TUBERCULOSIS IN ZOO ANIMALS 183

Int. Zoo Yb. (2011) 45: 183–202 DOI:10.1111/j.1748-1090.2011.00141.x

Mycobacterial infections in zoo animals: relevance, diagnosis and management* A. LE´CU1 & R. BALL2 1Parc Zoologique de Paris, M.N.H.N., 53 avenue de St Maurice, 75012 Paris, France, and 2Lowry Park Zoo, 1101 W Sligh Avenue, Tampa, Florida 33604, USA E-mail: [email protected]

While the world prevalence of (TB) is (Table 1). One-third of the human population increasing in the human population, TB infection remains is currently infected by TB (World Health a real concern in some animal populations all around the globe. Most mycobacteria of the TB complex are able to Organization, 2010) and in 2008 1 Á 8 million infect zoo and wildlife species, in which the pathogen- people died from the disease (United Nations, esis, receptivity and immune responses vary widely. The 2010). Recent increases of Multi Drug Resis- diagnostic tools usually applied in domestic animals tance (MDR-TB) or even Extreme Drug Re- show limited performance in zoo species, especially sistance (XDR-TB) strains, and co-infection when prevalence is low. Conversely, investigations of cell-mediated immunity through in vitro assay of g- with human immunodeficiency virus (HIV), interferon may have numerous advantages, as long as make the battle against TB more difficult the technical limits are known and can be improved upon. (Hanekom et al., 2010). TB is now placed on Furthermore, recent tools based on the investigation of the ‘most relevant diseases’ list in the health- humoral immunity seem very promising for the detection of antibodies directed against certain immunogenic my- related Millennium Development Goal 6 by cobacterial antigens in a wide range of species. All these the World Health Organization (WHO) and methods are currently evaluated in field studies, despite the United Nations (UN), together with ac- the difficulties to ensure rigorous validation. The devel- quired immune deficiency syndrome (AIDS) opment of these diagnostic tools is also impaired by the and malaria (United Nations, 2001, 2005; prevalence of mycobacteria other than TB also able to infect and create relevant disease in their host. Thus, World Health Organization, 2010). Vigorous decisions on positive and suspicious-animals issues action plans are applied to fight the disease in should be taken based on the evaluation of the risk of developing countries, although these coun- transmission to the rest of the zoological collection, the tries are also facing notable incidences of TB possible treatment options, animal welfare, conservation considerations and, of course, the zoonotic potential of within the animal population. this pathogen. The majority of the mycobacteria from the MTC have the ability to infect wild animals Key-words: interferon-g; mycobacteria; non-tuberculo- (Table 1), whereas the susceptibility, patho- sis mycobacteria; serology; tuberculosis; zoo. geny and immune responses towards myco- bacterial infection vary widely between INTRODUCTION mycobacteria (Mostowy et al., 2005) and Tuberculosis (TB) is the common name of the host-animal species. Although some mam- disease that could be caused by different mals species seem to show a lower incidence species of belonging to the Myco- rate (e.g. equids, New World monkeys), the bacterium tuberculosis Complex (MTC) predictability of infection outcome is still

*An earlier and partial version of this article was published as: EAZWV Tuberculosis Working Group (2010): Tuberculosis in zoo species: diagnostic update and management issues. In Transmissible diseases handbook (4th edn): 1–20. Kaandorp, J. (Ed.). Switzerland: European Association of Zoo and Wildlife Veterinarians Tuberculosis Working Group. This current manuscript is published with kind permission of the European Association of Zoo and Wildlife Veterinarians.

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MYCOBACTERIA OF THE MAJOR HISTORICAL KNOWN TUBERCULOSIS COMPLEX HOST OR BURDEN REPORTED WILD AND ZOO HOST M. tuberculosis human, non-human elephant, non-human primates, beisa oryx, addax, goats, primates birds, lowland tapir, giraffes, springboks, mongoose, rhinoceros, addra gazelle M. bovis cattle (1buffalo, bison) all ruminants, badgers, possums, meerkats, big cats, canids, rodents, non-human primates, wild boars elephants, camelids, rhinoceros, onager, horse, birds M. africanum human cattle, swine, non-human primates M. microti vole, camelids New World monkeys, big cats M. pinnipedii camel, tapir, big cats M. caprae goat, sheep, swine swine, cattle wild boars, red deer, white-tailed deer, camel, bison M. canetti human ? Dassie bacillus variant hyraxes meerkats

Table 1. Tuberculosis complex Mycobacteria and their reported hosts. hard to guess and, therefore, the eradication immunopathological mechanisms of lympho- of TB is potentially linked to the capacity of cytes in a way that allows them to stay within early diagnosis in domestic and wild host the macrophage they infect. There are four species. Genetic key factors [e.g. interferon- groups of T-helper lymphocytes (Th). The g (IFN-g) receptor or vitamin D genotypes] Th1 group participates in cell-mediated im- are likely contributing towards these differ- munity (CMI) by producing cytokines such ences (Rhodes et al., 2003; Schluger, 2005). as IFN-g, and then activating other immune The course of the disease and occurrence of cells participating in the fight against the latent infection versus active infection are mycobacteria. The Th2 group provides help variable among species, from extremely sen- to B lymphocytes, which are in charge of sitive Old World monkeys to apparently more producing humoral responses (immunoglo- resistant equids. bulins). During the course of the disease, the Species of mycobacteria out of the MTC Th2 pathway is also activated, although hu- and other than leprae (caus- moral protection seems to be poorly effective ing ) are known as mycobacteria other against this intracellular pathogen. These im- than tuberculosis (MOTT), non-tuberculous munological mechanisms normally lead to mycobacteria (NTM) or atypical mycobacter- the eradication or the containment of the ia. These are mainly environmental mycobac- mycobacteria, and their systemic local effects teria found in water and soil, but they are also could be tracked to assess previous or current able to contaminate vertebrates, although the presence of mycobacteria. When the Th1 occurrence of disease seems to be more pathway fails, it could lead to active disease related to the immune status of host or to very with active replication of mycobacteria in the close or repeated contact. surrounding cells (macrophage, dendritic Except for M. leprae, all mycobacteria are cells) or, more often, to containment within a facultative intracellular pathogens, often in- granuloma, which is the specific latency vading macrophages as the first host cell. phase of mycobacterial infection that is able Thus, a direct diagnostic will require cellular to last for years. The probability of latency samples and techniques to highlight the pre- depends on the host species, the mycobacteria sence of mycobacteria (stains) or their DNA. species, the infection dose and the route of For their survival, mycobacteria are driving transmission.

