2014 Keystone Veterinary Conference Discussions

PADLS-Pennsylvania Animal
Diagnostic Laboratory System
Find PADLS at the 8th Keystone
Veterinary Conference
Date: Thursday, August 14, 2014, 10:00am-3:30pm in Magnolia B, Hershey Lodge, Hershey PA
What can you learn
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10:00-10:15am—Welcome
10:15-10:30am—State of Animal Health in Pennsylvania—Craig Shultz, DVM
10:30-11:00am—PADLS Sample Submission Guidelines—Jenny Fisher, VMD
11:00-11:30am—Rabies in Pennsylvania and PADLS Submissions—Aliza Simeone, VMD
11:30am-12:00pm—Diagnostic Lab to Your Aid for Bovine Disease Diagnosis—Alex Hamberg, VMD, PhD
12:00-12:20pm—Diagnostic Lab and Small Animal Necropsies—Lore Boger, VMD, ACPV
12:20-1:00pm—Lunch Break
1:00-1:30pm—Animal Toxicosis: 5-year PADLS Review—Lisa Murphy, VMD, DABT
1:30-1:50pm—Parasitology PADLS Update—Jason Brooks, VMD, PhD, DACVP
1:50-2:10pm—Providing Veterinary Care for Backyard Poultry Producers—Eva Wallner-Pendleton, DVM, MS, ACPV
2:10-2:30pm—Johne’s and BVD Testing—Deep Tewari, BVSc, PhD, DACVM
2:30-2:50pm—Mastitis and Dairy Milk Quality—Bhushan Jayarao, BVSc, MVSc, PhD, MPH
2:50-3:20pm—Field Investigation in Diagnostics and Panel Discussion—Dave Wolfgang, VMD, MPH, DABVP-Dairy
3:20-3:30pm—Closing Remarks
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Craig Shultz, DVM
Director, Bureau of Animal Health and Diagnostic Services
 Existing Threats
 Foot and Mouth Disease
 New Challenges
 PEDv and PDCoV
 Changing Concepts in Disease Control
 Traditional quarantine and movement controls may not
be the best approach.
February 2014
• Index case diagnosed in
Ohio in May 2013
• 5500 cases in 28 states
nationwide by April 2014
• Would an introduction of
FMD be similar?
June 2014
 A First Line of Defense for Animal and Public Health
Animal Diagnostic
Laboratory
Penn State University
(PADLS-ADL)
Pennsylvania Veterinary
Laboratory
Harrisburg (PADLS-PVL)
New Bolton Center ,
University of Pennsylvania
School of Veterinary Medicine
(PADLS-NBC)
 Maintaining Healthy Livestock Populations
 Controlling Potentially Devastating Animal Disease
 Protecting Pubic Health (“One Health”)
 Diseases with Pandemic Potential (Influenza)
 Zoonotic Diseases
 Foreign Animal Diseases and Potential Impacts on the
Food Supply
 Maintaining Interstate and International Livestock
and Animal Product Commerce
The Lynchpin: Animal Diagnostic Laboratories
Jenny S. Fisher, MS, VMD
Animal Diagnostic Laboratory
The Pennsylvania State University
Recommended Supplies
 Sample Collection
 Insulated cooler with ice
 Sealable plastic bags (Zip-lock or Whirl-pak)
 Permanent marker
 Wide-mouth plastic jars (with and without formalin)
 String/suture
 Needles and syringes
 Red-top (no additive) and purple-top (EDTA)
Vacutainers
 Culture swabs with Amies medium
Basic Information to Include
 Complete History



Signalment
Clinical signs and duration of illness
Treatments administered
 Physical examination/Necropsy findings



PE findings (if still alive)
Condition of animal
Lesions detected (location, distribution, description)
 Photographs when appropriate

These can be invaluable for submissions of field necropsies
and ante mortem diagnostics
Basic Necropsy Procedure
Neurologic Cases
 Submission of entire brain is critical
 Rabies testing (TSEs)
 Removal of the brain
 Likely necessary only if neurologic history
 Three practical options:



Submit entire carcass to diagnostic lab
Remove head at atlanto-occipital junction and submit entire
head to diagnostic lab
Remove brain from head and submit whole brain
Sample Collection
 Basic principles of sampling
 Collect samples as soon as possible post mortem
 Collect cleanly and handle gently, especially for histology

Mucosal disruption, crush artifact
 Keep samples as cold as possible until arrival at lab, but
preferably not frozen


However, if a delay of 2 days or more is expected, frozen is better
than rotten
Keep formalin-fixed tissues at room temperature
 Keep all tissues separate and individually labeled
 Always use leak-proof containers!!!
Sample Collection
 Goals
 Collect necessary samples
 Preserve samples properly
 Package and ship samples properly
 If successful
 Collected appropriate samples of adequate quality to reach a
diagnosis
 Samples arrived safely at laboratory
 If unsuccessful
 Did not collect appropriate samples
 Samples are of poor quality
 Damaged samples or unsafe conditions during transit
Sample Collection
 Fresh tissues
 Solid organs
 Generally submit samples 2-3” in each direction
 Hollow organs
 Either remove and submit luminal content or tie off a loop with
string/suture to avoid contamination
 Fluids
 Collect aseptically into sterile plastic tube or sterile blood collection
tube without additive (red top)
 DO NOT submit in syringes…DO NOT submit with needles
 Swabs /culturettes with transport media
 Swabs in media can’t be tested for viruses
 Viral transport media and RNase inhibiting media can be used
 Use leak-proof containers!!!
Sample Collection
 What to collect and how to collect it
 Formalin-fixed tissues (histopathology)





Solid organs
 Samples should be no more than ¼” thick
 Interface of normal/diseased tissue is ideal
Hollow organs
 Open the organ and remove a 1” square portion of the wall taking
care not to damage the mucosal surface (rubbing, crush artifact)
Use 10:1 ratio formalin to tissue
Tissues from a single animal can be pooled (multiple animals may be
pooled if individual ID is not critical)
Use leak-proof containers!!!
Sample Collection
 What to collect and how to collect it
 Feces



Collect approximately ¼ cup in plastic sample jar or bag
Sample should be as fresh and clean as possible (free from soil,
bedding, etc)
Use leak-proof containers!!!
 Blood or serum
 Serum: Collect in red top (no additive) or marble top (serum
separator) tube
 DO NOT freeze marble top or red top prior to removing serum
 Whole Blood: Purple top (EDTA)
 Feed (nutrient analysis, toxicology/mycotoxins)
 Quart to gallon size Zip-lock bags
 Keep refrigerated
Sample Collection
Sample
Amount/Preparation
Colon content/feces
¼ cup in leak-proof container
Fresh small and large intestine
One loop of each tied closed with string
Fresh liver
One fist-sized piece
Fresh lung
One fist sized piece
Other fresh tissues (spleen, kidney, lymph node,
Separately bagged
rumen, abomasum, etc)
Formalin-fixed tissues (all of the above –
include multiple pieces of intestine)
One piece of each tissue in sealed formalin
container placed in a leak-proof bag
Any other tissues of interest
Fresh and formalin-fixed
Sample Packaging and Shipping
 Packaging
 All samples should be submitted in individually labeled
leak-proof bags, plastic sample jars, or tubes
 Use Zip-lock or Whirl-pak bags for fresh tissues
 Use ONLY plastic screw-top containers for formalin

Following 24-48 hours of fixation, excess formalin may be
removed and samples shipped on minimal formalin
 Double-bag groups of fresh tissues with absorbent
 Tape lids and then bag formalin-containing jars with
absorbent
Sample Packaging and Shipping
 Packaging
 Examples of UNACCEPTABLE containers


Fresh tissues
 Rectal sleeves
 Plastic grocery bags
 Improperly sealed Whirl-Pak bags
 Any bag sealed with twist ties or hand tied knots
Formalin
 Plastic bags of any kind
 Glass jars
 Petri dishes
 Jars with snap-on lids
Sample Packaging and Shipping
 Paperwork
 Thoroughly and legibly complete the proper lab
submission form making certain to:



Provide signalment
Provide clinical history/differential diagnosis
Indicate desired testing
 Place completed submission form in a separate, sealed
plastic bag and enclose with samples
Avoid common mistakes
 Excessively rotten tissues
 Leaking containers
 Lack of key tissues for diagnosis
 Diarrhea problem but no GI samples
 Pneumonia problem but no respiratory samples
 Toxicology: Liver is a great, general tissue (freeze)





