treatment.
Hypokalemia was found in peritoneal dialysis group while hemodialysis group had severe dehydration following
treatment. Blood gas analysis revealed metabolic acidosis in both groups. Urine outputs of dogs in peritoneal dialysis group were significantly increased more than those in hemodialysis group (p < 0.05).
Keyword : Hemodialysis, Peritoneal dialysis, Canine renal insufficiency.
108 
COMMON MALIGNANT TUMORS IN DOGS IN BANGKOK:
RETROSPECTIVE STUDY DURING JUNE 2010 TO MAY 2011
T. Mamom1*
1
Department of Pathology, Faculty of Veterinary Medicine, Mahanakorn University of Technology,
Bangkok, THAILAND.
In dogs, most studies on prevalence of canine tumors did not demonstrate the results regarding to tumor
malignancy. The aim of this study was to investigate the occurrence of malignant canine neoplasms in Bangkok
during June 2010 to May 2011. Canine biopsy samples (n= 248) clinically tentative diagnosis of tumor were
retrospectively investigated. From 248 samples, 54% (134/248) were classified as benign tumors, 35.5% (88/248)
were malignant tumors and 10.5% (26/248) were non-neoplastic lesions. Among 88 samples diagnosed as
malignant tumors, malignant mammary gland tumors were the most common malignant tumor (29.5% or 26/88)
followed by mast cell tumor grade II-III (17% or 15/88) and hemangiosarcoma (10.2% or 9/88). The forth and the
fifth most common malignant tumors in dogs were hemangiopericytoma (9.1% or 8/88) and squamous cell
carcinoma (8% or 7/88) respectively. For malignant mammary gland tumors, the average age of dogs affected was
8.8 (4-12) years and tubulopapillary carcinoma was the most common malignant mammary gland tumor observed
in this study. The average age of dogs affected with mastocytoma grade II-III, hemagiosarcoma, hemangiopericytoma and squamous cell carcinoma were 8.3 (3-12), 7.7 (3-10), 9.7(5-12) and 8.6 (4-12) years respectively. The size of
some common malignant tumors observed in this study was also shown in figure 1. The results of this study might
be useful for veterinarians in term of incidence of malignant tumors, in order to deal with canine oncologic
patients.
Keywords : Malignant tumors canine incidence retrospective
109
CASE REPORT: THE CANINE SYSTEMIC ATHROSCLEROSIS
Wallaya Phongphaew1*, Chareon Thongma1 and Wanida Laohasurayothin1
1
Department of Veterinary Pathology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
The six year-old, male cross breed dog was admitted to the Veterinary teaching hospital at Kasetsart
University with clinical signs of paresis, cyanosis of left forelimb and right hindlimb, and no femoral pulse could be
evaluated. The dog had undergone severe respiratory distress and eventually died. Necropsy and histopathological
examination revealed generalized-thickening and atheroma accumulation of the arterial walls. The completed
occlusion of the arterial lumen was found at the right femoral and left brachial arteries. Thyroid glands were
bilaterally atrophied. the histopatholocial evaluation of the thyroid showed follicles were absent and replaced with
fibrous tissue, adipose tissues, and lymphocyte infiltrations, suggesting severe lymphocytic thyroiditis. The dog was
diagnosed as the canine systemic atherosclerosis associated with lymphocytic thyroiditis.
Keywords : systemic atherosclerosis, hypothyroidism, lymphocytic thyroiditis
110 
INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) AND EPIDERMAL GROWTH FACTOR (EGF) IMPROVE THE DEVELOPMENTAL COMPETENCE OF FELINE EMBRYOS CULTURED SINGLY
Chommanart Thongkittidilok, Thanida Sananmuang, Theerawat Tharasanit, Mongkol Techakumphu*
Chulalongkorn University, Thailand
Single embryo culture has been used as a model to study the paracrine effect on developmental
competence of embryo. This technique may be applicable for embryo culture when the number of embryo is
limited. This study aimed to determine the effect of growth factors (IGF-1 and EGF) on developmental competence
of feline embryo cultured singly. Cumulus oocyte complexes were matured and fertilized in vitro with frozenthawed semen (day 0= day of fertilization). On day 2, cleaved embryos were randomly assigned to culture (50 µL
droplet/ 1 embryo) supplemented with growth factors as follows: (1) control, (2) IGF-1 25 ng/mL, (3) IGF-1 50 ng/
mL), (4) EGF 5 ng/mL, (5) IGF-1 25 ng/mL plus EGF 5 ng/mL, and (6) IGF-1 50 ng/mL plus EGF 5 ng/mL. Developmental competence was assessed by means of morula and blastocyst formation rate. Supplementation of IGF-1 at
25 and 50 ng/mL and EGF at 5 ng/mL significantly improved blastocyst rate compared with control (P<0.05).
However, a combination of these two growth factors adversely affected the blastocyst development.
It is concluded that supplementation of IGF and EGF during single embryo culture improves the
development of feline embryos, suggesting the paracrine role of these growth factors for promoting embryo development.
Keywords : Insulin-like growth factor-1, epidermal growth factor, feline, embryos, cultured singly
111
A RELEVANCE OF Oct 4-IMMUNOHISTOCHEMICAL STAINING PATTERNS TO THEIR RESPONSIBLE ON CANINE MAST CELL TUMORS GRADES: A PRELIMINARY STUDY
T. Eksiritrirat1, P. Lorsomsup1, K. Apisumputthangkur1, T. Nedumpun1, S. Wangnaitham2, A. Sailasuta2*
1
6th year student Academic year 2011,
2
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
*
Canine cutaneous mast cell tumors (MCT) are the second most prevalent cutaneous tumor in dogs. Recently, the cancer stem cell (CSC) hypothesis has been proposed to accumulate all tumor progression-driving genetic
elements that might involve in tumor progression and chemotherapeutic resistance. Several studies up to date also
exhibit that cancer stem cells can activate some stem cell phenotype-inducing transcription factors (TF), especially
Oct4. Oct4 is a member of POU (pit, oct, unc) transcription factor corresponding to a cellular transcription process.
The objective of this study was aimed to establish a protocol on Oct4-immunohistochemistry staining patterns that
might be corresponding to MCT’s grading. The study was performed in 15 MCT dogs which were histopathologically
and IHC-CD 117 classified into each grade. All specimens were routinely prepared by a standard method and IHC
was processed by Mouse antihuman Oct ¾ (c-10) antibody (Santa Cruz Biotechnology, CA) and EnvisionTM peroxidase (Dako, Denmark). The result has indicated that Oct4-Immunostaining pattern was intracytoplasmic, intracytoplasmic-nuclear, and intranuclear, relating to MCT grade I, II and III respectively. There were 2 cell subpopulations
in a MCT mass; Oct4-positive and –negative MCT cells. The Oct4 -positive cells might be defined as CSC, meanwhile
the Oct4-negative cells should be differentiated progeny cells from CSC. In conclusion, the Oct4-immunostaining
pattern of MCT in each grade is distinguishing from one another, relating to its responsible grade. The presence of
Oct4-positive cells might be a clue to indicate that a putative MCT cancer stem cell is obtainable.
Keywords : dogs, mast cell tumor, Oct4-Immunohistochemistry, cancer stem cells
112 
EXTERNAL AUDITORY CANAL IN POSTMORTEM INTERVAL (PMI) ESTIMATION
USING HIGH PRECISION THERMOCOUPLE: A PILOT STUDY
I.O Abdulazeez1 & M.M Noordin1*
1
Department of Pathology and Microbiology, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia.
*Corresponding author: Email : [email protected]
A close-to-accurate estimation of lapsed time since death is vital for the re-enactment of events occurring
prior to the death of a subject in every forensic investigation. There is a wealth of information on different
approaches to achieving such in medical forensics in contrast to its veterinary counterpart, which is still in its early
stages of development. This study attempts to generate data on the postmortem thermodynamics of the external
auditory canal being a readily accessible organ for forensic algor mortis purposes. Four healthy adult bitches weighing between 12 – 16kg were utilized under humane animal use and handling protocols. Thermocouple probes of
the series Pt100 were inserted into the external auditory canal of the right and left ear, post-euthanasia. Measurements were taken at 30 seconds intervals for a 24-hour period. Variations in ambient temperature were measured
as well during the study period frame. Results revealed that the ear canal is easily accessible at postmortem and
provides a good indicator of the core body temperature. This is useful in postmortem interval estimation. Data
obtained fitted a Multiple Multiplicative Factor model and can be possible utilized as a mathematical tool for field
use.
Keywords : dog, ear canal, postmortem, temperature, and thermocouple
113
SUBCUTANEOUS TISSUE GAS FORMATION;
A GOOD INDICATOR OF ELAPSED TIME SINCE DEATH.
1
Recent technical advances in, and awareness of forensic medicine, have opened up series of researches on
the myriad of phenomena after death in both man and animals. Veterinary forensic being in its infancy is yet to
obtain reference data for field use in this field of necro-radiology/virtual autopsy. Imaging techniques including two
-dimensional radiography are currently used in day-to-day forensic investigations in the medical field in contrast to
the veterinary counterpart. We utilized the 2-D radiography in assessing the reliability of observable changes in the
subcutaneous tissue for use in estimating time lapse since death in dog models over a course of twenty-four hours.
A significant change in the severity of postmortem subcutaneous emphysema was observed at the twenty-fourth
hour after death being a progression from the mildly severe state observable at the sixth hour after death. We
therefore concluded that postmortem radiographic assessment of subcutaneous tissue could be used in estimating
time lapse since death for the first twenty-four hours.
Keywords : dogs, gas, postmortem, radiography, subcutaneous tissue
114 
POSTMORTEM RADIOGRAPHIC DIAGNOSIS OF RADIO-OPAQUE FOREIGN BODY
IN THE PROXIMAL DUODENUM OF A CANINE CARCASS
Abdulazeez O.I1 and Noordin M.M1*
1
Clinically, two-dimensional radiographic diagnosis of abdominal foreign bodies are well documented and
easily interpreted. In the case of necropsy, most pathologists often do not have the liberty of obtaining thorough
anamnesis of the subject, as was the case of a 14 kg female mongrel dog submitted for routine necropsy examination. Two pins crossing at an angle of about 400 were observed at the pyloric segment of the duodenum on routine
radiography of the carcass. This report describes the postmortem radiographic signs of pyloric foreign body as
observed in this case. Postmortem radiography can thus be utilized in routine necropsy to identify foreign objects
prior to incision.
Keywords : Dog, foreign body, postmortem, and radiography
115
RIBONUCLEIC ACIDS CONCENTRATION IN POSTMORTEM INTERVAL (PMI)
ESTIMATION IN DOGS IN THE TROPICS
I.O Abdulazeez1 & M.M Noordin 1*
1
Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia,
43400 UPM Serdang, Malaysia.
*Corresponding author: [email protected]
The age-old methods used in routine veterinary pathology involving gross postmortem changes need a
revamp to provide non-biased forensic evidence/opinion in the court of law. Hence, a need for a standard
scientific approach that is repeatable, easily validated. The dearth of information on the behaviour of ribonucleic
acids after death in dogs in a natural tropical ambient condition inspired this research. We attempted and successfully extracted hepatic RNA from four euthanized adult bitches up to the fortieth hour postmortem. Results of
concentration of such extracted RNA did not reflect the postmortem interval hence is unreliable for use as a factor
for estimating time since death in dogs in the tropics.
116 
OPTIMIZED ELUTION OF CEFAZOLIN FROM CALCIUM SULFATE BEADS
W. Kangwansupapan1, N. Phaochoosak1, N. Tansakul1, P. Udomkusonsri1*
1
Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
*Corresponding author
Antibiotic beads are used to treat local bacterial infection. We studied the cefazolin elution from the calcium
sulfate beads and polymethylmethacrylate (PMMA) beads which prepared from different concentrations of
cefazolin. The calcium beads were made by mixing 0.25, 0.5 and 1g cefazolin injection formulation and 10 g
calcium sulfate. For PMMA beads, 1 and 2 g cefazolin were mixed with 10 g of PMMA. The beads were placed in
phosphate buffer saline and daily eluted cefazolin concentrations were determined by microbioassay for 8 days of
experimental period. It was shown that cefazolin-calcium sulfate beads released higher amounts of antibiotic than
cefazolin-PMMA bead significantly (P<0.05). In addition, the eluted cefazolin concentrations were higher than the
minimal inhibitory concentration (MIC) against 2 pathogenic bacteria species, Staphylococcus aureus (ATCC 25928)
and methicillin-resistant S. aureus (MRSA). The study showed the released cefazolin concentrations from 1g
cefazolin-calcium sulfate beads higher than 0.5 and 0.25 g cefazolin-calcium sulfate beads. The released cefazolin
from 2g cefazolin-PMMA bead higher than 1g cefazolin-PMMA bead. This study showed that cefazolin-calcium
sulfate and cefazolin-PMMA beads were both effective for treatment of local bacterial infection.
117
RAPID FROZEN OOCYSTS AGAINST CAECAL COCCIDIOSIS IN BROILERS
S. Sangmaneedet1,2*, P. Pata1, N. Phookrongta1, K. Duangprathum 1
1
Department of Pathobiology, Faculty of Veterinary Medicine, Khon Kaen University.
Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University. THAILAND.
2
Eimeria tenella is an obligate intracellular protozoan that causes severe form of caecal coccidiosis in
poultry. Chickens are infected by ingestion of sporulated oocysts contaminated with feed or water. Sporozoites and
merozoites are invasive stages that cause tissue damage and haemorrhage in the caecum. All stages of E. tenella
could be used to induce protective immunity in chickens. In this study sporulated oocysts rapidly frozen at -80C for
96 hours were investigated the anti-coccidial efficacy in prevention of E. tenella infection in broilers. The frozen
oocysts were orally given to chickens twice at age 1 and 2 weeks old prior to challenging with 30,000 oocysts at 3
weeks old. One hundred commercial broilers were divided into 5 groups (A-E) with 4 replicates. Group A was given
25,000 frozen oocysts at age 3 weeks old. Group B was given with 5 ppm diclazuril in drinking water for 7 days prior
to challenging. Group C was given 2,500 frozen oocysts followed by challenging. Group D was given 25,000 frozen
oocysts and then challenging. Group E was given only sporulated oocysts. Caecal lesions of all chickens were
examined, scored and statistically analyzed. Anti-coccidial medication, diclazuril, reduced damage of caecum from
E. tenella. Chickens given 2,500 or 25,000 frozen oocysts had the same caecal lesion score (1.2) which was
significantly (p<0.05) less than chickens inoculated with sporulated oocysts (2.0). The study indicates that
sporulated oocysts of E. tenella rapidly frozen at -80C may be used as a pretreatment to diminish the severity of
coccidiosis.
Keywords : Eimeria tenella, frozen oocyst, coccidiosis, broilers
118 
EFFECTS OF PELLETING METHODS ON FERTILITY RATE
OF SEMEN FROM THAI NATIVE COCKS
Sarawut Sringam1*, Adisak Sangkaew1, Prayong Sangsriruang1, Patchanee Sringam2
1
Department of Veterinary Surgery and Theriogenology, and 2Department of Veterinary Physiology,
Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, THAILAND.
Although, the fertility of poultry spermatozoa following freezing by the pellet method on dry ice has been
examined in a number of studies. However, dry ice may be not comfortable in some area of the study. To find out
the materials and methods that can substitute the use of dry ice for freezing semen should be done. In this study,
the efficacy of pellet frozen by using stainless steel and copper sheets were to compare to conventional method on
dry ice. Pool semen was collected from 8 fertile male Thai native cocks. The pooled semen was diluted 1:1 with
Beltsville Poultry Semen Extender (BPSE), and adjusted into the final concentration 1,500 X10 6 sperm/ml. The diluted semen was cooled within 20 min at 5 °C and then added with 6% dimethylacetamide and equilibrated for 1 min.
Each semen dilution was dropped ( 0.1 ml) into the well of three surfaces ( 1) dry ice, 2) stainless steel sheet
(0.3X12X12 cm), 3) copper sheet (0.1X12X12 cm)). The 2 nd and 3rd sheets were placed on 5 cm above liquid
nitrogen vapor. After 5 min, the resulted pellets were stored in liquid nitrogen until used for fertility test. Six hens
and 5 replications were intravaginal inseminated with semen from each pelleting method. Two pellets were
thawed in 0.2 ml BPSE in a 1.5 ml tube at 70 °C for 5-7 sec. The fertilization was assessed from the eggs of Days 2 to
3 post-insemination. Fertility rates were determined by candling eggs on Day 7 of incubation. The fertility rates obtained from pelleting frozen by stainless steel sheet and copper sheet method were not statistically significant
differences from dry ice method. This indicated that both metal sheets can use for pellet freezing instead of dry ice.
However, the varied on the thickness and type of metal sheet should be studied further.
Keywords : Fertility, pelleting method, Thai native cock, semen
119
SELECTION OF LACTIC ACID BACTERIA ISOLATED FROM DUCK
FOR PROBIOTICS ADJUNCTS
T. Kimprasit1, P. Songjinda2, S. Sukontasing3 and P. Amavisit4*
1
Center for Advanced Studies for Agriculture and Food, KU Institute for Advanced Studies,
2
Department of Farm Resources and Production Medicine,
4
Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine,
3
Faculty of Veterinary Technology, Kasetsart University, THAILAND.
Lactic acid bacteria (LAB) isolated from ducks’ feces (Anas platyrhynchos) were selected for potential
probiotics. The in vitro study including acid and bile tolerance test, inhibitory activity test against pathogenic
bacteria, and cell surface hydrophobicity test were performed. Three LAB isolates had strong inhibitory activity
against pathogenic bacteria and had high tolerance in stomach acidic condition and ox bile. Using the cell adhesion
evaluation, the isolates had high percentages of surface hydrophobicity at 54.59%, 54.26% and 59.74%, respectively. Though the LAB showed the promising probiotics, further study including sequence analysis and in vivo assay
should be examined for potential LAB confirmation.
Keywords : lactic acid bacteria, grazing duck, probiotics
120 
ANTIBODY RESPONSE TO ACTINOBACILLUS PLEUROPNEUMONIAE
IN CHRONICALLY INFECTED PIG FARMS IN THAILAND
Sukolapa Chiarasumran1, Sureerat Lopiroon1, Trisukhon Phongsayoikham1, Suraphan Boonyawatana2,
Sukuma Samngamnim3, Suphot Wattanaphansak3, Pornchalit Assavacheep3,*
1
Thai Foods Feed Mills Co., Ltd., 2 Intervet (Thailand) Ltd.,
3
Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University.
Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae, becomes a chronic and
silent health threatening disease to the intensive Thai pig producing industry. This investigation was conducted to
detect serum antibody against A. pleuropneumoniae in three chronically infected pig farms in Western part of
Thailand. Serum IgG was detected by an in-house indirect enzyme-linked immunosorbent assay. The antigen was
prepared from boiled whole cell of A. pleuropneumoniae serotypes 11 and 2, which were previously known as
predominant serotypes in all farms. A total of 210 blood samples at 8, 12, 16, 20 and 24 weeks of age were analyzed. The average ELISA-positive pigs at each sampling age were 7.0±9.5, 8.0±5.2, 35.0±37.2, 24.0±17.8, and
41.0±33.2%, respectively. Percentage of serological reactors in each farm was gradually increased, or slightly
declined during weeks 8-12. Increase of serological positive pigs was then found in two occasions during the
observation. The former peak was found at 16 weeks of age in two out of three farms, suggesting an immune
response to either vaccination, or early A. pleuropneumoniae infection. The latter antibody response, which
indicated a natural infection, was again enhanced at 24 weeks of age in all farms. This study demonstrated current
patterns of antibody response to A. pleuropneumoniae in chronic infected pig farms in Thailand, which can be
implemented for prevention and control program.
Keywords : antibody response, Actinobacillus pleuropneumoniae, pig, Thailand
121
EFFICACY OF THE GLUTARALDEHYDE-QUATERNARY AMMONIUM COMPOUND,
DUALGUARD 20®, ON DISINFECTING PIG BUILDING
Kochakorn Direksin*, Ketmanee Senapan, Maneenard Choodetwattana,
Kanokpan Boonpong, Pratchaya Prapaiwong
Department of Veterinary Medicine, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, THAILAND.
The aim of this study was to evaluate the efficacy of Dualguard 20® on disinfecting an evaporative building
of a commercial pig farm (10x50 meters2). Samples were drinking water, pen-washing water, floor swabs, and wall
swabs; pre-cleaning and 30 minutes post-spraying with the disinfectant. The water was tested for general qualities, Salmonella spp., E. coli, and total bacterial count (TBC). The wall and floor swabs (20x20 cm 2) of 5 locations
inside a building were evaluated for the present of Salmonella spp., E. coli, Streptococcus spp, and TBC. The water
used in the farm for diluting of the disinfectant was relatively good quality (pH = 7.0, no nitrate and nitrite,
CaCO3 = 100 ppm, NH4+ = 2 ppm). E. coli was detected in pen-washing water, but not in the other samples. There
was no growth of Salmonella spp. and Streptococcus spp. in every sample. TBC of drinking water, pen-washing
water, pen-washing water mixed with disinfectant, pre-cleaning, and post-disinfecting floor swabs were 0.046×104,
158×104, 0, 30.8×104, 3.6×104 CFU/ml., respectively. The Dualguard 20® was effective in reducing TBC on the floor
of the pig building. In farm conditions, however, it is not possible for the disinfectant to totally kill bacteria.
Efficacy of the disinfectant may be impeded by the presence of organic materials.
Keywords : pig, disinfectant, glutaraldehyde, quaternary-ammonium, total bacterial count
122 
SURVIVAL OF ACTINOBACILLUS PLEUROPNEUMONIAE UNDER
CONTROLLED LABORATORY CONDITIONS
Kornkaew Thongtaeng1, Miruntee Penroj1, Walaitip Wongthai1, Siriyaporn Thongwatchara1,
Sukuma Samngamnim2, Supot Wattanaphansak2, Pornchalit Assavacheep2*
1
2
Sixth year veterinary students, Faculty of Veterinary Science, Chulalongkorn University,
Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University
Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is a highly infectious
disease of pig. The disease is transmitted via direct contact and aerosol nasal discharge from infected pig. The
objective of this study was to investigate the survival of A. pleuropneumoniae under different environment
conditions in the laboratory: temperature, salinity, humidity, and on synthetic materials. Each determination of
A. pleuropneumoniae survival was calculated based on colony count from samples kept under different conditions.
The overall results showed that A. pleuropneumoniae remained viable outside pig more than 48 hours. In a low
temperature environment, A. pleuropneumoniae survived longer than the higher temperature. In the isotonic
solution, survival rate was prolonged. Under controlled humidity conditions, the survival rate was longer in
extremely dry, or wet than natural prevailing humidity. Survival on synthetic materials was further investigated, a
longer viability rate was found on absorbable material, cloth. A lesser extent on non-absorbable surface, rubber
boot was observed. In summary, this study showed physical environmental factors that could potentially affect the
survival of A. pleuropneumoniae included low temperature, salinity, non-humidity or saturated humidity condition,
and synthetic materials. To lessen the pathogen load, alteration of physical environment in pig farm infected with
A. pleuropneumoniae outbreak might be a relevant pleuropneumonia control policy.
Keywords: Survival, Actinobacillus pleuropneumoniae, laboratory conditions
123
INTERPRETATION THE RESULTS OF THE COMMERCIAL ELISA H1N1
AND H3N2 KITS AND HEMAGGLUTINATION INHIBITION ASSAY USING
LOCAL THAI SIV H1 AND H3 VIRUS ANTIGENS
Donruethai Sreta1,3, Sanipa suradhat1,2, Pravina Kitikoon1 , Alongkorn Amonsin1,2 and
Roongroje Thanawongnuwech1,2*
1
Emerging and Re-emerging Infectious Disease in Animals, Research Unit, Faculty of Veterinary Science
2
Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand,
3
Faculty of Veterinary Medicine, Rajamankala University of Technology Tawanok, Chonburi, Thailand
*
Two commercial ELISA tests (HerdChek H1N1 and H3N2 ELISA, Idexx Laboratories, Westbrook, Maine) and
the established in-house HI test using local Thai SIV isolates were evaluated with the swine sera collected from pigs
known to have specific H1N1 and H3N2 SIVs circulating in the selected farms from the 4 highest pig population
provinces of Thailand to compare the findings of the established HI test with the SIV commercial ELISA test kits for
H1 and H3. The results demonstrated that both sensitivity and specificity of the commercial ELISA test kits were
quit low when compared with the HI test using Thai SIV H1 and H3 antigens. Therefore, evaluation of the commercial test kits from other countries is necessary before use in our country. We suggest performing of the serological
diagnosis of Thai SIV using circulating SIV antigens and should be re-evaluated with genetic monitoring.
Keywords : Swine Influenza, ELISA, HI test, Thailand
124 
DETECTION OF MYCOPLASMA HYORHINIS FROM LUNG SAMPLES OF
SEROSITIS NURSERY PIGS
Nantawan Tangwatcharapong1 Panithan Jessadakarn1 Wiyada Winyuchonchareon1
and Athipoo Nuntaprasert 1*
1
The 16s rRNA genes of Mhyorhinis from five field outbreak lung samples were successfully amplified by
using this PCR technique and the resulting PCR products were 700 bp. (Fig.1 and Fig. 2) The sequence data of 16s
rRNA genes of Mhyorhinis from field isolates is on the process. In this study, this PCR technique of detection of 16s
rRNA genes will be useful for differentiation among other Mycoplasmas. Furthermore, this technique will be
developed for detection of this pathogen from other clinical samples such as saliva, nasal and tonsil swabs. This
study will be beneficial for control of this disease such as autogenous vaccine development.
Keywords : Mycoplasma hyorhinis, serositis, nursery pigs
125
EFFECTS OF DIETARY CHITO-OLIGOSACCHARIDE ADDITIVES ON
GROWTH PERFORMANCE AND ILEAL NUTRIENT DIGESTIBILITY IN WEANING PIGS
Sureerat Suthongsa1, Boonrit Thongsong2*, Sarinee Kalandakanond-Thongsong3, Rath Pichyangkura4
1
Program in Animal Nutrition, Department of Animal Husbandry, 2Department of Animal Husbandry,
3
Department of Veterinary Physiology, Faculty of Veterinary Science,
4
Department of Biochemistry, Faculty of Science, Chulalongkorn University, THAILAND.
*Corresponding author: email: [email protected]
Fifty weaned pigs were divided into 5 groups: receiving basal diet (control) and chitooligosaccharide (COS)
additive with three doses of 75, 150 and 225 mg/kg of diet, respectively and Lincomycin; 110 mg/kg of diet in basal
diet. They were used to determine the optimal dosage of COS derived from chemical hydrolysis of chitosan can
affect promoting growth performance and digestibility of nutrients in weaning pigs. The results showed that the
weaning pigs fed the 150 mg/kg COS were found significant increase (p<0.05) in both body weight gain and average
daily gain as well as feed conversion ratio improvement during some periods of time when compared to the control
and the antibiotic group. However, the average daily feed intake of the piglets did not differ among groups
throughout experimental period (56 days). Furthermore, the ileal nutrient digestibility of the weaning pigs fed the
150 mg/kg COS had significantly increased (p<0.05) in crude protein, fat and calcium compared to those in control
group at day 28th and day 56th of experimental period.
Key words : chitooligosaccharide, growth performance, nutrient digestibility, weaning pigs
126 
EVIDENCE OF HP-PRRS VIRUS WATER TRANSMISSION IN THAI SWINE FARM
1
PRRS virus (PRRSV) is a small, non-enveloped, circular, single-stranded RNA virus, belonging to genus
Arterivirus, which infects swine. Up to now, swine PRRS have been detected in serum, plasma, semen, nasal and
rectal swabs. HP-PRRSV appears to be widespread in the asian swine population, and the direct contact route is
considered the main route for viral dissemination and rapid transmission. Recently, studies in PRRS infected farms
have suggested that air, some insects or vehicles outside might contribute to transmission of swine PRRSV. The
objective of present study was determined by studying whether PRRSV can be survived in drinking water. Drinking
water samples from five different Thai swine herds in Ratchaburi province on Apil-August 2011 were collected.
Samples were filtered with a 125 mm filter papers (Whatman, USA) and papers were collected. Total RNA from
filtered paper sample was extracted using a commercial kit according to manufacturer’s instructions and detected
PRRSV using different specific PCR methods. Our results indicated that all five drinking water samples from five Thai
swine herd in Ratchaburi province were HP-PRRSV PCR positive. The detection of PRRSV in drinking water route
could provide evidence that PRRSV may enter into pig farms. From this result, the pig farmer should examine the
water supply for pigs in the farm regularly as well as should find the effective methods for treatment of
contaminated water before use in pigs. This study will be useful for control PRRSV as well as to understand the
epidemiology of PRRSV.
Keywords : HP-PRRSV, PRRSV, drinking water, PCR
127
DETECTION OF APXIV GENE IN A. PLEUROPNEUMONIAE
ISOLATES FROM THAI FATTENING PIGS IN 2011
1
Actinobacillus pleuropneumoniae (APP) causes pleuropneumonia which a severe mortal disease that
affects pig of all ages. There are four different Apx toxin genes have been found to be produced by the 15
serotypes: ApxI, ApxII, ApxIII and ApxIV. The ApxIV genes are species-specific and useful for detection all of 15 APP
serovars. The objective of this study was amplified ApxIV gene to detect APP isolates from Thai fattening pigs in
2011. Six fattening swine farms with APP history outbreak from six different regions of Thailand in 2011 (JanuaryAugust) were selected. The DNA was extracted from pure colonies using Total DNA Mini Kit (Geneaid, Taiwan). The
PCR which amplified ApxIV was performed with the specific primers. Our results showed that APP isolates were
successfully detected all serotypes and identified by PCR technique based on ApxIV gene. Three provinces (Nakhon
Sawan; Nakhon Pathom and Chonburi) were detected nine serotypes (1, 3, 4, 6, 9, 11, 12, 13 and 14) and Prachinburi province was detected only four serotypes (4, 6, 9 and 11). In contrast, two remaining provinces (Surin and
Yala) were found twelve serotypes (1, 2, 3, 5a, 5b, 7, 8, 10, 12, 13, 14 and 15). This study will be useful for understanding the epidemiology of an APP outbreak and the selection of vaccine for control of this disease in Thailand in
the future.
Keywords : APP, ApxIV, gene, serotype, PCR
128 
CURRENT INFORMATION ON PREVALENCE AND ANTIMICROBIAL
SUSCEPTIBILITY OF AEROMONAS SP. AND EDWARDSIELLA TARDA
ISOLATED FROM DISEASED GOLDFISH (CARASSIUS AURATUS)
Channarong Rodkhum1* Satidyos Soontronsatidpimon3, Panus Wongnonti3, Pilinporn Kanrai3,
Suphap Kumlangphaet1, Nopadon Pirarat2
1
Department of Veterinary Microbiology,2Department of Veterinary Pathology,
Faculty of Veterinary Science, Chulalongkorn University, Bangkok, THAILAND, and
3
Veterinarian in private institutes, THAILAND
Forty one (41) goldfishes from Thai ornamental fish shops suspected to have bacterial infection were
physically examined and necropsied. The fish organs were collected and isolated for the bacterial agents. The isolated bacteria were identified and analyzed for the prevalence and antimicrobial susceptibility. Fifty one (51) isolates of Aeromonas sp. and Edwardsiella tardar were successfully isolated from goldfishes including A. hydrophila
(39.22 %), A. sobria (33.33 %), A. caviae (5.88 %), A. salmonicida (11.77 %), and E. tarda (9.80 %). The antimicrobial susceptibility showed that 75% of A. hydrophila were susceptible to florfenicol and enrofloxacin and 85 %
were resistant to ampicillin, 88.24% of A. sobria were susceptible to enrofloxacin and 76.47 % were resistant to
ampicillin, 100 % of A. caviae were susceptible to florfenicol and 66.67 % were resistant to oxolinic acid, 83.33%
of A. salmonicida were susceptible to florfenicol and 66.67 % were resistant to ampicillin and oxolinic acid. Additionally, all E. tarda were susceptible to enrofloxacin and resistant to ampicillin. The results can be a preliminary
data that will be applied for selection of antimicrobial agents for control fish bacteria particularly Aeromonas sp.
and E. tarda.
Keywords: prevalence; antimicrobial susceptibility; Aeromonas sp.; Edwardsiella tarda; diseased goldfish
129
DETERMINATION OF SERUM CALCIUM CONCENTRATION
IN PERIPARTURIENT DAIRY COWS
Wandee Thiangtum1*, Niorn Ratanapob1, Samit Srisomrun2, Supada
Kananub3, Chanita Sujaritthanyatrakul3, Angkhana Khantaboot3
1
Department of Large Animal and Wildlife Clinical Sciences,
Kasetsart Veterinary Teaching Hospital –Kamphaeng Saen,
3
Kasetsart Veterinary Teaching Hospital- Nong Pho, Faculty of Veterinary Medicine,
Kasetsart University, THAILAND.
2
Serum calcium (Ca) concentration during periparturient or transition period of 1 month before and 1
month after calving is important for health and production in dairy cow, since most incidence of subclinical hypocalcaemia occurs in this period. The objective of this study was to investigate the serum Ca concentration of periparturient cows. Fifty three cows from 30 dairy farms were examined. Cows were divided into 2 groups by parity;
group 1 (1st and 2nd parity cows, n=27) and group 2 (>2nd parity cows, n=26). Each cow was collected blood samples
at d -30, d 0, and d 30 related to calving date and Ca concentration of the samples were determined. The average
serum Ca concentration at d 0 was significant lower than at d -30 and d 30 (P<0.05). Cows in group 1 had significantly higher Ca concentration than cows in group 2. The prevalence of subclinical hypocalcaemia were 48% and
61.5% in group 1 and group 2, respectively. The result indicated that dairy cows in advanced age were more likely
to develop subclinical hypocalcaemia.
Keywords : Dairy cows, Periparturient, Serum calcium, Subclinical hypocalcaemia
130 
PATHOGENICITY TO MEKONG GIANT CATFISH EGGS IN LABORATORY OF WATER MOULDS
ISOLATED FROM MEKONG GIANT CATFISH EGGS AND THE REARING WATER
Narong Abking,1 Wichukarn Fuangsawat 2 and Ong-ard Lawhavinit 2*
1
Department of Veterinary Technology, Faculty of Veterinary Technology,
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
2
Water moulds in the genera Achlya and Saprolegnia were isolated from eggs of the Mekong giant catfish,
Pangasianodon gigas, and the hatching tank water at the Inland Aquaculture Research Institute, Phra Nakhon
Sriayutthaya province, from 2008 to 2010. The optimal temperature of almost all isolates of the isolated was 30 °
C. The Achlya spp. and Saprolegnia spp. could tolerate a NaCl medium at 10 ppt and 25 ppt, respectively. An
exception was Saprolegnia sp. (E3/52-P2) which could tolerate NaCl up to 30 ppt. The isolates could grow in broth
at pH 4–11, while the optimal pH for Achlya spp. and Saprolegnia spp. was pH 5 and pH6, respectively. The study
on pathogenicity of the water moulds isolated in the laboratory was showed that the isolates were pathogenic to
the eggs are Achlya spp. (T.MCF1-02, E.MCF 2-001 and E4/52-10) and Saprolegnia sp. (E1/53-12).
Keywords : Eggs, Mekong giant catfish, Pangasianodon gigas, Achlya spp., Saprolegnia spp., pathogencity
131
LAPAROSCOPIC OVARIECTOMY IN NATIVE-SAANEN GOATS
Peerasak Suttiyotin 1*, Chowalit Nakthong2, Bunlue Kornmatitsuk2 and Somkiat Huaijantug2
1
Department of Preclinic and Applied Aanimal Science,
Department of Veterinary Clinical and Public Health, Faculty of Veterinary Science, Mahidol University, THAILAND.
2
Laparoscopic ovariectomy has been performed in many species but very few information was available for
goats. The aims of this study were to apply the technique to goats and to evaluate surgical time for removing of
ovaries. The animals were under general anesthesia during the surgery. Continuous ECG, blood gas analyser, pulse
oximetry and rectal temperature measurement were monitored during the surgery. The animals were placed in
dorsal recumbency in a 30° Trendelenburg position. Three-portal access technique was used, the main portal
positioned at midline, 3cm caudal to the umbilicus. The other 2 instrument portals were 5cm lateral to midline and
11cm caudal to the umbilicus. Grasping forceps and bipolar cauterizing forceps were used to manipulate,
coaagulate, disect and remove ovaries from abdomen.
We successfully performed laparoscopic ovariectomy in the goats. Exploration of abdominal space took
less than 1 minute. It took 4-8 min to grasp, cauterize and remove an ovary from abdominal cavity. Mean surgical
time was 23.8min, longer in the first animal (28.3 min) and shorter in the others. This indicates that training and
practicing may play important roles.
At best of our knowledge, this is the first study performed in goats in Thailand. We believe that the
laparoscopic overiectomy in goats will be beneficial to management and research in small ruminant.
Keywords : goat, laparoscopic ovariectomy, reproduction
132 
RISK FACTORS CONTRIBUTING TO ALCOHOL TEST POSITIVE IN SMALL DAIRY HERDS
IN SUPHANBURI PROVINCE, THAILAND
Pipat Arunvipas1* Tanasorn Tippayarak1 Adisorn Yawongsa1 Kitsada Bumrungkit1
1
Department of Large Animal and Wildlife Clinical Sciences, Faculty of Veterinary Medicine,
Kasetasrt University, Kamphaengsaen Campus, Nakhon Pathom 73140 THAILAND.
An investigation in to the reasons why milk rejection was high in small dairy herds in Bangblama district,
Suphanburi provinces was conducted in response to farmer’s complaints in August, 2010. The reason for milk
rejection was false positive results from the alcohol test. Herd data was collected by questionnaire and all milk
samples from 5 alcohol test positive herds were obtained and tested with the Alizarin test. All positive samples
were retested with the colt on boiling test. The data were analysed to determine the risk factors for these herds.
The result showed that late lactation cows (> 200 DIM), high lactation number and low quality of feed in farm were
risk factors of the false positive tests.
Keyword: Risk factors, milk rejection, alcohol test
133
SOMATIC CELL COUNT AND MILK COMPOSIITON OF DAIRY COWS KEPT BY
SMALL-SCALE HOLDERS IN CENTRAL THAILAND
S.Panneum1*, T.Rukkwamsuk2
1
2
Kasetsart University Veterinary Teaching Hospital Nhongpho,
Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140 THAILAND.
Corresponding author: e-mail: [email protected]
One thousand and five hundred thirty six milk samples were collected from 160 small-holder dairy farms
belonging to 6 Dairy Cooperatives and 2 Milk Collecting Centers from 8 provinces in the Central Thailand. All milk
samples were analyzed for somatic cell count and milk composition. Results showed that average fat, protein,
lactose and solid non fat content in milk were 3.67±2.63%, 3.10±0.47%, 4.68±0.41%, 8.49±0.62%, respectively.
These values were present study reviewed that average value of all milk contents were in normal value and were
similar with the previous study results which were of fat content, protein content, lactose content and solid not fat
content respectively. And milk composition of 8 different cooperatives were significantly difference due to the
difference of farmer’s feeding and management practices of each dairy cooperative. Natural logarithm of somatic
cell count (LNSCC) in this study was also in the normal value and tended to be lower as compared with the others
previous study results. The average LNSCC was 5.00±1.71.and were totally different among the cooperatives. This
probably might associated with milking hygiene and mastitis control program among farms and policy of each Diary
Cooperative to control of mastitis and udder health would be an major factor that influenced in difference
of LNSCC.
Correspondence mailing author: Kasetsart University Veterinary Teaching Hospital Nhongpho Ratchburi 70120.
Thailand. tel.0-3238-9293 fax.0-3238-9295
134 
INTRAMAMMARY INFECTION AT DRY OFF IN TWO LARGE DAIRY HERDS
Piyanat Prasomsri1*, Nirun Chunakoop2 , Kittisak Ajariyakhajorn1
1
Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, THAILAND.
2
Khumjareon Agricultural Co-operative, Nakorn Ratchasima, THAILAND.

Dry period is critical period of mammary gland involution in which dairy cow needs to revitalize mammary
gland tissue. Intramammary antibiotic infusion in all quarters at dry off is routinely used for dry cow management.
However, intramammary infection (IMI) at dry off is rarely revealed. This study, we determined IMI at dry off in two
large dairy herds during May to August 2011. We examined fifty three cows (208 quarters) for subclinical mastitis
using California Mastitis Test (CMT) and collected their quarter milk samples at 7 days before dry off and at dry off
for bacteriological culture. At 7 days before dry off and at dry off, results indicated that the number of CMT
negative quarters were 52.9% (110/208) and 50.0% (104/208), respectively. The uninfected quarters were
82.2% (171/208) and 79.8% (166/208), respectively. The most frequently bacterial pathogen associated with
IMI was coagulase negative staphylococci (CNS). These results indicated that most of studied quarters at 7 days
before dry off and at dry off were uninfected in which half of them have shown no sign of clinical and subclinical
mastitis. This informative data would be useful for reconsidering the intramammary antibiotic infusion in all
quarters at dry off in these studied herds.
Keywords : Intramammary infection, dry off, dairy cow
135
INFLUENCE OF COLOSTRAL QUALITY ON SERUM PROTEIN IN DAIRY CALVES RAISED IN
SMALL HOLDER FARMS
Suppada Kananub1, Theera Rukkwamsuk2, and Pipat Arunvipas2
1
Kasetsart University Veterinary Teaching Hospital Nongpho, Faculty of Veterinary Medicine,
2
Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140 THAILAND.
The objective of this study was to analyze the influence of colostral quality on serum protein of calves.
Samples were randomly selected from Veterinary Medical Teaching Hospital at Kampangsaen Campus and
Nongpho Animal Hospital visited farms in Nakhon pathom, Ratchaburi and Kanchanaburi province, in total 34 farms
contributed 160 samples. Total immunoglobulin of colostrum from calving date, 1 and 2 days after calving was
95.25±2.73, 37.37±2.73 and 17.56±2.77 mg/ml, respectively (P<0.01). Total immunoglobulin decreased with
milking times. The calves that fed high quality colostrum had higher serum protein, 7.49±0.13 g/dl, than calves fed
low quality colostrums, 6.4±0.22 g/dl (p<0.01). Increased serum protein after first colostrum feeding of high and
low quality colostrum was 1.55±0.13 g/dl and 0.81±0.18 g/dl (P<0.02).
136 
THE SURVIVAL OF BACTERIAL MASTITIS PATHOGEN IN MILK SAMPLES
Paveenuch Charoenkitroj1, Panyaphat Pomim1, Suthinee Krisnakupt1, Sukuma Samngamnim2,
Kittisak Ajariyakhajorn2*
1
Sixth year veterinary student, 2Department of Veterinary Medicine,
Faculty of Veterinary Science, Chulalongkorn University, THAILAND.
Mastitis is an important major loss in dairy industry. This costly disease is mainly caused by the bacterial
intramammary infection (IMI). Diagnosis of causing mastitis pathogens in dairy cows is the crucial information for
treatment, control and prevention strategies. However, fresh milk samples are not always immediately submitted
to laboratory. Condition of milk sample storage may influence the number of recovery bacteria and ability of
laboratory to identify mastitis pathogens. This study aims to determine the survival of bacterial mastitis pathogen
in milk samples at different temperature storage conditions. Firstly, we evaluated the survival of important IMI
bacterial pathogen including five isolates of each Staphylococcus aureus, Streptococcus agalactiae and Escherichia
coli in UHT milk. The tested milk samples were kept at three different temperatures (-20, 0 and 4oC) for 30 days.
The samples were determined for bacterial count at 0 (fresh sample), 1, 2, 7, 14 and 3 0 days. There were
significant different in bacterial count of Staphylococcus aureus in milk samples kept at 4 oC for 14 and 30 days
comparing to fresh samples (p <0.05). The bacterial counts of Escherichia coli were significant different for samples
that were kept for 30 days both at 4 oC and at 0 oC. This also occurred for samples kept for 7 and 14 days at -20 oC.
The second part of this study, eighteen field mastitic milk samples in which only single significant isolated IMI
pathogen was evaluated. Six mastitis pathogen groups were detected including CAMP-/ESCULIN+Streptococcus
spp., CAMP-/ESCULIN- Streptococcus spp., Staphylococcus aureus, Coagulase negative Staphylococci, Corynebacterium bovis and Yeast. Similar protocol as previous part was conducted. There were not significant different of
bacterial count at any temperature conditions for all samples kept for 30 days. These findings may influence by
the great variation of bacterial number in fresh samples at the beginning.
Keywords : Survival, Mastitis Pathogen, Milk
137
POLYMERASE CHAIN REACTION BASE-PREVALENCE OF
ENCEPHALITOZOON CUNICULI IN MEAT RABBITS
Preeda Lertwatcharasarakul1*, Attawit Kovitvadhi2, Kamonchanok Aiemsumaung2,
Boonchoo Piempinseat2, Patcharida Dittawong2, Waraporn Sinsuwonkwat3, Sakuna Phatthanakunanan4,
Siriluk Jala4, Aree Thayananuphat5, Pornchai Sanyathitiseree6
1
Department of Pathology, 2Sixth year’s student, 3Graduate student, 4Kampangsaen Veterinary Diagnostic Center,
5
Department of Companion Animals Clinical Sciences, 6 Department of Large Animal and Wildlife Clinical Science,
Encephalitozoonosis is an important protozoal disease in a rabbit. The predilection organ of an infection is
central nervous system, kidney and eye, and often causes a chronic latent disease. During November 2009 to July
2010, 84 rabbit’s brain samples were investigated from 2 slaughterhouses which supplied rabbits from Phetchaburi
and Nakhon Pathom province. The rabbits were divided into two groups: with neurological sign (group 1 suspected
Encephalitozoonosis) and without neurological sign (group 2 normal). Brain samples were extracted and amplified
by nested polymerase chain reaction. The positive result in group 1 and 2 revealed 42.9% (n=7) and 28.6% (n=77)
respectively. The positive result in Nakhon Pathom and Phetchaburi province presented 45% (n=11) and 25%
(n=66) respectively. The finding indicated that Encephalitozoon cuniculi was found in Thailand’s meat rabbit farms
and the appearance of DNA did not correlate with clinical sign.
Keywords : Encephalitozoon cuniculi, rabbit, PCR
138 
COMPARATIVE MORPHOMETRY AND ULTRASTRUCTURE OF HEPATOZOON
SPECIES INFECTED IN MANGROVE SNAKE (Boiga dendrophila melanota)
AND KING COBRA (Ophiophagus hannah)
T. Chalalai1*, C. Salakij1, P. Lertwatcharasarakul1, K. Prihirunkit1, T. Vasaruchapong2
1
2
Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok 10330, THAILAND.
Hepatozoon species are the most common intraerythrocytic parasite in reptiles. Heavy infection can lead to
anemia and blood cell morphology abnormality. The objective of this study was to performed comparative
morphometry and ultrastructure of Hepatozoon species that detected in Mangrove snakes (Boiga dendrophila melanota) and King cobras (Ophiophagus hannah). Blood samples were collected from two Mangrove snakes and six
King cobras from Queen Saovabha Memorial Institute. Light microscopy examinations, gamonts of Hepatozoon sp.
were observed in blood smear from all snakes. Gamonts were laterally displacement nucleus of erythrocyte. Some
gamonts were observed in plasma. The width and length of Hepatozoon gamonts were measured, there were
significant differences between Mangrove snakes and King cobras (p < 0.05). Gamonts from King cobra were larger
than gamonts from those of Mangrove snake. Transmission electron microscopic examination showed the gamonts
within parasitophorous vacuole membrane. The gamont was included with nucleus, conoid, rhoptries and
micronemes.
Keywords : hepatozoon, morphometry, ultrastructure,King cobra, Mangrove snake
139
RECENT TREND OF FOOD BORNE DISEASES IN JAPAN
Shigeki Yamamoto1*
1
National Institute of Health Sciences, MHLW, Japan.
Japan has two different surveillance systems. One is the data collection for food borne diseases and the
other is for infectious diseases. Food borne diseases are firstly reported from medical doctor to a local health
center. Food inspectors in the local health center investigate the cause of food borne diseases. Final results report
are sent to the Ministry of Health, Labour and Welfare. Food borne diseases are defined as the adverse effect to
humans after ingestion of food and drinks. Causative agents of food borne diseases are microorganisms such as
bacteria and viruses, chemicals such as poison of mushroom and fishes and toxic substances. Most cases of food
borne diseases are caused by microorganisms. The highest case during the decade is norovirus food poisoning, 250
to 500. Number of patients of norovirus is 8000 to 27000. Second is Campylobacter jejuni cases. These two food
borne diseases are very difficult to control. In case of norovirus, disease is transferred from human to human and
very few amount of agents can cause disease. In case of Campylobacter, disease is caused by chicken meat. More
than 80% chickens in some farm are infected with Campylobacter. Salmonella spp. and Vibrio parahaemolyticus
cases, popular food borne diseases in Japan are declined during the decade. Salmonella cases are mostly caused by
S. enteritidis in eggs.
Keywords : Food borne disease, Bacteria, Virus
140 
MOUSE STRAIN DIFFERENCES IN SUSCEPTIBILITY TO
DIARRHETIC SHELLFISH POISONING TOXINS, IN MOUSE BIOASSAY
Hodaka Suzuki 1*, Kenji Machii 1
1
Division of Biomedical Food Research, National Institute of Health Sciences, JAPAN.
*
The mouse bioassay has been used as the official method for detecting diarrhetic shellfish poisoning (DSP)
toxins in Japan for 30 years. This method, well known as Yasumoto’s method, has also been widely used in many
countries of the world. In the Japanese official protocol, ddY strain or ICR strain male mice weighing 16 – 20 g are
specified to be used in the assay. To the best of our knowledge, however, there have been no reports specifically
on the strain differences in susceptibility to DSP toxins. In this study, we investigated the strain differences of mice
to DSP toxins. A lethal dose of okadaic acid (OA), one of the representative DSP toxins, was injected intraperitoneally into mice. The mice were observed until 24 hours after injection. Five inbred strains (A/J, BALB/c, C3H/He,
C57BL/6, and DBA/2) and 2 non-inbred strains (ddY, and ICR) of mice were compared. All the mice were male,
weighed 16–20 g, and were 4–5 weeks old. The lethality of the mice was 90–100% in the A/J, BALB/c, ddY, and ICR
strains; 70–80% in the C3H/He and C57BL/6 strains; and 40% in DBA/2 strain. Survival analysis showed that the
BALB/c, C57BL/6, ddY, and ICR strains died earlier and the A/J, C3H/He, and DBA/2 strains survived longer. These
results indicate that significant mouse strain differences may exist in susceptibility to DSP toxins and that the
mouse strains used may be one of the crucial factors in the mouse bioassay.
Keywords : diarrhetic shellfish poisoning toxin, mouse bioassay
141
MICROBIOLOGICAL QUALITY AND ANTIBIOTIC RESIDUE OF BOILED MILK
IN BANGKOK AND METROPOLITAN REGION
Tirapa Rattanamatee1, Shunya Sanla1, Nichapat Yurayart1, Piyanat Prasomsri2,
Sukuma Samngamnim2, Thanasak Boonserm2, and Kittisak Ajariyakhajorn2*
1
Sixth year veterinary student, 2Department of Veterinary Medicine,
Faculty of Veterinary Science, Chulalongkorn University, THAILAND.
* Corresponding author : Email :
The microbiological quality and antibiotic residue of boiled milk sold by street vendors in Bangkok and
Metropolitan region were examined. One hundred samples of boiled milk were randomly collected from Bangkok
(n=50) and metropolitan areas (n=50) including Nonthaburi, Samut Prakan, Nakhon Pathom, and Samut Songkhram
provinces during April-July, 2011. We examined total plate count (TPC), laboratory pasteurized count (LPC), coliform counts, Escherichia coli count, Salmonella spp. Antibiotic residue was detected using microbial inhibitory assay. Our results revealed that an average of total bacterial count was 2.53±1.86 log colony-forming units (CFU) per
mL in which ranged 0.00 to 7.96 log CFU per mL. Laboratory pasteurized count ranged 0.00 to 5.42 log CFU per mL.
Coliform count ranged 0.00 to 5.51 log CFU per mL. Escherichia coli was found in only one sample in which the
count was 4.48 log CFU per mL. The number of samples that above Thai FDA pasteurized milk limitations were 23
and 11 samples for total bacterial count (≤ 10,000 CFU per mL) and coliform count (≤ 100 CFU per mL), respectively.
Salmonella spp. and antibiotic residue could not be detected in this investigation.
Keywords : Microbiological Quality, Antibiotic Residue, Boiled milk
142 
CULTIVATION OF JAVAN PORCUPINE (HYSTRIX JAVANICA) WHICH IS AN ORIGIN SPECIES
EXPECTED ANIMAL OF INDONESIA TO REINFORCE NATIONAL FOOD SECURITY
AND SOLVE THE PROBLEM OF ORGANIC WASTE.
Sheila 1, Risaar Siringo-ringo1, Supratikno 2, Srihadi Agungpriyono2
1
Undergraduate students of KH IPB 2Laboratory of Anatomy, Section of Anatomy,
Department of Anatomy, Physiology, and Pharmacology Faculty of Veterinary Medicine,
Bogor Agricultural University Jl. Agathis, Kampus IPB Dramaga Bogor (0251)8421865, Bogor,
Meat is an important animal protein sources for human but Indonesian meat consumption is still very low
because of it price. Meat prices become expensive due to Indonesia government still import meat. Furthermore,
meat import makes animal diseases (zoonoses) get easier to spread in Indonesia. Javan porcupine is an exotic
animal which is origin species of Indonesia that can be used as animal protein source. People in Central Java think
that porcupine is a pest. Therefore, they use porcupine as one of their meal. Some people who lived in Tawangmangu (Central Java, Indonesia) says that porcupine meat is really delicious and they believed that porcupine meat
have medicine effect to their body. Unfortunately, people who eat porcupine meat get it meat from hunted it in it
natural habitat. If this happened day by day it is possible if porcupine population will decrease and extinct. Beside
of porcupine meat, porcupine quills also can be used as a handicraft material and it quiet expensive product.
Porcupine management breeding also isn’t difficult. So, porcupine can be breed in small area or as family backyard
farming. Porcupine has minimal defecation and urination. Therefore, breeder only need once time a day to clean
porcupine cage. From feed aspect, porcupine can be feed by using market/ kitchen organic garbage like vegetables
and fruits. With using market/ kitchen organic garbage, can decrease the total of garbage in Indonesia. Porcupine
breeding is supports the government's efforts in alleviate the poverty problem, food security, and supports the
conservation efforts.
Keywords: exotic animal, Javan porcupine, food security
143
THE EVALUATION OF PERFORMANCE OF VETERINARY SERVICES (OIE PVS)
OF THAILAND: WHAT IS THE OIE PVS TOOL?
Thanawat Tiensin1*, Vilaiporn Wongphruksasoong1, Lamai Nammongkol1,
Wacharapon Chotiyaputta2 , and Sompop Chittapraphai1
1
Bureau of Disease Control and Veterinary Services, Department of Livestock Development, THAILAND,
2
Division of Foreign Livestock Affairs, Department of Livestock Development, THAILAND,
The World Organisation for Animal Health (OIE) has developed an Evaluation Tool for the Evaluation of
Performance of Veterinary Services (OIE PVS Tool) based on the chapters of the OIE Terrestrial Animal Health Code
and Aquatic Animal Health Code (the OIE Codes) relating to the quality of Veterinary Services and adopted by all
OIE Member Countries. The OIE PVS Tool is designed to assist Veterinary Services to establish their current level of
performance and to identify gaps and weaknesses in their ability and consistent with OIE international standards
and to comply with the agreement on the Application of Sanitary and Phytosanitary Measures (SPS Agreement) of
the World Trade Organization (WTO). Thailand’s Department of Livestock Development as a Permanent Delegate
of OIE has applied for the Evaluation of Performance of Veterinary Services based on OIE PVS Tool. The OIE PVS
evaluation team will tentatively visit Thailand by the end of 2011 for evaluation of Veterinary Services in the country. The objective of this report was to review the OIE PVS pathway, to form a shared vision with stakeholders
(including the private sector, academic institutions and professional organizations), and to establish priorities and
carry out strategic initiatives, and to have better understanding of OIE PVS Tool among all stakeholders. Active participation and investment by both the public and private sector is required in order to facilitate the strengthening
of Veterinary Services and their compliance with OIE international standards for quality and evaluation.
Key words : OIE, Evaluation, Performance of Veterinary Services, PVS, Thailand
144 
SEROLOGIC SURVEY ON BARTONELLA SPP. AMONG VETERINARY PROFESSIONALS
Nathatai Wanachalerm1, Fanan Suksawat1*, Orathai Pachirat2, Michael Y. Kosoy3, Jennifer Iverson3 Soawalak
Sripakdee4, Anusak Kerdsin4, Surang Dejsirilerk4, Pithai Kanbutra5, Pittaya Papirom6
1
Department of Veterinary Medicine, Faculty of Veterinary Medicine,
Department of Cardiology Medicine, Faculty of Medicine, Khon Kaen University ,Khon Kaen, Thailand,
3
Centers for Disease Control and Prevention, Fort Collins, Colorado, USA,
4
Department of Medical Sciences, Ministry of Public Health, Nontaburi Thailand,
5
Department of Veterinary Teaching Hospital, 6Department of Pathobiology,
Faculty of Veterinary Medicine, Khon Kaen University ,Khon Kaen, Thailand.
2
Bartonellosis caused in humans by Bartonella spp. is mostly zoonotic. At least 5 species of Bartonella are associated with human illnesses in Thailand: B. henselae, B. tamiae, B. tribocorum, B. elizabethae, and B. vinsonii
arupensis. Endocarditis and most other diseases are mostly reported from immunocompetent people. Ticks, fleas,
lice, and mites are confirmed or proposed as transmission vectors. Veterinary professionals are at risk due to the
regularity in contact with Bartonella spp. reservoirs. Therefore, objectives of this study were to investigate
Bartonella spp. in volunteering human subjects who works at veterinary clinics and to evaluate risk of Bartonella
spp. for veterinary professionals based on analysis their samples. Blood samples from 63 volunteers were collected.
Indirect fluorescent assay (IFA) was used for detection of specific antibodies or DNA. None of serum samples was
reactive to B. henselae, B. quintana, B. clarridgeiae, and B. vinsonii arupensis antigens. This study may indicate that
Thai veterinary professionals follow good veterinary practice and hygiene. However, molecular investigations are
required to determine the actual infection and to estimate the professional risk among veterinarians in different
parts of Thailand.
Keywords : Bartonella spp., Veterinary professinals, Poymeras chain reaction; PCR,Indirect fluorescein assay;IFA
145
ANTIMICROBIAL RESISTANT BACTERIA FROM DOGS ISOLATED
AT THE VETERINARY DIAGNOSTIC UNIT, KASETSART UNIVERSITY
P. Hanhaboon1, C. Bumrungpun2, S. Wongnarkpet3, and P. Amavisit1*
1
Department of Microbiology and Immunology, 2The Veterinary Diagnostic Unit,
3
Department of Public Health, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
* Corresponding author : email : [email protected]
The bacteria isolated from dogs brought to KU veterinary hospital in 2008 were reported for their antimicrobial resistance and originated sample. Top three collected samples including ear (543 isolates), skin (527 isolates), and urinary tract (488 isolates) were presented for their antimicrobial resistances against six drugs: amoxicillin, amoxicillin-clavulanic acid, cephalexin, ciprofloxacin, and enrofloxacin. In general, amoxicillin-clavulanic acid
resistant rates still half lower than amoxicillin resistant rates. This implies that the problem of extended spectrum
beta lactamese of bacteria isolated from dogs in this period was better than human isolates. The common bacterial
species isolated from ear samples were Pseudomonas spp, Staphylococcus spp. and E.coli that had different antimicrobial resistant profiles. Among them Pseudomonas spp had the highest resistant rates for amoxicillin, cephalexin,
amoxycillin/clavulanic acid, sulfamethoxazole/trimethoprim and doxycycline at 91%, 89%, 86%, 82%, and 78%
respectively. However different resistant rates were presented in Staphylococcus spp and E. coli. Therefore the antimicrobial of choice for otitis treatment in dogs should be concerned carefully following the species of bacterial
infection.
Keywords : antimicrobial resistance, dog, bacteria
146 
THE PREVALENCE AND GENOTYPES OF CRYPTOSPORIDIUM SPP.
FROM WATER BUFFALOES IN KHON KAEN PROVINCE
Fanan Suksawat1*, Somboon Sangmaneedet2 Tawin Inpankaew3, Wissanuwat Chimnoi3,
Nongnuch Pinyopanuwat3, Sathaporn Jittapalapong3
1
Department of Veterinary Medicine, 2Department of Pathobiology,
Faculty of Veterinary Medicine, Khon Kaen University ,Khon Kaen, Thailand,
2
Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University ,Bangkok, Thailand.
The prevalence and genotype of Cryptosporidium spp., from water buffalo (Bubalus bubalis) fecal samples in
Khon Kaen province were serologically and molecularly determined. A total of 275 fecal samples from water
buffaloes with average age 3.5 years in Khon Kaen were collected and examined by the acid fast stain technique
and polymerese chain reaction restriction fragment length polymorphism (PCR-RFLP). The prevalence of Cryptosporidium in buffaloes was 27 and 12 % by acid-fast staining and PCR, respectively. In this study, all PCR positive
samples were C. parvum (bovine genotype). This result indicated that Khon kaen might be endemic for
Cryptosporidium spp infection and water buffaloes were the carrier.
Keywords : hepatozoon, morphometry, ultrastructure,King cobra, Mangrove snake
147
MOLECULAR DETECTION OF TRYPANOSOMA EVANSI
FROM SLAUGHTERED COWS IN KHON KAEN PROVINCE
Fanan Suksawat1*, Somboon Sangmaneedet2, Marc Desquesnes3, Nongnuch Pinyopanuwat4
Wissanuwat Chimnoi4, Ketsarin,Kamyingkird4, and Sathaporn Jittapalapong4
1
Department of Veterinary Medicine, 2Department of Pathobiology,
Faculty of Veterinary Medicine, Khon Kaen University ,Khon Kaen, Thailand.
3
CIRAD-Bios, Systèmes Biologiques, UMR-Intertryp, Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Montpellier, F-34000 France ;
4
Department of Parasitology, Faculty of VeterinaryMedicine, Kasetsart University ,Bangkok, Thailand.
The aim of this study was to detect Trypanosoma evansi from cows at slaughter houses in Khon Kaen
province. A total of 204 blood samples were collected and examined by microhematocrit centrifugation technique
(MHCT), card agglutination test (CATT/T. evansi) and polymerase chain reaction. One T. evansi positive sample was
found by MHCT, 14 by CATT and 4 by PCR. All 4 PCR positive samples were from 14 CATT positives in this study.
This results indicated a possible risk of trypanosomiasis transmission among cattle and might be a source to
transmit to other nearby animals. This will need more investigations on strain diversity to conclude the potential
risk of T. evansi on human health.
Keywords : Trypanosoma evansi, cows, Khon Kaen
148 
A NEW GENOTYPE OF BARTONELLA ISOLATES FROM DEER
(RUSA TIMORENSIS) IN THAILAND
Decha Pangjai1*, Santaya Intachinda2, Soichi Maruyama3, Sumalee Boonmar4,
Hidenori Kabeya3,Wimol Petkanchanapong1, Wattanapong Wootta1, Piyada Wangroongsarb1,
Maskiet Boonyareth1, Poom Preedakoon1,Watcharee Saisongkorh1 and Pathom Sawanpanyalert1
1
National Institute of Health, Department of Medical Sciences, Thailand,
Nongkwang Livestock Breeding and Research Center, Ratchaburi Province ,Thailand,
3
Laboratory of Veterinary Public Health, Nihon University, Japan,
4
International Emerging Infections Program, Thailand MOPH-U.S. CDC Collaboration.
2
Genus Bartonella is known to be a variety of gram-negative, fastidious, hemotrophic bacteria in humans,
domestic and wild animals. Several Bartonella species are recognized zoonotic and transmitted by blood-sucking
arthropod vectors. The aim of the present study was to investigate the presence of Bartonella infections in deer in
Thailand. A total of 247 blood samples of deer (Rusa timorensis) were collected from Nongkwang Livestock
Breeding and Research Center, Ratchaburi Province, Thailand during February to March 2011. We investigated the
prevalence of Bartonella spp. by culture with blood agar. In total, 9 of the samples (3.6%) were positive for
Bartonella. Among 110 males and 137 females were 3 (2.7%) and 6 (4.5%) positive findings, respectively. Age of R.
timorensis was high prevalence in 2-4 year. Phylogenetic analysis of the citrate synthase (gltA), β-subunit of the
RNA polymerase (rpoB), cell division-associated protein (ftsZ) and 16-23S rRNA intergenic spacer (ITS) genes
indicated that all the isolates were classified into new genotype of Bartonella isolates from deer blood in Thailand.
Keywords : Bartonella,new genotype ,deer
149
THE EVALUATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
FOR Ehrlichia canis DETECTION
N. Tipmongkolsilp1 , N. Viseshakul2*
1
Clinical for Aquatic Animals, Faculty of Veterinary Medicine, Mahanakorn University, THAILAND,.
2
The Unit of Parasitology, Department of Pathology, Faculty of Veterinary Science,
Ehrlichia canis is a parasitic bacteria responsible for canine ehrlichiosis, an important tropical disease
transmitted by brown tick, Rhipicephalus sanguineus. For the evaluation of a new method of E. canis detection,
Loop-mediated isothermal amplification (LAMP), has been applied to the plasmids containing small ribosomal RNA
gene specific for E. canis, Anaplasma platys and Babesia spp. The comparison between PCR and LAMP using rRNA
specific primers showed no cross-reaction among each test. The LAMP results showed the same efficiency as
nested PCR technique. However, some advantages were verified for LAMP amplification to be more sensitive,
rapid, requires a simple equipment. This is a preliminary study which has a promising result in order to develop
an efficiently disease detection with the specificity and simplicity method.
Keywords: Ehrlichia canis, PCR, Loop-Mediated Isothermal Amplification (LAMP), Diagnostic method
150 
VILLI AND CRYPT RATIO OF PORCINE EPIDEMIC
DIARRHEA VIRUS (PEDV) INFECTED PIGLETS
Ruangurai Kitchodok 1 Natthanan Prasert 1 Somdech Tunboonjit 1 Sittikorn Traiyarach2
Paisan Tienthai 3 Komkrich Teankum2 Roongroje Thanawongnuwech2*
1
Sixth year students, Academic year 2011. 2Department of Veterinary Pathology
3
Department of Veterinary Anatomy, Chulalongkorn University, Bangkok 10330.
Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is probably one of
the most contagious disease that trigger serious economic loss in Asian swine industry. The objective is to investigate the virulence of Thai PEDV (0 8 NP02) isolated during 2007-2009 epidemical outbreaks compare with the
attenuated-type (53rd passages in Vero cells) and the wild type (1st passage) based on villi height and crypt depth
ratios (VH/CD). Twenty-eight one-day-old colostum – deprived piglets from the PEDV free farm were given 100
mg/kg body weight for Fe solution, 2mg/kg intramuscularly antibiotic injection and orally anticoccidial drug on the
first day of arrival before divided into 3 groups. Four piglets were mocked orally inoculated with 5ml mocked media
for the negative control group, twelve piglets were orally inoculated with 5ml of 10 5 TCID5 0 /ml of 53rd passages
PEDV for attenuated-type group and the remaining piglets were orally inoculated with 5ml of 10 5 TCID50 /ml of 1st
passages PEDV for wild type group. Clinical signs were observed and the whole piglets were euthanized at 12, 24,
36 and 72 hours after post inoculation using Pentobarbital Sodium (Nembutal ®, Ceva Sante Animate). The Lesions
of the small intestines particularly duodenum, jejunum and ileum were recorded and collected for histological examination and VH/CD ratios measurement. The VH/CD ratios were measured using by Micropublisher 5.0 connected with the light microscope. The results of swine showed that the wild type PEDV showed severe lesions not only
histopathologically lesions but also the lower VH/CD ratios in jejunum and ileum regions than those of the attenuated PEDV infected piglets significantly (p<0.05) compared with the negative control group. On the other hand,
both PEDV- inoculated groups were able to induce enteric lesions depending on the severity compatible with the
shortening of the villi. Moreover, 53rd passages of PEDV might help attenuating the pathogenicity in pigs with can
be applied for sow vaccination purpose.
Keywords : Porcine epidemic diarrhea(PED),villi height and crypt depth ratios(VH/CD)
151
EPIDEMIOLOGY OF BOVINE VIRAL DIARRHEA VIRUS INFECTION
IN DAIRY FARMS IN NORTHEASTERN THAILAND
T. Nilnont1,2*, A. Poopuak1, C. Piamsakun1, S. Aiumlamai3, K. Kanistanon4, J. Kampa5
1
Graduated student, Faculty of Veterinary Medicine, Khon Kaen University, Thailand,
Programed in Veterinary Technology, Faculty of Technology, Udon Thani Rajabhat University, Thailand,
3
Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, Khon Kaen University, Thailand,
4
Department of Physiology, Faculty of Veterinary Medicine, Khon Kaen University, Thailand,
5
Department of Pathobiology, Faculty of Veterinary Medicine, Khon Kaen University, Thailand
2
Bovine viral diarrhea virus (BVDV) causes losses in reproduction in the infected herds, which leading to substantial economic loss in dairy production. The objective of this study was to investigate present prevalence of the
infection in dairy populations of four provinces of the northeastern Thailand; Nakhonratchasima, Udon Thani,
Sakonnakhon and Khon Kaen, where the last investigation was done in 2008. Bulk tank milk samples (BTM) were
collected from dairy herds of 9 milk centers, total of 933 herds. BTMs were tested for specific antibodies to BVDV
by a commercial indirect ELISA. BTM results indicate a moderate to high herd prevalence, 50-80%, of the infection
in studied area. At present, BVDV prevalence (58.73%) Khon Kaen province was greater than the study in 2008,
when seroprevalence at the same cattle population was only at 35.23 %. This result indicated an active spreading of
BVDV in the area. Therefore, a practical BVDV control programme as a strict regulation on disease control and free
-BVDV in breeding practice has to be emphasized.
Keywords : BVDV, dairy cattle, bulk tank milk, prevalence
152 
RELEVANT EVIDENCES FOR THE DIAGNOSIS OF
ATYPICAL PESTIVIRUS INFECTION IN A CALF
Chinwiwat Piamsakun1, Suttisak Nopwinyoowong1,
Sirikachorn Tangkawattana1, Jaruwan Kampa1*
1
Department of Veterinary Pathobiology, Faculty of Veterinary Medicine,
Khon Kaen University, Khon Kaen 40002, Thailand
An 8-year-old calf, being suspicious of natural infection by atypical pestivirus as a cause of death, was
presented for necropsy at the Department of Veterinary Pathobiology, Faculty of Veterinary Medicine, Khon Kaen
University, Thailand. The apparent necropsy findings were severe mucosal erosion of the oral cavity through the
whole GI tract. The reduction in size of cerebellum was obvious and quite similar to the other reports concerning
the persistent BVDV-1 and BVDV-2 infections. Histopathology either with either routine or special stainings and
immunohistochemistry with uses of a specific antibody to bovine pestivirus could reveal general lymphocytic
infiltration and viral distribution in various tissues and organs, e.g. alimentary tract, skin and brain. Moreover,
RT-PCR assay with specific primers to the bovine pestiviruses could provide a molecular evidence to confirm the
etiologic virus. The above evidences were relevantly for supporting the diagnosis of atypical pestivirus as the mortal cause.
Keywords : atypical pestivirus, natural infection
153
IDENTIFICATION OF A PUTATIVE LRR CODING GENE IN L. INTERROGANS
Anthicha Kunjantarachot1, 2, Supachai Nitipan1, 2, Tepyuda Sritrakul1, 2,
Chattip Suphatpahirapol1, 2, Nuch Chotechuang2, Siriwan Prapong1, 2*
1
Interdisciplinary Graduate Program in Genetic Engineering, The Graduate School, Kasetsart University,
2
Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
Leptospirosis, caused by infection of Gram negative spirochetes of genus Leptospira, is an important reemerging zoonotic disease that has significant impacts on human and animal health worldwide including in Thailand. Now over than 200 serovars of pathogenic leptospires have been identified in which immunity mediated by a
particular serovar does not completely protect against heterologous serovars. Thus vaccine candidates that could
provide protective immunity against multiple serovar infection are needed. One of the potential subunit vaccine
candidates is leucine-rich repeat or LRR containing protein. LRR proteins identified in pathogenic bacteria are
shown to have various functions which usually involve in invasion mechanism, virulence and/or pathogenesis of the
bacteria in host cells. In this study, putative LRR coding gene was identified in L. interrogans serovar Autumnalis,
Canicola, Hebdomadis, and Pyrogenase by PCR amplification using primers designed from LA_1324. LA_1324 is annotated as gene coding for LRR containing protein. DNA sequence analysis revealed that putative LRR coding gene
from all serovars matched LRR coding genes, LA_1324 and LIC_12401 of L.interrogans, with over than 90% identity
and matched to LBJ_2271 gene located in chromosome I of L. borgpetersenii with 80% identity, suggested the conservation of this gene among pathogenic Leptospira serovars. Phylogenetic analysis also indicated that there is
evolutionary relatedness among this LRR coding gene in L. interrogans serovars. All results implied the possible significance of this putative LRR coding gene in virulence and pathogenesis of pathogenic leptospires.
Keywords : Leptospira interrogans, LRR proteins, Vaccine candidate,
Oral and Poster Presentation
Full Paper
Oral Presentation Full Paper
156 
CLONING AND CHARACTERIZATION OF SWAMP BUFFALO INTERFERON-g
U. Boonyaprakob*, S. Homsavart
Department of Physilogy, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
* Corresponding author
Introduction
Interferon-gamma (IFN-γ) is a cytokine that plays a central role in directing host immune responses to viral
and bacterial infection. After exposure to a particular antigen, IFN-γ is secreted predominantly from Th1 lymphocytes. It is also secreted by CD8 T cells, NK cells and other cell types (Schroder et al., 2004). Since a pronounced
IFN-γ secretion from peripheral blood mononuclear cells was observed in individuals infected with M. tuberculosis,
the IFN-γ release assays (a measurement of IFN-γ release by sensitized lymphocytes) was developed for the detection of M. tuberculosis infection (Rothel et al., 1990). A commercial IFN-γ release assay (BovigamÒ) was developed
for diagnosis of tuberculosis in cattle (Wood and Jones, 2001), and it was used for detection of TB in several bovine
species. However, Chuachan and others (2010) showed that the assay had a relative low sensitivity (72.6 %) and
specificity (66.7 %) in diagnosis of TB in Thai swamp buffalo, comparing to cattle (81.8% - 100% and 94-100%,
respectively). Although, reasons for the low sensitivity and specificity in the previous study were not investigated, it
may be due to the variations in sequences among different species. Thus, the purpose of this study was to clone and
sequence analyses of Thai swamp buffalo IFN-γ cDNA, and investigate the promoter region of the IFN-γ gene. The
molecular understanding of the IFNγ in the present study will support the development of the improved IFN-γ assay
in the swamp buffalo.
Materials and Methods
Blood was collected from 5 healthy Thai swamp buffalo (Bubalus bubalis carabensis). Total RNA was extracted from the buffy coat using TRIzol reagent (Invitrogen), and used for RT-PCR with primers B-IFNF/ B-IFNR
and conditions as described previously (Premraj et al., 2006). A fragment of 3' end cDNA of the gene was obtained
by 3'-RACE using the First Choice RLM-RACE kit (Ambion). DNA was extracted from the remaining Trizol fraction after isolation of RNA, and used for PCR amplification of the promoter region of IFN-γ gene. All primer sequences were provided in Table 1. The amplified fragments were separated on 1% agarose gel electrophoresis, purified by gel extraction kit (Qiagen), and cloned into the TOPO cloning vector (Invitrogen). The selected clones were
purified from transformed Mach1 competent cells (Invitrogen), and submitted to 1st BASE (Malaysia) for sequencing in both directions on an ABI 3730 genetic analyzer (Applied Biosystems).
Table 1. All primers used in the amplification
B-IFNF
B-IFNR
3' Gene Specific Primer 1
3' RACE Outer Primer
3' Gene Specific Primer 2
3' RACE Inner Primer
Forward CAACTACTCCGGCCTAACTCTCTC
Reverse AGGACCATTACATTGATGCTCTCCG
Forward ACCCAGCGCAAAGCCATAAA
Reverse GCGAGCACAGAATTAATACGACT
Forward AGGCCGGAGAGCATCAATGT
Reverse CGCGGATCCGAATTAATACGACTCACTATAGG
157
The sequences from Thai swamp buffalo, and the previously published IFN-γ sequences from other buffaloes, Indian and European cattle, goat, sheep, and human were aligned with the program ClustalW (Thompson et al.
1994). Phylogenetic trees were constructed using neighbor-joining by MEGA-4 software with bootstrapping of 1000
replicates.
Results and Conclusion
The full-length cDNAs was 1,141 bp in length, with a 31 bp 5′UTR and a 609 bp 3′UTR. The open reading
frame was 501 bp that translates into a 166 residues putative protein with a 23 amino acid signal peptide and a mature protein with 143 amino acids. Multiple sequence comparison showed a close amino acid homology (99.4–
100.0% identity) between the buffalo breed, while the homology between the Thai swamp buffalo and cattle was
lower (97.0-97.6%). Four amino acid substitutions between Thai swamp buffalo and European cattle were observed
at positions 19, 65, 105 and 143 of the mature protein. The phylogenetic tree based on the predicted IFN-g peptide
sequences showed the close relationship between Thai swamp buffalo and other bubaline breeds. The clustering of
sequences from buffalo and other large ruminants within the family Bovidae has bootstrap values less than 80%, indicating that buffalo IFN-g grouping with them, while sheep and goat forming a separate cluster with a bootstrap
value of 100%. Characterization of the IFN-g promoter showed that the major transcription factor binding sites were
highly conserved among the promoter sequences of Thai Swamp buffalo, Indian River buffalo, cattle, sheep, and
human, indicating that regulation of IFN-g expression across species may be similar.
In conclusion, a small difference in the amino sequence for IFN-g between Thai Swamp buffalo and cattle was reported. Effect of amino acid substitutions on the cytokine structures might be the reason behind the previous reports
on the lower success of the BovigamÒ IFN-γ test for Thai swamp buffalo. However, further investigation is needed
to qualify this conclusion.
Acknowledgements
This study was supported by the Kasetsart University Research and Development Institute. We are grateful to
the faculty of Veterinary Medicine, Kasetsart University for all facilities support.
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Schroder K, Hertzog PJ, Ravasi T, Hume DA. 2004. Interferon-gamma: an overview of signals, mechanisms and
functions. J Leukoc Biol 75:163-189.
Thompson, J., Higgins, D. and Gibson, T., 1994. CLUSTAL W: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22: 4673-4690.
Wood PR, Jones SL. 2001. BOVIGAM: an in vitro cellular diagnostic test for bovine tuberculosis. Tuberculosis
(Edinb). 81: 147-155.
158 
INVESTIGATION OF BOVINE LEUKOCYTE
ADHESION DEFICIENCY (BLAD) IN THAI HOLSTEIN CATTLE
Kalaya Kengvikkum1*, Nattanant Sirivattanatanyakul1, Buh-nga Chindawanichsakul1
1
Bureau of Biotechnology for Animal production, Department of Livestock Development
Introduction
BLAD is a specific Holstein autosomal recessive genetic disorder and results in death of homozygous animals.
It was firstly identified in North American Holstein (Shuster et al 1992). BLAD carriers were among the most popular top bulls of the Holstein breed such as Osborrndale Ivanhoe, Penstate Ivanhoe Star, and Carlin-M Ivanhoe Bell
(Shuster et al 1992, Powell et al 1996). Widespread use of Artificial insemination (AI) and multiple ovulation embryo transfer (MOET) techniques caused demanding semen from those elite Holstein bulls. Affected cattle with
BLAD were linked to common the ancestor sires that were proved to be carriers (Shuster et al 1992, Jørgensen et al
1993). Estimation of the BLAD allele frequency upon the PCR-RFLP test results showed that around 220 Thai Holstein cattle might have been affected with the recessive disorder.