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TRANSMISSION AND ZOONOTIC mycobacteria already reported within zoolo- RISK FROM ZOO ANIMALS gical collections, whereas ‘dassie bacillus’ was found in Meerkats Suricata suricatta Interspecific transmission can occur between and hyraxes Procavia sp but never reported taxonomically distant species as has been in humans. Atypical mycobacteria found in recorded in the literature (Lewerin et al., zoo species may also infect humans (espe- 2005; Jurcynski et al., 2007; Moser et al., cially if immunosuppressed) but they cannot 2008). Thus, silent extension of TB into a be named as truly ‘zoonotic’ as they are zoological collection owing to an increase of mainly acquired from the environment. latent-form cases should be a real concern for Third, the route of absorption will have a zoo managers. Transmission routes between bearing on the zoonotic risk of any mycobac- animals are directly associated with the loca- terial disease. Respiratory shedding of any lization of granulomas in shedding indivi- mycobacterial pathogens will have greater duals: pulmonary lesions generally lead to zoonotic potential than oral absorption of airborne transmission, whereas mesenteric mycobacteria. lymph-node lesions may lead to intestinal Fourth, exposure is another essential com- excretion of mycobacteria. Horizontal trans- ponent of any zoonotic spread of mycobac- mission is the most significant means of teria. Prolonged exposure and close contact contamination but vertical transmission could will increase the likelihood of zoonotic trans- also occur through placental or umbilical mission. The risk for TB transmission from infection (Kaneene & Pfeiffer, 2006). an animal with a case of active TB is higher The potential for a human to acquire any for daily handlers than for people with only mycobacterial disease from a zoo animal brief contact, such as members of the viewing requires a combination of several events. public. Trained elephants (Furley, 1997) and First, the animal that is actively infected with sea lions (Kiers et al., 2008) are classic a mycobacterial disease must be shedding examples of the zoonotic potential of myco- and it must be in a form that has a good bacterial disease from zoo animals, mainly potential for infection. This is most likely in because of the close contact between keepers the form of aerosolized droplets from respira- and animals during training sessions. Hous- tory secretions. Sputum from non-human ing conditions will also play an important role primates that have acquired the habit of in zoonotic potential and examples are re- spitting at people is also a potential source of ported of zoonotic transmission from rhino- infection. Other potential sources of infection ceros species (Stetter et al., 1995). A include urogenital or gastrointestinal shed- surveillance programme that targets early ding and the aerosolization of these materials detection in the more sensitive species (non- during routine husbandry and cleaning, such human primates, elephants, ungulates, sea as the use of a high-pressure hose (Dalovisio lions) has the potential to identify risk and et al., 1992). anticipate transmission to all people sub- Second, the species of mycobacteria and mitted to exposure. Improved diagnostics that the pathogenicity it has for humans must be raise the level of suspicion before actual considered. Among the MTC mycobacteria, shedding may prove to be extremely benefi- is likely to be the one cial to prevent extension of TB within hu- with the more relevant zoonotic burden (Co- mans and non-human inhabitants of zoos. sivi et al., 1998). Mycobacterium tuberculo- An important zoonotic potential exists in sis (Sternberg Lewerin et al., 2005), M. bovis post-mortem examinations of infected ani- (Stetter et al., 1995), Mycobacterium pinni- mals. It is critical to approach any suspect pedii (Moser et al., 2008), Mycobacterium case with caution and use appropriate protec- caprae (Pate et al., 2006) and Mycobacterium tive equipment. In some species, such as microti (Pattyn et al., 1970; A. Le´cu, unpubl. elephants, it may be advisable to have re- data) are examples of known zoonotic MTC spiratory protective equipment in use during

Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London 186 RESEARCH IN ZOOS: EPIDEMIOLOGY & CONSERVATION MEDICINE the entire examination. For the United States, domestic-animal populations and to keep its specific guidelines can be found at http:// prevalence below the ‘free of TB’ threshold. www.osha.gov/SLTC/tuberculosis/standards. The second is to avoid contamination from html, and the Centers for Disease Control and outside (livestock importations, wildlife). Prevention (CDC) for Guidelines for Prevent- Thus, the zoo community stands in a special ing the Transmission of Mycobacterium position, often keeping both domestic and tuberculosis in Health-Care Settings, 2005 wild species, with a constant need for animal (Jensen et al., 2005). transfers and exchanges. In addition to precautions in handling suspect post-mortem cases and a vigorous European national programmes and zoo ante-mortem TB surveillance programme for concerns zoo animals, an employee health plan is recommended. A regular TB survey of all All domestic species of cattle, buffalo and zoo personnel (through a skin test or a IFN-g wood bison in zoos and safari parks should be test) should be set in place in coordination subjected to routine testing as with local public-health authorities, likely often as the indicated testing interval for the associated with a vaccination schedule. Inter- area in which the zoo is located. As long as vals between controls are set based on current they stay within their original facility without national medical recommendations and local any close TB outbreak, wild species in cap- risk assessment. Mycobacterial infections are tivity are generally exempted from statutory opportunistic pathogens and the transition TB testing and, currently, there is no officially from infection to disease depends on the approved screening test for TB in species host’s immune competency. Educating staff other than bovines, deer and, occasionally, to identify the factors that will minimize primates (Cousins & Florisson, 2005; Defra, the risk of infection and reduce the risk of 2008). developing active disease (cigarette smoking, Several texts define sanitary policy for TB excessive alcohol consumption, high-fat within-country members [European Union diets) will also help to reduce the zoonotic (EU) Council Directive 64/432/EEC, and all potential of any mycobacterial disease, in following modifications; Council Directive, the same way that should be considered for 1964]. The EU policy towards TB in animal all opportunistic pathogens in the zoo health mainly focuses on the eradication of community. bovine TB and is based on two fundamental It should be noted that M. tuberculosis is principles: first, the Member States are pri- the main agent of TB found in humans, marily responsible for the eradication of whereas , M. bovis bovine TB and may receive community and account for o1% financial support for the eradication pro- of TB occurrence in humans (Olsen et al., gramme; second, eradication of bovine TB 2010). The risk of reverse zoonosis, from in the EU must be the final target and the humans to animals, also exists and has already Member States must consider eradication as been reported in several species, such as cattle the defined aim. (Ocepek etal., 2005), non-human primates Hence, most EU regulations apply only to (Michel & Huchzermeyer, 1998) and birds M. bovis, and sometimes M. tuberculosis, (Schmidt et al., 2008; Steinmetz et al., 2006). screening. Based on sequential Commission Decisions, 14 member states of the EU are NATIONAL REGULATIONS AND ZOO classified as free of bovine TB at the time of CONCERNS writing. Initially, Denmark, Germany, Lux- embourg, the Netherlands, Austria, Finland In all countries, legislation relating to animal and Sweden were classified as bovine-TB TB is implemented to achieve two main free (Commission Decision, 1999). In 2003, goals. The first is to eradicate TB from Belgium and France were added to the list