Copper: Kidney is best
Vitamin E: Serum
Selenium: Whole blood, serum
Zinc: Royal blue tube (trace metals), rubber stoppers will leach Zn
Vitreous fluid if nitrates or electrolytes (freeze)
Avoid common mistakes
 Samples that are not labeled
 Sample size too small
 Incomplete/absent history
 Incomplete/absent signalment
 No tests requested
Unfortunate Scenario
 Veterinarian euthanizes animal on farm and performs
necropsy
 Client submits complete set of fresh tissues but no
tissues in formalin
 Lab personnel immediately place portions of all tissues
in formalin for histopath
 Result
 Unable to be interpreted due to advanced autolysis
Sample Packaging and Shipping
 Shipping
 Pack samples in leak-proof insulated shipping container
with frozen gel packs and absorbent
 Be sure to protect fragile items such as blood tubes with
a rigid outer container
 Ship overnight delivery via courier (Fed Ex, UPS, DHL,
etc)
 Do not mail via US Postal Service
 Avoid mailing prior to weekends and holidays
 Follow IATA and US DOT regulations for hazardous or
infectious substances
Communicating with the Lab
 When in doubt, call the lab and ask for submission details
 If your submission form is properly completed and samples
are appropriately collected, packaged, and shipped, then
the lab should not need to contact you before beginning
analysis
 Typically allow at least 2 business days for preliminary
report
 In most cases allow 7-10 days for complete analysis and
final report
 Contact lab by phone at any time during business hours
 Accession number or owner’s name
Summary
 Field necropsies require a great amount of time and effort,
but a few extra minutes preserving, packaging, and
submitting samples could make the difference between a
solid diagnosis and a waste of time and money
 Do the necropsy promptly
 Collect sufficient samples and preserve them appropriately
 Thoroughly complete paperwork
 Package samples in individually labeled leak-proof containers
 Ship overnight on sufficient ice
PADLS LABS
 PADLS website http://www.padls.org/
 Penn State University
Pennsylvania Veterinary
 Animal Diagnostic Lab Laboratory
 Wiley Lane
2305 North Cameron St.
New Bolton Center
Veterinary Lab
382 West Street Rd
 University Park, PA
Kennett Square, PA
19348
 16802
 Ph: 814-863-0837
 FAX: 814-865-3907
 [email protected]
 http://adl.psu.edu
Harrisburg, PA
17110
717-787-8808
717-772-3895
[email protected]
610-925-6211
610-925-6822
[email protected]
For ADL Users
 Field necropsy mailers are available for $15 fee
 Includes field necropsy collection guide, sample collection materials, insulated
shipping container, and one-way shipping from ADL to your office
 Sample collection materials only (no shipper or shipping fee) are also available
for $5
 Carcass submission 24/7 walk-in cooler
 No live mammals for necropsy (no live animals after hours)
 Encourage clients to submit early in day
 Newly revised submission forms available on website or
from lab staff (please do not use old forms or state
regulatory forms)
References
 Necropsy
 Dairy Cattle Necropsy Manual. J Severidt, D Madden, G Mason, F
Garry, D Gould. Colorado State University, 2001.
http://www.cvmbs.colostate.edu/ilm/proinfo/necropsy/notes/inde
x.html
 Field Necropsy Techniques and Proper Specimen Submission for
Investigation of Emerging Infectious Diseases of Food Animals. J
Nietfeld. Vet Clin Food Anim, 26:1-13, 2010.
 The Necropsy Book. J King, L Roth-Johnson, D Dodd, M Newson.
CL Davis, DVM Foundation, 2005.
 Shipping Regulations
 International Air Transport Association (IATA).
http://www.iata.org/whatwedo/cargo/dangerous_goods/
 US Dept of Transportation.
http://www.phmsa.dot.gov/hazmat/regs
RABIES
SUBMISSIONS
IN PA
Dr. Aliza Simeone
Region 7 Veterinarian
PDA Bureau of Animal Health and
Diagnostic Services
[email protected]
PA, buried in Rabies!
Where does PA stand nationally?


#1 state for rabid
domestic animals
diagnosed in the U.S.
most years
Due to rabid cat
numbers
Submitting specimens
When there is any question of human or
domestic animal exposure, test!
 Please assist clients with accessing rabies
testing services—even if you don’t handle
the situation yourself, give them the
information they need!
 PLEASE don’t tell clients that your
submission service fees are for “rabies
testing”

Why Test for Rabies?
To prevent human cases of rabies
 To prevent unnecessary PEP for people
 To reduce human anxiety
 To prevent unnecessary euthanasia or
quarantine of potentially exposed domestic
animals

What animals do you submit for
rabies testing?

Mammals suspected of having rabies that
have potentially exposed a person or
domestic animal, e.g.:
 Bat
found in residence
 Raccoon that attacked dog

Mammals which have bitten or otherwise
exposed someone and died within the ten
day observation period under certain
circumstances
What PA labs perform rabies testing?

PVL—Harrisburg (PA Dept. of Agriculture)
 PADLS
rabies lab
 Rabies testing M-F

BOL—Lionville (PA Dept. of Health)
 Rabies
testing M-Sat AM + severe emergencies
on Sunday or holiday (determined by PA DOH)


Philadelphia Dept. of Public Health
 For human exposure cases within Philadelphia
Allegheny County Health Dept.
 Mostly for cases within Allegheny County
What sample is submitted?
MUST BE DEAD!
 PVL—any size specimen

A

brain, a head or even a whole cow
Lionville—only small samples allowed
 Small
animals OVER 3 lbs must be
decapitated and only the head submitted
 Submit brain only from livestock

NBC and ADL will perform that service

If you wish to submit partial brain
specimens, you MUST consult with the lab
FIRST to make sure you collect the
required sections
 Half
a brain will NOT work
Do not put specimens in any liquids or other
substances (e.g. no formalin or saline!)
 Try to avoid freezing specimens
 Keep specimens chilled throughout transit
 Bats should always be submitted whole

How does the specimen get to the
testing lab?






Hand delivery to lab
Overnight shipping
Courier service from vet practice
Hand delivery to certain PDA Regional offices
(by appt. only)
Via other PADLS Labs (ADL or NBC)
Municipality specific facilitated submission e.g.
Montgomery County, Philadelphia
Using the courier to submit from a
vet clinic

Currently the courier under Commonwealth contract is Quick
Courier


Specimens going to PVL:




1-800-355-1004
$15 flat rate (under 60 lbs) charged by PADLS to vet
Use code “Rabies Pickup Account PA-VETE23”
Address package to: PVL, 2305 N Cameron St. Harrisburg, PA 17110
Specimens going to Lionville:



Shipping rate varies—ask Quick Courier for the rate
Clinic must set up a payment method with Quick Courier
Address package to: PA Department of Health, 110 Pickering Way,
Exton, PA 19341

Avoid shipping over a weekend or holiday
 Hold

sample chilled until ready to ship
Packaging

prevent leakage
 maintain the integrity of the sample (ice
packs!!!)
 www.padls.org for further packaging details or
call the lab (717-787-8808)

Want results???
 Include
paperwork in the package (in a plastic
bag) with ALL relevant contact information for
parties who need the results.
Who do you call with rabies questions?