Material and Methods
Blood samples were collected from 220 cattle of Thai Holstein cattle’s jugular vein into EDTA containing
tubes. DNA was isolated using Axyprep Blood Genomic DNA Miniprep Kit and stored at 4°C until analysis. Genotyping for BLAD was carried out using PCR-RFLP methods. PCR was made in 20µl reaction mixture containing
Genomic DNA, 1U Taq polymerase, 0.2mM dNTP, 5 µM of each primer (F: 5’ GAA TAG GCA TCC TGC ATC
ATA TCC ACC A 3’ R: 5’ CTT GGG GTT TCA GGG GAA GAT GGA GTAG 3’) The primer were designed to
amplify a DNA fragment of 357 bp. (Meydan, et al. 2010) DNA was amplified after initial Denaturation at 94oC for
3 min, 33 cycles (94oC 30 sec, 66oC 30 sec, 72oC 30 sec) after the last cycle the samples were kept at 72oC for 5 min
and the PCR procedure was finished. The PCR products were analyzed by 2% agarose gel electrophoresis. The 357
bp DNA fragment was amplified by PCR. Aliquots of 2 µl of PCR product were subjected to restriction digestion
with TaqI in a 10 µl reaction volume. Using 0.2 µl TaqI restriction endonuclease without BSA per one sample, the
mixture was incubated at 65oC for 30 min. The digested amplified PCR product on 2% agarose gel yielded two
bands of 201 and 156 bp for normal animals and three bands of 201, 156 and 357 bp for carriers. The amplified
PCR product of 357 bp was for the BLAD locus. 2DNA of known carries of BLAD as a control was obtained from
Dr. Istvan Anton, Research Institute for Animal Breeding and Nutrition (ATK) Herceghalom, Hungary. The all genotypes were confirmed by DNA sequencing. After gel electrophoresis, the amplicons were purified using NucleoSpin® Extract II and sequenced by Big Dye Terminator chemistry on an ABI 3130. Automated DAN Sequencer.
159
The primer mentioned above successfully amplified the DNA fragments of 357 bp for BLAD. In figure 1,
there were five BLAD carriers: an example from Hungary as a control and four samples in Thai Holstein have three
fragments of 156 bp, 201 bp and 357 bp whereas unaffected cattle have only two fragments of 156 bp and 201 bp.
The BLAD positive and negative samples were sequenced by Big Dye Terminator chemistry on an ABI 3130 Automated DNA sequencer (Applied Biosystems, USA) The DNA sequences were analyzed using the Sequencing Analysis Software Version 3.3 (Applied Biosystems). Among five carriers’ allele, there were only two mutant alleles: a
control sample DNA from Hungary and one sample from Thai Holstein bull. The other three alleles (Figure 1: in the
column 5, 6, 7) were found to be false positive when confirmed by DNA sequencing method. To improve that PCRRFLP analysis should be a strong and reliable method for identification of BLAD. Many trials have been done in
both PCR and of TaqI enzyme conditions in order to find the optimum procedure. Finally, a proper condition of
TaqI enzyme was found by adding BSA 1 µl (0.1%) and reducing DDW (deionized distil water) from 6.8 µl to 5.8 µl
in total solution 10 µl. DNA samples from Thai Holstein cattle in column 5, 6, and 7 in Figure 1 were truly false positive when checking sequences. (Figure 2)
Figure1: Visualization of restriction
fragments produced by TaqI enzyme on
the PCR products.
Column M: 100 bp ladder.
Column 1-2: Positive control DNA from
Hungary cow and positive sample of
Thai Holstein cattle respectively.
Column 3-4: Negative DNA samples of
Thai Holstein cattle
Column 5, 6, 7: (false) positive DNA
samples of Thai Holstein cattle
Figure 2: Visualization of restriction
fragments produced by TaqI enzyme on
the PCR products.
Column M: 100 bp ladder.
Column 1-2: Positive control DNA
from Hungary cow and positive sample
of Thai Holstein cattle
Column 3, 4, 5, 6, 7: Negative DNA
samples of Thai Holstein cattle
160 
All genotypes were confirmed by sequencing method. The nucleotide sequence was in the GenBank with accession
number FJ853493 for BLAD. The result of sequencing for the mutant BLAD allele was confirmed a single point
mutation at the nucleotide 383 in the CD18 gene as the other reports (Figure 3) (Kriegesmann, et al 1997, Meydan,
et al. 2010). In this paper: the prevalence of BLAD carriers in Thai Holstein cattle was found 0.45% and the gene
frequency of CD 18, calculated according to the Hardy-Weinberg Law was only 0.01in the 220 samples. The other
incidence of BLAD carriers among Top sires was also found to be 23% in U.S.A. (Shuster et al 1992), 10% in
France (Tainturier et al 1995), 16% in Japan (Nagahara et al 1995), 3.33% in Iran (Norouzy et al 2005) and 4.76%
(Patel et al 2011)
Figure 3 Alignment of BLAD sequences from normal Thai Holstein cow on the first row,
mutant1 (positive control DNA from Hungary) and mutant 2 from normal Thai Holstein on
the second and third rows respectively. The mutation consists of a single point mutation of
nucleotide 383 in the CD18 gene. The box indicates the single point mutation site (A to G)
The incidence of BLAD in this paper is still low but the number of carriers could be higher in coming days. As the
selection within Holstein breed, its’ crosses and AI programs are major factors to spread of undesirable genetic
disorder, a routine screening of bulls and dams is required to reduce the recessive lethal gene in cattle population.
Acknowledgements
The author wish to thank Dr. Istvan Anton, Research Institute for Animal Breeding and Nutrition (ATK)
Herceghalom, Hungary for giving DNA of known carries of BLAD.
161
References
Jørgensen CB, Agerholm JS, Pedersen J, Thomsen PD. 1993. Bovine leukocyte adhesion deficiency in Danish
Holstein-Friesian cattle. I. PCR screening and allele frequency estimation. Acta Vet Scand. 1993; 34(3):2316.
Meydan H, Yildiz AM, Agerholm J. 2010. Screening for bovine leukocyte adhesion deficiency, deficiency of
uridine monophos synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency
in Holstein cows reared in Turkey. Acta Veterinaria Scandinavica. 52:56
Kriegesmann B, Jansen s, Baumgartner BG, Brening B. 1997. Partial genomic structure of the bovine CD18 gene
and the refinement of test for bovine leukocyte adhesion deficiency. J Dairy Sci 80: 2547-2549
Nagahara H, Tamoto K, Noda H, Kociba GJ. 1995. Expression and role of adhesion molecule CD18 on bovine
neutrophils. Can.J.Vet. Res. 59:1-7
Norouzy A, Nassiry MR, Eftekharishahrody F, Javadmanesh A, Mohammad Abadi MR, Sulimova GE. 2005.
Identification of bovine leukocyte adhesion deficiency (BLAD) carriers in Holstein Brown Swiss AI bulls in
Iran. Russ.J.Gen. 41(12):1679-1701
Patel M, Patel RK, Sing KM, Rank DN, Thakur MC, Khan A. 2011. Detection of genetic polymorphism in CD18
gene in cattle by PCR-RFLP. Wayamba Journal of Animal Science. http://www. Wayambajournal.com
Powell RL, Norman HD, Cowan CM. 1996. Relationship of bovine leukocyte adhesion deficiency with genetic
merit for performance traits. J. Dairy Sci. 79:895-899.
Shuster DE, Kehrli ME JR, Ackermann MR, Gilbert RO. 1992. Identification and prevalence of a genetic defect that
causes leukocyte adhesion deficiency in Holstein cattle. Proc.Natl. Acad.Sci. USA 89:9225-9229
Tainturier D, Grobet L, Brouwers, B, Bruyas JF, Fieni F, Battut I, Lecoanet J, Douart A, Breton I, Duclos P. 1995.
Observations on three cases of bovine leukocyte adhesion deficiency (BLAD) Revue-de Medecine Veterinaire 146(3): 189-195
162 
KAENG KA JAN DISTRICT, PETCHABURI PROVINCE, THAILAND
A. Sommanustaweechai1, B. Sangkarak1, D. K. Nugroho1, F. Wu1, H. Sinel1, L. L. Bo1, N. Kiry1, P. Boosom1, P.
Wongnark1, S. I. Jayme1, S. Sinthasak1, S. Urbenjapol1, S. Khuhapan1, T. Lamaisri1, T. Kedkhuntod1, T. Srisuvan1,
V. T. Le1, W. Posuya1, S. Theraverapanya2, K. Unjit3, M. Pattiyapong3, N. Ketusing3, K. Taweeseneepitch4, K.
Wongsathapornchai4, K. Chanachai4*
1
Participants of Veterinary Field Epidemiology in Action, Field Epidemiology Training Program for Veterinarian.
2
Petchaburi Provincial Livestock Office. 3 National Institute of Animal Health. 4 Field Epidemiology Training
Program for Veterinarian, Bureau of Animal Disease Control and Veterinary Services,
DEPARTMENT OF LIVESTOCK DEVELOPMENT, THAILAND. * Corresponding author
Introduction
Intestinal and blood parasites cause a lot of losses to dairy cattle due to parasitic
effect and carrier status of diseases especially in tropical countries. In Thailand, the
majority of dairy cattle are in small scale farming that may practice improper
parasitic control. High prevalence (54%) of intestinal parasite in dairy cattle in
Northern Thailand was reported. Trematodes was found to be the highest prevalence
(41%) following by nematodes (26%) and cestodes (4%) (Padungtod et al., 2001).
Blood parasites that were reported in a blood parasite survey of beef cattle are Theileria spp.(50%), trypanosome (2%), and microfilariae (1%) (Kaewthamasorn and
Wongsamee, 2006). In Petchaburi Province, middle part of Thailand, there are
approximately ten thousand dairy cattle in 350 small-scale dairy farms and half of
them are in Pa Deng Sub-district, Kaeng Ka Jan District (picture 1). Situation of
intestinal and blood parasites in this area have never been investigated. This study aimed to determine prevalence of
intestinal, blood parasites and their risk factors in dairy cattle farm in Pa Deng Sub-district, Kaeng Ka Jan District.
In February 2011, 68 out of 154 dairy cattle farmers willing to participate in this survey were selected for sample
collection. Five to six cattle were randomly selected from each farm for blood and feces collection. Intestinal parasites were determined by floatation and sedimentation techniques within 48 hours after sample collection, while
blood parasites were identified by thin blood smear and haematocrit centrifuge techniques. Microscopic examination
of the blood smears were done at National Institute of Animal Health, Department of Livestock Development,
Thailand. Data from farms including number of dairy cattle, farm management, and cattle health condition were observed and collected by using questionnaires.
Results and Conclusions
In total, 358 fecal and 382 blood samples were collected. Average age of sampled cattle was 65 months (SD=40.36).
The prevalence of overall intestinal parasite was 39 % (139/358, 95% CI 36-42) in animal level and 72% (46/64,
163
95% CI 67-78) in herd level. Species of parasiteinfestation were showed in table 1. Average packed cell volume
(PCV) was 27.20 % (SD=4.30). Theileria was the only blood parasite that was identified in 19% of blood sample
(71/382). Herd size (>20 cattle) significantly associated with intestinal parasite infestation (1.36, 95% CI 1.01-1.83),
while the presence of Theileria significantly associated with body condition score£2.5 (1.61, 95% CI 1.01-2.56)
(table 2).
Although most of farmers (96%) routinely deworm their cattle, the prevalence of intestinal parasite was still high.
High number of cattle in the farm may lead to higher chance of parasitic transmission and inappropriate parasitic
control. Association between Theileria infestation and body condition score suggested the possible impact to animal
health. The actual impact of intestinal and blood parasite infestation to animal health and production performance as
well as more effective control measures should be further investigated.
Acknowledgement
The study team would like to express sincere gratitude to Bureau of Animal Disease Control and Veterinary Services
and Petchaburi Provincial Livestock Officer who facilitated the field activities, Veterinary Faculty of Kasetsart University who gave technical input, National Institute of Animal Health who was responsible for sample testing and
Food and Agriculture Organization of the United Nations who provided budget support of this survey.
References
KAEWTHAMASORN, M. & WONGSAMEE, S. (2006) A preliminary survey of gastrointestinal and haemoparasites of beef cattle in the tropical livestock farming system in Nan Province, northern Thailand. Parasitol
Res, 99, 306-8.
PADUNGTOD, P., KANEENE, J. B., JARMAN, D., JONES, K., JOHNSON, R., DRUMMOND, A., DUPREY, Z.
& CHAICHANAPUNPOL, I. (2001) Enteric parasitosis in northern Thailand dairy heifers and heifer calves.
Prev Vet Med, 48, 25-33.
164 
MT. LOUKCHANG, PETCHABURI, THAILAND
F. Suksawat1*, S. Chantarachart1, P. Thonglon1, P. Papirom2, S. Sripakdee3, A. Kerdsin3, S. Dejsirilerk3
1
Department of Veterinary Medicine, 2Department of Pathobiology, Faculty of Veterinary Medicine, Khon Kaen
University, Khon Kaen, Thailand, and 3Department of Medical Sciences, Ministry of Public Health, Nontaburi,
THAILAND.* Corresponding author
Introduction
Wildlife poached from the forests is commonly found in Thailand (Wiek, 2008). They were previously exploited as pets or used for profit within the tourist industry. In many tourist destinations, one of unfortunate primates
that have been seen in big numbers, are monkeys. Old World monkeys of the Genus Macaca rescued at Wildlife
Friends Foundation Thailand (WFFT), Mt. Loukchang, Petchaburi Province, were the targets of this study. The objective of the current study was to microscopically identify intestinal parasites and blood parasites in monkeys for
medical care purpose during rehabitation prior to release them back to the wild.
Fecal and blood samples were randomly collected from 30 macaques at the average age of 10 years. There
were 7 pig-tailed macaques (Macaca nemestrina), 15 stump-tailed macaques (Macaca arctoides), and 8 long-tailed
macaques (Macaca fascularis). Simple direct smear and simple floatation methods were employed for intestinal parasitic detection, and thin blood smear was for blood parasitic infection (Wongsawad, 2009). Complete blood count
was done to support if any obvious clinical signs.
Twenty of 30 macaques (66.66%) were parasitized with Strongyle type (0, 14, 6 from pig-tailed macaques,
stump-tailed macaques, and long-tailed macaques, respectively), and one stump-tailed macaque was identified with
Trichuris spp (3.33%). High percentage of helminth infectivity corresponds to the studies in the New World monkeys (marmocets) (44.3%) (Diniz and da-Costa, 1995) and in the Old World monkey (green monkeys) (88.7%)
(Mutani et al., 2003). No evidence of blood parasites was seen. The result suggests that Strongyle type represents
major intestinal parasite among the studied macaques. In the near future, identification of round worm species and
the risk of parasitic infection should be performed for possible zoonotic disease indication. Pet and captive primates
can be carriers of many zoonotic diseases such as viral disease (e.g.the potentially fatal herpes B virus; measles, rabies), bacterial diseases (e.g. salmonellosis, tuberculosis); fungal diseases ( e.g. candidiasis, ringworm, cryptococcosis), and parasitic diseases (e.g. giardiosis, nematode and cestode infestation (Renquist and Whitney, 1987). Since
these could potentially cause severe consequences for the contact person, it is imperative that medical and veterinary
professionals collaborate to educate the public on the danger of the primate as a pet.
165
Acknowledgements
We thank the KKU Veterinary Medicine for research funding. Special thank goes to Mr. Edwin J. Wiek and
Dr. Prachya Engkhaninun for giving us a great opportunity to work with those affectionate macaques at the rescue
center in WFFT.
References
Diniz LS. and da-Costa EO. 1995. Health problems of Callithrix jacchus in captivity. Braz. J Med Biol Res. 28(1):61
-64.
Mutani A, Rhynd.K, Brown G. 2003. A preliminary investigation on the gastrointestinal helminths of the Barbodos
green .monkeys Cercopithecus aethiops sabaeus.Reu.Inst Med Tropic S Paulo. 45(4):193-195.
Renquist DM and Whitney RA Jr.1987.. Zoonoses acquired from pet primates Vet Clin North Am Small Anim
Pract. 17 (1):219-240.
Wiek. EJ. 2008.Wildlife Friends Foundation Thailand 2008 Annual Review.
Wongsawad C. 2009. Coprodiagnosis using smear and sedimentation techniques to detect intestinal parasites of Assameses, Macaca assamensis from Watt ham-pla, Chiang Rai Province.Trends Res Sci Tec.1(1):65-70.
166 
A COMPARISON OF FOOD-POISONING BACTERIAL CONTAMINATION
ON FOOD BETWEEN EUROPEAN COUNTRIES AND JAPAN
H. Suzuki1*, S. Yamamoto1
1
Divison of Biomedical Food Research, National Institute of Health Sciences, JAPAN
Introduction
“Shokuhin no shokuchudokukin osenjittai chosa (The national survey of food-poisoning bacterial contamination on food)” has been performed annually by the Ministry of Health, Labour and Welfare in Japan since 1998.
This surveillance is thought to be the useful baseline study of bacterial contamination on food in Japan. On the other
hand, “The community summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union” published by the European Food Safety Authority (EFSA) is the annual surveillance
report about zoonoses, zoonotic agents and food-borne outbreaks in the EU based on the Directive 2003/99/EC since
2005. The results of these annual surveillances were summarized for comparing the baseline of bacterial contamination on food between European countries and Japan.
Only Salmonella, Campylobacer and E. coli O157 contamination on food, which were examined in the both
“Shokuhin no shokuchudokukin osenjittai chosa” (MHLW 2009) and “The community summary report on trends
and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union” (EFSA 2006, 2007,
2009, 2010), were compared for 4 years (since 2005 to 2008).
The contamination levels of these food-poisoning bacteria on food were almost comparable between European
countries and Japan, but some exceptions. For example, in chicken meat (Table 1), the percentage of Salmonella
contamination was 6.4% in average in Europe but 46.7% in Japan and, in minced chicken meat (Table 2), the percentage of contamination was 6.6% in average in Europe but 36.6% in Japan. In contrast, the percentage of Salmonella contamination in sprouts (Table 3) was higher in Europe (2.1% in average) compared to Japan (0.1%).
Acknowledgements
This study was supported by Health and Labour Sciences Research Grants, Research on Food Safety from the
Ministry of Health, Labour and Welfare of Japan.
References
EFSA. 2009, 2010. The Community Summary Report on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks in the European Union in 2007, 2008. http://www.efsa.europa.eu/en/efsajournal/
pub/223r.htm, http://www.efsa.europa.eu/en/efsajournal/pub/1496.htm (accessed in March, 2011)
EFSA. 2006, 2007. The Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents,
Antimicrobial resistance and Foodborne outbreaks in the European Union in 2005, 2006.
http://www.efsa.europa.eu/en/efsajournal/pub/94r.htm, http://www.efsa.europa.eu/en/efsajournal/
pub/130r.htm (accessed in March, 2011)
MHLW. 2009. Heisei 20 nendo shokuhin no shokuchudokukin osenjittai chosa ni tsuite. http://www.mhlw.go.jp/
topics/syokuchu/kanren/yobou/060317-1.html (accessed in March, 2011)
167
Table 1 Salmonella in chicken meat
2008
Country
N
% pos
Austria
295
7.8
Belgium
88
11.4
Bulgaria
4,046
0.3
Estonia
Germany
993
10.8
Greece
64
15.6
Latvia
85
8.2
Lithuania
136
16.2
Luxembourg
101
5.9
Netherlands
1,408
7.7
Romania
295
2.4
Slovenia
315
0.6
Spain
195
3.6
Sweden
Switzerland
UK
Europe
8,021
4.0
Japan
30
46.7
2007
% pos
86
5.8
276
8.7
N
2006
% pos
N
2005
% pos
80
5.0
90
2.2
68
10.3
51
11.8
33
96
18.2
11.5
714
69
200
8.5
11.6
3.0
254
1,418
6.7
8.1
91
1,365
6.6
8.4
47
1,506
0.0
9.4
343
206
2.3
10.2
294
3.4
400
117
3.8
6.8
415
6.5
3,981
0
7.3
0.0
1,714
3,612
0
5.4
9.0
0.0
877
3,217
0
4.0
8.1
0.0
Table 2 Salmonella in minced chicken meat
2008
2007
Country
N
% pos
N
% pos
Belgium
70
12.9
Bulgaria
725
0.8
Hungary
258
13.6
Latvia
50
0.0
Poland
241
10.8
Romania
44
0.0
275
0.0
Europe
819
0.7
844
8.3
Japan
196
42.9
129
29.5
Table 3 Salmonella in sprouts
2008
Country
N
% pos
Austria
Czech
Estonia
7
0.0
Germany
229
5.2
Hungary
44
0.0
Ireland
Italy
Netherlands
Poland
Portugal
25
0.0
Romania
8
0.0
Slovenia
Europe
313
3.8
Japan
263
0.4
N
N
2007
% pos
17
2
135
101
4
0.0
0.0
2.2
0.0
0.0
581
149
26
4
1.5
0.0
0.0
0.0
1,019
251
1.1
0.0
N
2006
% pos
90
40.0
2,121
181
2,392
96
7.3
0.0
8.0
36.5
N
2006
% pos
20
60.0
2
0.0
5
0.0
114
3
4
52
66
0.0
0.0
0.0
0.0
0.0
30
296
251
0.0
4.3
0.0
2005
% pos
N
0
110
N
0.0
33.6
2005
% pos
56
0.0
22
4.5
45
123
310
0.0
0.8
0.0
Total
N
% pos
381
7.3
534
7.5
4,046
0.3
119
10.9
1,707
9.8
166
14.5
381
6.3
136
16.2
493
5.9
5,697
8.4
295
2.4
658
1.5
1,095
4.9
117
6.8
415
6.5
2,591
4.9
18,831
6.4
30
46.7
Total
% pos
70
12.9
725
0.8
348
20.4
50
0.0
2,362
7.7
500
0.0
4,055
6.6
531
36.6
N
Total
% pos
20
60.0
19
0.0
14
0.0
420
3.5
259
0.0
29
3.4
4
0.0
633
1.4
215
0.0
51
0.0
12
0.0
75
0.0
1,751
2.1
1,075
0.1
N
168 
CAMPYLOBACTER JEJUNI ISOLATED FROM BROILER IN CHIANG MAI, THAILAND
Nipa Chokesajjawatee1 Sarinya Pornaem1 Chomporn Chokboonmongkol2 Karl-Hans Zessin 3
Thomas Alter4 Prapas Patchanee5*
1
National Center for Genetic Engineering & Biotechnology, National Science & Technology Development Agency,
Pathumthani, THAILAND, 2 Animal Health and Technical Service Office, Bangkok Agro-Industrial Products Public
CO., LTD, Bangkok, THAILAND, 3Department Panel, Veterinary Public Health,4Institute of Food Hygiene, Free
University Berlin, GERMANY, 5Veterinary Public Health Center for Asia Pacific (VPHCAP), Faculty of Veterinary
Medicine, Chiang Mai University, THAILAND * Corresponding author
Introduction
Campylobacter (C) jejuni is one of the most common foodborne pathogen caused of human enteritis worldwide. Consumption of poultry product is an epidemiological factor for human campylobacteriosis which is also a
major concern in veterinary public health. Many molecular methods identifying a clonal differentiation between C.
jejuni have been reported. Multi Locus Sequence Typing (MLST) is an excellent subtyping method in the examination of global epidemiological study for foodborne pathogens. However, the cost and labor associated with the
MLST technique hinder its use in limited budget situation or in large-scale epidemiological survey. With the use of
regular PCR procedure, rep-PCR was adopted as an alternative subtyping technique for C. jejuni in this study. The
multiple amplicons of different sizes can be fractionated by electrophoresis generating DNA fingerprint patterns specific for individual bacterial strain. In this study, fingerprints generated by two repetitive elements, Enterobacterial
Repetitive Intergenic Consensus (ERIC) sequence and a tri-nucleotide repeats GTG, and the typing results from
MLST method of 16 C. jejuni isolated from broiler were analyzed and compared.
Bactarial strains and DNA preparation. Sixteen C. jejuni strains isolated from broiler’s cecum and skin
sample were used as representative strains in the study. The genomic DNA from each isolate was extracted using a
DNeasy tissue kit (Qiagen Inc., Valencia, CA).
Multilocus Sequence Typing (MLST): The system is based on PCR amplification of conserved 7 housekeeping genes following by DNA sequencing. PCR amplification was followed by the C. jejuni MLST protocol (Dingle
et al., 2001) for aspA, glnA, gltA, glyA, pgm, tkt and uncA genes. Each isolate was designated by 7 numbers, constituting an allelic profile or sequence type (ST) (Dingle et al., 2001). The sequence was compared to existing alleles in
the MLST C. jejuni database to determine allele numbers and STs (http://pubmlst.org). Clonal relationships between
genotypes were investigated by running a BURST analysis on all STs in the database and grouping isolates in clonal
complexes (CCs) on the basis of four or more shared identical alleles to a central allelic profile.
Rep-PCR fingerprinting: The rep-PCR fingerprints generated from primers specific for two different repetitive elements were used. The first set was ERIC-PCR with primers ERIC1 (5' ATG TAA GCT CCT GGG GAT TCA C 3')
and ERIC2 (5' AAG TAA GTG ACT GGG GTG AGC G 3'); and the second set was GTG-PCR with the use of a
single primer (GTG)5 (5' GTG GTG GTG GTG GTG 3') (Versalovic et al., 1994). For the PCR reaction conditions, a
long-range rep-PCR condition modified from Chokesajjawatee et al. (2008) was used. The fingerprint patterns were
analyzed using a computer software, GelCompar II, version 5.0 (Applied Maths BVBA, Belgium). Composite data
based on both ERIC and GTG fingerprints were calculated using similarity average from experiments with equal
weight assignment. The unweighted-pair group method using average linkages (UPGMA) were used as the clustering method.
169
Figure 1 Cluster analysis of C. jejuni isolates based on MLST (A) and rep-PCR (B).
A total of 9 different STs were identified by MLST. All allelic sequences were matched with existing databases. Nine C. jejuni isolates were further designated into 5 clonal complexes (CCs) whereas 7 isolates (ST 2276 and
2274) were remaining unassigned (Fig. 1A). Rep-PCR also provided 9 clonal clusters with a similarity index cut-off
value at 0.98 (Fig. 1B). Clonal identification of all 16 C. jejuni isolates was identical for both techniques. The repPCR typing method as shown in this study demonstrated an equal discriminatory power as that of MLST (0.8917). In
conclusion, rep-PCR can be used to differentiate among different strains of C. jejuni and provide sufficient epidemiological determination of both phylogenetic inference and clonal differentiation of Campylobacter strains.
Acknowledgements
We thank the CPF, CO., LTD for financial support and Veterinary Public Health for Asia Pacific and Central Laboratory, Faculty of Veterinary Medicine, Chiang Mai University.
References
Chokesajjawatee, N., Y. G. Zo, et al. (2008). "Determination of clonality and relatedness of Vibrio cholerae isolates
by genomic fingerprinting, using long-range repetitive element sequence-based PCR." Appl. Environ. Microbiol. 74(17): 5392-5401.
Dingle, K. E., F.M. Colles, et al. (2001). “Multilocus sequence typing system for Campylobacter jejuni.” J Clin Microbiol, 39: 14-23.
Versalovic, J., M. Schneider, et al. (1994). "Genomic fingerprinting of bacteria using repetitive sequence based PCR
(rep-PCR)." Methods Mol Cell Biol 5: 25-40.
170 
ROLE OF DOMESTIC ANIMALS IN CHIKUNGUNYA VIRUS ECOLOGY
IN AN ENDEMIC AREA IN THAILAND
Sonthaya Tiawsirisup1*, Woraporn Sukhumavasi1, Theerayudh Sukmee2, Mathirut Mungthin3, Saovanee Leelayoova3, Tawee Naaglor3, Sakultip Amsakul4, Roongroje Thanawongnuwech1
1
Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, THAILAND,
Department of Microbiology, Phramongkutklao College of Medicine, THAILAND, 3Department of Parasitology,
Phramongkutklao College of Medicine, THAILAND, 4Vector-borne Disease Control Center 11.3, Surat Thani, Office
of Disease Prevention and Control 11, Nakhon Si Thammarat, Ministry of Public Health, Thailand.
2
Introduction
Chikungunya virus (CHIKV) is an emerging or re-emerging mosquito borne virus that can be found in several countries in Asia and Africa. CHIKV belongs to Alphavirus genus of the Togaviridae family, and it is an enveloped, single-stranded, positive-sense RNA virus. It was first discovered in Tanzania, east Africa in 1952 and was
identified in Thailand in 1958. Transmission cycle of this virus involves human and amplifying mosquito vector.
Animals might serve as reservoir hosts for this virus in nature. This study was conducted to investigate the role of
domestic animals in the epidemiology of this virus in an epidemic area. Evidence of CHIKV infection in dogs, cats,
and monkeys was studied.
Animal serum samples were collected from Phangnga and Nakhon Si Thammarat province, southern Thailand during the epidemic of CHIKV in 2009. Viral nucleic acid was extracted from an individual serum sample by
using viral nucleic acid extraction kit II (Geneaid, Taiwan) according to the manufacturer’s recommendation, and
each of them was kept at -80 °C until tested. They were tested for CHIKV by using reverse transcription polymerase
chain reaction (RT-PCR) according to CV et al. (2007) and Theamboonlers et al. (2009) with slight modification. The
p r i m e r s w e r e D V R C h k - R 5’G G G C G G G T A G T C C A T G T T G T A G A 3 ’ a n d D V R C h k - F
5’ACCGGCGTCTACCCATTCATGT3’ (CV et al. 2007). RT-PCRs were performed in 25 µl-reaction. One and a
half µl of RNA was mixed with 12.5 µl of 2X-master mix (0.4 mM dNTP, 3.2 mM MgSO4) (Invitrogen, Carlsbad,
CA), 1 µl of forward and reverse primer (10 µM), 1 µl of SuperScript III RT/Platinum Taq Mix (Invitrogen, Carlsbad, CA), and 8 µl of ultrapure water (Invitrogen, Carlsbad, CA). After the reverse transcription step at 48°C for 30
min and the initial PCR activation step at 94°C for 5 min, the amplification was carried out for 35 cycles with the
following temperature cycling parameters: 94°C for 45 sec of denaturation, 56°C for 45 sec of annealing, and 72°C
for 1 min of extension. The final amplification cycle included an addition of 7 min extension at 72°C. RNA was amplified by using thermocycler (Perkin Elmer Cetus 9600, Perkin Elmer, Waltham, MA). The PCR product was mixed
with 6 µl of loading buffer (BlueJuiceTM Gel Loading Buffer, Invitrogen, Carlsbad, CA) and analyzed in 2% agarose
gel (UltraPure™, Invitrogen, Carlsbad, CA) with expected 330 base pair band.
171
A total of 106 serum samples were collected from Phangnga and Nakhon Si Thammarat province, southern
Thailand during the epidemic of CHIKV in 2009. Fifteen monkey sera, 38 dog sera, and 20 cat sera were collected
from Phangnga province. Nineteen dog sera and 14 cat sera were collected from Nakhon Si Thammarat province.
Viral nucleic acid were extracted from individual serum sample and tested for CHIKV by using reverse transcription
polymerase chain reaction; however, no CHIKV nucleic acid was detected from all tested serum samples. Since no
CHIKV nucleic acid was detected from these animals from the present study, they might not be involved in the
transmission cycle of this virus in nature in Thailand.
Acknowledgements
This work (HR 1160 A) was supported by the Higher Education Research Promotion and National Research
University Project of Thailand, Office of the Higher Education Commission.
References
CV MNK, Anthony Johnson AM, DV RSG. 2007. Molecular characterization of chikungunya virus from Andhra
Pradesh, India & phylogenetic relationship with Central African isolates. Indian J Med Res 126:534-540.
Theamboonlers A, Rianthavorn P, Praianantathavorn K, Wuttirattanakowit N, Poovorawan Y. 2009. Clinical and
molecular characterization of chikungunya virus in South Thailand. Jpn J Infect Dis 62:303-305.
172 
ANIMAL-ASSISTED THERAPY FOR PERSONS WITH DISABILITIES VIA
BRAIN-COMPUTER INTERFACE SYSTEM
Warangkhana Phanwanich1,2*and Yodchanan Wongsawat1
1
Department of Biomedical Engineering, Faculty of Engineering, Mahidol University, Thailand
2
Animal Supplement & Pharmaceutical Co., Ltd., Vet Products Group, Thailand
Introduction
According to the behaviors of animals (e.g. dogs), they naturally accept people as they are regardless of age or
physical ability (Cangelosi et al. 2006). This idea leads to the new science of Animal-Assisted Therapy (AAT)
which is known as an alternative medicine that employs the human-animal interaction to enhance the treatment of
patients and the independent living of the persons with mental and physical disabilities. To achieve these goals of
AAT, veterinary medicine, engineering as well as medical science need to be integrated. For example, AAT can distract the patients from acute or chronic pains. AAT is also used to reduce stress and anxiety of the patients in some
hospitals (Barker et al. 1998). Consequently, blood pressure can be reduced and sensory cortex can be automatically
further stimulated. These can obviously lead to the increasing in the survival rate. However, achieving the goals of
AAT for persons with severe disabilities (e.g. spinal cord injury, stroke, and amyotrophic lateral sclerosis) is one of
the challenging problems. In this paper, real-time canine emotional behavior interpretation is proposed together with
the brain-computer interface (BCI) system. BCI is used to command and reward the dogs without moving the body,
while fuzzy logic is used to develop the real-time canine emotional behavior interpretation as the feedback to the
persons with server disabilities.
(a)
(b)
(c)
Figure 1 (a) Overview of SSVEP system (b) Disable person when using the brain-computer interface (BCI) system
(c) Well-trained dog with 3-axis accelerometers for emotional behavior interpretation.
Proposed Methods
Brain-Computer Interface (BCI) is the technology that can help the persons with disabilities communicate directly from the brain to the target devices without moving the body. There are many types of BCI, however, in this
paper, for AAT, the steady-state visual evoked potential (SSVEP)-based BCI (Wongsawat et al. 2010) is employed
since it yields the high accuracy with less training time (Figure 1(a)). By staring at a specific frequency, the same
frequency will be appeared at the occipital lobe of the brain around O1 or O2 position (in EEG 10-20 system). Power
spectral density via periodogram at the four stimulated frequencies (6, 7, 8, and 13 Hz) and their harmonics are used
as the features of interest. According to the BCI system, the server disable person can command the well-trained animals (e.g. the dog in Figure 1(c))
173
via the proposed four designed commands by just looking at the specific frequency. Each frequency is corresponding
to each specific sound, i.e., 6 Hz is for calling the dog’s name, 7 Hz is for playing with the dog via greeting sound, 8
Hz is for giving the reward to the dog via the automatic feeding system, and 13 Hz is for telling the dog to call for
assistance. The BCI user is usually on the bed as shown in Figure 1(b).
(a)
(b)
©
Figure2. (a) Simulated dog with two 3-axis accelerometers on the back and tail. (b) Front panel of LabVIEW represents the tail signal of x-axis and y-axis of accelerometer in time and frequency domain. (c) Diagram of fuzzy classification modules.
Since the severe persons with disabilities cannot move at all, the proposed BCI system also integrates our
previously developed real-time canine emotional behavior interpretation system to enhance the two-way communication between the dog and the disable person (Phanwanich et al. 2011). Canine tail language is captured via two 3axis accelerometers (Figure 2(a)). Directions and frequencies are selected as our features of interests (Figure 2(b)).
New fuzzy rules and center of gravity (COG)-based defuzzification method are proposed in order to interpret the
features into three canine emotional behaviors, i.e., agitate, happy, and scare as well as its blended emotional behaviors as illustrated in Figure 2(c).
In this experiment, the subject can command the well-trained animal via SSVEP-based BCI system. Thirty
commands are taken into consideration. The accuracy that the dog acts in the correct motion with respect to the prerecorded sound in the BCI system is approximately 83.33%, The classification accuracy of selecting the correct
SSVEP frequencies is approximately 87.5% and the average emotional recognition rate in real dog is 93.75%. With
the high accuracy and the real-time implementation, the proposed BCI-based AAT system should be practical for the
persons with severe disabilities to have a chance to use the designed AAT system.
Acknowledgements
This project is partially supported by the National Research University (NRU) Funding of Mahidol University and the Vet Product Group.
References
Cangelosi PR, Embrey CN. 2006. The healing power of dogs: Cocoa's story. J Psychosoc Nurs Ment Health Serv 44
(1): 17-20.
Barker SB, Dawson KS. 1998. The effects of animal-assisted therapy on anxiety ratings of hospitalized psychiatric patients. Psychiatr Serv 49: 797-801.
Wongsawat Y, Punsawat Y. 2010. System and method for SSVEP based control of electrical devices: International
Patent No. PCT/TH2010/000036.
Phanwanich W, Kumdee O, Ritthipravat R and Wongsawat Y. 2011. Animal-assisted therapy for persons with disabilities based on canine tail language interpretation via fuzzy emotional behavior model. In proceedings of
174 
ESTABLISHMENT OF IN VITRO CULTURE OF SPERMATOGONIAL STEM CELL-LIKE
COLONY IN PUBERTAL DOMESTIC CAT (FELIS CATUS)
N. Tiptanavattana, M. Techakumphu and T. Tharasanit*
Chulalongkorn University, Bangkok 10330, Thailand
Introduction
Spermatogonial stem cells (SSCs) play a central role to maintain the spermatogenesis and also to transmit the
male genetic profiles throughout post-pubertal male animals. These SSCs have been successfully isolated and cultured in vitro from a number of domestic species such as mouse rat, hamster, bull and human (Aponte and de Rooij,
2008, Conrad et al. 2008, Hamra et al. 2005, Kanatsu-Shinohara et al, 2003;2008). In cat, optimization of in vitro
culture system for SSCs is prerequisite for in-depth study of male germ cells that can also be used as a model for
endangered wild cat species. However, successful isolation and culture of these SSCs in domestic cat have yet to be
reported. This study aimed at isolating and culture of SSCs from pubertal cat testes.
Six testes were obtained from pubertal cats (0.5-1.0 grams/testis) after castration at the Veterinary Public
Health Division of The Bangkok Metropolitan Administration, Bangkok, Thailand. They were transported in 0.9%
(w/v) normal saline solution at room temperature (approximately 30 °C) to the laboratory. Upon arrival, they were
decapsulated from the tunica albuginea in Hank’s balanced salt solution (HBSS). The SSCs and Sertoli cells were
enzymatically digested from testicular parenchyma (Anway et al. 2003) and subsequently cultured in vitro as previously described with some modifications (Kanatsu-Shinohara et al. 2003). Colonies were subpassaged onto mitomycin-treated CF-1 and Sertoli cells. The formation of SSC colony was daily observed under a phase contrast microscope. Fluorescent immunolabeling with monoclonal antibody against GFRa-1 (a specific marker for SSCs) was additionally performed.
Our culture technique could isolate the putative SSCs that demonstrated an intercellular bridge and germ cell
-like chain clusters (about 4-5 cells) by 7-14 days of culture. These colonies expressed modest intensity of GFRa-1,
which was localized at cellular surface. Therefore, we claimed that the observed colonies demonstrated a property of
SSC-like colonies. Although we could maintain the SSC morphology in vitro, these cells were arrested after culture
for approximately 54 days. In conclusion, we firstly reported that SSCs can be isolated from pubertal cat testis and
cultured in vitro, while other factors regulating the proliferation and senescence of SSCs in domestic cat remain to be
elucidated.
Acknowledgements
This study was financially supported by the Centenary Academic Development Project, Chulalongkorn University and Higher Education Research Promotion and National Research University Project of Thailand, Office of
the Higher Education Commission (HR1166I)
175
References
Anway M.D, Folmer J, Wright WW, Zirkin BR. 2003. Isolation of Sertoli cells from adult rat testes: an approach to
ex vivo studies of Sertoli cell function. Biol Reprod 68: 996-1002.
Aponte PM, de Rooij DG. 2008. Biomanipulation of bovine spermatogonial stem cells. Anim Reprod 5: 16-22.
Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Buhring H, Mattheus U, Mack A,
Wagner H, Minger S, Matzkies M, Reppel M, Hescheler J, Sievert K, Stenzl A, Skutella T. 2008. Generation
of pluripotent stem cells from adult human testis. Nature 456: 344-349.
Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE, Garbers DL. 2005. Self-renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc Natl Acad Sci USA. 102: 1743017435.
Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. 2003. Longterm proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69:
612-616.
Kanatsu-Shinohara M, Muneto T, Lee J, Takenaka M, Chuma S, Nakatsuji N, Horiuchi T, Shinohara T. 2008. Longterm culture of male germline stem cells from hamster testes. Biol Reprod.78: 611-617.
176 
COMPARISON OF LOW-COST LONG-TERM FREEZING MEDIA
FOR MICE PANCREATIC ISLET CELLS
Hathairat Chanphao1*, Suwicha Kasemsuwan2, Monchanok Vijarnsorn3 , Siriruk Chantrakru4, Narudee Kashemsant5
1
Center for Agricultural Biotechnology (CAB),Graduate school, 2Department of Veterinary Public Health Kamphaeng Saen campus, 3Department of Companion Animals Clinical Sciences 4Department of Anatomy,
and ,5Department of Physiology,Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
Introduction
Diabetes mellitus is an important endocrinopathy disease in companion pets. Nowadays, alternative treatments, such as pancreas and islet transplantation, replace daily insulin injections. Pancreatic islet cell banks are essential for the modern treatment of diabetes mellitus by islet transplant. To sustain the islet banks, a major requirement is the constant availability of donors. Lacking sufficient donors becomes a major problem because 3-4 donors
are required for a single patient (Omori et al. 2007). In this context, this study attempts a comparative evaluation of
three low-cost freezing medias (FM) which can extend the life span of islets collected from single donors: DMSO,
glycerol and ethyleneglycol. The results of this study in mice may also apply to the protocol of companion animals.
The proposed experiment determines the differences between these freezing medias, and recommends possible candidates for islet preservation.
The pancreas of 42 healthy ICR-mice was collected, and the islets were isolated using the collagenase method
(Lacy et al. 1967). Islets from each sample were cultured overnight in RPMI1640 culture medium and then they
were evaluated for glucose stimulated insulin secretion (GSIS) (Saleh et al. 2006), by Immulite insulin kit tests ( Immulite 1000® system), and intracellular ATP, by ATPlite Luminescence ATP Detection Assay System. Each sample
was divided into 3 groups, which were preserved in 3 different types of freezing media that consisted of 2M dimethylsulfoxide (DMSO) in DMEM, 3M glycerol in DMEM and 1.5M Ethylene glycol in DMEM respectively. All the
islets were evaluated for insulin release and intracellular ATP after one month of preservation. The results were analyzed statistically using one ANOVA.
The total insulin of the freeze-thaw islets decreases compared to the fresh islets (Fig. 1a), while the GSIS was
impaired. In addition, islets frozen in 3M glycerol in DMEM tend to have the highest intracellular insulin (Fig. 1b),
which suggests a high functionality. Among the three FM, the highest intracellular ATP was found in islets preserved using the same FM (Fig. 2). This indicates a high survival rate in this group. In conclusion, this study suggests that the FM 3 which consists of 1.5M Ethylene glycol in DMEM are the most promising candidates for cryopreservation. However, further experiments are required to explain the cause of impaired insulin release of the islets,
such as measuring intracellular Ca2+ and glucose transporter 2 proteins, thus clarifying the functional structure of
the frozen pancreatic islets
177
a)
b)
Figure 1 a) GSIS of fresh islet cells, compared with FM1, FM2 and FM3 after one month of preservation. b) Cell
GSIS when exposed to different concentrations of glucose. This indicates the suitability of the cells preserved with
FM2, which have the closest GSIS pattern to fresh cells. However, in terms of cell viability, FM3 provides the closest values to the fresh cells
Figure 2 Comparison between the ATP of fresh cells and from cells frozen with the poposed FM.
Acknowledgements
This work is supported by the Center of Excellence on Agricultural Biotechnology, Science and Technology
Postgraduate Education and Research Development Office, Office of Higher Education Commission, Ministry of
Education. (AG-BIO/PERDO-CHE)
References
Lacy PE, Kostianovsky M . 1967. Method for the isolation of intact islets of Langerhans from the rat p a n c r e a s .
Diabetes 16(1):35–39
Omori K, Valiente L, Orr C, Rawson J, Ferreri K, Todorov I, Al-Abdullah IH, Medicherla S, Potter AA,
Schreiner GF et al. . 2007. Improvement of Human Islet Cryopreservation by a p38 MAPK
Inhibitor. American Journal of Transplantation 7(5):1224-1232.
Saleh MC, Wheeler MB, Chan CB. 2006. Endogenous islet uncoupling protein-2 expression and loss of glucose
homeostasis in ob/ob mice. Journal of Endocrinology 190(3):659-667.
178 
VIRULENCE GENES PROFILE OF STREPTOCOCCUS AGALACTIAE STRAINS ISOLATED FROM
TILAPIAS (OREOCHROMIS SP.) AND THEIR CULTURING ENVIRONMENTS IN THAILAND
P. Kayansamruaj1, P. Nopadon2, C. Rodkhum1*
1
Department of Veterinary Microbiology, 2Department of Veterinary Pathology, Faculty of Veterinary Science,
Chulalongkorn University, THAILAND. * Corresponding author
Introduction
Streptococcus agalactiae (group B streptococcus; GBS) is a major cause of streptococcosis in tilapia in Thailand
(Suanyuk et al., 2008). This bacterium produces a massive mortality in fish species which making significant economic loss in aquaculture industry. Many virulence factors of GBS have already been identified since this bacteria
found as pathogen of many mammalian species including human as well. However, the genes encoding these virulence factors can loss and gain from their genome via horizontal transfer which may modify their virulence characteristics and pattern of host tropism (Franken et al., 2001). Thus, the purpose of this study is to investigate some important virulence genes contained in GBS from tilapia origin and their culturing environments.
Forty-four isolates of GBS were collected from tilapias and theirs culturing environments in Nakhonpathom, Prachinburi and Suphanburi provinces of Thailand during 2009 to 2011. All isolates were identified as GBS by standard
biochemical assays and species specific PCR (Martinez et al., 2001). Additionally, GBS isolates also classified as
molecular serotype Ia using multiplex PCR (Imperi et al., 2010). To investigate GBS virulence genes, primers were
newly designed for specific PCR. Information for the virulence genes target and the oligonucleotide primers using in
this study are described thoroughly in Table 1. Each virulence genes specific PCR were performed in PCR thermocycler starting with DNA denaturation at 95 °C for 5 min followed by 30 cycles of 95 °C for 1min, 56 °C for 1min
and 72 °C for 1 min, subsequently extension at 72 °C for 7 min in the last step.
Table 1 Detail of primer using in this study and their target genes
Target gene
Encoded protein
Forward primer (5’à3’)
Reverse primer (5’à3’)
Amplicon size (bp)
scpB
C5a peptidase
TGAGCCTCAGGCATCGCACC
CCGCTGTCGATCAAGAGCACGG
109
cspA
Serine protease
GGTCGCGATAGAGTTTCTTCCGC
AACGCCTGGGGCTGATTTGGC
104
fbsA
Fb-binding protein A
GTCACCTTGACTAGAGTGATTATT
CCAAGTAGGTCAACTTATAGGGA
85
fbsB
Fb-binding protein B
TCTGTCCAACAGCCGGCTCC
TTCCGCAGTTGTTACACCGGC
144
Lmb
Laminin-binding
TGGCGAGGAGAGGGCTCTTG
ATTCGTGACGCAACACACGGC
105
dltA
D-alanylation ligase
GTTTTTGGTAGGGCAAACAGGGTGC
CGCAAATGTTGGCTCAACCGCC
100
bibA
Immunogenic adhesin
AACCAGAAGCCAAGCCAGCAACC
AGTGGACTTGCGGCTTCACCC
127
gapC
GAPDH
AGACCGATAGCTTTTGCAGCACC
GATCCTTGACGGACCACACCG
100
PI-1
Pili-1 backbone
AACAATAGTGGCGGGGTCAACTG
TTTCGCTGGGCGTTCTTGTGAC
102
PI-2a
Pili-2a backbone
CACGTGTCGCATCTTTTTGGTTGC
AACACTTGCTCCAGCAGGATTTGC
128
PI-2b
Pili-2b backbone
AGGAGATGGAGCCACTGATACGAC
ACGACGACGAGCAACAAGCAC
175
179
The percentages of the virulence genes existence among the GBS isolates are showed in Table 2. Other important
virulence genes such as ß-hemolysin/cytolsin, CAMP factor and capsular polysaccharide encoding genes were excluded from this experiment because they have been characterized as an intrinsic virulence genes of GBS.
Table 2 The virulence genes existence among the isolated GBS strains (n= 44).
Virulence genes
Virulence genes existence in this study (%)
Information in other GBS strains
scpB
2%
Loss in GBS bovine strain (Franken et al., 2001)
cspA
100%
Found in all serotypes of GBS
fbsA
100%
fbsB
100%
lmb
2%
Loss in GBS bovine strain (Franken et al., 2001)
dltA
98%
bibA
100%
gapC
100%
PI-1
100%
Can be loss or found (Sorensen et al., 2010)
PI-2a
0%
PI-2b
100%
We found that only 2% of GBS samples have scpB and lmb contained while the pili backbone encoded gene (PI-1,
PI-2b) was presence in all GBS tilapia strain. A little variation of virulence genes profile among the isolated GBS
strains may indicated that these bacterial strains have common virulence genes pattern. The virulence genes possessed by the isolated GBS strains may indicated that all GBS strains isolated from this study are pathogenic strain
since non-pathogenic strains will not possess the important virulence determinant (Ochman, 2001).
Acknowledgements
We Thank the CU 72nd Anniversary scholarship and TRF for research funding.
References
Franken, C., Haase, G., Brandt, C., Weber-Heynemann, J., Martin, S., Lammler, C., Podbielski, A., Lutticken, R.
and Spellerberg, B. 2001. Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the
role of a putative composite transposon containing scpB and lmb. Mol Microbiol. 41(4): 925-935.
Imperi, M., Pataracchia, M., Alfarone, G., Baldassarri, L., Orefici, G. and Creti, R. 2010. A multiplex PCR assay for
the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae. J Microbiol Methods. 80
(2): 212-214.
Martinez, G., Harel, J. and Gottschalk, M. 2001. Specific detection by PCR of Streptococcus agalactiae in milk. Can
J Vet Res. 65(1): 68-72.
Ochman, H. 2001. Evolution of Bacterial Pathogens. In: Principles of Bacterial Pathogenesis. 1st ed. E.A. Groisman
(ed). California: Academic press. 2-29.
Sorensen, U.B., Poulsen, K., Ghezzo, C., Margarit, I. and Kilian, M. 2010. Emergence and Global Dissemination of
Host-Specific Streptococcus agalactiae Clones. MBio. 1(3).
Suanyuk, N., Kong, F., Ko, D., Gilbert, G.L. and Supamattaya, K. 2008. Occurrence of rare genotypes of Streptococcus agalactiae in cultured red tilapia Oreochromis sp. and Nile tilapia O. niloticus in Thailand--Relationship
to human isolates? Aquaculture. 284(1-4): 35-40.
180 
CONTRIBUTION OF THE MULTIDRUG EFFLUX SYSTEM MexXY-OprM IN AMINOGLYCOSIDES
RESISTANCE PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES FROM DOGS AND CATS
K. Poonsuk, S. Pagdepanichkit, R. chuanchuen*
Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, THAILAND.
*
Corresponding author
Introduction
Aminoglycosides (AMG) is one of the drugs of choice for treatment of Pseudomonas aeruginosa (PA) infection. AMG resistance has emerged through several mechanisms e.g. inactivation by AMG-modifying enzymes,
impair membrane permeability, expression of active efflux (El’Garch et al. 2007, Islam et al. 2009). Up to date, the
multidrug efflux systems in the Resistance-Nodulation Cell Division (RND) family have been known to play a crucial role in multi-resistance in PA. Among these, MexXY-OprM is the only efflux system that has been reported to
be associated with AMG resistance in PA (Poole 2005). However, the role of this system in AMG resistance in PA
isolated from companion animal has never been reported. The aim of this study was to reveal the contribution of the
MexXY-OprM efflux system and other associated chromosomal mechanisms in AMG resistance in PA isolated from
dogs and cats.
Materials and methods
Fourteen AMG resistant PA clinical isolates from dogs and cats were detected for expression of MexXY using
RT-PCR. The mexXY operon in the MexXY-overexpressing strains was deleted using alleric exchange mediating
gene replacement technique to generate Δ(mexXY)::FRT mutants (Chuanchuen et al. 2008). The successful deletion
was confirmed by complementation with mexXY containing plasmid, pAMR-1. Minimum inhibitory concentrations
(MICs) of 6 AMGs including gentamycin (GEN), amikacin (AMK), neomycin (NEO), kanamycin (KAN), streptomycin (STR) and spectinomycin (SPC) were determined in all Δ(mexXY)::FRT unmarked mutants and their corresponding parental strains. The mexXY regulatory gene (i.e. mexZ) and the mexZ-mexX intergenic region were sequenced for the presence of mutations. The integrity of nuoH was examined using RT-PCR. Mutations in galU and
rplY were investigated using DNA sequencing analysis.
Expression of MexXY was detected in all PA isolates. The mexXY operon was successfully deleted in 8 clinical isolates. The lack of mexXY resulted in reduction of all AMG MICs in Δ(mexXY)::FRT mutants in comparison to
their isogenic parent. The MIC value of GEN, AMK, NEO, KAN, STR and SPC was reduced 2-4 folds in Δ
(mexXY)::FRT mutants when compared to their corresponding parent strains. After complementation with pAMR-1,
the MIC value of all AMGs was restored. These results suggested the involvement of MexXY in AMG resistance in
PA from clinical cases of dogs and cats. The C98-G and A94-G base pair changes were identified in mexZ, resulting
in amino acid substitutions Ala33-Gly and Thr32-Ala in MexZ, respectively. Expression of nuoH located downstream to nuoG was detected in all isolates, indicating the integrity of membrane. No mutation was found in galU.
Two mutations G67-T and G367-T were found in rplY, resulting in Ala23-Ser and Ala123-Ser in RplY respectively.
The results obtained in this study confirmed that PA clinical isolated from dogs and cats used combination mechanisms in AMG resistance and MexXY plays a role in AMG resistance. Experiment is currently in progress to measure expression level of the mexXY efflux system.
181
Acknowledgements
This study was supported by a grant from Thailand Research Fund RMU 5380035 and K.P. is the recipient
of the Royal Golden Jubilee Ph.D. Program grant PHD/0014/2552.
References
Chuanchuen R, Wannaprasat W, Ajariyakhajorn K, Schweizer HP. 2008. Role of the MexXY multidrug efflux pump
in moderate aminoglycoside resistance in Pseudomonas aeruginosa isolates from Pseudomonas mastitis .Microbiol Immunol 52:392-398
El’Garch F, Jeannot K, Hocquet D, Barakat CL, Plésiat P. 2007. Cumulative Effects of Several Nonenzymatic
Mechanisms on the Resistance of Pseudomonas aeruginosa to Aminoglycosides. Antimicrob Agents
Chemother 51(3): 1016-1021
Islam S, Oh H, Jalal S, Karpati F, Ciofu O, HØiby N, Wretlind. 2009. Chromosomal mechanisms of aminoglycoside
resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Clin Microbiol Infect 15:60-66.
Poole K. 2005. Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 49(2):479487.
182 
ANTIBIOTIC RESISTANCE AND VIRULENCE GENES OF
ESCHERICHIA COLI ISOLATED FROM SWINE
K.K. Lay1, N. Chansong 2, C. Koowatananukul1 , R. Chuanchuen1*
1
Department of Veterinary Public Health,Faculty of VeterinaryScience, Chulalongkorn University, THAILAND.
2
Novartis (Thailand) Ltd. Animal Health Business Unit, Bangkok, Thailand.
Introduction
Swine is one of the main sources of food in Thailand and also a major reservoir of Escherichia coli. The
pathogen has been shown to play a role as reservoirs of antimicrobial resistance determinants, of which integrons are
the most commonly identified. Data on genetic characteristics of multidrug resistant E. coli including mechanisms
underlying antimicrobial resistance and virulence factors is still limited. Therefore, aims of the study were to investigate the characteristics of class 1 integrons and test for their transferability, determine the distribution of antimicrobial resistance genes and detect virulence genes in E. coli isolated from swine.
Three-hundred and forty-four E. coli isolates from fecal samples of swine were examined for susceptibility
to 9 antimicrobial agents including ampicillin (AMP), chloramphenicol (CHP), ciprofloxacin (CIP), erythromycin
(ERY), gentamycin (GEN), streptomycin(STR), sulfamethoxazole (SUL), tetracycline (TET), and trimethoprim
(TRI) by determination of Minimum Inhibitory Concentrations (MICs) using two-fold agar dilution technique according to the Clinical and Laboratory Standard Institute (CLSI) (CLSI, 2006). All the isolates were characterized
for class 1 integrons (Chuanchuen et al., 2007). All the isolates carrying intI1 gene were examined for the presence
of gene cassettes with PCR using a primer set 5ĆS and 3ĆS primers (Levesque et al., 1995). The purified PCR
products were submitted for DNA sequencing. Nucleotide sequences were analyzed by comparison to the published
sequence using NCBI blast search. All the isolates carrying class 1 integrons with resistance gene cassettes were tested for the possession of plasmids by using alkaline lysis method (Liou et al., 1999). Transferability test of class 1
integrons were conducted by conjugation method. The E. coli isolates carrying class 1 integrons with the resistance
gene cassettes were used as donors and the spontaneous rifampicin-resistant derivatives of Salmonella spp. with negative intI1 were used as recipients. Transconjugants were confirmed Salmonella by growing on Xylose Lysine Deoxycholate (XLD) agar with antibiotics and appeared black colonies on XLD agar. Plasmid DNA was extracted from
each transconjugant using alkaline lysis method and tested for the presence of intI1 with gene cassettes corresponding to those in the donors. All isolates were tested for the presence of 18 antimicrobial resistance genes based on
their resistance phenotypes, i.e AMP (blaPSE1, blaTEM), CHP (catA, catB, cmlA), GEN (aadA1, aadA2, aadB), STR
(aadA1, aadA2, strA, strB), SUL (sul1, sul2, sul3), TET (tetA, tetB) and TRI (dfrA1, dfrA10, dfrA12) (Chuanchuen
et al ., 2008). All the E. coli isolates were screened for the presence of 14 virulence genes including elt for heat labile
enterotoxin, estA and estB for heat stable enterotoxin, astA for enteroaggregative heat-stable enterotoxin, stx2e for
shiga toxin, faeG, fanA, fasA, fedA, fimF41 for fimbriae and eaeA, paa , sepA and aidA for attaching/effacing activity
using multiplex PCR with specific primer pairs (Bosworth and Casey, 1997).
183
Results and conclusion
All isolates were resistant to at least one antimicrobial agent and 98.3% of the isolates were resistant to at
least 3 different classes of antimicrobial agents and were defined as multidrug-resistant (MDR). The most commonly
found antimicrobial resistance pattern was AMP-CHP-CIP-ERY-GEN-STR-SUL-TET-TRI (15.7%). Eighty-six to
one hundred percent of the antimicrobial resistance isolates possessed at least one resistance genes corresponding to
their resistance phenotypes. Seventy-three percent of E. coli isolates have intI1 gene of which 22.3% carried gene
cassettes with size ranging from 650-2700 bp. Sequence analysis results showed 5 distinct integrons profiles (IPs) in
which partial sat, aadA1, aadA22, dfrA12, aadA2, sat and psp were present in variable regions. The gene cassette
aadA1 (26.8%) was most frequently found among the isolates. Class1integrons were detected on conjugative plasmid in 8 E. coli isolates. Ten virulence genes such as astA (34.88%), eaeA (13.08%), elt (57.85%), estA (0.58%),
estB (25%), faeG (4.65%), fedA (16.86%), fasA (98.26%), paa, (22.67%) and sepA (14.53%) were identified in E.
coli isolates. This study revealed that E. coli isolated from swine could serve as reservoirs for antimicrobial resistance determinants and virulence genes.
Acknowledgments
This work was partly supported by Chulalongkorn University Veterinary Science research fund 2010.
Khin Khin Lay is the recipient of “The Graduate Scholarship Program for Faculty Members from Neighboring
Countries”.
References
Bosworth BT and Casey TA. 1997. Identification of toxin and pilus genes in porcine E. coli using polymerase chain
reaction (PCR) with multiple primer pairs. In: Proceedings of the 97th Annual General Meeting, abstract B509. Am. Soc. Microbiol., Miami, Florida.
Chuanchuen R, Khemtong S, Padungtod P. 2007. Occurrence of qacE/qacEDelta 1genes and their correlation with
class 1 integrons in Salmonella enteric isolates from poultry and swine.Southeast asian J Trop Med Public
Health 38 (5): 855-862.
Chuanchuen R, Pathanasophon P, Khemtong S, Wannaprasat W, Padaungtod P. 2008. Susceptibilities to antimicrobials and disinfectants in Salmonella isolates obtained from poultry and swine in Thailand. J Vet Med Sci 70
(6): 596-601.
CLSI.2006. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of infrequently isolated for Fastidious Bacteria, Approved guide Line. In: CLSI document M45-A. Pennsylvania, Clinical and Laboratory
Standards Institute 12-13.
Levesque C, Piche L, Larose C, Roy PH.1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicro Agents Chemother 39 (1):185-191.
Liou JT, Shieh BH, Chen SW, Li C. 1999. An improved alkaline lysis method for minipreparation of plasmid DNA.
Prep. Biochem. Biotechnol. 29 (1) 49-54.
184 
CHARACTERIZATION OF CAMPYLOBACTER SPP. ISOLATED
FROM BROILER FLOCKS IN CHIANG MAI, THAILAND
Chomporn Chokboonmongkol1 Karl-Hans Zessin 2 Thomas Alter3 Prapas Patchanee4*
1
Animal Health and Technical Service Office, Bangkok Agro-Industrial Products Public CO., LTD, Bangkok,
Thailand ,2Department Panel, Veterinary Public Health, 3Institute of Food Hygiene, Free University Berlin,
GERMANY, 4Veterinary Public Health Center for Asia Pacific (VPHCAP), Faculty of Veterinary Medicine,
Chiang Mai University, THAILAND * Corresponding author: [email protected]
Introduction
Campylobacter spp. is one of the most important foodborne bacteria which cause gastroenteritis in human. Epidemiological studies have demonstrated that consumption of poultry product is risk associated for Campylobacter infection in human. The wide use of antimicrobial drugs in animal production is also a major public health concern since
it plays as a major role for selective pressure of resistant Campylobacter spp. and increasingly contributes of antimicrobial resistance pathogen strains in human via food chains (Senok et al., 2007). Multi locus sequence typing
(MLST) is a molecular typing technique used to characterize C. jejuni and generate comparable data worldwide via
http://pubmlst.org. The objectives of this study were to determine the prevalence of Campylobacter in broiler flocks
by using conventional microbiological, to determine antimicrobial resistance patterns and genotypic characterizations of C. jejuni using MLST.
Ninety-eight broiler flocks in Chiang Mai area were collected followed the EU COMMISION DECISION protocol
(2007/516/EC) including ten intact caeca and one whole carcass per slaughter batch, respectively. All samples were
examined according to the procedure for detection of Campylobacter spp. as described by ISO 10272-1. Campylobacter isolates were susceptibility tested for 5 antimicrobial agents, including erythromycin, ciprofloxacin, tetracycline, gentamycin and ampicillin. Campylobacter sequence data of 7 housekeeping genes was further compared to
allelic profiles in the MLST database to determine allele numbers and sequence types (STs) (http://pubmlst.org). A
clonal complex (CC) was assigned based on the basis of four or more shared identical alleles to a central allelic profile. All Campylobacter allelic profiles from Thailand were recovered and investigated by running a BURST analysis.
Campylobacter detection in broiler flocks demonstrated as a lower proportion comparing with the finding on broiler
carcasses. Campylobacter jejuni is a major Campylobacter species detected in both samples types. High percentage
of Campylobacter isolates was found as multidrug resistant (MDR) strains. Three new STs of C. jejuni were firstly
reported in Thailand by this study (ID 11486-7, 11489 and 11493-4). MLST analysis demonstrated the contamination of chicken carcasses in the slaughterhouse which is due to Campylobacter colonized in the intestine. Different
STs of C. jejuni isolated from skin samples reflect different routes of horizontal transmission during the slaughtering
process. However, further investigations on different STs disseminated along the slaughtering steps are needed to
substantiate the identification of critical control points in the broiler slaughtering process.
.
185
Table 1: Prevalence of Campylobacter in broiler flocks and broiler carcasses
Prevalence (%)
95% CI
Broiler flocks (caecal sample)
11.2 (11/98)
4.97-17.47
Broiler carcasses (skin sample)
51 (50/98)
41.12-60.92
Figure 2: Phylogenetic analysis and CC of C. jejuni ST data from Thailand using BURST algorithm calculation.
Satellite is excluded and not shown in the BURST analysis graphical display.
Acknowledgements
We thank the CPF, CO., LTD for financial support and Veterinary Public Health for Asia Pacific and Central
Laboratory, Faculty of Veterinary Medicine, Chiang Mai University.
References
Dingle, K. E., Colles, F. M., Wareing, D. R., Ure, R., Fox, A. J., Bolton, F. E., Bootsma, H. J.,
Willems, R. J., Urwin, R. & Maiden, M. C. 2001. Multilocus sequence typing system for Campylobacter
jejuni. J Clin Microbiol, 39, 14-23.
EU COMMISSION DECISION. 2007. Concerning a financial contribution from the Community towards a survey
on the prevalence and antimicrobial resistance of Campylobacter spp. in broiler flocks and on the prevalence
of Campylobacter spp. and Salmonella spp. in broiler carcasses to be carried out in the Member States.
Official Journal of the European Union. EC 516 (L190/29).
ISO. 2006. Microbiology of food and animal feed stuffs-Horizontal method for detection and enumeration of
Campylobacter spp. ISO/TS 10272-1:2006. International Organization for Standardization.
MLST. 2010. The Department of Zoology, University of Oxford, UK.
Available from: http://pubmlst.org [Accessed 05/09/10].
Senok, A., Yousif, A., Mazi, W., Sharaf, E., Bindayna, K., Elnima El, A. & Botta, G. 2007. Pattern of antibiotic
susceptibility in Campylobacter jejuni isolates of human and poultry origin. Jpn J Infect Dis, 60, 1-4.
186 
PARASITIC FOODBORNE ZOONOSES AS THREAT FOR PUBLIC HEALTH IN SOUTHEAST ASIA
R. Siringo Ringo 1*, Sheila 1, D. W. Lukman2
1
Undergraduate Student of Faculty of Veterinary Medicine, Bogor Agricultural University, 2Department of Animal
Diseases Science and Veterinary Public Health, Faculty of Veterinary Medicine, Bogor Agricultural University,
INDONESIA. * Corresponding author
Introduction
Southeast Asia countries have a large biodiversity as well as parasites species because of all of Southeast Asia
countries have tropical climate. Until now, parasitic diseases in animal are still neglected and the reporting of parasitic disease has not been applied properly. Besides that, in recent years, there has been a major change in food consumption patterns. For example, in some Southeast Asia region, hunting is a common way of acquiring meat. Many
people eat meat of wildlife (bush meat) because they believe that bush meat could increase man stamina, cure cancer, etc. The recent food safety system is still focusing in microbial foodborne diseases instead of parasitic ones.
Parasitic Foodborne Zoonoses
Unlike the emerging zoonoses which attract considerable international attention, the endemic parasitic zoonoses fall very much into the category of neglected diseases and some of them are now reemerging health problems.
They affect mainly the poorest communities and cause lowered productivity or death of livestock (WHO 2006).
Five neglected foodborne zoonoses in Southeast Asia are toxoplasmosis, cysticercosis, trichinellosis, cryptosporidiosis, and anisakiasis.
The prevalence of antibody against Toxoplasma in humans and animals ranged from 2-75% in Southeast
Asian countries including Bangladesh, Laos, Malaysia, Singapore, Thailand, Vietnam, and Indonesia (Terazawa et
al. 2003). Goat and chicken meat are considered as a potential sources of infection with the cyst form. The prevalence of infection among goats at a slaughterhouse in Jakarta was 48% (Iskandar 1996) and the prevalence among
chickens in Surabaya was 30% (Suwanti 2006).
Cysticercosis was reported in some provinces in Indonesia, i.e., North Sumatera, Bali, Papua, East Nusa
Tenggara, Lampung, North Sulawesi, Southeast Sulawesi, West Borneo, Jakarta, and East Java (Suroso et al. 2006).
The high prevalence of cysticercosis (23.5–56.9%) occured in the central highlands of Papua (Salim 2009).
Cryptosporidiosis prevalence in developing countries is predicted between 5-20%. In Indonesia, cryptosporidium cysts were found in stool samples of 10/474 subjects (2.1%) (Soetomenggolo 2008). In Thailand, a study
among 156 HIV-infected Thai patients who had acute diarrheal illness showed that 12.8% of them had cryptosporidiosis (Saksirisampant et al. 2002).
In Southern Java coast, a high prevalence of 97-100% of zoonotic Anisakis sp. infestation showed a high risk
of predatory fish to get infested (Jakob and Palm 2006). In Laos, a large outbreak of trichinellosis occured at least in
650 of estimated patients in Udomxay (northern Laos) in June 2005 (Anantaphruti 2001).
.Prevention and Control of Foodborne Zoonoses
Food handlers have a major role in the prevention of foodborne disease since they may cross contaminate
foods. Hence, food handler training is seen as one of the main strategies to improve food safety practices (Soon et
al. 2011). In general, the control measures of parasitic zoonotic diseases should involve the improvement of sanitary
infrastructures, strengthening of surveillance, public education on hygiene and handwashing, and health education to
children in order to promote long-terms change (Eddie et al. 2006). Furthermore, the community-based approach is
recommended to be developed and implemented in prevention and control of zoonoses since the communities play a
crucial role in prevention and control of zoonotic disease (FAO 2003).
187
Conclusion and Recommendation
There is an increasing risk to consumers of acquiring the infections of parasitic foodborne zoonoses in nonendemic areas because of growth of international trade and travel and an increasing interest in ethnic foods. Generally, foodborne parasitic zoonoses are associated with sociocultural practices and threat public health. Surveillance
and monitoring system including strict meat inspection by the authorities could prevent the parasitic foodborne zoonoses. Building the knowledge and awareness of community in prevention and control of zoonotic diseases should be
encouraged.
References
Anantaphruti MT. 2001. Parasitic contaminants in food. Southeast Asian J Trop Med Public Health 32 Supl 2: 218228.
Eddie C, Katalin B, Juan L, Wiliam A, Andrew S, Daniela B, Joseph D. 2006. Veterinary public health activities at
FAO: cysticercosis and echinococosis. Parasitol Int 55:S305-S308.
FAO. 2003. Expert final consultation on community based veterinary public health (VPH) systems. http://
www.fao.org/ag/againfo/ programmes/en/vph/info.html
Iskandar T, Partoutomo S, Beriajaya, Pratomo HW. 1996. A study on toxoplasmosis in sheep and goat at slaughter
house in Jakarta. P.205-208. In Veterinary Research Institute, Proceedings National Scientific Veterinary Meeting, Bogor, West Java (Indonesia).
Jakob E, Palm HW. 2006. Parasites of commercially important fish species from the southern Java coast, Indonesia,
including the distribution pattern of trypanorhynch cestodes. Verh Ges Ichthyol 5: 165-191.
Nurhayati APD, Hidayati D, Ressa P, Setiawan E. 2008. Pola distribusi Anisakis sp pada usus halus ikan kakap putih
(Lates calcarifer) yang tertangkap di TPI Brondong, Lamongan. Prosiding Seminar Nasional Basic Sains. Jurusan
Fisika FMIPA Universitas Brawijaya. 21 Februari 2008.
Saksirisampant W, Eampokalap B, Rattanasrithong M, Likanonsakul S, Wiwanitkit V, Nasingkarn A, Denmasae N.
2002. A prevalence of Cryptosporidium infections among Thai HIV-infected patients. J Med Assoc Thai 85
(Suppl 1):S424-S428.
Salim L, Ang A, Handali S, Tsang VCW. 2009. Seroepidemiologic survey of cysticercosis-taeniasis in four central
highland districts of Papua, Indonesia. Am J Trop Med Hyg 80(3):384-388.
Soetomenggolo HA, Firmansyah A, Kurniawan A, Trihono PP. 2008. Cryptosporidiosis in children less than three
years old in Ciliwung Riverside, Kampung Melayu Village, Jakarta, Indonesia. Paediatr Indones 48(2):99-103.
Soon JM., Singh H, Baines R. 2011. Foodborne diseases in Malaysia: a review. Food Control 22:823-830.
Suroso T, Margono SS, Wandra T, Ito A. 2006. Challenges for control of taeniasis/cysticercosis in Indonesia. Parasitol Int 55: 161-165.
Suwanti LT, Suprihati E, Mufasirun. 2006. Prevalensi Toxoplasma pada ayam di beberapa pasar di Kota Surabaya.
Media Kedokteran Hewan 22(1).
Terazawa A, Muljono R, Susanto L, Margono SS, Konishi E. 2003. High toxoplasma antibody prevalence among
inhabitants in Jakarta, Indonesia. Jpn J Infect Dis 56: 107-109.
WHO. 2006. Neglected zoonotic disease. WHO, Geneva
188 
CLONING AND SEQUENCING OF SIALOADHESIN AND CD163 CDNA FROM
THE FIELD PRRSV INFECTED PORCINE ALVEOLAR MACROPHAGES
1
*Corresponding author, E-mail: [email protected]
Introduction
An important analysis of virus receptors is gained much knowledge. For instance, the recognition of virus
receptor inhibitors of the viral receptor interaction has also useful for the study of viral infection. The basis data of
viral receptors may have the potential to design the drugs that limit the interaction between virus and its receptors at
the initiation of viral binding. The new therapy of viral diseases and the method to blocks viral infection into the target cell of host by using the other related viruses that use the similar receptor will be developed. Moreover, the siRNA functional genomics applications (Behlke, 2006) and the anti-receptor monoclonal antibodies may be useful for
inhibition of the replication of major viruses (Norkin, 1995). PRRS virus (PRRSV) can spread widely and cause the
severe diseases in swine around the world. Up to now, there are a few reports of the information about PRRSV specific receptors on the surface of the swine susceptible target cells. However, two essential receptors on porcine alveolar macrophages (PAM), as porcine Sialoadhesin (Sn) and CD163 have been reported for PRRS virus (PRRSV)
entry and uncoating, respectively. Recently, Sn and CD163 genes were examined to be related to the immunity of
pigs after PRRSV infection and may be used to improve PRRS immunity of pigs (Fengli Wang et al., 2011). The
understand ability of interaction between PRRSV and their receptors could be used to produce peptide mimetics and/
or specific antibodies to antagonize the envelope protein interactions with its receptors in an attempt to block
PRRSV entry and be necessary to inhibit infection of pigs with the PRRSV (Das et al., 2010). In this study, the Sn
and CD163 cDNA molecules were cloned and sequenced, their available data will be useful for expression and production of these recombinant proteins.
Six PRRS outbreak farms (2,000 sows on production) from the central part of Thailand were investigated based on clinical signs with specific PRRSV infection. The positive PRRSV nursery pigs were confirmed the
PRRSV strains from 18 sera (three sera per farm) using RT-PCR (Eglia et al., 2001). Six positive PAM samples of
nursery pigs with three PRRSV strains (EU, US and China) were collected by washing BAL with sterile PBS (-) and
were used for isolation of both Sn and CD163 cDNA. The total RNA was extracted from PAM using Total RNA
Mini Tissue Kit (Geneaid, Taiwan). The RT-PCR which amplified Sn and CD163 cDNA were performed with the
specific primers and followed the previous study (Van Gorp et al., 2008). The Sn and CD163 RNA were transcribed
to cDNA using SuperScript TM III First-Strand Synthesis System (Invitrogen, USA). Two pairs of primers of Sn
(EU003993) and CD163 (NM213976) cDNA were designed from GeneBank. Briefly, the CD163 primers are 5’TGCTGTAGTCGCTGTTCTCAG -3’ (forward), 5’- AAGGCTGAACTCACCAGGTT-3’ (reverse) and of Sn primers, 5’- CTCCTGGCTTCATCTGCTCT-3’ (forward), 5’- AGAGGTGGGCAGGATCAAA-3’ (reverse), respectively. The purified Sn and CD163 cDNA were then cloned into pCR-XL-TOPO® vector (Invitrogen, USA) and
then transformed into One Shot® TOP10 chemically competent E. coli. Single colony of transformants was selected
on LB agar plates containing kanamycin and was confirmed by direct colony PCR technique. The nucleotides and
deduced amino acids sequences of Sn and CD163 cDNA were sequenced by AITBIOTECH PTE LTD (Singapore)
and analyzed with BioEdit Sequence and Alignment Editor Software (version 7.0.5.3, USA).
189
Results and discussions
Fifteen sera samples from 18 nursery pigs were detected the presence of PRRSV strains and gel picture was
shown in Figure 1. There were 6 of US positive, 5 of EU positive and 4 of China positive sera samples, respectively. In
this study, six PRRSV positive PAM were selected from 15 PRRSV positive sera samples (two PRRSV positive PAM
samples per strain) to use for isolation both Sn and CD163 cDNA. Six cDNA of both Sn and CD163 were successfully
amplified from three PRRSV strains (EU, US and China) (Figure 2). Sn and CD163 cDNA were successfully cloned into
the pCR-XL-TOPO® vector. Ten positive clones of pCR-XL-TOPO-Sn (4 of US-Sn ; 3 of EU-Sn and 3 of China-Sn
clones) and twenty positive clones of pCR-XL-TOPO-CD163 (9 of US-CD163; 5 of EU-CD163 and 6 of China-CD163
clones) were positive transformants. The positive clone of pCR-XL-TOPO-US-CD163-001 was shown in Figure 3. The
sequence and deduced amino acids analysis of all studied Sn and CD163 are on the process.
Figure 1. Electrophoresis analysis of PRRSV strains from
sera samples. Lane M, 100 bp DNA Ladder (Fermentas,
USA). Lane 1, negative control. Lane 2 (EU), 3 (US), and 4
(China): positive controls. Lane 5 (EU), 6 (US) and 7
(China): Sera positive samples.
Figure 2. Electrophoresis analysis of Sn and CD163
cDNA expression from PAM samples. Lane M, 1 kb
DNA Ladder (BioLabs, USA). Lane 1 (US), 2 (China)
and 3 (EU): CD163 cDNA expression. Lane 4 (US), 5
(China) and 6 (EU): Sn cDNA expression.
Figure 3. Electrophoresis analysis of pCR-XL-TOPO-US-CD163-001 transformants from US strain. Lane M, 1 kb DNA Ladder
(BioLabs, USA). Lane 1, 2, 5 and 6, as negative transformants. Lane 3 and 4, as positive transformants.
Reference
Behlke, M.A. 2006. Progress Towards in Vivo Use of siRNAs. Mol. Therapy. 13: 645-666.
Das, P.B., Dinh, P.X., Ansari, I.H., de Lima, M., Osorio, F.A. and Pattnaik, A.K. 2010. The minor envelope glycoproteins GP2a and GP4 of porcine reproductive and respiratory syndrome virus interact with the receptor CD163. J.
Virol. 84: 1731-1740.
Eglia, C., Thür, B., Liua, L. and Hofmann, M.A. 2001. Quantitative TaqMan® RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus J. Virol. 98
(1): 63-75
Fengli Wang, Haifang Qiu, Qingde Zhang, Zhongzhen Peng and Bang Liu. 2011. Association of two porcine reproductive and respiratory syndrome virus (PRRSV) receptor genes, CD163 and SN with immune traits. Mol. Biol.
Rep. DOI 10.1007/s11033-011-1177-4.
Norkin, L.C. 1995. Virus Receptors: Implications for Pathogenesis and the Design of Antiviral Agents. Clin. Microbiol.
Rev. 8: 293-315.
Van Gorp, H., Van Breedam, W., Delputte, P.L. and Nauwynck, H.J. 2008. Sialoadhesin and CD163 join forces during
entry of the porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 89: 2943-2953.
