Untitled - Institut für Tierzucht und Tierhaltung - Christian

The Institute for Animal Breeding and Husbandry
of the Christian-Albrechts-Universität Kiel
Tuberculosis diagnosis and prevention
in West African Chimpanzees (Pan troglodytes verus)
at Tacugama Chimpanzee Sanctuary, Sierra Leone, West Africa
Dissertation
submitted for the Doctoral Degree
awarded by the Faculty of Agricultural and Nutritional Sciences
of the
Christian-Albrechts-Universität Kiel
submitted
M.Sc. Rupak Khadka
born in Lalitpur, Nepal
Dean: Prof. Dr. Dr. h.c. Rainer Horn
1. Examiner: Prof. Dr. Edgar Schallenberger
2. Examiner: Dr. habil. Silvia Wein
Day of Oral Examination: 29 January 2014
Schriftenreihe des Instituts für Tierzucht und Tierhaltung der
Christian-Albrechts-Universität zu Kiel; Heft 203, 2014
© 2014 Selbstverlag des Instituts für Tierzucht und Tierhaltung
der Christian-Albrechts-Universität zu Kiel
Olshausentraße 40, 24098 Kiel
Schriftleitung: Prof. Dr. Dr. h.c. mult. E. Kalm
ISSN: 0720-4272
Gedruckt mit Genehmigung des Dekans der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel
Dedicated to all the chimpanzees at Tacugama Chimpanzee Sanctuary, Freetown, Sierra Leone
i
Acknowledgements
I would like to express my sincere gratitude to my supervisor, Prof. Dr. Edgar Schallenberger,
Christian-Albrechts University, Kiel, Germany and my mentor, Mr. Bala Amarasekaran,
Director, Tacugama Chimpanzee Sanctuary (TCS), Sierra Leone for their guidance and
continuous support from the beginning of the project, continuous follow up, and invaluable
comments up to the final form of dissertation. All the pictures used in this dissertation were
provided from the sanctuary.
I would like to extend my special thanks to Dr. Rosa Garriga, Dr. Simona Papa, Dr. Jennifer
Jaffe, and Anita Mckenna on behalf of the sanctuary for their continuous technical support and
useful inputs for my research project. My kind regards to Mr. Bala’s family, all the staffs, and
volunteers from the sanctuary for their continuous support and love to accomplish my research
project in Sierra Leone. Thanks to the laboratory support from Choitram Memorial Hospital,
Sierra Leone and Animal Health and Veterinary Laboratories Agency, United Kingdom. Also
thanks to all my Nepalese friends who offered deep courtesy and delicious food during my stay
in Sierra Leone and Germany.
I am very much thankful to Dr. Steve Unwin and Dr. Chris Colin, Pan African Sanctuary
Alliance (PASA), to assist in editing and discussing tuberculosis survey questionnaire during
PASA veterinary workshop 2013 in Cameroon. Also I would like to express my deepest
gratitude to all the PASA member sanctuaries that participated and shared their information for
the survey. An additional special thanks to Dr. Chris Colin for her time to translate the survey in
French version.
Lastly, I would like to thank my family and friends, especially my father - Bhimsen Khadka,
mother - Ambika Khadka, brothers, sister-in-law, sister, nephew and my beloved wife Ms.
Samrita Thapa for their continuous love, support and encouragement to finish my abroad study.
ii
Table of Contents
Acknowledgements ......................................................................................................................... ii
Acronyms ...................................................................................................................................... vii
Tuberculosis ante-mortem diagnostics and therapy for field setting semi wild captive
chimpanzees ................................................................................................................................... 1
Abstract ........................................................................................................................................... 2
1. Introduction ................................................................................................................................. 3
1.1. Etiology and transmission.......................................................................................... 4
1.2. Pathogenesis .............................................................................................................. 4
1.3. Clinical signs and organs affected ............................................................................. 5
1.4. Chimpanzees and its status in Africa ......................................................................... 5
1.5. Tuberculosis in non human primates ......................................................................... 6
1.6. Tuberculosis diagnosis in non human primates ......................................................... 7
1.7. Tuberculosis treatment in non human primates ......................................................... 9
2. Materials and methods .............................................................................................................. 10
2.1. Tacugama Chimpanzee Sanctuary........................................................................... 10
2.2. History of chimpanzees ........................................................................................... 11
2.3. Anesthesia protocol ................................................................................................. 12
2.4. Tuberculin skin test.................................................................................................. 12
2.5. PrimaTB Stat Pak test .............................................................................................. 13
2.6. Nasogastric intubation ............................................................................................. 15
2.7. Chest radiographs .................................................................................................... 15
2.8. Tracheal wash sample collection ............................................................................. 16
2.9. Sample preparation for mycobacterial culture ......................................................... 17
2.10. DNA extraction and PCR ...................................................................................... 17
2.11. Anti-tuberculosis treatment ................................................................................... 18
2.12. Staff tuberculosis test ............................................................................................. 18
3. Results ....................................................................................................................................... 19
3.1. Skin and PrimaTB Stat Pak test in eighty chimpanzees .......................................... 19
iii
3.2. Diagnostic tuberculosis tests among ten suspected chimpanzees............................ 21
3.2.1. Skin and PrimaTB Stat Pak test ............................................................................ 21
3.2.2. Acid fast bacilli test .............................................................................................. 23
3.2.3. Chest radiograph and therapy ............................................................................... 23
3.2.4. Tuberculosis therapy protocol .............................................................................. 25
3.2.5. Culture and PCR ................................................................................................... 26
4. Discussion ................................................................................................................................. 28
5. Conclusion ................................................................................................................................ 33
References ................................................................................................................................ 34
Management of Tuberculosis under field conditions in non-human primates among Pan
African Sanctuary Alliance members ....................................................................................... 41
Abstract ......................................................................................................................................... 42
1. Introduction ............................................................................................................................... 43
2. Methodology ............................................................................................................................. 46
3. Results ....................................................................................................................................... 49
3.1. General information about the sanctuaries .............................................................. 49
3.2. Tuberculosis tests during quarantine period ............................................................ 50
3.3. Tuberculosis diagnostics.......................................................................................... 51
3.4. Tuberculosis medication .......................................................................................... 53
3.5. Sanctuary staff tuberculosis tests ............................................................................. 54
3.6. Husbandry and management practices .................................................................... 55
4. Discussion ................................................................................................................................. 58
5. Conclusion ................................................................................................................................ 62
References ................................................................................................................................ 63
General Summary ......................................................................................................................... 67
Allgemeine Zusammenfassung ..................................................................................................... 69
Appendix ....................................................................................................................................... 72
iv
LIST OF TABLES
Table 1: Number of chimpanzees and tuberculosis tests .............................................................. 11
Table 2: History and tuberculosis tests of ten suspected chimpanzees ......................................... 11
Table 3: Scoring system for intradermal skin test in primates eyes ............................................. 13
Table 4: Staff sputum tests ............................................................................................................ 18
Table 5: Skin and PrimaTB Stat Pak result before and during medication .................................. 22
Table 6: Acid fast bacilli test from eight chimpanzees ................................................................. 23
Table 7: Diagnostic tuberculosis tests with results in ten chimpanzees ....................................... 25
Table 8: Anti-tuberculosis medication of ten chimpanzees .......................................................... 26
Table 9: Tuberculosis tests after the completion of medications .................................................. 27
Table 10: List of twenty two sanctuaries accredited to PASA as of October 2013 ..................... 47
Table 11: Sanctuaries that responded to the survey ...................................................................... 49
Table 12: Tuberculosis tests for quarantine primates ................................................................... 51
Table 13: Tuberculosis tests for the sanctuary staffs .................................................................... 55
LIST OF FIGURES
Figure 1: Tuberculin skin test in the eyelid .................................................................................. 13
Figure 2: PrimaTB Stat Pak assay ................................................................................................ 14
Figure 3: Method of gastric intubation ......................................................................................... 15
Figure 4: Preparing for chest radiograph ...................................................................................... 16
Figure 5: Tracheal wash sample collection ................................................................................... 17
Figure 6: Percentage negative of Skin and Stat Pak tests ............................................................. 19
Figure 7: Negative skin test with PPD-A/B .................................................................................. 20
Figure 8: Skin test reaction with MOT and PPD-A/B .................................................................. 21
Figure 9: PrimaTB Stat Pak positive reaction............................................................................... 21
Figure 10: Normal chest radiograph ............................................................................................. 24
Figure 11: Chest radiographs before and during the treatment ..................................................... 24
Figure 12: Distribution of primate sanctuaries in twelve countries across Africa........................ 45
Figure 13: Types of tuberculosis diagnostics used in the sanctuaries .......................................... 52
v
Figure 14: Number of sanctuaries reporting for tuberculosis cases .............................................. 53
Figure 15: Response for tuberculosis positive cases..................................................................... 54
Figure 16: Hygiene and record keeping in the sanctuaries ........................................................... 56
Figure 17: Enclosure settings in the sanctuaries ........................................................................... 57
Figure 18: Accessibility of tuberculosis kits in the sanctuaries .................................................... 57
vi
Acronyms
AFB
Acid Fast Bacilli
AHVLA
Animal Health and Veterinary Laboratories Agency
AIDS
Acquired Immune Deficiency Syndrome
AP
Anterior Posterior
BCG
Bacillus Calmette-Guérin
b wt
body weight
CERCOPAN
Center for Education, Research and Conservation of Primates and Nature
CDC
Centers for Disease Control and Prevention
CD4
Cluster of Differentiation 4
CRL
Consolidated Right Lung
DNA
Deoxyribo Nucleic Acid
ET
Endotracheal tube
HIV
Human Immunodeficiency Virus
Ig
Immunoglobulin
ILAR
Institute for Laboratory Animal Research
IU
International Units
IUCN
International Union for Conservation of Nature
LHA
Left Hilar Adenopathy
MOT
Mammalian Old Tuberculin
NIH
National Institute of Health
NRC
National Research Council
NSF
No Specific Findings
PASA
Pan African Sanctuary Alliance
PCR
Polymerase Chain Reaction
PPD-A/B
Purified Protein Derivatives - Avian and Bovine
RHA
Right Hilar Adenopathy
SSC
Species Survival Commission
TB
Tuberculosis
TCS
Tacugama Chimpanzee Sanctuary
TST
Tuberculin Skin Test
vii
UK
United Kingdom
USA
United States of America
WHO
World Health Organization
mg
Milligram
ml
Milliliter
kg
Kilogram
µl
Microliter
µm
Micrometer
viii
Tuberculosis ante-mortem diagnostics and therapy for field setting semi wild
captive chimpanzees
1
Abstract
Tuberculosis is one of the most crucial diseases of humans and non-human primates worldwide.
Due to its zoonotic nature, it is very important to diagnose and prevent the spread of tuberculosis
between species. In this study, we aimed to understand the application of tuberculin skin and
PrimaTB Stat Pak tests for tuberculosis diagnosis in remote captive sanctuaries that have limited
resources and manpower. It also highlights to compare the results of ante-mortem diagnostics
and evaluate the reliability of these diagnostics when treatment was performed in suspected
chimpanzees. Data were collected from Tacugama Chimpanzee Sanctuary, Sierra Leone, from
April 2011 to October 2012. 80 out of 105 chimpanzees from different groups were tested for
tuberculin skin and PrimaTB Stat Pak tests. Ten tuberculosis suspected chimpanzees were tested
with tuberculin skin tests, Stat Pak tests, acid fast bacilli staining, chest radiographs, culture and
PCR and treated with anti-tuberculosis drugs. Among 80 chimpanzees, the combination of skin
and Stat Pak tests seem very promising as skin tests were 100% negative while almost 98% of
Stat Pak tests were negative. Tuberculosis tests among ten suspected chimpanzees with skin, Stat
Pak tests, acid fast bacilli staining and chest radiographs were inconsistent. All ten chimpanzees
received anti-tuberculosis drugs from three months to one year. After the completion of
treatment, all ten chimpanzees were tested negative with skin and Stat Pak tests. Furthermore,
five chimpazees were confirmed negative for tuberculosis with culture and PCR. Tuberculosis
can not be diagnosed with a single test but rather should apply different tests modalities. Antituberculosis treatment should be peformed considering the importance, costs, ethical and public
issues of animals. For remote sanctuaries, a combination of skin and Stat Pak tests can be very
useful and field friendly. Any suspicious or positive chimpanzess should be retested and further
diagnosis should be done with more reliable tests.
Keywords: Tuberculosis, Diagnostic tests, Chimpanzees, Anti-tuberculosis drugs, Sierra Leone.
2
1. Introduction
Tuberculosis is a leading cause of morbidity and mortality in the world. It is the world’s second
most leading cause of death from an infectious disease, after HIV/AIDS. According to WHO
estimates for 2012, about one third of the world’s population is estimated to be infected with TB
which resulted in 8.6 million new cases of active tuberculosis and 1.3 million deaths per year.
Among the 8.6 million global new cases of active tuberculosis, 24% of these occurred in Africa
(WHO, 2012). Only about 5-10% of tuberculosis infected individuals develop active disease and
become infectious while the remainder is either eliminated or existed as latent infection but do
not transmit infection (Gagneux, 2012; WHO; 2012). The populations that have co-infection of
tuberculosis with HIV/AIDS have a 50% chance of developing the disease and the existed latent
infection may accelerate the progression of HIV/AIDS (WHO, 2012; Mathema et al., 2006).
More than 95% of the death is concentrated in developing countries, particularly in Africa and
Asia. Southeast Asia has the highest mortality from tuberculosis while Africa has the highest
mortality per capita due to the increase in HIV/AIDS infection. Africa has the world’s highest
rates of human tuberculosis, ranging from 50 to over 300 new cases per 100,000 people while in
Sierra Leone, there were 674 new cases of tuberculosis per 100,000 people with a mortality rate
of 21.8 % (WHO, 2012). The increase of tuberculosis infection is strongly associated with the
prevalence of HIV infection in African continent. In the past two decades, tuberculosis
resurgence has been attributed to the HIV epidemic and lapses in public health programs but
more recently with the emergence of multi-drug resistant strains of tubercle bacteria (Mikota and
Maslow, 2011; Une and Mori, 2007). Tuberculosis control strategies are constantly requiring
improvement, including highlighting potential reservoirs that might play a big role in emergency
of resistant strains.