About submitting an animal for testing…
 PDA
(your regional office)www.agriculture.state.pa.us
or
 PA Vet Lab 717-787-8808 or
 PADOH lab in Lionville (610-280-3464)


About human exposure to rabid or potentially
rabid animal… PADOH (1-877-PAHEALTH)/
local health
About animal quarantines related to rabies
exposure…
 PDA

(your regional office)
About wildlife: PGC
RABIES
It’s No Tea Party
Getting The Most From Your
Diagnostic Lab
Alex Hamberg VMD PhD
PADLS
Overview
• Respiratory disease
• Neonatal disease
– Abortion
– Enteric
• Adult disease
– Enteric
Respiratory Investigation
• Clinical samples: $58.50
• Serum: $30 + accession fee
– (BRSV, BVD, IBR, PI3)
• Postmortem samples: $82.50 - 236.00
– e.g. field necropsy
• Autopsy:
– Ruminants over 250#: $150 - 302
– Ruminants under 250#: $82.50 - 236.00
Predisposing factors
• Viruses
• BVDV, PI3, BRSV, IBR
OSU Archive
– Bacteria
• Mycoplasma
– Stressors
•
•
•
•
Sharp changes in temperature
Management
New herd mates, transport
Toxic gases
Mycoplasma bovis
Common Pathogens
•
•
•
•
•
•
•
•
Arcanobacterium
pyogenes
Trueperella
Pasteurella multocida
Mannheimia hemolytica
Salmonella Dublin
Mycoplasma bovis
Histophilus somni
BRSV
IBR (fetal pneumonia)
What to Submit
• Live cattle (clinical samples)
– Nasal swab/secretions or tracheal wash
• PCR for IBR
• Cultures: Aerobic, Mycoplasma*
– Serum
• BRSV SN, BVD SN, IBR SN, PI-3 HI
What to Submit
• Postmortem
– Fresh
• Lung, Trachea
– PCR: BVD, IBR, BRSV
– Culture: Aerobic/Histophilus, Mycoplasma
– VI: PI3
– Formalin fixed
• Lung, Trachea
C.L. Davis
Turn around time
• 7 to 14 days to completion
– Necropsy report: 1 to 3 days
– Histology: 3 to 10 days
– PCR: 2 to 7 days
– Virus isolation: 1 to 3 weeks
– Culture: 3 to 10 days
• Mycoplasma 2 to 4 weeks
– M. bovis, M. dispar  ciliary impairment  predispose
– M. arginini may be synergistic
– Often complicated by Mannheimia hemolytica
Abortion Panels
• Cost
– Fetus panel: $136
• What to submit
– Fresh tissue
• Placenta, Liver, Lung, Spleen,
Abomasal Contents
– Formalin fixed tissue
• Placenta, Lung, Heart, Liver, Spleen, Kidney, Thymus,
Ileum, Tongue, Skeletal Muscle, Brain, Adrenal Gland, +/eyelid and thyroid
– Serum, optional (extra $30/sample)
Specimen Influence
• “The more of the placenta that can be examined, the
more accurate assessment of it is likely to be.”
• “Examination of a fresh fetus is 53 times more likely to
contribute a diagnosis than an autolyzed one.”
• “Submissions including the entire fetus, placenta and
serum are 4.28 times more likely to result in a
diagnosis.”
Maxie, G. Pathology of Domestic Animals, 5th ed, Vol 3, 2007. p. 481.
Points to Ponder
• Lymphoid tissue develops between 42 and 100
days of gestation.
• IgM is present as early as 59 days of gestation.
• IgG is present at about 150 days of gestation.
• Antigenic stimulation can stimulate lymphoid
tissue and Ig production at early stages of
gestation.
Schultz, R; Dunne, H; Heist, C. Ontogeny of the Bovine Immune
Response. Infection and Immunity, June 1973, p. 981-991
What does it mean
• More mature fetuses provide plenty of tissue and
can respond to stress and infections
– Autolytic/macerated fetuses can still help
• Even early fetuses can provide useful
information
– Diagnostic testing may be limited by the size of the
specimen
• Fetal serology is useful after 150 days of
gestation
Abortion panel tests
• Viruses
– BVD, IBR: PCR/IHC
• Leptospira: FA and/or PCR
• Neospora: depending on histologic
findings and history, PCR and/or IHC
• Culture
– Aerobic, Histophilus, Salmonella ± Fungal
• Serology (Dam or fluid from fetus)
– IBR SN, Leptospira, Neospora ELISA
Turn around time
• 7 to 14 days to completion
– Gross report: 1 to 3 days
– Histology: 3 to 10 days
– FA/PCR: 1 to 5 days
– Culture: 3 to 10 days
• Fungal 1 to 4 weeks
Neonatal Enteric Disease Panel
• Calves less than 5 weeks old
– Warranted if still nursing or on milk replacer
Neonatal Enteric Panel
• Clinical samples (feces): $136 +15
• Post-mortem samples: $61 - 215
• Necropsy: $82.50 - 236
What to Submit
• Live calves (clinical samples)
– Feces
• ELISA for Rotavirus, Coronavirus
• FA for Giardia and Cryptosporidium
• Cultures: Aerobic, Clostridium, Salmonella
– E. coli multiplex PCR
» Lt, sta, stx-1, stx-2, eae, K-88, K-99, and F-41
– Clostridium toxin testing/typing
What to Submit
• Post Mortem
– Fresh
• Ileum (ligated), Mesenteric Lymph Node, Spleen,
Liver, Feces and/or Cecal Contents
– ELISA: BVD, Rota, Corona, Giardia, Cryptosporidium
– Culture: Aerobic, Clostridium (ELISA), Salmonella
» Can add liver, spleen, etc. to Salmonella pool
– Formalin fixed
• Forestomachs, Duodenum, Jejunum, Ileum,
Cecum, Colon, Mesenteric Lymph Node, Spleen,
Liver
Lymph nodes
C
i
Educationalmodel.com
Turn around time
• 7 to14 days to completion
– Necropsy report: 1 to 3 days
– Histology: 3 to10 days
– FA: 1 to 3 days
– Culture: 3 to 14 days
– Virus isolation: 7 to 21 weeks
Common Pathogens
•
•
•
•
•
•
E. Coli
Cryptosporidium
Rotavirus
Coronavirus
Salmonella
Clostridium septicum/sordellii/perfringes
type A
• Sarcina sp.
Adult enteric panel
• Clinical Specimens (Feces): $136+15
– Mod. McMaster’s, Clostridium culture/Toxin
ELISA, Salmonella culture, Coronavirus
ELISA ± Mycobacterium paratuberculosis
culture/PCR
• Field necropsy: $61-215
• Necropsy: $150-302
What to Submit
• Live cattle (clinical samples)
– Feces
• ELISA for Coronavirus
• Cultures: Anaerobic, Salmonella
• Mycobacterium paratuberculosis culture/PCR as
appropriate
• Parasitology- Modified McMaster’s fecal floatation
What to Submit
• Necropsy
– Fresh
• Ileum (ligated), Mesenteric Lymph Node, Spleen,
Liver, Feces and/or Cecal Contents
– PCR: BVD
– ELISA: Corona
– Culture: Clostridium, Salmonella ± Mycobacterium
paratuberculosis
» Can add Liver, Spleen, etc. to Salmonella pool
– Fecal centrifugation: Feces or Cecal Contents
What to submit
– Necropsy
• Formalin fixed
– Forestomachs, duodenum, jejunum, ileum, cecum, colon,
mesenteric lymph node, spleen, liver
Specimen collection
C
i
Turn around time
• 7 to14 days to completion
– Necropsy report: 1 to 3 days
– Histology: 3 to 10 days
– PCR/ELISA: 1 to 5 days
– Culture: 3 to 14 days
• Mycobacteria – usually 7 to 42 days
– Parasitology: next business day
Questions?
Companion Animal
Autopsies at PADLS
Lore Boger MS, VMD, DACVP
Veterinary Pathology Services Manager
Pennsylvania Veterinary Laboratory
[email protected]
717-787-8808
71 dog and cat autopsies and field posts
58 dog
13 cat
Common reasons for submissions
 Suspected foul play/trauma/poisoning
 Suspected vaccine/drug reaction
 Intra or post operative/procedural deaths
 Abortion/neonatal mortality
 Sudden death
 Closure
 Work on agricultural species has priority
 Each PADLS laboratory reserves the right to
provide necropsy services for cats and dogs
only when resources allow
 Clients must call ahead for availability
 Dogs and cats are received for necropsy
only through a referring veterinarian
 Non-profit organizations are strongly
encouraged to supply a veterinarian’s name
for case records
 Before the animal is accepted and the
necropsy is performed, the veterinarian or
law enforcement officer must supply a
complete history
 All correspondence pertaining to the case
will be conducted only through the referring
veterinarian or law enforcement officer
 In cases of advanced autolysis, a necropsy
may not be performed
 No cosmetic necropsies will be performed
 Due to biosecurity concerns, remains may
be returned to the owner or referring
veterinarian at the discretion of the
laboratory performing the necropsy
 Discuss with individual laboratory to
determine if return of remains is possible
Necropsy fees:
$437.00 ($422 + $15) in-state cases
$563.60 ($548.60 + $15) out-of-state cases
 includes gross and histological exam
(additional fees for examination of the spinal cord)
Field necropsy fees:
$140.00 ($125 +$15) in-state cases
$177.50 ($162.50 +$15) out-of-state cases
 Charges include a $15 accession fee
 Ancillary testing is additional
 Fees must be submitted prior to necropsy
 Fees are the responsibility of the
owner, veterinarian or non-profit
organization as agreed to by all parties.
 For cases received from non-profit
organizations, the necropsy may be
performed and fees charged at
the discretion of the pathologist
Other services for companion animals:
 Biopsy
 Parasitology
 Toxicology
 Microbiology
For more information:
 www.PADLS.org
 Pennsylvania Veterinary Laboratory
717-787-8808
 Animal Diagnostic Laboratory
(Pennsylvania State University)
814-863-0837
 Veterinary Laboratory at New Bolton Center
(University of Pennsylvania)
610-925-6211
Lisa A. Murphy, VMD, DABT
PADLS New Bolton Center Toxicology Laboratory