Poster Presentation Full Paper
192 
PEGYLATED LIPOSOMAL DOXORUBICIN-INDUCED PALMAR-PLANTAR ERYTHRODYSTHESIA
(HAND-FOOT SYNDROME) IN A NASAL CARCINOMA DOG
S.Kunakornsawat1*, K. Imsilp2, S Jeamprapai1, S Poapolathep2, S Netramai3
1
Department of Companion Animals Clinical Sciences, 2Department of Pharmacology, and 3Veterinary Teaching
Hospital, Bangkaen campus, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
Introduction
The chemotherapeutic agent doxorubicin (unencapsulated doxorubicin) has been one of the most effective and
broadspectrum drugs available to veterinarians for the treatment of a variety of malignant tumors. The dose-limiting
toxicities of doxorubicin include myelosuppression and cardiotoxicity (Ogilvie 1989). Doxil ® is a pegylated
(polyethylene glycol coated) liposome-encapsulated form of doxorubicin. The polyethylene glycol coating allows
the doxorubicin to remain in the body for longer so that a greater amount of chemotherapy is delivered to the cancer
cells, while having fewer side effects on healthy tissue. When compared with doxorubicin, Doxil ® has proven to be
effective in decreasing cardiotoxicity, prolonging drug circulation times, and enhancing tumoricidal effects in a variety of tumor models (Vaage et al, 1993, Gabizon et al 1994a, Gabizon et al, 1994b, Lasic 1996). Nevertheless, severe and dose limiting mucocutaneous reactions occur, mostly the palmar-plantar erythrodysthesia syndrome
(PPES), more commonly known as hand-foot syndrome (Lotem et al, 2000, Nagore et al, 2000). Following administration of Doxil®, small amounts of the drug can leak from capillaries in the palms of the hands and soles of the
feet. The result of this leakage is redness, tenderness, and peeling of the skin that can be uncomfortable and even
painful. The aim of this report is increase clinical awareness of dermatological toxicity associated with administration of pegylated liposomal doxorubicin (Doxil®). Clinicians should be acquainted with this spectrum of mucocutaneous toxic effects, as this product is becoming better recognized and more widely precribed.
A four-year-old, male crossbreed dog was referred because of epitaxis of 12 week duration. On presentation, the dog
was loss of appetite, bloody nasal discharge, sneezing and stertorous breathing. The dog was finally diagnosed with
stage I nasal carcinoma. Subsequently, the dog received Doxil ® (0.5 mg/kg) intravenously, every 3 weeks for four
total treatments. The dog responded well to the first chemotherapy without development of any significant side effects (including no skin toxicity), and clinical signs ceased after three weeks. The second cycle of therapy of Doxil ®
(0.75 mg/kg) was administered intravenously. Cutaneous side effects (i.e. pain, lameness, edema, and/or ulceration)
that occurred during this period were documented. The dog was given antibiotic (cephalexin 30 mg/kg) orally and
applied topically anti-bacterial solution until all skin lesions were totally improved. The subsequent treatment was
delayed for a minimum of a week, and the Doxil® dose was decreased by 20%.
During the second chemotherapy, cutaneous reaction termed palmar-plantar erythrodysthesia (PPES) was observed.
A painful desquamating dermatitis characterized by skin changes, ranging from mild erythema, hyperemia, and alopecia to severe crusting, ulceration, and epidermal necrosis was noted. Lesions occurred primarily in areas of skin
contact, such as sternal, scrotal sac region and the skin surrounding the foot pads. Lameness associated with apparent paw discomfort while bearing weight has been found (Figures 1-2).
193
a
b
c
Figure 1 (a) macules and hyperpigmented patches on ventral abdomen (b) ulceration with exudate at sternal area
(c) erythema and ulceration at scrotal sac
a
b
Figure 2 (a) exudate at paw of forelimb (b) alopecia and ulceration at paw of hindlimb
The use of Doxil® at dose of 0.75 mg/kg developed cutaneous reaction in this dog. Early detection of PPES is crucial and can be achieved by the recognition of sings and symptoms. Moreover, it may be necessary to delay treatment or stop cytotoxic chemotherapy because PPES can have devastating consequences.
Acknowledgements
We gratefully acknowledge the staff, veterinarians, patients and owner-pets of the VTH,KU.
References
Gabizon A, Catane R, Uziely B, Kaufman B, Safra T, Cohen R, Martin F, Huang A, Barenholz Y. Prolonged
circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethyleneglycol coated liposomes. Cancer Res 1994a;54:987-992.
Gabizon A, Isacson R, Libson E, Kaufman B, Uziely B, Catane R, Ben-DOr CG, Rabello E, Cass Y, Peretz T,
Sulkes A, Chisin R, Barenholz Y. Clinical studies of liposome-encapulated doxorubicin. Acta Oncol
1994b;33:779-786.
Lasic DD. Doxorubicin in sterically stabilized liposomes. Nature 1996,380:561-562.
Lotem M, Hubert A, Lyass O, Goldenhersh MA, Ingber A, Peretz T, Gabizon A. Skin toxic effects of polyethylene
glycol-coated liposomal doxorubicin. Arch Dermatol 2000;136:1475-1480.
Nagore E, Insa A, Sanmartin O. Antineoplastic therapy-induced palmar plantar erythrodysesthesia (‘hand-foot’)
syndrome: incidence, recognition and management. Am J Clin Dermatol 2000;1:225-234
Ogilvie GK, Richardson RC, Curtis CR, Withrow SJ, Reynolds HA, Morris AM, Henderson RA, Klausner JS,
Fowler JD, McCaw D. Acute and short-term toxicoses associated with administration of doxorubicin to dogs to
dogs with malignant tumors. J Am Vet Med Assoc 1989;195:1584-1587.
Vaage J, Donovan D, Mayhew E, Abra R, Huang A. Therapy of human ovarian carcinoma xenographs using
doxorubicin encapsulated in sterically stabilized liposomes. Cancer 1993;72:367
194 
REFERENCE RANGE OF PROSTATIC SIZE AND VOLUME
IN NORMAL HEALTHY DOGS RELATED TO THE BODYWEIGHT
S. Ponglowhapan*, N. Sannamwong, N. Saengklub, P. Sriphutthachot
Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University,
Bangkok 10330 THAILAND. * Corresponding author
Introduction
Measurement of size and volume of the prostate via ultrasound scan images is beneficial to diagnosis of prostatic
diseases. The normal size, weight and volume of the canine prostate are known to be affected by age and bodyweight
(Barsanti and Finco, 1979); the latter is more correlated to prostatic volume (Atalan et al., 1999). To estimate whether the prostate of a dog is normal in size for a given age and/or bodyweight, formulae have been generated based on
both parameters (Ruel et al., 1998; Atalan et al., 1999). However, age and bodyweight of animal population included
in previous studies markedly varied, i.e. 9 months to 14 years old (Ruel et al., 1998) and weight between 4.5 and 65
kg (Atalan et al., 1999). In addition, some dogs had prostatic cysts (Ruel et al., 1998). These make the formulae previously published not appropriate for estimated volume of a normal-sized canine prostate. The objective of this study
was to determine the size and volume of prostates in relation to the bodyweight.
One hundred and one clinically healthy intact dogs, aged between 1.5 and 4 years, free from any prostatic diseases as
determined by clinical signs and transabdominal ultrasonographic examination were included. Dogs were divided
into 7 groups depending on their bodyweight (group I; >1-5, group II; >5-10, group III; >10-15, group IV; >15-20,
group V; >20-25, group VI; >25-30 and group VII; >30 kg). Prostatic size (length, width and depth) was measured
ultrasonographically and the volume was then calculated (Kamolpatana et al., 2000); prostatic volume (cm3) = [1/2.6
(length x width x depth)] + 1.8. The size and volume were compared among groups using ANOVA. Pearson coefficient correlation and multiple linear regressions were used to detect correlations between bodyweight and prostatic
volume. Results were considered statistically significant when P<0.05.
Results and conclusions
Statistical analysis showed no differences in animal’s age among groups (I-VII) . There were strong positive correlations between bodyweight and prostatic size as well as bodyweight and prostatic volume. Group VII had higher prostatic volume compared to any groups (P<0.05), and no difference in the volume was detected between group I and II.
The mean and range of prostaic size and volume were shown in Table 1. The regression equation was expressed as
Prostatic Volume (cm3) = 0.33 x Bodyweight (kg) + 3.28.
Development of the canine prostate can be divided into 3 phases, i.e. a phase of normality (1 to 5 years), a phase of
hyperplasia growth (6 to 10 years) and a phase of senile involution (11 years and over) (O’Shea, 1962). Age limit of
dogs was set between 1.5 and 4 years because at these ages the prostate is highly likely to be normal. Therefore, reference ranges of prostatic size and volume reported in this study could be useful in clincal practice to determine if,
based on a given bodyweight, the prostate is oversized. Furthermore, estimated volume of normal-sized prostate in a
dog could extrapolate from the formula [Prostatic Volume (cm3) = 0.33 x bodyweight (kg) + 3.28].
195
Table 1. Mean (±SD) and range of prostaic size and volume in dogs grouped by the bodyweight
Group
Bodyweight
(kg)
Prostatic size (cm)
length
width
Prostatic volume
(cm3)
depth
right lobe
left lobe
mean
I
1-5
1.98±0.47
(1.03-3.40)
2.12±0.29
(1.36-2.63)
1.56±0.30
(0.86-1.98)
1.55±0.34
(0.96-2.05)
1.56±0.31
(0.91-2.00)
4.51±1.32
(2.56-8.59)
II
>5-10
2.13±0.30
(1.56-2.76)
2.45±0.34
(1.62-2.88)
1.90±0.35
(1.23-2.54)
1.87±0.37
(1.15-2.65)
1.88±0.35
(1.19-2.59)
5.75±1.51
(3.43-8.63)
III
>10-15
2.39±0.48
(1.36-3.22)
2.68±0.49
(1.82-3.41)
2.12±0.45
(1.35-2.98)
2.09±0.47
(1.30-2.94)
2.11±0.46
(1.32-2.96)
7.25±2.29
(3.37-11.36)
IV
>15-20
2.32±0.46
(1.82-2.93)
2.77±0.58
(1.97-3.49)
2.15±0.52
(1.43-2.93)
2.11±0.50
(1.36-2.76)
2.13±0.50
(1.39-2.84)
7.60±3.15
(3.84-11.72)
V
>20-25
3.02±0.65
(2.34-4.24)
3.05±0.56
(2.23-3.90)
2.31±0.37
(1.72-2.64)
2.42±0.40
(1.91-3.07)
2.37±0.37
(1.81-2.85)
10.22±2.69
(7.59-13.40)
VI
>25-30
3.06±0.43
(2.38-3.68)
3.43±0.50
(2.86-4.40)
2.59±0.50
(1.84-3.58)
2.64±0.51
(2.02-3.79)
2.61±0.50
(1.99-3-68)
12.62±4.00
(8.30-20.07)
VII
>30
3.18±0.31
(2.65-3.60)
3.67±0.55
(2.86-4.45)
3.03±0.70
(2.19-4.43)
3.00±0.69
(2.12-4.40)
3.02±0.69
(2.22-4.41)
15.83±5.14
(8.29-22.29)
Prostatic volume (cm3) = [1/2.6 (length x width x depth)] + 1.8 (Kamolpatana et al., 2000)
Acknowledgements
This study was financially supported by the 6th year student project fund, Faculty of Veterinary Science,
Chulalongkorn University.
References
Atalan G, Holt PE, Barr FJ. 1999. Ultrasonographic estimation of prostate size in normal dogs and relationship to
body weight and age. J Sm Anim Pract 40:119-22.
Barsanti JA, Finco, D. 1979. Canine bacterial prostatitis. Vet Clin North Am Small Anim Pract 9:679-700.
Kamolpatana K, Johnston GR, Johnston SD. 2000. Determination of canine prostatic volume using transabdominal
ultrasonography. Vet Radiol Ultrasound 41:73-7.
O’Shea JP. Studies on the canine prostate gland. Factors influencing its size and weight. 1962. J Comp Pathol 72:321
-31.
Ruel Y, Barthez PY, Mailles A, Begon D. 1998. Ultrasonographic evaluation of the prostate in healthy intact dogs.
Vet Radiol Ultrasound 39:212-6.
196 
DETECTION OF METHICILLIN RESISTANT
STAPHYLOCOCCUS PSEUDINTERMEDIUS SCC MEC TYPEIII FROM CAT URINE
W. Tonpitak1* and C. Sornklein1
1
Department of Microbiology, Faculty of Veterinary Medicine, Mahanakorn University of Technology, THAILAND.
Introduction
Staphylococcus (S.) pseudintermedius is a novel species of Staphylococcus, which is the main causative
agent in canine pyoderma, otitis externa and in various infections in animal. Methicillin resistance is mediated by the
mecA gene located on the Staphylococcus cassette chromosome (SCC). Up to date SCCmec type of S. aureus are
classified as typeI to typeXI based on the arrangement of the SCC and the types of the cassette chromosome recombinaseA and B (ccrA and B) genes, however the type of S. pseudintermedius is classified according to S. aureus. The
isolations of the methicillin resistant S. pseudintermedius (MRSP) in various animals and human are reported but in
cat are rarely. The aim of the present study was to report the detection of S. pseudintermedius SCCmec typeIII from
urine cat. The finding is important to support the baseline of the epidemiology of MRSP in veterinary- and also may
be in human-medicine in Thailand and to be concerned the protection of the transmission between human and animals.
The methicillin resistant Staphylococcus aureus (MRSA) SCCmec typing control strains were included
strain NCTC10442 (typeI), N315 (typeII), 85/2082 (typeIII), 81/108 (typeIV) and WIS (typeV). Urine sample from
the cat was collected by voiding and cultured by standard technique. The suspected Staphylococcus sp. was identified by conventional biochemical method and then confirmed as S. pseudintemedius by the PCR-REA of the pta
gene restriction by MboI (Bannoehr et al., 2009). The methicillin resistant genotypic identification based on the mecA gene was detected by the PCR as previously described (Tonpitak et al., 2010). The SCCmec typing was performed according to Kondo et al. (2007) but only the multiplex PCR1 and PCR2 for identification the ccr genes
complex and the mec gene complex respectively were performed.
The viable bacterial count of urine sample was more than 105CFU/ml indicated bacterial urinary tract infection. Phenotypic characteristics of bacterial isolate were gram-positive cocci, oxidase negative, catalase test- and coagulase
test-positive. This isolate was diagnosed as coagulase positive Staphylococcus and selected for further specie identification based on the PCR-REA method. The isolate has one of the MboI restriction site in the internal fragment of
PCR product of the pta gene, therefore it was identified as S. pseudintermedius (data not shown). Then the mecA
gene PCR resulted in the expected fragment for MRSA reference strains as well as for the isolate (data not shown).
Based on SCCmec typing method described by Kondo et al. (2007); the isolate carrying the ccr gene complex type3
(ccrA3B3) and the classA mec gene complex (Fig 1) was identified as MRSP typeIII. The SCC mec type of this isolate was the same type as isolates in cat in Germany (Ruscher et al., 2009). However, SCCmec types of MRSP isolates from cats in the other reports from Germany and the countries in Europe are typeII-III; moreover, in Canada
and Portugal were typeV (Kadlec et al., 2010, Nienhoff et al., 2011, Pomba et al., 2010). This finding let us to concern the protection of MRSP widespread and transmission between humans and animals in Thailand.
197
Figure 1 Multiplex PCR 1 identifying the ccr genes (left) and multiplex PCR 2 identifying the mec class (right); M,
1 kb plus molecular standard (Invitrogen); 1-5, S. aureus reference SCCmec type I to V, respectively; N, negative
control; S, S. pseudintermedius isolate.
Acknowledgements
We are grateful to Prof. Ito, Jutendo University, Japan and Dr. Lulitanond, Khon Kaen University, Thailand
for kindly support the MRSA reference strains. This study is supported by Mahanakorn University of Technology.
References
Bannoehr J, Franco A, Iurescia M, Battisti A, Fitzgerald R. 2009. Molecular diagnostic identification of Staphylococcus pseudintermedius. J Clin Microbiol 47:469-471.
Kadlec K, Schwarz S, Perretten V, Anderson UG, Finn M, Greko C, Moodley A, Kania SA, Frank LA, Bemis DA,
Franco A, Iurescia M, Battisti A, Duim B, Wagenaar JA, van Duijkeren E, Weese SC, Fitzgerald JR, Rossana A, Guardabassi L. 2010. Molecular analysis of methicillin-resistant Staphylococcus pseudintermedius
of feline origin from different Europe countries and North America. J Antimicrob Chemother 65:1826-1837.
Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, Hiramatsu K. 2007. Combination of multiplex
PCRs for Staphylococcal cassette chromosome mec type assignment: Rapid identification system for mec,
ccr, and major differences in Junkyard regions. Antimicrob Agents Chemother 51:264-274.
Nienhoff U, Kadlec K, Chaberny IF, Versphol J, Gerlach GF, Schwarz S, Kreienbrock L, Nolte I, Simon D. 2011.
Methicillin-resistant Staphylococcus pseudintermedius among cats admitted to a veterinary teaching
hospital. Vet Microbiol doi:10.1016/j.vetmic.2011.05.045.
Pomba C, Couto N, Moodley A. 2010. Treatment of a lower urinary infection in a cat caused by multi-drug
methicillin-resistant Staphylococcus pseudintermedius and Enterococcus faecalis. J Feline Med Surg 12:802
-806.
Ruscher C, Lübke-Becker A, Wleklinski CG, Soba A, Wieler LH, Walther B. 2009. Prevalence of methicillinresistant Staphylococcus pseudintermedius isolated from clinical samples of companion animals and
aquidaes. Vet Microbiol 136:197-201.
Tonpitak W, Sornklein C, Suwannakhen S. 2010. Detection of Methicillin Resistant Staphylococcus sp. by simple
Polymerase chain reaction. In: Tonpitak W, Mamom T, Sotthibundhu P and Pavasutthipaisit S. editors.
Proceedings of the 4th MUT Veterinary Annual Conference; Nov 26; Bangkok, Thailand: 2010. pp 147-148.
198 
BLOOD COAGULATION FACTOR DEFECT IN CANINE EHRLICHIOSIS
A. Macotpet1*, K. Aiyaranoi1, J. Laoumanotham1, A. Kulilung1
E. Patarapanwichien2, S. Hoisung3, E. Barameechaithanan3
1
Department of Medicine, 2Department of Pathobiology and 3Veterinary Teaching Hospital, Faculty of Veterinary
Medicine, Khon Kaen University, THAILAND. * Corresponding author
Introduction
Ehrlichia canis is a Gram-negative, obligate intracellular bacterium which infected circulating monocytes and
mononuclear phagocytic cells in lymph nodes, spleen, liver and bone marrow (Sherding 2006). Hematological abnormalities include anemia, thrombocytopenia and leucopenia as well as hemostatic defect are common findings in
this disease (Bulla et al. 2004). Measurement of activated partial thromboplastin time (APTT) and prothrombin time
(PT) are performed in plasma and are the most commonly employed laboratory tests in patients suspected of having
a coagulopathy (Kummeling et al. 2006). APTT is a screening test for factors II, V, VIII, IX, X, XI and XII defect of
the intrinsic and common pathways, whereas PT is a screening test for factors II, V, VII and X defect of the extrinsic
and common pathways (Rizzo et al. 2008). The aim of this study was to identify a coagulation factor defect in Ehrlichia canis-infected dogs.
Blood samples were obtained from 19 Ehrlichia canis-infected and 16 clinically healthy dogs. E. canisinfected dogs were diagnosed with blood analysis using Canine SNAP 4Dx® test (IDEXX Laboratories, Westbrook,
ME, USA) or by a visible typical E. canis morula on a blood smear. Blood was collected from the cephalic vein and
transferred into two tubes containing EDTA and 3.8% sodium citrate. Platelet count was performed on EDTAanticoagulated blood using an automatic hematology analyzer (Diatron®, Austria). Citrated-anticoagulated blood
samples were centrifuged at 3000 rpm for 10 minutes. Plasma samples were divided into two aliquots to assess PT
and APTT by an automatic blood coagulation analyzer (Sysmex®, Japan). Results were presented as mean ± SD
(Table 1). The Mann-Whitney U test was used for comparisons between two groups. A P value < 0 .05 was considered
significant. If the prolongation of the PT or APTT was due to clotting factor defect, the clotting time of test plasma
mix with normal plasma should reduced more than 10% of test plasma clotting time alone. The Substitution experiments will be performed further using normal canine serum or normal adsorbed canine plasma and the results will be
interpreted as showed in table 1.(Mukherjee and Ghosh 2010)
Table 1 Interpretation of substitution experiment with the APTT test.
APTT of test plasma corrected with
Adsorbed Normal Plasma
Normal Serum
YES
NO
Interpretation
Factor VIII defect
NO
YES
Factor IX defect
YES
YES
Factor XI or XII defect
199
Table 2. Platelet count, APTT and PT of Ehrlichia canis infected and clinically healthy dogs.
Parameters
Ehrlichia canis infected dogs
Healthy dogs
(Mean ± SD)
(Mean ± SD)
numbers
19
16
Platelets (× 109/L)
123 ± 171
289 ± 98
0.001
APTT (seconds)
16.54 ± 2.21
15.18 ± 0.89
0.020
PT
7.28 ± 0.99
7.62 ± 1.60
0.260
(seconds)
P value
Same as previous study (Cortese et al. 2006) the reduction in platelet count was observed in canine monocytic ehrlichiosis. Therefore decreasing of platelet count may involve in the pathogenesis of canine monocytic ehrlichiosis that attributed with several mechanisms, acting together or alone. These include increased platelet consumption or sequestration, increased platelet destruction, suppression of platelet production, or immunologically or nonimmunologically mediated platelet destruction (Waner et al. 1995). Since APTT evaluates the intrinsic coagulation
pathway and PT involves the extrinsic coagulation pathway, it is likely that prolongation of APTT with normal PT is
related to defect of factors needed to trigger the intrinsic pathway, such as factors VIII, IX, XI, XII (Smith et al.
2005) and defective of these coagulation factors may involve in the pathogenesis of canine monocytic ehrlichiosis.
In this study, 2 of 19 cases that prolong APTT but normal PT were corrected with normal plasma but not corrected
with normal adsorbed plasma by substitution (APTT) experiments. So the cases were possible the Factor IX defect.
Factor IX defect, resulting either from reduced production of the functional factor IX protein or from production of a
defective protein, causes the bleeding disorder hemophilia B (Giannelli et al. 1997). 1 of 19 cases corrected with
both normal serum and normal adsorbed plasma. So this case was possible either the Factor XI or XII deficiency.
Factor XII deficiency individuals do not exhibit a symptomatic bleeding tendency (Littlewood 2000). However, coagulation defects in liver disease are often caused by reduced synthesis of coagulation factors or qualitative abnormalities in factor production (Kemkes-matthes et al 1991). Therefore coagulation factors defect in Ehrlichia canisinfected dogs may cause by liver damaged.
Acknowledgements
We thank the faculty of Veterinary Medicine, Khon Kaen University for research funding.
200 
References
Bulla C, Takahira RK, Araújo JP et al. 2004. The relationship between the degree of thrombocytopenia and
fection with Ehrlichia canis in an endemic area. Vet Res 35:141-146.
in-
Cortese L, Pelagalli A, Piantedosi D et al. 2006. Platelet aggregation and haemostatic response in dogs naturally
co-infected by Leishmania infantum and Ehrlichia canis. J Vet Med A 53: 546-548.
Giannelli F, Green GM, Sommer SS et al. 1997. Hemophilia B: Database of point mutations and short additions and
deletions. Nucleic Acids Res 25:133.
Kemkes-matthes B, Bleyl H and Matthes KJ. 1991. Coagulation activation in liver diseases. Thromb Res 64:253-261
Kummeling A, Teske E, Rothuizen J and Van Sluijs FJ. 2006. Coagulation profiles in dogs with congenital
portosystemic shunt before and after surgical attenuation. J Vet Inter Med 20:1319-1326.
Littlewood JD. 2000. Disorders of secondary hemostasis. In Day MJ, Mackin A and Littlewood JD (eds) :
BSAVA Manual of canine and feline hematology and transfusion medicine. Gloucester. British small animal
veterinary association. 209-215.
Mukherjee KL, Ghosh S. 2010. Medical laboratory technology: procedure manual for routine diagnostic test. Vol I
2nd ed. New Delhi: Tata Mc Graw Hill. 344-6.
Rizzo F, Papasouliotis K, Crawford E, Dodkin S and Cue S. 2008. Measurement of prothrombin time (PT) and
activated partial thromboplastin time (APTT) on canine citrated plasma samples following different storage
conditions. Res Vet Sci 85(1):166-70.
Sherding, RG. 2006. Rickettsiosis, Ehrlichiosis, Anaplasmosis, and Neorickettsiosis. In Birchard SJ and Sherding
RG (eds) : Saunders Manual of Small Animal Practice. 3rd edition. Elsevier Saunders. Missouri. 178-190.
Smith JW, Day TK and Mackin A. 2005. Diagnosing bleeding Disorders. Compend Contin Educ Pract Vet 27:
828-832.
Waner T, Harrus S, Weiss DJ et al. 1995. Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis. Vet Immunol Immunopathol 48:177-182.
201
NITRIC OXIDE LEVELS IN CANINE GRADE II MAST CELL TUMORS
E. Pattarapanwichien1*, A. Kanbanjong1, K. Yongwahish1, C. Prabwongsa1, A. Macotpet2, P. Wipoosak3
1
Department of Pathobiology, 2Department of Medicine and 3Veterinary Teaching Hospital, Faculty of Veterinary
Introduction
Mast cell tumors (MCTs) are one of the most common cutaneous neoplasms in dogs and are often aggressive
(Yamada et al. 2011). The cause of the tumor is unknown, although speculation exists that the cause may be a genetic component, virus or chronic inflammation (Grahan 2006). MCTs range from relatively benign to extremely
aggressive, leading to metastasis and eventual death from systemic disease (London et al. 2003). Nitric oxide (NO) is
a small, unstable, potentially toxic gas and is produced by nitric oxide synthase (Forstermann et al. 1995). Nitric
oxide has been invoked in nearly every normal and pathological condition including it has both positive and negative
affects in tumor biology (Ridnour et al. 2008). Therefore, factor determination indicative of increased NO radical
formation is of interest. The objective of this study was to compare nitric oxide levels between dogs with Grade
II mast cell tumors and healthy dogs.
Blood samples were obtained from 26 healthy dogs and 18 dogs suffering from spontaneous Grade II mast cell
tumors. The final diagnosis of Grade II mast cell tumor was made from histological findings. Griess test (Moshage et
al. 1995) was used in the determination of nitric oxide. This test is a chemical analysis test which detects the
presence of organic nitrite compounds. When sulphanilic acid is added, the nitrites form a diazonium salt. When
the azo dye agent is added a pink colour develops. The data were presented as mean ± SD. Non-parametric data were
determined using Mann-Whitney U test. P value of less than 0.05 was considered significant.
NO levels (micromolars)
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Normal
MCT
Groups
Figure 1 Comparison of the plasma NO levels between clinically healthy dogs (n = 26) and Grade II mast cell tumor
dogs (n = 18).
202 
Nitrite concentration in plasma were used as indicator for NO radical formation. In thi study, elevated levels
of NO were observed in mast cell tumor group compared to healthy group. Previous study has shown that NO biosynthesis was high in breast cancer compared to benign lesions and normal breast tissue (Thomsen et al. 1995). Reactive nitrogen species are not only responsible for mediating protein oxidation reactions under physiological conditions (Tuteja et al. 2004) but also the type of tissue and DNA damage produced by NO (Ohshima and Barthch 1994).
However, NO inhibits the proliferation of tumor cell by inducing cell differentiation and showing and antitumor effect (Bani et al. 1995). The mechanisms involved in cancer and cancer treatment are diverse and the effect of NO
depends on the tumor microenvironment as well as its concentration, spatial, and temporal constraints (Wink and
Mitchell 1998). The concentration of this radical can dictate the phenotypic response (Ridnour et al. 2006). Several
clinical and experimental studies indicate that the presence of NO in tumor microenvironment is detrimental
to tumor cell survival and metastasis. In contrast, numerous reports suggest that NO can have tumor-promoting effects. NO may exert a biphasic response, such that when NO levels go beyond a critical concentration that would be
suitable for tumor growth and survival, growth arrest and/or apoptotic pathways are initiated. These characteristics
of NO have been exploited therapeutically with impressive effects in pre-clinical models of cancer to slow tumor growth and to enhance the efficacy of both chemotherapy and radiotherapy (Singh and Gupta 2011).
Acknowledgements
We Thank the VET KKU for research funding.
203
References
Bani D, Masini E, Bello MG et al. 1995. Relaxin activates the L-arginine-nitric oxide pathway in human breast
cancer cells. Cancer Res 55:5272-5275.
Forstermann U, Kleinert H, Gath I et al. 1995. Expression and expressional control of nitric oxide synthases in
various cell types. Adv Pharmacol 34:171-186.
Graham JC. 2006. Soft tissue sarcomas and mast cell tumors. In: Birchard SJ, Sherding RG eds. Manual of small
animal practice. 3rd edition. Missouri. Saunder. 301-310.London CA. and Seguin B. 2003. Mast cell tumors in
the dog. Vet Clin North Am Small Anim
Pract. 33(3):473-489.
Moshage H, Kok B, Huizenga JR and Jansen PLM. 1995. Nitrite and Nitrate determinations in plasma: a critical
evaluation. Clin Chem 41(6):892-896.
Ohshima H and Bartsch H. 1994. Chronic infections and inflammatory processes as cancer risk factors: possible role
of nitric oxide in carcinogenesis. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis.
305(2):253-264
Ridnour LA, Thomas DD, Switzer C et al. 2008. Molecular mechanisms for discrete nitric oxide levels in cancer.
Nitric Oxide 19(2):73-76.
Singh S and Gupta AK. 2011. Nitric oxide: role in tumour biology and iNOS/NO-based anticancer therapies. Cancer
Chemo Pharmacol 67(6):1211.
Thomsen LL, Miles DW, Happerfield L et al. 1995. Nitric oxide synthase activity human breast cancer. Br J Cancer
72:41-44.
Tuteja N, Chandra M, Tuteja R and Misra M.K.. 2004. Nitric oxide as a unique bioactive signaling messenger in
physiology and pathophysiology. J. Biomed. Biotechnol 227–237.
Wink DA and Mitchell JB. 1998. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide Free Radic Biol Med 25:434– 456.
Yamada O, Kobayashi M, Sugisaki O et al. 2011. Imatinib elicited a favorable response in a dog with a mast cell
tumor carrying a c-kit c.1523A>T mutation via suppression of constitutive KIT activation. Vet Immunol
Immunopathol 142:101-106.
204 
GLUTATHIONE LEVELS IN CANINE MAMMARY CANCER
E. Pattarapanwichien1*, A. Kanna1, N. Tammasiri1
N. Manojai1, A. Macotpet2, P. Jaisil3
1
Department of Pathobiology, 2Department of Medicine and 3Veterinary Teaching Hospital, Faculty of Veterinary
Introduction
Mammary tumours are the most common tumors in the female dog (Kumaraguruparan 2005). Reports of the
occurrence of malignant forms vary from 26 to 73% (Pe´rez Alenza et al., 2000), carcinoma being the most common
malignant type (Morrison 1998). There are many evidence that reactive oxygen species (ROS) are involved in the aetiopathogenesis of many diseases (Szczubial et al., 2004) such as cancer (Diehn et al., 2009).
Glutathione (GSH) plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative
stress and may therefore play a key role in the pathogenesis of many diseases (Hultberg 2006). Glutathione is a
tripeptide with a structure of γ-L-glutamyl-L-cysteinyl-glycine, which is characterized by the γ-glutamyl peptide and
the reactive thiol group (Bannai et al. 1986). Glutathione scavenges free radicals, detoxifies heavy metals, helps
ferry amino acids into the cells, helps in bile production, and much more. Cellular GSH plays a central role in the
body’s defense against infection, free radicals and carcinogens (Bounous 2000). The objective of this study was
to compare the mean glutathione levels between dogs with mammary cancer and clinically healthy dogs.
Blood samples were obtained from 21 healthy dogs and 18 dogs suffering from spontaneous mammary cancer. The final diagnosis of mammary cancer was made from histological findings. Serum was separated by centrifugation of the clotted blood at 3000 rpm for 5 minutes. Serum glutathione level was determined by using DTNB reagent (Shigesawa et al. 1992). The glutathione concentration was calculated by comparing with standard glutathione
(Sigma, USA). The data were presented as mean ± SD. Non-parametric data were determined using Mann-Whitney
U test. P value of less than 0.01 was considered significant.
12
GSH (mg%)
10
8
6
4
2
0
healthy
mammary cancer
groups
Figure 1 the mean of reduced glutathione levels between healthy dogs (n =21) and dogs with mammary cancer
(n = 18)
205
The level of GSH depends first on the rate of synthesis and second on the rate of utilization (Arrick and Nathan 1984). In this pilot study, the GSH levels was significantly lower in cancer group than in healthy group (p <
0.01). Similar observation was made in a study of Kumara et al. (1995) showed significantly lower level of plasma
GSH in grade III cervical intraepithelial neoplasia and invasive cancer compared to controls. Furthermore, reduced
glutathione levels in the normal tissues were found to be higher than those in the tumoral tissues. Decreased tumoral
tissue GSH levels may be a consequence of the increased detoxification activity in the tumor cells (Saydam et al.
1997). Tumoral GSH levels reported in the literature show considerable differences, probably due to the heterogeneous tissue structure, the storage conditions of the samples or the methods used in analysis (Meister 1988).
Acknowledgements
We Thank the VM KKU for research funding.
References
Arrick BA, Nathan CF. 1984. Glutathione metabolism as a determinant of therapeutic efficacy. Cancer Res 44:
4424–4432.
Bannai S and Tateishi N. 1986. Role of Membrane Transport in Metabolism and Function of Glutathione in Mammals. J Membr Biol 89(1):1-8.
Bounous G. 2000. Whey protein concentration (WPC) and glutathione modulation in cancer treatment. Anticancer
Res 20:4785-4792.
Diehn M, Cho RW, Lobo NA et al. 2009. Association of reactive oxygen species levels and radioresistance in cancer
stem cells. Nature. 458(7239):780-783.
Hultberg M and Hultberg B. 2006. The effect of different antioxidants on glutathione turnover in human cell lines
and their interaction with hydrogen peroxide. Chem Biol Interact 163(3):192-198.
Kumara A, Sharma S, Pundirb CS and Sharmac A. 1995. Decreased plasma glutathione in cancer of the uterine
cervix. Cancer Letters 94:107-111.
Kumaraguruparan R, Balachandran C, Manohar B at al. Altered Oxidant-Antioxidant Profile in Canine MammaryTumours. Vet Res Commun 4:287-296.
Meister A. 1988. Glutathione metabolism and its selective modification. J Biol Chem 26:17205- 17208.
Morrison WB. 1998. Canine and feline mammary tumors. In: Morrison WB eds.Cancer in Dogs and Cats: Medical
and Surgical Management. Williams and Wilkins. Maryland. 591–598.
Perez Alenza MD, Peña L, del Castillo N, Nieto AI.. 2000. Factors influencing the incidence and prognosis of canine
mammary tumours. J Small Anim Prac 41:287–291.
Saydam N, Kirb A, Demir O et al. 1997. Determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione S-transferase levels in human lung cancer tissues.
Cancer Letters 119:13-19.
Shigesawa T, Sato C and Marumo F. 1992. Significance of plasma glutathione determination in patients with
alcoholic and non-alcoholic liver disease. J Gastroenterol Hepatol 7:7-11.
Szczubial M, Kankofer M, Lopuszynski W et al. 2004. Oxidative stress parameters in bitches with mammary gland
tumours. J Vet Med 51(7-8):336-40.
206 
DETERMINATION OF BLOOD TYPING POPULATION AND INCIDENCES OF
FELINE IMMUNODEFICIENCY VIRUS (FIV) AND FELINE LEUKEMIA VIRUS (FELV) IN 5 BREEDS
OF THAI DOMESTIC CATS
Chayakit Sinthusing1*, Monchai Lekjarernwong2, Wanchart Yippaditr3, Thunyakan Nithisakdiyanond4,
Lapapatr Phutdhikarnt4, Wanvisa Prasong4, and Atthariya Jiranaparat4
1
Department of Companion Animal Clinical Sciences, 2 Veterinary Teaching Animal Hospital Kamphaeng Saen
campus, 3 Veterinary Teaching Animal Hospital Unit Hua-Hin, 46th-year veterinary student, Faculty of Veterinary
Medicine, Kasetsart University, Kampeansan Campus, 73140 THAILAND.* Corresponding author
Introduction
The habit of the Siamese cat has the intelligence , there is the self-confident , think , know flatter , love a
house , love an owner , and love the freedom of oneself , from in the past continuously come to until from originally
that have 23 whole species , nowadays real Siamese cat species remain stays only 5 species, for example, Khao manee,
Black Bombay, Korat, Burmese cat, Royal Siamese cat. Feline inflectious diseases had been important to extinction
of Siamese cat species, for example ,FeLV(Feline leukemia virus) and FIV (Feline immunodeficiency virus) which
the prevention were difficult. Population of blood group have the important to study the risk of disease occurrence
such as Neonatal isoerythrolysis and use advantage in the blood transfusion. This study was investigated for determination of blood typing population and incidences of common infectious diseases in five Thai domestic cats such as
Khao manee, Black Bombay, Korat, Burmese cat, Royal Siamese cat.
Material and Method
The blood sample collected from 90 cats (each breed 18 cats) at 4 Thai domestic cats conservative institute
were determined blood typing and feline infectious diseases such as Feline immunodeficiency virus (FIV) and Feline
leukemia virus (FeLV) diseases by test kit. Three minlilite of blood samples would be devided to two parts for
determine blood typing and feline infectious diseases (FIV and FelV) and analysed population of blood typing from
each breeds of cats and determination incidences of FIV and FelV in five breeds of Siamese cat.
The result
The result showed majority of blood typing is A group (Figure 1.) and incidence of FIV was 33.34 % and
FeLV diseases was 28 % positive of all (Figure 2.).
207
Blood group
Figure 1 Blood typing population, majority of blood typing was A group follow by: Khao manee had 83.33% of A
group, Black Bombay 77.78% of A group, Korat 88.33% of A group, Burmese cat 66.67% of A group, and Royal
Siamese cat 83.33% of A group.
Feline leukemia virus
Feline immunodeficiency virus
Figure 2 . Graph showed incidences of FIV and FeLV infection in Five Siamese cat species. Incidences of FIV and
FeLV 33.33%, 22.22% Khao manee, 16.67%, 33.33% Black Bombay, 33.33%, 27.78% Korat, 16.67, 44.44% Burmese cat, 33.33%, 27.78% Royal Siamese cat.
Conclusion and Discussion
Although FIV and FeLV positive cats is lower but these diseases are important and need to control and prevent transmission because these diseases might be caused extinction of Siamese cat species in the future. Blood
group highest population was A group as same as the reported in Siamese mixed breed and should be advantaged for
blood transfusion in feline species.
References
Grace SF. Blood groups in cat. In: Nortsworthy GD, Crystal MA, Grace SF and Tilley LP, editors. The Feline
patient: Essential of diagnosis and treatment. 3rd ed. Lippincott Williams & Wilkins, 2006:589-592 2006.
208 
COMPARISON BETWEEN HEMODIALYSIS AND PERITONEAL DIALYSIS MANAGEMENT IN RENAL INSUFFICIENCY DOGS.
Chayakit Sinthusing1*, Nakrob Pattanapon2, Chamaiporn Sodawichit 2, Chinnawat Kasemmongkolchai 2,
Ketkaew Wasanasuk 3, Teerapat Rungnirundorn 3.
1
Department of Small Animal Clinical Practice, 2Veterinary Teaching Animal Hospital Kamphaeng Saen campus,
3
Veterinary Teaching Animal Hospital Bangkean campus, Faculty of Veterinary Medicine Kasetsart Univrtsity,
THAILAND. * Corresponding author.
Introduction
Healthy kidneys clean our blood by removing excess fluid, minerals, and wastes. They also make hormones
that keep our bones strong and our blood healthy. It controls our blood's pH by controlling which acids and bases to
keep and which to lose. It controls sodium, potassium, calcium, carbon dioxide, water balance, and more. When our
kidneys fail, harmful wastes build up in the body, blood pressure may rise, and body may retain excess fluid and not
make enough red blood cells. The wrong things are dumped, the wrong things are kept, toxins build up and the patient is sick. The state of toxicity that results is called uremia or uremic poisoning. Most every animal hospital can
provide diuresis, therapy in which extra fluid beyond what the patient can drink is provided, thus giving the kidney a
boost to remove toxic waste. In fact, dialysis may be another choice, though it is substantially more expensive than
diuresis and facilities that perform dialysis for pets are still few and far between (1-3). Twelve dogs had increased serum creatinine levels more than 7 mg/dl indicating renal insufficiency. They also had clinical signs of depresssion,
anorexia, vomiting, and oliguria.
Material and Method
Twelve dogs were divided into two groups (n = 6) to compare effectiveness of treatments using either hemodialysis or peritoneal dialysis. The peritoneal dialysis group was treated 6 times per day whereas the hemodialysis
group was treated 3 hours per day twice a week for 1 month Parameters used for determination were serum creatinine level, blood electrolytes, blood gas analysis, and complete blood count.
Peritoneal dialysis procedure (PD)
In PD, a soft tube called a catheter is used to fill dog’s abdomen with a cleansing liquid called dialysis solution. The walls of abdominal cavity were lined with a membrane called the peritoneum, which allows waste products
and extra fluid to pass from dog’s blood into the dialysis solution. These wastes and fluid then leave your body when
the dialysis solution is drained. The process of draining and filling was called an exchange and takes about 30 to 40
minutes. The period the dialysis solution in abdomen was called the dwell time. A typical schedule called for four
exchanges a day, each with a dwell time of 4 to 6 hours (1).
Hemodialysis procedure (HD)
HD group would be take blood from the jugilar vein and pass though hemodialyzer that priming with
dialysate solution and moved counter together. Blood from hemodialyzer would be pass though the cephalic vein and
back to body. The wastes product would be passed from blood to hemodialysate solution with ultrafiltration process.
The hemodialysis group was treated 3 hours per day twice a week (3).
209
The results
The result showed that serum creatinine levels of dogs in hemodialysis group (HD) were significantly reduced
more than dogs in peritoneal dialysis group (PD) (p < 0.05) after the first day of treatment unti 1 month (Figure 1.).
Hypokalemia was found in peritoneal dialysis group while hemodialysis group had severe dehydration following
treatment. Blood gas analysis revealed metabolic acidosis in both groups. Urine outputs of dogs in peritoneal
dialysis group were significantly increased more than those in hemodialysis group (p < 0.05).
Figure 1. Graph comparesion creatinine level between Peritoneal dialysis group (PD) and Hemodialysis group (HD).
Serum creatinine levels of dogs in hemodialysis group (HD) were significantly reduced more than dogs in peritoneal
dialysis group (PD) (p < 0.05) after the first day of treatment unti 1 month
Conclusion and Discussion
Dialysis process could be reduced wastes product such as creatinine and BUN from blood of the patients but
Hemodialysis could be higher performance to reduce them more than Peritoneal dialysis. Contraindication of
peritoneal dialysis should be carefull hypokalemia and other electrolyte imbalance while hemodialysis should be
careful hypovolemic condition during and after treatment. With dedicated 24-hour care, peritoneal dialysis is a viable
treatment option for dogs with acute, oliguric, or anuric renal failure. In dogs with ARF secondary to leptospirosis,
PD and HD can be a successful treatment option when aggressive medical therapy has failed (1).
References
Beckel NF, Labato MA, O’Toole TE, et al. Peritoneal dialysis in the management of acute renal failure in 5 dogs
with leptospirosis. J Vet Emerg Crit Care 2005;15(3):201–205. Lichtenberger, M. The many uses of peritoneal
dialysis. Proceedings, IVECCS 2007.
Kolff, W. J., and Berk, H. T. J. Artificial kidney, dialyzer with great area, 21:1944.
Weinreich T, De los Ríos T, Gauly A, Passlick-Deetjen J (2006). "Effects of an increase in time vs. frequency on
cardiovascular parameters in chronic hemodialysis patients". Clin. Nephrol. 6 (6): 433–9.
210 
COMMON MALIGNANT TUMORS IN DOGS IN BANGKOK: RETROSPECTIVE STUDY DURING
JUNE 2010 TO MAY 2011
T. Mamom1*
1
Department of Pathology, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, THAILAND. * Corresponding author
Introduction
In dogs, it was well known that skin tumors and mammary gland tumors are the first two most common
tumors reported in many studies (Brodey 1970, Finnie and Bostock 1979, Kasorndorkbua 2008, Mamom 2009, Rungsipipat et al. 2003). Different geographic location, breed popularity and environmental conditions may play an
important role in variation in the studies results. Most of the studies on prevalence of canine tumors did not demonstrate the results regarding to tumor malignancy. The aim of this study was to investigate the occurrence of malignant canine neoplasms in Bangkok during June 2010 to May 2011.
Canine biopsy samples (n= 248) clinically tentative diagnosis of tumor submitted to Mahanakorn Veterinary
Diagnostic Center during June 2010 to May 2011 were retrospectively investigated. All tissue samples were
reexamined histologically using H&E stained slides and additionally toluidine blue stained in some cases. Histopathological diagnosis was performed according to guideline for histological classification of tumor in domestic
animals edited by World Health Organization (WHO) (Goldschmitdt 1998, Hendrik 1998, Misdorp 1999). All data
were summarized and analyzed.
Type of malignant tumors
Malignant mammary gland tumor
Mast cell tumor grade II-III
Hemangiosarcoma
Hemangiopericytoma
Squamous cell carcinoma
Basal cell carcinoma
Malignant melanoma
Leiomyosarcoma
Apocrine carcinoma
Malignant histiocytic tumor
Osteosarcoma
Matrical carcinoma
Ceruminour carcinoma
Lymphangiosarcoma
Undifferentiated carcinoma
Undifferentiated sarcoma
NA = data not available
No. of samples
(n= 88)
26
15
9
8
7
5
4
4
2
2
1
1
1
1
1
1
Percentage
(%)
Average age range
(years)
Tumor size
(cm.)
29.5
17.0
10.2
9.1
8.0
5.7
4.5
4.5
2.3
2.3
1.1
1.1
1.1
1.1
1.1
1.1
8.8 (4-12)
8.3 (3-12)
7.7 (3-10)
9.7 (5-12)
8.6 (4-12)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3-13
3.5-12
1.5-10
3.5-30
2.5-15
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Figure 1: Common canine malignant tumors observed in this study including age affected and tumor size in the first
five most common malignant tumors.
211
From 248 samples, 54% (134/248) were classified as benign tumors, 35.5% (88/248) were malignant tumors
and 10.5% (26/248) were non-neoplastic lesions. Among 88 samples diagnosed as malignant tumors, malignant
mammary gland tumors were the most common malignant tumor (29.5% or 26/88) followed by mast cell tumor
grade II-III (17% or 15/88) and hemangiosarcoma (10.2% or 9/88). The forth to the tenth most common malignant
tumors in dogs were hemangiopericytoma (9.1% or 8/88), squamous cell carcinoma (8% or 7/88), basal cell
carcinoma (5.7% or 5/88), malignant melanoma (4.5% or 4/188), leiomyosarcoma (4.5% or 4/88), apocrine
carcinoma (2.3% or 2/88) and malignant histiocytic tumor (2.3% or 2/88). Other malignant tumors including osteosarcoma, matrical carcinoma, ceruminous carcinoma, lymphangiosarcoma, undifferentiated carcinoma and undifferentiated sarcoma were diagnosed as demonstrated in figure 1. The age range of dogs affected with some common
malignant tumors observed in this study was also shown in figure 1. For malignant mammary gland tumors, the
average age of dogs affected was 8.8 (4-12) years and tubulopapillary carcinoma was the most common malignant
mammary gland tumor observed in this study. The average age of dogs affected with mast cell tumor grade II-III,
hemagiosarcoma, hemangiopericytoma and squamous cell carcinoma were 8.3 (3-12), 7.7 (3-10), 9.7(5-12) and 8.6
(4-12) years respectively. The size of some common malignant tumors observed in this study was also shown in
figure 1. The results of this study might be useful for veterinarians in term of incidence of malignant tumors, in order
to deal with canine oncologic patients.
Acknowledgements
Special thanks were given to Mahanakorn University of Technology for granting this study, all clinics and
animal hospitals submitted the samples to MVDC, all the sixth year veterinary students joining this study and
laboratory scientist, Ms. Siriwan Theptien, for her excellent slide preparation.
References
Brodey RS. 1970. Canine and feline neoplasia. Adv Vet Sci Comp Med. 14:309-354.
Finnie JW and Bostock DE. 1979. Skin neoplasia in dogs. Aus Vet J 55:602-604.
Goldschmidt MH. 1998. Histological classification of epithelial and melanocytic tumors of the skin of domestic
animals. World Health Organization, Geneva. 106 p.
Hendrick MJ. 1998. Histological classification of mesenchymal tumors of skin and soft tissues of domestic animals.
World Health Organization, Geneva. 64 p.
Kasorndokbua, C. 2008. Diagnosis approach of canine cutaneous neoplasia. Proceeding of the 2nd MUT Veterinary
Medicine Annual Conference. MUT Press. Bangkok. pp. 1-6
Mamom T. 2009. Prevalence of Canine Cutaneous, Subcutaneous and Soft Tissue Neoplasms in Eastern Suburban
Areas of Bangkok Metropolis during 1997 to 2009. J Mahanakorn Vet Med. 4(2):32-45.
Misdorp W. 1999. Histological classification of mammary tumors of the dog and the cat. World Health Organization, Geneva. 59 p.
Rungsipipat A, Sunyasootcharee B, Ousawaphalangchai L, Sailasuta A, Thanawongnuwech R. and Teankum K.
Neoplasm of dogs in Bangkok. Thai J Vet Med 33(1):59-66.
212 
CASE REPORT: THE CANINE SYSTEMIC ATHEROSCLEROSIS IN A DOG
Wallaya Phongphaew*, Chareon Thongma* and Wanida Laohasurayothin*
*Department of Veterinary Pathology, Faculty of Veterinary Medicine, Kasetsart University
Introduction
Atherosclerosis is an important cardiovascular disease in human that causes severe complications including brain
and cardiac infarction as well as gangrene of extremities (Kumar et al., 2009). The pathogenesis of antherosclerosis
involves hyperlipidemia following metabolic syndrome and malnutrition. Canine atherosclerosis is rare in frequent,
however, it was shown to be associated with hypothyroidism in dogs (McGavin and Zachary, 2007).
Case history
The six year-old, male cross breed dog was presented to the Veterinary teaching hospital at Kasetsart University with
these following clinical signs; paresis, cyanosis of left forelimb and right hindlimb, and no femoral pulse could be
evaluated. During hospitalization, the dog had undergone severe respiratory distress and eventually died.
Necropsy findings
At necropsy, the dog was obese and exhibited large amount of subcutaneous and abdominal fats. The artery walls
were generalized-thickened and either lost elasticity or hardened. The affected arteries were presented in creamy or
yellowish plaques. The affected coronary arteries were prominent and appeared cord-like with thickened walls (Fig.
1A). The arterial lumens were narrowed and occlusions were found in the right femoral artery and left brachial artery. Thyroid glands were found with difficultly, as thyroid glands were bilaterally atrophied. Parathyroid glands
were located on adipose tissue (Fig. 1B). The lungs were consolidated and the trachea was filled with frothy exudates. Liver was markedly fragile, enlarged, and slightly pale-brownish in color. Both kidneys had rough surface,
were harden, and gave gritty feeling. Arcuate arteries were engorged and had thickened walls.
Figure 1: Prominent cord-like structure of coronary arteries (A). The parathyroid gland (arrow
head) was found on adipose tissue (B).
Histopathological findings
The principal lesions were found at the tunica intima and tunica media of arterial walls. The affected arteries demonstrated intima thickening, and accumulation of artheroma that composed of lipid, fibrous tissue and lipid-laden macrophages (Fig. 2A). In tunica media, shards of glass-like appearance were exhibited, suggesting cholesterol clefts.
Smooth muscle cells accumulated fat droplets within their cytoplasms. The thyroid follicles were absent and replaced with fibrous tissue, adipose tissues, and lymphocyte infiltrations, suggesting severelymphocytic thyroiditis
(Fig. 2B).
213
Figure 2: Accumulation of lipid in the intimal wall of affected arteries with infiltration of lipid-laden
macrophages (A), H&E, 60X. Severe lymphocytic thyroiditis, infiltrated lymphocytic
cells, thyroid follicles were absent and replaced with fibrous and adipose tissues. P: parathyroid gland. (B), H&E, 10X
Discussion and conclusion
According to necropsy and histopathological evaluation, the dog was diagnosed as the canine systemic atherosclerosis and severe lymphocytic thyroiditis. Lymphocytic thyrioditis is an autoimmune disorders that having to produce
immunoglobulin against self-thyroglobulin or self-thyroperoxidase (Gosselin et al., 1982). The affected thyroid follicles would be severely damaged by autoimmunity and that resulted in hypothyroidism (Benjamin et al., 1996;
Gosselin et al., 1982). Lymphocytic thyroiditis could possibly cause systemic atherosclerosis through initiating hyperlipidemia. As thyroid hormone is known to be important for many metabolic pathways, Thyroxin plays a major
role in lipid metabolism by enhancing activity of lipoprotein lipase (LPL). The LPL hydrolyzes triglycerides in the
chylomicrons to long-chain fatty acids and glycerol, to be readily uptake by cells (Xenoulis and Steiner, 2010; Johnson, 2005). Therefore LDL deficiency can lead hyperlipidemia. In such case, free cholesterols and triglycerides that
retained in blood vessels can then be deposited in tunica intima and tunica media of arterial walls, subsequently lead
to thickening of arterial walls and narrowing of arterial lumens. Clinical signs of ischemia can be noted when arteries
are completely occluded. In this dog, occlusions of right-femoral and left-brachial arteries were found to be remarkable from necropsy and that directly associated with paresis of the left fore-limb and right hind-limb.
Reference
Benjamin S, Stephen L, Hamilton B, Saunders W, Lee a, Angleton G and Mallinckrodt C. 1996. Associations between Lymphocytic Thyroiditis, Hypothyroidism, and Thyroid Neoplasia in Beagles. Vet Pathol 33:486494.
Gosselin S, Capen C, Martin S and Krakowka S. 1982. Autoimmune lymphocytic thyroiditis in dog. Elsevier Scientific Publishing Company 3:185-201.
Johnson M. 2005. Hyperlipidemia disorders in dogs. [Online available]. www.compendiumVet.com. (cited 2011
Aug 1). 361-370.
Kumar V, Abbas A, Fausto N and Aster J. 2009. Robbins and Cotran Pathologic basis of diseases. 8 th. Saunders.
Elsevier Inc. p345
McGavin M.D and Zachary J.F. 2007. Pathologic basis of veterinary diseases. 4th. Mosby Inc. p599-600, 721.
Xenoulis P and Steiner J. 2010. Lipid metabolism and hyperlipidemia in dogs. j.tvj. 183:12-21.
214 
INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) & EPIDERMAL GROWTH FACTOR (EGF) IMPROVE
THE DEVELOPMENTAL COMPETENCE OF FELINE EMBRYOS CULTURED SINGLY
Chommanart Thongkittidilok, Thanida Sananmuang, Theerawat Tharasanit, Mongkol Techakumphu*
Chulalongkorn University, Thailand * Corresponding author
Introduction
Single embryo culture has been used as a model to study paracrine effect on developmental competence of
mammalian embryos. In parallel, this technique can also be applicable for embryo culture when the number of embryos are limited (i.e. in the case of unexpected death of wild animal). A supplementation of growth factor such as
IGF-I in culture media help to increase the blastocyst rate and its quality (Neira et al., 2010; Shabankareh and Zandi, 2010; Herrler et al., 1998). Additionally, Epidermal growth factor (EGF) also plays role in mitogenic and cell
differentiation, thus resulting in the increase of blastocyst rate and also total cell number. (Sirisathien et al., 2003;
Shabankareh and Zandi, 2010; Harvey and Kaye, 1992; Paria and Dey, 1990). Moreover, there was a report indicated that a combination treatment of EGF and IGF-I stimulated the cumulus cell expansion, the metabolism of pyruvate and glutamine, and the nuclear maturation in cumulus-enclosed bovine oocytes in vitro (Rieger et al., 1998).
This study aimed at determining the effect of growth factors (IGF-1 and EGF) on developmental competence of feline embryo cultured singly.
Cumulus oocyte complexes were matured and fertilized in vitro with frozen-thawed semen (day 0= day of
fertilization). On day 2, cleaved embryos were randomly assigned to culture (50 µL droplet/ embryo) supplemented
with growth factors as follows: (1) control, (2) IGF-1 25 ng/mL, (3) IGF-1 50 ng/mL), (4) EGF 5 ng/mL, (5) IGF-1
25 ng/mL plus EGF 5 ng/mL, and (6) IGF-1 50 ng/mL plus EGF 5 ng/mL. Developmental competence was assessed
by means of morula and blastocyst formation rates.
A supplementation of IGF-1 at concentration of 25 and 50 ng/mL and EGF at 5 ng/mL significantly improved blastocyst rates (59.68, 62, and 56.14%, respectively) compared with control (40%) (P<0.05), however, a
combination of these two growth factors (IGF25ng/mL+EGF5ng/mL and IGF 50ng/mL+EGF 5ng/mL) adversely
affected the blastocyst development. (42.65 and 32.39%, respectively). It is concluded that either EGF or IGF-I independently enhances feline preimplantation embryos development. In addition, data accumulated from our studies
indicates that the IGF-1 and EGF promote ‘single embryo’ developmental competence similar to the results of
‘group embryo’ culture, suggesting an important role of paracrine/autocrine on feline embryos development.
215
Acknowledgements
This study was financially supported by the Centenary Academic Development Project, Chulalongkorn University and Higher Education Research Promotion and National Research University Project of Thailand, Office of
the Higher Education Commission (HR1166I) and RGJ-PhD grant.
References
Harvey MB, Kaye PL.1992. Insulin-like growth factor-1 stimulates growth of mouse preimplantation embryos in
vitro. Mol Reprod Dev. :31:195-9.
Herrler A, Krusche CA, Beier HM. 1998. Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis. Biol Reprod. 59:1302-10.
Neira JA, Tainturier D, Peña MA, Martal J. 2010. Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GMCSF, and LIF on the development of bovine embryos produced in vitro. Theriogenology. 73:595-604.
Rieger D, Luciano AM, Modina S, Pocar P, Lauria A. andGandolfi, F. 1998. The effects of epidermal growth factor
and insulin-like growth factor 1 on the metabolic activity, nuclear maturation and subsequent development
of cattle oocytes in vitro. J. Reprod Fertil. 112:123-30.
Paria BC, Dey SK. 1990. Preimplantation embryo development in vitro: cooperative interactions among embryos
and role of growth factors. Proc Natl Acad Sci U S A. 87:4756-60.
Shabankareh HK, Zandi M. 2010. Developmental potential of sheep oocytes cultured in different maturation media:
effects of epidermal growth factor, insulin-like growth factor I, and cysteamine. Fertil Steril. 94:335-40.
Sirisathien S, Hernandez-Fonseca HJ, Brackett BG. 2003. Influences of epidermal growth factor and insulin-like
growth factor-I on bovine blastocyst development in vitro. Animal Reproduction Science 77 : 21–32.
216 
EXTERNAL AUDITORY CANAL IN POSTMORTEM INTERVAL (PMI)
ESTIMATION USING HIGH PRECISION THERMOCOUPLE: A PILOT STUDY
1
Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia.
Introduction
An important piece of information needed for every forensic investigation is a close-to-accurate estimation of time
lapse since death of a subject (Henssge & Madea, 2005, 2007). Routine postmortem investigation in all medical
practice often requires the estimation of time of death estimation based on known gross postmortem changes (Swift,
2004). Tthese known postmortem changes provide vague non-standard estimates which are insufficient for forensic
investigative purposes. This study aims to observe the dynamics of algor mortis with specific attention to the external auditory canals in a tropical climate. The ear canal is as efficient as the rectum in humans for temperature cooling
models of estimating postmortem interval (Baccino et al, 1996).
Four adult bitches were obtained from the Kuala Lumpur city dog pound, were housed in the Faculty of Veterinary
Medicine Universiti Putra Malaysia dog kennels with appropriate feed and water prior to euthanasia with pentobarbitone (Dolethal® France). Immediately post-euthanasia, thermocouple probes (DE® Germany) connected to a computer (Kaliszan et al, 2005) were inserted into the right and left ear canals. The probes measurements were monitored
for twenty-four hours at thirty seconds intervals. Ambient temperature was measured during the course of the study
to obtain a mean for the period.
Out of the four bitches (eight probes) studied; two had complete readings for the twenty-four hour period (four
probes) because of technical glitches. The average ambient temperature was 30.4oC.
Temperature oC
217
Figure 1: the variations in the ambient temperature plotted with the mean.
Temperature oC
Figure 2: the graphical representation of drop pattern of the ear canal temperature postmortem.
The data extracted fitted a 4th degree polynomial model (r = 0.998 and S.E = 0.184). However, for ease of calculation
and routine use we chose the Multiple Multiplicative Factor Model (r = 0.996, S.E = 0.258) represented by the formulae below:
PMI = d√ [(Tb – ab) / (T – c)]
Where PMI = Postmortem Interval, a, b, c, and d are coefficients of the model (42.29, 514.2, 26.8, and 1.01 respectively).
This organ provides adequate information on algor mortis but requires further in-depth study.
References
Baccino, E, Martin, L.D.S, Schulier, Y, Guilloteau, P, Rhun, M.L, Leglise, J.F, et al. 1996. Outer ear temperature
and time of death. For Sci Int 83:133-146.
Henssge, C, Madea, B. 2004. Estimation of time since death in the early postmortem period. For Sci Int 144:167175.
Henssge, C, Madea, B. 2007. Estimation of time since death. For Sci Int 165:182-184
Kaliszan, M, Hauser, R, Kaliszan, R, Wiczling, P, Buczynski, J, Penkowski, M. 2005. Verification of the exponential model of body temperature decrease after death in pigs. Exp Phys 90(5): 727-738.
Swift, B. 2004. Timing of death. In Rutty, G.N (Ed.). Essentials of autopsy practice: New advances, trends and developments pp 189-213. London: Springer-Verlag London Limited.
218 
SUBCUTANEOUS TISSUE GAS FORMATION, AN INDICATOR OF ELAPSED TIME SINCE DEATH.
1
Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400
UPM Serdang, Malaysia.
*Corresponding author: [email protected]
Introduction
Forensic medicine has gained vital strides in the areas of technical advancements and sophistications in investigation
techniques/approaches (Thali, 2007., Dirnhofer, 2005). Radiography in essence is a common technique found and
used routinely in veterinary establishments for clinical purposes (O’Donnell & Woodford, 2008). Until recently, this
technique has been grossly under-utilized for veterinary pathology purposes especially in postmortem investigations.
The basic process of tissue degeneration and gas production begins immediately after the death of an organism but
progresses at different rates in different tissues (Swift, 2004). The cardiovascular system rich with stagnant blood
tissue at postmortem provides an optimum environment for bacterial colonization and subsequent gas production.
Such gases produced are easily picked up by 2-D radiography (Heng et al, 2008; 2009) to which we hypothesize that
the gas progresses in a manner directly related postmortem interval.
Four healthy adult dogs obtained from the Kuala Lumpur city pound were euthanized with pentobarbital sodium at
60mg/kg (Dolethal® France) following initial standardization and humane handling protocols. Lateral and ventrodorsal radiographs of the thorax were taken at six-hourly intervals for twenty-four hours. Specific technique chart for
such procedure were followed as established for the machine used (Shimadzu® Malaysia). Radiographs were read
for evidence of gas formation and/or accumulation in the subcutaneous tissue. Obtained images were graded 0 – 4,
for severity resulting from gas accumulation and tabulated against known time.
Number of animals
Hours/Severity
0
1
2
3
4
Total
0
4
-
-
-
-
4
6
3
1
-
-
-
4
12
1
3
-
-
-
4
18
-
-
3
1
-
4
24
-
-
-
2
2
4
Figure 1: the progression of severity of gas formation in the subcutaneous tissue of animals
Where the severity codes are interpreted as follows; 0 = Normal, 1 = Mild, 2 = Moderate, 3 = Severe, and 4 = Very
severe.
219
Gas began accumulating especially in the subcutaneous and neck muscle associated blood vessels by progressing
from normal (Figure 2) radiographic consistency to mild at the 12 th hour postmortem in a mild form, then slowly
progressed to very severe generalized subcutaneous emphysema (Figure 3) at the 24 th hour after death (Figures 3 &
4). This finding supports routine observation of gross and progressive swelling of the cutis at postmortem. Gas accumulation and thus rapidity of progression is however, affected by ambient conditions, animal’s body and nutritional
status as well as disease.
Figure 2: the radiograph image of the thorax of a dog at 0-hour postmortem showing normal radiographic
tissue intensities.
Figure 3: the radiograph image of the thorax of a dog at 24 hours postmortem showing severe generalized
subcutaneous emphysema.
References
Dirnhofer, R, Jackowski, C, Vock, P, Potter, K & Thali, MJ. 2006. VIRTOPSY: Minimally invasive imaging-guided
virtual autopsy. Radiographics, 26, 1305-1333.
Heng, HG, Teoh, WT & Sheikh-Omar, AR. 2008. Postmortem abdominal radiographic findings in feline cadavers.
Veterinary Radiology & Ultrasound 49(1), 26-29.
O’Donnel, C, & Woodford, N. 2008. Postmortem radiology – a new subspecialty. Clinical radiology, 63, 1184-1189.
Swift, B. 2004. Timing of death. In Rutty, G.N (Ed.). Essentials of autopsy practice: New advances, trends and developments pp 189-213. London: Springer-Verlag London Limited.
Thali, MJ, Jackowski, C, Oesterhelweg, L, Ross, SG, & Dirnhofer, R. 2007. VIRTOPSY – The Swiss virtual autopsy
approach. Legal medicine, 9, 100-104
220 
POSTMORTEM RADIOGRAPHIC DIAGNOSIS OF RADIO-OPAQUE FOREIGN BODY IN
THE PROXIMAL DUODENUM OF A CANINE CARCASS
Abdulazeez O.I1 and Noordin M.M1*
1
Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400
UPM Serdang, Malaysia.
Introduction and history
An adult 14 kg female carcass of a stray mongrel dog was submitted to the postmortem unit of the University Putra
Malaysia Faculty of Veterinary Medicine for routine necropsy and disposal. The only available history was that the
dog was found dead by the city hall stray dog control team and subsequently submitted the carcass while still warm
and fresh. Routine radiography was instituted (O’Donnell, 2008). External inspection of the carcass revealed an emaciated carcass with bulged eyes, severe pallor of mucus membranes and massive external parasitic infestation composing of lice and ticks, and matted perineum.
Two radio-opaque metallic foreign bodies were observed at the pyloric region of the duodenum crossed at an angle
of approximately forty degrees on right lateral positioning. Right lateral, dorso-ventral and thoracic views of the abdomen were obtained (Donchin, et al, 1994, Donald, 1998, Heng et al, 2008). The foreign bodies were identified as
pins from the image dimensions radiographically. There was no radiographic evidence of abdominal fat, supporting
the emaciated condition of the carcass. Gas formation in the intestinal lumen had already begun.
Postmortem examination
The dog was in very poor body condition with gross evidence of emaciation. On opening, the abdomen there was
little or no gross abdominal fat present the peritoneal sac. Two rusted pins were observed protruding at the gastric
end of the pylorus with a thickened consistency. They were about 2.3 cm and 1 cm respectively positioned at a 40 o
angle from each other.
221
Such foreign body protrusion through the depth of the organ often results into chronic irritation and fibrosis that ends
up with pyloric stenosis and possible tissue adhesion. We suspect that the animal foraged on garbage due to hunger
and inadvertently swallowed the pins. However, there is no concrete evidence as to the cause and manner of death.
Further laboratory analyses are required for to arrive at a definitive diagnosis of the cause and manner of death. Routine radiography is thus a useful pre-necropsy tool in diagnosing foreign bodies in dog carcasses.
References
Donald E.T. Diagnostic veterinary radiology. 1998. 3rd edition Oxford Press pp 35-38.
Heng, HG, Teoh, WT & Sheikh-Omar, AR. 2008. Postmortem abdominal radiographic findings in feline cadavers.
Veterinary Radiology & Ultrasound 49(1), 26-29.
O’Donnel, C, & Woodford, N. 2008. Postmortem radiology – a new subspecialty. Clinical radiology, 63, 1184-1189.
Donchin Y, Rivkind AI, Bar-Ziv J, Hiss J, Almog J, Drescher M. 1994. Utility of postmortem computed tomography
in trauma victims. J. Trauma, 37, 552-555.
222 
RAPID FROZEN OOCYSTS AGAINST CAECAL COCCIDIOSIS IN BROILERS
S. Sangmaneedet1,2*, P. Pata1, N. Phookrongta1, K. Duangprathum 1
1
Department of Pathobiology, Faculty of Veterinary Medicine, Khon Kaen University.
Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University. THAILAND.
*Corresponding author, e-mail: [email protected]
2
Introduction
Eimeria tenella is a protozoan parasite that causes caecal coccidiosis in poultry. The parasites mainly propagate in caecal mucosa leading to haemorrhage, thickening of caecum and death in infected animals (Innes et al.
2006). Prevention of coccidiosis occurs by use of anti-protozoal medication (Sangmaneedet et al. 2003) or vaccine
(Constantinoiu et al. 2008, Garcia et al. 2008). The purpose of this study was to investigate the anti-coccidial efficacy of oral treatment with frozen oocysts in prevention of E. tenella infection in broilers.
Unsporulated oocysts of E. tenella were collected from chickens and incubated in 2.5% potassium dichromate solution at room temperature for 72 hours until sporulation. Sporulated oocysts (SO) were then harvested by
floatation technique, washed, mixed in normal saline and immediately placed at -80C for 96 hours. The frozen oocysts (FO) were orally given to chickens twice at age 1 and 2 weeks old prior to SO inoculation (30,000 oocysts/
chicken) at 3 weeks old. One hundred commercial broilers were divided into 5 groups (A-E) with 4 replicates. Group
A was given 25,000 FO at age 3 weeks old. Group B was given with 5 ppm diclazuril in drinking water for 7 days
prior to SO inoculation. Group C was given 2,500 FO followed by inoculation with SO. Group D was given 25,000
FO and SO. Group E was given only SO. Caecal lesions of all chickens were examined and scored from 0-4 according to Conway and McKenzie method (1991) on day 5 after SO infection.
Table 1 Average caecal lesion score of chickens given frozen oocysts (FO) or diclazuril prior to inoculation with
30,000 E. tenella sporulated oocysts (SO).
Group
A
B
C
D
E
Treatment
25,000 FO
diclazuril,
SO
2,500 FO,
SO
25,000 FO,
SO
SO
Mean
lesion
score+SE
0.6+0.1a
0.9+0.1ab
1.2+0.1b
1.2+0.1b
2.0+0.2c
a,b,c
Different superscripts between means within row indicates significant difference (p<0.05)
Under microscopic observation, there was no morphological change of frozen sporulated oocysts after thawing at
4C. Oocysts, sporocysts and sporozoites were similar in appearance to normal sporulated oocysts. Chickens given
FO at dosage 2,500 or 25,000 oocysts per bird had the same average caecal lesion score (1.2), which were significantly (p<0.05) less than the groups inoculated with only SO (2.0). This indicates that treatment with FO may stimulate a protective
223
mechanism in chickens against sporozoite invasion. Diclazuril, an anti-coccidial chemical, administered for 7 days
before an infection occurred could also diminish severity of E. tenella; mean caecal lesion score was 0.9. Chickens
given 25,000 FO had some caecal lesions indicating that some sporozoites could survive for 96 hours at -80C. In
conclusion, sporulated oocysts of E. tenella rapidly frozen at -80C may be used as a pretreatment to diminish the
severity of coccidiosis.
Acknowledgements
We thank the Faculty of Veterinary Medicine, Khon Kaen University for research funding.
References
Constantinoiu, C.C., Molloy, J.B., Jorgensen, W.K. and Coleman, G.T. (2008). Antibody response against endogenous stages of an attenuated strain of Eimeria tenella. Vet Parasitol, 154, 193-204.
Conway, D.P. and McKenzie, M.E. (1991). Poultry Coccidiosis: Diagnostic and Testing Procedures. 2nd edn. Pfizer Inc. USA. 65 pp.
Garcia, J.L., Guimaraes, Jda, S., Jr., Headley, S.A., Bogado, A.L., Bugni, F.M., Ramalho, D.C and de Souza, L.M.,
(2008). Eimeria tenella: utilization of a nasal vaccine with sporozoite antigens incorporated into Iscom as
protection for broiler breeders against a homologous challenge. Exp Parasitol, 120, 185-190.
Innes, E.A. and Vermeulen, A.N. (2006). Vaccination as a control strategy against the coccidial parasites Eimeria,
Toxoplasma and Neospora. Parasitology, 133 Suppl, S145-168.
Sangmaneedet, S., Nopwinyouwong, S., Wongma, Y., Hinorn, W. and Padjuta, S. (2003). Use of diclazuril (VClazil®) for treatment of caecal coccidiosis in 6-week-old broilers. KKU Vet J, 13(1), 1-8.
224 
EFFECTS OF PELLETING METHODS ON FERTILITY RATE
OF SEMEN FROM THAI NATIVE COCKS
1
Sarawut Sringam *, Adisak Sangkaew1, Prayong Sangsriruang1, Patchanee Sringam2
1
Department of Veterinary Surgery and Theriogenology, and 2Department of Veterinary Physiology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, THAILAND
Introduction
Fertility of poultry spermatozoa following freezing by the pellet method on dry ice has been examined in a
number of studies. Dimethylacetamide (DMA) is the choice of the cryoprotectant for pellet freezing. However, dry
ice may be not comfortable in some area of the study. Method of Tseulitn et al. (1998) dropped fowl semen directly
in liquid nitrogen but the pellet samples were not easy to collect. To find out the materials and methods that can substitute the use of dry ice for freezing semen should be done. In addition, freezing method by using the pre-cooled
metal plate was successfully on ram semen before and was adapted to used in many species. The aim of the present
study was to compare the efficacy of various pelleting methods on the fertilizing ability of Thai native cock semen.
Animals and semen collection
Eight fertile Thai native cocks and 18 hens were selected and maintained under natural lighting and housed
individually in a cage. Feed was provided at 100 gm/day and water was available all time. Semen was collected
twice a week by abdominal massage.
Frozen procedures and fertility test
The pooled semen was diluted 1:1 with Beltsville Poultry Semen Extender (BPSE), gently mixed and adjusted
into the final concentration 1,500 X106 sperm/ml. The diluted semen was cooled within 20 min at 5 °C and then added with 6% DMA and equilibrated for 1 min. Each semen dilution was dropped ( 0.1 ml) into the well of three surfaces ( 1) dry ice, 2) copper sheet (0.1X12X12 cm), 3) stainless steel sheet (0.3X12X12 cm)). The 2 nd and 3rd sheets
were placed on 5 cm above liquid nitrogen vapor. After 5 min, the resulted pellets were stored in liquid nitrogen until
used for fertility test. Six hens and 5 replications were intravaginal inseminated with semen from each pelleting
method. Two pellets were thawed in 0.2 ml BPSE in a 1.5 ml tube at 70 °C for 5-7 sec for the insemination. The fertilization was assessed from the eggs of Days 2 to 3 post-insemination. Fertility rates (fertilized eggsX100/incubated
eggs) were determined by candling eggs on Day 7 of incubation.
225
Table 1 Fertility rates of 3 pelleting methods of semen from Thai native cocks
Pelleting methods
Fertility rate (number of fertiled egg/ total egg set)
Dry ice
42 (21/50)
Copper sheet
41.5(17/41)
Stainless steel sheet
44.7(21/47)
The fertility rates obtained from pelleting semen by stainless steel sheet and copper sheet method were not significantly differences from dry ice method. This indicated that both metal sheet can use for pellet freezing instead of
dry ice. However, the varied on the thickness and type of metal sheet should be studied further.
Acknowledgement
We thank the faculty of Veterinary Medicine, KKU for research funding.
References
Christensen, V.L. 1995. Diluents, dilution, and storage of poultry semen for six hours. In: Bakst MR and Wishart
GJ (Ed). Proc. First International Symposium on the Artificial Insemination of Poultry, Poultry Science
Association, Savoy, IL, pp. 90–106.
Donoghuea AM, Wishart GJ. Storage of poultry semen. Anim Reprod Sci. 62:213-32.
Salamon S.1970. The survival of ram spermatozoa following pellet freezing below -79°C. Aust. J. biol. Sci. 23,
459-68
Tselutin K, Narubina L, Maorodina T, Tur B. 1995. Cryopreservation of poultry semen. Br. Poult. Sci. 36: 805–811.
Tselutin K., Seigneurin F, Blesbois E, 1999. Comparison of Cryoprotectants and Methods of Cryopreservation
of Fowl Spermatozoa. Poult. Sci. 78:586–590
226 
SELECTION OF LACTIC ACID BACTERIA ISOLATED FROM DUCK FOR PROBIOTICS ADJUNCTS
T. Kimprasit1, P. Songjinda2, S. Sukontasing3 and P. Amavisit4*
1
Center for Advanced Studies for Agriculture and Food, KU Institute for Advanced Studies,
2
Department of Farm Resources and Production Medicine,
4
Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine,
3
Faculty of Veterinary Technology, Kasetsart University, THAILAND.
Introduction
Lactic Acid Bacteria (LAB) were found as an important group of benefit bacteria in poultry digestive tract.
LAB alter the intestinal microflora balance, inhibit the growth of harmful bacteria and increase resistance to
infection (Veldman 1992). LAB should survive in the gut and be tolerant to pH in the upper of digestive tract
(Kaushik et al 2009). Particularly probiotics that have inhibitory activity against pathogenic bacteria are benefit for
commercial uses (Zhang et al 2006, Nazef et al 2008 and Taheri et al 2009). The study was to select the potential
LAB from feces of ducks, test their acid and bile tolerance, and their inhibitory activity against pathogenic bacteria.
Furthermore cell surface hydrophobicities of the LAB were also examined.
LAB isolated from ducks’ feces (Anas platyrhynchos) were cultured in de Man Rogosa Sharp (MRS) broth
(Criterion, USA) and streaked onto MRS agar containing 0.5% calcium carbonate (CaCO3) and then were incubated at
370C for 24 h under anaerobic condition. They were characterized by Gram stain, catalase activity and cell morphology. The standard bacterial strains S. aureus ATCC25923 and the pathogenic bacteria including S. aureus,
E. faecium, and S. Typhimurium were chosen for inhibitory activity test. The inhibitory method was modified from
Kizerwetter-Swida and Binek, 2005. The inhibitory activity was scored as positive if a diameter of clear zone around
colony was equal or more than 5 mm.
The test of acid and bile tolerances were performed at pH 2, 3 and 4 with 0.2 N HCl and 3% ox bile. Spread
plating was done immediately (0 h) and 3 h following the method of Jin et al (1998). The survival percentages of LAB
were 3 h survival of LAB (log cfu/ml) x 100 / Initial of LAB (log cfu/ml).
The test for bacterial adhesion to hydrocarbon was modified from Kaushik et al 2009 using cell surface
hydrophobicity assay. The aqueous phase of bacteria grown in MRS broth was measured its absorbance (Abs) at 600
nm spectrophotometry. Percentages of surface hydrophobicity were 100 x [Abs (before mixing) – Abs (after mixing)]/
Abs (before mixing)
Results and Discussion
Of 27 isolated LAB, three isolates (B1-4, B2-4 and B4-2) had strong inhibitory activity and high tolerance
in stomach acidic condition and ox bile (Table 1). According to cell adhesion evaluation using cell surface hydrophobicity test, these isolates had high percentages of surface hydrophobicity at 54.59%, 54.26% and 59.74%, respectively. Theorically, the high percentages of surface hydrophobicity indicated that the bacteria have more chance to
adhere to host mucus because the hydrocarbon is a strong relationship to the epithelium of host gastrointestinal tract
(Garriga et al 1998).
227
Using the in vitro test, three LAB isolates that we have got from the study showed the suitable characteristics
of probiotics. As far as we known, LAB isolated from ducks's feces has not been reported yet. Different to other
domestic poultry, grazing ducks were raised in open areas. The diversity of LAB is expected to be isolated from this
animal species. However further study including sequence analysis and in vivo test should be performed for potential
LAB selection.
Table 1. Lactic acid bacteria (LAB) were examined for acid & bile tolerance test, adhesion ability test and inhibitory
pH 3
pH 4
82.33
78.40
73.13
93.68 94.92
82.17 93.88
93.21 93.39
3% ox bile
77.04
95.74
88.52
54.59
54.26
59.74
12.00±1.00 22.00±0.00
16.00±1.73 21.00±1.00
16.00±1.00 25.00±1.73
S. aureus
ATCC
25923
B1-4
B2-4
B4-2
pH 2
S. aureus
LAB
NO.