Human tuberculosis is caused by Mycobacterium tuberculosis. However, M. bovis – a zoonotic
species, is also responsible for an increasing proportion of human tuberculosis. M. bovis, unlike
M. tuberculosis, has a wide host range, often isolated from tuberculous cattle, and has several
wildlife maintenance hosts such as elephants, wild buffaloes, rhinoceros, badgers, non-human
primates and so on. The occurrence of tuberculosis in non-human primates is often caused by the
same organism that causes tuberculosis in humans and can spread rapidly from non-human
primates to humans and vice versa (Garcia et al., 2004; Bernacky et al., 2002).
3
1.1. Etiology and transmission
Tuberculosis is caused by tubercle bacillus belonging to the Mycobacterium tuberculosis
complex which consists of Mycobacterium tuberculosis, M. bovis, M. africanum, M. canettii and
M. microti. Mycobacteria are slowly growing, aerobic, non-motile, acid fast bacilli measuring
0.2 to 0.6 µm by 1.0 to 10 µm in size. It is a respiratory disease that is mainly contracted by the
inhalation of infectious droplets expelled from the respiratory tract of infected humans or
animals with active disease. The factors that determine the probability of transmission depends
on infectiousness of the source, host susceptibility of contacts, duration of exposure and the
environment in which the exposure takes palce (CDC, 2013). The principle vehicle of M. bovis
transmission to human is through the ingestion of contaminated milk. Infected individuals with
active tuberculosis may show no overt signs of the disease for weeks or months, during which
time they can transmit infection to others. In wild animals, airborne transmission occurs
primarily when there are interactions among humans, domesticated and wild animals.
1.2. Pathogenesis
Disease pathogenesis of tuberculosis infection in humans can be understood in four stages. The
lesions produced are generally similar in humans, non-human primates and other mammals
(Montali et al., 2001). In the first stage, about three to eight weeks after M. tuberculosis
contained in inhaled aerosols becomes implanted in the alveoli, the bacteria are disseminated by
the lymphatic circulation to regional lymph nodes in the lung, forming primary or Ghon
complex. At this time, conversion to tuberculin reactivity occurs. The second stage, lasting about
three months, is marked by haematogenous circulation of bacteria to many organs including
other parts of the lung; at this time acute and sometimes fatal disease can occur in the form of
tuberculosis meningitis or milliary tuberculosis. Inflammation of the pleural surfaces can occur
during the third stage, lasting three to seven months to two years and causing severe chest pain.
This condition is caused by either haematogenous dissemination or the release of bacteria into
the pleural space from subpleural concentrations of bacteria in the lung (Lopez, 2001). The free
bacteria or their components interact with sensitized CD4 T lymphocytes that are attracted and
then proliferate and release inflammatory cytokines (Kamholz, 1996). The last stage, when the
disease does not progress, may take up to three years. In this stage, more slowly developing
extrapulmonary lesions are seen in bones, joints, and frequent chronic back pain.
4
1.3. Clinical signs and organs affected
Tuberculosis is a chronic progressive disease that may take months to years to cause clinical
signs. The common clinical signs include anorexia, lethargy, irritability or change in behaviour,
low grade fluctuating fever, weight loss, coughing, chronic wasting, diarrhea, enlarged lymph
nodes, and acute to sudden death. Dyspnea, intermittent, hacking cough is observed when lungs
are extensively involved. Abdominal breathing is more often observed than coughing. Weight
loss is a symptom that usually occurs during the advance stage of disease. Tuberculosis can
affect all organs. Focal granulomatous or miliary tubercle lesions in any organ are suggestive of
tuberculosis, but lesions are most regularly observed in the lung, liver, lymph nodes and spleen.
It can also affect pleura, kidneys, abdomen, gastrointestinal tract, meninges, joints and bones.
1.4. Chimpanzees and its status in Africa
Chimpanzees (Pan troglodytes) are one of the four great apes. They are most closely related to
humans because they share more than 98% of their DNA with humans (Gagneux and Varki,
2001; King and Wilson, 1975). They are widely distributed across 21 Equatorial African
countries from Senegal in the west to Tanzania in the east, of which some of the countries are the
poorest in the world (Caldecott and Miles, 2005). The Species Survival Commission of the
International Union for Conservation of Nature has red listed chimpanzees as an endangered
species i.e. facing a high risk of extinction in the wild in the near future (IUCN, 2013; Oates,
2006). Current estimates of 150,000 to 250,000 chimpanzees population remain in the wild with
a population decline of at least 4.7% annually (Stiles et al., 2013; Walsh et al., 2003; Goodall,
2001). In the last three decades, their populations in the wild have been declined by more than
66% (Kormos and Boesch, 2003). A number of potential threats such as habitat loss, bush meat
trade, human disturbance, hunting, and spread of zoonotic and infectious diseases have resulted
in such a decline in chimpanzee population and if no further action is implemented to stop their
decline in population they are likely to extinct in the future (Kormos et al., 2003; Hilton-Tylor,
2002). The sub-species found in Sierra Leone are the Western Chimpanzees (Pan troglodytes
verus). Sierra Leone has confirmed estimates of 5,500 chimpanzees in the wild, the second
largest population in West Africa (Brncic et al., 2010). Several sanctuaries and rehabilitation
centers are established across Africa to rescue chimpanzees and other non-human primates that
are the victims of habitat loss, illegal trade and hunting. In Africa, PASA is an umbrella
5
organization of twenty two accredited sanctuaries across twelve countries that collectively care
for chimpanzees, gorillas, bonobos and different species of monkeys. As chimpanzees share
similar physiological and genetic characteristics, they transmit different zoonotic diseases with
humans and vice versa. Most of the transmission occurs in the conservation programs sanctuaries
or rescue centers where they live in close proximity with humans (Wallis and Lee, 1999).
Among various zoonotic diseases, tuberculosis has always been a threat to both humans and nonhuman primates.
1.5. Tuberculosis in non human primates
In non-human primates, tuberculosis is caused by Mycobacterium tuberculosis, M. bovis, and to
a lesser extent M. africanum, M. canettii, and M. microti, collectively included within the
Mycobacterium tuberculosis complex (Wilbur et al., 2012; Flynn et al., 2003; Butynski, 2001;
Walsh et al., 1996; Wayne and Kubica, 1986). It is a respiratory disease with serious zoonotic
concerns (De Leslie et al., 2002; Michel and Huchzermeyer, 1998; Chaloux, 1978; Tauraso,
1973), transmitted from humans to non-human primates and vice versa via aerosol route
(Ashford et al., 2001; Morris et al., 1994; Haberle, 1974) with rare digestive route (Sapolsky and
Else, 1987; Moreland, 1970). Outbreaks of tuberculosis in closed colonies of vervet monkeys
(Van Zijll Langhout et al., 2009), rhesus and cynomolgus monkeys (Garcia et al., 2004), rhesus
monkeys (Zumpe et al., 1980), African green monekys (Thorel, 1980) and baboons (Fourie and
Odendaal, 1983) have been reported. The transmission of tuberculosis to non-human primates,
especially in captive settings like sanctuaries and zoological gardens, is at high risk because of
close contact of non-human primates with humans and exposition to visitors (Michel et al.,
2003). The susceptibility varies among non-human primates, great apes are less susceptible to
tuberculosis than old world monkeys but are more susceptible than new world monkeys (Lerche
et al., 2008; Montali et al., 2001). Infected non-human primates with active tuberculosis can
easily transmit the disease to other healthy non-human primates and humans. On the other hand,
non-human primates with latent tuberculosis infections present a significant risk of reactivation
and the development of active tuberculosis (Capuano et al., 2003; Flynn and Chan, 2001).
Although animals with latent tuberculosis are not infectious, reactivation of latent infections that
were not detected using traditional screening methods during primary quarantine is emerging as
an important factor in the epidemiology of tuberculosis in non-human primates.
6
Recently, a new tuberculosis variant, closer to the human-associated lineage 6 (also known as M.
africanum West Africa 2) than to the other classical animal-associated tuberculosis strains was
discovered in the wild chimpanzees (Coscolla et al., 2013). Despite this findings, there is no data
on prevalence (or absence) of tuberculosis complex in the wild chimpanzee population. Most of
the non-human primate sanctuaries or rescue centers in Africa have close contact with humans
and are the potential candidates for reintroduction in the wild. The detection of even a few
suspect positive cases at these primate settings is of great concern, since these centres have large
numbers of non-human primates, which are usually housed in close proximity to each other and
with humans, increasing the risk of infectious disease transmission and thereby jeopardising the
conservation goals of the sanctuaries. Outbreaks of tuberculosis continue to occur in established
colonies of primates and can have severe consequences due to the loss of animals, transmission
to humans, disruption of re-introduction programms and costs associated with disease control.
1.6. Tuberculosis diagnosis in non human primates
In non-human primates, tuberculosis diagnosis is quite challenging because of lack of diagnostic
sensitivity and specificity among the available diagnostics and poorly defined clinical features of
the disease (Gibson, 1998). However, several diagnostic methods have been developed over time
in human medicine due to advancement of technology and molecular tools. Some of these
diagnostic approaches have been reviewed and practiced for ante-mortem diagnosis and
surveillance of tuberculosis in non-human primates (Lerche et al., 2008). No single test currently
meets all the requirements for accurate and efficient tuberculosis screening in non-human
primates. A combined application of several tests can increase the sensitivity and specificity of
screening and surveillance programs.
The tuberculin skin test (TST) using tuberculin purified protein derivatives (PPD) and
mammalian old tuberculin (MOT) have been most commonly used in humans and non-human
primates for many years and is the only ILAR/CDC approved method for tuberculosis testing for
non-human primates (Roberts and Andrews, 2008; NRC, 1980; Kennard et al., 1939). It is one of
the most frequently used ante-mortem tests for the diagnosis of tuberculosis in non-human
primates, however, significant shortcomings exist with this methodology (Lerche et al., 2008). It
is widely known that the specificity and sensitivity of the TST varies between MOT and PPD
7
preparations (avian and bovine), and that using this test alone increases the chances of false
negative diagnosis. MOT meets this requirement and has a greater reactivity than PPD and is
therefore preferred to PPD as the reagent to use in a TST to identify infected animals. However,
MOT is a crude culture of filtrate preparation that contains antigens common to many
mycobacterial species including those not associated with tuberculosis. Because of this antigenic
cross reactivity, TST suffers low specificity and false positive reactions are common. TST has a
number of limitations with regard to sensitivity and specificity that reduce its ability to be used
as a standalone test. False positive reactions may result from trauma associated during the test,
exposure to non-tuberculous environmental mycobacteria, prior vaccination with BCG and
antigenic cross-reactivity (Vervenne et al., 2004; Andersen et al., 2000; Dukelow and Pierce,
1987). False negative reactions may result due to latent infection, anergy associated with
progressive disease, therapy with immunosuppressive drugs, vaccination with polio, measles or
yellow fever (Motzel et al., 2003; Staley et al., 1998), incorrect injection/concentration and
misinterpretation in reading the test (Bushmitz et al., 2009). Limitations of TST described above
and especially its inability to detect animals with latent infections makes it an unsuitable tool to
use as a single, standalone test for tuberculosis surveillance in non-human primates.
Understanding TST has several shortcomings, a combination of TST with alternative
immunological assays has been suggested (Parsons et al., 2010; Bushmitz et al., 2009; Lin et al.,
2008). Therefore, new tuberculosis diagnostic algorithms are being developed, in which
serological assays may play an important role. This assay has been shown to have good
specificity and sensitivity for M. tuberculosis in non-human primates when used in conjunction
with TST (Lyashchenko et al., 2007). Stat Pak tests were promising for the diagnosis of
tuberculosis in other wildlife species such as elephants (Lyashchenko et al., 2012; Greenwald et
al., 2009), wild deers (Gowtage et al., 2009) and badgers (Chambers et al., 2008). Under field
settings like primate sanctuaries in Africa, this assay is of great advantage because it is portable,
more rapid, less labour-intensive and cheap.
Advancement of technology, molecular tools and the efficacy of diagnostic tests in humans has
provided useful information that has facilitated the diagnosis and prevention of tuberculosis in
non-human primates. When the screening tests like tuberculin, immune-assays or both became
reactive or suspicious or positive, it is always worth to obtain throat swabs or sputum or tracheal
8
wash samples for AFB and culture. To confirm the diagnosis of suspicious or positive case,
bacterial culture should be carried out in all suspected samples regardless of AFB results. Chest
radiographs can be used as an ancilliary tests for screeing of tuberculosis. Tracheal wash or
gastric lavage samples are very useful for ante-mortem tuberculosis testing in non-human
primates. Mycobacterial culture is considered as the golden standard test for the diagnosis of
tuberculosis (Bailey and Mansfield, 2010; Andersen et al., 2000; De Leslie et al., 2002; Mikota
and Maslow, 1997; Chadwick, 1982). It is reliable and specific but time consuming (takes 6-8
weeks). More recently, molecular technology like Polymerase Chain Reaction (PCR) has been
used to detect mycobacterial DNA for the diagnosis in non-human primates (Rocha et al., 2011;
Montali et al., 2001; Rock et al., 1995). Detection of TB infection by screening faeces or sputum
by PCR for mycobacterial DNA can be considered as a rapid and alternative diagnostic tool for
tuberculosis. Mycobacterial tuberculosis complex DNA has been isolated from the buccal swabs
of free ranging primates (Wilbur et al., 2012). In addition to detection of mycobacteria species by
PCR, identification and differentiation of mycobacteria to the species level had been
accomplished by sequence analysis of PCR products (Hillemann et al., 2006; Warren et al.,
2006; Alfonso et al., 2004). PCR is highly sensitive, accurate and significantly faster than
conventional culture method (one day versus 6-8 weeks) but it is very expensive and impractical
to field settings. However, the application of new diagnostic technology may allow more rapid
and accurate detection of tuberculosis.