ICP-MS (Inductively Coupled Plasma Mass
Spectrometry)
AA (Flame Atomic Absorption Spectrometry)
GC-MS (Gas Chromatography)
LC-MS-MS (Liquid Chromatography)
HPLC (High Performance Liquid
Chromatography)

July 2009-June 2014:
 5396 accessions requesting toxicology testing
 3178 cases (59% of the total) submitted just for
toxicology testing
350
Canine
300
Bovine
250
Goats
200
Equine
150
Camelids
Fish
Avian
100
Pigs
Sheep
50
Other
0
FY 2010
FY 2011
FY 2012
FY 2013
FY 2014
Vitamin E
1200
Lead
1000
Selenium
800
Copper
600
Serum mineral panels
400
Heavy metal panels
200
Complete mineral panels
0
FY 2010 FY 2011 FY 2012 FY 2013 FY2014
Nutritional mineral
panels

Anticoagulant rodenticides = 58
 33% positive





Ethylene glycol = 5
Ionophores = 10
LC/MS = 10
Tranquilizer screens = 4
Mycotoxins = 52 samples tested
 34 samples negative (65%)
 Of the 18 positive samples, only one exceeded FDA advisory
levels
 DON (vomitoxin) detected in 16 (31%)
 One positive for fumonisins
 4 positive for zearalenone
▪ 3 of these 4 also positive for DON
 Organophosphates/carbamates = 21
 Organochlorines/PCBs in birds = 10
 Pentobarbital = 19
▪ 14 associated with euthanized animals
▪ 4 deceased but unknown if euthanized or not
▪ 1 additional serum sample from a live dog
 Cantharidin = 6
 Caffeine = 3
 Acetaminophen = 2
 Other = 4


An adult Golden Retriever
presented with blindness,
abnormal mentation, and
tetraparesis
 Since the clinical signs started
an hour after returning home
from the family’s barn, the
clinician was concerned about
exposure to macrolide
endectocides such as
ivermectin or moxidectin
While the dog’s serum was
negative for these medications, it
instead tested positive for
pentobarbital
 Potential source for this dog
was an improperly disposed of
euthanized carcass

A 2-year old Chihuahua was
received following its death due
to a suspected malicious
poisoning
 In addition to diazepam and
pentobarbital administered
by the veterinary clinic,
oxycodone was detected is
multiple tissue samples and
bodily fluids along with
guaifenesin
 Guaifenesin is sometimes
used in dogs and cats and
more commonly in horses
 Oxycodone (OxyContin) is not
used therapeutically in dogs




A young dog was found
dead in its yard where a
water bowl is kept
When a second, bigger dog
drank from the same bowl
he soon began behaving
abnormally but recovered
When the owners dumped
out and then refilled the
water bowl they noticed
that the water immediately
turned cloudy
The water was submitted
for analysis and the
presence of a large
quantity of cocaine was
detected and subsequently
confirmed

A 4-year old Labrador retriever was
brought to a veterinarian with
generalized muscle tremors, ataxia,
hypersalivation, hyperesthesia, and
hyperthermia
 A tenant admitted poisoning the dog
after an altercation with the owner,
but would not say what had been used
 Luckily the dog was recovering well
with treatment

Submitted gastrointestinal contents
were positive for bifenthrin
 The toxicity of this pyrethroid
insecticide is generally low in
mammals

A bison cow collapsed and
died while donating blood
for her sick calf


The owner butchered the
animal afterwards and
following his veterinarian’s
advice submitted a sample
of ground meat for testing
The sedative xylazine, not
approved for use in foodproducing animals, and a
trace amount of ivermectin
were both detected
 This case was referred to the
Department of Agriculture’s
Bureau of Food Safety and
Laboratory Services in order
for them to contact the
owner to advise him not to
consume or sell the meat
from this animal
Within 24 hours of processing 128 360-pound
steer and bull calves 3 died and 10 developed
CNS signs of incoordination, hyperesthesia,
and the inability to rise without assistance
Approximately 40% of the animals exhibited
rapid respiration, salivation, lack of appetite,
and pressing around the water trough
 At processing the animals had received Express
5-PHM, 20/20 Vision, 4 cc Cydectin (moxidectin)
injectable, 3.5 cc Draxxin (tulathromycin), and
10 cc StandGuard pour-on (gammacyhalothrin)


Livers submitted from 2 of the animals were
negative for the gamma-cyhalothrin, but
toxic levels of moxidectin were detected
 Based on the information provided, it
appears that the animals received the 10%
Cydectin product instead of the 1% version,
resulting in a dose of moxidectin
approximately 2.5 times higher than the
recommendations on the product label






Three starlings were found dead on a property next to
a paintball facility
Birds had been previously observed eating paintballs
Paintball ingredients may vary depending on the
manufacturer, but may contain polyethylene glycol,
glycerol (glycerin), gelatin, sorbitol, dipropylene
glycol, and mineral oil
Dogs that ingest paintballs may develop severe
electrolyte disturbances, particularly increased
sodium levels (hypernatremia)
Serum sodium levels from the two of the dead birds
were markedly elevated


Each year livestock
(cattle, sheep, swine and
horses) develop seizures
and die acutely following
accidental or intentional
exposures to poisonous
substances in their
environment
Intoxications due to
pesticides or other organic
compounds may occur if
animals either have direct
access to them or they are
accidentally mixed with
feeds






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
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
Currently detectable using GC/MS
Anticholinesterase insecticides
Bromethalin
Metaldehyde
Organochlorines
Illicit stimulant drugs
Methylxanthines
Penitrem A
Roquefortine C
Strychnine
Many compounds elude detection by GC/MS due
to their chemical nature

Develop a highly
sensitive and specific
LC/MS screening
method to detect:
 Penitrem A
 Roquefortine C
 Strychnine
 Bromethalin



Improper storage of silage and other
foodstuffs can result in spoilage and
production of tremorgenic (seizure-causing)
mycotoxins
Signs usually appear within 30 minutes as
weakness, tremors, rigidity, hyperactivity
and panting
May quickly progress to seizures and
recumbency
Commonly used by homeowners, farm workers,
certified PCO’s, and animal control personnel for
rodent control
 Still available for over-the-counter purchase in
Pennsylvania
 Causes muscle spasms progressing to tetanic
convulsions within 10-120 minutes
 Death is due to hypoxia
 Detectable using the current PADLS GC/MS
screen, however many factors have made its
consistent identification difficult

January 2009
Dog found
dead
 Owner
suspects
neighbor has
killed multiple
dogs


Introduced as a
rodenticide in the
1980’s
 Causes seizures due to
the uncoupling of
oxidative
phosphorylation
 Cerebral edema and
spongy degeneration
of most CNS white
matter tracts occurs




Enhance the ability to
quickly detect toxic
substances capable of
causing seizures and sudden
death in animals
Provide accurate diagnoses
that will prevent additional
animal losses
Block the entry of
contaminated products into
the food chain that could
threaten both human and
animal health
One gram of feed was spiked at 0, 0.010, 0.100 and 1
ppm with a solution containing the 3 convulsants and
extracted with 4 mL of acetonitrile with 1% acetic
acid, vortexed for 15 seconds, shaken for 15 minutes,
and centrifuged for 5 minutes at 1400 RPM
 1 mL of extract was cleaned using the QuEChERS
method with PSA and C18 absorbents
 Supernatant was diluted 1:5 with acetonitrile with 1%
acetic acid and filtered with a 0.22 micron filter
 The method was validated using 3 different feeds

 Cat food, dog food and corn meal
 Each concentration was repeated in triplicate
Strychnine
Roquefortine C
Penitrem A

Both Real Kill (United Industries Corp., St.
Louis, MO) bait and dog food spiked at 2.5
ppm bromethalin were extracted and
prepared using the QuEChERS method