Inhibitory activity
(mm)d
S. Typhimurium
Bile tolerance
(%)b
E. faecium
Acid tolerance
(%)a
Hydrophobicityc
(%)
activity test.
10.00±1.00
16.00±1.00
27.00±1.00
13.00±1.73
21.00±1.00
22.00±1.00
a
Survival percentages of the isolates under stress condition
The percentages of attached isolates to 3% ox bile
c
The adhesion ability of the isolates to hydrocarbon (n-hexadecane)
d
The diameter of growth inhibition zone, shown as mean ± sd of 3 replicate experiments.
b
Acknowledgments
The study was supported by CASAF-A-01 grant, Kasetsart University.
References
Garriga M., Pascual M., Monford J.M. and Hugus M. (1998). Selection of lactobacilli for chicken probiotic adjuncts.
J Appl Microbiol. 84, 125-132.
Jin L Z, Ho Y W, Abdullah N and Jalaludin S (1998). Acid and bile tolerlance of Lactobacillus isolated from
chicken intestine. Lett Appl Microbiol. 27, 183-185.
Kizerwetter-Swida M and Binek M (2005). Selection of potentially probiotic Lactobacillus strains toward their
inhibitory activity against poultry enteropathogenic bacteria. Pol J Microbiol. 54, 287-294.
Kaushik J K, Kumar A, Duary R K, Mohanty A K, Grover S and Batish V K (2009). Functional and probiotic
attributes of an indigenous isolate of Lactobacillus plantarum. Plos one. 4, 1-11.
Nazef L, Belguesmia Y, Tani A, Prevost H and Drider D (2008). Identification of lactic acid bacteria from poultry
feces: Evidence on anti-Campylobacter and anti-Listeria activities. Poult Sci. 87, 329-334.
Taheri H, Tabandeh F, Moravej H, Zaghari M, Shivazad M and Shariati P (2009). Potential probiotic of
Lactobacillus johnsonii LT171 for chicken nutrition. Afr J Biotechnol. 8, 5833-5837.
Veldman A 1992. Probiotics. Tijdschr Diergeneeskd. 117, 345-348.
Zhang G, Ma L and Doyle M P 2006. Potential competitive exclusion bacteria from poultry inhibitory to
Campylobacter jejuni and Salmonella. J Food Prot. 70, 867-873.
228 
ANTIBODY RESPONSE TO ACTINOBACILLUS PLEUROPNEUMONIAE IN CHRONICALLY
INFECTED PIG FARMS IN THAILAND
Sukolapa Chiarasumran1, Sureerat Lopiroon1, Trisukhon Phongsayoikham1, Suraphan Boonyawatana2,
Sukuma Samngamnim3, Suphot Wattanaphansak3, Pornchalit Assavacheep3,*
1
Thai Foods Feed Mills Co., Ltd., 2 Intervet (Thailand) Ltd., 3 Department of Veterinary Medicine,
Faculty of Veterinary Science, Chulalongkorn University.* Corresponding author
Introduction
Porcine contagious pleuropneumonia is caused by Actinobacillus pleuropneumoniae (Gottschalk and Taylor,
2006). The production and economic impacts are thought to be a consequence of acute disease outbreak (Holmgren
et al., 1999, Assavacheep et al., 2003). Following such outbreak, it becomes a chronic and silent health threatening
disease to the intensive Thai pig producing industry. In the swine herd with chronic pleuropneumonia, bacterial isolation is often difficult leading to failure to identify disease carrier. To effective control and prevent the disease in
chronically infected herd, monitoring of antibody response is considered to be one of several useful strategies
(Beskow et al., 1989). Therefore, this investigation was conducted to detect serum antibody against A. pleuropneumoniae in three chronically infected pig farms in Western part of Thailand.
Three swine herds in the Western region of Thailand with history of pleuropneumonia lesion in fattening
pigs at slaughter house were chosen for present study. The serotyping was previously determined prior to the investigation. Predominant serotypes 11 and 2 were detected in all farms. An in-house indirect enzyme-linked immunosorbent assay using mixed boiled whole cell antigen was prepared to detect serum antibody from a total of 210 blood
samples at 8, 12, 16, 20 and 24 weeks of age. The antigen preparation method was according to (Gunnarsson, 1979).
The cut-off value determined by a ratio of optical density value of sample to positive was established as 0.3. Percentage of seropositive pigs was calculated at each sampling age.
The average ELISA-positive pigs at each sampling age were 7.0±9.5, 8.0±5.2, 35.0±37.2, 24.0±17.8, and
41.0±33.2%, respectively. Number of serological reactors in each farm was gradually increased during weeks 8-12 of
age. Increase of seropositive pigs was revealed in two occasions during the observation. The first peak was obviously found at 16 weeks of age in two out of three farms, suggesting an immune response to either vaccination, or early
A. pleuropneumoniae infection. The latter antibody response, which indicated a natural infection, was shown at 24
weeks of age in all farms (Figure 1). This study demonstrated a current pattern of antibody responses to A. pleuropneumoniae in chronic infected pig farms in Thailand, which can be implemented together with antimicrobial and
vaccine usage for control and prevention program.
229
Acknowledgements
This investigation was conducted at the Large Animal Hospital and Veterinary Diagnostic Laboratory-NP,
Chulalongkorn University, Nakhonpathom, Thailand.
References
Assavacheep, P., Persson, M., Luengyosluechakul, S., Watanaphansak, S., Laohasinnarong, D., Pungkhun, P. &
Wallgren, P. 2003. Actinobacillus pleuropneumoniae in Thai pig herds. Prevalence of serum antibodies and
relation to performance. J Vet Med B Infect Dis Vet Public Health, 50, 390-5.
Beskow, P., Soderlind, O. & Thafvelin, B. 1989. Actinobacillus (haemophilus) pleuropneumoniae infections in
swine: serological investigations and vaccination trials in combination with environmental improvements.
Zentralbl Veterinarmed B, 36, 487-94.
Gottschalk, M. & Taylor, D. J. 2006. Actinobacillus pleuroneumoniae. In: straw, B. E., Zimmerman, J. J., D'allaire,
S. & Taylor, D. J. (eds.) Diseases of swine. 9th ed. Iowa: blackwell publishing.
Gunnarsson, a. 1979. Evaluation of different antigens in the complement-fixation test for diagnosis of Haemophilus
pleuropneumoniae (parahaemolyticus) infections in swine. Am J Vet Res, 40, 1564-7.
Holmgren, N., Lundeheim, N. & Wallgren, P. 1999. Infections with Mycoplasma hyopneumoniae and Actinobacillus
pleuropneumoniae in fattening pigs. Influence of piglet production systems and influence on production
parameters. Zentralbl Veterinarmed B, 46, 535-44.
230 
SURVIVAL OF ACTINOBACILLUS PLEUROPNEUMONIAE UNDER CONTROLLED LABORATORY
CONDITIONS
Kornkaew Thongtaeng1, Miruntee Penroj1, Walaitip Wongthai1, Siriyaporn Thongwatchara1,
Sukuma Samngamnim2, Supot Wattanaphansak2, Pornchalit Assavacheep2*
1
Final Year Veterinary Students, 2 Department of Veterinary Medicine, Faculty of Veterinary Science,
Chulalongkorn University, THAILAND * Corresponding author
Introduction
Actinobacillus pleuropneumoniae is a causative agent of porcine contagious pleuropneumonia (Gottschalk,
2006). The disease is transmitted via direct contact and aerosol containing nasal discharge from respiratory tract of
A. pleuropneumoniae infected pigs (Shope, 1964; Velthuis, 2003). Once the bacterium is outside pig body, it tends
to survive for a period, depending on the harshness of environment (Gottschalk, 2006; del Rio, 2003; Diaz, 2006).
Investigation on the survival of A. pleuropneumoniae under controlled environment conditions would be basic
information for disease control and prevention on farm. Therefore, the objective was to investigate the viability of
Actinobacillus pleuropneumoniae under controlled laboratory environmental conditions.
A. pleuropneumoniae serotype 1 strain 4074 (Shope, 1964) was overnight cultured on TYE medium at 37°C
under 5% of CO2 condition for 24 hours (Garside, 2002). A loop of 2-3 colonies was suspended in 1 ml HEPES
saline or sterile water where appropriate. To determine the effect of environment, bacterial suspensions were kept
under different conditions including (1) temperature: stored at 4, 28, and 37°C, (2) salinity: suspended in HEPES
saline and sterile distilled water at room temperature, (3) humidity and synthetic materials: suspension on synthetic
materials (0.5X0.5 cm pieces of coverall cloth and rubber boots), and kept under dry, natural prevailing and saturated wet humidity conditions. The bacterial suspensions were collected every hour on day 1 and daily from days 2-3.
The samples from each time point were immediately submitted for colony count on agar plate. The viability was
calculated from 2 occasions of each experiment.
Viability of A. pleuropneumoniae outside pig body was examined under different laboratory conditions.
Overall A. pleuropneumoniae survival lasted for more than 48 hours with 107 CFU/ml at the beginning. Differences
in survival rates were observed under varying conditions. Regardless to salinity, at 4, 28 and 37°C the viability rates
were 12-48, 7-24, 4-6 hours, respectively. A. pleuropneumoniae survived better in HEPES saline (6 to >48 hours)
than distilled water (4-12 hours). Comparison of survival rate on material surface revealed that A. pleuropneumoniae remained viable on cloth (13-48 hours) longer than rubber boot (8-48 hours).
Survival rate was shortest under natural prevailing humidity (8-12 hours), followed by dry and wet conditions (24 to >48 hours), which are more consistent in humidity.
Under controlled laboratory conditions, A. pleuropneumoniae survived for more than 48 hours. Survival
rate of A. pleuropneumoniae were prolonged in lower temperature, presence of saline, under consistent humidity,
and on absorbable materials.
231
Acknowledgements
We sincerely thank the special project for final year veterinary student,Chulalongkorn University for partial
financial support, The Large Animal Hospital, Chulalongkorn University, Nakhonpathom for facility support.
References
del Rio, M.L., Gutierrez, B., Gutierrez, C.B., Monter, J.L., Rodriguez Ferri, E.F. (2003). Evaluation of survival of
Actinobacillus pleuropneumoniae and Haemophilus parasuis in four liquid media and two swab specimen
transport systems. American Journal of Veterinary Research, 64, 1176-1180.
Diaz, B., Schulze-Makuch, D. (2006). Microbial survival rates of Escherichia coli and Deinococcus radiodurans
under low temperature, low pressure, and UV-Irradiation conditions, and their relevance to possible Martian
life. Astrobiology, 6, 332-347.
Garside, L.H., Collins, M., Langford, P.R., Rycroft, A.N. (2002). Actinobacillus pleuropneumoniae serotype 1
carrying the defined aroA mutation is fully avirulent in the pig. Research in Veterinary Science, 72, 163-167.
Gottschalk, M. and Taylor, D.J. (2006). Actinobacillus pleuroneumoniae, In: Diseases of Swine, 9th edn, Eds. Straw,
B.E., Zimmerman, J.J., D'Allaire, S., Taylor, D.J. Blackwell Publishing, Iowa, pp. 563-576.
Shope, R.E. (1964). Porcine Contagious Pleuropneumonia. I. Experimental Transmission, Etiology, and Pathology.
Journal of Experimental Medicine, 119, 357-368.
Velthuis, A.G., De Jong, M.C., Kamp, E.M., Stockhofe, N., Verheijden, J.H. (2003). Design and analysis of an
Actinobacillus pleuropneumoniae transmission experiment. Preventive Veterinary Medicine, 60, 53-68.
232 
INTERPRETATION THE RESULTS OF THE COMMERCIAL ELISA H1N1 AND H3N2 KITS AND HEMAGGLUTINATION INHIBITION ASSAY USING LOCAL THAI SIV H1 AND H3 VIRUS ANTIGENS
Donruethai Sreta1,3, Sanipa suradhat1,2, Pravina Kitikoon1 , Alongkorn Amonsin1,2
and Roongroje Thanawongnuwech1,2*
1
Emerging and Re-emerging Infectious Disease in Animals, Research Unit, Faculty of Veterinary Science
2
Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
3
Faculty of Veterinary Medicine, Rajamankala University of Technology Tawanok, Chonburi, Thailand
*
Corresponding author
Introduction
For serological diagnosis of swine influenza virus (SIV) infection, hemagglutination inhibition (HI) test is a
gold standard recommended by WHO, but it requires a suitable representative virus from the local circulating strain
used as the HI virus antigen. In general, SIV is genetically unstable. Many sub-clusters have arisen within the different H1N1 and H3N2 SIV subtypes (Ma et al., 2009). For example historic data indicate that the H1N1 SIV
comprises of two major lineages; the North American- and Eurasian-swine lineages (Brown, 2000). Interestingly,
Genome of Thai SIV subtype H1N1 and H1N2 viruses are mix with both classic and Eurasian swine lineages.
While, subtype H3N2 belong to Human-like swine lineage. In addition, Thai SIV subtype H1N2 was reassorted
between H1N1 and H3N2 (Takemae et al., 2008; Sreta et al., 2009). Although, ELISA test is practical as it provides
rapid screening for the presence of SIV antibodies since the method can test 92 serum samples per plate in less than
2 hours (Webster et al., 2002). The commercial IDEXX ELISA H1N1 and H3N2 kits are used separately between
subtypes and are available worldwide. However, their capacity to detect antibodies of the Thai SIVs has not yet
been investigated. In this study, two commercial ELISA tests (HerdChek H1N1 and H3N2 ELISA, Idexx Laboratories, Westbrook, Maine) and the established in-house HI test using local Thai SIV isolates were evaluated with the
swine sera collected from pigs known to have specific H1N1 and H3N2 SIVs circulating in the selected farms from
the 4 highest pig population provinces of Thailand to compare the findings of the established HI test with the SIV
commercial ELISA test kits for H1 and H3.
During June 2008 to May 2009, 850 pig sera were cross-sectionally collected from pigs of various age
groups (gilts, sows, finishers, growers and weaning pigs) in those four provinces located in the highest pig density
in Thailand (http://www.dld.go.th/ict/yearly/yearly/50stock.50html). Total 12 pig farms facing with respiratory
symptoms from Ratchaburi, NakornPathom, Chonburi and Chachoengsao were obtained. The pig serum were
grouped based on known HI antibody titers against H1N1 (negative for H3N2); 0, 10, 20, 40, 80, 160, 320 and
≥640 were evaluated by a commercial ELISA H1N1 kit (HerdChek H1N1ELISA; Idexx Laboratories, Westbrook,
Maine). Similarly, sera of known HI antibody titers against H3N2 (negative for H1N1); 0, 10, 20, 40, 80, 160, 320
and ≥640 were evaluated by a commercial ELISA H3N2 kit (HerdChek H1N1ELISA; Idexx Laboratories, Westbrook, Maine). Ninety one pig sera were tested for H1N1 and 85 were tested for H3N2 according to the protocol
recommended by the manufacturer. All pig sera were stored in minus 20°C until used.
233
Known HI titers of pig sera were tested with the HerdChek H1N1 ELISA kit and in-house HI test. The twoby-two table of those tests was constructed and used for evaluation of sensitivity, specificity, false negative and false
positive (Table 1). The results demonstrated that both sensitivity and specificity of the commercial ELISA test kits
were quit low when compared with the HI test using Thai SIV H1 and H3 antigens.
Table 1. Results of HerdChek H1N1 and H3N2 ELISA kits compared with HI tests
ELISA H1N1
ELISA H3N2
Sensitivity (%)
89.36
70.83
Specificity (%)
77.27
91.89
False negative (%)
8.5
29.16
False positive (%)
22.7
8.11
The high sensitivity is related to the antigens used in both tests of H1N1due to sharing the same origin of
swine H1, classical swine H1in ELISA and local Thai H1N1 (Takemae et al., 2008; Sreta et al., 2009). However, the
swine H3 viruses found in both the United State and Thailand are human-like but both tests yielded low sensitivity
(70.83%). It should be noted that those H3N2 viruses are genetically different. Therefore, evaluation of the commercial test kits from other countries is necessary before use in our country. We suggest performing of the serological
diagnosis of Thai SIV using circulating SIV antigens and should be re-evaluated with genetic monitoring. In addition, specific Thai SIV diagnosis manual, suitable protocol for prevention and control, and community response
when facing with the disease outbreak should be performed and rehearsed routinely.
References
Brown, I.H. 2000. The epidemiology and evolution of influenza viruses in pigs. Vet. Microbiol. 74(1-2):29- 46.
Ma, W., Lager, K.M., Vincent, A.L., Janke, B.H., Gramer, M.R. and Richt, J.A. 2009. The Role of Swine in the
Generation of Novel Influenza Viruses. Zoonoses Public Hlth. 56(6-7):326-337.
Sreta, D., Kedkovid, R., Tuamsang, S., Kitikoon, P. and Thanawongnuwech, R. 2009. Pathogenesis of swine influ
enza virus (Thai isolates) in weanling pigs: an experimental trial. Virol. J. 6:34.
Takemae, N., Parchariyanon, S., Damrongwatanapokin, S., Uchida, Y., Ruttanapumma, R., Watanabe, C.,
Yamaguchi, S. and Saito, T. 2008. Genetic diversity of swine influenza viruses isolated from pigs during 2000
to 2005 in Thailand. Influenza Other. Respi. Viruses. 2(5):181-189.
Webster, R., Cox, N. and Stohi, K. 2002. WHO Manual on Animal Influenza Diagnosis and Surveillance. World
Health Organization. 15-67.
234 
DETECTION OF MYCOPLASMA HYORHINIS FROM LUNG SAMPLES OF SEROSITIS
NURSERY PIGS
Nantawan Tangwatcharapong1 Panithan Jessadakarn1 Wiyada Winyuchonchareon1 and
Athipoo Nuntaprasert 1 *
1
*
Corresponding author, E-mail: [email protected]
Introduction
Mycoplasma hyorhinis (Mhyorhinis) plays an important role of chronic secondary infection as enzootic
pneumonia in pig. This pathogen infects nursery pigs with clinical and pathologic findings of serositis (peritonitis,
pleuritis and pericarditis) (1) and is responsible for increased susceptibility of pigs to be infected with Haemophilus
parasuis (Hps) (2). Recently, there are many PRRS outbreaks in Thailand that causes high mortality rate in nursery
pigs and usually found the impact of a coinfection between Hps and Mhyorhinis infection. Therefore, this study is
focused on detection of 16s rRNA genes of Mhyorhinis from field lung samples of serositis nursery pigs using
Polymerase Chain Reaction (PCR) which is differential diagnosis of Mhyorhinis from other Mycoplasmas (2).
Lung samples (200 mg) of five serositis nursery pig were obtained from five PRRS infected farms in
Ratchaburi province on April-August 2011. All lung samples were submitted to Chulalongkorn University
Veterinary Diagnostic Laboratory to confirm PRRSV US strain using RT-PCR and were Hps negative results using
conventional bacterial culture. The appropriated lung tissue lesions were collected. The samples and positive
Mycoplasma hyopneumoniae (Mhop) antigen vaccines were extracted total DNA using Genomic DNA Mini Kit
(Geneaid, Taiwan). The two specific primers of 16s rRNA gene are forward primer (5' TAT CGC ATG ATG AGT
AAT AG 3') and reverse primer (5' GCT GCG TTA GTG AAA TTA T 3’) which based on sequences from GenBank
accession number CP002170. Mhyorhinis-specific PCR technique (3) was amplified using Taq DNA polymerase
(Fermentas, Canada). Initial denaturation at 94 °C for 2 min was followed by 34 cycles at 94 °C for 20 s, at 50 °C for
10 s, and at 72 °C for 50 s; a final elongation occurred at 72 °C for 5 min. The PCR products were analyzed on 1.5 %
agarose gel electrophoresis and readily visible by UV transillumination with an ethidium bromide-stained gel.
The 16s rRNA genes of Mhyorhinis from five field outbreak lung samples were successfully amplified by
using this PCR technique and the resulting PCR products were 700 bp. (Fig.1 and Fig. 2) The sequence data of 16s
rRNA genes of Mhyorhinis from field isolates is on the process. In this study, this PCR technique of detection of 16s
rRNA genes will be useful for differentiation among other Mycoplasmas. Furthermore, this technique will be developed for detection of this pathogen from other clinical samples such as saliva, nasal and tonsil swabs. This study
will be beneficial for control of this disease such as autogenous vaccine development.
235
Fig. 1 PCR analysis of 16s rRNA genes of Mhyorhinis. Lane M, 100 bp DNA Ladder (Invitrogen, USA). Lane 1,
negative control. Lane 2, positive Mhyorhinis. Lane 3 and 4, Mhop DNA .
Fig. 2 PCR against Mhyorhinis isolates. Lane M, 100 bp DNA Ladder. Lane 1, positive control. Lane 2, negative
control. Lane 3, 4, 5, 6, and 7, Mhyorhinis field isolates from PRRS infected farm 1, 2, 3, 4 and 5, respectively.
References
Oliveira and Pijoan. 2004. Vet Microbiol 99:1-12.
Kobayashi et al. 1996. J Vet Med Sci. Feb. 58(2):109-113.
Stakenborg et al. 2006. Vet Research Communications. 30: 239-249.
236 
EFFECTS OF DIETARY CHITO-OLIGOSACCHARIDE ADDITIVES ON GROWTH PERFORMANCE AND ILEAL NUTRIENT DIGESTIBILITY IN WEANING PIGS
S. Suthongsa1, B. Thongsong2*, S. Kalandakanond-Thongsong3, R. Pichyangkura4
1
Program in Animal Nutrition, Department of Animal Husbandry, 2Department of Animal Husbandry,
3
Department of Veterinary Physiology, Faculty of Veterinary Science,
4
Department of Biochemistry, Faculty of Science, Chulalongkorn University, THAILAND.
*Corresponding author: email: [email protected] Tel:+662-218-9685, Fax: +662-251-2582
Introduction
Pig is one of Thailand’s most important livestock. The demand of pork in the market has steadily increased
annually. Therefore, the pork production industry aims to increase the production to its optimum capacity. For instant, the reduction of time needs to wean piglets from milk as well as other activities in the production processes.
The down side that followed is the problem of health and high mortality rate of piglets by weaning, feed and feeding
change, social stresses and so on. Antibiotics were then introduced to the industry to reduce the problem of lost in
the production cycle. This brings into in consideration another problem which is the residual antibiotics in the animal
and meat product, which elevates the health risk in both the animal and consumers of the meat product. Consequently, the pig farms have turned to a natural and holistic approach. Furthermore, if natural and waste product from other agricultural industry can be used and applied to increase the performance of pigs; for example, chitin-chitosan
(Ravi Kumar, 2000) which has very good biological properties, non-toxic and biodegradability (Hejazi and Amiji.,
2003 and Liu et al., 2008), it would be of great interest. In this study, we hypothesized that optimal dosage of
chitooligosaccharide (COS) derived from chemical hydrolysis of chitosan can affect promoting growth performance
and digestibility of nutrients in weaning pigs compared to control and antibiotic additive group.
Animal procedures used in this study were approved by the Animal Care and Use committee, Faculty of
Veterinary Science, Chulalongkorn University. Fifty weaned pigs (Duroc x Large white x Landrace) at 21 day of age
were divided into 5 groups: receiving basal diet (control) and COS additive with three doses of 75, 150 and 225 mg/
kg of diet, respectively, and an antibiotic additive (Lincomycin, 110 mg/kg of diet) in basal diet. During 56 days of
experimental period, All piglets were housed individually in conventional nursery housing and freely accessed to
feed and water ad libitum. Individual feed intake and body weight parameter were determined daily and weekly, respectively to calculate body weight gain, average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR). Blood samples from 3 piglets from each group were collected via anterior vena cava puncture
every two weeks to determine blood urea nitrogen (BUN) and total serum protein (TP) (Tang et al., 2005). In addition, to collect ileal and stomach content, 3 piglets from each group were randomized and euthanized on day 28 th and
56 th of experimental period to determine chromic oxide as an indicator of nutrient digestibility (Huang et al., 2005)
and to measure pH in the stomach content.
237
The results showed that the weaning pigs fed the 150 mg/kg COS had significantly increased (p<0.05) in
body weight gain and average daily gain when compared to the control and the antibiotic group on day 42-49 (7 wk)
and the overall effects were found significant increase during day 28-56 (5-8 wk). The feed conversion ratio had significant improvement (p<0.05) found in comparing between them fed the 150 mg/kg COS and control group on day
21-28 (4 wk) and both during day 0-28 (0-4 wk) and during day 28-56 (5-8 wk). However, the average daily feed
intake of the piglets did not differ among groups throughout experimental period. The pH in stomach content of
weaning pig with COS additive groups trended to be lower than those in control and antibiotic group (p>0.05) at the
1 month (day 28) but the pigs fed the 225 mg/kg COS showed significant decrease (p<0.05) when compared to the
control and antibiotic group at the 2 months (day 56). There was no significant effect on blood urea nitrogen and total serum protein. Furthermore, the ileal nutrient digestibility of the weaning pigs fed the 150 mg/kg COS had significantly increased (p<0.05) in crude protein, fat and calcium compared to those in control group at the day 28 and 56
of experimental period. In conclusion, this present study provided the results of the effects of a chitooligosaccharide
dose at 150 mg/kg of diet that can be applied to substitute the antibiotic to get benefit of growth performance in the
weaning pigs.
Acknowledgements
This research supported by grants from the Office of National Research Council of Thailand (NRCT) and the
90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund).
References
Hejazi, R., Amiji, M. 2003. Chitosan-based gastrointestinal delivery systems. J. Control. Release. 89: 151-165.
Huang, R.L., Yin, Y.L., Wu, G.Y., Zhang, Y.G., Li, T.J., Li, L.L., Li, M.X., Tang, Z.R., Zhang, J., Wang, B., He,
J.H., Nie, X.Z. 2005. Effect of dietary oligochitosan supplementation on ileal digestibility of nutrients and
performance in broilers. J. Poul. Sci. 84: 1383-1388.
Liu, P., Piao, X.S., Kim, S.W., Wang, L., Shen, Y.B., Lee, H.S., Li, S.Y. 2008. Effects of chito-oligosaccharide supplementation on the growth performance, nutrient digestibility, intestinal morphology, and fecal shedding of
Escherichia coli and Lactobacilli in weaning pigs. J. Anim. Sci. 86: 2609-2618.
Ravi Kumar, M.N.V. 2000. A review of chitin and chitosan applications. Reactive and Functional Polymers. 46: 127.
Tang, Z.R., Yin, Y.L., Nyachoti C.M., Huanga, R.L., Li, T.J., Yang, C., Yang, X.J., Gonge, J., Peng, J., Qi, D.S.,
Xing, J.J., Suna, Z.H. and Fan, M.Z. 2005. Effect of dietary supplementation of chitosan and galacto-mannan
-oligosaccharide on serum parameters and the insulin-like growth factor-I mRNA expression in earlyweaned piglets. Domest. Anim. Endocrinol. 28(4): 430-41.
238 
EVIDENCE OF HP-PRRS VIRUS WATER TRANSMISSION IN THAI SWINE FARM
1
Introduction
PRRS virus (PRRSV) is a small, non-enveloped, circular, single-stranded RNA virus, belonging to
genus Arterivirus, which infects swine (1). Three distinct PRRSV strains of US, HP and EU strains have been recently
identified in Thai domestic pig. Up to now, swine PRRS have been detected in serum, plasma, semen, nasal and
rectal swabs (2 and 3). HP-PRRSV appears to be widespread in the asian swine population, and the direct contact route
is considered the main route for viral dissemination and rapid transmission. Recently, studies in PRRS infected farms
have suggested that air, some insects or vehicles outside might contribute to transmission of swine PRRSV (4, 5, 6, 7).
Therefore, the main objective of the present work was to gain further insight on potential route transmission of
PRRSV. Specifically, the role of PRRSV infection in the dense area of raising pigs that use the common natural
source of drinking water. This work was determined by studying whether PRRSV can be survived in drinking water.
Drinking water samples were collected from five different Thai swine herds in Ratchaburi province on
Apil-August 2011. Samples were filtered with a 125 mm filter papers (Whatman, USA) and papers were collected.
Total RNA from filtered paper sample was extracted using a commercial kit according to manufacturer’s instructions
and detected PRRSV using different specific PCR methods. PRRSV viral RNA was amplified by RT-PCR one step
(BioLine, USA). The multiplex PCR which amplified ORF1 and ORF7 was performed with the specific primers that
were described by previous study (8). The PCR products were analyzed on 1.5 % agarose gel electrophoresis and
readily visible by UV transillumination with an ethidium bromide-stained gel. The PCR product sizes of specifically
PRRSV strains are 756 bp (US strain), 666 bp (China strain) and 478 bp (EU strain), respectively.
All five drinking water samples from five Thai swine herd in Ratchaburi province were HP-PRRSV
PCR positive (Fig.1). In this sudy showed that there was the evidence of HP-PRRSV in drinking water of pigs in
Thai swine farms. This finding could be showed that PRRSV may entry and contaminate into pig farm by oral route.
Some study about the oral-transmitted PRRSV pigs by feeding muscle tissue obtained from recently infected pigs (3)
showed that all receiver pigs became viraemic by 6 days post-feeding. However, the transmission and pathogenesis
of PRRSV via drinking water remains to be elucidated machanism. The detection of PRRSV in drinking water route
could provide evidence that PRRSV may enter into pig farms. From this result, the pig farmer should examine the
water supply for pigs in the farm regularly as well as should find the effective methods for treatment of contaminated
water before use in pigs. This study will be useful for control PRRSV as well as to understand the epidemiology of
PRRSV.
239
Figure 1. Electrophoretic analysis of PRRSV. Lane M, 100 bp DNA Ladder (Fermentas, USA). Lane 1, negative
control. Lane 2 (EU), 3 (US), and 4 (China): positive controls. Lane 5, 6 and 7: positive samples from water.
References
1. Snijder, E.J. and Meulenberg, J.J. 1998. J. Gen. Virol. 79: 961-969
2. Darwin L. Reicks et al. 2006. J. Swine Health and Production. 14:258-264
3. Zimmerman, et al. 2006. Diseases of Swine. 9th edition: 387-417
4. FAO, 2011. 5: 1-8
5. An et al. (2010). Vet. Microbiol. 149, 104-112
6. Xu et al. (2010). Virol J. 7, 184
7. Wu et al. (2009). Arch. Virol. 154, 1589-1597 8.Vo Phong Vu Anh Tuan and Athipoo Nuntaprasert, 2011. The
5th, APVS P.38
240 
DETECTION OF APXIV GENE IN A. PLEUROPNEUMONIAE ISOLATES FROM
THAI FATTENING PIGS IN 2011
Vo Phong Vu Anh Tuan1 and Athipoo Nuntaprasert 1*
1
Introduction
Actinobacillus pleuropneumoniae (APP) causes pleuropneumonia which a severe mortal disease that affects
pig of all ages. This pathogen has serious impact on economy, ecology and animal welfare in the swine industry.
Recently, there are reported the outbreak of APP in fattening pigs which PRRS infection. The epidemiological
molecular study of APP serotypes may be useful for medication and vaccination control. Up to now, four different
Apx toxin genes have been found to be produced by the 15 serotypes: ApxI, ApxII, ApxIII and ApxIV (1 and 2).
The apxIVA gene are species-specific and useful for detection all of 15 APP serovars (3). In this study, we use PCR
technique based on ApxIV gene to detect A. pleuropneumoniae isolates from Thai fattening pigs in 2011.
Six fattening swine farms (3,000-5,000 pigs on production) with APP history outbreak from six different
regions of Thailand in 2011 (January-August) were selected (Fig. 1). All studied farms were confirmed US strain
PRRS infected using RT-PCR at Veterinary Diagnostic Lab, Faculty of Veterinary Science, Chulalongkorn University. Lung samples obtained from APP clinical sign pigs (40 % loss from respiratory diseases) after necropsy and
were cultured on chocolate and blood agar with NAD. The plates were incubated at 37oC for 24 hours under 5%
CO2. The DNA was extracted from pure colonies using Total DNA Mini Kit (Geneaid, Taiwan). The PCR was
carried out as previously described (4). The PCR which amplified ApxIV was performed with the specific primers.
The primer 1 (5’AAA GAC CAG ATG GAG GAG GA 3’) and primer 2 (5’GAG CTG AGT ATT TTG GGC GTG
3’) were designed based on sequence from ApxIVA gene (GenBank accession number AF021919). PCR products of
this studied apxIV genes will be categorized by their product patterns into four groups of all 15 serotypes (Han Sang
Yoo et al. , 2005). Group 1, consisted of serotype 4, 9 and 11, had the amplified product of 1,600 bp in size. Group
2, consisted of only serotype 6, had the amplified product of 2,000 bp in size. Group 3, consisted of serotype 1, 3,
12, 13 and 14, had the amplified product of 2,400 bp in size. Group 4, consisted of serotype 2, 5a, 5b, 7, 8, 10 and
15, had the amplified product of 2,800 bp in size.
APP isolates from Thai fattening pigs in 2011 were successfully detected all serotypes and identified by PCR
technique based on ApxIV gene (Fig. 2). The PCR products were divided into three patterns (table 1). Pattern 1 (3
provinces) was detected nine serotypes and pattern 2 (Prachinburi province) was detected only four serotypes. In
contrast, pattern 3 (2 remaining provinces) was found twelve serotypes. The distribution of APP serotypes in 2011
may cause by the movement of the replacement gilts or boars into the area. This study will be useful for understanding the epidemiology of an APP outbreak and the selection of vaccine for control this disease in Thailand in the
future.
241
Table 1. Serotypes of APP from the studied regions of Thailand.
Figure 2. Electrophoresis analysis of ApxIV gene. Lane M,
1.0 kb DNA Ladder (BioLab, USA). Lane 5, negative control.
Lane 1, 2, 3, 4 and 6 APP isolates from Thai fattening pigs
Figure 1. Map of regions to collect APP samples
References
1. Mittal et al., 1982. J. Clin. Micro. 15: 1019–1023
2. Schaller et al., 1999. Micro. 145: 2105–2116
3. Frey et al., 1995. Mol. Cell. Probes. 9: 277–282
4. Han Sang Yoo, 2005. J. Vet. Diagn. Invest. 17: 359–362
242 
DETERMINATION OF SERUM CALCIUM CONCENTRATION IN PERIPARTURIENT DAIRY COWS
W. Thiangtum1*, N. Ratanapob1, S. Srisomrun2, S. Kananub3, C. Sujaritthanyatrakul3, A. Khantaboot3
1
Department of Large Animal and Wildlife Clinical Sciences, 2Kasetsart University Veterinary Teaching HospitalKamphaeng Saen, 3Kasetsart University Veterinary Teaching Hospital- Nong Pho, Faculty of Veterinary Medicine,
Kasetsart University, THAILAND. *Corresponding author
Introduction
The periparturient period is the most critical time in the life of dairy cow. Subclinical hypocalcaemia is
important macromineral disorders that affect dairy cows in this period (Mulligan et al., 2006). Cows with subclinical
hypocalcaemia have no clinical signs of hypocalcaemia but may be more susceptible to other diseases such as
dystocia, retained placenta, abomasal displacement, mastitis and ketosis (Cutis et al., 1983). Age appears to be a
factor that predisposes cows to subclinical hypocalcaemia (Horst et al., 1997). The incidence of subclinical
hypocalcaemia increases in greater lactations (Cutis et al., 1984). The objective of this study was to investigate the
serum Ca concentration of periparturient cows.
The study was conducted on 30 dairy farms in Ratchaburi, Kanchanaburi and Nakhon Pathom province. Fifty
three cross-bred Holstein cows were enrolled at 1 month before the expected calving date. Cows were divided into 2
groups by parity; group 1 (1st and 2nd parity cows, n=27) and group 2 (>2nd parity cows, n=26). Blood samples were
collected from each cow at d -30, d 0 (within 3 days after calving) and d 30 related to calving date. Serum sample
were separated by centrifugation at 2000 rpm for 5 minutes then kept at -20°C until serum Ca was determined by
spectrophotometry (Liasys II). Variation was analyzed by the repeated-measurement. All statistical analyses were
performed by NCSS statistical software.
Results and discussion
Figure 1. Data represent the mean ± SD of serum Ca concentration at d -30, d 0 and d 30. Group 1 (1st and 2nd parity
cows, n=27) and group 2 (>2nd parity cows, n=26)
243
Figure 1 shows that the average serum calcium concentration during calving period was significant lower
than Ca concentration of cow in 1 month before and 1 month after calving. Serum Ca level of cows in group 1 were
higher than serum Ca level of cows in group 2. However, the average serum Ca level in pre and post calving in both
groups were in normal range (8.5-10 mg/dl). In contrast, the average blood Ca level during calving period in group 2
was dropped as result of subclinical hypocalcaemia (7.4 mg/dL), while the average serum Ca level in group 1 was
remained in normal range (8.1 mg/dL). As dairy cows become older, the incidence of subclinical hypocalcaemia increased. Similarly, DeGaris and Lean, 2009 reported that the incidence of milk fever tends to increase when cow get
older and the risk of milk fever was approximately 9% per lactation by increasing age Several factors that may
contribute to the age effect; For example, advancing age results in increasing milk production and a higher demand
for Ca (Horst et al., 1997). Aging also results in a decline in the ability to mobilize Ca from bone stores and decline
in the Ca transportation in the intestine as well as reduction in the number of receptors for 1, 25-dihydroxyvitamin D
in the intestine of cows (Horst et al., 1997).
Prevalence of subclinical hypocalcaemia (Ca 8-5.5 mg/dL) (DeGaris and Lean, 2009) in this study was 54.7%
(29/53). Prevalence of subclinical hypocalcaemia in group 1 and in group 2 was 48.2% and 61.5%, respectively.
Reinhardt et al (2011) reported that prevalence of subclinical hypocalcaemia of the US dairy herds in 1 st -2nd parity
cows was 33% and 50% in greater 2nd parity cows. The higher prevalence of subclinical hypocalcaemia may result
from the cow characteristics, nutrition, environment and management.
Acknowledgements
Authors would like to thank the dairy farmers and staffs of laboratory of Kasetsart university veterinary
teaching hospital Hua Hin and Nong Pho for cooperation and thank KURDI for research funding.
References
1.
2.
3.
4.
5.
6.
Curtis, C.R., Erb, H.N., Sniffen, C.J.and Smith, R.D.(1984). Epidemiology of parturient paresis: predisposing factors with emphasis on dry cow feeding and management. Journal of Dairy Science, 67, 817-825.
Curtis, C.R., Erb, H.N., Sniffen, C.J., Smith, R.D., Powers, P.A., Smith, M.C., White, M.E., Hillman, R.B.
and Pearson, E.J. (1983). Association of parturient hyocalcaemia with eight periparturient disorders in
Holstein cows. Journal of the American Veterinary Medical Association, 183, 559-561.
DeGaris, P.J.and Lean, I.J.(2008) Milk fever in dairy cows: a review of pathophysiology and control
principles. Veterinary Journal, 176, 58-69.
Horst, R.L., Goff, J.P.and Reinhardt, T.A.(1997).Buxton DR. Strategies for preventing milk fever in dairy
cattle. Journal of Dairy Science, 80, 1269-80.
Hulligan, F, O'Grady, L, Rice D and Doherty, M. (2006). Production diseases of the transition cow: Milk
fever and subclinical hypocalcaemia. Irish Veterinary Journal, 59, 697-702.
Reinhardt, T.A., Lippolis, J.D., McCluskey, B.J., Goff, J.P.and Horst, R.L. (2011). Prevalence of subclinical
hypocalcemia in dairy herds. Veterinary Journal,188, 122-4.
244 
PATHOGENICITY TO MEKONG GIANT CATFISH EGGS IN LABORATORY OF WATER MOULDS
ISOLATED FROM MEKONG GIANT CATFISH EGGS AND THE REARING WATER
Narong Abking,1 Wichukarn Fuangsawat 2 and Ong-ard Lawhavinit 2*
1
Department of Veterinary Technology, Faculty of Veterinary Technology,
2
Department of Microbiology and Immunology, Faculty of Veterinary Medicine,
Kasetsart University, THAILAND.*Corresponding author
Introduction
Fungal disease is a problem that occurs occasionally in fish farms and brood stock husbandry, it causes low
productivity of fry and low production of fish cultures (Kwanprasert et al., 2007). Diseases caused by water fungi
occur more often at the eggs stage. The mortality rate of incubated eggs due to water fungal infection sometimes
reaches 80–100% (Chukanhom and Hatai, 2004). Water moulds are classified in the order Saprolegniales which includes two families: the Saprolegniaceae (e.g. Achlya, Brevilegnia, Dictyuchus, Saprolegnia and Thraustotheca) and
Leptolegniaceae (Aphanomyces Leptolegnia and Plectospira), totaling 132 species in about 20 genera (Dick, 2001).
The Saprolegniales are the best-known group of water moulds. The most important water moulds in the order Saprolegniales are those water moulds in the genera Achlya and Saprolegnia that can be pathogens of many fish species
and their eggs (Willoughby, 1994). Normally, fungal diseases are a secondary infection following an injury
(Kitancharoen, 1995). At present, the number of Mekong giant catfish in the Mekong River and its branches is reducing to a critical level. Therefore, this species was put on the endangered species list under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (Poulsen et al., 2004). Control of any dissolved fungal infection problems provides a way to improve the egg hatching rate and conserve the Mekong Giant
Catfish in nature. Therefore, the purposes of this research were the isolation, and identification of the genera of water
moulds, and, the study of some of the morphological and, biological characteristics, and the pathogenicity of the isolated water moulds on eggs in the laboratory.
Isolation and identification.
Eggs of the Mekong giant catfish and water samples were collected in the spawning seasons from 2008 to
2010 at the Inland Aquaculture Research Institute, Phra Nakhon Sriayutthaya province Thailand. The samples were
collected during July to September, 2008 and May to August, in both 2009 and 2010. The number and type of samples are shown in Table 1.
Table 1 Sampling data of eggs of Mekong giant catfish and water from hatching tank at the Inland Aquaculture Research Institute, Phra Nakhon Sriayutthaya province, Thailand, from 2008 to 2010.
245
Sampling year
Type of sample
Number of eggs
Water from hatchery tank number
2008
85
16
2009
150
30
2010
150
30
The samples were isolated by inoculation or spreading on to glucose-yeast extract agar (GY agar) according
to Hatai and Egusa (1979) and incubated at 20 °C for 24– 48 h. For inhibition of bacterial growth, the addition of 500
µg/mL each of ampicillin and streptomycin sulphate to the medium was required. The purification process used a
zoospore spread technique according to Choi et al. (1999) and subcultured every month. For morphological observations, the fungi were inoculated into 50 mL of GY broth and incubated at 20 °C for 3– 4 days. The young hyphae in
the GY broth were washed five times in sterilized distilled water. Fungal isolates were identified to the genus level
by the different structures of asexual stage development and the zoospores discharged in sterilized tap water, according to Munchan (2003).
Effect of temperatures, NaCl and pH on fungal growth.
The advancing edges of fungal growth colonies were cut with a cork borer (8 mm in diameter) and placed
onto the center of Petri dishes (90 mm in diameter) that contained 20 mL of GY agar; the plates were incubated at
various temperatures: 10, 20, 30 and 35 °C. The other sets of fungal samples were placed on GY agar containing
NaCl (0, 5, 10, 15, 20 and 25 ppt) or GY medium with different pH levels (pH 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) and incubated at 20 °C. The radial growth of each colony was measured with vernier calipers and recorded for 3 days. All
experiments were performed in triplicate. Each experiment was compared with reference fungi such as Saprolegnia
diclina NJM 0501 and Achlya bisexualis NJM 0611. All reference strains of fresh water fungi used in this study were
supplied by Professor Dr. Kishio Hatai, Nippon Veterinary and Life Science University, Tokyo Japan. Statistical
analysis was carried out using the NCSS program (available source: www.ncss.com) by a one-way ANOVA to compare the average diameter of the fungal colonies at the 95% confidence level. The optimum growth was defined as
the examined fungal growth that produced an average diameter of the colony significantly (P<0.05) bigger than the
other groups.
Pathogenicity test of water moulds on eggs of Mekong giant catfish.
The pathogenicity test for the isolated fungi Achlya spp. T.MCF 1-02, E.MCF 2-001, E4/52-5, E4/52-8,
E4/52-10, E4/52-11, E4/52-12, Saprolegnia spp. E1/53-12, and A. bisexualis NJM 0611 was conducted. Zoospores
preparing by subcultured the isolated on GY agar for 2 days and cut into agar blocks (size 1×1 cm 2 ) 2– 3 pieces put
onto a plate with 20 mL GY broth and incubated at 20 °C for 2 days. Then, the hyphae were washed three times with
sterilized tap water and immersed in 30 mL sterilized tap water on a plate for 18– 24 h, and incubated at 20 °C. Zoospores that developed were released into the sterilized tap water on the plate. The zoospores were counted with a hemacytometer (Neubauer improved Bright– line; 0.0025 mm2) and the concentration adjust to 1×104 spores/mL. Then,
30 eggs of the Mekong giant catfish that had been artificially fertilized were immersed for 1 h in sterilized tap water
on a plate containing zoospores at 1×104 spores/mL. The control group was not immersed in the suspension of zoospores.
246 
All experimental groups were duplicated and the eggs were incubated at room temperature for 2 days. The
amounts of fry hatched were recorded and analyzed by the statistical program using a one-way ANOVA. The presence of infection was determined by the appearance of fungal hyphae developing on the egg surface. The non
hatched eggs were subcultured for fungal growth on GY agar. Some of the non hatched eggs were fixed with 10%
neutral buffered formalin solution, and then embedded into paraffin blocks and prepared histopathological slides for
examination of any histopathological characteristics.
Isolation and identification.
According to the morphological characteristics and zoospores released (Figure 1), as shown in table 2, the
isolated water fungi were identified as nine isolates of the genus Achlya and seven isolates of Saprolegnia
(Munchan, 2003). Fungal isolates from the genus Achlya had a puffy and whitish colony appearance and aseptate
hyphae. Furthermore, they took 3– 4 days to become fully grown on GY agar and break down GY agar in circular
shape. An asexual stage developed in 18– 20 h after immersion in sterilized tap water on a plate at 20 °C. The zoospores released was of the Achlyoid type. The zoospores occasionally discharged from a discharge pore and the
spore clusters usually accumulated at the opening of a zoosporangium (Figure 1A). On the other hand, the fungal
isolates from the genus Saprolegnia had a cotton-like, whitish colony appearance; their hyphae were aseptate and
showed fast growth on a GY agar plate and become to fully plate in 2 days. An asexual stage developed in 18– 20 h
after immersion in sterilized tap water on a plate at 20 °C. The zoospores released was of the Saprolegnoid type. The
zoospores were released and swam out from the opening of the zoosporangium (Figure 1B).
Table 2 Genera of water moulds isolated from eggs of Mekong giant catfish and water samples of the hatching tank
from 2008 to 2010.
Isolation year
Water moulds isolated from eggs
Water moulds isolated from hatching water
2008
Achlya spp. E.MCF1-02
Achlya spp. T.MCF2-001
2009
Achlya spp. (E4/52-2, E4/52-3,
E4/52-5, E4/52-8, E4/52-10, E4/52-11 and
E4/52-12)
Saprolegnia spp.(E3/52-P1, E3/52-P2, E3/52
-P3 and E3/52-P4)
Saprolegnia spp. E1/53-12
–
2010
–
Saprolegnia spp. (T3/53-3 and T3/53-5)
Note: E = water moulds isolated from eggs of Mekong giant catfish, T = water moulds isolated from water of
the hatching tank.
247
Figure 1 Arrow showing zoosporangium development of Achlya spp. (A) and Saprolegnia spp. (B) after zoospore
released (scale bar = 50 µm).
Effect of temperatures, NaCl and pH on fungal growth.
Water mould isolates of Achlya spp. (T.MCF 1-02, E.MCF 2-001, E4/52-2, E4/52-3, E4/52-5, E4/52-8,
E4/52-10, E4/52-11 and E4/52-12) and A. bisexualis NJM 0611 could tolerate a NaCl medium at 10 ppt but could not
grow in a higher NaCl medium at 15 ppt. Water mould isolates of the genus Saprolegnia spp. (E3/52-P1, E3/53-P3,
E3/52-P4, E1/53-12, T3/53-3 and T3/53-5) could tolerate a NaCl medium at 25 ppt but could not grow in a higher
NaCl medium at 30 ppt. Exception, Saprolegnia sp. E3/52-P2 and S. diclina NJM 0501 could tolerate a NaCl medium at 30 ppt but could not grow in a higher NaCl medium at 35 ppt. (Table 3).
The optimal temperature for growth of the isolated Achlya spp. and Saprolegnia spp. was 30 °C (Table 3)
which similar to the study of Chukanhom and Hatai (2004) who reported that the optimal temperature range for the
growth of S. diclina NJM 0208 and A. klebsiana NJM 2010 were 25– 30 °C and 30– 35 °C, respectively. Chukanhom
and Hatai (2004) also reported that S. diclina NJM 0208 could tolerate a high NaCl medium at 30 ppt but could not
grow in high NaCl medium at 40 ppt and A. klebsiana NJM 2010 could tolerate to a NaCl medium at 10 ppt but could
not grow in a NaCl medium at 20 ppt giving the same results as the present study. Almost all isolates could grow in
GY broth at pH 4– 11, while the optimal pH for Achlya spp. and Saprolegnia spp. was pH 5 and 6, respectively. The two
reference stains, A. bisexualis NJM 0611 and S. diclina NJM 0501, could grow in GY broth at pH 4– 11, while the optimal pH value was pH 5 and 6, respectively (Table 3). However, the results of the present study with the GY medium
at various pH levels contrasted with the study of Chukanhom and Hatai (2004) who reported that A. klebsiana NJM
0210 and S. diclina NJM 0208 could grow in GY medium at pH 4– 10, the optimal pH level were 6 and 7, respectively,
while in the present study, the fungal isolates of Achlya spp. and Saprolegnia spp. could grow in the GY medium at
pH 4– 11, with an optimal pH level of 5 and 6, respectively. Therefore, the results suggest that increasing the water temperature more than 30 °C, with 30 ppt NaCl and pH 6 could prevent fungal infection during the hatching process of
the Mekong giant catfish eggs. However, further study involving safety test for the eggs must be undertaken.
248 
Table 3 Optimal temperatures, salinity and pH effected on growth of isolated water moulds, compared with Saprolegnia diclina NJM 9219 and Achlya bisexualis NJM 0611.
Isolation year
Water fungus
Temperature (°C)
Salinity (ppt)
pH
2008
Achlya spp. T.MCF 1-02
30
10
5
Achlya spp. E.MCF 2-001
30
10
5
Achlya spp. E4/52-2
30
10
5
Achlya spp. E4/52-3
30
10
5
Achlya spp. E4/52-5
30
10
5
Achlya spp. E4/52-8
30
10
5
Achlya spp. E4/52-10
30
10
5
30
10
5
30
10
5
Saprolegnia spp. E3/52-P1
30
25
6
30
30
6
30
25
6
30
25
6
30
25
6
Saprolegnia spp. T3/53-3
30
25
6
Saprolegnia spp. T3/53-5
30
25
6
Achlya bisexualis NJM 0611
30
10
5
Saprolegnia diclina NJM 0501
30
30
6
2009
2010
Reference
Pathogenicity test of water moulds on eggs of Mekong giant catfish.
The infection and percentage of non hatch rates of Mekong giant catfish eggs after 2 days were recorded.
The results in table 4 show that Achlya spp. T.MCF 1-02, E.MCF 2-001 and E4/52-10 and Saprolegnia sp. E1/53-12
could be pathogenic to eggs of the Mekong giant catfish because no hatched eggs were found. Therefore, the control
group and Achlya spp. (E4/52-5, E4/52-8 and E4/52-11) could not be pathogens as no fungal hyphae were observed
on eggs. The non hatched eggs were infected and entangled with hyphae on the second day after challenge. The same
infected fungi used in the experiment were re-isolates from the non hatched eggs. The study on the histopathology of
eggs with fungal infection showed the invasion of water mould mycelia from the cell membrane into the egg yolk of
Mekong giant catfish which induced a gap in egg (Figure 2). These results, clearly demonstrated that experimental
fungi had a high pathogenicity to Mekong Giant Catfish eggs. Almost all of the hyphae in the eggs were died, because the protoplasm in the hyphae usually could not be observed.
249
Table 4 Percentage average hatched rate of eggs of Mekong Giant Catfish after 2 days infection with water fungus
zoospores.
Water fungus isolates
Percentage average hatched rate
Control group
8.3a
Achlya spp.T.MCF 1-02
0b
Achlya spp.E.MCF 2-001
0b
Achlya spp.E4/52-5
8.3a
Achlya spp.E4/52-8
8.3a
Achlya spp.E4/52-10
0b
Achlya spp.E4/52-11
8.3a
Achlya spp.E4/52-12
5.0a
0b
A.bisexualis NJM 0611
0b
Note: different superscripts indicate a significant differrence at P < 0.05.
Figure 2 Arrow showing the invasion by water mould mycelia (black color) in eggs of the Mekong Giant Catfish,
from cell membrane into egg yolk (green color) and inducing gap formation, staining with Grocott-Gomori methenamine-silver stain; power at 400× (scale bar = 50 µm).
250 
Solving the fungal infection problem is an alternative to improve the egg hatching rate and to conserve the
Mekong giant catfish in nature. Therefore, the purposes of this research were the isolation and, identification of the
genera of water moulds, study of some morphological and, biological characteristics, and the pathogenicity test of the
isolated fungi on eggs in the laboratory. The results showed that, the optimal temperature of almost all isolates of
Achlya spp. and Saprolegnia spp. was 30 °C. The Achlya spp. and Saprolegnia spp. could tolerate a NaCl medium at 10
ppt and 25 ppt, respectively. An exception was Saprolegnia spp. (E3/52-P2) which could tolerate up to 30 ppt. The isolates could grow in broth at pH 4– 11, while the optimal pH for Achlya spp. and Saprolegnia spp. was pH 5 and 6, respectively. The study on pathogenicity test of the water moulds isolated in the laboratory showed that the pathogens
were Achlya spp. T.MCF1-02, E.MCF 2-001 and E4/52-10 and Saprolegnia spp. E1/53-12. The non hatched eggs were reisolated in the same infected fungi used in the experiment. The histopathological detection on infected eggs results,
clearly demonstrated that experimental fungi had a high pathogenicity to Mekong giant catfish eggs.
Acknowledgements
The authors would like to thank the Inland Aquaculture Research Institute, Ayutthaya province, Thailand for
providing all the samples used in this study, Faculty of Veterinary Technology, Kasetsart University for instrumentation support, the Kasetsart University Research and Development Institute (KURDI) for financial support and Professor Dr. Kishio Hatai for supplying the reference stains of the fungi used in this study.
References
Choi YW, Hyde KD, Ho WH. 1999. Single spore isolation of fungi. Fungal Divers. 3: 29–38.
Chukanhom K, Hatai K. 2004. Freshwater fungi isolated from eggs of the common carp (Cyprinus carpio) in
Thailand. Mycoscience 45: 42–48.
Dick MW. 2001. Straminipilous Fungi. Kluwer Academic Publishers, Dordrecht. 670 pp.
Hatai K, Egusa S. 1979. Studies on pathogenic fungus of mycotic granulomatosis III development of the medium for
egg-fungus. Fish Pathol. 13: 147–152.
Kitancharoen N, Hatai K, Ogihara R, Aye DNN. 1995. A new record of Achlya klebsiana from snakehead, Channa
striatus, with fungal infection in Myanmar. Mycoscience 36: 235–238.
Kwanprasert P, Hanjavanit C, Kitancharoen N. 2007. Characteristics of Achlya bisexualis Isolated from Eggs of
NileTilapia (Oreochromis niloticus Linn.). KKU Research Journal 12(3): 195–202.
Munchan C. 2003. Some Biological and Pathological Characteristics of Saprolegnia and Achlya Isolated from
Ornamental Fishes. M. Sc. thesis, Khon Kaen University.
Poulsen AF, Hortle KG, Valbo-Jorgensen J, Chan S, Chhuon CK, Viravong S, Bouakhamvongsa K, Suntornratana,
U, Yoorong N, Nguyen TT, Tran BQ. 2004. Distribution and ecology of some important riverine fish species of the Mekong river basin. MRC Technical Paper. 10: 1489–1683.
Willoughby LG. 1994. Fungi and Fish Diseases. Pisces Press, Stirling, Scotland. 57 pp.
251
LAPAROSCOPIC OVARIECTOMY IN NATIVE-SAANEN GOATS
Peerasak Suttiyotin1*, Chowalit Nakthong2, Bunlue Kornmatitsuk2 and Somkiat Huaijantug2
1
Department of Preclinic and Applied Animal Science, 2Department of Clinical Sciences and Public Health,
Faculty of Veterinary Science, Mahidol University, THAILAND. *Corresponding author
Introduction
Laparoscopic ovariectomy has been proved to be beneficial as it reduces both time and pain. It has been performed in many species such as dogs (Davidson et al., 2004), cats (Van Nimwegen and Kirpensteijn, 2007), horses
(Murphy et al., 2005), donkeys (Aziz et al., 2008), sheep (Teixeira et al., 2011). Goats are one of the potential animals for both production and experimental purposes in Thailand. The aims of this study were to apply laparoscopic
ovariectomy with bipolar cauterizing probe to goats and to evaluate surgical time for removing of ovaries by this
technique.
Five clinically healthy Native-Saanan crossbred goats (1-2 years) were included in the study. Anesthesia was
induced with tiletamine and zolazepam (Zolitil® 100, Virbac, Thailand, 4.4 mg/kg IV). Endotracheal tube was
placed and anesthesia was maintained with isoflurane in oxygen. The animals were secured in dorsal recumbency in
a 30° Trendelenburg position. Continuous ECG, blood gas analyser, pulse oximetry and rectal temperature measurement were monitored during the surgery.
Three-portal access technique was used. Pneumoperitoneum was obtained with carbon dioxide via a Veress
needle. The first portal was placed 3 cm caudal to the umbilicus after a skin incision was made. A 10-mm, 57-cm, 0°
rigid laparoscope (Hopkins Karl Storz, Germany) connected with video camera was inserted to explore abdominal
organs. The other 2 portals were established, 5 cm lateral to midline and 11 cm caudal to the umbilicus, under laparoscopic guidance. Grasping forceps with locking handle (Karl Storz) was used to manipulate and grasp the ovary
via left instrument portal. Bipolar cauterizing forceps (right instrument portal) was used to cauterize and dissect
mesovarium to free the ovary from surrounding tissue. The ovaries were removed through left portal under laparoscopic guidance. The internal organs were observed for hemorrhage before the portals were closed in a standard
fashion. Time to remove each ovary and total surgical time were recorded.
We successfully performed laparoscopic ovariectomy in the goats. Exploration of abdominal space to get a
good view of ovary took less than 1 minute when precise locations of portal sites were assigned. It is important to
mark the portal sites so that viewing and approaching the ovary is easily achieved (Figure 1). It took 4-8 min to
grasp, cauterize and remove an ovary from abdominal cavity (Table 1). Surgical time was longer in the first animal
(28.3 min) and it reduced to approximately 20 min once the surgeon was comfortable and gained confidence in the
operation. This indicates that training and practicing may play important roles. This technique could be used in
goats, and can be beneficial to both research and reproduction management
252 
Figure 1 Laparoscopic ovariectomy in goats. With grasping forceps holding ovary, bipolar cautery forceps
coaagulated and cut through mesonarium and tissue around the ovary. Note that the ovary was held toward the
abdominal wall to avoid any damage to surrounding tissue.
Table 1 Goats included in the study.
Weight (kg)
BSC (1-5)
Time to remove ovary (min)
Left ovary
Right ovary
Surgical time (min)
Goat 1
21.5
Goat 2
20.8
Goat 3
17.2
Goat 4
16.5
Goat 5
13.5
Mean
17.9
3.5
3.0
3.0
3.5
3.0
3.2
8.6
8.3
28.3
8.2
4.2
25.1
8.1
6.6
24.9
6.1
4.2
19.7
4.3
4.8
20.7
7.1
5.6
23.8
Acknowledgements
This work was supported by Mahidol University. Laparoscopic equipment was provided by Dr.
Phingphol Charoonrut and Kosin Medical Supply Co.,Ltd. We thank staff at Prasupalun Livestock and Wildlife Hospital, Mahidol University for technical assistances.
References
Aziz DM, Al-Badrany MS, Taha MB. 2008. Laparoscopic ovariectomy in standing donkeys. Anim Reprod Sci
107:107-114.
Davidson EB, Moll HD, Payton ME. 2004. Comparison of laparoscopic ovariohysterectomy and ovariohysterectomy
in dogs. Vet Surg 33:62-69.
Murhpy J, Hendrickson DA, Hendrix S. 2005. Right flank laparoscopic ovariectomy of a regressing granulosa thecal
cell tumor of a pregnant mare: A case review. J Equine Vet Sci 25:309-311.
Teixeira PPM, Padilha LC, Motheoa TF, Silva MAM, Oliveira MEF, daSilva ASL, Barros FFPC, Coutinho LN,
Flôres FN, Lopes MCS, Rodrigues LFS, Vicente WRR. 2011. Ovariectomy by laparotomy, a video-assisted
approach or a complete laparoscopic technique in Santa Ines sheep. Sm Ruminant Res 99:199-202.
Van Nimwegen SA, Kirpensteijn J. 2007. Laparoscopic ovariectomy in cats: Comparision of laser and bipolar electrocoagulation. J Feline Med Surg 9:397-403.
253
INFLUENCE OF COLOSTRAL QUALITY ON SERUM PROTEIN IN DAIRY CALVES RAISED IN
SMALL HOLDER FARMS
Suppada Kananub1, Theera Rukkwamsuk2, and Pipat Arunvipas2
1
Kasetsart University Veterinary Teaching Hospital Nongpho, Faculty of Veterinary Medicine,
2
Introduction
The placenta of cow is not able to transfer immunoglobulin. Colostrum, consisting protein, fat, lactose,
minerals and vitamin, is the main nutrient source especially for immunoglobulin (Quigley and Drewry, 1998). A
correlation was found between serum immunoglobulin G (IgG) levels and serum proteins (Bagger and Eriksen,
2003). Nineteen percent of calves have IgG levels of less than 10 mg/ml (Filteau et al., 2003), which is known as
failure passive transfer (FPT), increasing the risk of pneumonia, diarrhea and death (NAHMS, 1992). Lower IgG
calves are 2 times more likely to contract pneumonia compare to higher IgG calves (Virtalaa et al., 1999) and more
likely to suffer from low growth rate (Bernard, 2006). There is little information available regarding colostral quality
and calves’ serum protein. Therefore the objective of this study was to analyze the influence of colostral quality on
calves’ serum protein.
Samples were randomly selected from Veterinary Medical Teaching Hospital at Kampangsaen Campus and
Nongpho Animal Hospital visited farms in Nakhon pathom, Ratchaburi and Kanchanaburi province. Thirty four
farms, 160 samples, 80 dams and 80 calves, consent to participate with this study. Colostrum, 300 ml, was collected
3 times, calving date, 1 and 2 days after calving. Calves’ blood, 5 ml, was collected 5 times, calving date, 1, 2, 7 and
14 days after calving. Total colostral immunoglobulin was analyzed by colostrometer. Serum was analyzed serum
protein by refractrometer. Statistical analyses were performed using one way ANOVA and repeated measurement
ANOVA. P-values <0.05 were considered to be statistically significant. All analyses were conducted using the
statistical software package STATA (version 8.2, Stata Corp., College Station, TX).
Average of total colostral immunoglobulin (total Ig) in first 3 days after calving was (mean±SEM)
95.25±2.73, 37.37±2.73 and 17.56±2.77 mg/ml respectively (p<0.01). Maximum total Ig is in the first milking after
calving and decreases in following milking times (Foley and Otterby, 1978 and Hostetler et al., 2003). Total colostral immunoglobulin indicating colostral quality of less than 50 mg/ml is considered to be of low quality and more
than 50 mg/ml is considered to be high quality colostrum (Kruse, 1970). The serum protein results are shown in
Table 1, grouped by colostral quality. Serum protein from first day after calving which were fed low quality
colostrum significantly differ from calves fed high quality (p<0.01). An increase of serum protein after first feeding
was 0.81±0.18 mg/ml in calves fed low quality colostrum, significantly different from calves fed high quality
colostrum, 1.55±0.13 mg/ml (p<0.02). This result corresponded to Stott and Fellah (1983) who found serum protein
and immunoglobulin were higher for calves fed high immunoglobulin colostrum. Results from this study emphasized the importance of colostral quality to calves’ serum protein, calves’ health.
254 
Table 1 Average of serum protein of calves in calving date, 1, 2, 7 and 14 days after calving, grouped by
colostral quality (0; low quality of colostrum and 1; high quality of colostrum)
Groupa
0
1
Age (day)
0
1
2
7
14
0
1
2
7
14
N
14
15
15
9
8
65
65
65
48
42
Mean
5.59
6.40
6.37
6.39
6.51
5.94
7.49
7.59
7.48
7.30
SEM
0.21
0.22
0.25
0.27
0.26
0.09
0.13
0.12
0.13
0.12
Min
4.60
5.20
4.70
5.20
5.90
4.80
5.20
5.60
5.20
5.90
Max
7.20
8.20
8.70
7.80
7.70
8.00
10.0
10.0
9.20
9.40
Acknowledgements
We thank KURDI for research funding and all dairy farmers who participate in this study.
References
Bagger, M., and Eriksen, L. (2003). Comparison of difference methods for measuring immunoglobulin content in
calf serum. Acta Vet. Scand., 44(1), 26.
Bernard, J.K. (2006). Developing quality dairy replacement heifers. Proceedings 3 rd Florida & Georgia Dairy Road
Show, 69-78.
Filteau, V., Bouchard, E., Fecteau, G., Dutil, L. and Tremblay, Du D. (2003). Health status and risk factors associated with failure of passive transfer of immunity in newborn beef calves in Québec. Can Vet J., 44, 907-913.
Foley, J.A. and Otterby, D.E. (1978). Availability, storage, treatment, composition and feeding value of surplus
colostrum. J. Dairy Sci., 61, 1033-1060.
Hostetler, D., Douglas, V.L., Tyler, J., Holle, J. and Steevens, B. (2003). Immunoglobulin G concentrations in tem
poral fractions of first milking colostrum in dairy cows. J Appl Res Vet Med., 1(2), 168-171.
Kruse, V. (1970). Yield of colostrum and immunoglobulin in cattle at the first milking after parturition. Anim. Prod.,
12, 619-626.
National Animal Health Monitoring System. (1992). Dairy herd management practices focusing on preweaned
heifers. USDA, Animal and Plant Health Inspection Service, Veterinary Services, Fort Collins, CO.
Quigley, J.D. and Drewry, J.J. (1998). Nutrient and immunity transfer from cow to calf pre and postcalving. J. Dairy
Sci., 81, 2779–2790.
Stott, G.H. and Fellah, A. (1983). Colostral immunoglobulin absorption linearly related to concentration for calves. J
Dairy Sci., 66, 1319-1328.
Virtalaa, A.M.K, GroÈhnb, Y.T., Mechor, G.D. and Erb, H.N. (1999). The effect of maternally derived immunoglobulinG on the risk of respiratory disease in heifers during the first 3 months of life. Prev. Vet. Med., 39,
25-37.
255
POLYMERASE CHAIN REACTION BASE-PREVALENCE OF ENCEPHALITOZOON CUNICULI
IN MEAT RABBITS
Preeda Lertwatcharasarakul1*, Attawit Kovitvadhi2, Kamonchanok Aiemsumaung2, Boonchoo Piempinseat2,
Patcharida Dittawong2, Waraporn Sinsuwonkwat3, Sakuna Phatthanakunanan4, Siriluk Jala4, Aree Thayananuphat5,
Pornchai Sanyathitiseree6
1
Department of Pathology, 2Sixth year’s student, 3Graduate student, 4Kampangsaen Veterinary Diagnostic Center,
5
Department of Companion Animals Clinical Sciences, 6 Department of Large Animal and Wildlife Clinical Science,
Faculty of Veterinary Medicine, Kasetsart University, THAILAND. * Corresponding author
Introduction
Encephalitozoon cuniculi is an obligate intracellular microsporidian protozoon parasite and primarily occurs in
rabbits. Urine contact is the usual route of transmission by exchanging of spores between the infected to normal animals. Transplacental and inhalation transmission has also been described. The most common sites of infection are
kidney, brain, spinal cord, liver, heart, and the lens. Encephalitozoonosis affected a rabbit in several forms such as
granulomatous nephritis and encephalitis leading to chronic renal failure and/or vestibular disease.
Spore staining in histological examination, serological method and polymerase chain reaction (PCR) were
normally used for E.cuniculi antigen detection (Csokai et al., 2009). PCR was proposed at present to be a good post
mortem method of organism detection. The aim of this report was represent of E.cuniculi infection in meat rabbits
and correlation between presence of protozoa DNA and clinical sign.
Four-month-old rabbit brain samples were collected from 2 slaughterhouses located in Phetchaburi and Nakhon Pathom province. The criteria for Encephalitozoonosis suspected cases are clinical signs of neurological system i.e. head tilt and hind limb paralysis. The rabbits were divided into two groups: suspect encephalitozoonosis and
without overt clinical sign. After slaughter process, 84 brain samples were taken and frozen immediately at -20°C
until required for nested PCR. Phenol-Chloroform method was used for DNA extraction. Nested PCR protocol followed by Csokai et al., (2009). The result data were statistically evaluated with chi-square test.
From observation of clinical sign can divided into group I for 7 samples and group II for 77 samples. The
positive result was presented of band on 550 base pairs by nested PCR method. Positive result in group I and II were
42.9 % (n=7) and 28.6% (n=77) samples respectively. The difference between the groups was not significant (p<0.05).
The positive results from Nakhon Pathom and Phetchaburi province presented 45% (n=11) and 25% (n=66) respectively.
Brain samples were investigated because this organ is the most detection by nested PCR when compared
with other organs (Csokai et al., 2009). Head tilt or hind limb paresis were used to be criteria for encephalitozoon
suspected. However, those clinical signs can cause by Pasteurellosis, Toxoplasmosis, CNS abscess, neoplasia, otitis
media or trauma. On the other hand, positive results from asymptomatic group may cause of most E.cuniculi infection don’t lead to clinical signs (Künzel and Joachim, 2010). Therefore, rabbits with neurological clinical signs cannot be absolutely concluded about infection.
256 
Up to now there are no any reports about zoonosis from contaminated meat or meat ingestion in human.
These protozoa are known as an opportunistic pathogen of immunocompromised hosts (Künzel and Joachim, 2010).
However, Ozkan et al. (2011) found E.cuniculi spore in rabbit farmers urine who worked in seropositive farm.
In conclusion, this was the important evidence E.cuniculi infection in meat rabbit farms of Thailand and
presence of organism DNA does not correlate with neurological signs.
Table 1 Positive results of E.cuniculi by nested PCR method. (n)
Group
Nakhonpathom
Phetchaburi
total
I
2(6)
1(1)
3(7)
II
5(11)
17(66)
22(77)
total
7(17)
18(67)
25(84)
Acknowledgements
This project was financed by Faculty of Veterinary Medicine, Kasetsart University, Thailand. Special thank to
Kasetsart University Veterinary Teaching Hospital Kamphaengsaen and colleagues of Kampheangsean Veterinary
Diagnostic Center of Faculty of Veterinary Medicine, Kasetsart University for research facilities. The author is grateful to rabbit farmers in Nakhonpathom and Phetchaburi province and slaughterhouse staff that allowed us to collect
samples.
References
Csokai, J., Joachim, A., Gruber, A., Tichy, A., Palozdy, A. and Künzel, F. (2009). Diagnostic markers for encephalitozoonosis in pet rabbits. Veterinary Parasitology, 163, 18-26.
Harcourt-Brown, F. and Holloway, H.K. (2004). Encephalitozoon cuniculi infection in rabbits. Seminars in Avian
and Exotic Pet Medicine, 13(2), 86-93.
Künzel, F. and Joachim, A. (2010). Encephalitozoonosis in rabbits. Parasitology Research, 106, 299-309.
Ozkan, O., Ozkan, A.T. and Zafer, K. (2011). Encephalitozoonosis in New Zealand rabbits and potential transmission risk. Veterinary Parasitology, 179, 234-237.
257
COMPARATIVE MORPHOMETRY AND ULTRASTRUCTURE OF HEPATOZOON SPECIES INFECTED IN MANGROVE SNAKE (Boiga dendrophila melanota) AND KING COBRA (Ophiophagus hannah)
T. Chalalai1*, C. Salakij1, P. Lertwatcharasarakul1, K. Prihirunkit1, T. Vasaruchapong2
1
2
Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok 10330, THAILAND.
Introduction
Hepatozoon species are the most common intraerythrocytic parasite in reptiles (Wozniak et al., 1996). The
high prevalence of Hepatozoon infection in King cobra (Ophiophagus hannah) were reported in Thailand (Salakij et
al., 2002). Although pathogenicity in infected snake is low (Wozniak et al., 1994) but in heavy infection can lead to
anemia and blood cell morphology abnormality and plasma membrane alteration (Wozniak et al., 1996). The objective of this study was to performed comparative morphometry and ultrastructure of Hepatozoon species that detected
in Mangrove snake (Boiga dendrophila melanota) and King cobra.
Blood samples of two Mangrove snakes and six King cobras from Queen Saovabha Memorial Institute
(QSMI) were collected from ventral caudal vein. Blood smear were prepared immediately after collection and
stained with Wright’s stain. The width and length of Hepatozoon gamonts were measured under light microscope
and DP72® software (Olympus®, Japan).
For statistical analysis each parameters obtained, data from each group of Hepatozoon sp. gamonts observed
in Mangrove snakes and King cobra were calculated for mean, variance and standard error by the independent sample T-test. The differences were considered significant when the probabilities values were <0.05.
For transmission electron microscopy (TEM), buffy coats from eight snakes were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4°C for 24 hours, post fixed with 1% osmium tetroxide and embedded in Spurr’s
epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate and examined using Jeol 1200
Ex electron microscope (Salakij et al., 2002).
Light microscopy revealed the gamonts of Hepatozoon sp. within erythrocytes in all snakes (Figure 1a, d).
Some gamonts were free from red blood cells. Gamonts were laterally displacement nucleus of erythrocytes (Figure
1a, d). There were significant differences between gamonts from Mangrove snakes and King cobras. Gamonts from
King cobras were large while gamonts from Mangrove snakes were smaller (Table 1).
Table 1 Comparative Hepatozoon gamonts of Mangrove snakes and King cobras in micrometer. (mean ± SE).
Mangrove snakes (N=200)
Gamont (width)
Gamont (Length)
King cobra (N=200)
p-value
4.22 ± 0.03
4.43 ± 0.07
0.009
15.56 ± 0.04
16.27 ± 0.04
< 0.001
258 
For TEM, the gamonts were within parasitophorous vacuole membrane (PVM), with electron-lucent
space between gamont and cytoplasm of erythrocyte (Figure 1c). The gamont was included with conoid, rhoptries
and micronemes (Figure 1b, e, f, g). Hook-liked formation of PVM to reached nuclei of red blood cells (Figure
1c, g). The phagocytosed Hepatozoon gamonts were easily detected in the cytoplasm of azurophil (Figure 1h) and
thrombocyte (Figure 1i). These evidences revealed that both azurophils and thrombocytes may play a major role
in eliminating Hepatozoon. The result from this study use to add the understanding of morphometry and ultrastructure of Hepatozoon sp. infected in Mangrove snakes and King cobras and may be beneficial for related study.
Figure 1
A
D
G
B
E
H
C
F
I
Figure 1 Morphological characteristic of Hepatozoon gamonts in Mangrove snakes (a-c) and King cobra (d-i) (a) Gamonts
of Hepatozoon, Wright stain (W) (b) Long-sectional TEM of gamont in erythrocyte, showing prominent nucleus of Hepatozoon laterally displacement erythrocyte nucleus. (c) TEM of denatured gamont within PVM. (d) Three gamonts within erythrocyte (W) (e-f) Long-sectional TEM of free gamont, including, nucleus, elongated rhoptries, micronemes and dense granule.
(g) Hook-liked formation of PVM to erythrocyte nucleus. (h) Gamonts in azurophil and thrombocyte (i).
259
Acknowledgements
This study was supported by Department of Pathology, Faculty of Veterinary Medicine, and Kasetsart
University.
References
Salakij C, Salakij J, Apibal S, Narkkong N, Chanhome L , Rochanapat N, and Suthunmapinunta P.2002.
Hematology, morphology, cytochemical staining and ultrastructural characteristics of blood cells in King
cobras (Ophiophagus hannah). Vet Clin Path. 31: 116-126.
Wozniak EJ, McLaughlin GL, Telford SR Jr. 1994. Description of the vertebrate stages of a hemogregarine spe
cies naturally infecting Mojave Desert sidewinders (Crotalus cerastes). Zoo and Wildlife Medicine 25:
103-110.
Wozniak EJ, Kazacos KR, Telford SR Jr, McLaughlin GL. 1996. Characterization of the clinical and anatomical
pathological changes associated with Hepatozoon mocassini infections in unnatural reptilian hosts.
International J Parasitology 26: 141–146.
260 
RECENT TREND OF FOOD BORNE DISEASES IN JAPAN
S. Yamamoto
Division of Biomedical Food Research, National Institute of Health Sciences, MHLW, Japan.
Introduction
September 2001, first BSE case was found and Japanese government lost its confidence about food safety
issue. To recover consumer confidence, Japanese government decided to make renewal of food safety policy. Japanese government introduced a frame work of risk analysis into food safety policy. July 2003, the food safety
commission was established in the Cabinet Office as an organization of risk assessment. Ministry of Health, Labor
(MHLW) and welfare and Ministry of Agriculture Forestry and Fisheries stay as risk management organization.
Consumer affairs agency was established to control food labeling in September 2009.
Japan has two different surveillance systems. One is the data collection for food borne diseases and the other
is for infectious diseases.
Food borne diseases are defined as the adverse effect to humans after ingestion of food and drinks. Causative
agents of food borne diseases are microorganisms such as bacteria and viruses, chemicals such as poison of mushroom and fishes and toxic substances. Most cases of food borne diseases are caused by microorganisms.
Food borne diseases are firstly reported from medical doctor to a local health center. Food inspectors in the
local health center investigate the cause of food borne diseases. Final results report to the main office to the local
government and these data are sent to MHLW (MHLW, 2000-2010).
The highest number of outbreak case during the decade is norovirus food poisoning, 250 to 500. Number of
patients of norovirus is 8000 to 27000. Second is Campylobacter jejuni outbreak (Figure 1, 2). These two food
borne diseases are very difficult to control. In case of norovirus, disease is transferred from human to human and
very few amounts of agents can cause disease. In case of Campylobacter, disease is mainly caused by chicken
meat. More than 80% chickens in some farm are infected with Campylobacter, and chicken meats are contaminated with it in the chicken processing plant.
Salmonella spp. and Vibrio parahaemolyticus cases, popular food borne diseases in Japan are declined during
the decade. Salmonella cases are mostly caused by S. Enteritidis in eggs. Japanese government introduced labeling
of sell-by-date to egg. In case of Vibrio, raw consumption of seafood should not contain more than 100 cfu/g ,
Vibrio spp.
261
Figure 1. Number of food poisoning patients by different pathogens for recent decade.
Figure 2. Number of outbreaks by different pathogens for recent decade.
References
MHLW, Food poisoning statistics. 2000-2010.
262 
MOUSE STRAIN DIFFERENCES IN SUSCEPTIBILITY TO DIARRHETIC SHELLFISH POISONING
TOXINS, IN MOUSE BIOASSAY
H. Suzuki1*, K. Machii1
1
Divison of Biomedical Food Research, National Institute of Health Sciences, JAPAN.
 Corresponding author
Introduction
The mouse bioassay has been used as the official method for detecting diarrhetic shellfish poisoning (DSP)
toxins in Japan since 1981 (Yasumoto, 1981). This method, well known as Yasumoto’s method, has also been
widely used in many countries of the world (FAO, 2004). In the Japanese official protocol (Yasumoto, 1981), the
extract from shellfish sample is injected intraperitoneally into 3 male, ddY or ICR strain mice weighing 16 – 20g,
then, if 2 or more mice die by 24 hours after injection, the sample is deemed positive (contaminated). To the best
of our knowledge, however, there have been no reports specifically on the strain differences in susceptibility to
DSP toxins. In this study, we investigated the strain differences of mice to DSP toxins.
