Tuberculosis in Nonhuman Primates, - an Overview ofDiagnostic Tools

Alex Lecu1, Sascha Knauf2, Kerstin Mätz-Rensing2, Franz-Josef Kaup2
1 Parc Zoologique de Paris, M.N.H.N, Paris, France;
2 Pathology Unit, German Primate Center, Göttingen, Germany;

1. Introduction
In order to assess the status of a nonhuman primate (NHP) toward tuberculosis infection,
several test methods with different informative value are available. The intradermal skin test
is still the most commonly used and the only ILAR/CDC-approved method for tuberculosis
testing of animals in import quarantine (Roberts and Andrews, 2008). However, there aresevere limitations that make this test unsuitable as a single stand-alone test for tuberculosis
surveillance in nonhuman primate (NHP) facilities. Advance in technology led to thedevelopment of new immune based screening assays, which should be included in the
diagnostic screening program, while performances of direct and molecular tools are also
improving. The use of several tests in combination can increase the sensitivity or specificity
of screening and surveillance programs. Here, we summarize common diagnostic techniques,
their application and limitations.

2.Non-specific approach
2.1. Clinical examination
Clinical status of infected animals may be altered, although this is more likely to happen in
the active form than in the latent form of the disease. Weight loss may occur after the firstmonth after infection, as well as initial hyperthermia (between 3 and 8 weeks after infection).
1Other clinical signs are scarcer, but may include cough or abdominal/pleural effusion. On
blood works, transient neutrophilia may occur within the first weeks. ESR, fibrinogen, Cprotein and other inflammation biomarkers may increase as well, while the albumin/alpha-2
ratio could decrease on plasmatic protein electrophoresis.
Associated to the clinical examination, imagery tools other than X-rays/CT-scan can help,
such as endoscopy or ultrasound, especially as they can help to visualize granulomas or
suspicious lymph nodes and to perform biopsies.

2.2. Chest radiographs
A posterior-anterior chest radiograph can be used to detect chest abnormalities. Lesions may
appear anywhere in the lung and may differ in size, shape, density, and the presence ofcavitations. Thoracic radiography may provide confirmation of pulmonary disease but cannot
distinguish between tuberculosis and other granulomatous or cavitary diseases of the lungs
such as nocardiosis, aspergillosis, or echinococcosis. Therefore, chest radiographs may be
used as an additional test procedure only for screening of TB. Although this additional test
has a limited sensitivity, it is valuable for the identification of animals that have a negative
tuberculin skin test (TST) reaction due to immunosuppression in an end-stage and fulminantdisease. Compared to other species, nonhuman primates rarely develop calcified lesions.
Therefore, chest radiographs can be difficult to interpret and ideally are presented to an
experienced radiologist or lung physician for interpretation. There are no pathognomonic
lesions, but enlargement of the bronchial lymph nodes may be an early albeit unspecific signof pulmonary mycobacteriosis. Larger tubercles or cavitations may be appreciatedradiographically. Abdominal radiography may help to identify or confirm splenomegaly or
mesenteric lymphadenopathy. Computed tomography scanning or magnetic resonanceimaging can give detailed real-time imaging of disease progression and reveal tiny
2

granulomas that may be overlooked despite regular X- ray (Lewinsohn et al. 2006).

3. Immunological based tools
3.1. Intradermal tuberculin skin test
In many facilities the in vivo diagnosis of tuberculosis is based upon the intradermal
tuberculin skin test (TST) using mammalian old tuberculin (MOT) or purified protein
derivates (PPD). The tuberculin is injected intradermally into an eyelid near its edge or into
the abdominal skin, or both. The use of 25-27 gauge and 1/2 to 5/8 inches needles isrecommended in order to reduce bruise risk. The test relies on the development of a delayed
hypersensitivity response against mycobacterial antigens. In nonhuman primates this delayedtype hypersensitivity develops as part of the adaptive immune cascade within 3-4 weeks after
infection. The protein fraction of the tuberculin is recognized by sensitized T-lymphocytes
causing release of cytokines, localoedema and local cellular infiltration by inflammatory cell
recruitment.