After multiple attempts with different LC/MS
modifications studies with spiked samples were
discontinued due to the instability of the
bromethalin standard and the difficulty in
obtaining it from the manufacturer
Our proposed alternative strategy is to use shelfstable commercial bromethalin bait products
paired with the analytical standard for
desmethyl bromethalin
Additional studies to better enable both
extraction and detection of bromethalin are
ongoing



Available since January 2014
Includes penitrem, roquefortine, and
strychnine
Appropriate sample types:
 Vomitus, GI contents, suspect bait material
 Liver for strychnine


Same price as other LC/MS screens = $72
Please let the laboratory know if you are
specifically concerned about bromethalin
Parasitology in PADLS
Jason W. Brooks, VMD, PhD, DACVP
Animal Diagnostic Laboratory
The Pennsylvania State University
Parasitology Tests
• Direct smear
• McMaster flotation
• ELISA (replacement for acid-fast/DFA)
– Cryptosporidium
– Giardia
• DFA
– Cryptosporidium and Giardia
• Double Centrifugation (replacement for Baermann)
McMaster Flotation
•
•
•
•
•
•
Quantitative flotation
Zinc sulfate
Sensitivity 100 eggs/gram
Most nematode eggs and coccidia
Will not float most trematode or cestode eggs
Will not reliably detect Cryptosporidium or
Giardia or larval nematodes
• Cost: $15
• Sample: 1 gram (4-5 pellets) fresh feces
• TAT: Same day
Clinical Use of the McMaster
Flotation
• Assess effectiveness of anthelminthic
drugs by FERT: fecal flotation for
nematodes on day 0 and 10-14 days later
– Expect 95% reduction in egg count
• Assess effectiveness of anti-coccidial
drugs by fecal flotation for coccidia on
day 0 and 7-10 days later
Centrifugation
• McMaster flotation may be combined with
centrifugation
• Centrifugation step following standard
McMaster flotation allows for greater
sensitivity
• Semi-quantitative
• Results reported as “<100 eggs/gram”
• Cost: $25
• Sample: Same as McMaster
• TAT: Same day
ELISA
• Commercial immunoassays for detection
of Cryptosporidium or Giardia in feces
• Verified for use in all non-human species
• Offers increased sensitivity and ease of
interpretation, and decreased technician
time compared to acid-fast staining
• Cost: $9
• Sample: 0.1 gram fresh or preserved feces
• TAT: 2-3 days (Tues and Fri)
DFA
• Commercial direct fluorescent antibody
assay for detection of Cryptosporidium
and Giardia in feces
• Cost: $15
• Sample: 1 gram fresh feces
• TAT: 2-3 days
Double Centrifugation
• Centrifugal fecal analysis for nematode
(lungworm) larvae
• Offers increased sensitivity and more
rapid TAT than Baermann
• Cost: $15
• Sample: 1-10 grams fresh or preserved
feces
• TAT: Same day
Parasitology Tests
• McMaster flotation +/- centrifugation
• ELISA
– Cryptosporidium
– Giardia
• DFA
– Cryptosporidium and Giardia
• Modified Double Centrifugation
Eva Wallner-Pendleton DVM MS DACPV
Animal Diagnostic Laboratory,
Pennsylvania State University, State
College, PA ([email protected])
Numerous calls to the diagnostic lab regarding health concerns
and basic needs.
 http://extension.psu.edu/animals/poultry/topics/urban-andsmall-poultry-production.


http://www2.ca.uky.edu/smallflocks/Management-chickenseggs.html

Small flocks kept for multiple reasons:





Produce eggs or meat for the family
Direct retail or farmer’s markets
Specialty retail: organic, pastured poultry
4-H project for children, hobby flocks for show and breeding
Alternative pets, personal enjoyment




Client education a must!!!
Do not behave like other
pets!
Clients rarely realize how
sick their bird actually is.
Encourage early
investigation of symptoms.
Assume possible systemic
illness that could spread to
entire flock and handle
accordingly.
Poultry are really
pretty hardy
creatures.
 The same 1o-20
illnesses tend to
reoccur and are easy
to learn.






Poor or incorrect diet
 Obesity, poor feathers, poor egg production, metabolic
bone disease
Social aggression (environment, diet, bird type)
Predation
 Birds extremely susceptible to P. multocida. septicemia,
all bites must be treated as emergency!
Miscellaneous hazards
 rodent bait, frost bite, dehydration in winter
Reproductive abnormalities:
 Egg binding, oviduct obstruction, tumors of the
reproductive tract, prolapse






Marek’s Disease
 Chicken herpes virus induced polyneuritis, lymphoid
neoplasm
Bumble foot
 Staph aureus infection of the foot pads.
Ascites syndrome
Intestinal coccidiosis
External and internal parasites
 Mites and lice, tapeworms, ascarids,
Respiratory Complex
 Infectious Laryngotracheitis, Infectious Bronchitis,
Mycoplasmosis
 Obtain stock from National Poultry Improvement Plan
participating breeders and hatcheries if possible.
▪ Salmonellosis outbreaks from small hatcheries!!
 Keep new birds isolated for 2-3 weeks
▪ Blood test screening for diseases
▪ Parasite check (internal and external parasites), especially if
bought at show or auction!
 Alert client to watch for any abnormalities, no matter
how slight.
▪ Birds hide their symptoms! Weight loss (or gain) difficult to
assess w/o handling!
▪ Access need for vaccinations.
▪ Will this be a closed flock or will client be taking birds to fairs,
farm show, 4-H??

Diets
 Many types of poultry rations available, meet
specific needs of poultry type and purpose:
▪ Egg-type chicken diets:
▪ starter (1-3 weeks),
▪ grower (3-16 weeks),
▪ Layer/breeder (first egg to molt)
▪ Meat-type chicken:
▪ starter (1-2 weeks),
▪ grower (2-5 weeks)
▪ finisher (last two weeks)
▪ Turkey and game bird diets:
▪ Similar to other diets, but protein content higher.
▪ Waterfowl:
http://www.healthybirds.umd.edu/files/raising%20waterfowl.
pdf
▪ Pigeon feeds: A mix of whole grains (milo, barley, wheat)
pellets, legumes such a peas (http://www.pigeonweb.net/).

Feeding Starter too long:
 Excess protein, too nutrient-dense, excessive growth rate, leg problems,
ascites in broilers.

Birds of any type given too much treats (bread, corn)
 Obesity, fatty liver, heat-stress, respiratory failure
Chicks or broilers fed layer rations:
 High calcium, vitamin D content: gout, renal failure
 Layers fed starter, grower, developer diets:
 Birds develop osteoporosis, poor egg shells
 Birds fed “poultry scratch” only: obesity, feather picking, poor
egg production; scratch is grain mix mostly, cannibalism, cloacal
prolapse, dystocia
 Egg type pullets fed meat bird rations

 Poor body frame, obesity, poor feathering, cannibalism

Provide adequate floor, feeder and
drinker space, nesting space, and places
to hide or get away (perches).

Provide a good quality feed, can add
kitchen/garden scrapes or grazing.

Many organic diets deficient in
methionine, increased feather picking
and aggression.

Reduce number of roosters (11:1)

If bird is injured remove immediately!!

Add more roughage (oats), good quality
alfalfa


Hay bales for getting away, perches
Beak trim if all else fails.
Often clients don’t look at
vent area and realize how
much trauma and blood loss
has occurred.
Vent gleet????
These animals must be
removed from the others for
treatment and healing.
Beak trimming may be
needed.
Be careful when adding new
birds to the flock.





Ascending infection 2°
to trauma or poor
nesting area sanitation
2° to systemic bacterial
infection, airsacculitis,
peritonitis.
E. coli, salmonella often
isolated.
Radiographs, CBC may
be helpful.
Hysterectomy









Lower oviduct egg-binding
usually visible, penguin-stance
Higher up: May need to
radiograph or ultrasound.
Early intervention very important:
Egg mass puts pressure on pelvic
organs (kidneys, ureters,
obstruction)
Immerse bird’s abdominal area in
warm water.
Lubricate area with K-Y, careful
digital pressure
Addition of calcium gluconate,
prostaglandins.
Ovocentesis /can be risky!!
There may be ectopic eggs, there
might be salpingitis, other
abdominal masses




Most common tumor of
older laying hens
Birds progressively get
thinner
Distended coelomic
cavity filled with clear,
straw-colored fluid
Trans-coelomic
implants throughout
the mesentery and
intestinal wall






Respiratory distress due
to shallow breathing,
poor heat tolerance
Fractured livers,
sudden death from
hemorrhaging
possible!!
Excessive caloric intake
Inadequate quantity or
quality protein
Mycotoxins or some
other liver anomaly
High egg production.
-Related to very rapid growth, skeletal muscle growth much faster,
outpaces heart and lung development and size.
-Seen in fast-growing broilers, cold early spring weather.
Birds fed 24/7, lights on 24/7
-After about three weeks of age, birds become cyanotic. Disease is not
reversible at this point!!
-May lose up to 50 % of flock, owner concerned are birds safe to eat? Not
an infectious condition but certainly not appetizing.
Solution:
Provide birds with at least 6 hours darkness starting ~ 3rd day.
Reduce feed intake, allow to clean up feeder.
Use dual-purpose strain birds that naturally grow slower.