1.7. Tuberculosis treatment in non human primates
Treatment of tuberculosis infections in non-human primates is generally discouraged and
euthanasia is recommended because of the public health hazard. However, rare and endangered
species like chimpanzees and other great apes can be treated assuring the public health hazard,
costs involved and ethical issues (Bushmitz et al., 2009; Montali et al., 2001). The animals must
be kept in strict quarantine conditions during the whole treatment period. Treatment has been
effective in non-human primates (Ward et al., 1985; Wolf et al., 1988) and elephants (Mikota
and Maslow, 2011) using human drugs like isoniazid, rifampin, pyrazinamide and ethambutol.
Due to the fact of lack of validated tuberculosis treatment protocol for animals, human
tuberculosis treatment method has been approached. Under the standard WHO tuberculosis
treatment system, the regimen for new patients consists of a 2 month intensive phase treatment
9
with isoniazid, rifampin, pyrazinamide and ethambutol, followed by a 4 month continuation
phase with isoniazid and rifampin. However, the duration of treatment for active tuberculosis
may last to 18 months and involves various types of antibiotics. The standard recomended doses
of anti-tuberculosis drugs for a human adult is isoniazid @ 5(4-6)mg/kg b wt, rifampin @ 10(812)mg/kg b wt, pyrazinamide @ 25(20-30) mg/kg b wt and ethambutol @ 15(15-20)mg/kg b wt
(WHO, 2010). Multidrug treatment helps to reduce the possibility of developing antibiotic
resistance and must be done on the basis of antibiotic sensitivity testing.
The main aim of this research is to understand a basic diagnosis method and focus on identifying
preventive measures to control tuberculosis in primate captive settings that have limited
resources and manpower. It highlights to compare the results of ante-mortem diagnostics and
evaluate the reliability of these diagnostics where treatment was performed in suspected
chimpanzees. This study also explains different tuberculosis diagnostics and preventative
measures which have been implemented for the management of tuberculosis in chimpanzees.
2. Materials and methods
2.1. Tacugama Chimpanzee Sanctuary
Tacugama Chimpanzee Sanctuary is located in the hills of Western Area Peninsula Forest
Reserve, Freetown, Sierra Leone. The sanctuary was founded through the joint effort of
conservationist Bala Amarasekaran, the Government of Sierra Leone and the Conservation
Society of Sierra Leone in 1995. The sanctuary is part of the large Sierra Leone Chimpanzee
Rehabilitation Program, playing a vital role in stopping the trade and preserving chimpanzees in
the wild. The overall aim of the sanctuary is to provide a safe home to endangered orphaned
chimpanzees, their rehabilitation into social groups with a view of re-introduction into wild and
observe the species in the wild by engaging with the public through education, environmental
sensitization and training programs in the community. The sanctuary cared 105 chimpanzees (48
males and 57 females) during April 2011 to October 2012. Their body weight ranges from 5 kg
to 55 kg divided in six different social groups and age varies from less than a year to twent six
years old. There was a separate quarantine facility for the new arrivals. Data were collected only
during normal health checkup and quarantine period of chimpanzees at the sanctuary premises.
10
2.2. History of chimpanzees
Case 1: 80 out of 105 chimpanzees from different groups were tested for skin and PrimaTB Stat
Pak tests during April 2011 to October 2012 (Table 1). Descriptive data analysis was performed.
Table 1: Number of chimpanzees and tuberculosis tests
Group
A
B
C
D
E
F
Qa
N
11
14
23
24
20
6
7
TST
11
10
18
13
16
5
7
PrimaTB Stat Pak
11
10
18
13
16
5
7
a
- 2 to 3 tuberculosis tests were performed in the quarantine; N is number
of chimpanzees in the group; TST is tuberculin skin test.
Case 2: Ten chimpanzees (three quarantine and seven residents) were suspected of tuberculosis.
Different tests were carried out in between March 2000 to December 2010. The history and
tuberculosis tests performed in these chimpanzees are presented in the Table 2.
Table 2: History and tuberculosis tests of ten suspected chimpanzees
ID
A
B
C
D
E
F
G
H
I
J
Location
Quarantine
Quarantine
Quarantine
Resident
Resident
Resident
Resident
Resident
Resident
Resident
Condition
Mild cough; nasal discharge
Mild cough; snotty nose
Mild cough; nasal discharge
Occasional cough
Occasional cough
Poor appetite
Poor appetite
Occasional cough
Occasional cough
Occasional cough
Tuberculin used
MOT
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
MOT, PPD-A/B
Stat Pak
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
AFB
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
X-ray
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Chimpanzees A-C stayed with the owner for 9 months, 1 year and 2 years respectively
before moving to the sanctuary while chimpanzees D-J stayed in close contact with
tuberculosis infected chimpanzee in the group; MOT is mammalian old tuberculin;
PPD-A/B is avian and bovine purified protein derivatives respectively; AFB is acid fast
bacilli test; X-ray is chest radiographs.
11
2.3. Anesthesia protocol
All the chimpanzees were anaesthetized for tuberculosis testing. The chimpanzees were
submitted to an overnight fasting before anesthetized. Blood samples were taken for serological
test once injected with tuberculin in the eyelid. Also routine health checkup was performed. They
were sedated with an intramuscular combination of medetomidine (Domitor® 1mg/ml - Pfizer,
UK and Zalopen® 10mg/ml - Orion Pharma, Finland) @ 0.3-0.4mg/kg b wt and ketamine
(Ketaset® 100mg/ml - Fort Dodge, USA and Imalgene® 100mg/ml - Merial, Spain) @ 5-7mg/kg
b wt (Unwin et al., 2009). As health checkup and collection were completed, medetomodine was
reversed with atipamezole (Antisedan® 5mg/ml - Pfizer, UK) @ 0.1-0.25mg/kg b wt (Unwin et
al., 2009). All chimpanzees were weighed during the procedure.
2.4. Tuberculin skin test
It is a very common method used in non-human primates which detects delayed type
hypersensitivity to tuberculin antigens. MOT (1,500 units; Synbiotics, USA) and avian and
bovine PPD (each 25,000 IU per ml; CZ Veterinaria, Spain) tuberculins were used to test the
chimpanzees during various occasions at the sanctuary (Figure 1). 0.1 ml of avian PPD and
bovine PPD tuberculin antigen was injected intrapalpebrally in the middle of the upper right and
left eyelid respectively while 0.1 ml of MOT was injected in either upper left or right eyelid. A
25 to 27 gauge ½ inch needle was separately used for each chimpanzee. The intrapalpebral route
of inoculation was preferred in non-human primates, as it removes the need for a second
manipulation of the animals to measure a possible induration in the skin of the abdomen. In most
of the occasions, intrapalpebral route of inoculation was performed. Only few skin tests were
performed in abdomen and forearm to cross check the reaction of eye tests. The
swelling/induration in both eyelids was observed for 24, 48 and 72 hours consecutively and the
inflammatory responses were scored (Ritcher et al., 1984). The scoring system for reaction and
interpretation is presented in Table 3.
12
Table 3: Scoring system for intradermal skin test in primates eyes
Reaction at 72 hours
Grade Interpretation
No reaction observed
0
Negative
Bruise only
1
Negative
Erythema without swelling
2
Negative
Erythema with minimal swelling, or slight swelling without erythema 3
Suspicious
Obvious swelling with drooping of eyelid and erythema
4
Positive
Swelling +/or necrosis with eyelids closed
5
Positive
Figure 1: Tuberculin skin test in the eyelid
Eyelid injection of avian purified protein derivatives in right eyelid of a 4 year old chimpanzee.
2.5. PrimaTB Stat Pak test
PrimaTB Stat Pak (Chembio Diagnostic Systems, USA) test was developed to detect IgG and
IgM antibodies against both M. tuberculosis and M. bovis in non-human primates. This lateral
flow system uses a cocktail of recombinant tuberculosis - specific antigens (ESAT-6, CFP-10,
MPB-83, and TBF-10) and a blue latex signal detection system (Lyashchenko et al., 2007).
13
Steps for PrimaTB Stat Pak test
•
Whole blood samples with a 5ml Vaccutainer® tube containing buffered sodium citrate
were drawn from the femoral vein.
•
The blood was centrifuged and serum was separated. Whole blood and plasma were also
used in some occasions.
•
The PrimaTB Stat Pak assay kit was removed from the pouch and placed in the flat
surface area.
•
The test units were labeled with the names of chimps, test time and date.
•
Using a micropipette or disposable pipette, 30 µl of serum or plasma specimen was added
onto the center of the sample ‘S’ well.
•
Once the serum/plasma specimen was applied to the sample ‘S’ well, then 3 drops (about
100 µl) of diluents was added slowly into sample ‘S’ well.
•
The result was read after 15-20 minutes after the addition of the diluents.
The appearance of a clear visible line or band in the test area along with the control line was
interpreted as positive. The appearance of blurred visible line or band in the test area along with
the control line was interpreted as suspicious. The absence of a line or band in the test area was
interpreted as negative.
Figure 2: PrimaTB Stat Pak assay
Comparing a negative result (on the left) and a positive result (on the right); the upper line with ‘C’ is a
control band and the lower band shown with an arrow in ‘T’ is the test band indicating a positive result.
14
2.6. Nasogastric intubation
The nasogastric intubation was performed to collect the gastric lavage samples for acid fast
bacilli (AFB) tests (Figure 3). The samples were tested using AFB staining method at Choitram
Memorial Hospital, Freetown, Sierra Leone. The sample fixed slide was stained with carbol
fuschin for 5-10 minutes under mild heat followed by a gentle rinse with tap water. The slide was
then decolorized with TB decoloriser (1% of acid alcohol), rinsed with tap water and
counterstained with methylene blue or malachite green. Finally the slide was rinsed with tap
water, dried and viewed under immersion oil lens. The presence of bright red rod bacilli in blue
background is considered as AFB positive otherwise AFB negative.
Figure 3: Method of gastric intubation
2.7. Chest radiographs
In non-human primates, chest radiographs are generally used only as an additional test to screen
for tuberculosis. It is especially important for skin test suspicious reacted or immunosuppressive
non-human primates. In this study, all suspected chimpanzees were sedated and taken to
Choitram Memorial Hospital in Freetown for chest radiographs. An anterior-posterior (Figure 4)
and lateral view chest radiographs were taken which was interpreted by human surgeons. The
problems associated with these tests were risk of taking chimpanzees to human hospital (almost
15
an hour drive from the sanctuary), costs involved and the efficacy of result interpretation.
Interpretation of chest radiographs was really difficult due to lack of consistency in reading the
radiographs from different surgeons. Chest radiographs taken during different period of
examinations are presented in the result section.
Figure 4: Preparing for chest radiograph
2.8. Tracheal wash sample collection
Five chimpanzees A, B, C, D and E were overnight fastened, sedated and positioned on their
back with neck extended. Anesthesia was monitored regularly and an intravenous access was
placed. A lignocaine lubricated endotracheal tube (ET) was intubated through the larynx up to
tracheas. Chimpanzees have very short tracheas before it bifurcates so one should be very
careful. A sterile human urinary catheter was inserted to the point down the ET. Approximately
20-30ml of sterile saline solution was infused through the syringe attached to the catheter inside
the trachea. The chimpanzees were gently shaken and the samples were then retrieved with the
syringe via catheter immediately after infusion. During the procedure, artificial oxygen was
supplied time to time from cylinder to ease the respiration. The collected samples were placed
into a sterile pot and stored at room temperature. The sterile pot was kept on wet ice inside the
polystyrene box and shipped to Animal Health and Veterinary Laboratories Agency (AHVLA) in
the United Kingdom for mycobacterial culture and molecular typing.
16
Figure 5: Tracheal wash sample collection
2.9. Sample preparation for mycobacterial culture
Samples were placed at 4°C on arrival at AHVLA, UK and processed for culture the following
morning. Each sample was assigned a unique laboratory reference number and the samples
processed in ascending numerical order with no other samples in a Class 1 cabinet within the
Class 3 laboratory suite. Tracheal aspirates required no decontamination step. Working with one
sample at a time, approximately 0.3ml of sample was inoculated onto set of media using a sterile
pastette®. The media sets comprised the following media: Stonebrinks egg based slope x1,
Lowenstein egg based slope plus glycerol x1, Lowenstein egg based slope plus pyruvate x1,
7H11 modified agar slope x1 and mycobacterial growth indicator tube (MGIT) x1.
2.10. DNA extraction and PCR
DNA was extracted from the untreated tracheal aspirates using the DNeasy Blood and Tissue
Kit® (Qiagen, UK) according to the standard protocol at AHVLA, UK (attached in the
Appendix). The extracted DNA was run through a Taqman® based Rotor-Gene JOE Real-Time
analysis PCR using a Corbett Rotorgene PCR thermocycler (Corbettlifescience.com) Taqman
IS1081 primers, probe and Sybr Green fluorescent dye. M. bovis known positive and M. avium
known negative controls were run with the chimpanzee samples.
17
2.11. Anti-tuberculosis treatment
We aimed to treat tuberculosis predisposed or newly arrived chimpanzees to prevent the
infection to others in the sanctuary. Prophylactic treatment was done for those chimpanzees that
were suspicious or positive to either skin or Stat Pak tests whereas full treatment for those that
showed abnormalities in chest radiographs in addition to suspicious or positive to skin or Stat
Pak tests. Chimpanzees A, B, D, F, G, I and J were given prophylactic course (2 - 3 months) and
chimpanzees C, E and H were given full course (6 months - 1 year). A daily medication record
sheet was maintained during whole period. Isoniazid (UCB Pharma®, Cemidon®), rifampin
(Sandoz®), ethambutol (Teofarma®), pyrazinamide (Almirall®) and ofloxacin (Teva®) were the
anti-tuberculosis drugs used for these chimpanzees. At least one or two or more than two
combinations of drugs was administered. Multidrug therapy had been considered which helps to
reduce the possibility of developing antibiotic resistance. Vitamin B6 was also given as
supplement therapy during the period because isoniazid is associated with pyridoxine deficiency.
2.12. Staff tuberculosis test
The staff sputum tests were performed using Acid Fast Bacilli (AFB) staining method at
Choitram Memorial Hospital and National Leprosy and Tuberculosis Control Program,
Freetown, Sierra Leone (Table 4). All the sanctuary staffs (n=20) directly or indirectly working
with chimpanzees were tested. The sputum fixed slide was stained with carbol fuschin for 5-10
minutes under mild heat followed by a gentle rinse with tap water. The slide was then
decolorized with TB decoloriser (1% of acid alcohol), rinsed with tap water and counterstained
with methylene blue or malachite green. Finally the slide was rinsed with tap water, dried and
viewed under immersion oil lens. The presence of bright red rod bacilli in blue background is
considered as AFB positive otherwise AFB negative.