3.1.2. Method
For the palpebral TST, 0.1 ml of MOT (2.500 IU) has to be injected intradermally as close as
possible into the edge of the upper eyelid using a sterile 25-27 gauge needle for each
nonhuman primate (Fig.1). For the abdominal TST, the hair should be shaved off without
traumatizing the skin, and the injection site should be marked with ink or permanent marker
to ease the reading. A volume of 0.1 ml MOT (2.500 IU) has to be injected intradermally into
the skin of the demarcated site on the abdomen. The abdominal skin test is most commonlyused when nonhuman primates suspected of TB infection after a first TST are retested. The
advantage of using the abdomen is that any indurations can be measured and a saline or buffer
control injection can be used. According to the experience of some working groups, theabdominal TST in nonhuman primates seems to be less sensitive than the palpebral test.
3

Fig. 1aFig.1b

Fig. 1a: Intradermal application of 0,1ml MOT as close as possible to the edge of the upper
eyelid.
Fig.1b: Directly after application a slight swelling of the upper eye lid is visible.

3.1.3. Reading TST
The animals must be observed for reactions at 24, 48, and 72 hours after injection under
optimal lighting conditions. Animals, which are suspected to be anergic, should be observed
after 2 to 8 hours and daily as “flash” reactions can quickly recede. A trained technician may
do the initial readings, but any reaction or suspected reaction needs to be diagnosed andinterpreted by an attending veterinarian. Close visual observation and even manual palpationof injection site in sedated animals may optimize the detection of a positive reaction(Panarella 2010). The following grading system is recommended:

Eyelid injection:
Score

Observations

Outcome0

No reaction

Negative

1

Bruise, extravasation of blood into the eyelid Negative4

associated with the injection of tuberculin
2

Varying

degreesoferythemaofthe Negative

palpebrum with minimal swelling
3

Moderate swelling with or without erythema

4

ObviousswellingoftheeyelidPositivewith Positive

drooping and varying degrees of erythema
5Marked swelling with necrosis and closed Positive
eyelid.

Abdominal injection

Score

Observations

Outcome0

No reaction

Negative

1

Moderate swelling

Negative

Height of induration 3-5 mm
2Moderate swelling

Doubtful

Height of induration 5-10 mm3

Obvious swelling

Positive

Height of induration >10 mm

5Fig. 2: Positive intracutaneous test, grade 4 reaction

Finally, again, visual scoring of the skin test is largely subjective promoting confusion
between negative and positive results. Therefore, alternative diagnostic approaches for antemortem TB testing are needed.

3.1.4. Frequency of TST
3.1.4.1. Before importation from non-European facility (= no agreement regarding directive
92/65 EEC):
Three negative tests with two-week interval are recommended. The last test should be done 10days before shipment. Prior to importation the supplier should provide data on tests made(tuberculin used, etc.).

3.1.4.2. In quarantine:
The minimal duration of quarantine is 42 days. During this time period, three tests are
required. The first test should be done one week after arrival to assure that the animals have ashort acclimation period. In case there were any reports of TB infections in the origin/sourceof the monkey within the past year, a prolonged quarantine period of 60 days including 2
additional tests with bovine PPD is advisable. Some dedicated primate centers (e. g. BPRC in
the Netherlands) install a three months (13 weeks) quarantine procedure regardless of theorigin/history of the animals.

3.1.4.3 Post quarantine:
The following interval for TST of species or groups of nonhuman primates is recommended
after quarantine for institutes with frequent animal transfer and close contact to humans:
macaques and vervets quarterly, baboons, prosimians and New World monkeys semi6

annually, great apes annually. A so-called closed institute holding monkeys with limited
exposure to humans may have a routine of TB testing every 12 months depending on thepolicy of the institute.