A lethal dose (4mg/ml/mouse) of okadaic acid (OA; LC Laboratories), one of the representative DSP toxins, was injected intraperitoneally into10 mice of each strain. The mice were observed every 10 minutes until 12
hours after injection, and then every 30 minutes until 24 hours after injection. C3H/He, C57BL/6, DBA/2 and ICR
strains of mice were compared in the 1st Experiment and A/J, BALB/c, ddY and ICR strains of mice were compared in the 2nd Experiment. ICR strain mice were used as control in both experiments. All the mice were male,
weighing 16 – 20g and 4 – 5 weeks old of age (Japan SLC, Inc.). Survival analysis was conducted using Log-Rank
test. The statistical analyses were performed using the statistical package R version 2.13.0 (R Development Core
Team, 2011).
The lethality was 90 – 100% in A/J, BALB/c, ddY and ICR strains, 70 – 80% in C3H/He and C57BL/6
strains and 40% in DBA/2 strain (Fig.1 & 2). Survival analysis, using Log-Rank test, clarified that C57BL/6, BALB/c, ddY and ICR strains died earlier and A/J, C3H/He and DBA/2 strains survived longer (Table 1 & 2). The results of ICR mice between 1st and 2nd Experiments were not significantly different (data not shown). These results
indicate that significant mouse strain differences may exist in susceptibility to DSP toxins and that the mouse
strains used may be one of the crucial factors in the mouse bioassay.
Acknowledgements
This study was supported by Health and Labour Sciences Research Grants, Research on Food Safety from
the Ministry of Health, Labour and Welfare of Japan.
263
Fig.1 Survival curves of mice in the 1st Experiment
No. of survived mice
10
8
6
4
2
0
0
3
6
9
12
15
18
21
24
Hours after inoculation
C3H/He
C57BL/6
DBA/2
ICR
Fig.2 Survival curves of mice in the 2nd Experiment
No. of survived mice
10
8
6
4
2
0
0
3
6
9
12
15
18
21
24
Hours after inoculation
A/J
BALB/c
Table 1 Survival analysis of the 1st Experiment
Strain
C3H/He
C3H/He
C3H/He
C57BL/6
C57BL/6
DBA/2
vs
vs
vs
vs
vs
vs
Strain
C57BL/6
DBA/2
ICR
DBA/2
ICR
ICR
Log-Rank test
NS
NS
***
*
NS
***
ddY
ICR
Table 2 Survival analysis of the 2nd Experiment
Strain
A/J
A/J
A/J
BALB/c
BALB/c
ddY
vs
vs
vs
vs
vs
vs
Strain
BALB/c
ddY
ICR
ddY
ICR
ICR
Log-Rank test
*
*
*
NS
NS
NS
References
Food and Agriculture Organization of the United Nations (FAO), 2004. 3. Diarrhoeic Shellfish
Poisoning (DSP). Marine Biotoxins. pp.53-95. http://www.fao.org/docrep/007/y5486e/y5486e00.htm
(accessed on June 24, 2011)
R Development Core Team, 2011. R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria
http://www.R-project.org/ (accessed on June 24, 2011)
Yasumoto, T., 1981. Method for the bioassay of diarrhetic shellfish toxin (in Japanese). Food
Sanit. Res. 31:515-522.
264 
SEROLOGIC SURVEY ON BARTONELLA SPP. AMONG VETERINARY PROFESSIONALS
Wanachalerm N1, Suksawat F.1*, Pachirat O2, Kosoy MY3, Iverson J3 Sripakdee S4, Kerdsin A4,
Dejsirilerk S4, Kanbutra P5, Papirom P6.
1
Department of Veterinary Medicine, Faculty of Veterinary Medicine,
Department of Cardiology Medicine, Faculty of Medicine, Khon Kaen University , Khon Kaen, Thailand,
3
Centers for Disease Control and Prevention, Fort Collins, Colorado, USA,
4
Department of Medical Sciences, Ministry of Public Health, Nontaburi Thailand,
5
Department of Veterinary Teaching Hospital, 6Department of Pathobiology, Faculty of Veterinary Medicine,
Khon Kaen University ,Khon Kaen, Thailand.* Corresponding author
2
Introduction
Bartonella spp. belong to the alpha 2 subgroup of Proteobacteria. Ticks, fleas, lice, sandflies and
mosquitoes are known or suspected vectors for bartonellosis (Rolain et al., 2003; Just et al., 2008). More than 24
described species belong to the genus Bartonella. Seventeen Bartonella sp. of these are currently considered
important causes of human diseases including Bartonella baciliformis, B. rochalimaea, B. quintana,
B. elizabethae, B. clarridgeiae, B. claridgeiae-like, B. grahami, B. kochleae, B. vinsonii subsp. berkhoffii (Bvb),
B. vinsonii arupensis, B. washoensis, B. henselae, B. alsatica, B. doshiae, B. tylorii, B. bovis and B. tamiae.
Seven species have been isolated from dogs and cats including B. henselae, B. clarridgeiae, B. koehlerae,
B. vinsonii subsp. berkhoffii, B. washoensis, B .elizabethae, B. quintana, and are zoonotic potential
(Breitschewerdt et al., 2010). At least 5 species of Bartonella are associated with human illnesses in Thailand:
B. henselae, B. tamiae, B. tribocorum, B. elizabethae, and B. vinsonii arupensis. The objective of this study was
to investigate Bartonella spp. in volunteering human subjects who works at veterinary clinics.
Blood samples from 63 volunteers were collected. Indirect fluorescent assay (IFA) was used for
detection of specific antibodies to B. henselae, B. tamiae, B. tribocorum, B. elizabethae, and B. vinsonii
arupensis antigens.
None serum samples was reactive to B. henselae, B. quintana, B. clarridgeiae, and B. vinsonii arupensis.
This result could be explained by 4 reasons as following: Firstly, in acute phase bartonellosis, significant
reduction of T lymphocyte and CD4+ and significant increment of IFN-gamma and IL-10 may cause nonresponsive cellular immunity (Ticona et al., 2010). Secondly, antibody may not be mounted at the detectable
level. As in the study by Dehio (2001), intraerytrocytic bacteraemia in B. tribocorum naturally infected mice
could soon recover in weeks 8-10. In B. grahamii infected murine model, transient immunocompetency and
persistent B-cell deficit was detected (Koesling et al., 2001). Thirdly the volunteers may be infected with other
Bartonella species. Lastly, pathogen may be phagocytosed before antibodies are stimulated.
265
This study was not corresponding to the serologic study on Bvb and B. henselae in 40 Thai veterinarians
by Suksawat (2006). There were 15 and 28 percents seroreactive to Bvb and B. henselae, respectively. That study
implied that veterinary professions could be at risk to Bartonella infection, whereas this study may indicate that
current Thai veterinary professionals follow good veterinary practice and hygiene. However, molecular
investigations are required to determine the actual infection and to estimate the professional risk among veterinarians in different parts of Thailand.
Acknowledgements
We thank the Graduate School, Khon Kaen University for research funding.
References
Breitschwerdt EB., Maggi RG, Chomel BB and Lappin MR. 2010. Bartonellosis: an emerging infectious disease
of zoonotic importance to animals and human beings. J Vet Emerg Crit Car, 20:8-30.
Dehio C 2001. Bartonella interactions with endothelial cells and erythrocytes. Trends Microbiol. 9(6):279-285
Just FT, Gilles J, Pradel I, Pfalzer S, Lengauer H, Hellmann K, Pfister K. 2008. Molecular Evidence for Bartonella
spp. in cat and dog fleas from Germany and France.Zoonoses Public Hlth. 55(8):514-520.
Koesling J, Aebischer T, Falch C, Schülein R and Dehio C. 2001. Cuttting Edge2001: Antibody-Mediated Cessation of Hemotropic Infection by the Intraerythrocytic Mouse Pathogen Bartonella grahamii1 J Imunol
167(1):11-14.
Rolain JM, Fournier PE, and Raoult D. 2003. First isolation and detection by immunofluorescent assay of Bartonella koehlerae in erythrocytes from a French cat. J Clin Microbiol. 41:4001–2.
Suksawat F. 2006. Serosurvey in Bartonella vinsonii subsp. berkhoffi and Bartonella henselae in Thai veterinarians.
Proc Thai Vet Assoc Congress 32th, Bangkok Thailand. 295-299.
Ticona E, Huaroto L, Garcia Y, Vargas L and Madariaga MG. 2010. The pathophysiology of
human bartonellosis resembles AIDS. Med hypotheses, 74:45-49.
the acute phase of
266 
ANTIMICROBIAL RESISTANT BACTERIA FROM DOGS ISOLATED AT THE VETERINARY
DIAGNOSTIC UNIT, KASETSART UNIVERSITY
P. Hanhaboon1, C. Bumrungpun2, S. Wongnarkpet3, and P. Amavisit1*
1
Department of Microbiology and Immunology, 2The Veterinary Diagnostic Unit,
3
Department of Veterinary Public Health,Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
* corresponding author
Introduction
Effects of antimicrobial drug resistant bacteria on clinical and public health are associated with increase
of illness and disease outbreaks. Resistance to important classes of antimicrobials will limit the choices of drugs
and lead to increasing costs of treatment. This could be public health problem as drug-resistant strains may easily
survive where antimicrobials are used, especially in hospitals. The antimicrobial resistant bacteria can simply
transmit between humans and animals. The occurrences of antimicrobial resistant bacteria from urinary tract, ears
and skin of dogs were reported in this retrospective study.
The collection of bacteria isolated from dog samples from October 2007 to September 2008 at the veterinary diagnostic unit, Faculty of Veterinary Medicine, Kasetsart University, was analyzed by NCSS program
(Hintze, 2000). Clinical samples were isolated on blood agar and MacConkey agar at facultative anaerobic condition following the method of Quinn et al (1994). Pure isolates were tested for antimicrobial resistances using Kirby Bauer disc diffusion method and their resistant break point were analyzed following Performance Standard for
Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals M100 -S17 (CLSI, 2007).
The antimicrobials were chosen for testing in each isolates depending on the bacterial species and suitable antimicrobials for clinical treatment. The available antimicrobials were ampicillin (10 µg), amoxicillin (10 µg ), amoxicillin
-clavulanic acid (30 µg ), ceftriaxone (30 µg), cephalexin (30 µg), cloxacillin (5 µg), oxytetracycline (30 µg)
doxycycline (30 µg), tetracyclin (30 µg), amikacin (30 µg), gentamycin (10 µg), tobarmycin (10 µg), azithromycin (15 µg), erythromycin (15 µg), clindamycin (2 µg), ciprofloxacin (5 µg), enrofloxacin (5 µg), norfloxacin (10
µg), trimethoprim-sulfamethoxazole (25 µg), chloramphenical (30 µg) and imipenem (10 µg).
Most of the isolates were derived from different clinical samples comprising respiratory tract, gastrointestinal tract, genital tract, urinary tract, mammary organ, blood, spinal fluid, and necropsy samples. Top three clinical samples from ear, skin and urinary tract (543, 527 and 488 isolates) were reported for antimicrobial resistances against six commonly used antimicrobial drugs including amoxicillin, amoxicillin/clavulanic acid, cephalexin,
ciprofloxacin, enrofloxacin and sulfamethoxazole/trimethoprim. We found that these isolates were mostly resistant to amoxicillin at 66%, 79%, and 72% respectively. However the resistances decreased to 36%, 44%,
and 34% against amoxycillin/clavulanic acid (Table 1). Therefore extended spectrum beta lactamase resistant
bacteria is olated from dogs in this period were not yet serious problems.
267
Ear specimens were the popular collected samples for microbiological test. In this study otitis were caused
by Staphylococcus spp at the highest rate at 35% (192/543). The resistant rates of bacteria from ear were different
in each bacterial groups (Table 2). Pseudomonas was highly resistant to betalactam drugs (amoxicillin, amoxicillin/
clavulanic acid and cephalexin) at 91%, 86%, and 89% respectively. However Staphylococcus spp were resistant to
gentamicin at 49% but resistant to amoxycillin/clavulanic only at 8.5%. Therefore the antimicrobial of choice for
otitis treatment in dogs should be concerned carefully following the species of bacterial infection. Commonly Pseudomonas spp were found in chronic otitis externa, while Staphylococcus spp were the causes of acute otitis externa.
Furthermore otitis is usually occurred from co-infection of both bacteria and yeast (Lyskova et al 2007, Müller E,
and Heusinger A.1994).
Antimicrobial resistant properties usually were encoded by mobile genetics elements that could horizontal
transfer among bacterial strains or move across their species. Antimicrobial resistant bacteria lead to severe
infection and treatment failure. Since clinical laboratories are first to encounter bacteria with new forms of drug
resistance, the report of antimicrobial resistant bacteria in veterinary hospital should be done routinely and consistently as one of the public health surveillance scheme.
Table 1 Antimicrobial resistant bacteria from urinary tract, skin and ears of dogs were tested against six
antimicrobial drugs.
Antimicrobials
Ear
Skin
Urinary
amoxicillin
349/528 (66%)
408/518 (79%)
333/465 (72%)
amoxicillin/clavulanic acid
194/532 (36%)
229/519 (44%)
162/476 (34%)
cephalexin
265/531 (50%)
316/522 (61%)
243/475 (51%)
ciprofloxacin
136/532 (26%)
232/523 (44%)
292/477 (61%)
enrofloxacin
179/532 (34%)
265/523 (51%)
309/468 (66%)
sulfamethoxazole/trimethoprim
308/491 (63%)
318/523 (61%)
326/453 (72%)
268 
Table 2 The antimicrobial resistant rates of three common bacteria isolated from ears of dogs brought to
Kasetsart University Veterinary Hospital in 2008
Bacteria
AMX AMC
CFL
CRO
Staphylococcus spp 108/187 16/188 63/188
41/132
57.8%
Pseudomonas spp
E.coli
8.5%
33.5%
31%
142/156 134/156 138/155 34/132
ENR
NOR
CIP
81/188 70/184
77/189
84/169
15/187
104/177
40.7%
49.7%
8%
58.8%
49/156 14/154
21/155
20/134 121/155 117/142
43%
38%
CN
DO
SXT
91%
85.9%
89%
25.8%
31.4%
9.1%
13.5%
14.9%
78.1%
82.4%
36/46
14/46
16/46
8/41
19/46
14/46
19/46
16/40
21/46
25/41
78.3%
30.4%
34.8%
19.5%
41.3%
30.4%
41.3%
40%
45.7%
61%
AMX, amoxicillin; AMC, amoxicillin/clavulanic acid; CFL, cephalexin; CRO, ceftriaxone; ENR, enrofloxacin;
NOR, norfloxacin; CIP, ciprofloxacin; CN, gentamicin; DO, doxycycline; SXT, sulfamethoxazole/trimethoprim
Acknowledgments
The study was funded by Kasetsart University Research and Development Institute (KURDI).
References
Clinical and Laboratory Standards Institute. 2007. Performance Standards for Antimicrobial Susceptibility Tests;
Seventeenth Informational Supplement. M100 -S17. Wayne, U.S.A.
Hintze, J. (2000). NCSS 2000, LLC. Kaysville, Utah, USA.
Lyskova P, Vydrzalova M and Mazurova J. 2007. Identification and Antimicrobial Susceptibility of Bacteria and
Yeasts Isolated from Healthy Dogs and Dogs with Otitis Externa. J. Vet. Med. A 54: 559–563
Müller E, and Heusinger A. 1994 Microbiological results of ear swabs from dogs and cats. Tierarztl Prax. 22:80-4.
Quinn, PJ, Carter, ME, Markey, B, and Carter GR 1994. Clinical Veterinary Microbiology. Wolfe publishing.
London
269
A NEW GENOTYPE OF BARTONELLA ISOLATES FROM DEER (RUSA TIMORENSIS) IN THAILAND
D. Pangjai1*, S. Intachinda2, S. Maruyama3, S. Boonmar4, H. Kabeya3, W. Petkanchanapong1,
W. Wootta1, P. Wangroongsarb1, M. Boonyareth1, P. Preedakoon1,
W. Saisongkorh1 and P. Sawanpanyalert1
1
National Institute of Health, Department of Medical Sciences, Thailand.
Nongkwang Livestock Breeding and Research Center, Ratchaburi Province,Thailand.
3
Laboratory of Veterinary Public Health, Nihon University, Japan.
4
International Emerging Infections Program, Thailand MOPH-U.S. CDC Collaboration.
* Corresponding author E-mail : [email protected]
2
Introduction
Genus Bartonella are small, gram-negative aerobic bacilli that are difficult to grow in culture, mainly transmitted by vector. These microorganisms infect erythrocytes of their mammalian hosts, and some species cause a
wide spectrum of human illness, such as chronic bacteremia, fever and endocarditis. Identification of the bacteria is
based on results of polymerase chain reaction (PCR) assay, followed by sequencing of several housekeeping genes
including citrate synthase (gltA), RNA polymerase beta subunit (rpoB), cell division-associated protein (ftsZ), heat
shock protein (groEL), riboflavin synthase alpha chain (ribC) and 16-23S rRNA intergenic spacer (ITS) genes.
Samples :
During February to March 2011, 247 blood samples from deer (Rusa timorensis) at Nongkwang Livestock
Breeding and Research Center, Ratchaburi Province, Thailand were collected form jugular vein into EDTA tubes.
These tubes were stored at -80 °C and tested at National Institute of Health, Thailand.
Isolation of bacteria :
The frozen blood samples were thawed at room temperature and centrifuged at 1,800 g for 60 min. After
centrifugation, the pellets were plated onto brain heart infusion agar (BHIA) plates containing 5% defibrinated
rabbit blood. The plates were incubated at 35°C under 5% CO2 for 4 weeks. Two or three of small and rough gray
colonies were picked from each plate, streaked out on fresh plates, and further cultured under the same conditions.
PCR amplification of gltA, rpoB, ftsZ and ITS:
Genomic DNA was extracted from each isolate of Bartonella spp. by using an Instagene matrix (Bio-Rad,
Hercules,CA.).The primers used for amplification and sequencing as following (1) gltA : BhCS.781p (5GGGGACCAGCTCATGGTGG-3), BhCS.1137n (5-AATGCAAA AAGAACAGTAAACA-3) (2) rpoB : 1400F
(5-CGCATTGGCTTACTTCGTATG-3), 2300R (5-GTAGACTGATTAGAACGCTG-3) (3) ftsZ : Bfp1 (5ATTAATCTGCAYCGGCCAG A-3), Bfp2 (5-ACVGADACACGAATAACACC-3) and (4) ITS : 16SF (5AGAGGCAGGCAAC CACGGTA-3), 23S1 (5-GCCAAGGCATCCACC-3). The current procedures for isolation
and PCR amplification followed by Inoue et al. (2008).
Specificity and sensitivity of PCR method were carried out under optimal reaction condition. 7 Bartonella spp.
strains and 20 other bacterial species were tested for evaluate the sensitivity and specificity. To study the detection
limit of PCR assay, serial 10 fold dilutions of
270 
B. henselae suspension in rang of 1x108 -10 0 CFU /ml were extracted DNA and performed. The sterile
distilled water and Francisella tularensis were used as negative control instead of the DNA template, B.henselae
was used as positive control in each reaction cycle. The PCR products were analysed by gel electrophoresis. The
limit of detection was defined as the lowest concentration of B. henselae that provided bands of PCR product.
phylogenetic analysis of gltA, rpoB, ftsZ and ITS :
The CLUSTAL_X program was used for the alignment of Bartonella sequences. The data that obtained in
this study were deposited in the GenBank/EMBL/DDBJ databases. A phylogenetic tree was drawn based on the
sequences of rpoB and gltA, using the neighbor-joining method with Kimura’s two-parameter distance method in
MEGA 5. Bootstrap analysis was carried out with 1,000 resamplings. A criterion of ≥ 96% homology to gltA,
rpoB, ftsZ and ITS was used to define groups.
The limit of detection was approximately 1-1.5 B.henselae CFU /ml. The sensitivity of the PCR by using
two targetting citrate synthase (gltA) and β-subunit of the RNA polymerase (rpoB) was 100% which showed the
positive results of all 7 reference Bartonella spp. strains. The specificity of the assay was verified by an absence of
amplification product when tested against 20 other bacteria. The primer rpoB and gltA have been well used for differentiating Bartonella spp. because of the much lower degrees of similarity between these genes in Bartonella spp.
In total, Bartonella spp. were obtained 9 deer (3.6 %). Among 110 males and 137 females were 3 (2.7%)
and 6 (4.5%) positive findings, respectively. Age of R. timorensis was high prevalence in 2-4 years. We revealed
that the isolated Bartonella were classified into new genotype of Bartonella by using four targetting gltA, rpoB,
ftsZ and ITS genes. These results are the first report in identification of new genotype of Bartonella from the deer
blood in Thailand. The prevalence of Bartonella in other animals are ongoing.
Figure.1
***
New genotype of Bartonella from Rusa timorensis was classified by phylogenic tree analysis using rpoB.
Acknowledgements
We appreciated the knowledge from Drs. Kai Inoue, Chi Shih-Hui, Shingo Sato and Naris Tengchaisri.
References
Inoue K, Maruyama S, Kabeya H, Yamada N, Ohashi N, Sato Y, Yukawa M, Masuzawa T, Kawamori F, Kadosaka T, Takada N, Fujita H, Kawabata H. 2008. Prevalence and genetic diversity of Bartonella species
isolated from wild rodents in Japan. Appl. Environ. Microbiol. 74(16): 5086–5092.
Matsumoto K, Berrada ZL, Klinger E, Goethert HK, Telford SR. 2008. Molecular detection of Bartonella
schoenbuchensis from ectoparasites of deer in Massachusetts. Vector Borne Zoonotic Dis. 8(4): 549–554.
Saisongkorh W, Rolain JM, Suputtamongkol Y, Raoult D. 2009. Emerging Bartonella in humans and animals in
Asia and Australia. J Med Assoc Thai. 92(5): 707-31.
271
THE EVALUATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR
Ehrlichia canis DETECTION
N. Tipmongkolsilp1 , N. Viseshakul2*
1
2
Clinical for Aquatic Animals, Faculty of Veterinary Medicine, Mahanakorn University, THAILAND.
The Unit of Parasitology, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University,
THAILAND. * Corresponding author
Introduction
Canine ehrlichiosis is an important canine disease in tropical area, including Thailand. The cause of this disease is
the obligate intracellular rickettsia Ehrlichia canis which transmitted by brown tick (Rhipicephalus sanguineus)
(Groves et al., 1975). Specific diagnosis of ehrlichiosis can be made using a blood smear, indirect immunofluorescence assay, ELISA, cultivation, Western blot, and polymerase chain reaction (PCR) (Cadman et al., 1994;
Iqbal et al., 1994; Hegarty et al., 1997). Recently, introduction of loop-mediated isothermal amplification (LAMP)
has resulted in development of another rapid, sensitive, and specific diagnostic method (Notomi et al., 2000). The
purpose of this study is to develop LAMP assay for alternative to standard PCR for routine diagnosis of E. canis.
Two sets of LAMP primers were designed against the specific region of E. canis strain Jake (accession:
NC_007354.1 version GI: 73666633). The LAMP reaction was conducted as described previously (Notomi et al.,
2000). To confirm that LAMP amplified the correct target, the product was electrophoresis in a 2% agarose gel
and visualized under a UV light after staining with ethidium bromide. The LAMP specificity was conducted using
each plasmid contained
E. canis, Anaplasma platys and Babesia spp. (Ariyawutthipan et al., 2008). The specific primers for compare specificity test, followed previous study which are E. canis using ECAN5 and HE3
(Murphy et al., 1998) and A. platys using EP2 and EP3 (Chang and Pan, 1996). The Babesia spp. primers were
designed from the consensus regions of five sequences of 18S ribosomal RNA gene (accession number: X59604,
AJ439713, AY072926, AY072925 and BOU16369) from GenBank. The PCR was performed as follows; in the
volume of 20 μl, there were 2 μl of template DNA, 2 μl of 10 mM forward primer, 2 μl of 10 mM reverse primer,
10 μl of 5U/μl i-Taq PCR master mix (Intron®, Korea) and sterile distill water was added to the volume of 20 μl.
The PCR cycles were applied accordingly (Murphy et al., 1998).
Amplification of LAMP specific primers to E.canis plasmid resulted in successful amplification of the target gene
whereby positive LAMP products demonstrated as a typical ladder pattern (Figure 1A). Similar results were observed in PCR with specific primers, where single bands of each plasmid were identified for A. platys (Figure 1B)
and Babesia spp. (Figure 1C). These preliminary study showed the possible to develop an efficiently disease detection with the specificity and simplicity method.
272 
(A)
(B)
(C)
Figure 1 Specificity of LAMP primers for the detection of Erhlichia canis (A). Lanes (M) 100 bp DNA ladder;
(1) E. canis plasmid; (2) Anaplasma platys plasmid; (3) Babesia spp. plasmid.
PCR amplification of A. platys plasmid (B). Lanes (M) 100 bp DNA ladder; (1) A. platys specific primer, EP2 and
EP3; (2) Specific LAMP outer primers, B3 and F3; (3) Specific LAMP inner primers, BIP and FIP. PCR amplification of Babesia spp. plasmid (C). Lanes (M) 100 bp DNA ladder; (1) Babesia spp. specific primer; (2) Specific
LAMP outer primers, B3 and F3; (3) Specific LAMP inner primers, BIP and FIP.
Acknowledgements
We would like to thank all staffs from the units of Parasitology and Biochemistry, Faculty of Veterinary Science,
Chulalongkorn University, Bangkok, Thailand.
References
Ariyawutthiphan, O., Shokshai-utsaha, K., Sananmoung, T., Chungpivat, S., Sarikaputi, M. and Viseshakul, N.
(2008). The microscopic and molecular detections of canine Ehrlichiosis. Thai Journal of Veterinary Medicine, 38, 29-36.
Cadman, H.F., Kelly, P.J., Matthewman, L.A. and Zhou, R. (1994). Comparison of the dot-blot enzyme linked
immunoassay with immunofluorescence for detecting antibodies to Ehrlichia canis. Veterinary Record, 135,
362.
Chang, W.L. and Pan, M.J. (1996). Specific amplification of Ehrlichia platys DNA from blood specimens by twostep PCR. Journal of Clinical Microbiology, 34, 3142-3146.
Grove, M.G., Dennis, G.L., Amyx, H.L. and Huxsoll, D.L. (1975). Transmission of Ehrlichia canis to dogs by
ticks (Rhipicephalus sanguineus). American Journal of Veterinary Research, 36, 937-940.
Hegarty, B.C., Levy, M.G., Gager, R.F. and Breitschwerdt, E.B. (1997). Immunoblot analysis of the immunoglobulin G response to Ehrlichia canis in dogs: an international survey. Journal of Veterinary Diagnosis Investigation, 9, 32-38.
Iqbal, Z., Chaichanasiriwithaya, W. and Rikihisa, Y. (1994). Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis. Journal of Clinical Microbiology, 32, 1658-1662.
Murphy, G.L., Ewing, S.A., Whitworth, L.C., Fox, J.C. and Kocan, A.A. (1998). A molecular and serologic survey
of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Veterinary Parasitology,
79, 325-339.
Notomi, T., Hiroto, O., Harumi, M., Toshihiro, Y., Watanabe, K., Nobuyuki, A. and Hase, T. (2000). Loopmediated isothermal amplification of DNA. Nucleic Acids Research, 28, e63.
273
IDENTIFICATION OF A PUTATIVE LRR CODING GENE IN L. INTERROGANS
A. Kunjantarachot1, 2, S. Nitipan1, 2, T. Sritrakul1, 2, C. Suphatpahirapol1, 2, N. Chotechuang2, S. Prapong1,
2*1
Interdisciplinary Graduate Program in Genetic Engineering, The Graduate School, Kasetsart University,
2
Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University, THAILAND.
Introduction
Infection with a group of pathogenic spirochetes of genus Leptospira leads to the illness called
leptospirosis, an important global re-emerging zoonosis. Current commercial leptospirosis vaccine could not provide
complete protection against heterologous serovars therefore attempts to identify conserved proteins among pathogenic leptospires that generate cross protection for subunit vaccines are being made. Leucine-rich repeat or LRR
containing proteins become an interesting vaccine candidates since they usually involve in invasion mechanism,
virulence and/or pathogenesis of the bacteria. Many LRR proteins have been reported in various pathogenic
bacterial species, for example Y. pestis (Kobe and Kajava, 2001). A study by Seepersaud et al. (2005) showed that
mice immunized with recombinant LRR protein, LrrG, from S. agalactiae were protected against lethal challenge.
In Leptospira spp., there is no officially report of LRR proteins. Like LrrG protein, leptospiral LRR proteins may
be essential for disease virulence as well as host cell attachment or disease pathogenesis and may serve as
potential subunit vaccine candidate. So the aim of this study is to identify LRR coding gene in L. interrogans
which related to L. borgpetersenii LRR coding gene.
Material and Methods
Genomic DNA of L. interrogans serovar Autumnalis, Canicola, Hebdomadis, and Pyrogenase were used as
DNA templates. LRR coding gene (Locus tag: LBJ_2271) of L. borgpetersenii serovar Hardjo-bovis strain JB197
was defined as template in blastn algorithm to find similar LRR coding genes in L. interrogans serovar Lai to be a
template for primer design. Gene LA_1324 was selected and primers were designed by MAC Vector ™ Suite
7.2.3. PCR was conducted to amplify putative LRR coding gene from gDNA of each serovar using gene-specific
primers. Then purified PCR products were subjected to sequence. Obtained DNA sequences were analyzed by
BLAST for matching with known nucleotide sequences deposited on NCBI databases. Sequences were further
aligned by ClustalW2 program to define any identity, similarity and difference among multiple sequences and
were translated to amino acid sequence by tools in Expasy Proteomic Server. Protein sequences were analyzed for
protein function. To examine evolutionary interrelation of LRR coding gene of all serovars presented in this
study, phylogenetic tree was built by MEGA4 program (Tamura et al., 2007).
PCR using forward 5′-CTCTTCCTGGTTGGATTGGAG-3' primer and reverse 5'-CTTGTATTTGGGA
GAACTGCTTG-3' showed a single DNA fragment amplified from each Leptospira serovars: a 470 bp fragment
from serovar Canicola, a 450 bp fragment from serovar Pyrogenase, and a fragment of 500 bp from serovar
Autumnalis and Hebdomadis. DNA sequences of all serovars obtained from sequencing were slightly shorter than
size of amplified products, about 410 bp in length, due to the presence of ambiguous bases in 5′ and 3′ end.
BLAST analysis demonstrated that putative LRR coding gene of all serovars matched LRR coding gene LA_1324
and LIC_12401 from L.interrogans with over than 90% identity and matched LBJ_2271 from L. borgpetersenii
with 80% identity.
274 
Multiple sequence alignment analysis of LA_1324 and all putative LRR coding genes showed that they have nucleotide identity over than 80%. This indicated that this gene is conserved among pathogenic serovars. All DNA
sequences were translated into amino acid sequence and scanned for functional or structural motifs in protein databases e.g. pfam, Prosite using protein characterization tools to confirm whether they are LRR coding gene. Distinct
LRR consensus sequence LxxLxLxxN/CxL was presented in all translated amino acid sequences and protein sequence analysis tools indicated the LRR motif within all queries. Phylogenetic analysis of conserved sequences
among LA_1342, LIC_12401, LBJ_2271, and four putative LRR coding genes (Figure 1) revealed close relatedness among this gene in L. interrogans serovars whereas gene of L. borgpetersenii has the highest distance when
compared to the group of LRR gene in L. interrogans. Among L. interrogans group, the nearest relative of
LA_1324 which is template for primer design is LRR coding gene of serovar Pyrogenase, while gene of serovar
Hebdomadis is the most evolutionary distant.
Figure 1 Phylogenetic tree of the LRR coding genes from multiple serovars of L. interrogans and similar representative gene of L. borgpetersenii using MEGA4 software.
In conclusion, there is similar LRR coding gene existed in L. interrogans as in L. borgpetersenii. The gene
is conserved among pathogenic L. interrogans serovars, suggesting the potential significance of this LRR coding
gene in virulence and pathogenesis therefore it is very interesting to investigate the presence of this gene in other
serovars of L. interrogans for its possibility to become leptospiral vaccine candidate.
Acknowledgements
We thank The program Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program
Thai Doctoral degree, the Office of the Higher Education Commission, Thailand for research funding. Partial
support is provided by the Interdisciplinary Graduate Program in Genetic Engineering, the Graduate School,
Kasetsart University.
References
Kobe B, Kajava AV. 2001. The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11(6):
725-732.
Seepersaud R, Hanniffy SB, Mayne P, Sizer P, Page RL, Wells JM. 2005. Characterization of a novel leucine-rich
repeat protein antigen from Group B Streptococci that elicits protective immunity. Infect Immun 73:
1671–1683.
Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software
version 4.0. Mol Biol Evol 24(8):1596–1599.
Oral and Poster
Presenting Authors Index
277
ORAL AND POSTER PRESENTING AUTHORS INDEX
A. Poopuak, 151
Buh-nga Chindawanichsakul, 74
A. Sailasuta, 111
Bunlue Kornmatitsuk, 131
A. Sommanustaweechai, 75
C. Bumrungpun, 145
Abdulazeez O.I, 114
C. Piamsakun, 151
Adisak Sangkaew, 118
C. Rodkhum, 85
Alongkorn Amonsin, 123
C. Salakij, 138
Alongkorn Kulilung, 102
C. Sornklein, 101
Angkhana Khantaboot, 129
Chailai Koowatananukul, 87
Anthicha Kunjantarachot, 153
Chaiyakorn Thitiyanaporn, 98
Anusak Kerdsin, 76, 144
Chamaiporn Sodawichit, 107
Arayaporn Macotpet, 102, 103, 104
Chanita Sujaritthanyatrakul, 129
Aree Thayananuphat, 96, 97, 137
Channarong Rodkhum, 128
Arjan Stegeman, 80
Chareon Thongma, 109
Arpapan Kanbanjong, 103
Chattip Suphatpahirapol, 153
Athipoo Nuntaprasert, 90, 124, 126, 127
Chayakit Sinthusing, 105, 107
Attapol Kanna, 104
Chayakrit Sinthusingha, 106
Attawit Kovitvadhi, 137
Chinnawat Kasemmongkolchai, 107
Atthariya Jiranaparat, 106
Chinwiwat Piamsakun, 152
B. Sangkarak, 75
Chommanart Thongkittidilok, 110
Boonchoo Piempinseat, 137
Chomporn Chokboonmongkol, 78, 88
Boonrit Thongsong, 125
Chonlada Prabwongsa, 103
278 
Chowalit Nakthong, 131
K. Imsilp, 92
D. Nilubol, 84
K. Kanistanon, 151
D. K. Nugroho, 75
K. Prihirunkit, 138
D. Nilubol, 73
K. Taweeseneepitch, 75
Decha Pangjai, 148
K. Unjit, 75
Denny Widaya Lukman, 89
K. Wongsathapornchai, 75
Donruethai Sreta, 123
Kalaya Kengvikkum, 74
Eakkasit Barameechaithanan, 102
Kamonchanok Aiemsumaung, 137
Ekkachai Patarapanwichien, 102, 103, 104
Kanchana Poonsuk, 86
F. Wu, 75
Kanokpan Boonpong, 121
Fanan Suksawat, 76, 144, 146, 147
Karl-Hans Zessin, 78, 88
H. Sinel, 75
Karn Yongwahish, 103
Hathairat Chanphao, 83
Karoon Chanachai, 80
Hidenori Kabeya, 148
Kawintra Aiyaranoi, 102
Hodaka Suzuki, 77, 140
Kenji Machii, 140
I.O Abdulazeez, 112, 113
Ketkaew Wasanasuk, 107
ISSARAPORN PHROMNAO, 100
Ketmanee Senapan, 121
J. Kampa, 151
Ketsarin,Kamyingkird, 147
Jaruwan Kampa, 152
Khin Khin Lay, 87
Jennifer Iverson, 144
Kittisak Ajariyakhajorn, 134, 136, 141
Jutanun Laoumanotham, 102
Kochakorn Direksin, 121
K. Apisumputthangkur, 111
Komkrich Teankum, 150
K. Chanachai, 75
Kornkaew Thongtaeng, 122
K. Duangprathum, 117
L. L. Bo, 75
279
Lamai Nammongkol, 143
Nantawan Tangwatcharapong, 124
Lapapatr Phutdhikarnt, 106
Naris Thengchaisri, 98, 105
M. Gholamia Ahangaran, 71
Narong Abking, 130
M. Pattiyapong, 75
Narong Tiptanavattana, 82
M.M Noordin, 112, 113
Narudee Kashemsant, 83
Maneenard Choodetwattana, 121
Nathatai Wanachalerm, 144
Marc Desquesnes, 147
Nattanant Sirivattanatanyakul, 74
Maskiet Boonyareth, 148
Nattapol Manojai, 104
Mathirut Mungthin, 79
Natthanan Prasert, 150
Michael Y. Kosoy, 144
Natthanet Sritrakoon, 96, 97
Mirjam Nielen, 80
Nichapat Yurayart, 141
Miruntee Penroj, 122
Nillawan Tammasiri, 104
Monchai Lekjarernwong, 105, 106
Niorn Ratanapob, 129
Monchanok Vijarnsorn, 83
Nipa Chokesajjawatee, 78
Mongkol Techakumphu, 82, 110
Nirun Chunakoop, 134
N. Ketusing, 75
Nongnuch Pinyopanuwat, 146, 147
N. Kiry, 75
Noordin M.M, 114
N. Phookrongta, 117
Nopadon Pirarat, 128
N. Rangsaz, 71
Nuch Chotechuang, 153
N. Saengklub, 99
Ong-ard Lawhavinit, 130
N. Sannamwong, 99
Orathai Pachirat, 144
N. Tipmongkolsilp, 149
P. Amavisit, 119, 145
N. Viseshakul, 149
P. Boosom, 75
Nakrob Pattanapon, 107
P. Hanhaboon, 145
280 
P. Kayansamruaj, 85
Piyanat Prasomsri, 134, 141
P. Lertwatcharasarakul, 138
Piyasak Wipoosak, 103
P. Lorsomsup, 111
Pongrat Jaisil, 104
P. Nopadon, 85
Poom Preedakoon, 148
P. Pata, 117
Pornchai Sanyathitiseree, 137
P. Songjinda, 119
Pornchalit Assavacheep, 120, 122
P. Sriphutthachot, 99
Prapas Patchanee, 78, 88
P. Wongnark, 75
Pratchaya Prapaiwong, 121
Paisan Tienthai, 150
Pravina Kitikoon, 123
Panithan Jessadakarn, 124
Prayong Sangsriruang, 118
Panpicha Sattasathuchana, 105
Preeda Lertwatcharasarakul, 96, 137
Panus Wongnonti, 128
PREENUN JITASOMBUTI, 100
Panyaphat Pomim, 136
Rath Pichyangkura, 125
Pareeya Udomkusonsri, 98
Risaar Siringo-ringo, 142
Patchanee Sringam, 118
Rissar Siringo Ringo, 89
Patcharida Dittawong, 137
Roongroj Thanawongnuwech, 79
Pathom Sawanpanyalert, 148
Roongroje Thanawongnuwech, 150
Paveenuch Charoenkitroj, 136
Roongroje Thanawongnuwech, 123
Peerasak Suttiyotin, 131
Ruangurai Kitchodok, 150
Pilinporn Kanrai, 128
Rungtip Chuanchuen, 86, 87
Pimchanok Thonglon, 76
S Jeamprapai, 92
Pithai Kanbutra, 144
S Netramai, 92
Pittaya Papirom, 76, 144
S Poapolathep, 92
Piyada Wangroongsarb, 148
S. Aiumlamai, 151
281
S. I. Jayme, 75
Shunya Sanla, 141
S. Khuhapan, 75
Sineenart Chantarachart, 76
S. Ponglowhapan, 99
Sirawit Pagdepanichkit, 86
S. Sangmaneedet, 117
Sirikachorn Tangkawattana, 152
S. Sinthasak, 75
Siriluk Jala, 137
S. Sukontasing, 119
Siriruk Chantrakru, 83
S. Theraverapanya, 75
Siriwan Prapong, 153
S. Urbenjapol, 75
Siriyaporn Thongwatchara, 122
S. Wangnaitham, 111
Sittikorn Traiyarach, 150
S. Wongnarkpet, 145
Soawalak Sripakdee, 76, 144
S.Kunakornsawat, 92
Soichi Maruyama, 148
Sakultip Amsakul, 79
Somboon Sangmaneedet, 146, 147
Sakuna Phatthanakunanan, 137
Somdech Tunboonjit, 150
Samit Srisomrun, 129
Somkiat Huaijantug, 131
Sanipa suradhat, 123
Sommai Homsavart, 72
Santaya Intachinda, 148
Sompop Chittapraphai, 143
Saovanee Leelayoova, 79
Sonthaya Tiawsirisup, 79
Sarawut Sringam, 118
Srihadi Agungpriyono, 142
Sarinee Kalandakanond-Thongsong, 125
Sukolapa Chiarasumran, 120
Sarinya Pornaem, 78
Sukuma Samngamnim, 120, 122, 136, 141
Sathaporn Jittapalapong, 146, 147
Sumalee Boonmar, 148
Satidyos Soontronsatidpimon, 128
Supachai Nitipan, 153
Sheila, 89, 142
Supada, 129
Shigeki Yamamoto, 77, 139
Suphap Kumlangphaet, 128
282 
Suphot Wattanaphansak, 120
Teerapat Rungnirundorn, 107
Supot Wattanaphansak, 122
Tepyuda Sritrakul, 153
Supratikno, 142
THANAKORN SRIRAT, 100
Surang Dejsirilerk, 76, 144
Thanasak Boonserm, 141
Suraphan Boonyawatana, 120
Thanawat Tiensin, 80, 143
Sureerat Lopiroon, 120
Thanida Sananmuang, 110
Sureerat Suthongsa, 125
THANIKUL SRITHUNYARAT, 100
Suthinee Krisnakupt, 136
Thaweesak Songserm, 80
Suttisak Nopwinyoowong, 152
Theerawat Tharasanit, 82, 110
T. Chalalai, 138
Theerayudh Sukmee, 79
T. Eksiritrirat, 111
Thomas Alter, 78, 88
T. Hoonsuwan, 84
Thunyakan Nithisakdiyanond, 106
T. Kedkhuntod, 75
Tipawadee Seidkhuntod, 96
T. Kimprasit, 119
Tirapa Rattanamatee, 141
T. Lamaisri, 75
TRASIDA PLOYNGAM, 100
T. Mamom, 108
Trisukhon Phongsayoikham, 120
T. Nedumpun, 111
Ukadej Boonyaprakob, 72
T. Nilnont, 151
V. T. Le, 75
T. Srisuvan, 75
Vilaiporn Wongphruksasoong, 143
T. Tripipat, 84
Vo Phong Vu Anh Tuan, 90, 126, 127
T. Vasaruchapong, 138
W. Posuya, 75
Taksaon Duangurai, 96
W. Tonpitak, 101
Tawee Naaglor, 79
Wacharapon Chotiyaputta, 143
Tawin Inpankaew, 146
Walaitip Wongthai, 122
283
Wallaya Phongphaew, 109
Wanchart Yippaditr, 106
Wandee Kongkaew, 80
Wandee Thiangtum, 129
Wanida Laohasurayothin, 109
WANPITAK PONGKAN, 100
Wanvisa Prasong, 106
Warangkhana Phanwanich, 81
Waraporn Sinsuwonkwat, 137
Watcharee Saisongkorh, 148
Wattanapong Wootta, 148
Wichukarn Fuangsawat, 130
Wimol Petkanchanapong, 148
Wissanuwat Chimnoi, 146, 147
Wiyada Winyuchonchareon, 124
Woraporn Sukhumavasi, 79
Yodchanan Wongsawat, 81
Invited Speaker Full Paper
285
Invited Speaker Full Paper Contents
SEROVAR IDENTIFICATION AND ANTIMICROBIAL RESISTANT PATTERN OF AVIBACTERIUM PARAGALLINARUM
………………………………………………………………………………………………………………………………………………………………………….286
EXTRA-LABEL DRUG USE IN EXOTIC PET…………………………………………………………………………………………………….……….290
HEMOGLOBIN VESICLE TRANSFUSION IN CANINE HYPOLVOLAEMIC SHOCK……………………………………………….……..292
EVIDENCE-BASED VETERINARY MEDICINE (EBVM)……………………………………………………………………………………….…… 295
DO AND DON’T IN EXOTIC PETS…………………………………………………………………………………………………………………….…..300
TREATMENT OF RABBITS WITH LONG BONE FRACTURES WITH TIE-IN CONFIGURATION INTRAMEDULARY PINS
ATTACHED TO EXTERNAL SKELETAL FIXATOR DEVICE: 25 CASES (NOVEMBER 2009- NOVEMBER 2010)
………………………………………………………………………………………………………………………………………………………………………....304
ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIAL ISOLATES OF PSITTACINE BIRDS IN THAILAND 2011………………..305
ADVANCED IMMUNOLOGY AND VACCINOLOGY FOR LIVESTOCK DISEASES
SECTION I: NATIVE DEFENSE MECHANISMS……………………………………………………………………………………………………....306
ADVANCED IMMUNOLOGY AND VACCINOLOGY FOR LIVESTOCK DISEASES
SECTION II: CELL MEDIATED IMMUNITY…………………………………………………………………………………………………………….310
REHABILITATION FOR ORTHOPEDIC SURGERY…………………………………………….……………………………………………………..317
NEUROLOGICAL EMERGENCIES…………………………………………………………………….……………………………………………………319
BECOMING A CLINICAL VETERINARY ONCOLOGIST – THE IMPORTANT THINGS TO KNOW………………………………..324
CLINICAL SMALL ANIMAL ONCOLOGY CASES……………………………………………………………………………………………………..329
BLOOD TRANSFUSION TECHNIQUE IN DOGS AND CATS: ICVS 2011 SCIENTIFIC PROGRAM (27TH OCT 2011)
………………………………………………………………………………………………………………………………………………………………………....337
UPDATE BOVINE HORMONE (AN UPDATE ON REPRODUCTIVE REGULATION IN ANIMALS)………………………………..345
EPIDEMIOLOGY OF MASTITIS IN DAIRY COWS…………………………………………………………………………………………………...347
UPDATE ON INFECTIVITY, TRANSMISSION, AND PATHOGENICITY OF FMDV AND RESEARCH NEEDS ………………...350
A MOLECULAR APPROACH TO STUDY GENETIC DIVERSITY………………………………………………………………………………...354
REVIEWS SEX IDENTIFICATION: BREAKTHROUGH IN MOLECULAR APPLIATIO…………………………......…………………...356
APPLICATION OF THE MITOCHONDRIAL DNA VARIATIO FOR CAPTIVE BREEDING MAAGEMENT IN ENANGERED
MALAYAN TAPIR (TAPIRUS INDICUS) IN THAILAND ……………………………………………………………………………...…………..358
GENETIC VARIATION IN WILD AND DOMESTICATED BUFFALOES IN THAILAND………………...……………………………….361
A NOVEL GENETIC MARKER FOR SUBSPECIES IDENTIFICATION AND GENETIC DIVERSITY IN TIGER……..……………. 363
MAMALIAN AND DNA BARCODING : MITOCHONDRIL CYTOCHROME B AS AN IDEAL MOLECULAR MARKER FOR
SPECIES IDENTIFICATION………………………………………..………………………………………………………………………………………… 365
ALTERNATIVE MEDICINE IN AQUACULTURE……………………………………………………………………………………………………….367
EPIDEMIOLOGY OF MASTITIS IN DAIRY COWS…………………………………………………………………………………………………...368
COMPARTMENTALISATION: WHERE ARE WE NOW?..............................................................................................369
PRRSV-HOST INTERACTION: INNATE IMMUNE RESPONSE………………………………………………………………………………….373
FMD VACCINE: RECOMMENDATIONS AND PROSPECTS OF IMPROVED VACCINE ……………………………………………...376
UPDATE ON ANTIGENIC VARIATION OF FOOT AND MOUTH DISEASE VIRUS IN SOUTH EAST ASIA……………………..383
ANTIMICROBIAL USAGES AND FOODBORNE ANTIMICROBIAL RESISTANCE IN ANIMALS.......................................388
AQUACULTURE FOOD SUSTAINABLE AND THREAT…………………………………………………………………………………………… 389
UPDATE MASTITIS: TREATMENT OF BOVINE MASTITIS DURING LACTATING PERIOD…………………………………………..394
286 
Serovar identification and antimicrobial resistant pattern of Avibacterium paragallinarum
Niwat Chansiripornchai and Kridda Chukiatsiri
Avian Heath Research Unit, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, email:
[email protected], Tel./Fax 02 252 9575
Abstract
Eighteen field isolates of Av. paragallinarum obtained from chickens were serotyped by the hemagglutination inhibition test. All of the isolates were NAD-dependent and serotyping revealed that 10, 5 and 3 isolates were
serovar A, B and C respectively. Seventeen antimicrobial sensitivity disks were used for disk diffusion assays. All
isolates were susceptible to amoxicillin-clavulanic acid and were resistant to cloxacillin and neomycin. There was a
high level of resistance to lincomycin and erythromycin.
Keywords: Avibacterium paragallinarum, disk diffusion test, infectious coryza,
Introduction Avibacterium paragallinarum (Blackall et al., 2005) is a Gram-negative, non-spore forming
bacterium which causes infectious coryza in chickens. The disease is an acute respiratory infection and has worldwide economic significance. The clinical signs of this disease are nasal discharge, facial edema, lacrimation, anorexia, diarrhea, retarded growth in young chickens and reduced egg production (10-40%) (Chukiatsiri and Chansiripornchai, 2007; Blackall et al., 2008). The Page scheme, the original scheme to serotype Av. paragallinarum, is based on
a slide agglutination test and recognizes three serovars - A, B and C (Page, 1962). There is no cross protection between serovars (Blackall et al., 2008). Partial cross protection within serovars A and C has been revealed but it has
been recognized that cross-protection within serovar B is not universal (Yamaguchi et al., 1991). This has resulted in
some commercial vaccines now containing several serovar B strains, giving a better protection against serovar B
than the same product with only one serovar B strain (Jacobs et al., 2003). Until now, Av. paragallinarum of
serovars A B and C has been recognized in Thailand in both commercial and native chickens, even though the chickens were vaccinated according to appropriate programs (Neramitmansuk and Neramitmansuk, 1985; Chukiatsiri et
al., 2009; 2010). The knowledge gained in serotyping studies provides the necessary information to select the appropriate vaccine. In addition to vaccination, an appropriate application of antibiotics for treatment and control of coryza is also important. An increase in the resistance to antibiotics by Av. paragallinarum has been reported with one
report of more than 75% of isolates being resistant to neomycin, streptomycin, and erythromycin (Hsu, 2007).
287
Culture media Blood agar plate with Staphylococcus hyicus as a feeder colony was used for observing satellitic growth. TM/SN agar supplemented with NADH, thiamine HCl, chicken serum and O-A complex was prepared as previously described (Blackall and Reid, 1982) and used for Av. paragallinarum isolation and disk diffusion
test. All agar plates incubated under 5% CO2.
Bacteria Av. paragallinarum isolates were obtained from chickens showing the typical clinical signs of infectious coryza (Chukiatsiri and Chansiripornchai, 2007; Chukiatsiri et al., 2010). The bacteria were growth on TM/
SN and blood agar, the latter with a nurse culture of S. hyicus. The suspected colonies were identified as Av. paragallinarum by a species-specific polymerase chain reaction (PCR) test (Chen et al., 1996).
Serotyping All isolates were typed according to the Page scheme (Page, 1962) using a hemagglutination-inhibition
(HI) method with specific antisera as previously described (Eaves et al., 1989). Briefly, the bacteria which growing
on TM/SN agar were harvested, washed and
treated with potassium thiocyanate (KSCN) for 2 hr. Then, the harvested bacteria were sonicated, centrifuged (5,000 g, 8 min) and washed with PBS. The antigen was re-suspended in phosphate buffered saline (PBS) with
0.01% thimerosal and kept at 4 C for 4 days. The specific antiserum was diluted in round bottomed microtitre trays
in a doubling dilution series using BSA-PBS (0.1% bovine serum albumin in PBS) to give dilutions of 1/2-1/512
(each well contained 50 µl of each diluted serum). The antigen (50 µl adjusted to contain 4 HA units) was added to
each well. The mixture was shaken well and allowed to stand for 20 min at room temperature. Lastly, 50 µl of 0.5%
(v/v) GA-fixed chicken erythrocyte was added and shaken well. The mixture was allowed to stand for 30 min at
room temperature before reading. The maximum serum dilution completely inhibiting hemagglutination was regarded as the HI titer. The isolate was assigned to the serovar corresponding to the antiserum that gave the highest titer.
Ten isolates were found to belong to serovar A, 5 isolates to serovar B and 3 isolates to serovar C.
Disk diffusion test The disk diffusion test was performed for 18 isolates of Av. paragallinarum as recommended by the Clinical Laboratory Standards Institute (CLSI, 2008) with some modifications. Briefly, a bacterial
suspension from an overnight culture was prepared in PBS at a 0.5 McFarland turbidity standard and spread over the
surface of TM/SN agar plates. The antimicrobial disks, all from Oxoid (Basingstoke, UK) except for tylosin which
was obtained from HuvePharma (Bangkok, Thailand), were placed on the agar surface by using a multi-disk dispenser (Oxoid, Basingstoke, UK). After incubation under 5% CO2 condition at 37 C for 24 hr, inhibition zones were
measured with a vernier caliper and the zones were recorded in millimeters. The susceptibility category (sensitive,
intermediate or resistant) was determined by comparing the zone of inhibition with the zone diameter breakpoint as
recommended by the CLSI (CLSI, 2008) except doxycyclin, oxytetracyclin, cloxacillin, amoxycillin, lincomycin,
neomycin, and tylosine which CLSI interpretive standard criteria are not presently available. The zone diameter
breakpoint of tetracycline has been used for doxycyclin and oxytetracyclin. Following CLSI recommendation, the
zone diameter breakpoint of clindamycin, and oxocillin have been used for lincomycin and cloxacillin, respectively.
For tylosine, amoxicillin and neomycin were interpreted according to the company recommend (HuvePharma and
Oxoid). The zone diameter interpretive criteria of each antibiotic were showed in table 1. All isolates were susceptible to amoxicillin-clavulanic acid and were resistant to cloxacillin, lincomycin and neomycin. More than 70% of the
isolates were susceptible to amoxicillin, ceftiofur, gentamicin, spectinomycin and tylosin. However, there was a high
prevalence of resistance to erythromycin.
288 
Table 1. Disk diffusion test of the antimicrobials against 18 field isolates of Av. paragallinarum
No of isolates classified as
Antibiotic (µg)
% Sensitive
Sensitive
Intermediate
Resistant
Amoxycillin (25)
13
0
5
72
Amoxycillin-Clavulanic
18
0
0
100
Acid (20+10)
Ampicillin (10)
11
1
6
61
Ceftiofur (30)
13
0
5
72
Cloxacillin (5)
0
0
18
0
Doxycyclin (30)
7
4
7
39
Enrofloxacin (5)
11
2
5
61
Erythromycin (15)
1
3
14
6
Gentamicin (10)
15
2
1
83
Lincomycin (2)
0
0
18
0
Neomycin (30)
0
0
18
0
Oxytetracyclin (30)
7
1
10
39
Penicillin (10)
8
5
5
44
Sulfamethoxazole6
0
12
33
trimethroprim (23.75+1.25)
Spectinomycin (100)
16
0
2
89
Tylosin (150)
17
1
0
94
References
Blackall, P.J., Christensen, H., Beckenham, T., Blackall, L.L. and Bisgaard, M. (2005).
Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium
and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium
paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb.
nov. International Journal of Systemic Evolutional Microbiology, 55, 353-362.
Blackall, P.J. and Reid, G.G. (1982). Further characterization of Haemophilus paragallinarum
and Haemophilus avium. Veterinary Microbiology, 7, 359-367.
Blackall, P.J. and Soriano, V.E. (2008). Infectious coryza and related bacterial infections. In:
Diseases of Poultry, 12 ed. Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K.
and Swayne, D.E. eds. pp 789-803. Blackwell Publishing Professional, Ames.
Chen, X., Miflin, J.K., Zhang, P. and Blackall, P.J. (1996). Development and Application of
DNA Probes and PCR Tests for Haemophilus paragallinarum. Avian Diseases. 40, 398-407.
Chukiatsiri, K., Sasipreeyajan, J., Neramitmansuk, W. and Chansiripornchai N. (2009). Efficacy
of Autogenous Killed Vaccine of Avibacterium paragallinarum. Avian Diseases, 53, 382-386.
Chukiatsiri, K., and Chansiripornchai, N. (2007). Case report : An outbreak of Infectious Coryza
in a layer farm. Journal of the Thai Veterinary Medical Association, 58, 98-107.
289
Chukiatsiri, K., Chotinun, S. and Chansiripornchai, N. (2010). An Outbreak of Avibacterium
paragallinarum serovar B in a Thai Layer Farm. Thai Journal of Veterinary Medicine. 40, 441444.
CLSI (2008). Clinical and Laboratory Standards Institute. Performance standards for
antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved
standard. 3rd ed. M31-A3. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute.
Eaves, L.E., Rogers, D.G. and Blackall, P.J. (1989). Comparison of Hemagglutinin and
Agglutinin Schemes for the Serological Classification of Haemophilus paragallinarum and
Proposal of a New Hemagglutinin Serovar. Journal Clinical Microbiology, 27, 1510-1513.
Hsu, Y.M., Shieh, H.K., Chen, W.H., Sun, T.Y. and Shiang J.H. (2007). Antimicrobial
susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains.
Veterinary Microbiology, 124, 209-218.
Jacobs, A., van den Berg, K. and Malo, A. (2003). Efficacy of a new tetravalent coryza vaccine
against emerging variant type B strains. Avian Pathology, 32, 265-269.
Neramitmansuk, W. and Neramitmansuk, P. (1985). Haemophilus paragallinarum from
infectious coryza in chicken. Journal of the Thai Veterinary Medical Association, 36, 133–140.
Page, L.A. (1962). Haemophilus infections in chickens. I. Characteristics of 12 Haemophilus
isolates recovered from diseased chickens. American Journal of Veterinary Research, 23, 85-95.
Yamaguchi, T., Blackall, P.J., Takigami, S., Iritani, Y. and Hayashi, Y. (1991). Immunogenicity
of Haemophilus paragallinarum Serovar B Strains. Avian Diseases, 35, 965-968.
290 
Extra-Label Drug Use in Exotic Pet
Benchapol Lorsunyaluck, DVM. Dip. of Cons. Med.
Wildlife Unit and Exotic Pet Clinic, Kasetsart University Veterinary Teaching Hospital Kampaengsaen, Faculty of
Veterinary Medicine, Kasetsart University, Thailand
All prescription drugs (pharmaceuticals) are regulated by the FDA, and to be released for sale and use, must be approved for specific use in humans and/or specific animals at specific doses. To be approved, each drug must go
through a series of testing and analysis to understand not only the benefits and effects, but also the side-effects and
risks of its use. This must be repeated extensively in each species for which the medication’s use is to be approved
in. At this time, no drug or pharmaceutical is approved by the FDA for use in any Wildlife, pet bird, reptile or most
small exotic mammals, and very few are approved in rats and rabbits.
Use of pharmaceuticals in species for which they are not approved is allowed, however, and is termed “Extra-Label
Use”. This does not mean that the use is experimental, and in many cases years of clinical use and research has
found a well-documented, reasonably safe protocol for the use of these medications in species for which no other
medical option is available. However, “Extra-Label Use” does mean that the full effect and side effects of the drug
have not been documented and there may be unknown risks involved with the use of the medication.
Most of the dosages used in exotic pets and wildlife are based on empirical data, observations, and experience. Because drug uptake depends on factors such as age, sex, physiology, disease state, diet, etc., it is important for us as
veterinarians to know some of the pharmacobiologic, physiologic, and anatomic characteristics of these species. It
should also be noted that most of the drugs used in exotic small mammals are extralabel.
This article will discuss some practical implications with an emphasis on common drug groups as extralabel use
which applied to the exotic pets and wildlife, including; antibiotics, non-steroid anti-inflammatory drug and antiparasitic drug.
Antimicrobial drugs
Most antimicrobials are used in an extra-label fashion. Antibiotics are probably the most commonly used medications in exotic pet medicine. Because pharmacokinetic studies are lacking in these pet species and are often empirical, it is helpful to know the basic pharmacologic features and the side effects of the drugs being used for maximum
safety and efficacy. For example; some exotic small mammals (rabbits, guinea pigs, chinchillas) are herbivorous
animals, their intestinal microflora consists mainly of gram positive bacteria and anaerobic bacteria. Antibiotic
choices in these animals are limited because many antibiotics suppress the normal flora and allow pathogens to proliferate, resulting in well documented enteric disorders. With pet birds, this is usually not a problem. Even so, vets
need to inform the owner and be conservative on usage: i.e., when prescribing drugs with a wide range before reaching toxic doses in tested species (like with many antibiotics), you may be able to go with higher published dosages.
With regard to drugs with known toxicities in some species (like the aminoglycosides) or with profound effects (like
CNS depressants and analgesics), the recommend dosing should be used conservatively at the lower ranges. Keep
the animal well hydrated and under close observation.
NSAIDs
Because exotic pets are increasingly considered by their owners to be part of the family unit rather than just possessions, more clients are expecting appropriate pain relief post-surgically, post-trauma, etc., for their pets. Likewise,
veterinarians are much more aware and proactive in providing pain management for their patients.
291
Analgesia results in smoother recoveries, a decrease in systemic stress and resultant stress-related diseases (i.e., gastric ulcers), and a more rapid return to normal behavior and function. Pre-emptive analgesia, or the administration of
analgesic drugs during premedication, is now the standard when performing painful procedures. The two main
groups of analgesic medications are opiates and non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are increasingly being used in exotic pets because of the analgesia they provide in response to pain associated with inflammation (i.e., arthritis and dental problems). However, NSAIDs are not considered adequate for treating severe pain
and are usually contraindicated in the patient that has received corticosteroids because of the potential for gastrointestinal ulceration or bleeding. Other characteristics of NSAIDs include their antipyrectic actions and many have a
long duration of action (i.e., at least 12-24 hours). Meloxicam, Caprofen and Tofenamic acid are probably the most
common NSAIDs used in exotic animals, and available in both oral and injectable forms. However, there are potential risks associated with the use of NSAIDs. The four most commonly reported clinical signs in domestic animals
are vomiting, anorexia, depression, and diarrhea. Less commonly, gastric ulceration, intestinal ulceration, renal failure, hepatic failure, and death may result.
Antiparasitic drugs
The commercial formulation of anti-parasitic drugs offers small animal practitioners an effective treatment for some
common, troublesome parasitic conditions of cats and dogs. A wide range of ectoparasites on exotic pets have been
successfully controlled with many drugs treatment such as ivermectin, selamectin, moxidectin, doramectin, Imidocloprid, Milbemycin oxime and lufenuron. Most avermectin compound treatments reported to date have been directed towards the treatment of ectoparasites, despite the evidence that avermectin compound and Milbemycin oxime
are efficacious against certain endoparasites that infest exotic species as well as dogs and cats. The macrocyclic lactone endectocide selamectin, approved for use in dogs and cats worldwide, has been shown to be safe and highly
effective in extra-label use for a range of ectoparasites and some endoparasites of a variety of small mammals and
birds. Furthermore Pyrethrum and cabaryl Powder sold for ectoparasites treatment on cat and dog is a safe and insecticide for many exotic pets and wildlife. Traditional routes of administration, for example injection, bathing, or instillation of acaricide into the ear canal, can be time-consuming, difficult, distressing, and painful, not to mention the
likelihood with some exotics of injury to the veterinarian or vet assistant. The use of some drugs applied as a spot-on
formulation on a single occasion for ectoparasite control provides a means of safely and effectively treating animals
with minimum stress and a maximum of owner compliance.
***This implies that a drug treatment of an exotic animal is always off-label and requires a rededication by the veterinarian. Thus the veterinarian has the full responsibility for the therapy and cannot rely on the liability of the pharmaceutical companies producing the drug. In addition, few pharmacological studies exist to develop a therapeutic plan
for exotic patients. Generally, The use of extralabel drug in exotic animal should rely on the few referral books, published case studies and a network of referral veterianary hospital with similar patients. There are many precautions to
using a drug extra-labelly, which is why it’s important to know the adverse effects and pharmacokinetics of the drug
in any species if not the species for which it is being used. Based on this information, proper precautions should be
taken. Additionally, knowing the metabolic differences between species may assist in proper administration of a given drug. Practical experiences and results suggest that the drug is effective against a variety of disease and problems.
So, most of the dosages are extra-label, based only on empirical data, observation and experience.
REFERENCE BOOKS FOR THE HOSPITAL LIBRARY
Following are several references listed for the most common exotic species seen in clinical practice. The books
which should be in any clinic that provides health care and choose the drugs to non-domestic patients.
Exotic Animal Formulary, 3rd edition. Carpenter, J. Elsevier, St. Louis, MO. 2005.
Zoo and Wild Animal Medicine. 5th edition. Fowler, ME et. al. (ed). Elsevier, St. Louis, MO 2003.
Textbook of Rabbit Medicine. Harcourt-Brown, F. Butterworth-Heinemann. 2001
Clinincal Avian Medicine. 2 vol. Harrison, G and Lightfoot, T. Spix Publishing. 2005
Etc.
292 
Hemoglobin vesicle transfusion in canine hypolvolaemic shock
Assit. Professor. Chayakit Sinthusing,
Department of Small Animal Clinical Practice, Faculty of Veterinary
Medicine, Kasetsart University.
Introduction
Since the discovery of blood type antigen by Landsteinter in 1900, allogeneic blood transfusion has developed
into a routine clinical practice; it has contributed to human and animal health and welfare. Infectious diseases such as
hepatitis and HIV have become widespread social problems in human while blood parasite, canine distemper, Rabies, FIP, Felv have become widespread problem in dog and cat after blood transfusion, but a strict virus test by nucleic acid amplification test (NAT) is extremely effective to detect trace presences of a virus to minimize infection
(although it is available mainly in a few developed countries). Even so, NAT poses problems such as detection limits
during a window period and limited species of viruses for testing. Emergence of new viruses (such as West Nile virus, avian influenza and Ebola) and a new type of pathogen, prions, also threaten humans and animals throughout the
world. The preservation period of donated red blood cells (RBCs) is limited to 3 weeks in blood bank. Immunological responses (such as anaphylaxis and graft-versus-host disease), and contingencies of blood type incompatibility
further limit the utility of blood products. There are many problems in the conventional blood transfusion. First of
all, it makes people susceptible to viral infections such as HIV or hepatitis type C and rabies or Felv in dog and cat.
Secondly even in case of emergency, blood type must be checked and identified before transfusion. Furthermore, red
blood cells cannot be stored for more than 21 days. On the other hand, with using blood substitutes which are free
from blood type, patients could get rid of the risk of infections. When considering the danger of blood-born pathogens, the diminishing donors for blood banks, and other limitations of blood transfusion, the necessity for the development of a blood substitute (oxygen carrier) is clear. Several hemoglobin based oxygen carriers (HBOC) are currently under clinical trials. Numerous reviews have been published on the biological actions and overall development
of HBOC’s. Different laboratories have used different approaches in developing their HBOC. Blood substitutes possessing sufficient stability could be readily available in any emergency. In these points of view, many attempts to
develop substitutes replacing native red blood cells have been made over the last 20 years, and perfluorocarbon
emulsions were first tried. Their use was, however, limited to certain surgical operations because their oxygen
transport capacity was insufficient. Then hemoglobin based blood substitutes had been developed, Research on oxygen carriers started at the end of World War II, when the military was burdened with an increase in demand of blood
transfusions for injured soldiers [3]. Hemoglobin (Hb) possesses both the
293
osmotic activity and the ability to transport and transfer oxygen. However, stroma free Hb was not directly considered as a viable oxygen carrier since it was observed to induce vasoconstriction and renal toxicity in mammals [2].
However, concentrated and encapsulated Hb is currently of interest as an alternative route of oxygen transport [4, 5].
The proposed particle-mediated Hb offers distinct advantages over the use of stroma free Hb. The foremost benefits
include: (i) to prevent vasoconstriction, (ii) to avoid renal toxicity and (iii) to prolong circulation time. When Hb
loaded particles are used as oxygen carriers, the long-term circulation and pore-connecting efficiency are considered
to be the most essential characteristics such as intermolecular cross-linked Hb, poly-ethylene glycol (PEG) conjugated Hb, and Transgenic Hb were currently used in clinical stage. However, hypertension and renal toxicity have
been observed as one of the side effects in clinical test. Then, liposomal Hb whose structure is similar to native red
blood cells has been paid attention to prevent this type of side reaction. In blood bank unit of Thai Red Cross Society
and Blood bank in Kasetsart Teaching Animal Hospital of Veterinary Medicine have tried to develop blood substitutes for reduce risk of infection, by AIDS, of haemophiliac or rabies, Felv patients who had received nonpasteurized plasma products. The requisites for artificial oxygen carriers that we develop should be not only effectiveness
for tissue oxygenation, but also the following:
1 No blood type antigen and no infection (no pathogens);
2 Stability for long-term storage (e.g. over 2 years) at room temperature for stockpiling for any emergency;
3 Low toxicity and prompt metabolism, even after massive infusion;
4 Physichochemical properties that are adjustable to resemble those of human blood and
5 Reasonable production expense and cost performance. Realization of such an artificial oxygen carrier will
revolutionize transfusion medicine.
When hemoglobin is encapsulated in the liposome, following features would be available. Denaturation of
hemoglobin is prevented, and high hemoglobin concentration will be performed together with low viscosity and low
oncotic pressure. However, there still remain some problems to be solved. The size distribution due to the fusion or
aggregation occurs not only during the storage but in the blood stream after injection (Djorjevich and Miller, 1980).
To solve these problems, we used polymerizable phospholipid, 1,2- bis(2,4-octadecadienoyl)-sn-glycero-3phosphocholine
(DODPC), to stabilize liposomes by minimizing mutual interaction of membrane lipids. After the hemoglobin was
encapsulated in lipid vesicles, phospholipids were polymerized by y-rays irradiation (5 kGy) at 4°C. The physical
stability of the ARC was significantly improved so that they could be stored for long-term with either the frozen
state or a dried powder form (Wang et al., 1992a). In this report, we have investigated the polymerization mechanism of liposomes encapsulating Hb, described the characteristics of ARC and in vivo study with rats.
To solve this problem of shear-induced liposome destruction, many previous studies investigated various strategies to increase the circulation persistence of liposomes by varying the composition of the lipid membrane to prevent neighboring liposomes from aggregating and fusing together. For example, cholesterol was incorporated into
lipid membranes to alter the membrane fluidity and surface charge, as well as decrease the oxidation of Hb [2]. In
the quest to provide mechanical strength to the vesicle membrane, several groups have demonstrated that vesicles
could be stabilizedby polymerizing unsaturated phospholipids in the bilayer, through the utilization of combined
redox type initiators and UV-irradiation leading to the formation of polymerized liposomes’ [3-5]. Work has also
been done to stabilize liposomes by coating them with polymers without introducing any permanent covalent linkages between the hydrophobic lipid tails. In fact, blood substitutes employing polymerizable phospholipids in the lipid
bilayer were first developed by Akama et al. [1].
The aim of developing an HBOC is to improve upon the limitations of whole blood by increasing the shelf life and
eliminating immune response (no blood typing necessary), while being economical to produce. Additionally,
transport of oxygen and car-bon dioxide should be similar to that of red blood cells (RBC). Numerous purification
procedures and chemical modifications of hemoglobin have been carried out to reduce toxicity and improve the efficacy of HBOC’s. The blood substitutes that are currently in clinical trials are predominantly hemo-globin based.
This review will focus on the purification and chemical modifications of hemoglobin in order to develop hemoglobin based blood substitutes.
294 
References
1. Akama et al., 1992 K. Akama, S. Tokuyama, F. Hosoi and H. Omichi, γ-Radiation-induced polymerization of
unsaturated phospholipid liposomes. Koubunshi Gakkai Yokousyu, 41 (1992), p. 3532.
2. Awasthi VD, Garcia D, Goins BA, and Phillips WT (2003) Circulation and biodistribution profiles of longcirculating PEG-liposomes of various sizes in rabbits. Int J Pharm 253:121–132.
3. Mudd S, Thalhimer W, eds. Blood substitutes and blood transfusion. Baltimore: Charles Thomas Books,
1942:156.
4. Sakai H, Hamada K, Takeoka S, Nishide H, and Tsuchida E (1996) Physical properties of hemoglobin vesicles
as red cell substitutes. Biotechnol Prog 12:119–125.
Sakai H, Takeoka S, Park SI, Kose T, Nishide H, Izumi Y, Yoshizu A, Kobayashi K, and Tsuchida E (1997) Surface
modification of hemoglobin vesicles with poly(ethylene glycol) and effects on aggregation, viscosity and blood flow
during 90% exchange transfusion in anesthetized rats. Bioconjug Chem 8:23–30.
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Evidence-based Veterinary Medicine (EBVM)
Adisorn Yawongsa1
Abstract
Evidence-based veterinary medicine (EBVM) has become more and more important over the past decade, as
it is a new approach to the practice of veterinary science. EBVM is the gathering of research evidence and integration of the results to obtain the best information for use in the veterinary sciences. EBVM consists of systematic reviews and provides one clear and precise result. There are systematic review, a qualitative systematic review and a
quantitative systematic review. EBVM can be used in the various fields of veterinary medicine. Currently practicing
veterinarians require the skills to efficiently utilize EBVM to its full potential.
Key words: Evidence base veterinary medicine, Systematic reviews, Meta-analysis
Department of Large Animal and Wild Life Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University,
Kampangsaen, Nakhon-Pathom, 73140.
E-mail: [email protected] (A. Yawongsa)
Evidence-based Veterinary Medicine (EBVM)
Evidence-based veterinary medicine (EBVM) has become of great importance over the past decade. There
has been an explosion of evidence from veterinary research being performed, more so than ever before. These findings are accumulating rapidly and becoming increasingly available to both veterinarian and their patients. The name
EBVM is in reference to evidence-based medicine (EBM) by Sackett et al. (1996). They define EBM as follows:
“Evidence based medicine is the conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients. The practice of evidence based medicine means integrating individual
clinical expertise with the best available external clinical evidence from systematic research”. The Evidence-based
veterinary medicine association (EBVMA) explains that evidence-based veterinary medicine is the incorporation of
EBM principles and the basic concepts into the veterinary profession. The task facing EBVM is monumental as veterinary medicine covers several species of animals. As such, there is the need for clinically relevant research to support them all (EBVMA, 2009). There is a lot of information and research available, some of which is reliable and
some of which is not. Veterinarians want to know the best practices or be able to make the best decision for their
patients (Cockcroft and Holmes, 2003). There is a need by veterinarians, owners, and policymakers to integrate information in an objective manner so that it is both comprehensible and useful.
Systematic reviews are an integral part of EBVM. Nowadays, there is a lot of research being published and
sometimes the objective or purpose of the research is similar. However, the results of these studies may be inconsistent or contradicting. Some of the studies have a small number of samples, making the results unreliable. Systematic reviews summarize the information from the literature and analyze the results of different clinical trials relating
to a specific topic to formulate concise and advanced conclusions (Yawongsa, 2010).
296 
The reliability of research is important. The highest reliability level of evidence is obtained from systematic
reviews and meta-analyses of randomized controlled clinical trials (RCT) or single RCTs (Holmes and Ramey,
2007). Cockcroft and Holmes (2003) have compiled a list rating the reliability of evidence from research from high
to low.
1. Systematic reviews
2. Meta analysis
3. Blinded randomized controlled studies
4. Cohort studies
5. Case-control studies
6. Case series
7. Single case reports
8. Ideas, editorials, opinions, consensus report
9. Comparative animal research
10. In vitro "test tube research"
Systematic reviews
Systematic reviews are a part of the process in EVBM. A systematic review is more transparent than the traditional narrative review. Narrative reviews are a descriptive overview by experts of a selection of studies with published findings (Williams 1998). Biased descriptions of narrative reviews are common and it is well recognized that
studies and the author’s opinion are reported selectively (Gotzsche 1987). However, narrative reviews are a very
popular way to access information. Unfortunately, some research studies are poorly designed and executed (Altman
DG 1994). To avoid unbiased advice, veterinarians should look at information detail from research studies.
Systematic review is a tool used in EVBM that will provide one conclusion from multiple studies. In the diagnosis and treatment of animals the veterinarian requires reliable scientific evidence to support their decisionmaking. Veterinarians obtain scientific information from such sources as published and unpublished articles, narrative reviews and veterinary textbooks (Holmes and Ramey, 2007; Yawongsa, 2010). Systematic reviews must contain sufficient articles from primary studies to ensure quality. The data must be suitable and can be synthesize by
statistical analysis (meta-analysis, meta-regression). The results of systematic reviews are combined to obtain one
result. Systematic reviews without using statistical methods to combine the outcome for results is called qualitative
systematic reviews whereas systematic reviews using statistical methods such as meta-analysis to combine the results is called quantitative systematic review (Malinee, 2001; Greenhalgh, 1997; Yawongsa, 2010).
The process of a systematic review
The procedure of a systematic review has been described by many authors, the general steps are as follows
(Cockcroft and Holme, 2003; Hemingway and Brereton, 2009; Yawongsa, 2010).
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1. Research questions
Research questions are important as the first step as it is also the hypothesis for the systematic review. The
research questions should be specific and in the form of a clear and structured question. The questions for the review
must define the details of the primary articles such as the populations, tests, reference standards or study designs.
2. Procedures and processes for literatures searching
Procedures and processes for literatures searching have to design before the start of the study review. Criteria select for research such as study type, study design, sample type, intervention type, outcome or purpose of the
questions. Researches will have to search for literature using a variety of sources such as computerized bibliographic
databases of published and unpublished research, conference or symposia proceedings, dissertations, books, granting
agencies, manual journal searching. Generally, electronic databases for veterinary are Pubmed Medline, CAB direct,
Sciencedirect, Scopus, IVIS, VetGate and NetVet. Relevant literatures (Primary studies) are selected on the basis of
their relevance to the research question through the setting of objective inclusion and exclusion criteria.
3. Inclusion and Exclusion Criteria
It must be decided if primary study is of relevance for systematic review or not. Two issues relevant to the review
are question and methodological quality. Relevance to the research question is most important because all potential
primary studies that deal with the topic must be identified. Thus, inclusion criteria revolve around the research question being asked. Once inclusion criteria for primary studies have been determined, the next step is to define exclusion criteria that are based on methodological quality.
4. The critical appraisal of research evidence
Literatures must have their quality evaluated following evaluation procedures. The quality of the research
ought to be identified as high or low and should have two or more persons evaluating to reduce bias. The procedures
of Chalmers et al. (1981) and Jadad et al. (1996) are using for assessing the quality of a randomized control trial of
research in EBM. Selected studies should then be subjected to a more refined quality assessment. The minimum acceptable level of quality can be crudely defined by study design during question formulation and study selection. To
aid in the prevention of bias, blind reviewers should evaluate the selected studies without knowledge of the origin or
authors of the articles. The reviewers should have extensive prior knowledge of the existing literature in the field and
at least one member of the review team has expertise in the specific topic of the review. For quantitative systematic
reviews, these detailed assessments can be used to explore heterogeneity, to inform decisions regarding suitability of
statistical analysis (meta-analysis).
5. Extracting the Data
Studies have been selected, and full-text articles on these studies are available, the pertinent data to answer
the review questions must be extracted. Study selection is sensible to use more than one researcher to extract data
independently in order to check consistency. Study characteristics, quality and results should be tabulated first. Data
synthesis may then use statistical methods for exploring differences between studies (heterogeneity) and combining
their accuracy (meta-analysis) appropriately. If an overall meta-analysis is not feasible, subgroup meta-analysis may
be feasible and may provide clinically useful answers.
298 
6. Analysis, Interpreting and Presentation of Results
Systematic reviews will have been completed the study table. In general, systematic reviews are valuable to
describe, often in simple terms, the findings of the review with respect to the results and methodological features in
the articles selected (which by inference describes the features of those articles that were rejected). Systematic quantitative reviews may be desirable to perform a meta-analysis. However, meta-analysis depends on the results of study
characteristics and the nature of the data.