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(Commission Decision, 2003), and by 2009, nearby forests or TB in livestock on a nearby the Czech Republic, Slovakia, Slovenia and farm). Poland were also listed as bovine-TB free Even if they do not provide compulsory (Commission Decision, 2009a). Outside but content, valuable guidelines and recommen- close to the EU, Norway and Switzerland also dations are also produced by the professional have the status ‘free of bovine TB’ (de la zoo community, such as the European Asso- Rua-Domenech, 2006). Member states can ciation of Zoos and Aquaria position state- also apply to the EU to classify part of their ment on the Animal Health Strategy for the regions as free of TB, such as the UK EU (EAZA, 2009), led by the European obtained for Scotland (Commission Decision, Association of Zoo and Wildlife Veterinair- 2009b). The main criterion for keeping the ans (EAZWV), or the European Endangered ‘officially free of TB’ status for a country or a Species Programme (EEP) or Taxon Advi- region is to maintain the prevalence of bovine sory Group (TAG) recommendations on pre- TB in cattle herds below 0 Á 1% (OIE, 2010a) transfer health requirements for managed per year for six consecutive years (Council species. Directive, 1964). The Balai Directive (EU Council Directive US states programmes and zoo concerns 92/65/EEC; Council Directive, 1992) re- quires that all ruminants traded between The Animal and Plant Health Inspection institutions must come from officially bo- Service (APHIS) Uniform Methods and Rules vine-TB-free herds, as mentioned in Trade (UM&R) have produced the minimum stan- Control and Expert System (TRACES) health dards for the maintenance of TB-free accre- certificates. Moreover, to be ‘Balai-ap- dited herds of cattle and bison, and the proved’, an institution must be free of ‘bovine maintenance of state or zone status in the TB’ (as listed in Annex A of the Directive), US Department of Agriculture’s (USDA) TB for at least 3 years, or free of ‘TB’ (this term eradication programme (USDA, 2004). These includes all mycobacteria of the TB complex) minimum standards do not preclude the adop- for primates, felidae and ruminants, if the tion of more stringent standards by any state or Member State has a control monitoring pro- zone. Each individual state has various regula- gramme regarding these particular species. tions with regard to the testing requirements During a transfer between two approved for exotic ruminants before importation and bodies, TB testing is not required by the Balai several have no specific requirements at all. Directive, but many member states are al- The current status of each state can be checked lowed to add TB testing as an additional at the APHIS website. Within the zoo commu- requirement when importing ungulates, what- nity in North America, the Association of Zoos ever institutions they come from (e.g. re- and Aquariums (AZA) and American Associa- quested by UK, Sweden). Nevertheless, zoo tion of Zoo Veterinarians (AAZV) recommend mammals are still being exchanged in na- TB testing for zoo ungulates, but no standard tional and international transfers within Eur- protocols are in place. Regulations are similar ope without appropriate individual testing. to the EU in that M. bovis and M. tuberculosis TB prevalence in both domestic species are reportable disease. In a confirmed diagno- and free-ranging wildlife that surrounds zoo sis of M. bovis the state veterinarian, USDA premises and animal enclosures are also a Veterinary Services and Animal Care (for major factor that can modify the compulsory facilities licensed under the Animal Welfare TB requirements of a zoo. Any TB monitor- Act), public health officials and other relevant ing/action programme on neighbouring wild- regulatory agencies should be promptly noti- life or livestock should impact zoo TB policy fied. If M. tuberculosis is diagnosed, local and must take into consideration the relation- public-health officials are notified first. ship between zoo animals and exogenous All livestock herds, within a 10 mile (16 km) species (e.g. badgers Meles sp, deer within radius, will be tested within 6 months of any

Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London 188 RESEARCH IN ZOOS: EPIDEMIOLOGY & CONSERVATION MEDICINE diagnosis of bovine TB in livestock or free- radiographs, the size of animal patients, ranging wildlife (USDA, 2004). anaesthesia considerations and availability of the devices, except for ultrasounds, often OVERVIEW OF CURRENT prevent their use in zoo species. DIAGNOSTIC METHODS For species such as birds, laparoscopy remains one of the most useful tools (Pavlas Diagnostic methods are seldom homogenous, et al., 1993; Speer et al., 1999), followed by are rarely validated for zoo species and can biopsy of suspect granulomas or abnormal hardly ever be transposed from one species to tissue for further direct examinations another. As population size and the nature of [stains, polymerase chain reaction (PCR) and wild species would often prevent decent culture]. validation for these tests (M. Miller, 2008), zoo veterinarians must be aware of the actual diagnostic tools available, their limits and the Limits of direct examination current progression of scientific knowledge in Culture stands as the ‘gold standard’ method TB diagnostic means. (M. Miller, 2008) but requires from 2 weeks to 3 months for culture to occur (Liebana Limits of an unspecific diagnosis et al., 1995), depending on whether the Clinical signs of TB are rarely seen before mycobacteria belong to the slow-growing death in zoo animals (Montali et al., 2001; group (e.g. all mycobacteria from MTC) or Lyashchenko et al., 2006). If a cough and rapidly growing group (e.g. Mycobacterium dyspnoea are noticed, this is always in a very abcessus or ). Var- late and irreversible stage of the pulmonary ious modifications and improvement have form of disease. The most frequent sign been added to culture methods (i.e. use of noticed among mammals is chronic weight liquid or semi-liquid culture media) in order loss. to reduce the delay to below 30 days (White- Different imaging techniques can play a law & Strum, 2009). Some mycobacteria role in TB diagnostics, but they are only species hardly grow on these alternative informative on localization and aspect of the media (e.g. M. pinnipedii, M. L. Boschiroli, lesions. Although radiography is a regular pers. comm.) and some of these culture step in human diagnostic procedures, its techniques are still not validated in veterinary performance may be restricted by the limita- medicine. Hence, faster testing still relies on tions of animal species in zoo veterinary microscopic examination and mycobacterium medicine: the size of the thoracic cage of DNA amplification methods. some animals may prevent the use of X-rays The threshold number of bacilli needed to (e.g. ungulates over 250 kg), reference obtain a positive result by microscopic exam- images are often missing and lesions may be ination of Acid Fast Bacilli stained with the too subtle for radiographic detection. More- Ziehl Neelsen method (ZN) is around over, in some species, calcified lesions are 5000 bacilli ml À 1 and is dependent on the very rare (e.g. felids and non-human pri- reader. Other bacteria also share the Acid Fast mates). resistant property, and also react to ZN dyes, High-frequency ultrasonography, compu- such as Nocardia sp. Detection limit could be ter tomography (CT), magnetic resonance enhanced using fluorescence microscopy imagery (MRI) and even positron emission (FM), although access to this technique is tomography (PET) scans could also be useful not as widespread as ZN. The usual staining to detect granulomas or lesions in non-palp- techniques (ZN and modified ZN) allow the able lymph nodes, when they are in the thorax detection of bacilli loads superior to 103–104 or because animal conformation prevents colony-forming units (CFU) per ml: as a easy palpations, such as in pinnipeds (Jur- comparison, fewer than ten CFU of M. tuber- cynski et al., 2007; Lacave et al., 2009). For culosis per ml are enough to infect a

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Cynomolgus macaque Macaca fascicularis techniques, they still cannot be used as a (Lin et al., 2006). This means that a sample broad and primary screening tool as de- may indicate an infection source while its scribed in Table 2. It should be noted that microscopic results remain negative. those DNA-based techniques show the best Some biological samples may contain very sensitivity and specificity values when ap- few bacilli (Martin-Hernando et al., 2007); plied on pure culture samples, even those that for example, because of highly calcified le- are early stage (Fitzgerald et al., 2000). sions (Capuano et al., 2003) or intermittent When there is sufficient DNA (rich sample shedding in biological fluids, such as bron- or culture), molecular typing should be per- chial secretions or milk. As an example, 15– formed. Spoligotyping or other methods [e.g. 20% of active pulmonary forms are not even variable number tandem repeat (VNTR)] al- confirmed by any culture in humans (Frieden low researchers to identify strains of myco- et al., 2003). Therefore, while positive culture bacteria and these techniques can be a very provides evidence of disease, negative culture useful tool to track the epidemiological cir- results may not rule out infection in exposed cuit of the TB. In recent years, several TB or suspect animals. strains from zoos were spoligotyped and their The detection of DNA material directly fingerprint can be compared with other circu- from raw biological samples is sometimes lating strains, either from captive or from performed by some laboratories. The biologi- feral animals. Whenever a TB mycobacter- cal media issued from zoo animals could be ium is discovered, this kind of typing should broncho-alveolar lavage (Flynn et al., 2003), always be performed, or at least samples must gastric lavage, saliva from oral swab and be kept frozen ( À 25 or À 80 1C) until further stools or elephant trunk wash (Mikota et al., typing analyses are available. The standard 2000). Amplification methods (e.g. PCR) are use of all molecular techniques is application now well developed and can help to specify on pure culture material. However, PCR the mycobacteria with the use of selected techniques, and sometimes spoligotype, probes. In the recent years, sensitive and could also be applied to raw material (la- specific PCR probes became available, some- vages, tissues), which decreases the delay times associated with rapid processing (e.g. before the results are available, although this Genoquicks) able to provide species identi- application may compromise sensitivity and fication of mycobacteria or antibioresistance specificity. profile within a few hours. However, some of these biological samples are likely to host CMI-based diagnostics many other bacteria or biological compounds as inhibitors that are impairing PCR efficacy. During the CMI process, mycobacterial anti- Even with the simplification of molecular gens are presented by macrophages to Th

TB TEST (E.G. PCR) SE 5 72% DISEASE NO DISEASE SP 5 96% (ACTIVE TB) (LATENT OR NO TB)

Prevalence 5% 5 95 POSITIVE PREDICTIVE VALUE PPV 5 3 Á 6/(3 Á 613 Á 8) 5 49%

PCR1 3 Á 6 3 Á 8 NEGATIVE PREDICTIVE VALUE PCR À 1 Á 4 91 Á 2 NPV 5 91 Á 2/(91 Á 211 Á 4) 5 98%

Table 2. Example of polymerase chain reaction (PCR) sensitivity in human sputum, with 5% tuberculosis (TB) prevalence situation: from N. Veziris, pers. comm. With the given sensitivity (SE) and specificity (SP), a positive TB test result means an even probability (49%) of a TB-infected or a TB-free animal. The lower the prevalence, the lower the positive predictive value

Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London 190 RESEARCH IN ZOOS: EPIDEMIOLOGY & CONSERVATION MEDICINE lymphoctes, which become activated and still missing within the European Economic then produce various cytokines, inducing Community, despite a common regulatory their own replication and the activation/mito- definition from resolution 64/432/EEC sis of other ‘helped’ cells. Memory of Th cells (Council Directive, 1964). In the United regarding previous contact with Mycobacter- States, tuberculin is standardized. ium can be assessed (Chapel et al., 2006) by Although the worldwide source for tuber- presenting selected antigen(s) to them, either culin production is M. bovis strain AN5 in vivo [tuberculin skin test (TST) also called (Schiller et al., 2010), there is an important the ‘Mantoux’ test] or in vitro [lymphocyte discrepancy between PPD quality, leading to proliferation assay (LPA) or IFN-g tests]. a relevant variation in the TST results (Ran- TST is far from being validated for zoo gel-Frausto et al., 2001; Good et al., 2007). species, as there is considerable variation Although named ‘purified’, tuberculin PPD between tegument and dermal structure with- still contains a very large panel of different in species, compounded by a lack of under- antigens (Borsuk et al., 2009) in non-predict- standing of immunophysiology across able proportions. Reading methods may also various taxa. The test relies on local inflam- vary between veterinarians when a ‘distant’ matory cell recruitment (Ciftci et al., 2005; opinion about TST local reactions is applied Olsen et al., 2010), which can be low or to avoid repeated immobilizations. Therefore, absent for many reasons, such as tegument whenever possible, close examination, palpa- cellular organization, immunosuppressive tion and caliper measurement of tegument are status or superficial temperature. TST sensi- strongly recommended in order to avoid tivity is often poor; for example, 70–90% in false-negative results. Skin test interpretation humans and 50–90% in zoo hoofstock (Cou- guidelines exist only for humans (Katial, sins & Florisson, 2005). Specificity is also 2003), non-human primates (Bushmitz et al., dependent on species-specific features (e.g. 2008), cattle (OIE, 2010a), deer (Clifton- orangutans Pongo sp prone to an unspecific Hadley & Wilesmith, 1991; OIE, 2010b) and TST-positive reaction) or individual condi- tapir (Gomis et al., 2008). Visualization is tions, such as malnutrition, medical treat- often used to read intrapalbebral TST per- ments or unrelated inflammatory status, that formed on primates and, while not official for have previously been shown to affect the regulatory purposes, this method is used results of tuberculin testing, potentially re- within zoological facilities and as a means of sulting in false negatives (Kiwanuka, 2005). surveillance. However, close eyelid evalua- The co-infection by other NTM (e.g. avium tion and palpation is also recommended in complex) may also lead to false-positive these species, as it could enhance the sensi- results: specificity is influenced by the kind tivity of TST (Panarella & Bimes, 2010). All of antigen (tuberculin) injected intradermally; regulatory testing must be read by palpation. for example, mammalian old tuberculin To increase the specificity of TST as well (MOT) is a crude culture filtrate preparation as in cases of doubtful or inconclusive results, that contains antigens common to many my- a comparative TST is strongly recommended, cobacterial species; therefore, cross reactions with the use of both bovine and avian tuber- owing to NTM sensitization sharing these culin (Cook, 1993; Clifton-Hadley et al., antigens could be expected. Other tuberculin 2001). This comparative test is still the only have more selected contents [protein-purified official test allowed by some national regula- derivate (PPD)], either for M. bovis screening tions for species such as deer (Sockett, 1993). (bovine PPD) or for Mycobacterium avium A time interval between two TSTs is required complex (MAC) screening (avian PPD). to overcome the desensitizing effect of tuber- The materials and methods of application culin and is usually set to a minimum of 42 of the skin test vary between countries and days (de la Rua-Domenech et al., 2006). veterinarians. For bovine tuberculin produc- In vitro tests of cellular immunity rely on tion, a standardized quality of tuberculin is (re)stimulation of T lymphocyte memory.

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Thus, the first mandatory step is to keep cells large range of exotic Bovidae, but also some alive until they reach the laboratory. Blood animals outside this family (e.g. Giraffidae). samples should arrive at the laboratory for However, artiodactyls may respond poorly with stimulation no later than 8–10 hours after the standard positive control antigens (unspeci- collection and should be kept at ambient fic mitogen) or their IFN may not be detected room temperature (below 22 1C), avoiding by the ELISA (Riquelme, 2009). It is likely that temperatures that are too cold (below 4 1C) similar problems occur with the primate-re- or too high (more than 25 1C) (Waters et al., ferred test; although a list of ‘validated’ species 2007) in order to keep lymphocytes alive is provided on its leaflet, field studies showed until they arrive at the laboratory to be incu- contrasting results between different species bated. Particular attention must be paid to (Le´cu & Riquelme, 2008; Riquelme, 2009; S. immediate, homogenic and full mixing of Hoby & C. Wenker, pers. comm.); for example, blood and anticoagulant (heparin) at collection. the IFN-g of certain species may trigger a These CMI in vitro tests are performed in greater optic signal through the ELISA in- two steps, for a total run of 48 hours mini- cluded QUANTIFERONs rather than the one mum. The first step is to incubate T cells with of the PRIMAGAMs. a selected mycobacterial antigen (usually In order to overcome the problem of speci- bovine and avium PPD; sometimes purified ficity in ELISA detection of IFN-g, in-house antigen, such as ESAT-6 or CFP10), leading modified tests can be created. One solution is the lymphocytes to produce cytokine and to the detection of mRNA coding for IFN-g, proliferate if they had previously been ex- which seems to have broad nucleotid se- posed to the same mycobacterial antigen(s). T quences, shared by several different mammal cells are usually issued from blood centrifu- species; moreover, mRNA IFN-g sequences gation and separation techniques, but other from several species are now available in relevant fluids (pleuretic or abdominal effu- gene banks, and these can be used to develop sion fluids) could also be considered (Ariga & specific in-house ELISA tests (Harrington Harada, 2008) as long as their cellular content et al., 2007). This option is currently devel- is elevated. The second step is to reveal either oped for some zoo or wild species, although the LPA rate through radio-isotopic techni- the entire procedure can only be performed ques or the amount of relevant cytokines (e.g. by experienced laboratories. An alternative IFN-g) produced through an enzyme-linked solution is to design a specific IFN mono- immunosorbent assay (ELISA) or an en- clonal antibody to be used instead of the zyme-linked immunosorbent spot (ELISPOT, regular ELISA, as is currently being devel- which is more sensitive). Once the cells have oped for elephants and rhinoceros (Morar been stimulated to produce IFN-g, then the et al., 2007; Rutten, 2007). At the time of ELISA or ELISPOT can be delayed if the test writing, these dedicated tests are still not wells are frozen. available for routine use. LPA is less available to current practice The duration of the CMI is rather unknown than IFN-g tests, owing to the use of radio and its behaviour over the course of infection isotopes in their process, while commercial is only theoretical. All studies concerning kits for the IFN-g test are available, designed long periods of time (Vervenne et al., 2004) for cattle (BOVIGAMs: Wood & Jones, and work on bacille Calmette–Gue´rin (BCG) 2001), primates (PRIMAGAMs), deer vaccination protection length suggest that (CERVIGAMs, production discontinued at stimulation tends to revert to a baseline level, this time) and humans (QUANTIFERON which remains unknown beyond a year. GOLDs, T-SPOT-TBs). Recent studies show that the use of these tests can be Humoral immunity-based diagnostics extended to some exotic species (Grobler et al., 2002; Riquelme, 2009); for example, a Serodiagniosis of TB suffered from a bad cattle-designed test will detect IFN-g of a reputation in human TB medicine as early

Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London 192 RESEARCH IN ZOOS: EPIDEMIOLOGY & CONSERVATION MEDICINE trials assessed antibodies against single my- PAKs, DPPs VetTB) or primates (PrimaTB cobacterial antigens, which resulted in tests STATPAKs) also look promising in non- that were low in sensitivity and specificity target species, such as deer (Lyashchenko (Pottumarthy et al., 2000). This has changed et al., 2008; Greenwald et al., 2009), Black in the last few decades as humoral tests began rhinoceros Diceros bicornis (Duncan et al., to look at multiple mycobacterial antigens, 2009; Espie et al., 2009), tapirs and camels resulting in improved sensitivity and specifi- (Moser et al., 2008) or South American sea city. lions Otaria byronia (Jurcynski et al., 2007). During the course of an infection, Th1 The sensitivity and specificity of these activity (the CMI pathway) is thought to be serological assays are encouraging. However, initially greater than Th2 (the humoral route) these values are often issued from descriptive in order to control and confine infection studies performed on either very sensitive (Chapel et al., 2006). An inversion of this species (i.e. prone to start disease soon after Th1/Th2 balance control is often associated infection) or under very clear enzootic condi- with a recrudescence or with active disease tions, perhaps jeopardizing the real sensitivity and extensive pathology (Welsh et al., 2005; and specificity values (Chambers, 2009). Ser- Doherty & Rook, 2006). Although seeking ological profiles of animals in long-term antibodies may appear to be of little help quiescent infection status are still poorly when screening for latent infected animals, it known, and these are likely variable along a is becoming more relevant in the detection lifetime and then able to decline below detec- and monitoring of active, ‘sick’ and shedding tion thresholds (Lerche et al., 2008) of cur- individuals. It has also been clearly noted in rent serological tests during certain periods of humans (Abebe et al., 2007), cattle (Welsh latent infection. et al., 2005) and in wildlife (Chambers, 2009) Repeated testing seems to be the only way that some antibody elevations occur during to adapt with the actual limitations, as anti- the shift from latent to active disease, so that a body titre is thought to arise when relapse is prognostic value may be added to certain about to occur. As zoonotic risk increases serological results. considerably within this shifting time, the A panel of mycobacterial antigens are able detection of early serological change may be to elicit persistent humoral responses in hu- relevant to trigger a deeper screening (direct mans (Lyashchenko et al., 2000) and in a examination) and preventive measures for wide range of species (Haagsma & Eger, staff, the public and animals in the vicinity. 1990; M. Miller, 2008). Mainly assessed in non-human primates (Khan et al., 2008), Booster or no booster? most relevant antigens have been screened through different techniques (Cranfield et al., In both humoral- and cell-mediated immunity, 1990; Lyashchenko et al., 2000), and selected the phenomenon of anamnestic rise could be to design ELISAs and rapid lateral flow considered to enhance the sensitivity of tests: technology tests, with good results in the through a TST, the injection of mycobacterial species for which they have been validated, antigens (tuberculin) may initiate a boost of such as elephants (Lyashchenko et al., 2006; the immune response of host and then in- Greenwald et al., 2009) and primates (Lyash- crease antibody production and re-launch chenko et al., 2007). However, the panels of cellular-mediated activity (i.e. IFN produc- immunostimulant antigen will remain di- tion) if the subject was previously exposed to rectly linked to the type of mycobacterium mycobacterial infection. This ‘booster effect’ involved, host species, time frame of disease has been noticed in several studies in both the and also individual condition. Some commer- humoral and the cellular response of cattle cial tests validated for Asian elephants Ele- (Palmer et al., 2006) and in a humoral phas maximus and African elephants response of elephants (W. Shaftenaar, pers. Loxodonta africana (ElephantTB STAT- comm.). In human medicine, this

Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London TUBERCULOSIS IN ZOO ANIMALS 193 phenomenon is even used in a ‘two-step TB fected individuals and their monitoring. For Skin Test’ (Menzies et al., 1994; Katial, this purpose, CMI tests are used. The applica- 2003), with two skin tests performed at a 3 tion of screening methods focuses the atten- week interval. If the second test is negative, tion of all zoo stakeholders and encourages then the subject is considered uninfected; if priority monitoring of animals with doubtful the second test is positive, then the subject results, which is the first essential step to may be considered to have a ‘boosted’ reaction preventing the silent extension of TB within to an infection that occurred a long time ago. such a multispecific animal population. However, in zoo animals, the possibility of In the United States, a Tuberculosis Sur- producing a false positive because of a PPD veillance Plan for Non-Domestic Hoofstock injection (e.g. direct immune stimulation of has been developed by the National Tubercu- PPD antigens in the absence of any infected losis Working Group for Zoo and Wildlife background) should remain a real concern. Species (2001). This group identified the Studies on tuberculin injection effects on the following guidelines to prevent the transmis- immune response of uninfected animals are sion of TB between institutions. First, identi- under progress (A. Le´cu, unpubl. data). Thus, fy the TB status of the sending institution. the use of a booster effect should be carefully Avoid animal exchanges with facilities that applied as an ancillary tool and must be do not have a TB surveillance plan in place. interpreted with caution. Maintain farm animals separate from non- domestic ungulates in the collection. The CURRENT SCREENING transfer of domestic livestock from zoos to RECOMMENDATIONS farms should be avoided when possible to minimize the potential risk of transmission Within some species, current captive popula- from the zoo to the livestock industry, as is tions show more than 10% of infected herds the case between wildlife and domestic stock (e.g. elephants; Mikota et al., 2000). To draw a (Artois et al., 2004; Wilson et al., 2009). An parallel, for a country to keep the TB-free institutional surveillance plan should be de- status regarding bovine TB, the OIE (2010a,b) veloped and instituted. Pre-employment and requires that the percentage of herds confirmed annual tuberculin testing are recommended infected with M. bovis has not exceeded 0 Á 1% for employees. All animals that are shipped per year for three consecutive years. should be tuberculin tested on the premises of The entire zoo community should make an the sending institution as part of pre-shipment effort, even if available tools are still – and clinical examination. Tuberculin tests should will likely remain – unvalidated. Using the be read by palpation. Test results should be three diagnostic modalities (i.e. direct, CMI interpreted taking into consideration the his- and serological techniques) available for TB tory of the individual animal enclosure, the screening in combination for each individual previous movement history of the animal and case reduces the limitations of each type of the TB risk status of the institution. All test, reducing the likelihood of false negatives animals should have a permanent identifica- and false positives. tion (transponder or tattoo). Ungulates should Zoological managers have the duty to en- be quarantined upon arrival according to the sure the protection of the visitors and staff, as guidelines established by AZA (Quarantine well as welfare of their animal collection. Procedures Recommended for AZA Accre- Detecting TB in zoo species has two impor- dited Institutions: R. E. Miller, 1995) and tant aims. The first is to protect staff (keepers, AAZV (Preventive Medicine Recommenda- veterinarians) and visitors from zoonotic con- tions: Junge, 1995). Animals not screened for tamination. Serology and PCR are aimed at TB before shipment should be tested during this predictive purpose, focusing on the active quarantine at the receiving institution. (i.e. excretion) phase of the disease. The Immunological in vitro tests are often the second aim is the detection of latently in- easiest to perform because they only require a