Caused by breaks in skin
or secondary to
bacterial septicemia
Must differentiate from
gout with clinical
chemistry or cytology
http://www.harrisonsbirdfoods.com/avmed/a
mpa/10.pdf






Poorly designed
perches
Concrete flooring
Rocky terrain.
Manure build-up in
coop, dirty, wet slats
Sharp objects in
bedding
Early treatment more
successful.






Respiratory viruses
Mycoplasma
gallisepticum
E. coli
Hemophilus
paragallinarum
(Coryza)
Pasteurella multocida
(Cholera)
Aspergillosis
 Blood samples, choanal,
tracheal or sinus swab in
VI media or dry swab for
PCR or submit dead
and/or sick birds
(preferred)
Herpes virus, carrier state exists.
Disease characterized by high
mortality, slow spread, hemorrhagic
or caseous tracheitis.
 Outbreaks after shows, exhibitions.
 Necropsy, histopath, virus isolation
to diagnose.
 Treatment:


 Vaccinate in face of outbreak!! Yearly
booster, at least one month before a
show.
 Supportive care for secondary infections

http://www.jefferspet.com/vaccines/camid/LIV/cc
/3501/c2c/ln

http://www.firststatevetsupply.com/store2/
Classic gross and histopath
lesions
Vaccination: Cell-culture
origin vaccine preferred,
conjunctival drop. All birds
in flock must be immunized.
Use vaccine up in 2 hours or
less.





Most common cause
of “colds” in chickens.
Birds are coughing,
egg production drops
Birds will often
develop secondary E.
coli infections
Virus isolation, paired
serology used to
diagnose.
Vaccines are available

http://www.jefferspet
.com/vaccines/camid/
LIV/cc/3501/c2c/ln

http://www.firststatev
etsupply.com/store2/v
accines/killedvaccines.html
COUGHING, SNEEZING, CATARRHAL
TRACHEITIS, SPREADS RAPIDLY!
WRINKLED EGGS, THIN SHELLS OR
NO SHELLS, LOSS OF PIGMENT



Cause respiratory illness
(sinusitis, tracheitis,
airsacculitis), reduce egg
production, are spread
horizontally AND
vertically.
Chickens often subclinical
carriers. Turkeys always
get sick.
Broilers: Develop CRD
 Chronic respiratory disease

Infected birds not suitable
for breeding stock!
1. Once infected, always infected.
2. No amount of drugs will completely eliminate
mycoplasma infections, remission of symptoms
only.
 3. Mycoplasma infected breeders always a risk for
producing mycoplasma-infected offspring.
 4. Economic impact: reduced eggs, reduced
livability in offspring, CRD, increased carcass
downgrades, increased susceptibility to any other
bacterial or viral respiratory disease or vaccine.
 Large percentage of backyard chickens are
infected!



Prevention:





Buy only from NPIP MG-Clean breeders and hatcheries.
Pre-purchase exam: screen for mycoplasma
Practice biosecurity, closed flock.
Vaccines available, prevent economic loss.
Treatment
 If diagnosed on farm, antibacterial treatments include:




tylan, tetracyclines, Aivlosin, Erythromycin.
Meat animals: Best to butcher ASAP.
Egg-layers: Careful about drug residues!!!
Strictly pets: Other medication options available.
Chronic sinusitis: Exudate becomes hard and inspisated:
may need surgical removal.

A herpes virus






Causes inflammatory
and lymphoid
neoplastic lesions in
the central and
peripheral nervous
system.
Visceral and ocular
neoplasia
Lifelong carrier state
Vaccine must be
given at hatch ideally.
Affected birds rarely
recover.
Request hatchery to
vaccinate or purchase
vaccine





Fecal parasitology
suggested on every
poultry disease workup.
Medicated feed:
1 lb./ton amprolium,
often not enough!
Sulfa meds effective,
but cannot be used in
layers, or organic birds!
Coccidiosis vaccines
available.


A big issue with small flocks but fortunately
most show minimal pathogenicity
Guideline:
 Worm monthly.
 Most pathogenic:
 Gapeworm (syngamus trachea), severe
respiratory signs.
 Cappillaria (threadworm, causes significant
weight loss)
Outstretched necks, labored breathing, ruffled
feathers, weight loss
Attach to the lining of the trachea and feed on blood


Syngamus egg
Ascarid Eggs
Cappilaria egg


Intestinal blockage
Enteritis due to heavy
larval migration
 Can be seen on
scrapings

Liver spots: Can be a
problem in processed
birds.
A problem in turkeys
primarily, but has
been observed in floor
layers, esp. organic
birds and broiler
breeders, pea fowl,
guineas, gamebirds.
 Can be introduced by
cecal worms infected
with the protozoa.

Bird consumes
infected cecal worm
egg or earthworms
transporting cecal
worm eggs.
 Direct transmission
can occur as well
(absorbing
contaminated feces
through cloacal
contact).

 If diagnosed HISTOSTAT 50 medicated premix
(0.01875% nitarsone) can be prescribed in nonorganic birds. This organic arsenical is still legal
in the US, not in the EU.
 Worming regularly.
 Organic birds: Oregano, diatomaceous earth,
separating area where infected birds first
discovered.
 Metronidazole strictly prohibited in the US in
poultry.



Piperazine
Diatomaceous Earth???
Hygromycin B (Rooster Booster)
 http://www.jefferspet.com/rooster-booster-
multi-wormer/camid/liv/cp/16800/
 Can be used in laying hens, multiple species.

Fenbendazole
 Approved for turkeys in feed.

Mites and Lice:
 Feather mites (blood





suckers)
Northern Fowl Mite
Red Mite
Must apply
treatment to birds
and environment at
least 3X 1-2 weeks
apart.
Scaly leg mites
Be careful with
residue issues in
eggs, meat

Yellow Jacket wettable
sulfur

 Feed at 3-4 lbs/ton for 3
 For placing in the boxes,
weeks
 Also reduces ammonia
production in a house
litter, can be used to dust
birds


Carbaryl (Sevin)
Diatomaceous earth

 Can be placed in litter,
Spinosad insecticide
 Can be sprayed on birds
with mites
Tetrachlorvinphos
(Rabon)
 organophosphate.
 Can be sprayed on
nest boxes, scratch boxes

Poultry Shield FG
birds.

Approved pyrethrins

$40.00 for complete testing (highly
subsidized for in-state cases)
 Necropsy, bacteriology, histopathology, virus
isolation, toxicology, serology, PCR tests
 http://www.padls.org/
 4 Board-certified avian pathologists available at
the Penn State (814-863-0837) and New Bolton
Center (610-444-5800) Laboratories










http://www.jefferspet.com/products/triple-actionmulti-wormer
http://www.randallburkey.com/Wormers/products/42/
http://www.firststatevetsupply.com/store2/vaccines/liv
e-vaccines.html
http://www.elanco.com/pdfs/electorpsp-new-claimpress-release-_04-29-11.pdf
http://www.jefferspet.com/categories/poultry/healthwellness/poultry-vaccines

http://vbs.psu.edu/adl
 Submission forms
 Avian Submission forms
 Avian Necropsy forms