Table 4: Staff sputum tests
Health Organisations in Sierra Leone
n
Choitram Memorial Hospital
13
National Leprosy/Tuberculosis Control Programm
n is the number of staffs at the sanctuary
18
7
3. Results
3.1. Skin and PrimaTB Stat Pak test in eighty chimpanzees
The overall performance with the combination of skin and Stat Pak tests seemed very promising
(Figure 6). Skin test results with PPD-A/B were 100% negative while almost 98% of Stat Pak
test results were negative. A digital image for all the tests (TST and Stat Pak) had been captured.
Following the skin test, the score was read in 24, 48 and 72 hours. Two of the chimpanzees
showed positive reaction with Primat TB Stat Pak test while all were negative with TST. After
six months both chimpanzees, which showed positive reaction with Stat Pak test in the previous
tests, were again tested with Stat Pak along with TST. Both results were negative for the tests.
All the sanctuary staffs (n=20) were negative to acid fast bacilli staining tests. The tests were
carried out at two different health centers in Freetown, Sierra Leone.
Figure 6: Percentage negative of Skin and Stat Pak tests
19
a
b
c
Figure 7: Negative skin test with PPD-A/B
Skin test reading with PPD-A/B for 24 hours (a), 48 hours (b) and 72 hours (c) from an 11 years old
chimpanzee (photo taken on 10-12, January 2012).
20
3.2. Diagnostic tuberculosis tests among ten suspected chimpanzees
3.2.1. Skin and PrimaTB Stat Pak test
All ten chimpanzees were tested with PPD-A/B, MOT and PrimaTB Stat Pak tests over different
period. Details of the tests performed are presented in Table 5.
a
b
Figure 8: Skin test reaction with MOT and PPD-A/B
MOT negative reaction in October 2009 in chimpanzee A (a) and PPD-A/B suspicious reaction
in May 2009 in chimpanzee E (b).
Figure 9: PrimaTB Stat Pak positive reaction
21
Table 5: Skin and PrimaTB Stat Pak result before and during medication
Tuberculin used
PrimaTB Stat Pak
ID
A
B
C
D
E
F
G
H
I
J
MOT
PPD-A
PPD-B
Site
Result
Date
Result
Aug 09
Sep 09
Oct 09
Dec 09
Jan 10
Feb 10
X
Oct 07
Nov 07
Dec 01
Mar/Jun 02
Jun 02
Jul/Aug 02
May 07
X
Jun 09
Nov 01
Jan 02
Jun/Jul 02
X
X
Apr 07
Feb 08
X
Oct 01
Mar 07
X
Jun 09
Sep 01
May 07
X
Jun 09
Apr 01
X
X
Jun 09
Jun 01
Oct 07
X
X
Jun 09
Mar 00
Aug 08
X
Jun 09
X
X
X
X
X
X
Apr 10
X
X
X
X
X
X
X
May 09
X
X
X
X
Apr 07
X
X
X
May 09
X
X
May 09
X
X
X
May 09
X
X
Apr 08
Apr 09
X
X
X
Dec 07
May 09
X
X
X
Apr 09
X
X
X
X
X
X
X
Apr 10
X
X
X
X
X
X
X
May 09
X
X
X
X
Apr 07
Apr 07
X
Feb 08
May 09
X
Mar 07
May 09
X
X
May 07
May 09
X
X
Apr 08
Apr 09
X
X
X
Dec 07
May 09
X
X
X
Apr 09
X
Re
Le
Re
Re
Le
Re
Re/Le
Re
Le
Re
Re
At
Re
Re
Re/Le
Le
Re
Le / At
Lfa/At /Re
Re/Le
At
Le
Re/Le
Re/Le
Re
Le / At
Re/Le
Re
Re
Le / At
Re/Le
Le
Re
Re/Le
Re/Le
Le
Re/Le
Re
Re/Le
Re/Le
Re
Re
Le
Re/Le
Re
+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/-
Aug 09
Sep 09
Oct 09
Jan 10
X
X
X
X
X
May 09
X
X
X
X
X
X
Dec 08
May 09
X
X
X
X
X
X
May 09
X
X
X
May 09
X
X
X
Aug 08
Apr 09
X
X
Dec 08
May 09
X
X
X
Aug 08
Apr 09
X
X
+/+/X
X
X
X
X
X
X
X
X
X
X
+
X
X
X
X
X
X
X
X
X
X
X
X
X
X
+/X
X
X
X
X
MOT is mammalian old tuberculin; PPD- A and PPD - B are avian & bovine purified bovine derivatives
respectively; + is positive; - is negative; +/- is suspicious; X is respective tests were not performed; Re,
Le, At and Lfa were the tests performed on right eyelid, left eyelid, abdomen & left forearm respectively.
22
3.2.2. Acid fast bacilli test
Nasogastric lavage samples were collected from chimpanzees A, D, E, F, G, H, I and J over
different period of time (Table 6). All samples were analyzed at human laboratory. Only sample
from chimpanzee H was reported to be doubtful but rest samples were reported as AFB not seen.
Table 6: Acid fast bacilli test from eight chimpanzees
ID
Date
Frequency
Result
A
D
E
November 2009
June 2009
April 2007
May 2009
June 2009
June 2009
June 2009
June 2009
June 2009
March 2010
One
One
One
One
One
One
One
One
One
One
Negative
Negative
Negative
Negative
Negative
Negative
Suspicious
Negative
Negative
Negative
F
G
H
I
J
3.2.3. Chest radiograph and therapy
Nine chimpanzees except chimp B were taken for their chest radiographs after suspicious skin
and Stat Pak tests. Chimpanzees A, D, F, G, I and J had no significant findings on chest
radiographs so considered normal (Figure 10). Chest radiograph from chimpanzee C showed
consolidation of right lung and she was put on full course of anti-tuberculosis drugs for nine
months. Chest radiograph following nine months of treatment showed no lesions in the lungs.
Chimpanzee E had been taken for several chest radiographs after and during the medication
period. Different changes and interpretation had been done. She was given first course of
treatment for eight months in 2007 following her suspicious skin tests results and close contact
with infected chimpanzee. After six months of treatment, radiographs showed left peri-hilar
inflammation. In early 2008, she had tuberculosis adenopathy lesions and again put on second
course of treatment for nine months. After the completion of second course of treatment, chest
radiograph was confirmed clear of tuberculosis. In 2009, she was tested positive for Skin and
Stat Pak tests. Chest radiographs could not revealed conclusive results and started third course of
23
treatment for one year. Chest radiographs for chimpanzee H showed adenopathy of right hilum
(Figure 11a) so put on full tuberculosis therapy. After the completion of six months of
tuberculosis treatment (Figure 11b), chest radiograph showed no lesions in the lungs.
Figure 10: Normal chest radiograph
a
b
Figure 11: Chest radiographs before and during the treatment
Adenopathy of right hilum (a) and radiograph with no lesions after six months of treatment (b)
24
The overall diagnostic tests performed in ten chimpanzees are presented in Table 7. Skin test
results are classified as PPD-A, PPD-B and MOT. These were the tests performed before
tuberculosis treatment started.
Table 7: Diagnostic tuberculosis tests with results in ten chimpanzees
ID
PPD-A
PPD-B
MOT
Stat Pak
AFB
Chest X-ray
A
-
-
+/-
+
-
NSF
B
+/-
+/-
+/-
+
X
X
C
-
-
+/-
-
X
CRL
D
-
+/-
+/-
-
-
NSF
E
-
+/-
+/-
+
-
LHA
F
+/-
+/-
+/-
-
-
NSF
G
-
-
+/-
-
-
NSF
H
+/-
+/-
+/-
-
+/-
RHA
I
-
-
+/-
+/-
-
NSF
J
+/-
-
+/-
-
-
NSF
PPD-A and PPD-B are avian and bovine purified bovine derivatives respectively
MOT is mammalian old tuberculin
AFB is acid fast bacilli test
+ is positive; - is negative; +/- is suspicious
X is respective test not performed
NSF is no significant findings
CRL is consolidation of right lung
LHA is left hilar adenopathy
RHA is right hilar adenopathy
3.2.4. Tuberculosis therapy protocol
All the chimpanzees received at least one or two or more than two combinations of drugs during
their course. As tuberculosis could not be neglected, all of them were treated with either
prophylactic or full anti-tuberculosis drugs even though some of them had normal radiographs
and were negative to acid fast bacilli but were suspicious with skin or Stat Pak tests in some
occasions. The drug therapy protocol for ten chimpanzees is presented in Table 8.
25
Table 8: Anti-tuberculosis medication of ten chimpanzees
ID
Sex
Agea
Weightb
Anti-tuberculosis drugs
Year
Duration
A
B
C
M
F
F
4
4
4
10.6
9.6
10.7
D
E
F
F
10
7
36
21.3
8
24
9
30
I1 + R1
R1 + P2
I1 + R1 + E1 + P1
I1 + R1
R1 + P2
I1 + R1 + E1 + P1
I1 + R1
I2 + R1 + E2 + P2
I2 + R1 + E2
R1 + E2 + P2 + O1
R1 + E2 + O1
R1 + E2
R1 + P2
I1
I1 + R1 + E1 + P2
I1 + R1
I1
R1 + P2
2009/10
2010
2007/08
2008
2009
2007
2007
2008
2008
2009
2009
2009/10
2009
2009
2009
2009
2009
2009
3
2
2
7
2
6
2
3
6
2
5
5
2
2.5
2
4
2.5
2
F
G
H
M
F
F
9
9
10
28
36
36
I
J
F
F
13
10
34
33
a
and b are respective age (in years) and weight (in kilograms) during medication
I1 and I2 are Isoniazid @ 5 and 10mg/kg respectively
R1 is Rifampicin @ 10mg/kg
P1 and P2 are Pyrazinamide @ 25 and 30mg/kg respectively
E1 and E2 are Ethambutol @ 20 and 25mg/kg respectively
O1 is Ofloxacin @ 10mg/kg
M is male and F is female
Duration in months
3.2.5. Culture and PCR
Mycobacterial culture and PCR was done for chimpanzees A, B, C, D and E at AHVLA
laboratory in the United Kingdom. The inoculated media sets were incubated for six weeks at
37°C and preliminary reported as no growth was evident in the solid or liquid media. The media
sets were incubated for a further eight weeks (fourteen weeks in total) to allow time for the
slower growing mycobacteria species to emerge. The cultures were negative for mycobacterial
growth at fourteen weeks. The DNA was run through a Taqman® based Rotor-Gene JOE RealTime analysis PCR using a Corbett Rotorgene PCR thermocycler (Corbettlifescience.com)
26
Taqman® IS 1081 primers, probe and Sybr Green fluorescent dye. M. bovis known positive and
M. avium known negative controls were run with the chimpanzee samples. All chimpanzee
samples were a clear negative whilst the M. bovis and M. avium showed the analysis expected for
those controls.
All ten chimpanzees were tested negative with skin and Stat Pak tests after the completion of
tuberculosis treatment in 2010/11/12. PPD-A/B and MOT tuberculin were used in chimpanzees
and were negative. Stat Pak tests were also carried out simultaneously with skin tests. All the
chimpanzees except chimpanzee E were negative with Stat Pak. Chest radiographs for
chimpanzees C, E and H (after the completion of treatment) were taken. Tracheal wash samples
from chimpanzees A, B, C, D and E confirmed negative for tuberculosis by mycobacterial
culture and molecular typing. The diagnostic tests carried out are presented in Table 9.
Table 9: Tuberculosis tests after the completion of medications
ID
TST
Stat Pak
Chest X-ray
PCR
A
-
-
x
-
-
B
-
-
x
-
-
C
-
-
-
-
-
D
-
-
x
-
-
E*
-
+
?
-
-
F
-
-
x
x
x
G
-
-
x
x
x
H
x
-
-
x
x
I
-
-
x
x
x
J
-
-
x
x
x
TST is tuberculin skin test
- is negative; + is reactive
? is inconclusive
x is respective tests not performed
* second Stat Pak test done after 6 months was negative
27
Culture
4. Discussion
Application of Skin and PrimaTB Stat Pak tests for field settings
Most of the sanctuaries in Africa have access to only simple diagnostic facilities. In the
conservation settings like sanctuaries and rehabilitation or rescue centers it is not always easy to
test the animals unless there is a major concern of disease outbreak or serious illness or injuries.
In our study, there was no positive reaction with skin tests but PrimaTB Stat Pak test produced
two apparent blurred visible line or band in the test result. That is, when the subjects were
retested with PrimaTB Stat Pak after six months, there was no evidence of clear visible line or
band in the test. The skin tests remained negative in this occasion. Thus, while Stat Pak test has a
(albeit and non-significant) chance of producing false positives while no such effect was found
for the skin test. Our study reported testing with specific number of chimpanzees from one only
sanctuary. Even though TST is the mainstay tool for the ante mortem diagnosis of tuberculosis in
non-human primates, it should not be used alone for the screening as it has its own limitations.
The combination of TST with other immunological assays can increase the sensitivity and
specificity for the screening of tuberculosis in non-human primates (Lerche et al., 2008).
The combination of skin test and PrimaTB Stat Pak test, in the context of remote sanctuaries, is
the best way for the early detection of tuberculosis. Being so simple, reliable, accurate and
portable, PrimaTB Stat Pak assay would be a very good diagnostic tool in conjunction with skin
test for the remote field settings. PrimaTB Stat Pak assay had successfully detected the
antibodies with advanced tuberculosis infected monkeys where skin tests were consistently
negative. For the early and reliable detection of tuberculosis, a combination of skin test and
PrimaTB Stat Pak test gives the best testing algorithm in non-human primates (Bushmitz et al.,
2009; Lyashchenko et al., 2007). Intradermal skin test along with PrimaTB Stat Pak test had
been a good diagnostic tool for tuberculosis in rhesus, cynomolgus and African green monkeys
with high diagnostic sensitivity - 90% and specificity - 99% (Lyashchenko et al., 2007).