Generally, journals published in English are more likely than other languages to be registered in various
electronic databases and are more likely to be published rapidly and repeatedly, they are also more likely to be cited
(Egger 2001). This situation creates a language bias. Typically, the research results of a study with results or outcomes found to be statistically significant will have a greater opportunity to be published than studies that did not
result in finding anything of statistical significance. In addition, some researches are reprinted more than once. Caution should be taken as low-quality research can lead to a low quality systematic review and biases of publications
need to be explored for the reasons behind the heterogeneity and if they contain information that could help to determine if any practical inferences can be generated. All recommendations should take into account the strengths and
weaknesses of the evidence.
Figure 1. Forest plot (Hintze, 2007)
The results of systematic quantitative reviews by meta-analysis often present as Forest plot as shown in the
table (Fig. 1). The results of quantitative review show each study result as symbol in the Forest plot and the samples
size of each study is demonstrated by its symbol size. The arm of the symbol in Forest plot indicates confidence intervals of each study result. The final result is the combination of all the research, and is represented by a diamond
(Bax et al. 2006; Hintze, 2007).
In summary, EVBM is the combination of reliable evidence gathered from research and integration of the results in
order to provide the best information to use in veterinary science. The results are then applied to the various fields of
veterinary science. The results of systematic literature review are clear and precise and it allows for a single conclusion from research in which results of studies differ or conflict. The accuracy and reliability of the study are based on
the quality of the literature in the study.
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Thus, the results of a systematic review need to assess its reliability. Before deciding to apply the systematic reviews
result, one should consider its suitability for implementation. However, it should be noted, one should be careful not
to dismiss expert opinion, even if anecdotal, experts may know something worthwhile and useful.
References
Altman, D.G. 1994. The scandal of poor medical research. BMJ. 308: 283-284.
Bax, L., Yu, L.M., Ikeda, N., Tsuruta, H., Moons, K.G.M. 2006. Development and validation of MIX: comprehensive free software for meta-analysis of causal research data. BMC Medical Research Methodology. 6, 50.
Chalmer, T.C., H. Smith, B. Blackburn, B. Silverman, B. Schroeder, D. Reitman and A. A. Ambroz. 1981. A
method for assessing the quality of a randomized control trial. Control. Clin Trials. 2: 31-49.
Cockcroft, P. D. and M. A. Holmes. 2003. Handbook of Evidence-Based Veterinary Medicine. Iowa State
University Press. 210.
Ebvma.org. 2009. What is evidence-based medicine? Evidence-Based Veterinary Medical Association
(EBVMA). Mississippi State, MS. Available at: http://www.ebvma.org/
Egger, M. and G.D. Smith. 1998. Bias in location and selection of studies. Br. Med J. 316: 61-6.
Egger, M., Dickersin, K., Davey-Smith, G. 2001. Problems and limitations in conducting systematic reviews. In: Egger, M., Davey Smith, G., Altman, D.G., eds. Systematic reviews in health care. 2nd ed. London, England: BMJ Publishing Group. 43-68.
Greenhalgh, T. 1997. How to read a paper: Papers that summarise other papers (systematic reviews and meta
-analyses). BMJ. 315: 672-5.
Hemingway, P. and N. Brereton. 2009. What is a systematic review? London: Hayward Medical Communications.
Hintze, J. 2007. NCSS User's Guide IV: Statistics. LLC. Kaysville, Utah, USA. p. 455-457.
Holmes, M., Ramey, D. 2007. An introduction to evidencebased veterinary medicine. Vet Clin North Am:
Equine Pract. 23, 191-200.
Jadad, A.R., R.A. Moore, D. Carrol, C. Jenkinson, D.J. Reynolds, D.J. Gavaghan, H.J. McQuay. 1996. Assessing the quality of reports of randomized clinical trials: is blinding necessary. Control. Clin Trials. 17: 1-12.
Laopaiboon, M. 2001. Quantitative Systematic Review: Meta-analysis (In Thai). Journal of Health Science.
10: 151-71.
Sackett, D.L., Rosenberg, W.M., Gray J.A., Haynes, R.B., Richardson, W.S. 1996: Evidence based medicine: what it is and what it isn’t. Brit Med J. 312, 71-72.
Williams, C.J. 1998. The pitfalls of narrative reviews in clinical medicine. Ann Oncol. 9: 601-605.
Yawongsa, A. 2010. Evidence-based veterinary medicine (EBVM) (In Thai). Kasetsart Veterinarians. 20: 41
-51.
300 
Do and don’t in exotic pets
Pornchai Sanyathitiseree
Department of Large Animal and Wildlife Clinical Science
Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsaen
Campus, Nakornpathom province, 73140, THAILAND
E-mail: [email protected]
Nowadays exotic pet patients is still increasing, a small animal practitioner find it difficult for treatment and
management. Not only exotic pet medicine is challenging science but also it is a mysterious medicine. Moreover, the
exotic pet veterinarian is responsible many diverse species. This purpose of this article is to provide the practitioner
to a clinical tip in each group of exotic animal (not include fish) in clinical medicine, husbandry and basic biology in
the practice. A do’s and don’ts list will give suggestion the practitioner guideline for operating an exotic pet practice.
A new species of exotic pet introduces into a pet market and veterinary clinic. It would be difficult for any
veterinarian in accustomed to wildlife/exotic animal standard of care. Actually, successful exotic pet practitioner requires attention care in the same degree as in small animal clinic. Our clients in the field of exotic pet have been
known that they are master in masking clinical signs. The growing in experience of exotic pet medicine is increasing
in the past 10-15 years ago. From these clinical experiences can become possible to predict the outcome of exotic
patients consistently. This paper is designed to provide an overview of topic do and don’t for consideration when
manage exotic patients successfully. The author provides information from many sources of references and my experience. Again, the list only serves an outline in exotic animals practice. The outline includes general practice, history
taking, physical examination, drug usages and contraindication, nutrition and treatments.
Do
Don’t
1. First do no harm
2. Treat for the treatable
3. Should be a lifetime student
4. Must be a keen observer
5. Do staff training
6. Review thoroughly of husbandry practices management,
hygiene, and nutrition before a standard physical examination
7. Provide available information on exotic disease
8. Be familiar with the exotic animal literatures
9. Being familiar with exotic pet standard of care
10. Obtain a complete history (especially in avian cases)
11. Consistent physical examination in all cases
12. Think about common causes occur commonly
13. Understanding the masking phenomenon
14. Understanding exotic pathology
15. Understanding the whole life story of animal
16. Understanding the impact of (intrinsic and extrinsic factors) predisposing factors to any disease of exotic pets
17. Proficient in the anesthesia, anatomy and radiographic
anatomy1
8. Have enough time for treatment and consultation
19. Educating the owner
1. Do not characterized by optimism and
avoidance of the unknown
2. Do not tell “nobody knows anything” before searching thorough exotic literatures
3. Do not fear for a variety of exotic species. There is only a
few dozen species likely to be encountered with regularity in
clinical practice
4. Do not dominate the conversation in history taking
5. Do not diagnoses by looking
6. Do not treat by laboratory test result
7. Do not assume any assumption
8. Do not postpone today
9. Do not give your patient a disease
10. Do not consider food giving correlate with appropriate nutrition
11. Do not maintain eye contact with prey species
12. Do not contact, moved or handled during moulting or ecdysis process
13. Do not handling invertebrate animal far above a soft surface
14. Do not handling the extremities of invertebrates
15. Do not use latex gloves with powder for invertebrate, fish
and amphibian patients
16. Do not restraint any delicate tail of animal
301
Do
Don’t
20. Assessment the tolerance of animal for handling, sample
diagnosis taking and providing care
21. Being familiar with normal characteristic for the exotic
species
22. Know the biologic data of the common species
23. Knowledge of common exotic species syndromes
24. Preventive medicine is the cornerstone in exotic pets
management
25. Apply basic concepts of therapy of small animal medicine
to exotic pet medicine
26. Keep notes and record the results of any treatments
27. Beware of extra-label drugs usage
28. Quarantine all new acquisitions
29. Wash hands after handling to prevent zoonotic diseases
transmission
30. Perform oral cavity examination at the end of physical
examination
31. The basic concepts of disease may be using the
"VITAMINDP" and “HECK” scheme
32. Treat the exotic pet, not just the disease
33. Stock with towels in a variety of sizes to aid in patient
restraint in examination room
34. Do support the immune system in treating an ill reptile or
amphibian
35. Understanding the external environment and basic biology of amphibian
36. Do gently opening the amphibian’s mouth
37. Replace amphibians in water or physiological saline bath
periodically during prolonged exam or prolonged procedure
38. Clean and rinse chamber/container before using with amphibian patients
39. All reptile patients must be well hydrated before and during administration of all medications
40. The diversity within the class Reptilia necessitates the
description of generalities rather than species specifics
41. Localize any lesions or clinical signs and formulate a list
of differential diagnoses in reptiles
42. Keeping reptile patients at PBT during treatment process
43. Beware of darkening of skin or sloughs at the site of some
medication injection in reptiles
44. Be patience in assessment and handling chelonians
45. Give warm fluid therapy to exotic patients (especially
reptile) based on ideal body temperature range
46. Bathing reptiles with lukewarm water for stimulating
water drinking and also to pass feces and urines
47. Low oxygen saturation is require to stimulating breathing
in anesthesia reptiles
48. Should clean a skin before injection to reduce the risk of
abscess formation in reptiles
49. Use horizontal mattress suture of non absorbable material
to close the skin or reptiles
50. Live invertebrates food may attack the reptiles
17. Do not use deionized or distilled water as natural water in
amphibian and fish
18. Do not use tap water for fish and amphibian husbandry
19. Do not administer intracolemically if suspected spaceoccupying lesion, pneumonia, obstipation, egg retention or
preovulatory follicles in reptile patients
20. Do not be tipped upside down in turtles
21. Do not housed any bird in sight of any raptor
22. Do not treat critically ill birds
23. Do not consider a ferret is a cat or a dog
24. Do not rush into handling a bird patient and put it down if
you are not sure
25. Do not touch the lip and vibrissae of a rabbit at first
26. Do not grasp lizards by tail
27. There is no single treatment regimen
28. Do not give up resuscitation efforts too early in reptile
patients
29. Do not administer a large volume of fluid therapy in
snake by stomach tubing
30. Do not discard any biological specimens
31. Do not mixed guinea pigs with rabbits
32. Do not use over medication
33.Corticosteroids are not indicated in rabbits
34. Do not use fipronil in rabbits
35. Do not use ivermectin in chelonians
36. Do not use penicillins (especially ampicillin, amoxicillin)
cephalosporins, macrolides (erythromycin, tylosin, spiramycin), lincosamides (lincomycin, clindamycin), tetracyclines
(especially tetracycline, chlortetracycline, oxytetracycline),
bacitracin and vancomycin in strictly herbivorous small
mammals (i.e. rabbit, guinea pig, hamster)
302 
Do
Don’t
51. Reptiles often require longer treatment periods than any
other animals
52. Septicemia and bacteremia should be considered in any
bird that is severely depressed
53. Understanding key morphologic groups of birds (i.e. sexual dimorphism, species-specific behaviors)
54. Perform any therapeutics only in a stable or withstand the
stress of treatment in avian patients
55. The acronym for avian examination is ABC
56. Assessment avian body condition score at pectoral region, over the abdomen, flanks, thighs and neck areas
57. Attention to a strong physiologic response to stressful
situations in rabbits
58. Hospitalized exotic patients should be weighed at a regular each day
59. The three first aid essentials are heat, fluids and food for
exotic patients
60. Small herbivore mammals are susceptible to dysbiosis
and enterotoxemia with some antibiotics usage
61. Determine appropriate compounding drugs for use in
exotic pets
62. Accurate weighing is important
63. Draw from the abundance of knowledge in small animal/
large animal/horse/poultry/aquatic medicine when managing
unusual or difficult exotic animal cases
64. Double check all drug dosage and twice calculate
65. Study and consider the entire radiographic film/picture,
not only just the lesion
66. Always be personable with your patients and owners
67. The answer is on autopsy
68. Expect some miracle for biological life
69. Be honest with your common sense
Note: Abbreviation in the table
ABC = Appearance, behavior and coprology
PBT = Preferred body temperature
VITAMINDP (V = vascular; I = infectious, immune [autoimmune or immunodeficiency], inflammatory; T = toxic;
A = anomaly; M = metabolic; N = nutrition, D = degenerative; P = Physiological, physical, psychological, or pharmacological)
HECK (H = husbandry, E = experience level of the owner, C = cleanliness, K = knowledge of the natural history
Summary
The field of exotic pet medicine is like in the other medicine which ever changing and expanding fields.
However, the cornerstone of good practitioner exotic medicine is still the careful evaluation of the patients. There is
no substitute for looking at the animal and observing its lesion with physical examination for making a clinical diagnosis. Excellence in exotic clinical practice results from the successful combination of continuing education and
critical thinking. This “do and don’t list” can provide some guidance and framework for newcomer or experienced
veterinarian. Finally, the challenge to exotic veterinarians today is to continue to grow and improve.
303
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Mayer, J. and Martin, J. 2005. Barriers to exotic animal medicine. Vet Clin North Am Exotic Anim Pract. 8:487496.
Powers, L.V. 2006. Techniques for drug delivery in small mammals. J. Exotic Pet Med 15(3):201-209.
Romagnano, A. 1999. Examination and preventive medicine protocols in psittacines. Vet Clin North Am Exotic
Anim Pract. 2:333-355.
Rosenthal, K.L. 2004. Therapeutic contraindications in exotic pets. J. Exotic Pet Med 13(1):44-48.
Rosenthal, K.L., Forbes, N.A., Frye, F.L. and Lewbart, G.A. (ed.) 2008. Rapid review of exotic animal medicine
and husbandry: pet mammals, birds, reptiles, amphibians and fish Manson Publishing/The Veterinary Press,
London.
Rosskopf, Jr. W.J. (ed.) 2003. Species syndromes. Sem Avian Exotic Pet Med 12(3):123-182.
Schaer, M. 2008. Clinical signs in small animal medicine. Manson Publishing, London.
Schmidt, R.E. 2003. Practical gross pathology. Sem Avian Exotic Pet Med 12(2):59-61.
Walker, I.D.F. 2000. Amphibian therapeutics. Vet Clin North Am Exotic Anim Pract. 3:239-255.
Williams, B.H. 2000. Therapeutics in ferrets. Vet Clin North Am Exotic Anim Pract. 3:131-153.
Worell, A.B. (ed.) 2010. Standard of care. J. Exotic Pet Med 19(1):3-81.
304 
Treatment of rabbits with long bone fractures with tie-in configuration intramedulary pins attached to
external skeletal fixator device: 25 cases (November 2009- November 2010)
T. Sirun¹, W Chantornvong¹, M Vijarnsorn2
¹Kasetsart veterinary teaching animal hospital
2
Department of Companion Animal, Faculty of Veterinary Medicine, Kasetsart University
Abstract:
Objective: To evaluate outcome and complications of treatment tie-in configuration intramedulary pins attached to
external skeletal fixator (ESF) device
Design: Retrospective case series.
Animal: client-owned 25 rabbits with long bone fractures
Procedures: Medical records of rabbits with long bone fractures during November 2009- November 2010 were reviewed. Rabbits undergone tied-in intramedulary pins attached to ESF device were included in this study.
Results: Rabbits of both sexes aging 7.8 ± 7.5 months were reviewed. Fractures were commonly observed on tibia
(84%), femur (8%) and humerus (8%). Duration to the time of operation was 1.9 ± 1 day. The time to heal was defined as the time where a fracture line was no longer seen on the radiographs and the external fixator frames were
removed. The time to heal was 39.1 ± 10.4 days. Complications observed were External fixators get caught on the
cage or other housing
Conclusions: Tie-in configuration intramdeullary pins attached to ESF device provides excellent outcome and is economical. It is an outstanding alternative to plate and ESF device as it provides minimal invasive surgery with good
stability and anti-rotational property.
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Antimicrobial Susceptibility of Bacterial isolates of Psittacine Birds in Thailand 2011
Taksaon Duangurai a, Kaset sutasha a , Chunyapatt Bomrungpan a, and Chanyaporn Prasatpornchai a
a
Kasetsart Animal Hospital, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand 10900.
Abstract
One hundred and three samples of hospitalized pet birds (14 species) in Family Psittaciformes such as parrots, parakeets, conures, cockatoos and macaws were investigated for pathogenic bacterial infection and contamination. The birds were the outpatients during January to August 2011 from the Avian and Exotic Pet Clinic, Veterinary
Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart University, Thailand. The common clinical signs of
hospitalized pet birds were anorexia, diarrhea, abnormal color of dropping, purulent nasal discharge, dyspnea and
regurgitation. The common specimens were derived from crop, cloacal, choanal swab and organ lesions by necropsy.
Ten bacteria species from one hundred and eighty three isolates were Escherichia coli (27.32%), Enterobacter spp
(8.19%), Klebsiella pneumoniae (15.30%), Proteus mirabilis (3.28%), Pseudomonas aeruginosa (9.29%), Acinetobacter spp. (8.74%), α-Streptococcus spp. (13.66%), Staphylococcus spp. (8.74%), Bacillus spp. (4.37%),and Pasteurella spp. (1.09%). The isolates were tested for their antimicrobial sensitivity against eleven drugs, using disc diffusion assay. The isolates sensitivity results were amikacin (84.70 %), gentamicin (80.87 %), ciprofloxacin (69.95
%), tobramycin (67.76 %) , enrofloxacin (66.67 %) sulfa-trimethoprim (61.75 %) azithromycin (61.20 %),
cephalexin (61.75 %), doxycycline (59.02 %) , amoxicilin-clavulanic acid (58.47%),, and amoxicillin (31.69%), respectively. This study aims to provide the update information to guide the proper antimicrobial drug selection and
report recent antimicrobial sensitivity profile of pet bird pathogens in Thailand.
Keywords : antimicrobial sensitivity, pet birds , pathogenic bacteria, disc diffusion assay
306 
Advanced Immunology and Vaccinology for Livestock Diseases
Section I: Native Defense Mechanisms
James A. Roth *, D.V.M., Ph.D., DACVM
Director, Center for Food Security and Public Health
Executive Director, Institute for International Cooperation in Animal Biologics
Distinguished Professor, Department of Veterinary Microbiology and Preventive Medicine,
College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, USA
Phone: 515-294-8459, Fax: 515-294-8259 email: [email protected]
www.cfsph.iastate.edu
Abstract
Native defense mechanisms are those that are always active and do not require previous exposure to protect the animal from infection. They can be activated within seconds to minutes after microbial invasion. The major native defense mechanisms include phagocytic cells (neutrophils and macrophages), the complement system, natural killer
cells, antimicrobial peptides, and production of native defense cytokines by sentinel cells. Various stressors tend to
reduce the efficacy of the native defense mechanisms leading to increased susceptibility to infection. The activity of
all of these native defense mechanisms is enhanced if the animal has developed acquired immunity (antibody and T
cell-mediated immunity) to a specific pathogen. Since this series of presentations focuses on advances in immunology and vaccinology, the only aspect of native immunity to be discussed in detail will be the role of sentinel cells in
early detection of microbial invasion, activation of native defense mechanisms and induction of acquired immunity.
307
Introduction
Native defense mechanisms are those that are always active and do not require previous exposure to protect the animal from infection. They can be activated within seconds to minutes after microbial invasion. The major native defense mechanisms include phagocytic cells (neutrophils and macrophages), the complement system, natural killer
cells, antimicrobial peptides, and production of native defense cytokines by sentinel cells. Various stressors tend to
reduce the efficacy of the native defense mechanisms leading to increased susceptibility to infection. The activity of
all of these native defense mechanisms is enhanced if the animal has developed acquired immunity (antibody and T
cell-mediated immunity) to a specific pathogen. Since this series of presentations focuses on advances in immunology and vaccinology, the only aspect of native immunity to be discussed in detail will be the role of sentinel cells in
early detection of microbial invasion, activation of native defense mechanisms and induction of acquired immunity.
Production of pro-inflammatory cytokines by Sentinel Cells
Macrophages, dendritic cells, and mast cells are important sentinel cells, which play major roles in early detection of
microbial invasion and the induction of the immune response. These cells are concentrated at or under epithelial surfaces and have receptors, called toll-like receptors (TLRs), which are capable of binding molecules that are unique to
infectious agents. These molecules include bacterial lipopolysaccharide (endotoxin), flagellin, bacterial DNA (which
is rich in CpG motifs), and bacterial lipoproteins. These microbial molecules are considered to be danger signals and
the sentinel cells respond rapidly by secreting pro-inflammatory cytokines (Interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNFα)). These molecules produce local inflammation at the site of production and activate neutrophils, macrophages, natural killer cells, and endothelial cells to have enhanced activities required for efficient
elimination of a local infection. If the proinflammatory cytokines are secreted in high enough concentrations, they
will begin to circulate in the blood stream. When they reach the hypothalamus, they induce fever, loss of appetite,
and lethargy. They act on the bone marrow to induce neutrophilia and cause the liver to produce large quantities of
acute phase proteins. These actions enhance the ability of the native defense mechanisms to control an infection. The
lethargy and loss of appetite reduce the animal’s interaction with other members of the same species, thereby reducing the spread of infection. Fever slows the replication of some viruses and enhances some native defense killing
mechanisms. This response requires extra energy and protein synthesis. Since the animal has a reduced appetite and
is not eating, the amino acids for the response are mobilized from the muscle and other tissues. Very high concentrations of the pro-inflammatory cytokines can be induced by high concentrations of microbial molecules, such as endotoxin, leading to septic shock.
Induction of the proinflammatory cytokines is essential to induce an immune response. Therefore, every time that an
animal is vaccinated, proinflammatory cytokines are produced. Typically, a single vaccine will only induce low levels of cytokines and no clinically apparent reaction is observed. However, if multiple vaccines are administered at
the same time, especially if some of them are Gram negative bacterins containing free endotoxin, enough cytokines
may be released to cause fever, lethargy and loss of appetite lasting for a few days. The animal may already have a
low-grade infection causing low levels of the cytokines to be induced. The additional small amount produced by a
vaccine may be enough to induce clinical signs.
308 
Role of dendritic cells, macrophages, and B cells in Antigen Processing and presentation to T lymphocytes
T helper (CD4+) lymphocytes can only recognize peptide antigens presented on major histocompatability class II
(MHC II) molecules. These peptides come from proteins that are endocytosed, enzymatically degraded into short
peptides, and bound to MHC II molecules, which are then transported to the cell surface for presentation to T helper
cells. T cytotoxic (CD8+) lymphocytes can only recognize peptide antigens presented on MHC I molecules. These
peptides come from proteins that are synthesized in the cytoplasm of the antigen presenting cell. A sampling of all
proteins synthesized within a cell are processed through a proteosome in the cytoplasm and transported into the
endoplasmic reticulum where short peptide fragments become bound to MHC I molecules. There are no cytotoxic
T cells that recognize normal self proteins because the healthy animal is tolerant to normal self proteins. If a cell becomes infected with a virus, or mutates and starts producing foreign proteins in its cytoplasm, peptides from those
proteins will be presented to cytotoxic T cells. The cytotoxic T cell will respond by killing the cell that is
synthesizing foreign protein. It takes 7 to 10 days for the cytotoxic T cells to become efficient at controlling virus
infection after initial exposure. They respond very rapidly on second exposure to the virus.
Antigens from both killed vaccines and live vaccines can be endocytosed, processed, and presented on MHC II
molecules to T helper cells. However, only live vaccines can replicate and cause the production of pathogen proteins
in the cytoplasm of infected cells. Therefore, live vaccines are typically considered to be more effective at inducing
cytotoxic T cells in response to vaccination. Now that this endogenous pathway of antigen presentation is understood, researchers have developed methods to deliver vaccine antigens into the cytoplasm with out using typical
modified live vaccines. One method is to use adjuvants, such as ISCOMS (immune stimulating complexes) which
fuse with the cell membrane and can deliver protein antigens into the cytoplasm for processing and presentation on
MHC I molecules to cytotoxic T cells. Another method is to use recombinant virus vectors which have one or more
genes of the pathogen inserted into their genetic material so that when the vector infects a cell, it causes the
production of pathogen proteins in the cytoplasm for processing and presentation on MHC I molecules.
The
advantages of using a viral vector to deliver the protein, instead of a typical modified live virus are that the vector
may be easier to grow, or safer to use than the MLV vaccine. It may also break through maternal antibody more
easily.
Dendritic cells, macrophages, and B lymphocytes are the cell types that play a major role in processing and presenting antigen on MHC II molecules to T helper cells. Dendritic cells are the major antigen presenting cells in all
primary immune responses. Macrophages and B cells play a major role in antigen presentation to T helper cells in
secondary immune responses. Dendritic cells are found in many tissues of the body, especially in the epithelium and
in lymphoid tissues. Their primary function is to trap antigen, process it and present it to T helper lymphocytes.
Dendritic cells in the tissues will capture antigens on their dendrites, phagocytose the antigens, break the proteins
associated with those antigens down into small peptides, bind the peptides to MHC II molecules in endosomes and
present those peptides attached to MHC II molecules on their cell surface. While they are doing this, they will
migrate from the tissues where they found the antigen and drain with the lymphatics into lymph nodes where they
will interact extensively with T helper lymphocytes capable of recognizing the foreign peptides bound to their
MHC II molecules.
Initial exposure to antigen will cause the B cells and T helper cells, which recognize that antigen to undergo mitosis
resulting in the production of more B and T cells that recognize the same antigens. This clonal expansion occurs in
the lymphoid tissues. After clonal expansion, B cells become a major antigen presenting cell to T helper cells. The
B cells will capture antigen using its antigen receptor. The B cell antigen receptor is a membrane bound form of the
antibody molecule they will secrete in response to antigenic stimulation with the same antigenic specificity. The B
cell will endocytose the antigen it has captured, process the proteins associated with that antigen into small peptides,
bind the peptides to MHC II molecules, and present those molecules on its surface for T helper cells to find. T helper
cells, which are capable of recognizing the specific peptides presented by the B cell will use the T cell receptor to
bind to the MHC II molecule and peptide in an immunological synapse which also involves accessory and adhesion
molecules. This interaction occurs in the lymphoid tissues.
309
The T helper cell will then secrete cytokines which direct the B cell to undergo mitosis, switch from IgM to one of
the other classes of antibody (IgA, IgE, or IgG depending on the characteristics of the pathogen), and eventually to
become a plasma cell which secretes large quantities of antibody.
The initial antigen processing and presentation to cytotoxic T cells in a primary immune response occurs predominantly in the lymphoid tissue where naïve T cells and antigen presenting cells are concentrated. After the cytotoxic T
cells have undergone clonal expansion and maturation in the lymphoid tissues, they circulate between the blood and
the tissues monitoring for the presence of the foreign peptide from the antigen, which they recognize on tissue cells.
If they do not find the antigen, they drain with the lymphatics to lymph nodes and eventually return to the blood
through the thoracic duct. If they do find the antigen that they recognize, the will kill the cell synthesizing the foreign
peptide. Thereby preventing the virus infected cell from producing more virus.
There are a complex set of interactions and signals, which act on T helper cells to allow them to direct the induction
of the best type of immune response for that particular pathogen. Natural infection generally induces the correct set
of signals to produce the correct type of immune response. Modified live vaccines, which replicate at the natural site
of infection generally induce signals similar to those induced by natural infection, resulting in the correct type of
immune response. Killed vaccines with adjuvants that are delivered by injection induce mostly artificial signals for
the T helper cell. Those artificial signals may be the correct signals and induce good immunity, or they may be the
wrong signals and cause the induction of the wrong type of immune response, which is only partially protective or
not protective. There are even examples of killed vaccines that enhance the disease process through induction of the
wrong type of immune response.
References
Abbas, A.K., Lichtman, A.H., Pillai, S. (2007). Cellular and Molecular Immunology. 6 th edition.
Saunders, Philadelphia, PA. 566 pages.
Tizard, I. R. (2009). Veterinary Immunology: An Introduction, 8th edition. Saunders, Philadelphia, PA 574 pages.
Murphy, K., Travers, P., Walport, M. (2008) Janeway’s Immunobiology. 7th Edition. Garland Science, New York.
887 pages.
310 
Advanced Immunology and Vaccinology for Livestock Diseases
Section II: Cell Mediated Immunity
James A. Roth *, D.V.M., Ph.D., DACVM
Director, Center for Food Security and Public Health
Executive Director, Institute for International Cooperation in Animal Biologics
Distinguished Professor, Department of Veterinary Microbiology and Preventive Medicine,
Phone: 515-294-8459, Fax: 515-294-8259
email: [email protected] www.cfsph.iastate.edu
Ratree Platt, D.V.M., Ph.D.
Veterinary Specialist, Center for Food Security and Public Health,
Department of Veterinary Microbiology and Preventive Medicine,
Phone: 515-294-3750, Fax: 515-294-8259 email: [email protected]
Abstract
Cell-mediated immunity (CMI) is a rather simple label for a broad and complex set of mechanisms that require
T-cell activity. Effecter T cells are induced against pathogens in response to infection or vaccination; some activated
T cells eventually differentiate into memory T cells that can expand and exert antimicrobial activity very rapidly in
the event of secondary exposure. The major subsets of T cells are the T helper 1 and 2 (Th1 and Th2) cells, cytotoxic
T cells (CTLs), and gamma delta (gd) T cells. B cells and the subsets of T cells recognize antigen differently and
respond to antigen differently. Thorough evaluation of vaccine efficacy requires the evaluation of T cell responses to
vaccination in populations of animals over time. Evaluation of T cell activation using specific monoclonal antibodies
with compatible fluorochrome conjugates and flow cytometry has proven to be a useful tool for monitoring the
ability of a vaccine to induce T cell-mediated immunity in experiments with large number of animals. Multiparameter flow cytometry (MP-FCM) has been used in our laboratory to simultaneously identify the three major T
cell subsets, and the percentage of each T cell subset expressing CD25, IFNg, and IL-4. The changes in their
expression indicate the memory responses to the antigens tested and can be compared among treatment groups. We
have used these techniques to monitor T cell mediated immunity in cattle, swine, horses, and dogs. The details of
several CMI evaluations of various pathogens and vaccines are presented.
311
Introduction
Cell-mediated immunity (CMI) is a rather simple label for a broad and complex set of mechanisms that require Tcell activity. Effecter T cells are induced against pathogens in response to infection or vaccination; some activated T
cells eventually differentiate into memory T cells that can expand and exert antimicrobial activity very rapidly in the
event of secondary exposure. The major subsets of T cells are the T helper 1 and 2 (Th1 and Th2) cells, cytotoxic T
cells (CTLs), and gamma delta (gd) T cells. B cells and the subsets of T cells recognize antigen differently and respond to antigen differently (see table).
Lymphocyte Type
Antigen Recognition
Response to Antigen
B cells
Intact, unprocessed cell surface
molecules
Produce Antibodies
T helper 1 (Th1) cells
(CD4+)
Exogenous peptides processed and Secrete cytokines which activate phagocytes
presented on MHC II
and natural killer cells
T helper 2 (Th2) cells
(CD4+)
Exogenous peptides processed and Secrete cytokines which help antibody producpresented on MHC II
tion (primarily IgA and IgE)
T cytotoxic cells (CTLs)
(CD8+)
Endogenous peptides processed
and presented on MHC I
Cytotoxic for cells synthesizing foreign proteins
Gamma delta (gd) T cells
Intact, unprocessed cell surface
molecules
Secrete cytokines and/or cytotoxic for cells
with abnormal surface antigens
T helper 1 and 2 cells
Helper T cells coordinate the overall adaptive immune response by modulating the activities of many other immune
cells, both through direct cell-cell contact and secretion of cytokines. In recent years, the balance of immune regulation between Th1 and Th2 cells has been a major area of investigation in immunology. Successful defense against
the majority of bacterial and viral infections requires an early Th1 cell-mediated inflammatory response. Interferon
gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFα) are particularly important Th1 cytokines. Th2 responses are generally characterized by IgA or IgE antibody production and often by recruitment of
eosinophil and mast cell activity. Of the Th2 cytokines, IL-4, IL-10, and IL-13 are among the most dominant and
best characterized. One purpose for Th2 responses appears to be mediation of immunity against helminth infections.
However, when Th2 cell-driven mechanisms dominate early in infection, microbial replication may proceed unchecked. This, as well as antibody-mediated hypersensitivity, can enhance the severity of disease. Importantly, cytokines from Th1 responses tend to suppress Th2 responses, and vice versa, so that one or the other response predominates at a given time in an animal. This may have an impact on vaccine efficacy. If an animal is mounting a strong
Th2 response to internal or external parasites when it is vaccinated; it may not be able to mount an effective Th1
response to the vaccine. The primary immune response seems to be more susceptible to misdirection than a secondary response. Therefore, it would be best to treat young animals for parasites, then wait two or three weeks before
312 
Measurement of cytokine production by Th cells is a useful measure of vaccine efficacy against infectious agents of
animals. Much of the research that has shaped the Th1 versus Th2 theory has involved studies in mice. Human helper T cell responses also polarize toward Th1 or Th2 profiles, though the definitions seem to be less rigid. There is
considerable evidence to support this paradigm in numerous domestic animal species, as well. However, it is important to be aware that the effects exerted by a cytokine in one species may not be identical to those exerted by its
homologue in another species.
Cytokines can be categorized by the many different functional activities they mediate, and any cytokine may have
multiple effects. Some cytokines activate innate immune cells such as phagocytes and natural killer cells and stimulate their participation in inflammatory processes, whereas others suppress or resolve inflammation. Several cytokines promote the activation of B cells and antibody production, as well as antibody class switching by B cells. Certain cytokines have effects further upstream in the ontogeny of cells, in that they direct the maturation and mobilization of distinct classes of cells. Chemokines are chemotactic cytokines that are able to induce chemotaxis and control
immune cell traffic in at least 2 ways. They attract the specific sets of immune cells that bear corresponding receptors and activate the integrin molecules that mediate leukocyte adhesion to vascular endothelium around the site of
an infection.
Cytotoxic T cells
Cytotoxic T lymphocytes (CTLs), which express the CD8 molecule on their surface, have an important role against a
wide range of intracellular pathogens. These lymphocytes are programmed to kill infected cells when their T cell
receptors (TCRs) recognize pathogen-derived peptides that have been processed and displayed on MHC I molecules
via the endogenous pathway. The endogenous pathway is most efficient at processing and presenting peptides, which
are synthesized in the cytoplasm of the infected cell. Cytotoxic T cells require seven to ten days after initial exposure
to develop an effective response capable of controlling a viral infection. If an animal was previously vaccinated with
a vaccine that stimulated CTL formation, those memory CTLs should rapidly respond and kill virus infected cells
early in secondary infection and help stop virus replication before clinical signs appear or before they become severe.
Gamma delta T cells
Gamma delta (γδ) T cells are the other major T cell subset. The T cell receptor for γδ T cells is a heterodimer comprised of a γ chain and a δ chain, which are structurally distinct from the α and β chains that form the T cell receptors
of CD4+ and CD8+ T cells. Antigen recognition by γδ T cells is not usually MHC-restricted. Certain host cell markers, such as stress-induced heat-shock proteins, can activate γδ T cells. The functions of γδ T cells in response to
pathogens are not well understood, but their prominent distribution in mucosal surfaces is consistent with a role in
surveillance and rapid response to invaders. There is also evidence that γδ T cells modulate inflammatory responses
and promote tissue repair.
Evaluation of Cell-Mediated Immunity
Past efforts to evaluate immune responses in domestic animals typically focused on humoral immunity. One reason
for this is the relative ease with which serum antibody responses can be monitored, by use of ELISAs and virus neutralization and hemagglutination inhibition assays. Immunological studies in recent years have highlighted the substantial roles of cell-mediated immunity in defense against human and animal infections. This has accentuated the
importance of examining cell-mediated immunity as part of any thorough effort to characterize an immune response
to infection or vaccination.Thorough evaluation of vaccine efficacy requires the evaluation of T cell responses to
vaccination in populations of animals over time. The cell-mediated immunity (CMI) evaluation requires assays that
can be performed on large numbers of out bred animals. Some assays for T cell activity are too cumbersome to use
for this purpose, for example, assays for T cell cytotoxicity. Other assays for T cell activity are inherently more practical.
313
Evaluation of T cell activation using specific monoclonal antibodies with compatible fluorochrome conjugates and
flow cytometry has proven to be a useful tool for monitoring the ability of a vaccine to induce T cell-mediated immunity in experiments with large number of animals.
Multi-parameter flow cytometry (MP-FCM) has been used in our laboratory to simultaneously identify the three major T cell subsets (CD4+, CD8+, and gamma delta T cell receptor+ (γδ TCR+) T cells), and detect the expression of
the activation marker CD25 (IL-2 receptor) on the surface of activated cells, intracellular interferon gamma (IFNg, a
T helper 1 cytokine) and intracellular interleukin 4 (IL-4, a T helper 2 cytokine). Assays are conducted by isolating
peripheral blood mononuclear cells (PBMC) from experimental animals, adding cells in microtiter wells with or
without the antigens of interest, incubating the cells for 4-6 days. Protein transport inhibitor is added at the conclusion of the incubation to accumulate the intracellular cytokines inside the cells, then staining for the cell surface
markers and permeabilizing the cell membranes to allow antibody into the cytoplasm before staining for the intracellular markers. The antibodies against CD4, CD8, and the gd TCR will identify the type of T cell subset. The antibodies against CD25, IFNg, or IL-4 will detect the expression of each of the activation markers. The results provide the
percentage of each T cell subset expressing CD25, IFNg, and/or IL-4. The changes in their expression indicate the
memory responses to the antigens tested and can be compared among treatment groups of experimental animals. Our
laboratory has used these techniques to monitor T cell mediated immunity in cattle, swine, horses, and dogs.
T-cell activity is a critical component of immunity to bovine respiratory syncytial virus (BRSV). We tested the effects of immunization by modified-live (MLV) and inactivated BRSV vaccines on cell-mediated and humoral immunity in young calves. The two forms of vaccine stimulated similar serum neutralizing antibody production, although the early kinetics of those responses differed. CD4+, CD8+, and γδ TCR+ T cells were analyzed before and
after immunization for BRSV-specific recall responses, as evaluated by CD25 upregulation measured by flow cytometry. Modified-live BRSV primed each of the three T cell subsets for statistically significant responses to antigen. Inactivated vaccine also primed each T cell population for significant antigen-driven CD25 upregulation, including responses by CD4+ and γδ T cells that were stronger and last longer than those primed by MLV. Monoclonal antibody was used in additional assays to block MHC class I during incubation of BRSV antigen with PBMC
from an animal in the inactivated vaccine group. The recall response by CD8+ T cells was more inhibited by this
treatment than the other subsets, further suggesting that the inactivated vaccine had primed antigen-specific CD8+ T
cells. In summary, the data indicate that balanced BRSV-specific T cell responses can be induced by inactivated, as
well as modified-live, conventional vaccines, which may implicate an alternative pathway of MHC class I antigen
presentation. (Sandbulte and Roth, 2003)
In another BRSV study, Modified-live BRSV MLV vaccine was administered to calves with no detectable neutralizing antibodies against BRSV. The MP-FCM was performed to detect recall responses to BRSV before and after vaccination. Significant T cell recall responses were detected in some of these seronegative calves before vaccination.
This suggested that they were presumably exposed to BRSV earlier while they still had maternal antibody, which
inhibited them from developing an active antibody response. Following vaccination, the virus-specific IgG, virus
neutralizing antibody, and T cell recall responses to BRSV were all elevated more rapidly in these calves than in the
calves with no T cell recall response before vaccination. This demonstrates that exposure to BRSV can induce both
T and B cell memory in young calves without developing seroconversion. (Sandbulte and Roth, 2002)
Bovine viral diarrhea virus (BVDV) is one of the most important respiratory viruses in cattle. Passive antibody to
BVDV by colostrum intake may interfere with the development of a protective immune response. We did a MPFCM study to determine if calves with a high level of maternal antibody and calves without maternal antibody to
BVDV would develop protective T cell responses to BVDV. Two groups of calves were fed with colostrum with
high BVDV antibody titer. Two other groups were fed milk replacer without maternal antibody. Two groups (one of
each feeding group) were challenged with virulent BVDV at 2-5 weeks of age. Milk replacer-fed calves died after
the challenge. Colostrum-fed calves with high maternal antibody levels did not become ill after the BVDV challenge. They did not develop a measurable neutralizing antibody but demonstrated specific T cell responses to BVDV
and higher expression of IFNg compared to the colostrum-fed, non-challenged calves.
314 
When the maternal antibody had waned, all remaining calves were challenged with the same virus. After the second
challenge, only the calves that were exposed to BVDV while they had maternal antibody were protected. These data
indicate that calves exposed to BVDV while maternal antibody levels are high can develop antigen specific CD4+,
CD8+, and gd T lymphocytes responses and protective immunity in the absence of an active antibody response.
(Endsley et al., 2003, 2004, Ridpath et al., 2003)
An MLV vaccine containing BVDV types 1 and 2 was shown to induce CD4+, CD8+, and γδ TCR+ T cells specific
for both types of BVDV as measured by CD25 upregulation and production of IFNg. In contrast, an inactivated vaccine that contained BVDV types 1 and 2 induced CD4+ and γδ TCR+ T cell but not CD8+ T cell specific responses
for both types of BVDV. This is probably because the inactivated vaccine was not capable of infecting the cells and
expressing virus protein in the cytoplasm for processing and presentation on MHC I molecules. (Platt et al., 2008)
It is noteworthy that a killed BRSV vaccine induced CD8+ cytotoxic T cells for BRSV, but a killed BVDV vaccine
did not induce CD8+ T cells that recognized BVDV. A potential explanation for the difference between these two
viruses is that the attachment protein (G) and fusion protein (F) on the killed BRSV virus may still be functional and
may carry the killed BRSV virus into the cytoplasm where some of the viral protein may have been processed and
presented on MHC I molecules.
In a BHV-1 (Bovine Herpesvirus 1 / IBRV- Infectious Bovine Rhinotracheitis virus) vaccine experiment, calves immunized with an MLV vaccine containing BHV-1 developed memory CD4+, CD8+, and γδ TCR+ T cells that recognized BHV-1 antigen for at least 6 months after vaccination. The CD4+, CD8+, and γδ TCR+ T cells also produced IFNg in response to BHV-1 at 2 months after vaccination, but the response to IFNg was undetectable by 6
months after vaccination. When the calves were challenged with BHV-1 at 6 months after vaccination they were protected from clinical signs and had a strong anamnestic response to BHV-1 as detected by both CD25 upregulation
and intracellular IFNg production. (Endsley et al., 2002)
An experiment was conducted to evaluate the ability of a pentavalent (BVDV type 1, BVDV type 2, BHV-1, BRSV,
and PI-3) modified live virus (MLV) vaccine to induce antibody and T cell responses when administered to three age
groups (1-2 week, 4-5 week, and 7-8 week old) of calves with maternal antibody and to evaluate protection from
BVDV type 2 challenge. In each age group, 8 calves were vaccinated and 4 calves served as non-vaccinated controls.
All calves received a virulent BVDV type 2 intranasal challenge on day 84 after vaccination. Serum virus neutralizing (SVN) antibody and antigen specific T cell subset responses (expression of CD25, IFNg, and IL-4 by CD4+,
CD8+, and γδ TCR+ T cells evaluated by MP-FCM were evaluated for all 5 viruses. Hematology and clinical scores
were evaluated for 14 days after challenge. Maternal antibody titers to all 5 viruses were measured on the day of vaccination. Maternal antibodies declined in spite of vaccination for all groups of calves for all viruses (except for the
titer to BVDV type 1 in the calves vaccinated at 4-5 weeks of age). The SVN titers for both types of BVDV in the
calves vaccinated at 4-5 and 7-8 weeks of age (but not in calves vaccinated at 1-2 weeks of age) had an anamnestic
response after challenge. The SVN antibody titers to BVDV type 2 of the 2 older groups were significantly higher
(p<0.05) than the youngest group after challenge. All three subsets of T cells of all three groups vaccinated at different ages responded to in vitro re-stimulation with BVDV types1 and 2 by increasing expression of one or more of the
activation markers (CD25, IFNg and IL-4). In contrast, there was no consistent evidence of T cell subset recall responses to BHV-1, BRSV, or PI-3 after vaccination. All three age groups of vaccinated calves had significant protection from BVDV type 2 challenge as determined by hematology, rectal temperature, and clinical scores. One week
after BVDV type 2 challenge, the vaccinated calves from all three age groups generally continued to have higher T
cell subset recall responses to BVDV types 1 and 2 than the non-vaccinated calves. In conclusion, the pentavalent
MLV vaccination induced both B cell and CD4+, CD8+, and gd TCR+ T cell memory responses to BVDV types 1
and 2 when given to calves with maternal antibody between 1 and 8 weeks of age, and protected them from challenge with virulent BVDV type 2. In contrast, there was no evidence of induction of antibody or T cell responses to
BHV-1, BRSV, or PI-3 after vaccination in any age group of calves with maternal antibody. Since the calves were
not challenged with any of these three viruses it is not known whether they would have had anamnestic antibody or
T cell responses to these viruses or if they would have been protected from challenge. (Platt et al., 2006/1)
315
Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in cattle. Cell–mediated immunity
is thought to be the major protective mechanism to prevent infection with MAP from producing chronic Johne’s disease. A vaccine containing Fruend’s incomplete adjuvant plus MAP when administered to young calves has been
shown to reduce the incidence and severity of Johne’s disease. We evaluated the ability of the MAP vaccine given to
calves 0 to 3 months old and heifers 9 to 12 months old to induce memory CD4+, CD8+, and γδ TCR+ T cells as
detected by expression of CD25 after exposure to MAP purified protein derivative (ppd) in vitro. Lymphocytes from
both age groups of animals responded strongly to MAP ppd by up regulating CD25 and by secreting IFNg into the
supernatant. These responses correlated well with the intradermal skin test using MAP ppd. (Platt et al., 2006/2)
Multi-parameter flow cytometry has proven to be a useful tool for analysis of the induction of cell mediated immunity by vaccines through detection of T cell subset responsiveness to specific antigens. However, this technology has
some important drawbacks and limitations. Since these are live cell assays, they have significant day-to-day and animal-to-animal variability. This requires that large numbers of animals be used and that they be sampled repeatedly.
The procedure is labor and equipment intensive and is therefore expensive. Lymphocytes should be isolated and set
up in the assay as soon as possible after blood collection. Overnight shipment of blood samples to the laboratory can
work, but it may introduce additional variability. Because of the day-to-day variability inherent in the assay, valid
comparisons can only be made between lymphocytes from groups of animals assayed in parallel on the same day.
References
Barry, M. and Bleackley, R.C. (2002). Cytotoxic T lymphocytes: all roads lead to death. Nature Reviews Immunology. 2, 401-409.
Brown, W.C., Rice-Ficht, A.C. and Estes, D.M. (1998). Bovine type 1 and type 2 responses. Veterinary Immunology
and Immunopathology. 63, 45-55.
Carding, S.R. and Egan, P.J. (2002). Gamma Delta T Cells: Functional Plasticity and Heterogeneity. Nature Reviews
Immunology. 2, 336-345.
Doherty, P.C. (1996). Cytotoxic T cell effector and memory function in viral immunity. Current Topics in Microbiology and Immunology. 206, 1-14.
Endsley, J.J., Quade, M.J., Terhaar, B., Roth, J.A. (2002). BHV-1-Specific CD4+, CD8+, and gammadelta T cells in
calves vaccinated with one dose of a modified live BHV-1 vaccine. Viral Immunology.15, 385-93.
Endsley, J.J., Roth, J.A., Ridpath, J., Neill, J. (2003) Maternal antibody blocks humoral but not T cell responses to
BVDV. Biologicals. 31, 123-5.
Endsley, J.J., Ridpath, J.F., Neill, J.D., Sandbulte, M.R., Roth, J.A. (2004). Induction of T lymphocytes specific for
bovine viral diarrhea virus in calves with maternal antibody. Viral Immunology. 17, 13-23.
Fischer, T., Buttner, M. and Rziha, H.J. (2000). T helper 1-type cytokine transcription in peripheral blood mononuclear cells of pseudorabies virus (Suid herpesvirus 1) primed swine indicates efficient immunization. Immunology. 101, 378-387.
Platt, R., Burdett, W., Roth, J.A. (2006/1). Induction of antigen-specific T-cell subset activation to bovine respiratory
disease viruses by a modified-live virus vaccine. American Journal of Veterinary Research. 67,1179-84.
316 
Platt, R., Roth, J.A., Royer, R.L., Thoen, C.O. (2006/2). Monitoring responses by use of five-color flow cytometry in
subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine. American Journal of Veterinary Research. 67, 2050-8.
Platt, R., Coutu, C., Meinert, T., Roth, J.A. (2008). Humoral and T cell-mediated immune responses to bivalent
killed bovine viral diarrhea virus vaccine in beef cattle. Veterinary Immunology Immunopathology. 122, 8-15.
Ridpath, J.E., Neill, J.D., Endsley, J., Roth, J.A. (2003). Effect of passive immunity on the development of a protective immune response against bovine viral diarrhea virus in calves. American Journal of Veterinary Research.
64, 65-9.
Sandbulte, M.R., Roth, J.A. (2002). T-cell populations responsive to bovine respiratory syncytial virus in seronegative calves. Veterinary Immunology Immunopathology. 84,111-23.
Sandbulte, M.R. and Roth, J.A. (2004). Methods for analysis of cell-mediated immunity in domestic animal species.
Journal of the American Veterinary Medical Association. 225, 522-530,
Seder, R.A. and Ahmed, R. (2003). Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nature Immunology. 4, 835-842.
Spellberg, B. and Edwards, J.E. (2001). Type 1/Type 2 immunity in infectious diseases. Clinical Infectious Diseases.
32, 76-102.
Tirado, S.M. and York, K.J. (2003). Antibody-dependent enhancement of virus infection and disease. Viral Immunology. 16, 69-86.
317
Rehabilitation for Orthopedic Surgery
Napaporn Senarat
The idea of applying rehabilitation principles and techniques to animal is not new. In fact, many of treatment
protocols for humans were developed and continue to be developed using animal models. The use of dogs as a research models has linked traditional physical therapy practice and veterinary medicine. Currently, protocols that
have been developed and studied in humans undergoing rehabilitation are being adapted for use in animal physical
rehabilitation.
Rehabilitation is the science of the application of physics, biomechanics, anatomy, physiology and psychology. And rehabilitation is an integral component of physiotherapy and is the process of helping and individual achieve
the highest possible level of function, independence and quality of life following illness or injury.
Physiotherapy is concerned with physical function, and regards movement and optimal function as central to
the health and well-being of individuals. A practical healthcare profession, physiotherapy involves the assessment,
diagnosis and treatment of disease and disability through physical means. Based upon the principles of medical science, it is regarded as within the sphere of conventional (rather than alternative) medicine.
The goals of Physiotherapy are to eliminate the cause of the dysfunction, to reduce pain and inflammation,
increasing and maintain muscle strength and joint flexibility, prevent or minimize atrophy of muscles, cartilage,
bone, tendons and increasing cardiovascular fitness.
Some of the physical therapy methods that can be adapted to canine rehabilitation include :
Thermotherapy (or Heat and cold application) is the therapeutic use of physical agents or means to heat or
cool the body. Effects of superficial heat are dilating of blood vessels, increase the extensibility of fibrous tissue,
pain relief. This technique is useful for chronic arthritis, muscle tension, joint stiffness after surgery. For cryotherapy, usually use in case of acute trauma, post operative joint and bone fracture, pain relief and reduce inflammation.
Ultrasound therapy, ultrasound frequencies of 0.5 to 5 MHz are used in physical therapy. Mechanical energy
in tissue is converted to heat and is able to deliver heat to deeper tissue structures. The degree of tissue warming decreases with distance to the transducer. Clinical applications for ultrasound therapy are pain relief and increase blood
flow in case of tendinitis and bursitis, chronic joint disease and muscle tension which usually occur after surgery
joint and bone fracture. Cancer, localized tissue or bone infections are contraindicated for treatment with therapeutic
ultrasound.
Low-level laser therapy (LLLT) is a medical and veterinary treatment that uses low-level lasers or lightemitting diodes to alter cellular function. Low-level laser therapy is considered to be especially effective for: wound
management, joint conditions (including arthritis), soft-tissue injuries (including strains, sprains, tendonitis and tenosynovitis, capsulitis and bursitis) and chronic pain.
318 
Electrical stimulation uses an electrical current to cause a single muscle or a group of muscles to contract. By
placing electrodes on the skin in various locations the physical therapist can recruit the appropriate muscle fibers.
Contracting the muscle via electrical stimulation helps strengthen the affected muscle. Electrical stimulation can be
used in dogs with neurological disorders for the purpose of muscle strengthening, retarding muscle atrophy, muscle
re-education, relief of contractures, relief of muscle spasm and trigger points, and pain relief.
Therapeutic exercise is an exercise planned and performed to attain a specific physical benefit, the benefits
are to : improve the rate of recovery, improve the quality and quantity of movement and enhance performance, conditioning, and endurance. Passive exercise motion imparted to a segment of the body by another individual, machine, or other outside force, or produced by voluntary effort of another segment of the patient's own body. Active
assistive exercise voluntary contraction of muscles controlling a part, assisted by a therapist or by some other
means. Active exercise motion imparted to a part by voluntary contraction and relaxation of its controlling muscles.
Aquatic therapy or Hydrotherapy, the physical properties of water can be utilized with great efficiency for
physical therapy and rehabilitation. The potential uses for aquatic therapy are many-fold, for example : rehabilitation
after orthopedic surgery (such as fracture, OCD, CCLR), rehabilitation after neurological injury (such as after intervertebral disc surgery, degenerative myelopathy) and muscle strengthening and improved joint function.
Pain, inflammation, joint stiffness, muscle atrophy, etc. can happen in almost case of orthopedic surgery.
Rehabilitation and physical therapy should be considered as part of the postoperative management to prevent muscle
atrophy, increase muscle mass and strength, and increase stifle joint flexion and extension range of motion. The most
important thing for dog undergoing any joint surgery is early mobilization.
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Neurological emergencies
Pakthorn Lewchalermwong, DVM
Neurology clinic
Kasetsart University
Bangkok, Thailand
Introduction
Neurological emergencies require rapid and accurate decision-making and treatment. Inappropriate management in the early stages of disease can have catastrophic consequences for the animal. Studies on acute head injury
and on status epilepticus in humans show that having an emergency protocol in place improves the outcome. Thus,
to ensure optimal treatment in an emergency situation, it is recommended that emergency protocols that take into
account the availability of staff and equipment for each clinic are developed pre-emptively.
Traumatic Brain Injury (TBI)
Evidence of head trauma among the physical findings should always be treated as a significant. An important early goal is to determine if the trauma to the head has resulted in traumatic brain injury (TBI). This may
seem a matter of semantics, but in fact, this is an important distinction. The term “head trauma” is quite inclusive –
ocular trauma, jaw fractures, broken teeth, epistaxis are all indicative of trauma to the head, without necessarily involving the brain. With TBI, there is neurological dysfunction attributed to the traumatic event and this necessitates
measures to minimize further injury.
Another important distinction is between primary and secondary brain injury. Primary injury occurs at the
time of impact and is caused by the forces of acceleration, deceleration and torque. Unfortunately, there is little that
can be done to treat primary brain injuries and recovery is related to the severity of initial injury. Examples of primary brain injuries include intraparenchymal haemorrhage, contusions, diffuse axonal injury, and less commonly, subdural haematomas. Secondary brain injury refers to pathologic processes that occur after initial injury (e.g., cerebral
oedema, ischemia) but progress and potentiate brain injury. Secondary brain injury results in progressive neuronal
damage at sites both locally and distally from primary injury. The aim of treatment of patients that have sustained
TBI is to prevent or limit secondary brain injury.
320 
Medical treatment
Significant injuries to the cardiovascular, respiratory and central nervous systems take priority over other injuries. Supplemental oxygen is always beneficial so as long the animal is not struggling against it. Oxygen provided
via flow-by, or oxygen mask is usually readily accepted. The key goal to keep in mind is the maintenance of systemic and cerebral perfusion. Therefore, if the animal is deemed to be hypovolumic, fluid therapy is almost always required to restore intravascular volume to maintain tissue perfusion. Ideally, the goal should be to maintain MAP > 70
mm Hg. It is important to titrate fluid therapy to physical findings – i.e. improvement of pulse quality, heart rate, etc.
While under resuscitation should be avoided, over-resuscitation with isotonic solution may also predispose to cerebral oedema. Therefore, fluid therapy in patients with TBI deserves particular attention and proper monitoring.
The hypertonicity of this fluid offers an additional osmotic effect at the level of the brain and may assist in
lowering ICP. Hypertonic saline has also been shown to
modulate pro- and anti-inflammatory mediators, regulate neutrophil-endothelial cell interactions, and attenuate polymorphonuclear neutrophil cytotoxicity in vitro. These benefits may explain the improved outcome when patients
with TBI are resuscitated with hypertonic saline.
The most commonly misused drug in veterinary patients with TBI is glucocorticoids. Currently, there is overwhelming evidence against its use in the setting of TBI and therefore, steroids should be considered contraindicated
both in shock and head trauma. Among other deleterious effects, its tendency in promoting or worsening hyperglycaemia.
In traumatised patients, hypoventilation can be a significant problem, especially if it leads to hypercarbia in
patients with TBI. The increase in CO2 will lead to cerebral vasodilation and increase ICP. Therefore blood gas analyses are very useful in guiding therapy. Patients with TBI that neurologically deteriorate should be intubated and
hand ventilated until their PaCO2 or end-tidal CO2 is normalized to 35 mmHg.
Both pain and anxiety lead to increases in ICP, therefore the use of analgesics is recommended.
Surgical tratment
A description of the surgical techniques for intracranial surgery can be found elsewhere. Although it is rare
that surgery is indicated in head injury cases, there are several specific abnormalities that can be associated with an
episode of head trauma that may warrant the consideration of surgical treatment: Acute extra-axial hematomas, Calvarial fractures, Acute intraparenchymal hematoma, Hemorrhagic parenchymal contusions, Intracranial hypertension (ICH)
Spinal cord injury (SCIs)
Acute spinal cord injuries (SCIs) are an extremely common problem in veterinary medicine and can lead to
permanent, sever neurological deficits affecting motor, sensory, and autonomic function. The most common causes
of acute SCI in dogs are acute intervertebral disk herniations, fibrocartilaginous embolism (FCE), and trauma. Cats
suffer from similar diseases but at different frequencies, with trauma being more common than intervertebral disk
herniations and thromboembolic events much more common than FCE.
The events that follow acute SCI are typically divided into primary and secondary damage, with the secondary
damage developing over acute (0-48 hours), subacute (48 hours-2 weeks), and chronic phases. Vascular injuries bypass the primary damage caused by physical trauma but induce the same secondary injury pathways.
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Medical treatment
Medical treatment of acute spinal cord concussion and ischemia is aimed at limiting the final extent of secondary tissue damage. Minimizing secondary injury is generally achieved by ensuring adequate perfusion and oxygenation of the animal and administration of neuroprotective agents. Treatment must be initiated as soon as possible
after injury, as the majority of secondary tissue damage occurs within 24 hours of the primary injury.
It should contemplate the systemic complications of acute spinal cord injuries, and includes the administration of intravenous fluids (colloids and cristalloids) to maintain a good mean arterial pressure (MAP) that ensures
adequate spinal cord perfusion.
The main goal of this treatment is to increase spinal cord perfusion. It must be remembered that local autoregulatory mechanisms are lost and spinal cord microcirculation is altered in many spinal cord injuries. This will lead to
additional spinal cord ischemia which will potentiate and worsen the secondary mechanisms of spinal cord injury. In
animals, the maintenance of an adequate circulation volume which ensures adequate spinal cord perfusion is considered critical for prognosis.
Administration of glucocorticoids is the most controversial aspect in the treatment of patients with spinal cord
injuries. Clinical trials have not been performed in domestic animals to demonstrate the effectiveness of MPSS treatment for spinal cord injury, and the secondary effects or complications of this treatment can be highly detrimental
(gastrointestinal ulceration and perforation, pancreatitis, secondary infections).
Besides aggressive fluid therapy, the standards for recumbent patient care: soft, padded, clean and dry bedding, urinary bladder care (manual emptying or catheterization), eye lubrication, etc. Non-surgical treatment of spinal fractures and luxations depends on whether the injury is considered to be unstable based on the threecompartment model. If there is no instability, cage rest for 6 weeks is an adequate treatment. If the fracture or luxation is considered unstable but the owners decline surgery, an external splint can be placed immobilizing the damaged area of spine.
Surgical treatment
Surgical treatment should be performed urgently in cases of acute spinal cord compression (herniated discs)
and in cases of unstable vertebral fractures/luxations. Acute disc herniations should be managed performing spinal
cord decompression and removal of the herniated disc material from the vertebral canal. Vertebral fractures and/or
luxations require surgical decompression, re-alignment of the displaced vertebral fragments and fixation.
Status epilepticus and cluster seizures
Status epilepticus (SE) can be defined as continuous seizure activity lasting for 30 minutes or longer, or repeated seizures with failure to return to normality with in 30 minutes. Cluster seizures describe the occurrence of
multiple seizure events within a 24-hour period. SE and severe cluster seizures can cause permanent neurological
sequelae or even death.
322 
Treatment protocol
The goals of treating SE are simple: stop the seizures, protect the brain from further damage, and allow full
recovery from the episode of SE.
Fluid therapy should be provided to guarantee cerebral perfusion using a maintenance dose unless there has
been a major loss of bodily fluids or hyperthermia. In cases of hyperthermia, the body temperature should be brought
down by wetting the animal or increasing the administration of fluids. If increased intracranial pressure is suspected,
mannitol may be administered as this is effective in treating intracellular oedema and it inhibits peroxide formation.
Furosemide has a stabilising effect on the membranes, so it may potentiate the effects of the anticonvulsants and the
effects of mannitol. The use of steroids in cases of status epilepticus is not recommended.
Levetiracetam,widespread safety and the absence of hepatic metabolism, recommended in cases of hepatic
encephalopathy. Its mode of action is not fully elucidated, although it would appear to act on the synaptic vesicle
protein SV2A reducing both the excitatory and inhibitory activity in the presynaptic membranes. Recent studies have
demonstrated that a dose of 60 mg/kg IV administered over 2 minutes is capable of maintaining serum levels for at
least 8 hours. The maintenance dose is 10-20 mg/kg q8h.
Drug treatment of the refractory SE patient
Status epilepticus that does not respond to a benzodiazepine or phenobarbital is considered refractory and
requires more aggressive treatment. Potential reasons for resistant seizure activity include inadequate anticonvulsant
doses, an uncorrected metabolic abnormality, a cerebral mass such as a tumour, or encephalitis such as Pug dog encephalitis.
Propofol: Propofol has barbiturate- and benzodiazepine- like effects and can suppress eNS metabolic activity. Propofol can be administered by intravenous bolus (4-8 mg/kg, to effect) or by constant-rate infusion (0.1-0.6
mg/kg/min or 6-12 mg/kg/h). However, this drug should be used with caution, preferably in settings where definitive
airway control and haemodynamic support are possible, as hypoxaemia secondary to apneoa is a primary side effect,
as is myocardial depression.
Barbiturates: Thiopental and pentobarbital have potential, though unproven, cerebral protective effects in
the management of SE. These drugs will almost always control the physical manifestations of seizures but.with negligible anticonvulsant properties. Pentobarbital should be given to effect rather than at a specific dose
(2-15 mg/kg i.v.) as there is tremendous individual variation in response.
Inhalational anaesthetics: These are recommended only as a last resort in cases of resistant SE. Isoflurane,
an inhalational general anaesthetic agent, may be efficacious in the treatment of resistant SE but may need to be used
for an extensive period of time. Obviously, isoflurane therapy necessitates ventilation and intensive-care monitoring,
and hypotension may occur during therapy.
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References
1. Olby NJ: Spinal dieases. Vet Clin North Am Small Anim Pract 2010; 40(5): 791-802
2. Laurent, S. Garosi. Diseases of the Brain; Vet Clin North Am Small Anim Pract 2010; 40(1)
3. Richard, A. LeCouteur. The Ultimate Small Animal Neurology Seminar. In: Proceeding Continuing Education
Program, The Veterinary Practitioner Association of Thailand, 2010 ; 110-119
4. Sande A, Chad W : Trauamtic brain injury: a review of pathophysiology and Management, Journal of Veterinary
Emergency and Critical Care 20(2) 2010; 177-190
5. Elisa M, Treat of Head and Spinal Trauma: Proceeding of the Congreso Latinoamericano de Emergencia y
Cuidados Intensivos LAVECCS,2009
6. Podell M, How treat status epilepticus. In : Proceeding of the Southern European Veterinary Conference & Congreso Nacional AVEPA, 2009
7. Chan D, Management of Traumatic Brain Injury. In : Proceeding of the Southern European Veterinary Conference & Congreso Nacional AVEPA, 2008
8. Daniel L, Head Trauma Management. In: European Veteriney Conference Voorjaarsdagen, 2008; 70-71
9. Platt SR, Treatment options for head trauma patients. In: Proceeding of the 33 rd World Small Aniaml Veterinary
Congress, 2008 ;498-500
10. Anor S, Spinal Cord Injury. In: Proceeding of Southern European Veterinary Conference, 2007
Platt SR, Olby NJ: Neurological emergencies. In: Platt SR and Olby NJ (ed): BSAVA Manual of Canine and Feline
Neurology, Gloucester, British Small Animal Veterinary Association, 2004; 320-336
324 
BECOMING A CLINICAL VETERINARY ONCOLOGIST – THE IMPORTANT THINGS TO KNOW
Rodney C. Straw BVSc., Dip AVCS*
Brisbane Veterinary Specialist Centre
Cnr Keong and Old Northern Rds
Albany Creek 4035 Queensland AUSTRALIA
Introduction
What separates a surgical oncologist from other surgeons is awareness and proficiency of some unique cancer operations and most importantly, solid grasp of tumor biology and a close interaction with other cancer specialties
(chemotherapy, radiation therapy, surgical pathology, diagnostic imaging, immunotherapy, etc.). An oncology surgeon must be able to apply SURGICAL ONCOLOGY THINKING. To impart this concept, I tell my students, residents and fellows what I was told years ago years ago, and that is to inwardly ask these questions before attempting a
surgical treatment for cancer:
1. What am I treating? Does the biopsy fit the clinical situation? For instance a fibroma in the oral cavity does not
erode bone, so if this is occurring then maybe it is something else such as a fibrosarcoma.
2. What is the known biology of this cancer? Is it highly invasive, what is the metastatic potential, does histological
grade predict outcome, are there any other prognostically significant variables, etc.?
3. Is a cure possible? If so, aggressive interventions are justified based on a projected long-term survival.
4. What is the proper surgical dose? Just as chemotherapy and radiation are described by dose, surgical procedures
can be described by dose: intralesional, marginal, wide or radical. Each of these "surgical doses" has an appropriate
time and place. (Table 1)
5. What are my alternatives to surgery? A careful multidisciplinary approach to the individual patient is best
planned from the outset, rather than after a poorly performed or timed surgery.
Complete surgical removal of localized cancer cures more patients (human and animals) than any other form of treatment. To attain this ideal goal, surgeons must have a thorough understanding of anatomy, physiology, resection and
reconstruction options for all organs, expected tumor behaviour and the various alternatives or adjunctive treatments.
Surgical oncologists should not only be good technicians (cancer carpenters) but dedicated tumor biologists. Surgery will play a role at one point or another in the management of most cancer patients. This surgery may include
any of the following: diagnosis (biopsy), resection for cure, palliation of symptoms, debulking and a wide variety of
ancillary procedures to enhance and compliment other forms of treatment.
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Most patients with cancer are "old". Old is a relative term. It is much more important to know the physiologic age
of the patient than its chronologic age. An "old" dog or cat with normal measurable organ function should not be
denied treatment simply on the basis of age. I am aware of no cancer where increasing age worsens tumor related
prognosis. In fact the opposite is sometimes true; in some instances young animals have a worse prognosis than older animals with the same histological tumor type. "Old" animals will tolerate aggressive surgical intervention as
well or as poorly as "young" patients.
Surgery For Diagnosis
Biopsy principles will be covered in the next section, however it bears emphasizing that properly timed, performed
and interpreted biopsies are one of the most crucial steps in the management of the cancer patient. Not only does the
surgeon need to procure adequate tissue to establish a diagnosis but also the biopsy must not compromise subsequent
surgical resection.
Surgery For Cure
Before a surgeon can be in a position to provide the optimal operation for the patient with cancer, he or she should
be able to answer the following questions:
1. What are the type, stage, and grade (if this has bearing on outcome) of cancer to be treated?
2. What are the expected local and systemic effects of this tumor type and stage?
3. Is a cure possible?
4. Is an operation indicated at all?
5. What are the options for alternative treatment?
A recurring theme in surgical management of cancer is that the first surgery has the best chance of cure. Several
mechanisms for this improvement in survival have been advanced. Untreated tumors have had less chronologic time
to metastasize than recurrent cancer. Untreated tumors have near normal anatomy, which will facilitate operative
maneuvers. Recurrent disease may have had seeding of previously noninvolved tissue planes requiring wider resection than would have been required on the initial tumor. An ill-defined negative aspect of recurrent cancer is likely
related to changes in vascularity and local immune responses. Regardless of the mechanism, curative intent surgery
is best performed at the first operation.
The actual surgical technique will obviously vary with the site, size and stage of the tumor. Some general statements
that need to be emphasized with cancer surgery are:
1. All incisional biopsy tracts should be excised in continuity with the primary tumor, since tumor cells are capable
of growth in these wounds. Fine needle aspiration cytology tracts are of little concern while punch biopsy tracts are
of intermediate concern. With this in mind, all biopsies should be positioned in such a manner that they can be removed at surgery.
2. Early vascular ligation (especially venous) should be attempted to diminish release of large tumor emboli into the
systemic circulation. This is probably only clinically meaningful for those tumors with a well-defined arterial and
venous supply such as splenic tumors, retained testicles and lung tumors. Small numbers of cancer cells are constantly being released into the venous circulation by most tumors. Larger, macroscopic cell aggregates may be more
dangerous, however, and these may be prevented from vascular escape with early venous ligation.
3. Local control of malignant cancer requires that an adequate margin of normal tissue be removed around the tumor.
Resection can and should be classified in more detail than radical vs. conservative. Tumors with high probability of
local recurrence (soft tissue sarcoma, malignant mast cell tumors, feline mammary adenocarcinoma, etc) should have
2 to 3 cm margins removed 3-dimensionally. Tumors are not flat and wide removal in one plane does not ensure
complete excision.
326 
Fixation of malignant cancer to adjacent structures mandates removal of the adherent area in continuity with the tumor. This is commonly seen with oral cancer that is firmly adhered to the underlying mandible or maxilla. Invasive
cancer should not be peeled out, shelled out, enucleated, curetted or whittled on if a cure is expected. A pseudo capsule surrounds some malignant cancers. This capsule is composed of compressed and viable tumor cells, not healthy
reactive host cells. The pseudo capsule is therefore not a plane for dissection if cure is the goal. If a malignant tumor is opened at the time of resection, that procedure is reduced to no better than a large biopsy. A level of dissection that is one tissue plane away from the mass should be strived for. Invasion of cancer into the medullary cavity
of a bone requires subtotal or total bone resection and not curettage. Surgery may be measured in terms of dose,
much like chemotherapy and radiation therapy (Table 1).
4. Tumors should be handled gently to avoid risk of breaking off tumor cells into the operative wound where they
may grow. Copious lavage of all cancer wound beds will help mechanically remove small numbers of exfoliated
tumor cells but should not replace gentle tissue handling.
Table 1 Classification of wound margins.
TYPE OF SURGERY
PLANE OF DISSECTION
RESULT
Intracapsular
Tumor removed in pieces or curetted:
"debulking"
Macroscopic disease left behind
Marginal
Removal just outside or on the pseudo
capsule
Usually leaves microscopic disease
Wide
Tumor and capsule not entered, cuff of
normal tissue surrounds the specimen
Possible skip lesions
Radical
Entire compartment or structure removed
No local residual cancer
(E.g. amputation)
The aggressiveness of resection should only rarely be tempered by fears of wound closure. It is better to leave a
wound partially open with no cancer than closed with residual cancer.
Lymph Node Removal
A great deal of controversy surrounds the surgical management of regional lymph nodes draining the primary tumor
site. As a general rule, epithelial cancers are more likely to metastasize to lymph nodes than are mesenchymal cancers. However, any enlarged regional lymph node requires investigation. Lymphadenomegaly may be from metastasis of cancer (firm, irregular and sometimes fixed to surrounding tissue) or from hyperplasia and reactivity to various tumor factors, infection, or inflammation. The former cause is a poor prognostic sign and the latter may be a
beneficial host response. Enlarged lymph nodes as a result of cancer metastasis and invasion are generally uniformly
effaced by tumor cells and can often be diagnosed by fine needle aspiration. Positive lymph nodes usually are a sign
of impending systemic metastasis. Lymph nodes should be removed under two general circumstances:
1. If the lymph node is positive for cancer and not fixed to surrounding normal tissues, it may be possible to remove
the node with some therapeutic intent. Frequently however, many lymph nodes drain a primary tumor site (e.g. oral
cavity) and lymphadenectomy is incomplete (e.g. neck dissection). Although it is usually not practical, removal of
the primary tumor, intervening lymphatic ducts and draining lymph node has been recommended (en bloc resection).
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En bloc resection may be possible for a malignant toe tumor with metastasis to the popliteal lymph node, but is usually only accomplished with amputation. Few other anatomic sites are routinely amenable to this therapy.
2. Normal appearing lymph nodes which are known to drain a primary tumor site should be sampled (biopsy or cytology) to gain further staging information. This is particularly important if adjunctive treatment decisions
(irradiation or chemotherapy) would be predicated on confirmation of residual cancer. Intrathoracic or intraabdominal lymph nodes are perhaps most crucial since they are not readily accessible to follow-up examination.
Lymph nodes are not removed under two general circumstances:
1. Lymph nodes in critical areas (retropharyngeal, hilar, mesenteric) which have eroded through the capsule and become adherent (fixed) to surrounding tissues cannot be curatively removed without serious harm to the patient. They
are best biopsied and left alone or treated with other modalities. The occasional exception is metastasis of limb and
foot tumors to prescapular and popliteal lymph nodes that can be removed with amputation (radical en bloc resection).
2. Prophylactic removal of "normal" draining lymph nodes or chains of lymph nodes (as opposed to sampling for
stage) is of no benefit and may be harmful. Regional lymph nodes may in fact be the initiator of favorable local and
systemic immune responses and elective removal has been associated with poor survival in certain human cancers.
Palliative Surgery
Palliative surgery is an attempt to improve the quality of the patient's life (pain relief or improved function) but not
necessarily the length of the patient's life. This type of surgery requires very careful consideration of the expected
morbidity of the procedure versus the expected gain to the patient and the client. In essence, it comes down to a decision of when to give up. One of the most difficult decisions in surgical oncology is the decision not to operate.
Treatment of any kind should never be worse than no treatment.
Certain situations do exist, however, where palliative surgery may be beneficial. If an infected and draining mammary tumor in a patient with asymptomatic lung metastasis is the limiting factor in the patient's life, mastectomy
may still be a logical procedure. Splenectomy for haemangiosarcoma is commonly performed but probably has little
impact on survival and can be considered palliative, since it will stop the threat of immediate haemorrhage.
Debulking Surgery
Incomplete removal of a tumor (planned or unplanned) is referred to as debulking or cytoreductive surgery. It is
commonly performed but rarely indicated. Its theoretical indication is to enhance the efficacy of other treatment modalities. Debulking is a practical consideration prior to cryosurgery to decrease the amount of tissue to freeze and
the time it will take. It may also help the treatment planning and dosimetry with certain forms of irradiation but the
improved cancer control achieved is more a result of geometric considerations than removal of a few logs of tumor
cells. Removing 99.9% of a one-centimeter tumor (1 x 109 cells) still leaves a million cancer cells behind. Immunotherapy and chemotherapy could theoretically be helped by tumor volume reduction. If deep-seated tumors are
debulked with the anticipation of postoperative radiation therapy, the margins of known tumor or the operative field
should be marked with radiopaque metal clips to allow proper treatment planning from radiographs.
Surgery And Chemotherapy
The combined use of chemotherapy and surgery is becoming more commonplace in veterinary oncology. Many
chemotherapy agents will impede wound healing to some extent. In spite of this risk, few clinically relevant problems occur when surgery is performed on a patient receiving chemotherapy. General recommendations are to wait 7
to 10 days after surgery to begin chemotherapy, especially for high-risk procedures. The use of intraoperative or
perioperative chemotherapy is receiving increased attention and could have greater implications to wound healing.
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Surgery And Radiation
Theoretical advantages can be advanced for both pre- and postoperative radiation. Either way, some impairment of
wound healing potential will exist. Radiation damage to normal tissues (stem cells, blood vessels and lymphatics) is
more permanent than chemotherapy damage. As radiation dose and field size increase, the potential complications
(with or without surgery) increase. If radiation therapy is given preoperatively, surgery can be performed after acute
radiation reactions have resolved. Postoperative radiation is recommended to start immediately postoperative or after a two to three week delay. In spite of the theoretical problems, surgery can be safely performed on irradiated tissues. Complications may occur but are not prohibitive.
Prevention Of Cancer
Certain common cancers in dogs and cats can be prevented. It is well known that spaying before one year of age will
reduce the risk of mammary cancer in the dog (and to a lesser degree, the cat), by 200-fold over intact bitches. Castration of the male dog will help prevent perianal adenomas and, obviously, testicular cancer. Elective removal of
cryptorchid testes is another example of preventative surgery as these teticles have a high risk of developing sertoli
cell tumours.
Miscellaneous Oncologic Surgery
With greater use of regional intraarterial chemotherapy, surgeons may be called upon to place long-term vascular
access catheters.
Surgeons and radiotherapists may work together for the operative exposure of nonresectable cancer so that large doses of irradiation may be delivered to the tumor or tumor bed after exclusion of radiosensitive structures
(intraoperative radiation therapy).
Discussion
It is clear that surgery will be the mainstay of cancer treatment in veterinary medicine for many years to come. It is
also clear that just because a surgical procedure is possible is not the best reason to do it. Although rhinotomy and
curettage of the canine nasal cavity can be performed, it does not improve survival over untreated patients. Likewise, simple versus radical mastectomy in the dog does not influence survival but it may in the cat. More surgery is
not always better surgery. Long term follow-up of well-staged and graded tumors with defined surgical technique is
necessary to demonstrate the true value of any operation. A great deal of progress in surgical technique and surgical
thinking needs to take place before the role of surgery in cancer management can be optimized. A better understanding of expected tumor biology and more precise staging methods (angiograms, CT scans, etc.) will hopefully facilitate more precise surgical operations to be performed. In spite of these anticipated advances in technology and biology, the most difficult aspect to learn is surgical judgment.
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CLINICAL SMALL ANIMAL ONCOLOGY CASES
Rodney C. Straw BVSc., Dip AVCS*
Brisbane Veterinary Specialist Centre
Cnr Keong and Old Northern Rds
Albany Creek 4035 Queensland AUSTRALIA
Introduction
This session will be a compilation of clinical cases. These notes form background information from which the cases
will be selected to represent.
Nasal Planum Tumours
Tumours of the nasal planum are rare in the dog, but more common in the cat. The most common nasal planum tumour is squamous cell carcinoma (SCC). SCC appears to be an actinic (UV exposure-associated) malignancy in
cats and dogs, as evidenced in part by the fact that it appears more commonly in light-coated cats. These tumours can
be very invasive locally, but rarely metastasise.
The early stages of nasal planum SCC may simply show mild crusting or scabbing of the nasal planum. Over time
(months to years), the lesions can enlarge and become more deeply erosive, erythematous and ulcerated.
Lymphadenopathy is rarely appreciated in these cases, and pulmonary metastasis is extremely rare. However, any
appreciated lymphadenopathy should be investigated via needle aspiration cytology, and thoracic radiography is reasonable prior to undertaking an expensive or aggressive therapeutic approach (see below).
A diagnosis can be achieved by means of a simple Keyes-type skin punch biopsy, which can be obtained using
quick-acting injectable sedation. Imprint or aspiration cytology is typically unrewarding for achieving a diagnosis.
Limiting sun exposure (i.e. restricting access to the outdoors) may arrest the progression of very early lesions.
Numerous treatments have been demonstrated to be effective for superficial SCC, including cryotherapy, photodynamic therapy, radiotherapy (RT), hyperthermia, and intralesional chemotherapy (i.e. direct injection into the tumour) with cisplatin or carboplatin. Deeply invasive lesions are typically less responsive to these types of treatment.