http://www.padls.org/

http://www.vet.upenn.edu/veterinaryhospitals/NBC-hospital/diagnosticlaboratories/new-bolton-center-padls/padlslaboratory/avian-pathology-laboratory
BVD and Johne’s disease
Deepanker Tewari
BVSc PhD DACVM
PADLS Keystone Vet Conf 2014
BVD –Bovine viral diarrhea
-Acute disease
-Persistent infection
-Mucosal disease (mix of Cytopath and Non-Cytopath)
BVD infection
Dam Not
always BVD
PI
BVD infection-PI
BVD –Bovine viral diarrhea
Tests we offer
•
•
•
•
•
•
•
Antigen ELISA (ACE)
Immunohistochemistry
Pool/Indv. serum and ear notch PCR ($1-$2/animal)
Bulk tank milk PCR- 50 ml milk sample
Microplate assay
SN test (only Type 1)- Types 1a, 1b and II
Tissue PCR
Sampling issues
∗ Rinsing the ear-notcher between animals properly
∗ Careful vaccine use or make sure sample is not
contaminated with vaccine
∗ Making sure ear is cleaned
∗ Collection Guidance available from lab (red top tube)
Sampling issues
Serum based assays (antigen ELISA, microplate assay) not
recommended for animals 3 months and younger
Maternal antibody complexing with antigen and raising
the possibility of false negatives.
Missed PI
BVD and milk testing option
PCR BVD milk/tissue-$35
PCR Milk submission Requirement
1) 50 ml shipped cold
2) 200 animals in a string/bulk tank
3) Vaccination with modified vaccine wait 3 wks
4) ELISA can’t be used on bulk tank milk
PI animals
• Poor doers
• Like Typhoid Mary
• Can be Hard to
recognize
JOHNE’S DISEASE:
PADLS Testing options
CLINICAL SIGNS
Johne’s is a Chronic,
Debilitating disease of
Cows. It results in :
•1) Diarrhea
•2) Emaciation
•3) Weight loss
Diagnosis of Disease in the Laboratory
•Enzyme linked immunosorbent assay (ELISA)
(Serum and milk)
The interpretation is valid if herd prevalence is above 0
IDEXX ELISA TEST (old):
S/P ratio >1.2
High
+
>0.6-1.19
Moderate +
>0.25-0.59
Low
+
<0.15-0.24
(Neg) Least
IDEXX ELISA TEST: (current- serum)
S/P ratio >1.0
High
+
>0.7-0.9
Moderate +
>0.6-0.7 Low
+
<0.6
Neg
Organism based tests
Liquid culture
Culture individual animal ($17) - 7wks
Discounts for program participation
Machine flags samples positive based on Pressure changes in head space.
Acid fast Staining and PCR confirmation
Organism based tests
∗ PCR pools (5)
culture individual in a pos pool, results
in a wk for negative ($20).
∗ Pos addl cost and time.
∗ PCR individual ($20)- results in a week
Pooled testing
∗ Feces are pooled in lab (5 per pool)
∗ PCR testing is performed on pools (Proficiency tests)
∗ Pos pools are tested by Liquid culture
∗ Do All pos pools turn up culture positive?
Indv Culture and PCR Pools
∗ 100% correlation for High and Medium pos on PCR
∗ 60% correlation for Low pos (Ct 35-42)
∗ Pass through, dead organisms, test sensitivity issues
∗ Culture high (<25 days)
∗ moderate (25-35 days)
∗ low shedders (35-42 days)
PCR High <30 Ct
PCR med 30-35 Ct
PCR low >35-42 Ct
Best testing approaches
Herd monitoring involves rotational testing
-Milk/Serum ELISA-follow up culture/PCR
-Fecal pooling/Indv culture
∗ Environmental monitoring in herds of unknown status
Acknowledgments
PVL: Anna Martin Hays, Corey Zellers, Donna Krouse, Eric Hue
Janelle Hippensteele, Willard Feria,
Receiving and Billing team
PSU and NBC team
PADLS-Penn State
Department of Veterinary & Biomedical Sciences
The Pennsylvania State University
Antimicrobial Susceptibility of Mastitis Pathogens
Isolated from Milk Samples in Pennsylvania from
2004-2011
Bhushan Jayarao, Subhashinie Kariyawasam, Valerie Lintner, Traci Pierre, Tammy Mathews
Introduction
• A study was conducted to determine whether
antimicrobial susceptibility patterns of major
mastitis pathogens isolated from milk samples
of dairy cows have changed over time (20042011).
• 1714 bacterial isolates (Escherichia coli,
Klebsiella species, Serratia marcescens,
Staphylococcus aureus subspecies aureus,
coagulase-negative Staph. species,
Streptococcus dysgalactiae subspecies
dysgalactiae, Strep. agalactiae and Strep.
Uberis) obtained from quarter milk samples
submitted to the Penn State University’s Animal
Diagnostic Laboratory for diagnostic
bacteriologic testing
Approach
•
Antimicrobial susceptibility testing was performed with the
Sensititre® automated system using the mastitis plate format.
•
Antibacterial agents
–
–
–
–
–
–
–
–
–
–
•
Penicillin
Ampicillin
Cephalothin
Ceftiofur
Penicillin + novobiocin
Erythromycin
Oxacillin+2% NaCl
Pirlimycin
Tetracycline
Sulfadimethoxine
Trend analysis and linear regression analysis were performed
to determine whether the percentage of isolates that were
resistant was associated with year and if the resistance or
susceptibility to a given antibiotic was observed over a eightyear period.
Gram positive cocci
Ampicillin
Change p value
No change
Increased
No change
No change
Staph. warneri
No change
S. haemolyticus
No change
Staph. xylosus
Strep. dysgalactiae No change
Strep. agalactiae No change
No change
Strep. uberis
Staph. aureus
CNS
Staph. hyicus
0.13
0.017
0.193
0.427
0.59
0.371
0.749
0.66
0.997
Penicillin
Change
Staph. aureus
CNS
Staph. hyicus
Staph. warneri
S. haemolyticus
S. xylosus
Strep. dysgalactiae
Strep. agalactiae
Strep. uberis
p value
No change 0.17
No change 0.35
No change 0.179
No change 0.427
No change 0.654
No change 0.725
No change 0.934
No change 0.659
No change 0.86
Ceftiofur
Change
p value
No change
No change
No change
NA
No change
No change
NA
NA
No change
0.641
0.774
0.197
NA
0.286
0.554
NA
NA
0.669
Penicillin/
Novobiocin
Cephalothin
Change
p value
No change
No change
No change
NA
No change
No change
NA
NA
No change
0.893
0.774
0.133
NA
0.286
0.554
NA
NA
0.392
Pirlimycin
Erythromycin
Change
p value
No change
No change
No change
No change
NA
No change
No change
No change
No change
0.792
0.895
0.776
0.322
NA
0.67
0.317
0.479
0.222
Sulphadimethoxine
Oxacillin + 2% NaCl
Change
p value
No change
NA
NA
NA
NA
NA
No change
NA
No change
0.559
NA
NA
NA
NA
NA
0.554
NA
0.35
Tetracycline
Change
p value
Change
p value
Change
p value
Change
p value
No change
No change
No change
No change
NA
NA
No change
NA
No change
0.559
0.726
0.559
0.554
NA
NA
0.171
NA
0.584
No change
No change
No change
No change
No change
No change
No change
No change
No change
0.745
0.076
0.315
0.491
0.281
0.054
0.853
0.444
0.978
Increased
No change
Decreased
No change
No change
No change
No change
NA
No change
0.029
0.291
0.042
0.794
0.25
0.327
0.227
NA
0.959
Decreased
No change
Decreased
No change
No change
No change
Increased
Increased
No change
0.007
0.523
0.035
0.967
0.355
0.843
0.024
0.001
0.287
Gram negative rods
Ampicillin
Change p value
Ceftiofur
Change p value
Cephalothin
Change
p value
Sulphadimethoxine
Change
p value
Tetracycline
Change
p value
E. coli
No change 0.175 No change 0.426 No change
K. pneumoniae No change 0.133 No change 0.261 No change
0.078
0.449
No change
Increased
0.219
0.029
No change
No change
0.822
0.746
K. oxytoca
No change 0.133 No change 0.309 No change
NA
S. marcescens No change 0.723 No change 0.652
0.185
NA
Increased
Increased
0.002
0.006
No change
No change
0.937
0.846
Results and Implications
• Overall, over a eight-year period there was no change in
the susceptibility to most of the antimicrobials tested--– with the following exceptions:
• CNS overtime have become more susceptible to ampicillin,
• Staph. aureus and Staph. hyicus have become more susceptible to
sulphadimethoxine and tetracycline,
• K. pneumoniae and S. marcescens have become more susceptible
to sulphadimethoxine
• Strep. dysgalactiae and Strep. agalactiae showed more susceptible
to tetracycline.
• In summary, these results suggest that mastitis pathogens have not
developed resistance to antimicrobials approved in the United
States for dry cow treatment and mastitis over the study period.
Suggestions
•
•
•
•
Mixed cultures - care during sampling.
Sample transportation - enough ice in the box.
If you need explanation of results- call…