However this test has not been validated with great apes and is still under investigation (Unwin
et al., unpublished data). Only few diagnostic tests have been validated for the use on samples
collected from great apes. In most of the cases, validation is often complicated due to difficulties
in obtaining the samples. However, PrimaTB Stat Pak assay still needs to be validated by testing
its reliability for tuberculosis detection in infected populations of chimpanzees.
28
We chose PPD-A/B tuberculin antigen to MOT because of its standardized content, well defined
preparation and specific reaction pattern (Bushmitz et al., 2009; Miller, 2008). Moreover, the
comparative PPD-A/B tests not only help to differentiate reactions between tuberculosis
mycobacteria and mycobacteria other than tuberculosis but also increase the specificity of the
skin tests. We did not perform skin tests in the abdomen because intradermal abdominal tests
may be less accurate and difficult to score without sedation in non-human primates (Capuano et
al., 2003) but it is most commonly used when suspected non-human primates needs retesting
after the first test (Bushmitz et al., 2009). From the zoonotic point of view, we tested the
sanctuary staffs with tuberculosis tests at human health centers to make sure that everyone is free
of the disease. Staff test was done only with acid fast bacilli staining tests. All the relevant
history of the staff concerning tuberculosis was assessed before they joined the job at the
sanctuary. An annual check for staff tuberculosis tests had also been established. All the
volunteers are required to submit their tuberculosis test report before visiting the sanctuary. The
visitors do not have close contact with chimpanzees at the sanctuary.
Ante-mortem diagnostics and therapy in suspected chimpanzees
A variety of ante mortem tests were performed and treated accordingly for ten chimpanzees with
respect to its history and previous tests. Interpretation of these tests and results was quite
challenging so it was difficult to neglect the presence or absence of the disease. A combination
of comparative tuberculin testing with MOT and PPD, serological test - PrimaTB Stat Pak, chest
radiographs, AFB tests, mycobacterial culture and PCR of tracheal wash samples were
performed to rule out the disease from the ante mortem diagnostic point of view. MOT and PPDA/B were the two kind of tuberculins used to test the status of tuberculosis in this study over
different period. Serological test with Stat Pak were done as it can detect the antibodies against
the antigens. Chest radiographs and AFB tests were carried out following skin and Stat Pak tests
which helped to diagnose the disease in conjunction with other tests.
In our study, suspected quarantine chimpanzees stayed with people in the village for a reasonable
amount of time before moving to the sanctuary so we assumed that they might have been in close
contact with tuberculosis infected people but did not develop the disease. These quarantine
chimpanzees had been tested with skin and Stat Pak with suspicious results. All chimpanzees in
29
quarantine must go through at least two skin tests at an interval of 4 to 6 weeks and the result of
the first skin test can determine the following tests considering that no suspicious or positive case
is detected (Unwin et al., 2009; NIH, 2007). The presence or absence of tuberculosis with those
people could not be confirmed in this study as these chimpanzees come from different regions of
the country. It could be possible that as they were freely living they might have exposed to
environmental or non-tubercular mycobacteria (Watchman et al., 2011, Georoff et al., 2010).
The resident chimpanzees were exposed or lived with tuberculosis infected chimpanzee since
late 2006 in the sanctuary. Generally, chimpanzees are tested annually (Unwin et al., 2009; NIH,
2007) but in case of outbreak tests should be performed as early as possible. All of them had
undergone skin tests when they were received in the beginning and were negative. Detection of
positive animals is very difficult in early period of infection and in the advance stage of the
disease may be non-responsive. A comparative testing with MOT and PPD-A/B was performed
in all chimpanzees to differentiate the non-specific responses or sensitization. Understanding that
skin test alone is not enough to diagnose tuberculosis, we performed testing with a combination
of TST and Stat Pak simultaneously but the results were not satisfactory. In only one case, both
TST and Stat Pak tested positive. With remaining chimpanzees, either TST was suspicious or
Stat Pak was suspicious. The study in three species of monkeys showed that skin test along with
Stat Pak had a good diagnostic tool for tuberculosis with high diagnostic sensitivity and
specificity (Lyashchenko et al., 2007) but this test has not been validated with great apes and is
still under investigation (Wenker et al., 2012; Unwin et al., unpublished data). Chest radiographs
and AFB tests were carried out with all chimpanzees which helped to diagnose tuberculosis in
conjunction with other tests. Individuals, humans and animals, reacting to skin tests with no
previous history of reaction are highly recommended for chest X-ray (NIH, 2007; Whitey et al.,
1973). This test is very valuable to detect non-human primates that have negative skin tests due
to immunosuppression at the end stage of the disease. However, differential daignosis should be
confirmed with pneumonia, nocardiosis or aspergillosis (Kunimoto and Long, 2005; Malaga et
al., 2004). In non-human primates, calcified lesions are rarely developed and even if present it is
very difficult to observe, possibly due to the relatively small size of lesions (Wenker et al., 2012;
Capauno et al., 2003). Therefore, radiographs are more difficult to interpret and must be
consulted to an experienced radiologists or lung physicians for better interpretation.
30
With endangered animals like chimpanzees, treatment with the intention of curing the disease by
detecting it at an early stage is preferable. The standard protocol of two months of isoniazid,
rifampin, pyrazinamide and ethambutol followed by four months of isoniazid and rifampin was
practiced in wildlife (Daly et al., 2006). Chimpanzees were treated with anti-tuberculosis drugs
due to suspicious results and exposed with tuberculosis infected chimpanzee. The disease status
in those exposed or suspected chimpanzees were not confirmed as the test results were
inconsistent. Despite all these tests, tuberculosis could not be totally ignored and risk could not
be taken, all the chimpanzees were treated accordingly for tuberculosis. The protocols for the
treatment of tuberculosis in these chimpanzees were based on the assumption that chimpanzees
with suspicious or exposed tuberculosis were treated similarly to humans who were exposed or
lived together with tuberculosis infected people. For the individuals who are exposed to
tuberculosis but have no signs of active disease, the length of treatment ranges from six months
to one year typically done with a single drug. However, multiple drug therapy with isoniazid,
rifampin, pyrazinamide and ethambutol is recommended so as to prevent from the emergence of
resistant bacteria (American Thoracic Society, 2003).
Orangutans (confirmed or highly suspected tuberculosis positive) had been treated with isoniazid
@ 5mg/kg b wt, rifampin @ 10mg/kg b wt, ethambutol @ 22mg/kg b wt and pyrazinamide @
24mg/kg b wt as per WHO recommendations (Calle, 1999). In our study, full treatment course
ranges from six months to one year and prophylactic course ranges for two to three months only
because the outcome and efficacy of treatment of non-human species is not clearly known
(Mikota and Maslow, 2011). During the treatment period, skin test with Stat Pak and chest
radiographs were taken regularly to see the improvement. Non-human primates receiving anti
tuberculosis treatment give false negative result with tuberculin tests because these drugs are
immunosuppressive (Theon, 1993; Gibson et al., 1971). A rhesus monkey with confirmed
tuberculosis (after necropsy) showed 120 consecutive negative tuberculin tests which received
isoniazid and ethambutol for nine years (Dillehay and Huerkamp, 1990). Non-human primates
that have received anti tuberculosis treatment may still have the disease and should screen those
animals with other ancillary tests.
31
Irrespective of skin, Stat Pak, AFB tests and radiograph results, we performed mycobacterial
culture and PCR from tracheal wash samples from five suspected chimpanzees. Collecting
tracheal wash samples for mycobacterial culture and PCR is the most convienent method in nonhuman primates (Wenker et al., 2012; Unwin et al., 2009). Mycobacterial culture remains the
golden standard test for the diagnosis of TB (De Lisle et al., 2002). Polymerase chain reaction
detects mycobacterial DNA in any biological samples and intrinsically has the advantage of
being much quicker than the conventional culture methods of diagnosis. Non-invasive samples
such as saliva, urine and faeces have been used to detect the infection by PCR for mycobacterial
DNA (Calvignac-Spencer et al., 2012; Koendgen et al., 2010; Leendertz et al., 2006). Hence, it is
considered as a rapid and alternative diagnostic tool for tuberculosis. Application of new
diagnostic technology may allow more rapid and accurate detection of tuberculosis in nonhuman primates. Availability of kits and funds will continue to be a major obstacle for diagnostic
technology in the sanctuaries. Tuberculosis diagnosis in primate sanctuaries can be improved by
creating an international expert network which can monitor the current facilities and provide
funds for mycobacterial culture and PCR in suspected cases. The network can co-ordinate to
assess future diagnostic advances such as faecal or urine based PCR which can detect
tuberculosis early and be field friendly. No diagnostic method is accurate on its own and as many
techniques as possible must be combined to reduce ‘false negative’ results.
Due to technical difficulties and costs involved, we could not perform mycobacterial culture and
molecular typing for all chimpanzees. However, confirmed negative results with those performed
indicate there was no existence of tuberculosis with other chimpanzees at the sanctuary. Neither
antibiotic sensitivity tests nor the serum level of the drugs was determined during the treatment
peroid due to unavailability of the specific laboratory tests in Sierra Leone. Although blood
serum levels of drugs were not analyzed, we believe those multi drug therapies in these
chimpanzees have resulted for the destruction of the microorganisms.
32
5. Conclusion
There is no ante mortem diagnostics which is 100 % reliable for the diagnosis of tuberculosis.
For the early and reliable detection of tuberculosis, a combination of skin test and Stat Pak assay
gives the best testing algorithm in non-human primates especially for remote sanctuaries. Any
suspicious or positive cases with skin or Stat Pak tests should be retested and further diagnosis
should be done with more reliable tests like other blood assay tests, diagnostic microbiology,
chest radiographs, mycobacterial culture and PCR. The treatment should be strictly monitored.
Mycobacterial culture and isolation of mycobacteria remain a golden standard for the
tuberculosis diagnosis. Molecular techniques like PCR detects mycobacterial DNA as an
alternative diagnostic tool for tuberculosis but these diagnostics are generally expensive,
impractical and inaccessible in remote field settings. Tuberculosis cannot be diagnosed with one
tool but rather should be confirmed with other available tests. To rule out the disease, there
should be a well-established protocol in the sanctuary to test tuberculosis for both humans and
non-human primates in regular interval of time.
33
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40
Management of Tuberculosis under field conditions in non-human primates
among Pan African Sanctuary Alliance members
41
Abstract
Disease management is an important task in the primate sanctuaries which primarily care
different species of primates for rehabilitation and reintroduction. A number of zoonotic diseases
exist within the primate species and tuberculosis is one of them. A survey questionnaire was
conducted among 22 Pan African Sanctuary Alliance (PASA) members across 12 African
countries, from which 13 sanctuaries replied. The main aim of this study was to determine the
strategies currently adopted by PASA sanctuaries members for the management of tuberculosis
in non-human primates. Screening for tuberculosis to all new arrivals and resident primates with
skin test was practised in all sanctuaries; skilled veterinarians or vet technicians performed these
tests. Intrapalpebral inoculation of tuberculin was a quite popular method of testing in most of
the sanctuaries. Only few other diagnostics had been applied as they were generally
impracticable and inaccessible for field setting sanctuaries. Staffs were continuously tested for
tuberculosis before joining and during the job period though varies in timing and type of tests
performed. Only few sanctuaries responded to provide details about the status of tuberculosis and
its medication either they were reluctant to share or were not on top of tuberculosis management
at their sanctuaries. Regarding record keeping and hygiene management, all the sanctuaries
maintained a similar trend to keep data on tuberculosis testing for future reference. We
understood that tuberculosis management in field sanctuaries is complicated by difficulties with
detection, medication and prevention in both humans and non-human primates. With this study,
we provided the evidences of tuberculosis testing and the strategies to handle this disease at
different primate sanctuaries in Africa. The results of this survey could enable sanctuaries to
develop and improve strategies for better handling and management of tuberculosis and
potentially other infectious diseases in future, through learning from each other.
Keywords: PASA, Tuberculosis testing, sanctuary, non-human primates, Africa.
42
1. Introduction
A global review of the world's primates found that 49% of species are threatened or face
extinction (IUCN, 2013). More than 40% of African primates are threatened with extinction due
to illegal pet trade, bush meat trade, deforestation, infectious disease and climate change (Stiles
et al., 2013; Mittermeier et al., 2009; Oates et al., 2008; Chapman et al., 2006; Grubb et al.,
2003). Over the last ten years in Africa, there has been an alarming increase in the number of
non-human primates, especially chimpanzees, being influx to the sanctuaries or rescue or
rehabilitation centers as a result of the bust meat hunting, habitat lost and human wildlife
conflict. Most of the by products and/or victims of such activities are orphan babies which end
up reaching the sanctuaries either by confiscation or handed in voluntarily or after being
sensitized about the wildlife law (Beck, 2010; Kabasawa, 2009; Farmer, 2002). It is estimated
that for every live captive ape, the poachers killed 5 to 15 apes which includes the mother and
often the other members of the social group for bush meat or to retrieve the infants as a pet and
not all the infants survive (Kormos et al., 2003; Teleki, 1989; Goodall, 1986). The number of
orphaned primates in the sanctuaries or rescue centers have increased and continued to increase
(Faust et al., 2011). This increasing number of orphaned primates has led to the establishment of
an umbrella organization across Africa called the Pan African Sanctuary Alliance - PASA in
2000 (Mills et al., 2005; Farmer, 2002).
PASA is an alliance of 22 accredited sanctuaries across 12 countries in Africa (Figure 12) caring
a variety of non-human primates which includes chimpanzees, gorillas, bonobos and different
species of endangered monkeys (Table 10). 19 out of 22 sanctuaries house an ape that includes
chimpanzees, gorillas and bonobos. PASA member sanctuaries are committed to provide the best
possible facilities and care to captive African primates, while working towards the protection and
conservation of the species by engaging them in community outreach and education programs
with a view of reintroduction to wild. Veterinary care and disease management is a challenging
aspect to maintain sound health and to prevent entering disease in the sanctuary. In most of the
sanctuaries necessary measurements have been applied to prevent the dissemination of diseases.