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1 3 2 Unspecific Direct Immunological screening diagnosis

2-1 2-2 Imagery Stains (ZN, CMI based tests Humoral based fluorescence, ...) tests Bloodworks PCR In vivo ELISA Chemistry Culture TST MAPIA Clinical signs In vitro (e.g. weight loss, Molecular biology Rapid Flow Test cough, ...) (spoligotype, ...) LPA IFNg Other multiplex Gross necropsy Pathology systems

Fig. 1. Any positive results from boxes 1, 2-1, 2-2 or 3 should trigger the use of an ancillary test from another box to complete the testing process: CMI. cell mediated immunity; ELISA. enzyme-linked immunosorbent assay; IFNg. interferon-g; LPA. lymphocyte proliferation assay; MAPIA. multi-antigen print immunoassay; PCR. polymerase chain reaction; TST. tuberculin skin test; ZN. Ziehl–Neelsen stain. blood sample but any repeated positive out- to deal with. Zoos that engage in treatment come in this immunological investigation must strictly comply with these rules and seek should trigger a secondary confirmation test permission for treatment from the official that uses direct techniques, in order to deter- authorities. When considering treatment op- mine the shedding status of the animal tions, a collection must be aware of the (Fig. 1). If direct examination is positive, potential sequela resulting from a failure in measures should be taken to avoid contam- treatment and the production of resistant ination of staff, surrounding animals and strains of mycobacteria (Lyashchenko et al., premises. However, it should be considered 2006). It is important to consider that the that in a known outbreak of TB (i.e. preva- result of a successful TB treatment is rarely lence is higher), the positive predictive value eradication, but bringing the animal back to a of testing is increasing and then a positive latent stage, which means that reactivation result may be sufficient when used in con- will always be a possibility at any stage of its junction with current history to confirm life when treatment is discontinued. Thus, TB-infected status. treated animals should be monitored closely for the rest of their life. It could be recom- Management considerations for positive or mended that treated animals remain in the premises where they have been treated or are suspected animals transferred to an institution with at least the The choice for euthanasia or treatment should same level of monitoring abilities. be considered carefully in concordance It is recommended that any animal with a with veterinary, animal management, occupa- culture positive for M. tuberculosis or M. tional physician, government officials and bovis should be euthanized owing to the the respective studbooks of the species in- potential for zoonotic transmission, transmis- volved [e.g. EEP, Species Survival Plan sion to conspecifics and the documented (SSP), TAG]. development of MDR-TB. Treatment might Treating an animal for TB implies follow- be encouraged in the case of sero-positive ing strict rules of drug administration, phar- animals or other ancillary diagnostics, but macokinetic controls, observance and only if the clinical evidence supports the excretion follow-up, which are very difficult potential for latent TB without any excretion.

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NTM at the University of Wisconsin reports isolat- ing the organism from 9 Á 1% of US zoos There are currently over 140 species of NTM, submitting faecal samples in a 2 year period which are a widely diverse group of organ- (1998–2000). Diagnosis in non-domestic isms with a broad spectrum of virulence and hoofstock is complicated by the fact that potential for causing disease in humans and serologic assays have not been validated in animals. The NTM share an important char- these species and, therefore, culture is still the acteristic in that they are all found in some standard. niche in the environment. Zoonotic risk is, There are NTM species, such as Mycobac- therefore, not as great with NTMs as animals terium kansasii and , and humans may have an equal opportunity that are almost always associated with sig- for environmental exposure. The notable ex- nificant disease when isolated from respira- ception is in a post-mortem examination of an tory specimens in humans (Griffith, 2010) animal that has disseminated NTM disease, and have been recognized recently as signifi- allowing potential exposure of the pathologist cant pathogens in zoological collections. to unusually high levels of mycobacteria. Mycobacterium kansasii is particularly pro- Diagnostically, this proves challenging when blematic in that is closely resembles M. bovis mycobacterial disease is suspected and an infection in ruminants. Disease from this NTM is cultured. organism has been reported in Bontebok MAC consists of two major groups of slow- Damaliscus pygargus (Terrell et al., 2009) growing acid-fast bacilli: M. avium and Myco- and llama (Johnson et al., 1993), and has been bacterium avium intracellulare. The route of seen in Cape buffalo Syncerus caffer and infection is typically via the gastrointestinal Addax Addax nasomaculatus (R. Ball, unpubl. tract and it is typically shed via faeces and will data). Western blot analysis was useful in the contaminate soil. MAC has produced disease case of the Addax to differentiate it from M. in numerous species of birds and mammals, bovis. Mycobacterium szulgai was recently and is an important pathogen for respiratory implicated as the cause of osteoarthritis and disease in humans (Griffith, 2010). Captive pneumonia in African elephants (C. Lacasse marsupials seem to be pre-disposed to infec- et al., 2007), but could also create severe liver tion more than their wild counterparts and lesions in amphibians (Chai et al., 2006) and disease is typically from MAC. Clinical dis- pneumonia in crocodiles (Roh et al., 2010). ease often manifests itself as osteomyelitis is a water-borne (Richardson & Read, 1986; Bush et al., atypical Mycobacterium species that com- 1995). Marsupials appear to be highly suscep- monly infects fish and amphibians. The in- tible to specific mycobacterial infections that fection is typically limited to the skin, mostly may be linked to deficiencies in their cellular involving limbs, but spread to deeper struc- immunity. Histopathological inspection of tures (joints) has been reported (Petrini, affected tissues indicates that, unlike most 2006). At least 150 fish and frog species eutherians, marsupials are unable to wall off (Talaat et al., 1997), aquatic mammals (e.g. infection sites, resulting in the formation of dolphins) and snakes are known to acquire satellite lesions and generalized disease (Bud- natural M. marinum infection. Individuals who dle & Young, 2000). fish or work with aquariums are at an increased Johne’s disease is an infectious disease of risk of exposure (A. Lacasse et al., 2009). ruminants caused by Mycobacterium avium One complication of NTMs is the possibility paratuberculosis (MAP). The organism pri- of producing false positives in some TB testing, marily affects the small intestines, resulting in especially TST, as they share several molecular poor digestion, diarrhoea and chronic weight components with the TB complex mycobacter- loss. Johne’s disease has been diagnosed in ia. To counter this, some humoral screening captive non-domestic hoofstock and in free- tools now use more specific recombinant anti- ranging wildlife. The Johne’s Testing Center gens, and IFN-g tests now also rely on specific

Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London 196 RESEARCH IN ZOOS: EPIDEMIOLOGY & CONSERVATION MEDICINE proteins’ stimulation to differentiate the im- the diversity of response to mycobacterial mune reaction between NTMs and the MTC. infection are powerful sources of knowledge As mentioned previously, orangutans con- for TB science. A large proportion of pub- sistently produce false positives on TST and lished literature on the ‘newest’ TB diagnostic other diagnostics must be utilized to deter- tools research involves wild species, both free mine the status of mycobacterial infection ranging and captive, demonstrating that the with regard to the MTB complex. Orangutans zoo community is not only following scien- are well-known reactors to intradermal skin tific improvements on TB diagnostics but also testing of essentially any tuberculin (Calle actively participating in developing them. et al., 1989; Wells et al., 1990) without any evidence of mycobacterial disease. One con- sideration for this sensitization is that there is PRODUCTS MENTIONED IN THE TEXT less exposure to the soil-borne organism in the BOVIGAMs: in vitro test for the diagnosis of bovine natural history of such arboreal animals and TB, manufactured by Prionics AG, CH-8952 Schlieren- Zurich, Switzerland. that in managed situations they become sensi- CERVIGAMs: whole-blood assay for the cytokine tized. In the case of the Cape buffalo with M. (production discontinued), manufactured by Prionics kansasii mentioned above, TST was positive AG, CH-8952 Schlieren-Zurich, Switzerland. to bovine tuberculin on one occasion. DPP VetTBs: Dual Path Platform, rapid serological assay detecting antiboody against mycobacterial antigens MPB83 and ESAT6/CFP10 on two separated lines, CONCLUSION manufactured by Chembio, Medford, NY 11763, USA (not available at the time of writing). MTC has existed for more than 40 000 years, ELEPHANT-STATPAKs: rapid serological assay de- and obviously benefits from human and ani- tecting specific antigens to MTC in elephants, manufac- tured by Chembio, Medford, NY 11763, USA. mal dispersion (Wirth et al., 2008). It is now GENOQUICK-MTBs: molecular genetic assay for the classified as a ‘re-emerging’ disease, with a rapid direct detection of the MTC, manufactured by Hain strong burden in developing countries. Lifescience GmbH, Hardwiesenstrae 1, 72147 Nehren, Germany. Zoo collections should not consider them- s selves out of reach from the TB threat. In the PRIMAGAM : in vitro test for the diagnosis of TB in non-human primates, manufactured by Prionics AG, CH- absence of serious surveillance, MTC myco- 8952 Schlieren-Zurich, Switzerland. bacteria are able to spread silently within a PRIMATB-STATPAKs: rapid serological assay detect- zoo as latency forms, multiplying potential ing specific antigens to MTC in elephants, manufactured sources of excretion for the remaining by Chembio, Medford, NY 11763, USA. QUANTIFERON GOLDs: whole-blood test for diag- zoo inhabitants, including visitors and staff. nosing MTC in humans, manufactured by Cellestis The diagnostic techniques available are not as GmbH, D-64293 Darmstadt, Germany. reliable as stand-alone tools but when used in T-SPOT TB: whole-blood test for diagnosing MTC in combination they facilitate a reduction in the humans, manufactured by Oxford Immunotec, Abindon, risk of keeping or importing infected animals. Oxforshire, OX14 4RY, United Kingdom. TUBERCULIN OTs: prepared from culture filtrates of Long incubation periods and latency of any Mycobacterium tuberculosis (strains Pn, C, and Dt), mycobacterial disease require that surveil- which are heat-inactivated, manufactured by Synbiotics lance plans are established on a long-term Corp., 11011 Via Frontera, San Diego, CA 92127, USA. basis in order to be effective. An important aspect to remember in all mycobacterial infections and diseases is that REFERENCES ABEBE, F., HOLM-HANSEN, C., WIKER,H.G.& BJUNE,G. the host is often in a compromised state; then (2007): Progress of serodiagnosis of Mycobacterium additional efforts should be directed towards tuberculosis infection. Scandinavian Journal of Immu- discovering what is allowing opportunistic nology 66: 176–191. bacteria to cause disease. ARIGA,H.& HARADA, N. (2008): Evolution of IGRA It should be noted that the variety of researches. Kekkaku 83: 641–652. ARTOIS, M., LOUKIADIS, E., GARIN-BASTUJI, B., THOREL, species in zoos, the apparent naı¨ve status M. F. & HARS, J. (2004): Infection des mammife`res towards mycobacteria for some of them and sauvages par Mycobacterium bovis, risques de

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Int. Zoo Yb. (2011) 45: 183–202 c 2011 The Authors. International Zoo Yearbook c 2011 The Zoological Society of London