Before you sample, what to consult? call- 814-8635939
– leave a number to call or send an email to [email protected]
FIELD INVESTIGATIONS
HOW TO ENSURE A REASONABLE EVALUATION OF FIELD
BASED PROBLEMS
PADLS AT 8TH KVC, HERSHEY, PA 08/14/2014
Patricia Dunn, DVM, MS
Eva Wallner Pendleton, DVM, MS
Robert Van Saun, DVM, MS, PhD
Ernest Hovingh, DMV, PhD
David Wolfgang, VMD, MPH
Department of Veterinary and Biomedical Sciences, Penn State University
New Bolton FI Team
• Swine
–
–
–
–
–
• Dairy
– Linda Baker, VMD, MS
Gary Althouse, DVM, PhD
– James Ferguson, VMD, MS
Thomas Parsons, VMD, PhD
– David Galligan, VMD, MBA
Ines Rodriguez, VMD, MS
Meghann Pierdon, VMD
• Avian
Seth Dunipace, VMD
– Sherrill Davison, VMD, MBA
– Donna Kelley, DVM, MS
How Problems are Identified
• Farmer or ag specialist identifies an issue in
production or health
• Local ag professionals seek assistance or ask
for help solving problem
• Diagnosticians identify a complicated problem
from samples already submitted
• Economics or situation dictate a more holistic
approach
Integrate what
is at farm level
with Diagnostic
Laboratory
Integration
•
•
•
•
•
Systems-based approach
Cannot solve complicated problems alone
Team concept
Visualize the big picture!
Appreciation for other areas of herd health
picture
– Economics, Environment, Pathogen/toxin and
Management
Conduit for Diagnostic Samples
• Herd samples vs. individual samples
• Appropriate samples
– Type and number of samples
– Delicate tissues
– Subclinical or incubation stage vs. chronically
infected
– Pooled or individual samples
– Confounding situations
• Strategies to sample herds vs. individuals
What do we do?’
• Visit farms to investigate animal disease /
production / welfare issues
• As a result of:
– request by the herd or regional vet
– request by the farm owner/manager
– request by a consultant or herd advisor
– samples submitted to the lab
What do we do?
• Investigate:
– Chronic, persistent, problems
– Complicated, multifactorial problems
– Acute and/or dramatic increase in disease/death
– Problems involving multiple herds
– Unusual clinical signs/symptoms
– Possible new/emerging/zoonotic disease
– Common disease problem, but looking for another
opinion
The process
• Contact herd owner/manager & veterinarian
• Gather existing data/test results/reports, etc.
• Make preliminary analysis/assessment
– Infectious vs. non-infectious?
– Chronic vs acute disease?
– Emergency vs routine response?
• Decide upon appropriate course of action:
– Collect more samples/data prior to visit?
– Make visit as soon as possible?
The visit
• Usually invite herd vet or other pertinent
individuals
• Take one or more of our technicians to farm
• Often bring back diagnostic samples with us
– Feces, blood, milk, feed, water, etc…
– Who do we want to test? Why?
The visit
• Usually try to examine the whole farm/
management system, if possible
– Assess risk of introducing and spreading disease
• Observe appropriate biosecurity protocols
– Proper clothing, disinfection, etc.
– Evaluate ‘risk’ (susceptibility) level of animal
groups/ages
“Disease”
• Non-infectious disease:
– Nutritional / metabolic, traumatic,
neoplasia, poisoning, low production
• Infectious disease: “Invasion & multiplication
of infective agents / microorganisms in body
tissues”
– Bacteria, viruses, fungi,
mycoplasmas, parasites, etc...
Outbreaks
• When to be concerned •
– Higher numbers
– Drop in production
– Rapid spread
– Unusual ages
– Mixed species
– Unusual clinical signs
– Zoonotic spread
Factors
– New animals
– Poor animal health
– Intensive production
– Stressful environment
– Poor animal sanitation
– Poor nutrition
– Overlap with wildlife
“States” of infectious disease
UNEXPOSED
EXPOSED to infectious agent
UNINFECTED
INFECTED
Subclinical
Chronic, subclinical (or
clinical) infection
Mild
Clinical
Recovered / uninfected
Severe
Clinical
Dead
What to sample and when? Strategy?
•
•
•
•
•
•
•
•
•
•
Tissue
Secretions
Blood
Antibodies
Pathogen
Feed
Water
Environment
Acute case
Chronic case
Collecting & testing samples
• Want:
– the correct sample…
– a high quality sample…
– from the correct animal(s)…
• In order to properly determine the infection
status of an animal
– Know how to interpret the results!
• What can…and can’t…the test tell you?
• Cost efficient while maintaining sensitivity
Pooled Serum with PCR
• Clean whole blood sample (similar strategy –mycoplasma)
• Take early in week, keep cool, send over night
• Lab will spin and pool serum (need accurate quantities)
– In theory can pool up to 400
– Most cost efficient pool of 30 (bias towards + pool)
• Run PCR on pool cost ~ $60
• + pools checked with microplate PI test-$5/animal
1000 animals
1000 animals
PI test X $5/animal =
$5000
33 pools X $60 = $1980
$2430
3 + pools 90 X $5 = $450
The follow-up
• Attempt to respond in a timely manner – esp.
when urgent/acute problem!
• Integrate our observations, pre-existing test
results, data, and results from our samples, to
make recommendations
– Further diagnostics by us or others?
– Intervention/treatment/management of problem?
• Include vet and other appropriate people
Assessment of biosecurity risk posed by visitors
Low Risk
Moderate Risk
High Risk
One farm per day with minimal
animal contact
Visits more than one farm
per day, some animal
contact
Visits many farms or
auctions with animal
contact
Same clothing not worn to
different farms without
laundering or sanitation
Unsoiled coveralls & boots.
Boots cleaned between
sites.
Clothing not cleaned or
sanitized between sites.
Dropping off materials
or supplies
.
Materials or supplies are left in
areas without animal or feed
contact
Materials or supplies may be
left in area with some animal
or feed contact
Materials or supplies are
left in animal or feed
contact areas
Contact w/ potentially
infected animals
Contact with healthy animals
but no contact with infected
animals
Contact with healthy animals
and limited contact with
infected animals
Works with highly infected
or exposed animal groups
Work in animal or feed
contact areas
Does not work in animal
or feed contact areas
Minimal exposure and when
in contact areas wears clean
protective clothing
Works in animal or feed
contact areas without clean
equipment or clothing
Does not travel out of the US
or Canada
Limited travel outside of US
or Canada without animal
contact
Travel to foreign countries
with animal contact in those
countries
Understands and promotes
Not an advocate
Little appreciation
Number of farms
visited per day
Clothing
Foreign Travel
.
.
.
Biosecurity Knowledge
Flow within a livestock setting
Susceptibility of Animals
•Newborns
•Nursery
•Maternity
•Weaning aged
•Heavy production
•Growth
•Aged breeding
•Cull animals
Manure
Airflow
Concentration of
Pathogens
Pathogen
Gm +
Gm-
TB-like
Fungi
Virus
Best pH
Activity in organic
material
+
+
Some
+
Most
Wide range
Good
EPF
Formaldehydes
and aldehydes
++
++
++
++
++
Wide range
Good
EPF
Chlorine and
Chloramines
++
++
Some
++
+
Acid
Very poor
CE
Iodophors
++
++
Some
++
+
Acid
Fair to poor
CE
Sodium Hydroxide
++
++
Some
++
++
Alkaline
Good
P
Quaternary
ammoniums
++
+
Little
Some
Some
Alkaline
Fair
CE
Phenols
++
++
+
Some
Some
Acid
Good
EPF
Potassium peroxymonosulfate
++
++
+
+
Most
Wide range
Good
EPF
Disinfectants
Chlorhexidene
Compound
*
C - Clean equipment & hard surfaces E - Equipment F - Footbath P – Premises
Common
uses*
Conclusions
• It’s important…
– to know how to detect the disease status of
animals (eg. subclinical, carrier animals) and herds
– to collect the “right” samples, from the right
animals
– to look at the WHOLE picture, not just an
individual test result
– to evaluate and apply non-specific as well as
targeted biosecurity/prevention practices
Penn State is committed to affirmative action,
equal opportunity and diversity of its workforce.