However, the sanctuaries are not completely resistant to spreading of zoonotic or infectious
diseases because non-human primates live in close proximity to humans and there is always a
high risk of disease transmission between non-human primates and humans and vice versa. From
43
the reintroduction point of view also management of infectious or zoonotic disease is very
important before they are being introduced to the wild as 11 sanctuaries are engaged in
reintroduction programs (Faust et al., 2011). Infectious or zoonotic diseases are also likely to
spread if non-human primate species are reintroduced into wild without proper screening of such
diseases. Several epidemic outbreaks which caused massive mortality of non-human primates in
the wild population have been reported. Among various zoonotic and infectious diseases,
tuberculosis has always been a threat to human, domestic animals and non-human primates
(Garcia et al., 2004; Lerche et al., 2008).
Tuberculosis is one of the most concerning diseases found in non-human primates worldwide,
because of its high incidence, zoonotic potential and high mortality rates. Due to its zoonotic
nature, it is very important to diagnose and prevent the spread of tuberculosis between human
and non-human primates. It becomes more important in the context of captive settings such as
sanctuaries and rehabilitation centers, where they live in groups and are in close proximity with
humans. Understanding that no single test is 100% reliable, a wide range of diagnostics has been
developed over time. In non-human primates, tuberculosis diagnosis is quite challenging because
of lack of diagnostic sensitivity and specificity among the available diagnostics and poorly
defined clinical features of the disease (Gibson, 1998). Infected non-human primates with active
tuberculosis can easily transmit the disease to other healthy non-human primates and humans. On
the other hand, non-human primates with latent tuberculosis are not infectious but always
possess a significant risk of reactivation and progress to active tuberculosis (Capuano et al.,
2003; Flynn and Chan, 2001). It is very important to have an early, sensitive and accurate
diagnostic tool for the surveillance and control of tuberculosis so that further spread of the
disease can be prevented.
44
Photo courtesy: PASA
Figure 12: Distribution of primate sanctuaries in twelve countries across Africa
Cameroon, Congo and DR Congo have three sanctuaries per country; Gabon, Kenya, Nigeria and
South Africa have two sanctuaries per country; Gambia, Guinea, Sierra Leone, Uganda and
Zambia have one sanctuary per country.
In captive field settings, tuberculosis diagnosis is further complicated because of unavailability
of well-equipped diagnostic facilities. The tuberculin skin test is the most common and basic
tuberculosis screening test practiced in non-human primates. However, we know very little about
which diagnostics and preventative measures are followed for different non-human primate
species, and in different sanctuaries, when considering tuberculosis. This research will help to
45
establish a basic and effective tuberculosis diagnostic method and preventative measures,
particularly for a small captive setting where sophisticated tests and equipments are not easily
accessible. No overview of the subject has been conducted before, and a survey of what
strategies the individual sanctuary apply would be interesting to conduct, in order to define and
assess the similarities (or otherwise) in approaches for the management of tuberculosis across
sanctuaries. The results of such survey could enable sanctuaries to develop and improve
strategies for the handling and management of tuberculosis, through learning from each other in
future. The knowledge shared and gained in this field will not only increase the health and
welfare of non-human primates, but will also create valuable ties among the PASA members
across Africa continent.
The main aim of this paper is to identify and share the strategy or strategies which have been
implemented by different PASA sanctuaries across Africa for the management of tuberculosis.
We hope this survey will provide useful information for all PASA members in the future to
better manage tuberculosis and potentially other infectious diseases.
2. Methodology
A survey questionnaire both in English (sample attached in the Appendix) and French version
was created to study the monitoring and management strategies of tuberculosis in non-human
primates from PASA member sanctuaries in Africa. The survey questionnaire was distributed
among the veterinarians and directors or managers from different sanctuaries during PASA
veterinary workshop held on 18-22th February 2013 at Limbe Wildlife Center in Cameroon. We
chose to distribute the questionnaire during the workshop as most of the sanctuaries do not have
easy or frequent access to internet. Prior to this, the questionnaire was first mailed out via email
by PASA veterinary director on 4th February 2013 to all sanctuaries directors, managers and
veterinarians. We requested them to review it before coming to the workshop. For those who are
not the sanctuary directors, or managers, we asked them to get their permission to fill it in on the
sanctuary's behalf at the workshop. We received good responses from different sanctuaries
during the workshop. The final mailed out was circulated via email to those who could not fill
out during the workshop on 23rd June 2013.
46
47
1997
1995
1983
1997
1974
1991
2001
1990
2006
2006
1993
1994
2002
1998
1988
1988
1999
1994
1995
1992
1993
Centre pour Conservation des Chimpanzees
CERCOPAN
Chimpfunshi Wildlife Orphanage
Colobus Conservation Limited
Chimpanzee Rehabilitation Project
Drill Ranch
Fernan-Vaz Gorilla Projet
Habitat Ecologique et Liberte des Primates
Jane Goodall Institute - Chimpanzee Eden
Jeunes Animaus Confisques au Katanga
Limbe Wildlife Center
Lola ya Bonobo
Lwiro Primate Rehabilitation Center
Ngamba Island Chimpanzee Sanctuary
Projet Protection des Gorilles
Projet Protection des Gorilles
Sanaga-Yong Chimpanzee Rescue Center
Sweetwaters Chimpanzee Sanctuary
Tacugama Chimpanzee Sanctuary
Tchimpounga Chimpanzee Rehabilitation Center
Vervet Monkey Foundation
South Africa
Congo
Kenya
Sierra Leone
Gabon
Cameroon
Congo
DR Congo
Uganda
Cameroon
DR Congo
South Africa
DR Congo
Congo
Nigeria
Gabon
Kenya
The Gambia
Nigeria
Zambia
Guinea
Cameroon
Country
Monkeys
Chimpanzees and Monkeys
Chimpanzees
Chimpanzees
Gorillas
Chimpanzees
Gorillas
Chimpanzees and Monkeys
Chimpanzees
Chimpanzees, Gorillas and Monkeys
Bonobos
Chimpanzees
Chimpanzees
Chimpanzees
Chimpanzees and Monkeys
Gorillas
Monkeys
Chimpanzees
Monkeys
Chimpanzees and Monkeys
Chimpanzees
Gorilla and Chimpanzees
Primates in the sanctuaries
Retrieved from www.pasaprimates.org on 23 October 2013; Farmer, 2002.; DR is Democratic Republic.
1996
Ape Action Africa
a
Founded
Name of the sanctuaries
Table 10: List of twenty two sanctuaries accredited to PASA as of October 2013 a
The survey questionnaire was distributed along with cover letter and guidelines to facilitate the
completion in time without any difficulties. The responders had always an option to ask if they
were not clear with any subject matters. Even the responders were asked to notify in case they do
not want to participate in this survey. The survey consists of 6 sections, each containing specific
relevant questions under particular topics of interest.
Section A: It consists of general background of the sanctuary along with current number and
species of non-human primates.
Section B: It consists of 8 questions about the tuberculosis testing procedures during the
quarantine period. We assumed that all PASA member sanctuaries followed its guidelines for
tuberculosis testing with respect to primate species they hold.
Section C: It consists of 10 questions seeking information on current tuberculosis diagnostic
procedures being followed and the tests available in the respective sanctuaries.
Section D: It is a sensitive section with 5 questions where the sanctuaries were requested to
provide information on tuberculosis medication in case they had tuberculosis outbreak in the past
and were subjected to the therapy. All the members were assured that their data will remain
confidential for this section.
Section E: It consists of 5 questions to know about primate care staff's tuberculosis testing.
Section F: It consists of 8 questions on husbandry and management practices such as information
on record keeping, hygiene, enclosure setting and the availability of tuberculosis kits.
We analysed the data based on the information provided by the sanctuaries to the questionnaire.
Most of the questions were answered and in case it was not answered the reason was provided.
Two sanctuaries: Chimpanzee Rehabilitation Project in the Gambia and HELP - CONGO could
not fill out completely because this questionnaire was not really applicable as they no longer
accept chimpanzees. We got responses from the 13 sanctuaries. Although PASA sanctuaries
house a variety of non-human primates that includes chimpanzees, gorillas, bonobos and
different species of monkeys, 12 out of 13 responded sanctuaries housed chimpanzees. We did
not get responses from the sanctuaries which housed gorillas and bonobos. Therefore, most of
the information provided in this study reflects tuberculosis management data from chimpanzees
and monkeys. As our questionnaire survey was distributed among a small number of sanctuaries,
we did descriptive analysis of our data which was considered to be the most appropriate.
48
3. Results
3.1. General information about the sanctuaries
In total, 13 PASA sanctuaries responded spread in 10 African countries (Table 11). Two
sanctuaries responded that due to their sanctuary management policy they could not share the
information regarding the infectious diseases and other 7 sanctuaries did not respond.
Interestingly, 12 out of 13 reported sanctuaries housed 849 chimpanzees. The questionnaire
reporting persons were 6 veterinarians, 3 directors or managers and 4 both veterinarians and
directors or managers. Two sanctuaries (Lwiro Primate Rehabilitation Center and Tchimpounga
Chimpanzee Rehabilitation Center) housed both monkeys and chimpanzees while Vervet
Monkey Foundation housed vervet monkeys only. We could not get the population of gorillas
and bonobos for this study as the sanctuaries holding this species did not respond to our
questionnaire.
Table 11: Sanctuaries that responded to the survey
Sanctuary*
Location
N
Primate species
Centre pour Conservation des Chimpanzees (CCC)
Guinea
43
Chimps
Chimpfunshi Wildlife Orphanage (CWO)
Zambia
126
Chimps
Chimpanzee Rehabilitation Project (CRP)
The Gambia
103
Chimps
Habitat Ecologique et Liberte des Primates (HELP)
Congo
42
Chimps
Jane Goodall Institute - Chimpanzee Eden (JGI)
South Africa
34
Chimps
Jeunes Animaus Confisques au Katanga (JACK)
DR Congo
38
Chimps
Lwiro Primate Rehabilitation Center (LPRC)
DR Congo
55+75 Chimps+Monkeys
Ngamba Island Chimpanzee Sanctuary (NICS)
Uganda
48
Chimps
Sanaga-Yong Chimpanzee Rescue Center (SYCRC)
Cameroon
72
Chimps
Sweetwaters Chimpanzee Sanctuary (SCS)
Kenya
39
Chimps
Tacugama Chimpanzee Sanctuary (TCS)
Sierra Leone
98
Chimps
Tchimpounga Chimpanzee Rehabilitation Center (TCRC)
Congo
151+8 Chimps+Monkeys
Vervet Monkey Foundation (VMF)
South Africa
540
* Abbreviations are used for the following notifications
N is the current number of primates during reporting
DR is Democratic Republic
49
Monkeys
All chimpanzees from Chimpanzee Rehabilitation Project in the Gambia live freely in 4 social
groups in 3 islands for more than 10 years. They do not intervene with chimpanzees and no
longer accept chimpanzees so this questionnaire did not really applicable to this sanctuary.
However, they have tuberculosis tested all their chimpanzees and provided the information what
they did before releasing them free in the islands. HELP - Congo sanctuary also no longer
accepts new orphan chimpanzees since many years so they do not deal with tuberculosis tests.
Their adult chimpanzees live freely in the forests and on the islands. All new arrivals they
recommend to Tchimpounga Chimpanzee Rehabilitation Center, Congo. Some sections of this
questionnaire were not really applicable to this sanctuary but they provided the responses which
was relevant to them. Rests of the sanctuaries are continuously accepting the new arrivals even
though they are over their holding capacity.
3.2. Tuberculosis tests during quarantine period
All sanctuaries reported that every new arrival passes through the quarantine period irrespective
of the species and the origin. Except HELP – Congo sanctuary, other twelve sanctuaries provided
information on their tuberculosis tests during the quarantine (Table 12). HELP - Congo project
no longer accepts new chimpanzees so they do not deal with tuberculosis tests. The Chimpanzee
Rehabilitation Project in the Gambia also no longer accepts chimpanzees but provided the
information on tuberculosis tests based on what they performed before.
Six sanctuaries (50%) reported that the duration of quarantine period for tuberculosis testing was
three months. Four sanctuaries reported that they finished it in less than three months. One
sanctuary each finished it in more than three months and in three months or more than three
months. Seven of the sanctuaries (58%) performed skin test only one time during quarantine
period while three sanctuaries (25%) and two sanctuaries (17%) performed three and two skin
tests respectively. All the sanctuaries agreed that skin test reading was done in every 24 hours
following the test day. There was mixed results about the time period or interval between the
tests. Three sanctuaries reported that they could not specify the exact time. Three sanctuaries
maintained a month of interval time between the tests and the other two maintained 1.5 months.
Two sanctuaries reported that they repeat the tests if it becomes suspicious. One sanctuary
50
performed at every 15 days. All sanctuaries isolated their new arrivals from rest of the existing
groups and used separate feeding bowls or utensils.
Table 12: Tuberculosis tests for quarantine primates
Sanctuary
Duration§
Frequency*
Interval#
Who performed?
CCC
Three
One
Wait and see†
Vet/Manager
CWO
Three β
One
-
Vet
CRP
Three
One
-
Vet/Manager
JGI
Three
One
2 months
Vet
JACK
Three
One
Wait and see†
Vet
LPRC
Three β
One
-
Vet
NICS
Threeµ
One
1 month
Vet
SYCRC
Three
Two
1.5 months
Vet/Vet Tech
SCS
Three
Three
1 month
Vet
TCS
Three¥
Three
1 month
Vet
TCRC
Three β
Three
15 days
Vet/Vet Tech
VMF
Three β
Two
1.5 months
Vet Tech
§
- Length of quarantine period in months
- Performed in less than three months
µ
- Required more than three months
¥
- Three months for healthy but more than three months for suspicious
* - Number of skin tests performed during the quarantine period
#
- Time period between the successive tests in the quarantine
†
- If suspicious, repeat in one month
β
In all the sanctuaries, tuberculosis test was done by recognized veterinarians or veterinarian
technicians. Only in few cases, the managers themselves performed the tests as they were well
trained. In some sanctuaries the veterinarians themselves are the managers.
3.3. Tuberculosis diagnostics
All twelve sanctuaries provided information on the diagnostics used for tuberculosis in their
sanctuaries (Figure 13). Besides tuberculosis tests in quarantine period, seven sanctuaries tested
51
their non-human primates ‘opportunistically’ – refers when non-human primates are sedated for
other reasons’’; three sanctuaries tested them biannually and two tested annually. All sanctuaries
used tuberculin antigen for the skin tests.