However, complete excision of the nasal planum (“nosectomy”) can be achieved with acceptable functional and cosmetic results in both cats and dogs. The goal of this therapy is to achieve tumour-free surgical margins, thereby preventing local recurrence. RT has been employed in the post-operative setting for the treatment of residual disease
after surgery, and has been associated with a good outcome.
Lung Tumours
Primary lung tumours are relatively rare in dogs and cats, especially when compared with the astronomical tobaccoassociated incidence of such tumours in humans. Metastatic lung cancer is much more common than primary lung
cancer in both the dog and the cat. The vast majority of lung cancers in domestic animals are malignant, with carcinomas arising from the respiratory epithelium being most common. Again, some studies have suggested that dogs
living in an urban environment may be at increased risk when compared with dogs living in a rural environment
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The most common presenting complaints in animals with lung tumours are (in decreasing frequency) cough, dyspnea, lethargy, and weight loss. (Other differentials for cough and dyspnea?) Approximately 25% of cases will be
discovered incidentally, without any evidence of clinical signs. In cats, a percentage of animals will present with
signs referable to a mass at an external site (e.g. digits, face, etc.). Thoracic radiographs then reveal a pulmonary
mass later confirmed to be primary pulmonary neoplasia.
Physical examination may reveal increased respiratory rate and effort, or dullness on auscultation. Thoracic radiographs provide the most important information in obtaining a diagnosis of lung cancer. They will usually demonstrate a solitary, spherical mass, often in the caudal lung lobes. Occasionally, pleural effusion or evidence of tracheobronchial lymphadenopathy may be observed. Advanced imaging (e.g. CT scan) can better delineate the extent of
the mass, and may yield evidence of intrapulmonary metastasis or lymphadenopathy that is not detectable by conventional radiographs. Needle aspiration cytology (often performed under ultrasound guidance) can often confirm a
diagnosis of carcinoma. Often, a histologic diagnosis is only reached at the time of definitive therapy.
For patients with a solitary lung mass without evidence of metastasis, thoracotomy with complete lung lobectomy is
the treatment of choice. The tracheobronchial lymph nodes and all lung lobes should be carefully evaluated at the
time of surgery, and biopsy should be performed of any enlarged lymph nodes.
The median survival time after surgery for dogs with primary lung tumours is approximately one year. The major
prognostic factors for survival are: 1) Presence or absence of lymph node metastasis; 2) Presence or absence of clinical signs at presentation; 3) Histologic grade of the tumour.
Although adjuvant treatments such as chemotherapy and RT are effective in the management of human lung cancer,
the role of these modalities in the treatment of lung tumours in domestic animals has been poorly evaluated. In patients with high-risk disease (e.g. high histologic grade or lymph node metastasis), the addition of chemotherapy may
be reasonable. Recently, the UW Oncology Service investigated the role of post-operative inhaled chemotherapy on
the outcome in dogs with lymph node-positive lung cancer, in which the prognosis is extremely poor (median survival time = 60 days with surgery alone). The addition of inhaled chemotherapy significantly extended survival in these patients, when compared to historical controls treated with lung lobectomy alone.
Management of Lateral Thoracic Wall Masses in Dogs
Masses involving the lateral chest wall of dogs are seen infrequently in general veterinary practice. Such lesions are,
however, usually primary malignant tumours of ribs. They pose a serious clinical problem because full thickness
chest wall resection is necessary for successful management. The work-up, surgical procedure, after-care, adjunctive
therapy, follow-up, and potential complications will be presented.
I reviewed 295 dogs reported in the literature with chest wall tumours and of these 201 had documented long term
follow-up. Chondrosarcoma, osteosarcoma, haemangiosarcoma and fibrosarcoma are the common tumours affecting
the ribs with the first two mentioned histological types being the most common. Dogs with chondrosarcoma of the
ribs have the best prognosis following resection with reported median survival times up to 250 weeks. Dogs with
malignant rib tumours treated conservatively without surgery have a poor prognosis with reported median survival
times of 2 to 15 weeks depending on the histology. Wide surgical margins including at least one unaffected rib cranial and caudal to the lesion and dorsal and ventral margins allowing 2-3 cm of grossly unaffected rib are necessary
for successful en block resection of malignant primary rib tumours. Reconstruction involves the use of polypropylene mesh, diaphragmatic advancement, latissimus dorsi muscle flaps, or a combination of these with or without augmentation using omental pedicle grafts. Analgesia can be effectively provided pre-emptively, intra-operatively and
postoperatively by intercostal nerve blocks, opiates either delivered by systemic administration (constant rate infusion or intermittent intravenous or subcutaneous injections) or by transdermal patches, epidural morphine, intrapleural local anaesthesia, and non-steroidal anti-inflammatory drugs as well as newer and alternative analgesic strategies.
Adjuvant chemotherapy drugs are usually recommended for dogs with osteosarcoma.
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Carboplatin, cisplatin and doxorubicin either as single agents or in some combination have demonstrated efficacy in
treating dogs with osteosarcoma. The efficacy of chemotherapy drugs in the adjuvant setting for treating dogs with
chondrosarcoma has not been demonstrated. In the author’s experience, dogs with haemangiosarcoma of the ribs
have very poor prognoses, as metastatic disease, either measurable or occult, is almost invariable. However, two
dogs in one study lived 48 and 52 weeks after surgery and survival times as long as 112 weeks have been reported.
Dogs with rib fibrosarcoma have been reported to have a median survival of 26 weeks with a range of 16-64 weeks.
Metastatic disease has been reported in dogs with chest wall tumours especially those with haemangiosarcoma and
osteosarcoma. Recurrence is not common after wide resection with reported incidences of 10-15%. Histologically
determined complete surgical margins confer an improved probability for local control.
Early wide surgical excision is recommended in the management of tumours of the canine lateral thoracic wall. This
treatment has been reported to be associated with low long-term morbidity and excellent survival probabilities for
dogs with certain histological types of primary rib cancer.
Soft Tissue Sarcomas In Dogs & Cats
Soft Tissue Sarcomas (STS) are tumors of mesenchymal tissue. Most STS arise from the skin, subcutaneous tissue,
or palate, and represent malignant or transformed mesenchymal cells.
The etiology is generally not known with some notable exceptions such as those associated with infestations with the
parasite Spirocerca lupi, those induced by the feline sarcoma virus and those thought to be associated with feline
vaccinations (Vaccine Associated Sarcomas, VAS). Sarcomas can also be induced by ionizing radiation and high
grade STS are rare, serious, late complications of radiation therapy.
Although STS develop from a variety of mesenchymal tissues we usually consider them as a group for two main reasons. One is that pathologists often disagree about the histiogenesis (fibrosarcoma vs. neurofibrosarcoma vs. haemangiopericytoma) and secondly, the biological behaviour of most solitary canine STS shares many common features.
They may arise from any anatomic site in the body. They tend to appear as pseudo encapsulated fleshy tumors but
have poorly defined histological margins and can infiltrate facial planes. Local recurrence after conservative surgical
excision is common. STS tend to metastasize via the haematogenous route although the rate is low (10-25%). They
generally have a poor response to systemically administered chemotherapy or radiation therapy for clinically detectable disease.
Fine needle cytology is an extremely valuable, easily performed, screening test to rule out common differential diagnoses such as lipoma, mast cell tumor, abscess etc. For definitive diagnoses a carefully procured and prepared biopsy
for histopathology is essential. Care must be taken to place the biopsy in location so the entire biopsy tract can be
removed with definitive surgery or included in a radiation field. The biopsy therefore should be performed atraumatically, aseptically with excellent hemostasis and preferably by the same surgeon who is to perform the definitive surgery. Biopsy wounds should not be drained and seromas avoided by following good surgical technique.
If the mass is fixed to bone, regional radiographs must be performed. Computerized tomography or magnetic resonance imaging may also be useful to help accurate local staging especially for tumors especially those located on the
head and pelvis. Thoracic radiography is important for clinical staging. Serum biochemistry, hematology, and urinalysis are useful for overall health evaluation but may not lend diagnostic information. Some dogs with STS are
hyperglycemic (intra-abdominal leiomyosarcoma).
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Forming the basis for clinical staging is tumor size, fixation, site, status of the regional lymph node and detectable
metastases. All too commonly inadequate surgical excision is performed. These tumors are often loosely separated
from adjacent structures by a pseudo capsule, which deceives the surgeon into electing this margin for resection. The
result is a marginal resection. The pseudo capsule is made up of viable tumor cells and is not a fibrous response by
host cells. The viable tumor cells project from the pseudo capsule into surrounding tissue and these cells are left behind by marginal resection. The result is a very high probability of local tumor recurrence, usually within 6 months.
After subsequent marginal or intralesional resections, the probability and rate of recurrence increases and often the
recurring tumor is of higher histological grade than the original lesion. The risk of metastasis is, therefore also increased. Cure rests with accurate preoperative diagnosis and staging and the performance of complete tumor removal
by properly performed first time surgery. Excision with wide (2 to 3 cm margins) or radical margins is the treatment
of choice.
Biopsy tracts must be excised in continuity with the mass. Any areas of fixation including bone and fascia must be
excised en bloc with the mass. A recurrence free survival rate of approximately 90% can be achieved at 12 months
with complete surgical resection. The margins of the resected specimen should be carefully evaluated by the
pathologist because dogs with STS removed with incomplete margins are over ten times more likely to suffer recurrence than dogs with completely excised tumors. Histological grade is also of prognostic significance.
Radiation therapy alone is generally not considered optimal treatment for dogs with STS but marginal resection in
conjunction with megavoltage radiation therapy results in a local recurrence rate of approximately 20-30%. Where
wide surgical margins cannot be achieved, adjuvant radiation therapy may be considered.
Cats are similar to dogs when it comes to STS except for the fact that nerve sheath tumors and synovial cell sarcomas are extremely rare in cats compared to dogs. There are also several unique fibrosarcoma settings in cats. Cats
have been reported to suffer from a virally induced multicentric fibrosarcoma; they can develop a vaccine associated
fibrosarcoma; and there have been reports of fibrosarcoma developing in the orbit of cats with pthysis bulbi, ocular
trauma or ocular foreign bodies.
The cause of nonvirally induced, non –vaccine associated and non-ocular fibrosarcomas is generally not known.
These tumors are seen in cats 8 to 12 years of age with no sex or breed predilection. Over 50% of these fibrosarcomas occur on the limbs making limb salvage curative surgery very difficult. These solitary STS’s are slow growing,
low grade and can be permanently controlled with properly performed surgery. Wide or radical surgical margins are
indicated for the management of these tumors in cats.
Sarcomas following ocular trauma occur most often in cats 7 to 15 years of age and the latency following trauma
averages 5 years. Damage to lens and chronic uveitis may be risk factors. These tumors are often advanced by the
time they are diagnosed with extension into the bone of the orbit and the optic nerve. Therefore I recommend removal of the phthisical feline eye or cat eyes that are blind and have been severely traumatized or chronically inflamed.
Feline sarcoma viruses (FeSV) are true hybrids and result from the rare recombination of feline leukemia virus
(FeLV) DNA provirus with cat proto-oncogenes.
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Cats that have FeSV are always FeLV positive. It is thought that only 2% of fibrosarcoma of cats are virally induced. The FeSV-induced tumors are multicentric and mostly found in young cats. These tumors are generally very
fast growing with metastatic rates reported of 30% or so. Prognosis is very poor in these cats. Although some authors indicate chemotherapy with radiation therapy may be tried, recurrences and progressive disease usually ensues.
Vaccine-associated sarcomas consist of cells that are morphologically and immunohistochemically compatible with
fibroblasts and myofibroblasts. The precise pathogenesis of vaccine-associated sarcomas is unknown but may involve stimulation of these cells by highly immunogenic and persistent adjuvants or other vaccine components resulting in inflammation that alone or in association with unidentified carcinogens or oncogenes leads to neoplastic transformation and tumor development. Transition zones from inflammatory granuloma to sarcoma have been identified
and strongly suggest that the inflammatory response to vaccination is antecedent to sarcoma development in cats.
Vaccine-associated feline sarcomas are highly invasive and, often, rapidly growing neoplasms that require aggressive treatment, which may include a combination of surgery, radiation therapy, and chemotherapy. Metastases may
develop, and the metastatic rate increases with survival time. Because vaccine-associated sarcomas often mimic benign postvaccinal injection site granulomas, differentiating these lesions is critical.
Reconstructive Surgery
Like many other cancer therapies, curative surgery for patients with solid tumours is achieved at the risk of normal
tissues. Where the advantage gained by aggressive, wide surgical margins outweighs the disadvantages there is
“therapeutic gain”. The surgical oncologist must consider this in a global sense before embarking on “radical” surgical exercises. There are, however, many settings where complete surgical excision may be curative and it is paramount that surgical margins are not compromised for the sake of ease of closure.
Soft tissue defects may be managed in several ways and it is the prepared surgeon who will be able to offer his or her
patients the best opportunity for low morbidity, rapid recovery and return to function after excisional surgery for
cancer.
Besides being familiar with the technology of plastic and reconstructive surgery (the “nuts and bolts”), I cannot overemphasise the importance of solid knowledge of wound healing, wound management and atraumatic surgical technique.
Although a thorough review of wound healing and wound management is beyond the scope of this particular seminar series, I will take time at the start of these seminars to present some principles of good tissue handling. “Poor
tissue handling can defeat the best surgical plans.” (Mike Pavletic)
An understanding of the blood supply to the skin of dogs and cats is essential. The subdermal plexus is the major
vascular network supporting the skin. The vessels of this plexus are in the fatty subcutaneous tissue sometimes
called areolar tissue on the deep surface of the dermis in the middle to lower portions of the limbs. Where there is a
panniculus muscle (cutaneous muscle) the subcutaneous plexus lies deep and superficial to this muscle. So it is
clear, surgeons must take care not to disrupt this crucial element of vascular support to the skin. Skin must be elevated deep to the subdermal plexus if the vascular supply to the elevated section is to be preserved. If the tissue between the panniculus muscle and the skin is severed there will be complete necrosis of the overlying skin. The skin
should always be undermined in the fascial plane beneath the cutaneous musculature or, where there is no panniculus
muscle, undermine in the fascial plane well below the dermal surface.
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Another important element of canine and feline cutaneous vascular anatomy is the location of the primary cutaneous
arteries. These are reliable large vessels that provide blood to the subdermal plexus. They can be manipulated to
provide vascular pedicle grafts very useful in reconstructive surgery. Several of these will be discussed with case
examples in the seminar.
I have found several things helpful for successful reconstruction of large skin defects:
Prepare a wide area of skin so you can “change your mind” and go with plan b.
Position the animal so the skin is relaxed and not “twisted” under the weight of the recumbent patient
(particularly for large dogs).
Use a sterile skin-marking pen.
Use skin hooks and pointed towel clamps to hold, move and temporarily stretch and secure skin.
Keep exposed dermis and cutaneous musculature moist with saline
Test skin tension of recipient and donor sites by “pinching” skin with your fingers prior to making skin incisions.
Use opened surgical sponges as templates to help design flaps.
Use analgesics! Have the animal comfortable in post op so as to minimize the risk of self-trauma.
Use closed suction drains.
Keep animals under close, in hospital, supervision for up to 5 days post op for involved, larger defect reconstructions so adequate analgesics can be given and strict rest enforced.
Minimally bandage flaps and observe them for signs of complications frequently.
Wound tension is one of the major enemies of skin closure. Excessive tension during wound closure often results in
circulatory compromise to the wound resulting in skin necrosis and wound breakdown. There is considerable variability of skin mobility between breeds of dogs, cats and dogs, and individuals pending on the degree of obesity and
so on. With experience, the surgeon can determine whether there is excessive tension for a wound to be closed directly. In the face of excessive tension choices can be made:
1. Undermining skin edges
2. Suture patterns to help offset and distribute tension
3. Releasing incisions
4. V-Y advancement
5. Z.-plasty
6. Skin stretchers
7. Tissue expands
Using skin flaps or grafts
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Lines of tension have been determined by studies using canine cadaver skin and stab incisions. Separation of the
wound edges indicated the direction of tension "bands". The anatomical maps derived serve as the general guidelines to the small animal surgeon when closing skin. Wounds should be closed parallel to these tension lines however moderate-sized skin defects can often be closed perpendicular to these tension lines where ample loose skin is
available for wound closure. For example, tension lines of the limbs would suggest that wound apposition be performed parallel to these bands to facilitate closure. However, there is more loose skin around the circumference of
the limbs compared with the proximal-distal direction. When defects on the back, ventral abdomen, or lateral surface
of the trunk of a large animal are closed, undermining and advancement of the skin may be impaired by the animal's
weight, which can pin mobile skin against the surgery table. Sandbags, beaded "vacuum" bags, or rolled-up towels
can be placed in front of and behind the surgical region, to elevate the skin "trapped" against the table.
There can be and dramatic improvement in the ability to mobilise skin using the appropriate positioning of the patient. This is most commonly noted in larger dogs during unilateral or bilateral mastectomy. The surgeon can further reduce skin tension in the inguinal, axillary and sternal regions by loosening rope ties used to secure the respective hind limbs or fore limbs.
The loose skin over the neck and trunk of the dog and cat permits the surgeon to close many skin defects by undermining alone. The key to successful elevation of skin is preserving its blood supply. This requires preservation of
the direct cutaneous vessels and associated subdermal plexus. The following guidelines will help successful undermining of the skin in small animals:
1. Skin should be undermined below the panniculus muscle layer, when present, to preserve the subdermal plexus
and associated direct cutaneous vessels.
2. Skin without an underlying panniculus muscle (middle and distal portion of the extremities) should be undermined in the loose areolar fascia beneath the dermis to preserve the subdermal plexus.
3. Direct cutaneous arteries and veins should be preserved whenever possible during undermining.
Skin closely associated with an underlying muscle should be elevated by including a portion of the outer muscle fascia with the dermis rather than undermining between these structures. This may help minimise injury
to the subdermal plexus.
Transposition Flap
This is a rotating flap with many uses I have found helpful where direct closure or skin tension relieving techniques
just won’t work. It is applicable to most body regions. It is based on the subdermal plexus not a direct cutaneous
artery so to ensure the blood supply to the flap is adequate, adherence to the geometry and relative measurements of
the skin flap is important.
Using a technique of grasping skin adjacent to the defect the surgeon can find areas of accessible movable skin to
transpose into a defect (“borrow form Peter to pay Paul”). The width of the flap equals the width of the defect so a
line is drawn with sterile marking pen parallel to the base of the flap. Where this line finishes away from the defect
is the pivot point. The flap length is the distance from the pivot point to the most distant part of the defect. A line is
drawn from the pivot point at 90 degrees to the baseline for a distance equal to that measured from the pivot point to
the most distant part of the defect (flap length). The flap is elevated in the standard way taking care not to amputate
the blood supply either at the level of the subdermal plexus or by making the base of the flap too narrow. The flap is
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rotated into the defect then sutured with simple interrupted sutures. The donor site should be able to be closed directly without tension in a similar fashion.
Axial Pattern Flaps
For the large defects created after wide excision of tumours such as soft tissue sarcomas and mast cell tumours, I
commonly use axial pattern flaps which are flaps utilising the primary cutaneous arteries supplying the subdermal
plexus. The following is an overview of the flaps I use and the information is paraphrased from Mike Pavletic’s
Textbook:
Cervical Cutaneous Branch of the Omocervical Artery:
Potential uses: Facial defects, ear reconstruction, cervical defect, shoulder defect and axillary defect.
Reference incisions: Caudal incision is spine of scapula in a dorsal direction; cranial incision is parallel to the caudal
incision equal to the distance between the scapular spine and the cranial scapular edge (cranial shoulder depression);Flap length is variable but may extend to the contralateral scapulohumeral joint.
Thoracodorsal Artery:
Potential uses: Thoracic defects, shoulder defects, forelimb defects axillary defects.
Reference incisions: Cranial incision is spine of the scapula in a dorsal direction; Caudal incision is parallel to the
cranial incision equal to the distance between the scapula and the scapular spine and the caudal scapular edge (caudal
shoulder depression); Flap length is variable and can survive ventral to the contralateral scapulohumeral joint.
Superficial Brachial Artery
Potential uses: Antebrachial defects, elbow defects.
Reference incisions: Incision lines are so the flap base includes the cranial one third of the flexor surface of the elbow and the lateral and medial incisions are parallel to the humeral shaft. The flap is progressively tapered approaching the greater tubercle. The flap length can be variable up to one ending at the level of the greater tubercle.
Caudal Superficial Epigastric
This is my favourite axial pattern flap!
Potential uses: Inner thigh defects, flank defects, stifle area, perineal area, prepucial area and even up as far as the
dorsal lumbar skin.
Reference incisions: Medial incision is made on the abdominal midline, in the male dog; the base of the prepuce is
included in the midline incision to preserve the adjacent epigastric vasculature. The lateral incision is parallel to the
medial incision and at an equal distance from the mammary teats. The flap length is variable and may include the
last four glands and adjacent skin in the dog and the last three in the cat.
Genicular Artery
Potential uses: Lateral or medial aspect of the lower limb from the stifle to the tibiotarsal joint.
Reference Incisions: Base of the flap is 1 cm proximal to the patella and 1.5 cm distal to the tibial tuberosity laterally. The flap borders extend caudodorsally parallel to the femoral shaft. The flap terminates maximally at the level
of the greater trochanter.
There are other useful axial pattern flaps including the cranial superficial epigastric, the caudal auricular, the reverse
saphenous (an axial pattern variant), the deep circumflex iliac (dorsal and ventral branches) and the lateral and caudal arteries but the ones I have described are the most commonly used in my practice.
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Blood Transfusion Technique in Dogs and Cats: ICVS 2011 Scientific program (27th Oct 2011)
Selapoom Pairor, DVM
Kasetsart Pet Blood Bank, Kasetsart University Veterinary Teaching Hospital
The Faculty of Veterinary Medicine, Kasetsart University,
Bangkok, THAILAND 10900
Blood transfusion has become an increasingly important part of veterinary emergency and critical care
medicine. The goals of transfusion therapy including:
Restore or maintain oxygen-carrying capacity of blood
Maintain blood volume
Maintain haemostasis
Restore essential blood compenents concentration
Blood transfusion is indicated in patients with many different critical conditions, including anemia, hemorrhage, coagulopathy, and hypoproteinemia. Although potentially life saving, it does carry some risk. In addition to
selecting the appropriate blood product, several steps need to be completed to prepare the product for administration
and the patient for receiving a transfusion.
Main indications for transfusion therapy
Anemia
Anemia is the major indication for blood transfusion in veterinary practice. It can be the result of blood loss,
hemolysis or a lack of red blood cell production (eg, bone marrow disease).
In anemic patients, red cell containing products are transfused to increase blood oxygen carrying capacity.
Even the hematocrit can guide the transfusion decision, the individual patient’s clinical circumstance is the
most important determinant for transfusion. Clinical indications for red cell transfusion include pallor, weakness, exercise intolerance, tachycardia and tachypnea at rest.
Disorders of haemostasis
Patients with ongoing blood loss due to severe thrombocytopenia may require platelets containing product transfusion in order to prevent life-threatening hemorrhage. Transfusion to supply platelets is often effective in temporarily preventing or controlling hemorrhage caused by acquired or inherited thrombopathia or
aplastic disorders. It is not indicated for Immune Mediated Thrombocytopenia.
Plasma contains coagulation factors and proteins are used most commonly for coagulation-factor replenishment in patients with acquired or congenital coagulopathies.
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Deficiencies of specific plasma components
Hypoproteinemia caused by increased protein loss or by production failure can be temporarily
improved by plasma transfusion. Fresh frozen plasma transfusion has been advocated in treating pancreatitis. The
potential hope is to replenish depleted proteins and naturally occurring antiproteases such as AT III, antichymotrypsin, and α-macroglobulin.
However, there have been no prospective studies evaluating the effectiveness of fresh frozen plasma in the treatment
of canine acute pancreatitis.
Dosage and Rate of Administration
Red cell containing products
 In canine patient, as a general rule:
 2 ml/kg whole blood (FWB, SWB) for every 1% desired increase in PCV
1 ml/kg pRBCs for every 1% desired increase in PCV.
by assuming that there is not ongoing blood loss or hemolysis.
In feline patient, most of them will initially receive 1 unit of WB.
Administration over 1-4 hours
In normovolemic anemic patient; rate should be set at 0.25 mL/kg for the first 30 minutes. If no adverse affects
are witnessed, then the rate can be increased. The maximum rate is 10-20 mL/kg/hr
 May give more rapidly when treating hemorrhagic shock.
 In patients where circulatory overload is concerned (eg, patient with congestive heart failure), rate should not
exceed 4 mL/kg/hr
Plasma products
 For coagulation factor replacement: 10-20 mL/kg, over 1-4 hours, q 8-12 hr or until bleeding stops. Long transfusion times will compromise the viability of coagulation factors and result in potentially ineffective transfusion.
 For albumin supplementation: 45 mL/kg to increase serum albumin concentration by 1 g/dl. If plasma is being
administered for replacement of albumin, it may be delivered over a longer period of time.
Cryoprecipitate
 1 unit/10 kg q4 – 12 hr or until bleeding stops
 Cryoprecipitate should be administered as rapidly as possible, (maximum rate of 3-6 mL/minute for a mid size
dog) in order to achieve therapeutic levels of haemostatic proteins.
Platelet rich plasma and Platelet concentration
1 unit/10 kg; give over 1-2 hours. At this dosage, the platelet count should elevate 40,000/uL when counted 1-2
hours post transfusion.



Route and Method of Administration
Ideally, blood products should be given into the cephalic or jugular vein via an intravenous catheter. In some
critical situations eg, in severely hypotensive or pediatric patients, the blood can be given into the proximal femur
using an 18–20G intravenous needle or a spinal needle placed in the trochanteric fossa. Extraction of blood from the
marrow cavity into the blood stream is highly efficient and is almost as effective as direct intravenous infusion.
Blood is absorbed from the marrow at a rate of one drop per minute, but may be administered at a faster rate by using an infusion pump. Intraperitoneal administration of blood is an inefficient method of administration, achieving
only a 40% extraction of blood given.
339
Blood can be administered concurrently with isotonic solutions. The concurrent administration of blood and
hypotonic solutions via the same catheter can also result in red cell lysis.
Blood should not be administered concurrently (through the same catheter) with intravenous fluids containing calcium or glucose. Administration of intravenous medical preparation concurrently with blood administration
must be avoided.
A filtered giving set specifically designed for the certain blood products should be used for blood administration to reduces the risk of microthrombi entering the circulation. This may be particularly important when blood
has been stored following collection.
Transfusion related concerns
Transfusion reactions, a spectrum of metabolic and immunologic changes concurrent or subsequent to a
transfusion, can be immune-mediated and non-immune mediated in nature, and can happen immediately, or can be
delayed after a transfusion. Clinical signs of a transfusion reaction depend on the amount of blood transfused, the
type and amount of antibody involved in the reaction, and whether the recipient has had previous sensitization.
Basically, severity of most transfusion reactions depend on dose; thus, early recognition of a problem can
avert disaster. Patients should be carefully observed, especially during the first 30 minutes of the transfusion. The
benefit of the prophylactic use of antihistamines and glucocorticoids before blood transfusion has been debated.
Monitoring parameters include measurements of body temperature, heart rate, pulse quality and synchronicity, and respiratory rate. If a reaction is suspected, the transfusion must be stopped immediately.
Severe reactions are rare when patients have been correctly typed and crossmatched. In dogs, cross matching
is ideally essential for second and subsequent transfusions (if > 4-5 days have elapsed since the first transfusion).
Cats must be transfused with type compatible blood.
Conclusion
Currently, although many enhancements in transfusion medicine have improved the safety of blood transfusions, the basic considerations of transfusion in veterinary practice still be remaining as ever (a) avoiding blood
transfusions unless specific indications are met; and (b) using transfusion alternatives if medically prudent; because
blood products are not used to treat disease; they are supportive therapies given to correct deficiencies in the patient
until the underlying disease process can be treated.
References available from author upon request.
340 
Update Bovine Hormone
(An Update on Reproductive regulation in animals)
Somchai Sajapitak
Department of Large Animal and Wildlife Clinical Science, Faculty of Veterinary Medicine,
Kasetsart University, Thailand
Abstract
The secretion of luteinizing hormone (LH) is controlled by two different regulatory pathways; the tonic and
the surge pathways. The tonic pathway is responsible for a pulsatile pattern of LH secretion in mammalian species
that stimulates follicular maturation and steroid synthesis, while the surge pathway induces ovulation in female animals. Each LH pulse corresponds to a gonadotrophin-releaseing hormone (GnRH) pulse, which in itself is controlled
by metastin (kisspeptin-54) neuron or kisspeptin/neurokinin B/dynorphin (KNDy) neuron pathways. Thus, the pulsatile LH secretion is regulated by the brain mechanism called the “GnRH pulse generator” located in the mediobasal
hypothalamus. On the other hand, LH surge is likely to require inputs from more anterior regions such as the preoptic area and suprachiasmatic nuclei. It has been suggested that the pattern of pulsatile GnRH or LH secretion is the
most reliable parameter to monitor the activity of the brain mechanism regulating the gonadal axis. In addition, central mechanisms that control the reproductive system are inhibited by energetic challenges. Energetic challenges inhibit hypothalamic gonadotropin releasing hormone secretion, which in turn precludes pituitary LH secretion, retards
follicular development and prevents ovulation and estrous behaviour. This review paper focuses on the role of kisspeptin or KNDy neurons and energy availability in controlling mammalian reproduction in order to apply the understanding of this mechanism to the manipulation of animal reproduction.
Introduction
The activity of reproductive system is largely dependent on environment. Of all the known environmental
factors controlling reproductive activities, food availability has been considered to play the most important part in
reproductive regulation and probably acts as a proximate regulator of reproductive functions. In fact, stress and energy availability have been shown to be a significant hindrance to reproduction. Specifically, energy availability has
been shown to influence the secretion of reproductive hormones (Krulich et al., 1974; Blake, 1975; Du Ruisseau et
al., 1978; Kawakami and Higuchi, 1981; Higuchi et al., 1986; Ohkura et al., 2004), which can have an effect on a
reproductive function.
Previously, the hormonal network that is responsible for the control of the reproduction is gonadotrophic
axis. This gonadotrophic axis is mainly composed of three major hierarchical elements such as: (i) the hypothalamic
gonadotrophin-releaseing hormone (GnRH), (ii) the pituitary gonadotrophins (luteinizing hormone, LH and Follicular stimulating hormone, FSH), (iii) sex steroids and other hormones produced by gonads.
341
The estrous cycle in mammals is controlled by a cascade of neuroendocrine events that involved the activation of the hypothalamo-pituitary-gonadal axis. In this context, the understandings of mechanisms that are responsible for the physiological control of the reproductive axis and its physio-pathological alterations have been recently
revolutionized by the identification of the fundamental role of kisspeptins and Kisspeptin/neurokininB/dynorphin
(KNDy). KNDy is a key hypothalamic neuropeptide which is placed immediately in the upstream of GnRH neurons
and relays the peripheral steroidal information to GnRH neurons to control an estrous cyclicity. This paper focuses
on the role of kisspeptin, KNDy neurons and energy availability in controlling mammalian reproduction and the potential practical uses of this newly discovered knowledge in the manipulation of animal reproduction.
The concept of energy availability sensing in the brain
Energy availability information is conveyed to and integrated in the brain. The integrated information is used
in the regulation of reproduction which leads to the control of LH secretion. The LH secretion is mediated by the
adjustment of the GnRH pulse generator that facilitating or suppressing neuron or humoral pathways (Nishihara et
al., 1999; Grill and Kaplan, 2002; Parhar et al., 2002). Furthermore, the energy availability sensor in the lower brain
stem may also adjust the reproductive function. Behavioral and metabolic mechanisms that ensure the whole body’s
energy homeostasis are largely controlled by the energy supply to the brain, which is detected by specific central
glucose-sensing cells, as demonstrated by the effects of brain glucoprivation induced by intracerebroventricular 2DG injection (Muller et al., 1973; Nagatani et al., 1996a). The administration of energy suppression into the fourth
ventricle (4V) suppressed pulsatile LH secretion in rats (Murahashi et al., 1996) and sheep (Ohkura et al., 2000).
These results support the idea that energy availability sensing mechanisms may be located in the lower brain stem to
control reproductive function and feeding behavior. Moreover, this sensor could be present in the lower brain stem
(ependymocytes and serotonergic neurons) as observed by increased [Ca2+]i in ependymocyte induced by glucose
availability (Moriyama et al., 2004) in vitro study.
Mechanism of suppression of Reproductive function by energy deficient conditions through the neuronal
pathways in animals
Energy availability has been found to be a key regulator of mammalian reproduction (Wade and Schneider
1992). Disruption of metabolism has been proven to be a form of stress capable of suppressing pulsatile luteinizing
hormone (LH) secretion. This disruption can be induced by administration of different pharmacological agents that
can mimic either 2-deoxy-D-glucose (2DG)-induced glucoprivation or 2-sodium mercaptoacetate (MA); for example
a pharmacological blockade of the b-oxidation of fatty acids that induced lipoprivation (Cagampang et al., 1990;
Maeda and Tsukamura, 1996; Nagatani et al., 1996a; Tsukahara et al., 1999; McManus et al, 2002; Sajapitak et al.,
2008a; Sajapitak et al., 2008b).
It has been found in our studies that the neuroendocrine mechanism of energy deficiency caused by pharmacological reduction of glucose utilization, 2DG (Nagatani et al., 1996a; Tsukahara et al., 1999) or blockade boxidation, MA (Shahab et al., 2006), or administration of ketone body (Iwata et al., 2010) induced suppression of
pulsatile LH secretion in female rats. The mechanism of central energy deficiency such as glucoprivation or lipoprivation-induced suppression of LH secretion (Ohkura et al., 2000; Murahashi et al., Sajapitak et al., 2008a; Sajapitak
et al., 2008b), to understand the regulatory mechanism are detected by the lower brain stem specifically ependymocytes around the 4V [Pancreatic-type glucokinase, an enzyme playing a critical role in sensing blood glucose level in
pancreatic B cells, is localized in ependymocytes of the lower brainstem (Maekawa et al., 2000) and intracellular
calcium levels in ependymocytes taken from the hindbrain respond to a change in extracellular glucose levels
(Moriyama et al., 2004)] and subsequently activates the noradrenergic input to the paraventricular nuclease (PVN).
342 
The release of noradrenergic (NE) from the neuron terminals needs binding with a1- and a2-adrenergic receptors to
activate the corticotrophin-releasing hormone (CRH) neurons projecting from PVN which causes an increased CRH
synthesis and release in the medial preoptic area (mPOA)/ median eminence (ME) to regulate LH secretion in female
rats (Nagatani et al., 1996b; Tsukahara et al., 1999; Sajapitak et al., 2008b) [Fig. 1.].
Fig. 1. Schematic summary drawings of the neuroendocrine pathway indicating lipoprivation induced suppression of
pulsatile release of gonadotropin-releasing hormone GnRH)/luteinzing hormone (LH) release in female rats. The
availability of energy is detected by the lower brainstem, specifically ependymocytes around the fourth ventricle
(4V). The energy information is conveyed to the solitary tract nucleus (NTS) to activate noradrenergic (NE) neurons
in the A2 region of the nucleus of the solitary tract (NTS) that project to the paraventricular nuclease (PVN) through
the noradrenergic pathway by α1- and α2-adrenergic receptor subtypes and suppress the pulsatile LH secretion in
female rats.
Note: mPOA, medial preoptic area; ME, median eminence; CRH, corticotrophin-releasing hormone; MA, mercaptoacetate; 2DG, 2-deoxy-D-glucose; KB, Ketone body.
343
The kisspeptin neuron: A new player in the GnRH pulse-generating mechanism
Kisspeptins is the peptide products of the KiSS-1 gene isolated from the human placenta and the putative
receptor of this gene (GPR54 in the neuroendocrine) regulates the reproduction. Metastin (kisspeptin-54) directly act
on GnRH neurons to stimulate GnRH and LH release because GnRH neurons have been reported to express GPR54
mRNA (Messager et al., 2005). Metastin/kisspeptin-GPR54 signaling has been suggested to control ovarian cyclicity
through two regulating modes of GnRH release. A population of metastin/kisspeptin neurons is located in the anteroventral-peri-ventricular nucleus (AVPV), which is considered to trigger GnRH surge and also mediated the estrogen
positive feedback action on the release of GnRH. The other hypothalamic population of metastin/kisspeptin neurons
is located in the arcuate nucleus (ARC) but not in the anterior pituitary in the rat (Adachi et al., 2007; Kinoshita et
al., 2005; Smith et al., 2005). The metastin (kisspeptin-54)-GPR54 system has attracted the interest of reproductive
scientists because of its key role in puberty (Seminara et al., 2003) and potent stimulatory effect on GnRH/LH release in rats (Matsui et al., 2004; Navarro et al., 2004), mice (Gottsch et al., 2004), monkeys (Shahab et al., 2005)
and goat (Wakabayashi et al., 2010). This part is believed to be involved in generating GnRH pulses and mediating
negative feedback action of estrogen on the release of GnRH. In addition, it has been reported that diminished expression of hypothalamic KiSS-1 mRNA may be associated with suppression of reproductive function in fooddeprived prepubertal rats (Castellano et al., 2005) or ob/ob mouse (Smith et al., 2006).
Kisspeptin/neurokininB/dynorphin (KNDy) neurons: a hypothesis for the GnRH pulse generator
The ARC kisspeptin neuronal population is that the majority of the cohort co- expresses neurokininB (NKB)
and dynorphin A (Dyn) (Goodman et al., 2007; Navarro et al., 2009; Wakabayashi et al., 2010), and are therefore
referred to as KNDy neurons (Lehman et al.,2010). Accumulating evidences suggest that the KNDy neurons act as a
GnRH pulse generator. It is possible that the GnRH pulse generation could be controlled by manipulation of the
KNDy neuronal activities with specific agonists or antagonists for NKB and Dyn receptors (Noritake et al., 2011).
Therefore, GnRH neurons express mRNA for GPR54 (metastin/kisspeptin receptor) and closely associated with the
terminal of KNDy neurons. However, the precise mechanism of peptide regulation by two modes of GnRH release is
still unclear and needs to be further determined.
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Epidemiology of mastitis in dairy cows
Veerasak Punyapornwithaya
Clinics for ruminant, Faculty of Veterinary Medicine, ChiangMai University
Introduction
Bovine mastitis is one of the most significant diseases of dairy herds. The disease has a negative impact on
farm economics due to reduction in milk production and treatment costs (Hogeveen et al., 2011). Numerous mastitis
researchers used epidemiology as a tool to determine the distribution of mastitis pathogens, health related stage of
host, risk factors, the occurrence of mastitis and mastitis control. Epidemiologic studies in mastitis significantly provide a better understanding in the relation between pathogen, cows and environment. In this article the epidemiology
of mastitis including 1) current mastitis trend, 2) the using of mathematical mastitis model, 3) the identifying of risk
factors and 4) the applying of molecular technique for mastitis studies are briefly described.
Trend of mastitis prevalence
The trend of mastitis has been changed as the prevalence of mastitis caused by environmental pathogen increases whereas mastitis due to contagious pathogen decreases or continues ongoing. The streptococcal species and
the coliform bacteria have increased in relative importance as a source of both clinical and subclinical mastitis. In
addition, Mycoplasma spp. and coagulase-negative Staphylococci (CNS) have been increasingly considered as an
important pathogen.
The operating of mastitis control program aiming to reduce the occurrence of contagious mastitis is successful
in reducing incidence of contagious pathogen especially Streptococcus agalactiae and the significant reduction in
Staphyloccous aureus and Streptococcus dysagalactiae. Concurrent with the change, the increasing of mastitis incidence due to Streptoccus uberis was found. The main organisms associated with clinical mastitis in England and
Wales are E. coli and S. uberis (Zadok and Fitzpatrick, 2009). In New Zealand the most common pathogen associated with mastitis is S. uberis (McDougall, 2003). However, in some countries contagious pathogen is still important.
Data showed that S. aureus and S. dysgalactiae are the most common causes of clinical mastitis in Norway (Whist et
al., 2007). In the Netherland, S. aureus is the main pathogen followed by E. coli and S. dysgalactiae (Barkema et al.,
1998).
Mycoplasma mastitis has been increasingly recognized as an important disease. Many case reports for mycoplasma disease, especially for mastitis, in US and UK were published in the last 5 years. An US national study
showed that the prevalence of Mycoplasma spp. in bulk tank milk is 7.9 percent. However, the true prevalence may
be higher because the microbiological culturing of bulk tank milk has a moderate sensitivity. Mycoplasma bovis is
the most important pathogen that causes mastitis and other disease such as pneumonia and arthritis.
348 
The prevalence of CNS mastitis varies among countries (Pyorala and Taponen, 2009). Interestingly, CNS are
one of the most commonly isolates organisms from milk samples of cows with subclinical mastitis in many countries. These organisms can cause persistent infections, which result in increased somatic cell count. Generally, the
CNS mastitis is not a therapeutic problem because cure rates after antimicrobial treatment are usually high. This
mastitis can be controlled by using a mastitis control program for contagious mastitis pathogens such as post-milking
teat disinfection.
The using of mathematical model for mastitis research
Mathematical models are developed to explain transmissions and effects of mastitis pathogen at population
level. The models have been constructed and described in many mastitis researches. For example, an ordinary differential equation model was constructed to estimate the basic reproduction ratio to determine the effect of herd management on mastitis control (Zadoks et al., 2002). This method is used to determine the effect of lactation therapy for
subclinical mastitis (Barlow et al., 2009).
The classical mathematical model for describing infectious diseases is SIR model which has 3 compartments
including susceptible hosts, infectious hosts and recovered hosts.
This model and its modification have been
used in mastitis researches. For example, the SIR model is used to quantify the effect of post-milking disinfection on
the transmission of S. aureus (Lam et al., 1996). The SIR model is modified to SEIR models that include susceptible,
latent, infectious and recovery compartment respectively. A primary advantage of SEIR models is that such models
incorporate the dependent effect of population level pathogen prevalence and the transmission parameter of pathogens. These models can be used in quantifying the overall impact of intervention such as vaccination and antibiotic
treatment implementation (Barlow et al., 2009).
Identification of factors associated with mastitis
Mastitis is the result of interactions of pathogens with their hosts in a specific environment. Therefore, an understanding of host and environmental factors that affect this interaction is essential to mastitis control and
prevention. Contagious mastitis pathogens can be controlled through use of proper management practices. However,
environmental mastitis pathogens are not well controlled by these methods and are, in several countries, the most
frequent cause of subclinical and clinical mastitis in both lactating and non-lactating cows, particularly on wellmanaged farms (Bradley, 2002). Therefore, in general, milking time hygiene practices are highly associated with
contagious mastitis whereas environmental sources such as bedding are more related with environmental mastitis.
Numerous studies have investigated factors for clinical mastitis and subclinical mastitis (Dufour et al., 2011). However, risk factors identified from various studies may be similar or different depending on geographical location,
type of study, criteria for defining mastitis or subclinical mastitis, type of management practices and analysis
method. Conclusively, knowledge of risk factors that are associated with the occurrence of mastitis is critical to the
prevention and control of mastitis.
Molecular epidemiology of mastitis
The use of molecular strain typing in the evaluation of transmission dynamics of mastitis pathogen provides
more understanding of mastitis epidemiology. Using molecular or DNA-based methods, isolates belonging to bacterial species can be differentiated at strain level, allowing for determining sources and transmission routes of pathogens. In addition, the identification of virulent factor genes provides more important information about the interaction between the pathogens and host responses see review (Zadoks and Schukken, 2006).
349
Mastitis pathogens are divided into two major groups including contagious and environmental pathogen
based on the source of transmission. In general, the finding of multiple bacterial strains of a single species causing
mastitis in a population of cows suggests these infections arise from one or more environmental sources that harbor
with many strains. In comparison, the finding of a limited number of strains is consistent with cow to cow transmission. However, a molecular typing of S. uberis from mastitis outbreak herds demonstrated the possibility of cow to
cow transmission within dairy herd (Zadoks et al., 2003). Data from several outbreaks showed that the predominant
strain of S. uberis rather than a variety strains causes mastitis in dairy herds (Zadoks et al., 2003). This finding challenges the common understanding that the source of S. uberis transmission is environment rather than udder to udder
and the new conceptual that strains of S. uberis may has a potentially transmission ability like contagious bacteria is
proposed (Zadoks and Schukken, 2006).
The using of strain typing method to differentiate bacterial strains demonstrated that the degree of mastitis
virulence depends on strain of bacteria. In other words, for example, not all S. aureus has the same pathological ability to cause mastitis but some strains of S. aureus are more likely to cause clinical mastitis than others.
In conclusion, the prevalence of environmental mastitis has been increased in many countries. However,
contagious mastitis is still a major problem in some countries. CNS and mycoplasma mastitis are more concern. The
using of mathematical models and molecular epidemiology in mastitis researches provide a better understanding for
disease transmission, interaction between host and pathogen and pathogenic virulence of agents which are important
for developing control strategies.
References
Bradley, A. 2002. Bovine mastitis: An evolving disease. Vet J. 164:116–128.
Barkema, H. W., Y. H. Schukken, T. J. Lam, M. L. Beiboer, H. Wilmink, G. Benedictus, and A.
Brand. 1998. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk somatic
cell counts. J Dairy Sci 81(2):411-419.
Barlow, J. W., L. J. White, R. N. Zadoks, and Y. H. Schukken. 2009. A mathematical model
demonstrating indirect and overall effects of lactation therapy targeting subclinical mastitis in dairy herds.
Prev Vet Med 90(1-2):31-42.
Dufour, S., A. Frechette, H. W. Barkema, A. Mussell, and D. T. Scholl. 2011. Invited review: effect of udder health
management practices on herd somatic cell count. J Dairy Sci 94(2):563-579.
Lam, T. J., M. C. DeJong, Y. H. Schukken, and A. Brand. 1996. Mathematical modeling to
estimate efficacy of postmilking teat disinfection in split-udder trials of dairy cows. J Dairy Sci 79(1):62-70.
Hogeveen, H., Huijps, K. and Lam, J.G.M. Economic aspects of mastitis: New developments. NZ
Vet Journal. 59:1, 16-23
McDougall, S. 2003. Intramammary treatment of clinical mastitis of dairy cows with a
combination of lincomycin and neomycin, or penicillin and dihydrostreptomycin. N Z Vet J 51(3):111-116.
Pyorala, S. and S. Taponen. 2009. Coagulase-negative staphylococci-emerging mastitis
pathogens. Vet Microbiol 134(1-2):3-8.
Whist, A. C., O. Osteras, and L. Solverod. 2007. Streptococcus dysgalactiae isolates at calving
and lactation performance within the same lactation. J Dairy Sci 90(2):766-778.
Zadoks, R. N., H. G. Allore, T. J. Hagenaars, H. W. Barkema, and Y. H. Schukken. 2002. A
mathematical model of Staphylococcus aureus control in dairy herds. Epidemiol Infect 129(2):397-416.
Zadoks, R. N., B. E. Gillespie, H. W. Barkema, O. C. Sampimon, S. P. Oliver, and Y. H. Schukken.
2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy
herds. Epidemiol Infect 130(2):335-349.
Zadoks, R. N. and Y. H. Schukken. 2006. Use of molecular epidemiology in veterinary practice.
Vet Clin North Am Food Anim Pract 22(1):229-261.
350 
Update on infectivity, transmission, and pathogenicity of FMDV and research needs
Jef M. Hammond
Donald King, Nick Knowles, Jemma Wadsworth, Bob Statham,Yanmin Li, Pip Hamblin, Ginette Wilsden, Geoff
Hutchings, Valerie Mioulet, Miki Madi, Begona Valdozo, Nigel Ferris and Elizabeth Wilson
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 ONF,
United Kingdom.
Abstract
Foot-and-mouth disease (FMD) is highly contagious, infects a wide variety of domestic and wildlife hosts and occurs
as multiple non-cross-protective virus serotypes. Its presence restricts trade opportunities for endemic countries and
poses a constant threat to those countries free of the disease. The morbidity of FMD is high in infected adult livestock. The disease is rarely fatal in adult animals, but often high mortality is seen in young animals due to myocarditis. Following infection, the incubation period is 2 to 21 days and large amounts of virus are excreted by infected
animals before clinical signs are evident. The World Reference Laboratory for FMD (WRLFMD) at the Institute for
Animal Health, Pirbright, regularly receives samples for FMD diagnosis and virus isolates made in reference laboratories from many parts of the world. The in-vitro antigenic properties of selected isolates are assessed for vaccine
matching and nucleotide sequencing allows precise characterisation of new isolates and tracing of their origin by
comparison with viruses held in the extensive WRLFMD collection. This analysis assists the monitoring of emergence and spread of FMD virus globally.
Introduction
FMD is one of the most infectious viral diseases known and has had devastating economic, social and environmental
impacts. FMD is caused by a virus of the family Picornaviridae, genus Aphthovirus (the FMD virus [FMDV])
which has seven immunologically distinct serotypes (O, A, C, SAT1, SAT2, SAT3 and Asia 1). FMDV is transmitted by direct or indirect contact from animate and inanimate vectors, and may spread over great distances with movement of infected or contaminated animals, products, objects, and people. Airborne spread may occur up to 60km (40
miles) overland and 300km (190 miles) by sea, especially in temperate zones.
Infectivity, transmission, and pathogenicity
FMDV is highly contagious to bovidae, cattle, sheep, goats, swine, all wild ruminants and suidae. The morbidity of
FMD is high in infected adult livestock. The disease is rarely fatal in adult animals, but often high mortality is seen
in young animals due to myocarditis. Following infection, the incubation period is 2 to 21 days (average 3 to 8) and
large amounts of virus are excreted by infected animals before clinical signs are evident. Infected animals exhibit
blisters and ulcers on the mouth, tongue, lips, feet and udder. Clinically, animals salivate excessively, have fever,
sore feet, lose weight and stop producing milk.
351
On recovery from FMD, at least 50% of ruminants become ‘carriers’ with persistent sub-clinical infection. These
animals present a critically important risk to susceptible animals as reservoirs of the infection. Unfortunately, currently available vaccines do not protect animals from carrier status.
The current understanding of the FMDV infectious process in naïve animals is summarized in Figure 1.
Research needs
Current research needs can be divided into 4 broad areas; New generation vaccines and diagnostics, understanding
the immune response in vaccinated and infected animals, understanding the risk posed by the carrier state and investigating the potential of anti-viral approaches to controlling disease spread.
New Generation FMD Vaccines
A new generation of FMD vaccines with the following properties are the major objectives of a number of research
initiatives:





Longer duration of protection;
Faster onset of protection;
Broader spectrum of protection against different strains;
More potent immunity to prevent viral replication and development of viral carriers;
Better discrimination of vaccinated animals that go on to become infected;
Thermostable, safer to make and easier to administer.
Understanding the immune response to FMDV
Understanding about the protective memory immune response to FMDV is scant.
352 
The development and analysis of alternative vaccination and other control strategies can be greatly enhanced by
identifying the components of the virus recognized by the immune system and developing mechanisms to exploit
this knowledge. The five epitopes (regions) neutralized by antibody on the surface of FMDV have been mapped in
atomic detail. In contrast, very little is known about the FMDV epitopes recognized by T-cells in either ruminants
or other susceptible species. The search for these epitopes will reveal which FMDV proteins are important for stimulating cell-mediated responses. It will also underpin the development of technology to study the role of T-cells in
protection against FMDV.
There is virtually no information on the role of T-cells as effectors of immunity to FMDV. Limited previous work
focused on identification of viral proteins and epitopes recognized by CD4+ (helper) T-cells, with a view to exploiting them in vaccines to provide help for antibody production. However, there is very little information on CD8+
(effector) T-cell responses to FMDV. CD8+ T-cells represent an important effector arm of the immune response
against viral infections, by virtue of their ability to kill virus-infected cells and to deliver signals that inhibit virus
replication.
The risk posed by the carrier state
In terms of trade, FMD vaccination has two critically important limitations: firstly, individually vaccinated animals
cannot always be readily distinguished from infected animals, and secondly, vaccination does not protect animals
from becoming FMDV carriers. Therefore, widespread vaccination does not result in the lowering of trade barriers.
Although all controlled studies performed to date have failed to demonstrate transmission of FMDV from carriers to
susceptible animals, it is the potential or supposed threat presented by carriers that needs to be mitigated. Over 50%
of ruminants exposed to live FMDV become carriers. The establishment of the carrier state depends on the strain
and serotype of FMDV and the duration of the carrier state depends on the species and individual. Carrier animals
eliminate the virus very slowly, which suggests that FMDV has a means of preventing the development of (or eluding) an effective immune response. It is possible that virus infection blocks the secretion of cytokines and/or
proteins involved in virus elimination. Further investigation is required to understand the molecular basis of
persistence. An improved understanding of persistence will allow the rational development of vaccination and antiviral strategies to prevent the carrier state.
There are three key questions with respect to the carrier state that require better understanding:


How does the carrier state arise and how is it maintained?
How important are carriers for initiating new outbreaks?
How can carrier detection be improved?
Antiviral Agents for the Treatment of FMD
The highly infectious nature of FMD demands immediate action to halt an outbreak. While it is theoretically possible to develop vaccines that would prevent infection within 24 hours of administration, antiviral agents are more
likely to have utility in this setting than vaccines. It is recognized that antiviral agents could play a critical role in
two areas:
1. Directly reducing the viral load in infected animals and thus disease burden and spread of the virus
Developing more rapid immune protection through compounds that enhance host immunity or directly retard the
activity of the FMDV.
353
Ideally, these antivirals would have the following properties:
Rapidly active (24 hours to reduce clinical signs and shedding);
Multiple-serotype protection (active against all serotypes);
Cheap to produce and easy to administer;
Long-lasting, with an active duration of 5-10 days;
Stable and do not interfere with diagnosis and vaccine-induced immunity.
Summary
FMD is probably the most infectious disease known. Much has been discovered about its infectivity, transmission
and pathogenesis, however far more remains to be determined. Recent advances in genetics, proteomics, immunology, vaccine design and delivery and chemotherapeutics offer an unprecedented opportunity to develop new technologies to reduce the risk posed by FMD and improve the management of outbreaks. It has been recognized that a
global, coordinated research strategy is required to capitalize on these developments with the Global FMD Research
Alliance (GFRA) carrying the banner for this initiative. Specifically, the objective of the Alliance is to reduce the
economic risk of FMD to developed nations and improve the management tools for nations with endemic FMD. The
goals of the GFRA crystallize current research needs and are summarized below:
Development of improved killed or new-generation vaccines with rapid onset and longer duration of protection,
longer stability, broad spectrum and prevention of carrier state through improved understanding of T-cell and
local immune responses.
Development of antiviral agents that inhibit FMDV infection through retarding virus binding or replication, thus
reducing herd viral load and spread of the virus.
Enhancement of an understanding of the mechanism of FMDV persistence and the nature of the threat posed by
carrier animals.
Improvement in the specificity of tests to distinguish carrier animals.
Determination of the implications for future FMD-control policies and strategies from the introduction of a series of
new vaccine, diagnostic and antiviral technologies.
Research and development regarding many of these questions is currently ongoing at the Institute for Animal Health,
Pirbright Laboratory and many other institutions around the world.
354 
A MOLECULAR APPROACH TO STUDY GENETIC DIVERSITY: AS POWERFUL TOOLS FOR CAPTIVE BREEDING PROGRAM IN SARUS CRANE REINTRODUCTION PROJECT
J. Insee1, S. Kamolnoranath2, S. Baicharoen2, S. Chumpadang2, W. Sawasu3,
W. Wajjwalku1,4*
1
Interdisciplinary Graduate Program in Genetic Engineering. Faculty of Graduate School, Kasetsart University,
Bangkok, Thailand. 2Conservation Research and Education Division, Zoological Park Organization, Thailand.
3
Nakornratchasima Zoo, Nakornratchasima, Thailand.
4
Faculty of Veterinary Medicine, Kasetsart University Kamphaeng Saen Campus, Nakhonpathom, Thailand.
Introduction
The sarus crane (Grus antigone) is vastly distributed across five continents. Four subspecies including Indian (Grus a.
antigone), Eastern (Grus a. sharpii), Australian (Grus a. gillae) and the extinct Philippine sarus (Grus a. luzonica; Blanford,
1896; Meine and Archibald, 1996). In Thailand, the eastern sarus crane is the protected wild animal according to Thai Wildlife
Preservation and Protection Act, B.E. 2535 (1992). Nowadays, the breeding program zoological park organization, Thailand, has
successfully increased the number of individuals to be more than one-hundred. This population is prepared for future
reintroduction (Tanee et al., 2009). Therefore, understanding genetic diversity of maternity and paternity are important to
improve the captive management with the goal of species survival and reintroduction into the wild.
Blood samples from 24 females and 21 males of eastern sarus crane were collected from Nakhonratchasima Zoo,
Khaokheaw Open Zoo and Bang-Pha Research Water Bird Center in 2009. Genomic DNA was isolated from blood samples
using phenol/chloroform method. PCR amplification of the Cytochrome b, ND6 and D loop of female samples and male samples
were amplified using Z-chromosome-specific primer: ALDOB, CHD, BRM and SPIN-Z. The PCR products were purified by
GeneJETTM Gel Extraction Kit. The amplification product were sequence by 1 st base Co, ltd. The multiple alignments and
diversity analysis were performed using Bioedit program version 7.0.5.2.
Genetic diversity of mtDNA in founder female cranes
The genetic diversity of twenty-four founder female population was base on cytochrome b, ND6 and D-loop. The length
of cytochrome b, ND6 and D-loop sequence alignment are 1,143, 522 and 1,004 bp, respectively. The number of variable sites
within cytochrome b, ND6 and D loop region were four, four, and 38 sites, respectively. These sites were group into four, three
and 20 haplotypes base on cytochrome b, ND6 and D-loop, respectively. We analyzed the composite sequences derived from
cytochrome b, ND6 and D-loop. Phylogenetic tree base on maximum likelihood among the female founder from the combined
2,904 bp mtDNA showed 20 haplotypes of Grus antigone shapii (Table1.).
Genetic diversity of Z chromosome
We amplified a 1,520 bp long fragment of the aldolase B fructose-bisphosphate (ALDOB) gene, 512 bp of the chromohericase binding protein (CHD) gene, 399 bp of the brahma (BRM) gene and 246 bp of the Spindle Z (SPIN-Z) gene. The results
show that ALDOB gene has no variation. However, we found two, six and four haplotypes in CHD, BRM and SPIN-Z gene, respectively. We analysis the combined sequences of ALDOB, CHD, BRM and SPIN-Z gene and found 11 haplotypes of male
Grus antigone shapii. The segregation sites and position are shown in table 1.
Currently, there are seven maternal and nine paternal lines have already given offspring. Twelve young cranes of offspring generation were reintroduction including TSCf1-1-1, TSCf1-1-2, TSCf1-1-3 and TSCf2-1-18 haplotypes base on mtDNA
and TSCm1-1-1-1-1, TSCm1-1-1-1-2, TSCm1-1-1-1-4, TSCm1-1-1-2-2 and TSCm1-1-1-3-3 haplotypes base on Z chromosome.
355
Table1. The haplotype of 24 captive founder female Grus antigone shapii in three areas base on mtDNA sequences of
cytochrome b, ND6 and D-loop with polymorphic sequence nucleotide
Table2. The haplotype of 21 captive founder male Grus antigone shapii in three areas base on Z chromosome sequences of
CHD, BRM and SPIN-Z with polymorphic sequence nucleotide
Conclusion
Our results indicate that, base on our founder sampling, D loop is the most variable region in mitochondrial DNA and
BRM gene is the most variable site in Z chromosome of Grus antigone shapii. We found 20 haplotypes in founder females and
11 haplotypes in founder males. But there are only seven maternal and nine paternal lines have already given offspring. This
research was useful to the mating plan to maintain the genetic diversity in offspring generation which will be released to the
wild.
References
Blanford, W.T. (1896). A note on the two sarus cranes of the Indian region. Ibis, 2, 135-136.
Meine, C. and Archibald, G. (1996). The Cranes: Status Survey and Conservation Action Plan. 1st Edn., IUCN, Gland, Switzerland and Cambridge, UK., pp. 294.
Tanee, T., Chaveerach, A., Anuniwat, A., Tanomtong, A., Pinthong, K., Sudmoon, R. and Mokkamul, P. (2009). Molecular analysis for genetic diversity and distance of introduced Grus antigone shapii L. to Thailand. Journal of Biological Sciences,
12, 163-167.
356 
REVIEWS
SEX IDENTIFICATION: BREAKTHROUGH IN MOLECULAR APPLICATION
M. Sukmak1, 2 W. Klinsawat3 W. Wajjwalku4*
1
Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140.
Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok, Thailand.
3
Conservation Biology, College of Food, Agriculture and Natural Resource Sciences, University of Minnesota.
4
Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen Campus,
Nakhon Pathom, 73140.
2
To understand distribution pattern, mating pattern and affecting population growth rate, sex ratio is required
as a key parameter. Sex identification using non invasive samples has been useful for wildlife management, by
identifying sex ratio and inferring social structure. The most common technique for sex identification in mammal is
polymerase chain reaction (PCR) to amplify the specific region of Y-specific SRY (Sex determining region of the Y
chromosome) locus. However, amplification failure can lead to false negative results when using low-quality DNA
fecal samples (Dallas et al., 2000). Therefore, a positive PCR control is needed to verify that no amplification truly
indicates male, and not due to allelic dropout. Molecular sex identification can be categorized into 4 categories.
The first category is designing primers which specific to Y-chromosome (Male specific regions). SRY gene is
Y-specific, intronless gene and no homologous region comparing X chromosome. As a male specific marker, SRY
has been increasingly applied to sex various family of mammals: human, soricidae, squirrel and marten. However,
using only male specific region to verify sex might not provide an accurate result, consequently, positive PCR
control using other gene fragments are needed. For example, in elephant, Sry was used as a male-specific region and
fragment of mitochondrial DNA (mtDNA) region as a positive PCR control (Gupta et al., 2006). However, there are
debates about mtDNA as a sexing control due to unequal quantity between mtDNA and nuclear DNA. Compared to
nuclear DNA, mtDNA has more quantity and easy to amplify. Duplex PCR was developed for sexing six bovid
species. GAPDH housekeeping gene-specific primer was used along with Sry-specific primers as a duplex PCR
condition (Prasant et al., 2008). This method can increase PCR accuracy and avoid any doubts about PCR
amplification failure.
Second category is similar to the first Y-specific primers but X-specific primers are used as a positive PCR
control. The third category is using only one primer pair which can amplify both X and Y homologous region
identical in length, follow by differentiation using restriction enzyme. PCR-RFLP (restriction fragment length
polymorphism) of point mutation which differ in sequence between X and Y homologues are significantly useful for
sexing. The main application is Zfxy gene (zinc finger protein, X and Y linked) which encode zinc finger protein. This
gene is widely used in many mammalians such as human and ungulate, cetacean, canine, elephant, fox, mustelid and
rabbit. However, this method is not appropriate for degraded DNA especially which PCR product size is less than
1 kb make it too difficult to amplify and use the restriction enzyme.
357
The last category is also using only one primer pair similar to third group but their PCR product was differ in
size between males and females. AmelXY (amelogenin gene, X and Y linked) encode an enamel protein and their gene
structure is conserved among mammals. This gene presents on both the X and Y chromosome, but AmelX and AmelY
are difference in lengths due to substitution/deletion. For example, in humans, AmelX has 6 base pairs deletion
comparing to AmelY (Sallivan, 1993). In cattle, AmelY has 63 base pairs deletion comparing to AmelX. These
differences can be observed clearly on gel electrophoresis. Thus, this gene has been used widely for identifying sex
in cattle, wild ruminant, red deer (Cervus elaphus), sika deer (Cervus nippon) and panda (Ailuropoda melanoleuca).
Thus, this gene has been widely used for sex identification in Cervidae Zfxy gene is also widely used in many
species such as canine, marine mammal and primate. In white-tailed deer, 50 bp deletions were found in Y
chromosome allele in 930 base pairs intron (Shaw et al., 2003). However, these fragments were quite large, and
therefore, application toward noninvasive sample is limited. The shorter Zfxy fragments were successfully amplified
in white-tailed deer (Lindsay and Belant, 2008). Moreover, there are other genes which are widely used in primate
such as Utxy gene (ubiquitously transcribed tetratricopeptide repeat protein gene) and Dbxy gene (dead box
polypeptide).
However, in some mammals, their length difference between X and Y homologues are not sufficient enough
to be observed on gel electrophoresis (Sukmak et al., 2011). In these cases, fragment analysis plays an important role
in molecular sexing. For example, Zfx and Zfy between male and female African rhinoceros (Diceros bicornis) are 7
bp differences in sizes. Males were characterized by 95 base pairs of X chromosome and 102 base pairs of Y
chromosome while females were characterized only 95 base pairs of X chromosome. Thus, fragment analysis using
labeled primers to amplify these homologous regions is useful for verifying the differences (Peppin et al., 2010).
Recently, new application which is a combination between Real time PCR and high resolution melting analysis
(HRM) has available. This application has been used for detecting DNA variations including single nucleotide
polymorphism (SNPs), insertion, and deletion (Montgomery et al., 2007) through their melting temperature. This approach provides a high sensitivity and has been previously applied to identify sex in many species of avian (Morinha
et al., 2011; Brubaker et al., 2011).
References
Sallivan KM, Mannuci A, Kimpton CP, Gill P. 1993. Biotechniques. 15: 636-641.
Dallas JF, David NC, Freda M, Klaus-Peter K, Hans K, Stuart BP, Phillip JB. 2000. Conserv. Genet. 1:181-183.
Shaw CN, Wilson PJ, White BN. 2003. J. Mammal. 84: 123-128.
Gupta SK, Thangaraj K, Singh L. 2006. J. Forensic Sci. 51: 805-807.
Montgomery J, Wittwer CT, Palais R, Zhou L. 2007. Nat. Protoc. 2: 59-66.
Lindsay AR, Belant JL. 2008. Conserv. Genet. 9: 443-447.
Prashant, Gour DS, Dubey PP, Jain A, Gupta SC, Joshi BK, Kumar D. 2008. J. Appl. Genet. 49: 379-381.
Peppin L, McEwing R, Ogden R, Hermes R, Harper C, Guthrie A, Carvalho GR. 2009. Conserv. Genet. 11:11811184.
Sukmak M, Dejchaisri S, Thongtipsiridech S, Wajjwalku W. 2011. KKU. Vet. J. 20:11-20.
Morinha F, Magalkaes P, Ferro A, Guedes-Pinto H, Rodrigues R, Bastos E. 2011. Ann. Zool. Fennici. 48:00-00.
Brubaker JL, Karouna-Renier NK, Chen Y, Jenko K, Sprague DT, Henry PFP. 2011. Mol. Ecol. Res. 11:415-417.
358 
Application of the Mitochondrial DNA Variation for Captive Breeding Management in Endangered
Malayan Tapir (Tapirus indicus) in Thailand
W. Tipkantha1,3,4*, W. Wajjwalku2, Y. Muangkram 2, S. Kamolnorranath1 and B. Siriaroonrat1
Bureau of Conservation, Research and Education, Zoological Park Organization,
2
Faculty of Veterinary Medicine, Kasetsart University,
3
Center for Agricultural Biotechnology ,Kasetsart University, Thailand,
4
Center of Excellence Agricultural Biotechnology, Kasetsart University, Thailand
* corresponding author
1
Introduction
Malayan tapir (Tapirus indicus) is a reserved species under Thai Wildlife Preservstion and Protection Act
(1992) and listed as endangered (EN) species by IUCN Red list of Threatened Species (2008). The wild population
is now highly fragmented and continuously decline due to the deforestation and overexploitation (Mace et al., 2000;
IUCN, 2011). Little is known about their current population status in the wild in Thailand. At present, the ex situ
conservation plays important role in conserving the endangered Malayan tapir. The recent global captive population
of Malayan tapir is 261 (115.145.1) specimens in 79 institutions worldwide (Prastiti, 2010) and 42 individuals are
kept in Thailand at Zoological Park Organization zoos and the Safari World, Bangkok. With a small population size,
genetic diversity of captive Malayan tapir in Thailand is likely to be low. Genetic management should be applied to
achieve the goal of maintaining genetic diversity in the population. Many molecular markers such as microsatellite,
minisatellite and mitochondrial DNA (mtDNA) have been increasingly applied for biodiversity analysis and conservation for endangered species including Tapiridae. It has been applied to determine a variation of maternal linage of
population using mitochondrial control region and cytochrome b markers. In this study, we have applied mitochondrial control region to investigate the maternal-linage variation in 25 captive Malayan tapirs with the goal of developing effective captive breeding plan in this species.
The biological samples including whole blood, hairs and feces of 25 captive Malayan tapirs from 5 zoos of
Zoological Park Organization (ZPO) were collected for DNA extraction. Genomic DNA was extracted using the
DNeasy Tissue or Blood kit (Qiagen Inc.,USA). The silica-base method was used for hair and fecal extraction. PCR
amplification of a portion of the cytochrome b to displacement control region (D-loop) using four primers [CB0
(5’-CATGACTAATGATATGAAAAACC-3’) 2) HkkRe (5’-TGGTCTTGTAAACCAGAAAAG-3’) 3) D-loopFw
(5’-CACCCATCAACACCCAAAGCT-3’) and 4) D-loopRe (5’-CCTGAAGAAAGAACCAGATGC-3’)] with
DreamTaq® DNA Polymerase (Fermentas International Inc., Canada). Direct double-stranded sequencing of the
1690 bp mtDNA PCR products from 25 Malayan tapirs were performed using Dye Terminator Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA) for the extension-dideoxy-chain terminator method (Sanger et al. 1997) and
analyzed by a 373-A sequencer (Applied Biosystems). The cytochrome b and D-loop sequences were aligned by the
CLUSTAL W algorithm (Thompsom et al. 1994). The maximum likelihood phylogenetic tree were constructed
using MEGA5.
Results and Discussions
Of 1690 bp PCR product, 1140 bp nucleotide sequenced were cytochrome b. The analysis of this gene marker differentiates three haplotypes (TI1, TI2 ans TI3) of captive Malayan tapir sampled. The result was in accordance
with a previous investigation by Muangkram et. al (2010). In addition, 520 bp sequence analysis of the mitochondrial D-loop region from this study divided animals into nine haplotypes (Table 1).
359
Table 1 The haplotype of 25 captive Malayan tapirs in ZPO base on Cytochrome b and D-loop with polymorphic
sequence nucleotide
Haplotypes
Cytochrome
b
TI1
TI2
TI3
Dloop
TI1-1
TI1-2
TI1-3
TI2-1
TI2-2
TI2-3
TI2-4
TI2-5
TI3-1
1
4
0
1
4
1
1
4
2
1
4
3
1
4
4
Variable site of D-loop
1
1
1
1
4
4
4
4
6
6
7
7
4
G
.
.
.
.
.
.
A
A
9
A
G
G
G
G
G
G
G
G
6
G
.
A
A
.
A
A
A
A
5
C
.
.
.
.
.
T
.
.
6
G
.
.
.
A
.
A
.
A
3
G
.
.
.
.
.
.
.
A
9
T
.
.
.
.
.
.
.
C
0
A
.
.
.
.
G
.
.
.
1
T
.
.
.
T
.
.
.
.
1
4
9
1
5
2
1
5
7
1
6
0
1
C
.
.
.
.
.
.
C
C
5
A
.
.
.
.
.
.
G
G
9
G
A
A
A
A
A
A
A
A
7
C
.
.
T
T
T
T
T
T
From population record, five maternal lines have already given offspring to-date. However, the other three
groups (TI1-2, TI2-3, and TI2-4) have no living female and TI2-1 consist of only geriatric females(over 30 years
old). These results provide the genetic data for population manager to make a suitable decision to manage and exchange bloodlines for appropriate breeding program for captive Malayan tapir. To assess the population genetic
diversity, more information is needed from other mtDNA gene markers and nuclear DNA that could provide insight
into both paternal and maternal evolutionary history. The male genetic diversity in this population also suggests
further investigation for better understanding of paternal population diversity.
Table 2 Current captive Malayan tapir population and breeders in 5 zoos divided by mtDNA D-loop diversity
The number in parentheses show a current breeding animal
Conclusions
Genetic analysis has been shown to be a valuable tool for small population management and can be applied in
captive Malayan tapir. Mt DNA analysis from this study showed 9 haplotypes using D-loop sequence comparison.
To maintain and maximize genetic diversity, an effective genetic management must be applied in this species
through collaboration, breeding loan and genetic exchange within Thailand, in Southeast Asian region and at the
global zoo community.
360 
Acknowledgement
Authors would like to thank National Research Council of Thailand and Royal Thai Government for the approval and funding support on this project.
References
Avise, J.C.1995. Conservation Biology. 9(3). 686-690.
DeSalle, R and Amato, G. 2004. The expansion of conservation genetic. Nature (5), 702-712.
Frankham et al., 2003. Introduction to Conservation Genetics. Cambridge Univ. Press
Ashley et al. 2005. J. Mam. Evol. 3:315-326
Mace, G.M. and A. Balmford. 2000. Priorities for the conservation of Mammalian Diversity
Muangkram, Y. 2010. ICVS conference 2010
Prastiti, S. 2010. International studbook keeper of Malayan tapir (Tapirus indicus). Bogor. 1-34
361
GENETIC VARIATION IN WILD AND DOMESTICATED BUFFALOES IN THAILAND
M.Yindee*, W.Wajjwaku†, W.Manatchaiworakul†, N.Kowlim†,
S.Duangjuntrasiri¥, B.Colanbender£ and J.A.Lentra£
* Faculty of Veterinary Science of Mahidol University, 999 Phuttamonton, Salaya, Thailand.
† Department of Pathology, Faculty of Veterinary edicine, Kasetsart University, Kamphaengsaen, 73140
Nakhornpathom, Thailand.
‡ Department of Obstetrics Gynecology and Reproduction, Faculty of Veterinary Scine, Chulalongkrn University,
Heri-Dunant road, Pathumwan 10300, Bangkok, Thailand.
¥ Khao-Nang-Ram Wildlife Research Station, Haui-Kha-Kheang Wildlife Sanctuary, Department of National Parks,
Wildlife and Plant Conservation, Thailand.
£ Faculty of Veterinary Medicine, Yalelaan 2, 3648CM Utrecht, The Netherlands.
The water buffalo (Bubalus bubalis) has been divided is two distinct subspecies, the swamp type (Bubalus
bubalis carabensis; 2n=48), and the river type (Bubalus bubalis bubalis; 2n=50). Both types have been evolved from
ancestral buffalo-like animals approximately 1 million year ago (Kikkawa et al., 2003). They are supported to have
been domesticated separately in the Indus valley and China, respectively (Cockrill, 1981; Kumar et al., 1994; Lau et
al., 1998; Lei et al., 2007; Yindee et al., 2010). A clear separated of the river and swamp was confirm based on the
mitochondrial DNA (mtDNA) sequences, microsatellites (Zhang et al., 2007) and, more recently, Y-chromosomal
sequences (Yindee et al., 2010). In the Chinese swamp buffalo population, two mtDNA haplotypes A and B were
observed (Lau et al., 1998). There were estimated to have diverged about 18000 yr ago, but do not have distinct
geographical ranges. Lineage A has undergone a population expansion and is predominant north of the Yangtze River. The largest haplotype diversity was found near the coast south of the Yangtze River (Lau et al., 1998).
The available data (Groeneveld et al., 2010) do not allow a comparison of the mtDNA diversity patterns
among different counties. This precludes a more definite localization of the site of domestication. Here we report the
mtDNA sequences of Thai swamp buffaloes from the northern, northeastern, middle and southern regions. Our data
reveals a complex haplotype pattern in Thailand with the diversity higher than the previous study in China region
(Lei et al., 2007). This result is consistent with a domestication of swamp buffalo in Southeast Asia.
Sixty-four blood samples were obtained from domestic buffaloes throughout Thailand, and fifteen blood
samples from domestic buffaloes in three restricted areas. Forty-four fecal samples were collected from wild buffalo
in Haui-Kha-Kheang Wildlife Sanctuary (HKK). All samples were extracted following phenol-chloroform extraction
and ethanol precipitation standard procedures. Primers used for PCR amplification and sequencing of the mtDNA
cytb gene and control region were designed on the basis of the complete water buffalo mtDNA sequence (Genbank
AF547270 entry). Sequences were aligned and checked using the Seqman program from DNAstar 5.0 package.
Insertion/deletions in aligned sequences were excluded in the analyses. Haplotype diversity for the control region
segment 15792-363 in Thailand (this study) and China (Lei et al., 2007) was calculated using the Arlequin3.1
program (Excoffier et al., 2005). Reduced median networks and the pairwise mismatch distributions (cytochrome b
segment 14530-15690, 1160 bp, and control regions segment 15811-16721, 910 bp) were constructed via the
program Network 4.5.1.6 (Bandelt et al., 1999).
362 
Results and discussion
Mitochondrial gene fragment of 123 cytochrome b and 63 control regions were amplified and sequenced from
DNA extracted from 79 captive blood and 44 wild fecal samples. The alignment shows that haplotypes are divided in
two haplogroups. These correspond to the A and B lineages defined by Lei et al. (2007) and are represented by 19
and 11 control region haplotypes and 9 and 6 cytochrome b haplotypes, respectively. Only four control region haplotypes (AH1-AH4) were also found in the 119 Chinese swamp buffaloes (Lei et al., 2007). The ratio of A and B haplotypes does not correlate clearly with the geographical origin. This argues against a separate origin of the A and B
haplogroups. Haplotype diversities were calculated for the control region. The nucleotide diversity index (π) as alternative measure of the mtDNA variation in this dataset is large determined deviations. Haplotype diversity across
Thailand is higher than the reported value from the Chinese populations (Lei et al., 2007). Median networks as visualization of haplotypes relationships, with the cytochromes b haplotypes allowing a clustering of D-loop haplotypes,
most of which were observed only in one animal. Both the cytochrome b and the D-loop networks illustrate the separation of the A and B haplogroups with haplotypes IT1 in an intermediate position. The Thai network is more complex than the Chinese pattern, in which high frequencies of AH1 and BT5 suggest a population expansion (Lei et al.,
2007). The network does not indicate a differential geographic distribution of haplotypes within Thailand. This result
of no genetic structure is contrast to the Y-chromosomal haplotypes (Yindee et al., 2010), which indicates spatial
clustering.
Our haplotype analysis does not support China as primary site of domestication as proposed earlier (Lei et
al. 2007). Although more definite conclusions would require a more comprehensive sampling from other regions in
Indochina mainland, the available data suggest a relatively recent origin of the Chinese population. Especially the
predominance of the AH1 haplotype indicates a smaller base population than that for the Thai animals. This is most
outspoken north of Yangtze River and in the western regions south of the Yangtze, suggesting that gene flow along
the Chinese coast was more intensive than from the coast to the west or across the Yangtze River. To assess the direction and level of of gene flow between the Chinese and Southeast Asian population, nuclear markers such as microsatellite, are needed to investigate both paternal and maternal evolutionary histories.
In case of 44 fecal samples of wild buffaloes, only two haplotypes were found. Most are the same as domestic Haikou buffalo sequence (NC006295 entry), but only two samples were the same as domestic buffaloes sequence
which from three restricted areas. These results indicate wild buffaloes are the same species as domestic buffaloes.
References
Bandelt HJ, Forster P, Rohl A, 1999:Mol.Biol.Evol. 16 37-48.
Cockrill WR, 1981: The water buffalo: a review. Br.Vet.J. 137 8-16.
Excoffier L, Laval G, Schneider S, 2005: Evol.Bioinform.Online. 1 47-50.
Groeneveld LF, Lenstra JA, Eding H, Toro MA, Scherf B, Pilling D, Negrini R, Finlay EK, Jianlin H, Groeneveld E,
Weigend S, 2010: Anim Genet. 41 Suppl 1 6-31.
Kikkawa Y, Takada T, Sutopo, Nomura K, Namikawa T, Yonekawa H, Amano T, 2003: Anim Genet. 34 96-101.
Lei CZ, Zhang W, Chen H, Lu F, Liu RY, Yang XY, Zhang HC, Liu ZG, Yao LB, Lu ZF, Zhao ZL, 2007: Anim
Genet. 38 97-102.
Yindee M, Vlamings BH, Wajjwalku W, Techakumphu M, Lohachit C, Sirivaidyapong S, Thitaram C, Amarasinghe
AA, Alexander PA, Colenbrander B, Lenstra JA, 2010: Anim Genet PMID: 20546176.
Zhang Y, Sun D, Yu Y, Zhang Y, 2007: Anim Genet. 38, 569-575.
363
A NOVEL GENETIC MARKER FOR SUBSPECIES IDENTIFICATION AND GENETIC
DIVERSITY IN TIGER.