Figure 13: Types of tuberculosis diagnostics used in the sanctuaries
All twelve sanctuaries used tuberculin to test their non-human primates. Five sanctuaries used
both purified bovine derivatives – avian/bovine (PPD-A/B + MOT), four sanctuaries used only
purified bovine derivatives – avian and bovine (PPD-A/B) and three sanctuaries used only
mammalian old tuberculin (MOT). Seven sanctuaries (58%) performed tuberculin skin test along
with Stat Pak kit. Four sanctuaries (33%) performed others tests like Acid fast bacilli tests, Chest
X-ray, mycobacterial culture and PCR if they have suspicious or positive case (Figure 13).
Interestingly, ten sanctuaries reported that they had encountered with false positive or negative
reaction with skin tests. All the sanctuaries preferred eyelid as the best site to perform tuberculin
tests. Three sanctuaries preferred both eyelid and abdomen. Seven sanctuaries routinely
performed necropsies of tuberculosis suspected or positive non-human primates, two were
unresponsive for necropsy and nine preferred to collect samples from the necropsied ones. Three
sanctuaries reported they neither perform necropsy nor collect samples from necropsied ones.
52
3.4. Tuberculosis medication
We got responses from thirteen sanctuaries for this section. As this section seeks information
about suspicious or positive tuberculosis and medication in case they had tuberculosis outbreak,
we assured the sanctuaries that their data will remain confidential for this section.
Figure 14: Number of sanctuaries reporting for tuberculosis cases
Seven of the sanctuaries (54%) reported that they had suspicious cases of TB while six (46%)
reported they did not have it (Figure 14). All these seven sanctuaries agreed that the suspicious
cases should be retested until it was confirmed and two of them recommended that suspicious
non-human primates must be put under prophylaxis treatment.
Four of the sanctuaries (30%) had tuberculosis positive cases in their sanctuaries (Figure 14) and
three of them had successfully treated with multi drug therapy (Isoniazid + Rifampin +
Pyrazinamide + Ethambutol). The remaining one did not share their treatment history. Nine of
the sanctuaries (70%) reported that they did not have tuberculosis positive case.
53
Figure 15: Response for tuberculosis positive cases
Six sanctuaries recommended for full tuberculosis treatment, three recommended for euthanasia
and four could not recommend in case if tuberculosis turns out to be confirmed positive at their
sanctuary in the future (Figure 15).
3.5. Sanctuary staff tuberculosis tests
Thirteen sanctuaries provided data about their staff tuberculosis testing (Table 13). Most of the
sanctuaries reported that their staffs were vaccinated with BCG when they were child. Nine of
the sanctuaries (70%) tested their staffs before recruiting to work with non-human primates at the
sanctuary. Twelve of the sanctuaries (92%) performed their staff’s tuberculosis tests (Mantoux or
sputum or thoracic radiographs) either in the sanctuary or took them to the human hospitals.
Most of the sanctuaries performed these tests either annually or semi-annually. Ten sanctuaries
reported that they had not come across with suspicious tuberculosis case with their staff while
working at the sanctuary while only three had such cases for which they recommend for recheck
until it is confirmed. Surprisingly, two of the sanctuaries reported that they had tuberculosis
positive staffs and they were put under tuberculosis treatment.
54
Table 13: Tuberculosis tests for the sanctuary staffs
Sanctuary
Before recruit At sanctuary
How often?
Suspicious/Positive
CCC
No
No*
-
No/No
CWO
Yes
Yes
Annually
CRP
JGI
No
Yes
†
No/No
Yes
M
Others
Yes
S
Annually
No/No
M+S+R
Annually
Yes§/No
No/No
JACK
Yes
Yes
HELP
No
YesR
OthersѰ
No/No
LPRC
Yes
YesS
Semi-annually
Yes§/Yes‡
NICS
Yes
YesM
Annually
No/No
SYCRC
No
YesM+S+R
Others#
Yes§/Yes‡
SCS
Yes
YesS
Semi-annually
No/No
TCS
Yes
YesS
Annually
No/No
TCRC
Yes
YesM+R
Annually
No/No
VMF
Yes
YesR
Annually
No/No
*- Planned to check with thoracic radiograph
- Rechecked until confirmed
M
- Mantoux test
S
- Sputum test
R
- Thoracic radiograph
†
- Tested only once when they used to handle the chimpanzees
#
- Annually radiographed for skin positive staffs and biannually for other staffs
Ѱ
- Radiographed only once during recruiting for the job
‡
- Put under tuberculosis treatment
§
3.6. Husbandry and management practices
All thirteen sanctuaries kept records about the previous history of non-human primates on arrival
and for each of tuberculosis tests performed once arrived at the sanctuary. All of their primate
care takers use protective clothings, boots or masks while working with non-human primates.
Twelve of the sanctuaries (92%) used bleach as a disinfectant in their sanctuaries (Figure 16).
55
Figure 16: Hygiene and record keeping in the sanctuaries
Seven of the sanctuaries cleaned their dens or cages once in the morning per day and four
cleaned it twice in the morning and the evening. All of them cleaned once their non-human
primates went outside in the enclosures. Two sanctuaries Chimpanzee Rehabilitation Project in
the Gambia and HELP - Congo have free living chimpanzees so they did not have to clean their
cages or dens.
Nine sanctuaries had their enclosure attached to each other, two had completely isolated from
each other while two sanctuaries reported that they have both attached and isolated enclosure
settings (Figure 17).
56
Figure 17: Enclosure settings in the sanctuaries
Ten of the sanctuaries (77%) used to bring their tuberculin or blood assay kits from abroad or
through PASA (Figure 18). Only three sanctuaries (23%) from South Africa, Uganda and Kenya
have easy access to buy tuberculin or other kits in their own country. Nine of the sanctuaries
(70%) maintained the tuberculin vials in the cold chain system at the sanctuary.
Figure 18: Accessibility of tuberculosis kits in the sanctuaries
57
4. Discussion
This study was aimed at determining the strategies currently adopted by PASA sanctuaries
members across Africa for the management of tuberculosis in non-human primates. The
population of non-human primates in the wild is decreasing while the population in captivity like
sanctuaries, rescue centers is increasing. PASA sanctuaries rescue and care for thousands of
African primates confiscated from the illegal bush meat or pet trade or habitat destruction.
Though PASA was founded in 2000, the establishment of primate sanctuaries in Africa dated
back in the mid-1980s. Since then there has been an increase in the number of primate
sanctuaries in Africa. In the beginning, 11 sanctuaries from 10 African countries were affiliated
with PASA (Farmer, 2002) while this number increases to 22 accredited sanctuaries across 12
African countries in 2013 (www.pasaprimates.org). The increase in number of primate
sanctuaries is a clear indicative of loss of primate habitat and increase in bush meat or pet trade.
In most of the cases, the victims of such illegal activities are primate orphans who ended up
reaching to the sanctuaries or rehabilitation centres. The increase of chimpanzee sanctuaries in
Africa is a direct consequence of increasing in number of orphanage chimpanzees (Cox, 2005).
The questionnaire was designed to gain information on all species of non-human primates from
the sanctuaries. PASA sanctuaries housed chimpanzees, gorillas, bonobos and a variety of
monkeys. Majority of the sanctuaries are largely focused on caring chimpanzees. Our result
showed that 13 out of 22 PASA sanctuaries housed 849 chimpanzees and 623 monkeys.
According to (Faust et al., 2011), 15 out of 22 PASA sanctuaries housed a population of more
than 877 chimpanzees. The trend of orphanage non-human primates incoming to the sanctuaries
is increasing day by day. Apart from the illegal bust meat trade or habitat loss, this increase in
number is due to the implementation of strict wild life laws, confiscation procedures and primate
conservation awareness programs in African countries (Kuhar et al., 2012; Humle et al., 2011).
All sanctuaries provide the best possible care to the resident non-human primates with only the
primates that have cleared all quarantine are allowed to join the other social group. All the new
arrivals must be kept isolated in quarantine to ensure proper examination and disease screening
before introducing to other at the sanctuary. The sanctuaries receive different species of primates
from different places and of origin so there is always a high risk of transmission of infectious or
58
zoonotic diseases. Tuberculosis is one the serious zoonotic diseases that all sanctuaries screen
during their quarantine period. The length of quarantine period is dictated by the disease of
concern and health status of rescued non-human primates. Normally three months is referred as a
minimum period to allow investigating the disease of concern (Farmer et al., 2009; Boardman et
al., 2004). Irrespective of origin and health status, all new arrivals should undergo the quarantine
period. If non-human primate is in good condition, then all the tests can be carried out in time
otherwise it will prolong the quarantine period and so the tuberculosis testing. The prolonged
period might be due to poor health condition of non-human primates or suspicious tuberculosis
tests results or known exposure to tuberculosis (Beck et al., 2007).
Chimpanzees, gorillas and bonobos in quarantine must go through at least two skin tests at an
interval of 4 weeks whereas monkeys must go through three to five skin tests at an interval of 2
weeks (Unwin et al., 2009; NIH, 2007). The result of the first skin test can determine the
following tests considering that no suspicious or positive case is detected. After the quarantine,
the recommended skin testing for great apes is annually and for monkeys is either quarterly or
semi-annually. The gap between two skin tests also depends on non-human primate species.
Normally great apes are tested at an interval of a month while the monkeys are tested in every 15
days until 3 to 5 negative tests are performed. Skin tests results may be affected by false positive
or negative reactions so one should be careful during recording the reaction on the following
days. False positive reactions occur from trauma associated during the test, exposure to nontuberculous environmental mycobacteria, prior vaccination with BCG and antigenic crossreactivity (Vervenne et al., 2004; Andersen et al., 2000; Dukelow and Pierce, 1987). False
negative reactions occur due to latent tuberculosis infection, anergy associated with progressive
diseases, therapy with immunosuppressive drugs, vaccination with polio, measles or yellow fever
(Motzel et al., 2003; Staley et al., 1998), incorrect injection or concentration and
misinterpretation while reading the test.
Tuberculin has been the mainstay for tuberculosis testing in non-human primates (Roberts and
Andrews, 2008; NRC, 1980; Kennard et al., 1939). We reviewed two different types of
tuberculin – MOT and PPD-A/B. MOT shows a high degree of cross reactivity with atypical or
non-tubercular mycobacteria. PPD-A/B tuberculin antigen has standardized content, well defined
59
preparation and specific reaction pattern (Bushmitz et al., 2009; Miller, 2008). Moreover, the
comparative PPD-A/B tests not only help to differentiate reactions between tuberculosis
mycobacteria and mycobacteria other than tuberculosis but also increase the specificity of the
skin tests. Intrapalpebral (upper eyelid) test is the most preferred method for tuberculosis testing
in non-human primates as it is relatively easy to observe the delayed hypersensitivity response
against the antigens in unrestrained or awake non-human primates. Intradermal abdominal test is
difficult to score without sedation in the non-human primates but it is most commonly used when
suspected non-human primates needs retesting after the first test (Capuano et al., 2003).
For the field conditions, a sero diagnostic test, PrimaTB Stat Pak has been used which is based
on lateral flow immunochromatographic method. A combination of skin test and PrimaTB Stat
Pak assay gives the best testing algorithm in non-human primates (Lyashchenko et al., 2007).
Skin test along with PrimaTB Stat Pak had been a good diagnostic tool for tuberculosis in rhesus,
cynomolgus and African green monkeys with high diagnostic sensitivity - 90% and specificity 99% (Lyashchenko et al., 2007). However, this test has not been validated with great apes and is
still under investigation (Unwin et al., unpublished data). Under the field conditions especially in
Africa, other tuberculosis diagnostics like chest radiographs, AFB tests, mycobacterial culture
and PCR are not accessible and very costly. However, such sophisticated tests could be carried
out in other countries if the samples are preserved for future analysis.
Treatment of tuberculosis infections in non-human primates is generally discouraged and
euthanasia is recommended because of the public health hazard. However, rare and endangered
species like chimpanzees and other great apes should be treated assuring the public health
hazard, costs involved and ethical issues (Bushmitz et al., 2009; Montali et al., 2001). The
animals must be kept in strict quarantine conditions during the whole treatment period.
Treatment for tuberculosis infections has been effective in non-human primates (Wolf et al.,
1988; Ward et al., 1985) using human drugs like isoniazid, rifampin, pyrazinamide and
ethambutol. Isoniazid and rifampin are the primary drugs whereas pyrazinamide and ethambutol
are the secondary. Multidrug therapy may be considered which helps to reduce the possibility of
developing antibiotic resistance and must be done on the basis of antibiotic sensitivity testing
(Lyashchenko et al., 2006).
60
There is a high level of interaction between humans and non-human primates in the sanctuaries
which vows potential risks of disease transmission. The increasing frequency of human
tuberculosis in developing countries presents a serious threat to non-human primates in
sanctuaries or rehabilitation centres or zoological collections (Michel et al., 2003). If the disease
management protocol is not well addressed or followed, there is always a high risk of disease
transmission between non-human primates to human staffs and vice versa. The sanctuaries must
have strict protocols to implement and minimize the risk of disease spread. Before recruiting to
work with primates, all staffs should be tested with tuberculosis and then after recruiting at every
six months (Unwin et al., 2009). Ideally, the newly recruited staff should not have any contact
with non-human primates for the first week of employment. As most of the sanctuaries are
located in an endemic area to tuberculosis and vaccinated with BCG, staffs will test positive on
Mantoux tests (Andersen and Doherty, 2005; Brock et al., 2004; Habroe, 1981). Further tests like
sputum test and chest radiographs will help to confirm the disease status. In case, if staff turns
out tuberculosis positive, then he/she should not be allowed to work with non-human primates
and should not live in the proximity of the sanctuary. Instead they must be relieved from the
work and put under tuberculosis treatment. All volunteers/researchers/visitors entering the
sanctuary are required to submit the evidence of recent tuberculosis testing.
In order to reduce the risk of transmission of zoonotic diseases like tuberculosis in the
sanctuaries, a high level of personal hygiene should be maintained among the primate caretaker
staffs. All PASA sanctuaries maintained proper standard of hygiene, in respect to the personal
staff hygiene and that of the dens or cages, food storage and preparation room, clinics to ensure
well-being of non-human primates and staffs. Bleach containing 50grams per liter available
chlorine is an effective disinfectant to kill most of the pathogens including mycobacterium
species (WHO, 2012). Being easily available and cheap, most of the sanctuaries have used
bleach to clean the cages. All the sanctuaries primate caretaker staffs used protective clothing
while working with non-human primates. Such practices help to reduce the risk of mechanical
transmission of infectious agents. Keeping records of arrival and tuberculosis testing is
extremely important to assess the history of the primates. The pattern of enclosure setting is an
important task to minimize the direct or indirect contact between the different social groups
within the sanctuaries (Cox, 2005). It will help to prevent the direct contact and spread of
61
infectious diseases among non-human primates in other social groups. As tuberculosis spread
through air transmission, sanctuary management should give proper attention in designing the
enclosures. All the sanctuaries were using different types of diagnostics for tuberculosis and
tuberculin testing seemed to be practiced widely. Most of them reported that they got tuberculin
vials from abroad because of unavailability in their own countries. Some sanctuaries got it
through the coordination of PASA. As most of the sanctuaries are located in remote settings,
keeping the available tuberculin vials in freeze is also challenging due to lack of electricity.
As the influx of non-human primates to the sanctuaries is increasing, there is also a huge
challenge to tackle with the management of zoonotic diseases. Sanctuaries perform their
primate’s health check during quarantine and post quarantine period. This allows to screen and
tests for different zoonotic diseases including tuberculosis. All the sanctuaries have established
protocols for tuberculosis screening, in particular, strict tuberculosis testing during quarantine,
annual or biannual screening in post quarantine period and its prevention through strict human
screening followed with improved husbandry practices.
5. Conclusion
In field sanctuaries, it is tough to manage tuberculosis due to difficulties with detection,
unavailability of kits and lack of preventive measures in both humans and non-human primates.
Each sanctuary is more or less equipped to diagnose and screen tuberculosis. All the sanctuaries
have established protocols for tuberculosis screening. Strict quarantine checking for new arrivals,
annual or biannual screening in post quarantine period and strict human screening should be
followed with improved husbandry practices. Chimpanzees, gorillas and bonobos in quarantine
should be tested at least two times with intradermal skin tests at an interval of four weeks
whereas different species of monkeys should be tested three to five times at an interval of two
weeks. Apart from tuberculin testing, other field kits which can detect tuberculosis very quickly
and efficiently should be recommended. The information on tuberculosis management should be
shared and discussed among the sanctuaries improve the strategies for better handling and
management of tuberculosis and potentially other infectious diseases in future.
62
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General Summary
Tuberculosis is a highly contagious and zoonotic disease, its diagnosis and prevention at right
time is very important in any non-human primate settings. Chimpanzees (Pan troglodytes verus)
are among the most threatened and endangered non-human primates in Africa. Habitat loss, bush
meat trade, human exploitation and potential zoonotic diseases are the main causes of a sharp
decline in the population of chimpanzees in the wild. Several conservation programs are initiated
throughout Africa to preserve chimpanzees. However, chimpanzees in the wild and in
conservation settings are always a threat to infectious and zoonotic diseases like tuberculosis. It
has been proven that tuberculosis is one of the main diseases for massive mortality of
chimpanzees in captive settings such as conservation areas and sanctuaries. In non-human
primates, tuberculosis is a respiratory disease caused by M. tuberculosis, M. bovis and M.
africanum which is transmitted within and between species by aerosol route. Lack of proper
diagnosis and prevention can cause a high mortality within captive settings because chimpanzees
are social animals and live in close proximity to human beings. In such a scenario, proper
diagnosis of tuberculosis and preventive measures against it in a captive setting is very
important. Furthermore, diagnosis and prevention of tuberculosis becomes more difficult in a
captive setting with minimal resources i.e. few trained manpower and lack of sophisticated
diagnostic mechanisms. This research aimed at addressing aforementioned challenges by
identifying a basic tuberculosis diagnosis method and feasible prevention measures to control an
outbreak of tuberculosis in a captive setting that has minimum resources. In this research,
Tacugama Chimpanzee Sanctuary was chosen as a captive setting.
Intra-dermal tuberculin skin test (TST) using both avian and bovine purified protein derivatives –
PPD-A/B and PrimaTB Stat Pak test, a serological assay, were used to screen and diagnose
tuberculosis in chimpanzees at the sanctuary. A total of 80 out of 105 chimpanzees from
different social groups were tested with PPD-A/B and Stat Pak, respectively, during April 2011
to October 2012. Our results showed that all tested chimpanzees were negative with TST and
only two showed positive reaction with Stat Pak. After six months, these two were tested
negative by Stat Pak. Our study was done with a specific number of chimpanzees from one
sanctuary but still it holds promising results. We suggest the combination of skin test and Stat
Pak assay, in the context of remote sanctuaries, is the best way for the early detection of
67
tuberculosis as it is easily available, economical and simple. We analysed ante mortem
tuberculosis diagnostic tools and tuberculosis therapy from ten suspected chimpanzees. The ante
mortem diagnostics used were skin tests, Stat Pak, acid fast bacilli test and chest radiographs,
culture and PCR. All these chimpanzees were either exposed to tuberculosis infected chimpanzee
or recently arrived to the sanctuary. Tuberculosis tests performed in these chimpanzees did not
reveal consistent results. As the risk could not be taken of infecting the disease to others,
chimpanzees were put on anti-tuberculosis treatment based on human tuberculosis treatment
approach. Isoniazid, rifampin, ethambutol and pyrazinamide were the drugs used to treat these
suspected chimpanzees from three months to one year over different periods. We concluded that
tuberculosis cannot be diagnosed with one tool alone but rather should be confirmed with other
available tests. Culture and PCR remain the gold standard to confirm the disease as we were able
to do in five chimpanzees. Considering the importance, costs and ethical issues of animals, we
successfully diagnosed and treated ten chimpanzees which are living together in the group.
In order to investigate diagnostic and preventive measures currently being practiced in minimally
resourced captive settings, a survey questionnaire was conducted among the members of the Pan
African Sanctuary Alliance - PASA. It is an umbrella organization of 22 accredited sanctuaries
across 12 African countries caring a variety of non-human primates with majority focused on
chimpanzees. 13 sanctuaries shared their strategies for the management of tuberculosis in nonhuman primates. Most of the sanctuaries have adopted a basic method of diagnostic and
preventive methods for tuberculosis management both with non-human primates and humans.
Most of the sanctuaries remain reluctant to share the presence or absence of status of tuberculosis
with their non-human primates in the sanctuaries; probably they are not on top tuberculosis
management. The survey data could enable sanctuaries to develop and formulate guidelines for
better management of tuberculosis and potentially other infectious diseases in future, through
learning from each other in captive non-human primate sanctuaries in Africa.
In general, tuberculosis can be diagnosed with skin tests and simple blood kits when
sophisticated tests are not available. In field settings, it is difficult to detect tuberculosis.
Availability of kits and prevention in both humans and non-human primates is essential.
Sanctuaries must follow a strict protocol to prevent and control the spread of the disease.
68
Allgemeine Zusammenfassung
Tuberkulose ist eine hoch ansteckende bakterielle Erkrankung, die innerhalb von und zwischen
Spezies übertragen werden kann, sie ist also eine typische Zoonose. Ihre Diagnose und
gegebenenfalls Therapie ist in Primatenzentren äußerst wichtig. Schimpansen (Pan troglodytes
verus) gehören zu den gefährdetsten Menschaffen in Afrika. Der Populationsrückgang in der
Wildnis hat mannigfaltige Ursachen, wichtig sind Verluste an traditionellem Lebensraum,
Eindringen von Menschen in Urwald und Savanne, illegales Jagen zum Fleischverzehr, sowie
möglicherweise Ausbreitung zoonotischer Erkrankungen. Etliche Arterhaltungs-Initiativen
werden in Afrika durchgeführt.
Menschenaffen im Busch und noch stärker in Auffangstationen sind von Infektionskrankheiten
wie Tuberkulose bedroht. Ein hoher Anteil an Verlusten von Schimpansen in Schutzgebieten und
in Primatenzentren wird in verschiedenen Feldstudien der Tuberkulose zugeschrieben. Diese
Erkrankung
meist
des
Respirationstrakts
wird
mittels
Tröpfcheninfektionen
durch
Mycobacterium tuberculosis und M. bovis übertragen. In Auffangzentren können aufgrund der
größeren Nähe zum Menschen und des oft eingeschränkteren Platzangebots sowie
eingeschränkter Diagnose- und Therapiemöglichkeiten vermehrt Todesfälle durch Tuberkulose
auftreten.
Für
Primatenzentren
sind
stringente
Vorsorgeuntersuchungen
und
Krankheitsvermeidungs-Strategien zwingend notwendig. In vielen dieser Zentren in Afrika
bestehen aber leider oft sehr eingeschränkte Möglichkeiten der Diagnose und Therapie aufgrund
schlecht ausgebildeter Mitarbeiter und nicht verfügbarer aufwändiger Untersuchungsverfahren.
Unsere Untersuchungen wurden unter Feldbedingungen und bei minimalen Ressourcen im
Tacugama Chimpanzee Sanctuary in Sierra Leone durchgeführt. Es sollten einfache und
verlässliche Tuberkulose Diagnostik- und Vorbeugungsmaßnahmen entwickelt und getestet
werden, um die Gefahr von Tuberkuloseausbrüchen zu vermindern.
In den Untersuchungen im ersten Teil verwendeten wir einen Intrakutan-Tuberkulin-Hauttest
(mit hochgereinigtem Geflügel-/Rinder-Tuberkulin, PPD-A/B) und einen neu entwickelten BlutSchnelltest (PrimaTB Stat Pak), um ein Tuberkulose-Screening bei vorher zu anästhesierenden
Schimpansen der Auffangstation auszuführen. Dabei wurden zwischen April 2011 und Oktober
69
2012 aus unterschiedlichen Gehegegruppen 80 von 105 Tieren mittels Haut- und Bluttest
untersucht. Alle 80 getesteten Schimpansen erwiesen sich im Hauttest, der in die oberen
Augenlider appliziert wurde, als negativ. Zwei mit Bluttest untersuchte Tiere zeitigten ein
positives Resultat, allerdings bei einer Nachuntersuchung nach sechs Monaten ein negatives.
Unsere Untersuchungen wurden zwar nur an einer limitierten Stichprobe an Schimpansen eines
einzigen Zentrums durchgeführt, trotzdem glauben wir, klare Aussagen treffen zu können. Im
Falle weit abgelegener Primatenzentren verspricht eine Kombination aus Intrakutan-Test und
Blut-Schnelltest
eine
vertretbar
kostengünstige
und
relativ
einfache
Methode
zur
Tuberkulosetestung zu sein.
Bei zehn Tuberkulose verdächtigen Schimpansen, welche entweder nachweislich in engem
Kontakt mit infizierten Affen oder mit Menschen standen, verglichen wir aufwändigere
Tuberkulose - Diagnoseansätze. Bei diesen Tieren wurden die oben beschriebenen zwei
Verfahren
ergänzt
durch
eine
spezifische
Blutausstrich-Färbemethode,
eine
Röntgenuntersuchung, der Tuberkulose-Erregerkultur und einem PCR- Erregernachweis. Wir
mussten erkennen, dass keine Einzeltestung, sondern nur die Kombination mehrerer Verfahren
belastbare Ergebnisse zeitigt. Der direkte Erregernachweis kombiniert mit PCR sind zwar
aufwändig, können aber als Goldstandard angesehen werden, wie wir bei fünf Schimpansen
aufzeigen konnten. Die zehn Tuberkulosen verdächtigen Schimpansen wurden vorsorglich einer
Tuberkulose -Behandlung unterzogen, um eine mögliche Gefährdung anderer Tiere oder des
Betreunungspersonals zu minimieren. Der theapeutische Ansatz ähnelte der Vorgehensweise
beim Menschen. Isoniazid, Rifampin, Ethambutol und Pyrazinamid wurden in unterschiedlichen
Kombinationen erfolgreich drei bis zwölf Monate lang angewendet.
Im zweiten Teil der Arbeit wird dargestellt, wie eine sehr detaillierte und umfangreiche Umfrage
unter allen Primatenzentren der ‘‘Pan African Sanctuary Allianc‘‘ konzipiert und ausgeführt
wurde, um erstmals Tuberkulose-Schutzmaßnahmen und Untersuchungs- sowie eventuell
Behandlungsverfahren zu erfassen und Maßnahmen zu koordinieren. Die Umfrage erfasste alle
22 bekannten Primatenzentren in 12 afrikanischen Ländern, die sich alle mit den
unterschiedlichen Menschenaffenspezies beschäftigen. Dreizehn Zentren kooperierten und gaben
Einblick in ihre Tuberkulose Management-Strategien, die sowohl die Menschenaffen als auch
70
das Bereuungspersonal einschließen müssen. Viele Verantwortliche waren sehr skeptisch, den
Tuberkulosestatus ihrer Zentren offenzulegen, vielleicht auch, weil befürchtet wurde, dass somit
Management- und Strategiemängel offenkundig wurden. Die Fülle an Rückmeldungen sowie der
daraus resultierende Datenaustausch ermöglichten es, klarer formulierte Handlungsanweisungen
zu formulieren und zu implementieren, um nicht nur Tuberkulose, sondern auch andere
Infektionskrankheiten besser zu bekämpfen. Hier konnten relativ einfach erhebliche
Synergiepotentiale erschlossen werden.
Als grundlegende Untersuchungsverfahren zum Tuberkulose-Nachweis bei Menschaffen haben
sich der Intrakutan-Test und ein neuer einfacher Blut-Schnelltest unter Feldbedingungen
bewährt. Um die hochansteckende Zoonose Tuberkulose wirksam zu bekämpfen, sollte ein strikt
kontrolliertes Untersuchungsprogramm eingehalten werden, gerade wenn in abgelegenen
Primatenzentren nur sehr limitierte Untersuchungsverfahren zum Einsatz kommen können.
71
Appendix
72
Management of Tuberculosis (TB) under field conditions in non-human primates among
Pan African Sanctuary Alliance (PASA) Members
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