1, 3*
W Buddhakosai , W Wajjwalku1,2, N Kaolim2, D Thongthainun4
1
Interdisciplinary Graduate Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkhen
campus, Bangkok, 2Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen campus, Nakhon Pathom, 3Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, 4Khao-kheaw Open Zoo,
Chonburi, THAILAND. * Corresponding author
Introduction:
The tiger (Panthera tigris) population has been declining by several causes including illegal trading and habitat loss.
Six existing subspecies are in threatened state (Morell, 2007; Chundawat et. al., 2010). Integrated with ecological
data, subspecies identification and genetic diversity play a vital role in tiger recovery and persistence. Effective conservation planning and captive breeding program need information of genetic variation. Characterizing the mitochondrial DNA (mtDNA) sites containing such highly informative single nucleotide polymorphisms (SNPs) allow more
precise assignment of individuals into their subspecies. Previous study (Luo et. al., 2004) assessed tiger evolutionary
history and population genetic structure by combining shorter mtDNA fragment (less than 600 bp) together. The use
of longer DNA fragment will facilitate the discovery of new SNP site, and therefore new haplotype. Here, we propose a newly designed mtDNA primer pair which encompasses NADH dehydrogenase subunit 6 (ND6), tRNA Glu,
and partial cytochrome b (Cyt b). Data of subspecies specific SNPs derived from more extended sampling of captive
tigers in Thailand provide and insight into captive breeding program with the goal of enhancing the species survival
in the wild.
Material and method:
Based on Luo et. al. (2004), subspecies specific SNPs located in several gene fragments, especially, Cyt b and
surrounding fragments. We downloaded the sequences from GenBank including cytoplasmic mitochondrial DNA
(Cymt) (Accession number DQ151550, NC_014770, NC_010642, AY736634 – AY736658, AY736784 –
AY736808) and nuclear copy of mitochondrial DNA (Numt) (Accession number DQ151551, AF053053, AF053054)
which is a common finding in felidae (Cracraft et. al., 1998; Kim et. al., 2006). Multiple alignments were performed
using Bioedit program. To design primers, the fragments containing the conserved sequence among Cymt and the
mutation sites of Numt were preferred. We placed the 3’end of each primer at the mutation sites on Numt to avoid the
hybridization of primer to Numt. Each primer (forward and reward primer) composed of 22 – 23 nucleotides. We
extracted DNA from blood, tissue, hair and cell culture (22, four, three and one sample, respectively) by the phonolchloroform method. PCR reactions were performed under the 60°C annealing temperature and 90 seconds extension.
The PCR product were further sequenced and analyzed by BioEdit and MEGA5 program.
Results and Discussion:
Comparison with the Numt reference sequences confirms that our primers exclude amplification of Numt. 30
samples (including hair, tissue, cell culture and blood samples) were classified to three haplotypes of P. t. altaica,
four haplotypes of P. t. corbetti, two haplotypes of P. t. jacksoni and two haplotypes of P. t. tigris. 14 SNPs were
newly found in this study as shown in Table 1. Totally, 26 SNPs located in this fragment, including 6 subspecies specific SNPs. This result showed that only one PCR reaction of this novel primer pair can resolve the subspecies assignment as well as explore new haplotypes. We also test this condition with the DNA template extracted from fresh
feces and the primers successfully amplified the targeted fragment under the same PCR condition (data not show in
the table). However, subspecies identification can be confirmed by the SNPs located in other locations of mitochondrial DNA, in combination with the use of microsatellite loci, nuclear DNA markers.
The overall results reflect the high potential of the novel primers and the high diversity of genetic in the captive tigers of Thailand. This novel primer can decrease the time, cost of genetic study and be suitable for subspecies
screening. To assess fine-scale genetic structure and infer both paternal and maternal evolutionary history, nuclear
DNA should be integrated in the study.
364 
Table 1 shows only the new found variation sites from this study compared to the previous report by Luo et. al.
(2004), Luo et. al. (2008). Total 14 sites distribute across ND6 and Cyt b (three and 11 sites, respectively)
SNPs position corresponding to cat complete mitochondrial DNA (Lopez et. al, 1996)
ND6
Haplotype
Number of
individual
Cyt b
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
4
5
5
5
5
5
5
5
5
5
5
5
6
5
7
0
0
1
1
2
3
4
5
8
9
9
0
2
5
0
8
0
7
6
2
0
5
1
4
9
3
2
A
•
•
•
•
•
•
•
•
9
C
•
•
•
•
T
T
•
•
1
T
•
•
•
•
•
•
C
•
2
T
•
•
•
•
C
•
•
•
4
G
•
•
A
A
A
A
•
•
8
T
•
•
•
•
•
•
•
C
5
C
•
•
•
•
•
•
T
•
8
C
•
T
•
•
•
•
•
•
6
C
•
•
•
•
•
•
•
•
9
C
•
T
•
•
•
•
•
•
1
C
•
•
•
•
•
•
•
•
5
C
T
T
T
T
T
T
T
T
4
G
•
•
•
A
•
•
•
•
6
A
•
•
•
•
•
•
G
G
ALT A
ALT B
ALT C
COR A
COR B
COR C
COR D
JAX A
JAX B
2
2
3
3
12
1
6
1
2
TIG A
2
•
•
•
•
•
•
•
•
T*
•
•
T
•
G
TIG B
1
G
•
•
•
A
•
•
•
•
•
T
T
•
G
• represent the identity to the nucleotide of the top row haplotype (ALT A)
*Base with the asterisk is similar to a haplotype of Bengal tiger from India (Mondol et.al. 2009)
References:
Chundawat, RS. , B. Habib, U. Karanth, K. Kawanishi, J. Ahmad Khan, T. Lynam, D. Miquelle, P. Nyhus, S. Sunarto, R. Tilson and S. Wang. 2010. Panthera tigris. In: IUCN 2010. IUCN Red List of Threatened Species.
Cracraft, J., J. Feinstein, J. Vaughn and K. Helm-Bychowski. 1998. Sorting out tigers (Panthera tigris): mitochondrial sequences, nuclear inserts, systematics, and conservation genetics. J of Anim Conservation. 1; 63 - 74
Kim, J., A. Antunes, S. Luo, J. Menninger, W.G. Nash, S.J. O’Brien and W.E. Johnson. 2006. Evolutionary Analysis
of a Large mtDNA Translocation (numt) into the Nuclear Genome of the Panthera Genus Species. Gene.
366: 292 – 302.
Lopez, J.V., S. Cevario and S.J. O’Brien. 1996. Complete Nucleotide Sequence of the Domestic Cat (Felis catus)
Mitochondrial Genome and a Transposed mtDNA tandem repeat (Numt) in the Nuclear Genome. Genomics.
33(2), 229 – 246.
Luo, S., J. Kim and W.E. Johnson. 2004. Phylogeography and Genetic Ancestry of Tigers (Panthera tigris). PLoS
Biology. 2(12): 2275 – 2293.
Luo, S., W.E. Johson and J. Martenson. 2008. Subspecies Genetic Assignments of Worldwide Captive Tigers Increase Conservation Value of Captive Populations. Current Biology. 18: 592 – 596.
Mondol, S., K. Ullas Karanth, U. Ramakrishnan. 2009. Why the Indian Subcontinent Holds the Key to Global Tiger
Recovery. PLos Genet 5(8): e1000585. doi:10.1371/journal.pgen.1000585.
Morell, V. 2007. Can the Wild Tiger Survive?. Science. 317: 1312 – 1314.
365
MAMMALIAN DNA BARCODING: MITOCHONDRIAL CYTOCHROME B AS AN IDEAL MOLECULAR MARKER FOR SPECIES IDENTIFICATION
Y. Muangkram1, 2, W. Klinsawat3, C. Salakij2, W. Wajjwalku2*
1
The Graduate School, Bangkaen campus,Bangkok 2Department of Pathology, Faculty of Veterinary Medicine,
Kamphaengsaen campus, Nakorn Pathom, Kasetsart University, THAILAND. 3 Conservation Biology, College of
Food, Agriculture and Natural Resource Sciences, University of Minnesota.
Introduction
Biodiversity of entire species create the individual specific might differ each other by anatomical appearance.
However, in morphologically cryptic species, biodiversity assessment has become much more difficult. DNA barcoding facilitates their identification as well as enhances discovery of new species. Interspecific genetic variation on
short DNA fragments allows assignment of individual to species. The large scale screening for donor species of noninvasive sample has strong implication toward conservation planning and wildlife monitoring. The markers consist
of mitochondrial DNA (mtDNA) and nuclear DNA marker. The variation analysis of nuclear marker is useful for
DNA fingerprint for individual. On the other hand, the mtDNA marker is commonly used and served as tools for
species identification to infer evolutionary relationship and biodiversity assessment. Studies of DNA barcoding in
mammalian mostly based on mitochondrial cytochrome c oxidase subunit I (COI) (Hebert et al. 2003b), providing
sufficient resolution and robustness in some groups of organisms (Hebert et al. 2003a). However, amplification of
long DNA fragments does not applicable to forensic samples with low-quality DNA such as feces and hair. Based on
many genes of mtDNA which have been described in many species, the deceasing order of conserved sequences are
12S rDNA, 16S rDNA, cytochrome b, and control region, respectively (Arif and Khan 2009). This study proposes a
new DNA barcoding marker, a partial cytochrome b gene, and compares its efficiency in mammalian species identification with previously proposed partial 12S rDNA, 16S rDNA.
Table 1 The universal primer of each gene.
Gene
12S rDNA
16S rDNA
Cytochrome b
Primer
Forward
Reverse
Forward
Reverse
Forward
Reverse
Sequences
L1085
H1259
L2513
H2714
195Fw
195Re
CCCAAACTGGGATTAGATACCC
TTTGCTGAAGATGGCGGTA
GCCTGTTTACCAAAAACATCAC
CTCCATAGGGTCTTCTCGTCTT
TTYGCATACGCAATCYTACGATC
GTTGMCCTCCYATTCATGTYAG
Length (bp)
173
200
154
Mammalian sequences covering the amplicon of 12S and 16s fragment from Kitano et al. 2007 and of cytochrome b fragment from this study were downloaded from GenBank. In total, 350 species out of 200 genera met this
criterion. The reconstruct phylogenetic tree using neighbor joining (NJ) and maximum composition likelihood analysis implemented in MEGA5 (Tamura et al. 2011) with confidence values which were establishes by 10,000 bootstrapping.
366 
The results showed the identification ability in genera and species of each gene (Table 2). Compared among
the selected markers across Mammalia, cytochrome b gene could identify 100% of genera and 98.86% for species
identification. However, the partial cytochrome b gene of this study cannot differentiate Bos indicus from B. taurus
and Ursus arctos from U. maritimus.
Table 2 The summary showed three partial specific sequences of each gene.
Gene
Sized
Variation
12S rDNA
16S rDNA
Cytochrome b
180 bp
204 bp
154 bp
93 (51.67%)
90 (44.12%)
102 (66.23%)
Genera identification
80.95%
85.71%
100%
Species identification
82.86%
88.57%
98.86%
We propose that 154 bp of mitochondrial cytochrome b gene could served as the excellent DNA barcoding
for mammalian species identification. The application of this primer toward conservation and forensics includes primary screening for donor species of unidentified fecal and hair samples or other animal parts. Integration of mitochondria cytochrome b gene with the other region of mtDNA and/or nuclear DNA could provide more reliable information for species identification.
Acknowledgements
We thanks for my colleague, the department of Pathology, faculty of Veterinary Medicine, Kamphaengsaen
campus, Nakorn Pathom, and the graduate School, Bangkaen campus, Bangkok, Kasetsart University, THAILAND.
References
Arif I.A. Khan H.A. 2009. Molecular markers for biodiversity analysis of wildlife animals: a brief review. Animal
Biodiversity and Conservation. 32.1: 9-17.
Kitano T., Umetsu K., Tian W., Osawa M. 2007. Two universal primer sets for species identification among vertebrates. International Journal of Legal Medicine. 121: 423-427.
Hebert P.D.N., Cywinska A., Ball S.L.,deWaard J.R. 2003a. Biological identification through DNA barcodes. Proc
R Soc Lond Ser B 270: 313-321.
Hebert P.D.N., Ratnasingham S, deWaard J.R. 2003b. Barcoding animal life: cytochrome c oxidase subunit I divergences among closely related species. Proc R Soc Lond B (Suppl 1) 270: S96-S99.
Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011. MEGA5: Molecular evolution genetics
analysis using maximum, evolutionary distance, and maximum parsimony methods. Molecular Biology and
Evolution.
Thompson J.D., Higgins D.G., Gibson T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Research 22 (22): 4673-4680.
367
ALTERNATIVE MEDICINE IN AQUACULTURE
Jumroensri Thawonsuwan
Coastal Aquatic Animal Health Research Institute, Department of Fisheries
130/2 Pawong, Muang, Songkhla, THAILAND
Diseases hamper the growth of aquaculture, the fastest growing food producing sector that contributes
shrimps, fishes and molluscs for human consumption. Maintaining and restoring the health status through preventing
and treating illnesses is usually done with the help of chemotherapeutants. Antibiotics play an important role in
countering bacterial infections. Continuous use of antibiotics is associated with the development of drug resistant
pathogens and accumulation of antibiotic residues in the products for human consumption. Therefore, finding alternative substances having high potential to prevent and control diseases will benefit aquaculture. In this context, medicinal herbs, natural immunostimulants and probiotics are apt choices. Several herbs act directly as antimicrobial
agents against invasive pathogens including bacteria, virus, parasites and fungus. Some traditionally used plants are
known to possess immune-stimulatory properties, while others work entirely as anti-stress agents. Garlic, turmeric,
Fahtalaijone and Chinese ginger are some such medicinal herbs that have been proven to have antimicrobial activities. The most practical way to obtain the desired benefits from such herbs, in aquaculture, is by incorporating them
in aquafeeds as the herbs are available as aqueous and solvent extracts, and in powder form. Application of some of
the above-mentioned medicinal herbs for the benefit of farmed aquatic animals will be presented.
368 
Epidemiology of mastitis in dairy cows
Veerasak Punyapornwithaya
Clinics for ruminant, Faculty of Veterinary Medicine, ChiangMai University
Introduction
Bovine mastitis is one of the most significant diseases of dairy herds. The disease has a negative impact on
farm economics due to reduction in milk production and treatment costs (Hogeveen et al., 2011). Numerous mastitis
researchers used epidemiology as a tool to determine the distribution of mastitis pathogens, health related stage of
host, risk factors, the occurrence of mastitis and mastitis control. Epidemiologic studies in mastitis significantly
provide a better understanding in the relation between pathogen, cows and environment. In this article the epidemiology of mastitis including 1) current mastitis trend, 2) the using of mathematical mastitis model, 3) the identifying of
risk factors and 4) the applying of molecular technique for mastitis studies are briefly described.
Trend of mastitis prevalence
The trend of mastitis has been changed as the prevalence of mastitis caused by environmental pathogen increases whereas mastitis due to contagious pathogen decreases or continues ongoing. The streptococcal species and
the coliform bacteria have increased in relative importance as a source of both clinical and subclinical mastitis. In
addition, Mycoplasma spp. and coagulase-negative Staphylococci (CNS) have been increasingly considered as an
important pathogen.
The operating of mastitis control program aiming to reduce the occurrence of contagious mastitis is successful
in reducing incidence of contagious pathogen especially Streptococcus agalactiae and the significant reduction in
Staphyloccous aureus and Streptococcus dysagalactiae. Concurrent with the change, the increasing of mastitis
incidence due to Streptoccus uberis was found. The main organisms associated with clinical mastitis in England and
Wales are E. coli and S. uberis (Zadok and Fitzpatrick, 2009). In New Zealand the most common pathogen associated
with mastitis is S. uberis (McDougall, 2003). However, in some countries contagious pathogen is still important.
Data showed that S. aureus and S. dysgalactiae are the most common causes of clinical mastitis in Norway (Whist et
al., 2007). In the Netherland, S. aureus is the main pathogen followed by E. coli and S. dysgalactiae (Barkema et al.,
1998).
Mycoplasma mastitis has been increasingly recognized as an important disease. Many case reports for mycoplasma disease, especially for mastitis, in US and UK were published in the last 5 years. An US national study
showed that the prevalence of Mycoplasma spp. in bulk tank milk is 7.9 percent. However, the true prevalence may
be higher because the microbiological
369
Compartmentalisation: Where are we now?
Pennapa Matayompong
Department of Livestock Development, Ministry of Agriculture and Cooperatives
69/1 Phaya Thai Road, Bangkok, Thailand 10400, [email protected]
The outbreak of avian influenza (AI) in Thailand in 2004 caused extremely severe socio-economic impacts. In 2005,
the World Organisation for Animal Health (OIE) developed a compartmentalisation concept. Thailand has adopted
the OIE concept and started the establishment of poultry compartmentalisation for avian influenza since July 2006.
The main objectives are to prevent and control an avian influenza epizootic; to strengthen the biosecurity in poultry
farms and maintain Notifiable Avian Influenza (NAI) free status; to reduce risk of NAI reoccurrence in poultry farms
while improving the overall health status on farms; and to facilitate international trade of fresh poultry meat.
Under this program, a compartment is limited to broiler rearing farms (chickens and ducks) managed in a common biosecurity management system, with a traceability system, surrounded by a 1 km radius buffer zone, and with
a well defined surveillance for NAI within the compartment and the respective buffer zone. The implementation of
NAI free compartment is voluntary; however, a strong partnership between government and private sectors is
required. The Department of Livestock Development (DLD) as the Veterinary Authority of Thailand is responsible
for auditing and certifying NAI free compartment, designating buffer zone, setting up sampling protocol for NAI
surveillance in compartment and buffer zone and conducting NAI surveillance in buffer zone. The private sectors are
responsible for managing biosecurity and conducting NAI surveillance in their compartments. The surveillance programme contains a first phase of sampling criteria to certify as NAI free compartment and a second phase of sampling criteria to maintain of such status.
The OIE, since 2007, has selected Thailand as a pilot country for the compartmentalisation project to be used as a model
for other OIE members in the control and eradication of animal disease as well as to facilitate trade where the disease eradication cannot be achieved in a short period. The OIE experts have provided technical advice for Thailand poultry compartmentalisation.
Following the OIE expert’s advice, the DLD, since January 2011, has expanded the compartmentalisation concept to
include breeder farms, hatcheries, feed mills and slaughterhouses in order to achieve the certification of compartment
for the entire integrated poultry system. The NAI free compartments for breeder farms and hatcheries are operated
and certified on the same criteria applied for the one of broiler farms. Feed mills and slaughterhouses are NAI free
compartment certified on the criteria of Good Manufacturing Practices (GMP) and Hazard Analysis and Critical
Control Point (HACCP) relevant to the risk mitigation of NAI exposure, traceability system and contingency plan in
the event of a change in the level of exposure. Compartment manager is required to supervise all actions carried out
in the compartment to assure the NAI free status of compartment and to liaise with the DLD on the compartment
activities. The on-line data base of all poultry compartments has been established to provide the list of certified compartments including the detailed information and surveillance history of each compartment.
Meanwhile, Thailand has been free from AI for nearly 3 years. Vaccination against AI is still prohibited. Although
the recognition of Thailand poultry compartmentalisation by the international community will still be a long process,
the compartment made thus far is a significant measure to maintain AI free status of Thailand and provide safety and
sustainability for the Thai poultry meat production.
370 
culturing of bulk tank milk has a moderate sensitivity. Mycoplasma bovis is the most important pathogen that causes
mastitis and other disease such as pneumonia and arthritis.
The prevalence of CNS mastitis varies among countries (Pyorala and Taponen, 2009). Interestingly, CNS are
one of the most commonly isolates organisms from milk samples of cows with subclinical mastitis in many
countries. These organisms can cause persistent infections, which result in increased somatic cell count. Generally,
the CNS mastitis is not a therapeutic problem because cure rates after antimicrobial treatment are usually high. This
mastitis can be controlled by using a mastitis control program for contagious mastitis pathogens such as post-milking
teat disinfection.
The using of mathematical model for mastitis research
Mathematical models are developed to explain transmissions and effects of mastitis pathogen at population
level. The models have been constructed and described in many mastitis researches. For example, an ordinary differential equation model was constructed to estimate the basic reproduction ratio to determine the effect of herd management on mastitis control (Zadoks et al., 2002). This method is used to determine the effect of lactation therapy for
subclinical mastitis (Barlow et al., 2009).
The classical mathematical model for describing infectious diseases is SIR model which has 3 compartments
including susceptible hosts, infectious hosts and recovered hosts.
This model and its modification have been
used in mastitis researches. For example, the SIR model is used to quantify the effect of post-milking disinfection on
the transmission of S. aureus (Lam et al., 1996). The SIR model is modified to SEIR models that include susceptible,
latent, infectious and recovery compartment respectively. A primary advantage of SEIR models is that such models
incorporate the dependent effect of population level pathogen prevalence and the transmission parameter of pathogens. These models can be used in quantifying the overall impact of intervention such as vaccination and antibiotic
treatment implementation (Barlow et al., 2009).
Identification of factors associated with mastitis
Mastitis is the result of interactions of pathogens with their hosts in a specific environment. Therefore, an understanding of host and environmental factors that affect this interaction is essential to mastitis control and
prevention. Contagious mastitis pathogens can be controlled through use of proper management practices. However,
environmental mastitis pathogens are not well controlled by these methods and are, in several countries, the most
frequent cause of subclinical and clinical mastitis in both lactating and non-lactating cows, particularly on wellmanaged farms (Bradley, 2002). Therefore, in general, milking time hygiene practices are highly associated with
contagious mastitis whereas environmental sources such as bedding are more related with environmental mastitis.
Numerous studies have investigated factors for clinical mastitis and subclinical mastitis (Dufour et al., 2011). However, risk factors identified from various studies may be similar or different depending on geographical location, type
of study, criteria for defining mastitis or subclinical mastitis, type of management practices and analysis method.
Conclusively, knowledge of risk factors that are associated with the occurrence of mastitis is critical to the prevention and control of mastitis.
Molecular epidemiology of mastitis
The use of molecular strain typing in the evaluation of transmission dynamics of mastitis pathogen provides
more understanding of mastitis epidemiology. Using molecular or DNA-based methods, isolates belonging to bacterial species can be differentiated at strain level, allowing for determining sources and transmission routes of pathogens. In addition, the identification of virulent factor genes provides more important information about the interaction between the pathogens and host responses see review (Zadoks and Schukken, 2006).
371
Mastitis pathogens are divided into two major groups including contagious and environmental pathogen
based on the source of transmission. In general, the finding of multiple bacterial strains of a single species causing
mastitis in a population of cows suggests these infections arise from one or more environmental sources that harbor
with many strains. In comparison, the finding of a limited number of strains is consistent with cow to cow transmission. However, a molecular typing of S. uberis from mastitis outbreak herds demonstrated the possibility of cow to
cow transmission within dairy herd (Zadoks et al., 2003). Data from several outbreaks showed that the predominant
strain of S. uberis rather than a variety strains causes mastitis in dairy herds (Zadoks et al., 2003). This finding challenges the common understanding that the source of S. uberis transmission is environment rather than udder to udder
and the new conceptual that strains of S. uberis may has a potentially transmission ability like contagious bacteria is
proposed (Zadoks and Schukken, 2006).
The using of strain typing method to differentiate bacterial strains demonstrated that the degree of mastitis
virulence depends on strain of bacteria. In other words, for example, not all S. aureus has the same pathological ability to cause mastitis but some strains of S. aureus are more likely to cause clinical mastitis than others.
In conclusion, the prevalence of environmental mastitis has been increased in many countries. However, contagious mastitis is still a major problem in some countries. CNS and mycoplasma mastitis are more concern. The using of mathematical models and molecular epidemiology in mastitis researches provide a better understanding for
disease transmission, interaction between host and pathogen and pathogenic virulence of agents which are important
for developing control strategies.
References
Bradley, A. 2002. Bovine mastitis: An evolving disease. Vet J. 164:116–128.
Barkema, H. W., Y. H. Schukken, T. J. Lam, M. L. Beiboer, H. Wilmink, G. Benedictus, and A.
Brand. 1998. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk somatic
cell counts. J Dairy Sci 81(2):411-419.
Barlow, J. W., L. J. White, R. N. Zadoks, and Y. H. Schukken. 2009. A mathematical model
demonstrating indirect and overall effects of lactation therapy targeting subclinical mastitis in dairy herds.
Prev Vet Med 90(1-2):31-42.
Dufour, S., A. Frechette, H. W. Barkema, A. Mussell, and D. T. Scholl. 2011. Invited review: effect of udder health
management practices on herd somatic cell count. J Dairy Sci 94(2):563-579.
Lam, T. J., M. C. DeJong, Y. H. Schukken, and A. Brand. 1996. Mathematical modeling to
estimate efficacy of postmilking teat disinfection in split-udder trials of dairy cows. J Dairy Sci 79(1):62-70.
Hogeveen, H., Huijps, K. and Lam, J.G.M. Economic aspects of mastitis: New developments. NZ
Vet Journal. 59:1, 16-23
McDougall, S. 2003. Intramammary treatment of clinical mastitis of dairy cows with a
combination of lincomycin and neomycin, or penicillin and dihydrostreptomycin. N Z Vet J 51(3):111-116.
Pyorala, S. and S. Taponen. 2009. Coagulase-negative staphylococci-emerging mastitis
pathogens. Vet Microbiol 134(1-2):3-8.
372 
Whist, A. C., O. Osteras, and L. Solverod. 2007. Streptococcus dysgalactiae isolates at calving
and lactation performance within the same lactation. J Dairy Sci 90(2):766-778.
Zadoks, R. N., H. G. Allore, T. J. Hagenaars, H. W. Barkema, and Y. H. Schukken. 2002. A
mathematical model of Staphylococcus aureus control in dairy herds. Epidemiol Infect 129(2):397-416.
Zadoks, R. N., B. E. Gillespie, H. W. Barkema, O. C. Sampimon, S. P. Oliver, and Y. H. Schukken.
2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy
herds. Epidemiol Infect 130(2):335-349.
Zadoks, R. N. and Y. H. Schukken. 2006. Use of molecular epidemiology in veterinary practice.
Vet Clin North Am Food Anim Pract 22(1):229-261.
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PRRSV-host interaction: Innate immune response
Win Surachetpong
Department of Veterinary Microbiology and Immunology
Kasetsart University, 50 Ngan Wong Wan Road, Ladyao, Chatujak, Bangkok, THAILAND 10900 Tel: (662)
9428436 Email: [email protected]
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major causative agents of swine
diseases. Currently, vaccines and good management practices have been implemented for effective PRRSV control.
To prevent PRRSV infection, the porcine innate immune system utilize cells such as macrophages and dendritic cells
together with humoral proteins such as cytokines and serum proteins that work coordinately to limit virus multiplication. During viral entry into porcine cells, the pattern recognition receptors such as TLR3 and RIG-I recognize
PRRSV nucleic acid and subsequently trigger the signaling pathways that lead to the production of type I IFN. To
establish infection, PRRSV employs multiple strategies to evade or subvert the host innate immune system including
antigenic and genetic diversity of the viruses, mechanisms that dampen the innate immune response and interfere antigen presentations. For example, the non-structural proteins of PRRSV including nsp1, nsp2, and nsp11 have been
involved in inhibition of type I IFN production. Thus, the knowledge gained from basic understanding of host innate
immune system and virus interaction can lead to a novel adjuvant or vaccine design that provide protective immunity,
but still prevent viral shedding with high safety. Such a new vaccine will be applied as a tool for PRRSV control in
the field.
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging disease of swine and leading
causes of economic losses to the swine industry worldwide. PRRSV causes a reproductive failure in sows and respiratory distress in young pigs (Xiao et al., 2010). During 2006 to 2007, the emerging of highly pathogenic PRRSV in
China raised great concern among farmers and scientists. With the significant economic losses and the emerging of
highly pathogenic PRRSV, the immunologists need to revisit the roles of immune system in PRRSV and identify
mechanisms that provide protective immunity in pigs. To prevent PRRSV infection, the porcine immune system utilizes both cellular and humoral responses in innate and adaptive immune system to prevent virus multiplication. Notably, the innate cellular defenses that involve in anti-PRRS comprises of NK cells, macrophages and dendritic cells.
For the humoral responses, cytokines especially type I IFN play a critical role in anti-PRRSV immunity. Other cytokines including IL-10 and pro-inflammatory cytokines such as IL-1, IL-8 and TNF-a have been contributed to
PRRSV infection (Darwich et al., 2010).
Host pattern recognition receptors detect PRRSV
Multiple evidences suggested that the intracellular Toll-like receptor 3 (TLR3) which detects dsRNA is a major protein that recognizes PRRSV in porcine innate immune cells (Darwich et al., 2010). Other TLRs including TLR
4 and TLR7 are highly expressed in PRRSV infected lymphoid tissues and tracheobronchial lymph nodes. However,
the definite roles of these TLRs require further studies. In addition to the TLRs, the cytosolic nucleic sensors RIG-I
have been involved in PRRSV recognition in porcine alveolar macrophages.
374 
Activation of these pattern recognition receptors and signaling pathways influences innate immune response
that determines the outcome of adaptive immunity through the effect of cytokines and chemokines.
Mechanisms of PRRSV inhibit host innate immune response
To establish infection, PRRSV employs multiple strategies to subvert the host innate immune system.
Among the evading strategies, PRRSV uses multiple non-structural proteins (nsp) to interfere with type I IFN
production (Figure 1). For example, nsp1a inhibited IFN-b through blocking of NF-kB activation, IRF3 and IFN
promoter activities. Moreover, nsp11 suppressed IFN production through inhibition of RIG-I and IRF3 signaling and
NF-kB activation (Yoo et al., 2010). Such examples emphasize the roles of viral proteins in modulation of host
innate immune response to benefit virus infection.
Figure 1 Mechanisms of PRRSV recognition by host innate immune system and multiple viral proteins that interfere
these signaling pathways and inhibit type I IFN production.
Future challenges for PRRSV vaccine design
Limitations of current PRRSV vaccines include the inconsistence of inactivated vaccines on immune protection and the safety concern and efficacy of modified live vaccine against heterologous challenge (Murtaugh and Genzow, 2011). As the innate immune system determines the magnitude and quality of adaptive immune response
through production of cytokines that govern the specific T cell subsets and antibody production from B cells. Recent
advances in transcriptomic, proteomic and system analysis of host cell response to a specific pathogen will provide
complete understanding of the complex process of host innate immune system during PRRSV infection. Together,
the fundamental findings of PRRSV and porcine cells interaction could lead to new targets for a novel vaccine design
that enhances better immune response and assists PRRSV control in pig farms.
375
References
Xiao, S. Wang, Q. Jia, J. Cong, P. Mo, D. Yu, X. Qin, L. Li, A. Niu, Y. Zhu, K. Wang, X. Liu, X. and Chen Y.
(2010). Proteome changes of lungs artificially infected with H-PRRSV and N-PRRSV by two-dimensional
fluorescence difference gel electrophoresis. Virology Journal, 7, 107
Darwich, L. Diaz, I. and Mateu, E. (2010). Certainties, doubts and hypotheses in porcine reproductive and
respiratory syndrome virus immunobiology. Virus Research, 154, 123-132.
Yoo, D. Song, C. Sun, Y. Du, Y. Kim, O. and Liu, HC. (2010). Modulation of host cell
responses and evasion strategies for porcine reproductive and respiratory syndrome virus. Virus Research, 154,
48-60.
Murtaugh, M.P. and Genzow, M. (2011). Immunological solutions for treatment and prevention of porcine
reproductive and respiratory syndrome (PRRS). Vaccine, 29, 8192-8204.
376 
FMD vaccine: recommendations and prospects of improved vaccine
Jef M. Hammond
Donald King, Nick Knowles, Jemma Wadsworth, Bob Statham,Yanmin Li, Pip Hamblin, Ginette Wilsden, Geoff
Hutchings, Valerie Mioulet, Miki Madi, Begona Valdozo, Nigel Ferris, David Paton and Elizabeth Wilson
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 ONF,
United Kingdom.
Abstract
Foot-and-mouth disease (FMD) is highly contagious, infects a wide variety of domestic and wildlife hosts and occurs
as multiple non-cross-protective virus serotypes. Its presence restricts trade opportunities for endemic countries and
poses a constant threat to those countries free of the disease. FMD vaccines formulated from inactivated live virus
mixed with an adjuvant, form the basis of FMD control strategies in endemic regions. Many countries free of disease
maintain stocks of FMD antigens in vaccine banks for use in case of an outbreak. The World Reference Laboratory
for FMD (WRLFMD) at the Institute for Animal Health, Pirbright, regularly receives samples for FMD diagnosis
and virus isolates made in reference laboratories from many parts of the world. The in-vitro antigenic properties of
selected isolates are assessed for vaccine matching and nucleotide sequencing allows precise characterisation of new
isolates and tracing of their origin by comparison with viruses held in the extensive WRLFMD collection. This analysis assists the „real time‟ monitoring of emergence and spread of FMD virus globally and advises on appropriate
vaccine strains for use internationally. A number of initiatives are also underway to develop next generation FMD
vaccines, which it is hoped will provide the platform for improved progressive control of this most infectious of
diseases.
Global FMD Situation and current vaccine recommendations
FMD is endemic in Africa, Asia, the Middle East and South America (figure 1). Recently we have determined the
global clustering of FMD viruses and identified 7 virus pools, where multiple serotypes occur but within are topotypes that are mainly confined to that pool. We have defined 3 pools covering Europe, the Middle-East and Asia, 3
pools covering Africa and 1 pool covering the Americas. This enables a regional approach to be taken to assist global
control of FMD. An increased regional knowledge of FMD outbreaks and identification of these within particular
reservoirs or pools of FMD activity can lead to globally informed regional FMD control programmes.
377
Such programmes should use the most up-to-date information, and would allow control strategies to be developed
based upon greater regional knowledge. These seven pools can be considered as containing multiple serotypes but
with subtypes/topotypes mainly confined to that pool. This then allows for better-informed control measures to be
implemented within that pool, but in the context of a real-time map of global FMD status. It also follows that if
vaccination is to be a major tool for control, each pool could benefit from tailored or more specific vaccines relevant
to the topotypes present in that pool, and the pools will not necessarily be reliant on the currently available generic
vaccines. Recently there has been a notable increase in the incidence of FMD outbreaks reported in Asia and the
Middle East and a concurrent spread of the serotypes O Pan-Asia 2 and A Iran 05 strains. In 2010, both Japan and the
Republic of Korea reported FMD outbreaks losing their status as countries listed by OIE as FMD-free without
vaccination. The majority of viruses have been isolated from samples submitted from Africa and Asia which remain
the major reservoirs for the FMD virus and more than 80% of isolates were shown to be serotype O (figure 2). In
Southern America, FMDV circulation has mainly been detected in Ecuador and Venezuela.
Movement of live animals still constitutes by far the greatest risk for spread of FMD, followed by trade in animal
products. In parts of Africa, the Cape buffalo provides an important reservoir for the maintenance of certain FMD
virus (FMDV) serotypes. FMDV continues to evolve, giving rise to new strains that cause periodic upsurges in the
number of cases and increase the risk of spread into new areas. A World Organisation for Animal Health (OIE)/Food
and Agriculture Organization of the United Nations (FAO) network of FMD Reference Laboratories has been established to help track the emergence and distribution of different FMD virus variants, to make recommendations on
vaccine strains needed in different parts of the world, and to raise standards of laboratory testing. In many parts of
the world, the main barriers to FMDV spread operate at a regional rather than a national level, consistent with
attempts to establish regional disease control programmes.
Issues with Current Vaccines for FMD
Vaccines currently available for FMD control and eradication are based on preparations of whole virus that are
derived from cell culture, chemically inactivated and blended with suitable adjuvant, and as such are far from ideal.
As the disease is caused by seven different serotypes of virus, it is often necessary to include a combination of strains
in the vaccine used to ensure protection. This is further complicated by the evolution of new subtypes of virus. The
more virus serotypes in a vaccine, the more expensive it becomes, restricting use in many developing countries.
The need for multiple vaccine types has led to the development of “vaccine banks” holding large stocks of
concentrated antigens prepared from live virus. Preparation of material for these banks Intermediate, sporadic
Endemic Free Free. Virus present in game parksFree with vaccination Countries with multiples zones:FMD-free, free
with vaccination or not freeWRLFMD requires specialist high level bio-security facilities and, consequently, is
limited to a few manufacturers.
378 
Furthermore, the immunity offered by current vaccines is short lived, so revaccination is necessary, again limiting
the role of vaccines in developing effective population immunity levels. There is also an absolute requirement for a
cold chain, making widespread vaccination in developing countries a particular problem.
The immunity offered by current vaccines takes several days to develop, by which time new animals have become
infected, all excreting high levels of virus into the environment. Furthermore, vaccinated animals may become undetectable carriers of virus, providing a potential covert source of infection to susceptible animals. Because of this risk,
a six-month interval is required between the last vaccination and return of the disease-free status. Any delay in reestablishing FMD-free status can inflict major economic costs on a country through the imposition of trade embargoes.
Figure 1. Conjectured status of FMD
379
It should also be noted that currently there are varying degrees of effort to identify improved vaccines in different
regions. There are relatively few for use in Africa, while the developed world‟s vaccine banks have a good stock of
vaccines destined for emergency use. Such a global approach for progressive control would require that each region
or pool would need laboratory support for typing and vaccine matching work. Obviously regional Reference Laboratories and WRLFMD can provide major support, but certain regions will need development of their own regional
laboratory, perhaps utilising support from twinning programmes encouraged by both FAO and OIE.
Threat Analysis and Vaccine Recommendations
A threat analysis has been carried out utilising data collected over the period January 2009 to May 2011 by
WRLFMD. There are seven serotypes of FMD and multiple subtypes within those serotypes. Below is an analysis of
their current circulation, risk of spread outside their current distribution and vaccine recommendations for control.
Detailed information is available from the Head of WRLFMD- Dr Jef Hammond.
Serotypes O and A still present the greatest threat for spread into FMD free areas with the lineages/genotypes A-Iran
-05 and O PanAsia 2 still presenting the greatest risk of further spread. Through continued analysis WRLFMD
demonstrated gaps in cover with emerging A-Iran-05 field isolates and the A22 Iraq vaccine strain.
Figure 2. FMD serotypes isolated in 2010: 80% were serotype O and no type C or SAT3 were detected.
380 
However, new vaccines released in 2009 by both Intervet and Merial specifically for A-Iran-05 appear to provide
much better protection, but limited information is available on the ability of these vaccines to cross-protect against
other A field strains as A22 Iraq has been shown to do. Antigenic variability of serotype O viruses is less than the A
serotype and O1 Manisa has been recommended for many years as a suitable vaccine for viruses of the ME-SA topotype. However, some recent O isolates have shown poor r values by VNT. This situation is being closely monitored.
A newly available vaccine from Intervet „O PanAsia 2‟ has shown consistently higher r values against recent field
isolates of the PanAsia 2 genotype within this topotype. Asia 1 Shamir has given a good antigenic match to most
strains within the Asia 1 serotype for many years. However, one isolate from Pakistan in 2009 gave a very low r value by VNT and several isolates from Bahrain, Pakistan and Iran isolated in 2011 have also failed to match with Asia
1 Shamir vaccine. The situation is also being carefully monitored. There is need to maintain a close watch on the incidence and variation with the SAT1 and SAT2 serotypes due to possible incursions from various trade routes out of
Africa, but there is confusion over which vaccine strains to recommend as these are limited and there is very little
information on the protection that they could offer against current field strains. SAT 3 appears to be mainly associated with African buffalo and is not considered a real threat for broader spread and serotype C has not been reported
since 2004.
New Generation FMD Vaccines
New generation of FMD vaccines with the following properties are currently being developed in a number of laboratories around the world:
Longer duration of protection;
Faster onset of protection;
Broader spectrum of protection against different strains;
More potent immunity to prevent viral replication and development of viral carriers;
Better discrimination of vaccinated animals that go on to become infected;
Thermostable, safer to make and easier to administer.
WRLFMD Vaccine Recommendations
The recommendations made by the WRLFMD are drawn principally from a list of vaccine strains for which master
seed vaccine viruses are believed to be available within the portfolios of vaccine suppliers able to fulfil the quality
requirements for use in Europe. The ranking of the utility of the viruses is based on the results obtained by the
WRLFMD from in vitro serological tests to match these vaccine viruses to recent field isolates. As such, the
WRLFMD can only recommend vaccine virus strains for which it has received supplies of both the vaccine virus and
the homologous antiserum. Since these vaccine strains are chosen to protect against threats from outside of Europe, it
can be anticipated that the vaccines should also be useful to counter such threats at source. However, other vaccine
viruses may have been produced, for example by vaccine manufacturers located in the regions from which the threats
arise and using local isolates, that would also provide an equivalent or even better antigenic match to the field isolates that pose the threat.
381
HIGH PRIORITY
O Manisa
O BFS or O Campos
PanAsia 2*
A24 Cruzeiro
Asia 1 Shamir
A Iran 05
A22 Iraq
SAT 2 Saudi Arabia or equivalent
MEDIUM PRIORITY
A Eritrea
A Iran 96
SAT 2 Zimbabwe
A Iran 87 or A Saudi 23/86
SAT 1 South Africa
A Malaysia 97
A Argentina 2001
O Taiwan 97 (or equivalent pig-adapted strain)
A Iran 99
LOW PRIORITY
A15 Bangkok related strain
A87 Argentina related strain
C Noville
SAT 2 Kenya
SAT 1 Kenya
SAT 3 Zimbabwe
A Kenya
NB Strains are not listed in order of importance within each priority grouping.
*Very recent introduction: PanAsia 2 vaccine use will be monitored carefully during 2011
382 
Summary
FMD is present in many regions of the world and the highest risk of spread is through the movement of live animals
and animal products. Currently available inactivated FMDV vaccines can confer serotype specific protection in 5 –
10 days depending on quality and potency, however they do not induce long lived protective immunity, resulting in
the need for multiple regular vaccinations in endemic settings. Among the limitations of such vaccines are the requirement for production of very large quantities of live FMDV in high biosecurity containment facilities, a relatively short shelf life of formulated product, and the absolute requirement of a strict cold chain throughout distribution
and delivery in the field. Next generation FMD vaccines will need to provide broader and longer duration protection
and ideally not have absolute reliance on the cold chain for distribution. The WRLFMD at the centre of the OIE/
FAO FMD Reference Laboratory Network provides a global „real time virus map’ for the implementation of better
informed control measures for FMD. This network provides a vital contribution to the global control of FMD and
provides opportunities and expertise for developing and sustaining laboratory capacity and capability, exchange of
materials and technologies, harmonising approaches to diagnosis and supporting complementary research. Laboratories within the network regularly receive samples for FMD diagnosis from many parts of the world. The in-vitro antigenic properties of selected isolates are assessed for vaccine matching and nucleotide sequencing allows precise
characterisation of new isolates and tracing of their origin by comparison with viruses held in virus collections. This
analysis assists the monitoring of the „real time‟ emergence and spread of FMD virus globally. The clustering of
FMD viruses into 7 virus pools, with 3 pools covering Europe, the Middle-East and Asia, 3 pools covering Africa
and 1 pool covering the Americas, is now enabling a targeted approach to progressive FMD control through the
combined activities of OIE and FAO and the regional authorities.
However, it is of great importance to acknowledge that a major combined effort both National and Global is needed
for control to be successful.
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Update on antigenic variation of foot and mouth disease virus in South East Asia
Wilai Linchongsubongkoch* Kingkarn Boonsuya Seeyo and Janya Samanit
Regional Reference Laboratory for FMD in Southeast Asia, National Institute of Animal Health,
Department of Livestock Development, Pakchong, Nakhonratchasima 30130, Thailand
Corresponding author: Tel & fax : +66 44 313869 ; email: [email protected]
Abstract
The Regional Reference Laboratory for foot and mouth disease (FMD) in South East Asia (SEA),
Pakchong, Thailand has been recognized as an OIE Reference Laboratory for FMD since May 2009, the laboratory
has been served for FMD diagnosis in South East Asia (SEA) and OIE member countries. The diagnostic test has
been carried out by standard ELISA typing and virus isolation on field samples submitted for type identification,
these samples were further investigated for antigenic variation. During the past 2 years (2010-2011), a number of
field viruses type O and A causing outbreak in Thailand, Cambodia, Lao PDR, Myanmar and Vietnam have been
studied. The result of r-value was expressed as the serological relationship between the ratio of serum titer against
heterologous field strain and homologous vaccine strain. The r-values of FMDV type O from Thailand, Cambodia,
Lao and Vietnam showed close antigenic relationship to vaccine strains (O189/87), it is therefore suggested that the
existing seed vaccine strains for type O need not to be changed. In contrast, the r-value of type A causing outbreak in
Thailand was demonstrated close antigenic relationship to A/Sakolnakorn/97 than A118/87 current vaccine strain.
This study indicated that recent type A viruses gave antigenic diverse from A118/87 vaccine strain, therefore, it was
recommended to put an additional of A/Sakolnakorn/97 vaccine in the existing trivalent vaccine in order to give a
wide broader protection of type A outbreak in the field. In conclusion, the recent field outbreak of type O in SEA
indicated that no antigenic variation has been found, while the antigenic variation of type A has been found only in
Thailand, due to no FMD sample of type A from SEA country was submitted to the laboratory. However this study
would be useful in supporting the selection of appropriate virus vaccine strain for production of high efficacy vaccine
and enhancing the efficiency of the strategic planning for FMD control in SEA region.
Keywords: foot and mouth disease virus, r-value, sequencing ,LP ELISA
Introduction
Foot and mouth disease, a highly contagious disease of cloven-hoofed animals, is important in South East
Asia region. Serotypes O, A and Asia1 are considered endemic in Thailand and South East Asia, causing significant
economic losses primarily due to lower production of affected animals and subsequent constrain of international
trading. Rapid and accurate diagnosis plays an important role in the prevention of disease spread and can ensure that
appropriate vaccines are selected for use against circulating filed strains.
384 
Standard ELISA typing test for type identification of field samples (Roeder and Le Blance Smith, 1987) and other
serological tests including virus neutralization (VN) test (Rweyemamu,1978), liquid phase blocking ELISA (LP
ELISA) (Hamblin et al.,1986) in parallel with ELISA-non structural protein (ELISA-NSP) (Linchongsubongkoch et
al., 2008), can be used to support disease surveillance and sero-monitoring of vaccinated and infected animals. In
addition, strain differentiation investigation by antigenic variation study of field viruses and reference vaccine
strains become a very important in supporting a basic information of the evolution in FMD field outbreak strains.
This study was carried out by determination of the serological relationship (r-value) between filed virus strains and
the reference vaccine strains and is useful for selecting the appropriate virus strain for vaccine production and
enhancing the strategic action plan of FMD control at the national and regional level. Linchongsubongkoch et al.
( 2008) reported that the r-value of type O causing outbreak during 1997-2007 indicated that the serological relationship was very close to O189/87 vaccine strain, while type A showed antigenic changes from time to time, it was
indicated that the type A field viruses causing outbreak during 2001-2007 and during 2008-2009 (data not published) were closely related to A118/87 vaccine strain. Therefore, in this present study, a number of field isolate
viruses types O and A causing outbreaks in SEA region during 2010-2011 were studied for updating the FMD antigenic variation by determining the serological relationship between the reference virus vaccine strains and field
virus strains which would be useful to support the selection of appropriate virus for vaccine production and control
of FMD in SEA region.
Reference viruses and field viruses
Reference viruses type O189/87, A118/87 and A/Sakolnakorn/97 were obtained from current seed
vaccine strains. Field samples from FMD infected animals submitted for laboratory diagnosis which were subjected
for typing identification using standard ELISA typing test and maintained by inoculating to primary lamb kidney
cell for 2-3 passages and further 4 or 5 passages in BHK-21 cell line. The serotype of the cell culture supernatant
fluid was again confirmed by antigen typing test as described by Roeder and Le Blanc Smith (1987). The reference
vaccine strain and field isolate viruses were titrated by indirect sandwich ELISA method (Kitching et al., 1988)
and selected the optimal dilution for virus to use in the liquid phase blocking ELISA (LP ELISA) test
(Linchongsubongkoch et al., 2000).
2. Bovine antisera
The reference sera used in the LP ELISA, including bovine anti FMD type O189/87, A118/87 and A/
Sakolnakorn/97 were prepared from experimental cattle that have been vaccinated with reference homologous
virus. Blood from vaccinated animals were taken at 21 days post vaccination for immune sera.
Liquid phase blocking ELISA (LP ELISA)
Bovine antiserum against homologous vaccine strain was used to determine antibodies to FMD virus by
LP ELISA . The bovine serum was diluted into two fold dilution series, and then a fixed concentration of reference
vaccine strain and field isolate viruses giving an optical density (OD) in the range of 1.0 –1.5 were reacted with bovine post vaccination serum with homologous virus of each type. The antibody titer to FMD virus was determined
as described by Hamblin et al. (1986), Kitching et al.(1988) and Linchongsubongkoch et al. (2000).
385
4. The serological relationship (r-value)
The LP ELISA method has been used to examine the serological relationship between field isolate viruses and the reference vaccine strains which was expressed as r-value.
r-value = Serum titer against heterologous field strain
Serum titer against homologous vaccine strain
The guideline suggestion for r-value obtained by LP ELISA and criteria of interpretation were described
by Samuel et al. (1990) as this follows.
r = 0-0.19
highly significant serological variation from the reference strain
r = 0.20-0.39
significant difference from the reference strain, but protection may
be satisfactory if using a sufficiently potent vaccine.
r = 0.40-1.0
not significantly difference from vaccine strain.
Results
Table 1. Situation of FMD outbreak in SEA during 2010-2011, diagnostic assay using ELISA typing
Test for FMD serotype identification in samples submitted to the Regional Reference Laboratory.
Year
2010
2011
Country
No. of sample
Type identification by ELISA typing
O
A
Asia1
NVD*
Thailand
48
23
4
-
21
Cambodia
20
12
-
-
8
Myanmar
3
-
2
-
-
Vietnam
27
26
1
-
-
Cambodia
31
16
-
-
15
Lao PDR
20
16
-
-
4
Sri Lanka
4
2
-
-
-
Thailand
64
13
37
-
14
217
108
44
-
62
Total
NVD = no virus detected
386 
Table 2. Result of r-value of FMD type O field viruses in Thailand, Cambodia, Lao PDR and Vietnam during
2010-2011, using O189/87 as a homologous system.
Country
Year
Total sample
% r-value range of type O
0-0.19
0.20-0.39
0.40-1.0
Cambodia
2010
3
0
0
3
Vietnam
2011
2010
2
5
0
0
0
0
2
5
Lao PDR
Thailand
2011
2010
3
7
0
0
0
0
3
7
2011
5
25
0
0(0%)
0
0(0%)
5
25(100%)
Table 3. Result of r-value of FMDV type A field viruses in Thailand and Myanmar during 2010-2011, using
A118/87 and A/ Sakolnakorn/97 as homologous system.
Year/
Country
Total sample
r-value range type A118/87
0-0.19
0.20-0.39
2010 Thailand
3
no binding reaction
Myanmar
2011Thailand
2
NOT DONE
33
no binding reaction
38
0.40-1.0
r- value range type A/Sakolnakorn/97
0-0.19
0.20-0.39
0.40-1.0
0
0
3
0
0
33
0(0%)
0(0%)
36(100%)
Remark: FMD type A of Myanmar was not investigated for r-value, due to no virus could be adapted in cell culture
387
Conclusion and discussion
The antigenic variation of FMDV in SEA during 2010 -2011 was investigated by determining the
serological relationship between the reference vaccine viruses and field viruses, the result was expressed as r-value.
Table 2 showed that 100% of filed viruses type O from Cambodia, Lao PDR, Vietnam and Thailand gave an r-value
greater than 0.40 indicated that the serological relationship was very close to virus vaccine strain, therefore the current vaccine of O189/87 could give a protection to the circulating viruses in Thailand and the region. While the situation of type A was different, the r-value in table 3 showed the 100% of field viruses from Thailand gave an r-value
greater that 0.40 to A/Sakolnakorn/97 than A118/87 system. Similarly result was found in the FMD type A profiling
test demonstrated that no binding reaction to the A118/87 system but showed high binding reaction to A/
Sakolnakorn/97 system (data not published). Therefore, the recent situation of type A field viruses during 20102011 have antigenic changed from current vaccine of A/118/87 to A/ Sakolnakorn/97 that might be resulting from
disease outbreak from time to time. By the history of type A seed vaccine selection, A/Sakolnakorn/97 was selected
as a new seed vaccine strain and being used from 1997-2001. Then, again a new seed vaccine strain of A118/87 was
selected in 2001 and being used up to the present (Linchongsubongkoch et. al., 2008). Although, the antigenic variation of type A occurred during 2010-2011 was showed close related to A/Sakolnakorn/97. In this regard, it not
necessary to select a new vaccine strain, it was recommended to use a monovalent vaccine of A/Sakolnakorn/97 to
add in the existing trivalent vaccine in order to give a wide broader protection to the field outbreak viruses. However, the nucleotide sequencing of FMD field outbreak were also investigated and analyzed as phylogenetic tree (data
not published). The result demonstrated that type O in SEA region was defined as South East Asia (SEA) topotype,
Myn98 strain. For type A viruses in Thailand and Vietnam was defined as only one topotype of ASIA. This studied
would be useful for tracing back to the original virus causing outbreak and supporting the seed virus selection for
vaccine production in enhancing the action plan for FMD control in the region.
Acknowledgements
The authors wish to thank the RRL staffs for their help and participation in the successful implementation of this study.
References
Hamblin, C., Barnett, I.T.R. and Hedger, R.S. (1986). A new enzyme linked immunosorbent assay (ELISA) for
the antibodies against foot and mouth disease virus. II. Application. J. Immun. Methods, 93, 122-129.
Kitching, P., Rendle, R. and Ferris, N.P. (1988). Rapid correlation between field isolates and vaccine strains of foot
and mouth disease virus. AFRC Institute for Animal Health, Pirbright, Working, Surrey, UK.
Linchongsubongkoch,W., Rumlumdoan,S., Kamolsiripichaiporn,S. and Janukit,T. 2000. Antigenic variation of
foot and mouth disease viruses from field outbreak in Thailand. Proceedings in 38 th Kasetsart University An
nual Conference, 207-214.
Linchongsubongkoch,W., Udon, R., and Samanit, J. (2008). Antigenic evolution of foot and mouth disease virus
strains in Thailand. J. Thai Vet. Med.Assoc. 59, 80-89.
Roder, P.L. and Le Blanc Smith, P.M. (1987). Detection and typing of foot and mouth disease virus by enzyme
linked immunosorbent assay : a sensitive, rapid and reliable technique for primary diagnosis . Res. Vet. Sci.
43, 225-232.
Rweyemamu, M.M. 1978. The selection of vaccine strains of foot and mouth disease virus. Br. Vet. J. 134, 63-67.
Samuel, A.R., Ouldridge E. J., Arrowsmith, A.E.M., Kitching, R.P. and Knowles, N.J . (1990). Antigenic analysis
of serotype O of foot and mouth disease virus isolates from the Middle East 1981 – 1988. Vaccine. 8 , 390396.
388 
Antimicrobial Usages and Foodborne Antimicrobial Resistance in Animals
Thongchai Chalermchaikit (DVM, MPH, PhD)
Faculty of Veterinary Science, Chulalongkorn University, Bangkok
“Antimicrobial resistance (AMR) is a major global public health concern and a food safety issue.....The use
of antimicrobial agents in food producing animals/crops provides a potentially important risk factor for selection
and dissemination of AMR microorganisms and determinants from animals/food crops to humans via the consumption of food.” (The CODEX AdHoc Intergovernmental Task Force on Antimicrobial Resistance, 2011)
Global animal health antimicrobial market is worth about 5 billions USD per year. Around 16,000 tons of
antimicrobial was annually used in the USA; only 1,280 tons (8 %) was used in human, the rest was used in livestock 77 % and others (companion animals, aquaculture, etc.) 15 % (Benbrook, Meeting American Society for Microbiology, 2001). The use of antimicrobials in food animals may cause problems in the selection for resistance.
Since animals frequently harbor pathogenic bacteria for man in their intestinal tracts, i.e. Salmonella, Campylolobacter, Yersinia, Listeria and entero-haemorhargic Escherichia coli. For example, the use of enrofloxacin in poultry production was followed by emergence of fluoroquinolone-resistant Campylobacter species among both poultry and humans. Good farm practices, educational programs and law implementation for control of antimicrobials
use in food-animals are important keys of AMR containment. For example, AMR situation on some antimicrobials
in Thailand had been declined (Figure 1) as well as sale of antimicrobials (Figure 2).
AMP = Ampicillin
% antimicrobial resistance
60
2002-2003
50
2008
AMX = Amoxycillin
2009
CHL = Chloramphenicol
40
GEN = Gentamicin
30
NAL = Nalidixic acid
20
NOR = Norfloxacin
10
0
TCY = Tetracycline
AMP AMC
CHL
GEN
NAL
NOR
TCY
SUL
TMP
SXT
Figure 1 Antimicrobial resistance patterns
SUL = Sulfonamides
TMP = Trimethoprim
SXT = Sulfamethoxazole+Trimethoprim
45
4,885 MB (40.2 %)
40
% Market Share
35
2002
4,995 MB (28.9 %)
30
2009
2010
5,376 MB (28.5 %)
115 MB
(0.9 %)
25
1,965 MB (10.4 %)
20
2,861 MB (16.5 %)
15
1,455 MB (8.4 %)
2,719 MB (14.4 %)
158 MB
(1.3 %)
10
5
0
Vitamins &
Minerals
Antimicrobials Coccidiostats
Growth
Feed additives
Vaccines
Supportives
Anthelmintics
Disinfectants
Others
promoters
Figure 2 Market shares of animal health products in Thailand. (Data from: Animal Health
Products Association, Thailand) (MB = Million baths)
389
Aquaculture food sustainable and threat
Visanu Boonyawiat
Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine,
Kasetsart University, Thailand, [email protected], Tel: 6634351901, Fax: 6634351405
ABSTRACT
Aquaculture contributed 50 percent of aquatic animal food for human consumption in 2008. Moreover it is
expected to increase production to meet the future demand. However, the rapid grown of aquaculture generated
adverse effects to the environmental and socio-economic impacts. The concept of sustainable development was
introduced by FAO in 1991 for conservation of the natural resource base and environment issue parallel with the
rapid grown of aquaculture system. The major constraining the development and sustainability of the aquaculture
sector are the limitation of natural resource inputs (e.g. water, culture sites, feed and energy), the transboundary
aquatic animal pathogens/diseases and the food safety issue. Many shrimp viral pathogens (e.g. White spot virus,
Taura syndrome virus and Infectious myonecrosis virus) were instances for transboundary diseases that pathogens
can travel alongside the movement of their hosts. Recently epidemic disease that might be transboundary disease in
Southeast Asia region are Francisellsis in Tilapia fish and gregarine infection in whiteleg shrimp. Usually, aquaculture farms were sited in freshwater and costal area that has more risk of contamination by chemical and biological
agents than in open seas. Thus, any food safety concerns are becoming increasing on aquaculture producing.
Keyword: aquaculture, current status, constraining, transboundary disease, food safety
Current status of global aquaculture
Fisheries and aquaculture products are globally important sources of much needed, high quality, aquatic
animal proteins. Global aquaculture has grown dramatically over the past 50 years to around 52.5 million tonnes
and worth US$98.5 billion (not including aquatic plant) in 2008. This production amount is accounting 50 per cent
of the world’s fish food supply, approximately. Asia countries were dominating this production with China the
leading worldwide. Their product was accounting for 89 per cent by volume and 79per cent by value in 2008
(Figure 1). The rapid growth in this region has been driven by a variety of factors, including pre-existing aquaculture practices, population and economic growth, relaxed regulatory framework and expanding export opportunities.
(Bostock et al., 2011).
Figure 1. Global aquaculture production by region. Source: FAO (2010). (a) Aquaculture by quantity 2008
(excluding aquatic plants). (b) Aquaculture by value 2008 (excluding aquatic plants).
Three hundred and ten of aquatic species (excluding aquatic plants) were recorded by FAO as cultured in 2008.
However, the top 10 of culture species was account for 53 % of total production quantity and 45% by value. Freshwater fish production is dominated by various species of carp, although tilapia and later pangasius catfish has become more importance (Table 1).
390 
Table 1. Most significant of freshwater fish species by quantity. Developed from Fishstat data FAO (2010). nei,
not elsewhere included.
species
number of
countries
production quantity
(tonnes)
growth rate (%)
2008
Average (1999-2008)
silver carp
50
3,848,258
5.5
2.8
grass carp (¼white amur)
57
3,775,267
4.3
4.0
common carp
100
3,000,529
5.5
3.7
Nile tilapia
74
2,334,432
8.6
22.2
bighead carp
28
2,321,513
7.2
6.3
catfish
5
2,281,838
7.9
31.1
pangas catfishes nei
5
1,380,702
52.4
220.1
freshwater fishes nei
97
1,247,859
225.1
21.2
Coastal shellfish aquaculture primarily comprises oyster, scallop and whiteleg shrimp, with Atlantic salmon as the
leading intensively farmed marine fish (Table 2).
Table 2. Most significant coastal aquaculture species by quantity. Developed from Fishstat data FAO (2010). nei,
not elsewhere included.
species
growth rate (%)
number of
countries
production quantity
(tonnes)
2008
cupped oysters nei
18
3,385,382
24.5
3.1
Japanese carpet shell
13
3,141,851
3.2
13.4
whiteleg shrimp
36
2,259,183
21.7
106.7
Atlantic salmon
31
1,456,721
5.6
11.2
scallops nei
7
1,137,379
22.4
9.9
Average (1999-2008)
Development and sustainable aquaculture
The development of aquaculture depends on the interaction of many factors. The initial factors are market demand
on the target species and availability of environmental resource. Additional, the infrastructure, technical capability
(i.e. hatchery production, feed, e.t.c.), investment in culture system and technical research, human resource and institutional system were driven the progression of aquaculture (Muir and Young, 1998). Although rapid growth of
aquaculture has enabled the world fisheries supply to keep pace with population growth (Boyd et al, 1998), it generated adverse environmental and socio-economic impacts. Therefore, the concept of sustainable development was
introduced by FAO in 1991. The sustainable development has been well-defined as “the management and conservation of the natural resource base, and the orientation of technological and institutional change in such a manner
as to ensure the attainment and continued satisfaction of human needs for present and future generations. Such development conserves land, water, and plant and animal genetic resources, is environmentally non-degrading, technically appropriate, economically viable, and socially acceptable” (FAO, 1991). However, the sustainable development and management of aquaculture and fisheries systems can only occur if these activities are well planned and
integrated into the natural and social resource, ecosystems, and farming systems.
391
Threat factors for aquaculture
Natural resource inputs: The growth of aquaculture is increasing pressure on natural resource inputs, noticeably
water, sites, feed and energy. Aquaculture, especially in coastal zones, is frequently in competition with other activities that uses the same resource (e.g. tourism and port developments). Much of the crustacean farming, most
marine species and other intensive fish aquaculture require a higher quality diet, usually containing fish meal and
often fish oil. The aquaculture sector consumed 3,724 thousand tonnes of fish meal (68.2% total global fish meal)
and 835 thousand tonnes of fish oil (88.5% total reported fish oil) in 2006 (Tacon and Metain, 2008). Unless the
substitution of the protein source from fish meal in aquafeed, aquaculture will continue to consume the majority of
fish meal and oil produced. Therefore, fish meal and fish oil will not be sufficient to meet the global demands (e.g.
livestock, aquaculture) (Bostock et al., 2011). Moreover, the energy cost and carbon emissions of aquaculture activities are obtain greater attention.
Infectious diseases: Infectious diseases are constraining the development and sustainability of the aquaculture sector through direct production losses and increased operating costs and indirectly, through restrictions on trade and
impacts on biodiversity. Aquaculture is faced with transboundary aquatic animal pathogens/diseases. These include
increased globalization of trade and markets; the intensification of fish-farming practices through the movement of
broodstock, postlarvae, fry and fingerlings; the introduction of new species for aquaculture development. The
shrimp viral pathogens (e.g. White spot virus (WSV), Taura syndrome virus (TSV) and Infectious myonecrosis
virus (IMNV)) are the classical instance of transboundary diseases that pathogens can travel alongside the movement of their hosts (Bondad-Reantaso et al., 2005). Moreover the current epidemic disease that might be transboundary disease in Southeast Asia region are Francisellsis in Tilapia fish and gregarine infection in whiteleg
shrimp.
- Francisella spp. is current emergent fish pathogens in several cultured fish species worldwide (Chen et al., 1994;
Chern and Chao,1994; Mauel and Miller, 2002; Nylund et al., 2006; Hsieh et al., 2007; Soto et al., 2009). Mortality
in fingerling tilapia had been report up to 90% (Soto et al., 2009). In 2010, cage culture tilapia in Thailand was
demonstrated to infection with Franciscella speies (Boonyawiwat et al.,2011) and the phylogenetic study of 16S
rRNA sequence showed 99.9 identical to Francisella noatunensis subsp. orientalis that had been report in Taiwan
(unpublished data). Francisellosis is an acute to chronic disease caused by different Francisella species. Noticeable
features are presence of multiple whitish nodules on several visceral organs, especially kidney and spleen(Figure2).
Figure 2. Tilapia fish affected from Francisellosis; (a) Prominent of anterior kidney with white granulomatous
nodule. (b) Splenomegaly with white granulomatous nodule on the surface.
392 
- White feces syndrome; The severely affected shrimp showed yellowish to whitish discoloration of midgut (Figure
3). Populations of severely shrimp were affected due to the reduction of feed consumption, growth rates, and average daily growth (ADG); however, feed conversion ratios (FCR) was increasing. White feces syndrome is caused
by multiple factors such as filamentous blue green algae (Schizothrix calcicola and other benthic forms) (Brock
and Main, 1994), rancid and poor quality of lipid in feed pellets, Vibrio infection (Linsuwan, 2010) as well as
gregarine infections (Brock and LeaMaster, 1992). There have been concerns regarding these gregarines cause economic losses due to lower survival and growth of shrimp in ponds (Lightner, 1993). There are two large suborders
of Eugregarinidae, the members of which are distinguished morphologically by the number of body divisions
(Levine 1976). Gamonts of members of Septatorina are divided into two distinct compartments. Other way round,
gamont stage of Aseptatorina is comprised of a single compartment (Jones, 1994). Septate gregarines are parasites
commonly found in midgut of both natural and cultured shrimp. In particular, the genera Cephalobus and Nematopsis have distributed worldwide in cultured penaeids (Lightner, 1993). Additionally, aseptate gregarine
(Paraophiodina scolecoides) has been reported infecting the midgut of cultured larvae and post-larvae specimens
of P. vannamei (Jones, et al., 1994). White feces syndrome was a prominent problem of shrimp culture in Thailand
and southeast Asia since 2009. The survey was found the relationship of new unidentified aseptate gregarine infection (Figure 4) with white feces syndrome (Boonyawiwat and Pulpipat, submitted).
Figure 3. Shrimp affected from White feces syndrome showed whitish discoloration of midgut (black arrow). Figure 4. New aseptate gregarine was found in midgut content by wet preparation technique; nucleus located near the
posterior end (black arrow).
Food sefety: The role of aquaculture in food safety has been a major concern of the sector for many years. Fisheries products are generally regarded as safe and nutritious foods, but sometimes products from aquaculture have
been associated with certain food safety issues, Due to the risk of contamination by chemical and biological agents
is greater in freshwater and coastal ecosystems than in open seas. The food safety hazards of aquaculture products
are vary according to culture system, management practices and environment. Foodborne disease associated with
pathogenic bacteria, parasites, residues of pesticide, insecticide and veterinary drugs, and heavy-metal contamination have all been identified as hazards. The origins of such food safety concerns are diverse, ranging from inappropriate aquaculture practices to environmental pollution and cultural habits of food preparation and consumption.
Thus, as aquaculture makes its transition to a major food-producing sector, proper assessment and control of any
food safety concerns are becoming increasingly important (FAO/NACA/WHO., 1997).
393
Reference
Bondad-Reantaso, M.G., Subasinghe, R., Arthur, J.R., Ogawa, K., Chinabut, S., Adlard, R., Tan, Z. and Shariff, M. (2005).
Disease and health management in Asian aquaculture. doi:10.1016/j.vetpar.2005.07.005
Boonyawiwat V, Pulpipat T, Kamlangdee A, Butrdee O, and Wajjwalku, W. (2011). Detection of Francisella-like Organism
(FLO) in culture tilapia (Oreochromis spp.) bu in situ hybridization. Proceedings of 16th Federation of Asian Veterinary
Associations Congress 2011 and 78th PVMA Annual Convention & Scientific Conference. Cebu city. Philippines. p.268.
Boonyawiwat, V and Pulpipat, T. (submitted). Finding of a new aseptate gregarine in cultured Pacific white shrimp affected
from white feces syndrome in the eastern and middle part of Thailand.
Bostock,J., McAndrew,B., Richards, R., Jauncey,K., Telfer, T., Lorenzen, K., Little, D., Ross, L., Handisyde, N., Gatward, I.
and Corner, R. (2010). Aquaculture: global status and trends. Phil. Trans. R. Soc. B., 365, 2897-2912.
Brock, J.A. and LeaMaster, B. (1992). A look at the principle bacterial, fungal and parasitic diseases of farmed shrimp. In:
Proceedings of the special session on shrimp farming. Ed Wyban, J. pp. 212-226. World Aquaculture Society, Baton
Rouge, LA, USA.
Brock, J.A. and Main, K.L. (1994). A guide to the common problems and diseases of cultured Penaeus vannamei. pp.146-147.
World Aquaculture Society, Baton Rouge, Louisiana, USA
Boyd, C.E., Massaut, L. and Weddig, J.L.(1998). Towards reducing environmental impacts of pond aquaculture. INFOFISH
International, 2/98:27-33.
Chen S.C., Tung M.C., Chen S.P., Tsai J.F., Wang P.C., Chen R.S., Lin S.C. and Adams A. (1994). Systemic granulomas
caused by a rickettsia-like in Nile tilapia, Oreochronuis niloticus (L). from southern Taiwan. Journal of Fish Diseases. 17,
591-599.
Chern S-C. and Chao C.B. (1994). Outbreaks of a disease by rickettsia-like organism in cultured tilapias in Taiwan. Fish
Pathology, 29, 61-71.
FAO, (1991). Elements for strategies and agenda for action. Strategies and tools for sustainable agriculture and rural development. FAO/Netherlands Conference on Agriculture and the Environment, held 15-19 April 1991 in S-Hertogenbosch, The
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FAO/NACA/WHO. (1997). Joint study group on Food Safety Issues Associated with Products from Aquaculture. Bangkok,
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Jones, T.C., Overstreet, R.M., Lotz, J.M. and Frelier, P.F. (1994). Paraophioidina scolecoides n. sp., a new aseptate gregarine
from cultured Pacific white shrimp Penaeus vannamei. Dis Aquat Org. 19, 67-75.
Hsieh, C.Y., Tung, M.C., Tu, C., Chang, C.D. and Tsai, S.S. 2006. Enzootics of visceral granulomas associated with Francisella-like organism infection in tilapia (Oreochromis spp.), Aquaculture, 254, 129–138.
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394 
Update Mastitis: Treatment of Bovine Mastitis during Lactating Period
Sukolrat Boonyayatra*
*Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University
Abstract
Bovine mastitis is an important animal health disease leading to significant economic losses to the dairy industry
throughout the world. Bovine mastitis is characterized by an inflammation of the mammary gland usually caused
by an intramammay infection (IMI) of a wide variety of microorganisms. Pathogens including Staphylococcus aureus, Streptococcus agalactiae, Coliform bacteria, Coagulase-negative Staphylococci and Strep. uberis are commonly isolated from clinical mastitis cases and usually concerned for treatment. Various therapeutic strategies have
been developed to control IMI and reduce losses due to clinical and subclinical mastitis. Antimicrobials have been
one of the most common treatment plans for therapy of bovine mastitis. Because cure rates are highly depended on
the pathogen, treatment of mastitis should be based on bacteriological diagnosis. If treatment decision has to be
made without results of bacteriological diagnosis, such as in acute mastitis, the concept of evidence-based medicine
should be applied and incorporated with herd data to initiate a proper treatment plan. Some popular practices for
treatment of clinical mastitis, such as frequent milk-out and oxytocin injection, have limited supporting evidences
and should be recommended with caution. Treating subclinical mastitis is generally not economical during lactation because of high treatment costs and poor efficacy. Because bovine mastitis is the most common reason for the
use of antimicrobials in dairy herds, treatment of bovine mastitis should be supervised by veterinarians.
Introduction
Bovine mastitis is an important animal health disease leading to significant economic losses to the dairy industry
throughout the world (Seegers et al., 2003). Various therapeutic strategies have been developed to control intramammary infection (IMI) and reduce losses due to clinical and subclinical mastitis. Antimicrobials have been one
of the most common treatment plans for therapy of bovine mastitis. Cure rates are highly depended on the causal
pathogen (McDougall et al., 2007). This article focuses on the use of antimicrobials and some other common practices for treatment of bovine mastitis caused by different micro-organisms.
Therapy of mastitis caused by Staphylococcus aureus
Treatment results of Staph. aureus IMI during lactating period is usually disappointing due to its resistance to host
immune response. In addition, the efficacy of therapy of mastitis caused by Staph. aureus can also be varied because of the variation of its antimicrobial resistance, particularly to penicillin G (Olsen et al., 2006).Cure rates for
mastitis caused by penicillin-resistant strains of Staph. aureus are lower than those of mastitis due to penicillinsusceptible strains (Taponen et al., 2003). The reason of this difference is still unclear. It may be due to pharmacologic problems of the antimicrobials, or the association between virulence factors and β-lactamase gene of the
resistant isolates. It is recommended that, before treatment, decision for the therapy of Staph. aureus IMI should be
made according to the resistance to penicillin G using β-lactamase test.
Therapy of mastitis caused by Streptococcus agalactiae
Unlike Staph. aureus mastitis, treatment of Strep. agalactiae mastitis using intramammary infusion products usually results in high cure rates (Tyler, 1992). A general
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recommendation is to use penicillin or its derivatives because Strep. agalactiae isolates are highly susceptible to
penicillin (Messier et al., 1994). Because Strep. agalactiae can hardly survive in the environment, eradication of
Strep. agalactiae to be a Strep. agalactiae-free herd can possibly be achieved. Two treatment options are available
when trying to eliminate Strep. agalactiae from the herd. The first protocol is called “blitz therapy” which is a program of treating all quarters of all cows with a penicillin-based intramammary antimicrobial for 3 consecutive
milkings. Another treatment scheme is a selective treatment based on culture results of all cows in a herd. The difference between the two programs is the cost of discarded milk versus the cost of additional bacteriological cultures. It is generally agreed that treating Strep. agalactiae IMIs is economically beneficial.
Therapy of mastitis caused by Coliform bacteria
Because of a high rate of spontaneous bacteriological cure, treatment of coliform mastitis has mainly focused on
the symptomatic treatment using anti-inflammatory agents such as nonsteroidal antiinflammatory drugs
(McDougall et al., 2009). Several studies have shown that using antimicrobials did not significantly reduce either
the duration of infection or the severity of clinical signs (Pyörälä et al., 1994; Pyörälä and Pyörälä, 1998). However, systemic administration of antimicrobials is still recommended for severe cases to reduce the risk of bacteremia
which consequently improves the reduction of milk production and mortality rate of infected cows (Wenz et al.,
2001). Systemic therapy using antimicrobials in the group of cephalosporins (such as ceftiofur and cefquinome)
(Erskine et al., 2002; Shpigel et al., 1997) and fluoroquinolones (such as enrofloxacin, danofloxacin and marbofloxacin) (Soujala et al., 2010; Poutrel et al., 2008; Schneider et al., 2004) have shown to be more effective in
shortening the duration of clinical signs compared to trimethoprim-sulfonamide (Pyörälä et al., 1994), gentamicin
(Jones and Ward, 1990) and colistin (Pyörälä et al., 1994). Thus, systemic antimicrobial treatment, with or without
symptomatic or supportive therapy, should be recommended for therapy of severe coliform mastitis.
Therapy of mastitis caused by Coagulase-negative Staphylococci (CNS)
Treatment protocols for CNS mastitis are varied depending upon the specific characteristic of the causative species. Even though some cases of persistent IMI of CNS have also been reported, it is generally assumed that the
spontaneous cure rate of CNS mastitis is high (Wilson et al., 1999). Thus treating subclinical and mild clinical
mastitis due to CNS has been usually considered unnecessary. Beta-lactam antimicrobials have probably been
drugs of choice for treatment of CNS (McDougall et al., 2007). Similar to what has shown with Staph. aureus IMI,
the phenomenon of lower cure rates when cows were infected with β-lactamase producing CNS compared to those
of cows infected with β-lactamase negative strains can be observed (Pyörälä and Pyörälä, 1998). Therefore, treatment decision based on β-lactamase test results is also recommended for CNS mastitis.
Therapy of mastitis caused by Streptococcus uberis
Different classes of antimicrobials have been tested for their efficacy against Strep. uberis IMI during lactation.
Even though the antimicrobial susceptibility of streptococci associated with bovine mastitis have demonstrated a
trend of resistance to macrolides and lincosamides (Loch et al., 2005), satisfactory responses of treating Strep.
uberis IMI using a macrolide antimicrobial called tylosin (McDougall et al., 2007) and a lincosaminide antimicrobial called pirlimycin have been reported (Gillespie et al., 2002). In contrast to macrolides and lincosamides, levels
of susceptible to penicillin G by environmental streptococci have remained high (Loch et al., 2005) and consequently related to a high cure rate of Strep. uberis IMI when penicillin and its derivatives were used for treatment
(Taponen
396 
et al., 2003). Furthermore, cephalosporins including cephapirin sodium (Apparao et al., 2009), cefuroxime
(Wraight, 2003), ceftiofur (Oliver et al., 2004) and cefquinome (Milne et al., 2005), have also been applied to treat
Strep. uberis IMI. However, treatment duration using cephalosporins may need to be extended to achieve a high
bacterological cure rate.
Emptying infected mammary glands using frequent milk-out or oxytocin injection
Frequent milk-out (FMO) with and without oxytocin is a popular recommendation for the treatment of clinical
mastitis. The logical reason supporting this recommendation is that FMO can increase the removal of abnormal
secretions, infectious agents, toxins and inflammatory mediators accumulated in milk of an infected quarter. However, evidences supporting this practice are very limited and some even suggested detrimental effects of this practice (Roberson et al., 2004). According to the concept of evidence based medicine, FMO should be recommended
with caution for treatment of clinical mastitis.
In addition to FMO, the use of oxytocin to help emptying the mammary gland has also been recommended for
treatment of clinical mastitis. The cure rates of clinical mastitis when oxytocin is used alone do not different from
the cure rates of mastitis when intramammary infusion of antimicrobials is used alone (Guterbock et al., 1993).
However, when oxytocin injection is combined with intramammary infusion of antimicrobials, bacteriological cure
rate is significantly improved (Hillerton et al., 1999). Thus, it can be inferred that oxytocin should be used in combination with intramammary infusion of antimicrobials in order to achieve a high cure rate of clinical mastitis.
Treatment of Subclinical Mastitis
Treatment of subclinical mastitis during lactation is not commonly recommended due to an economical standpoint.
In the last decade, dairy producers have been encouraged to maintain a low level of bulk milk SCC turning into an
increased profit. The use of effective therapy of subclinical IMI during lactation is therefore essential if it may increase the bacteriological cure, decrease SCC, and effectively complement preventive measures (Swinkels et al.,
2005). Treatment of subclinical mastitis has been focused on IMIs caused by Gram-positive bacteria based on their
higher prevalence compared to those of Gram-negative bacteria. Treatment of subclinical mastitis can be performed using either intramammary treatment or systemic treatment. Intramammary infusion of various antimicrobials, such as amoxicillin (Wilson et al., 1999), pirlimycin hydrochloride (Gillespie et al., 2002), ceftiofur (Oliver
et al., 2004), cefquinome (Kasravi et al., 2011), penethamate hydriodide (Sandgren et al., 2008), have been shown
to be able to successfully cure IMI and reduce SCC of subclinically infected cows. In addition, a number of antimicrobials including penethamate hydriodide (Salat et al., 2008) and peniclillin (Sandgren et al., 2008) have been
reported for systemic treatment to successfully treat subclinical mastitis. When profitable effects of intramammary
treatment were compared to those of systemic treatment, the later may be more preferable based on beneficial responses observed with uninfected quarters.
References
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