Because of a number of variables, the facility veterinarian may choose TST at less frequent
time intervals. If nonhuman primates are shipped from a facility with less frequent TB testing
than advised, the receiving veterinarian must be notified about the deviating procedure before
the animals are transferred.
It should be considered that intervals and frequency of TST may interfere with the outcome
due to mechanisms of immunological sensitization or desensitization. So far, neither of theseactions has been demonstrated in NHP, but a booster effect by repeated TST is sometimes
elicited in human practice to reveal latent infections in elderly people. Moreover,desensitization through repeated tuberculin injection was described in cattle. TST overuseshould therefore be avoided, especially within a shorter period of time.

It is important that each NHP tuberculin test is accurately recorded for each animal. An
animal’s clinical record should also include information about the building and/or room, inwhich it was kept, its social housing partners, and any movement (when it applies) during
quarantine. A digital image and exact measurement of a suspect skin reaction is a useful tool
to compare test results in repeated tests.

3.1.5. Interpretation issues
3.1.5.1. Tuberculins
The amplitude of the hypersensitivity response and, therefore, the accuracy of the TST
reading may correlate with the number of (replicating) tubercle bacilli but also depends on
various factors including the amount of circulating, primed, antigen-specific T-cells and the7amount of specific antigen within the tuberculin preparation that is used for the screening.
Mammalian old tuberculin (MOT) is a poorly defined preparation composed of various
mycobacterial antigens that are known to be highly cross-reactive regarding sensitization ofhosts by environmental mycobacteria. In comparison, the purified protein derivatives (PPD)
are a precipitated fraction of culture filtrates, but also comprise a mixture of mycobacterial
antigens. MOT is less purified, but it holds more tuberculin units than PPD. In nonhumanprimates, MOT has greater reactivity than PPD and is therefore preferred to PPD as thereagent to use in a TST to identify infected animals.
When using PPDs, one should be aware that potency of different PPDs differs. After MOT,
the recommended PPD is bovine PPD (purified from M. bovis) at the usual dose of 2000 UI /0.1 ml. M. tuberculosis (“human”) PPD, also called RT23, could be used but reduces thesensitivity of the TST compared to bovine PPD. Moreover, it appears that tuberculin units
needed to trigger TST reaction are 100 to 1000 times greater in primates than in humans, thus
the use of RT23 could be questionable in terms of dosage.
A comparative TST could be performed using two PPDs such as avian PPD and bovine PPD
injected simultaneously but into two separated sites (e. g.: one eyelid each). Differential
analysis of reactions on the two sites gives information on the predominant immunologicalrecognition by host. This TST is meant to differentiate between reactions to TB complex
mycobacteria and mycobacteria other than tuberculosis (MOTT) and then increases the
specificity of the skin test. However, it should be known that avian and bovine PPDs share atleast 21 common antigens. Anecdotally, some other “in-house” tuberculins have beendeveloped, like M. scrofulaceaum related ones, which could be used only in very specificstudies or suspicion.
At least, quality, conservation, or suboptimal concentration of the tuberculin can profoundly
influence results of TST usually producing false negative reactions. Despite OIE or EEC
regulations discrepancies between the same tuberculin categories in different countries may
8

still occur, thus in-house re-testing remains of primordial importance.

3.1.5.2. Interactions
The TST is limited in its efficacy as animals with early or advanced infection may give false
negative reactions because of a latency period upon infection or immunosuppression in caseof progressive TB disease, respectively. The method relies on local immune cell recruitment.
Therefore, the test will turn out negative, if the amount of local or systemic immune cells isdecreased (Mahanta et al 1994). Concomitant diseases like measles or fungal infections may
also result in a false negative TST reaction due to immunosuppression (Ganaway 1974,
Potkay 1966, Tauraso 1973). Vaccination with polio, measles or yellow fever vaccine may
have the same effect (Ott 1979, Staley 1995). Therapy with isoniazid or undergoing treatment
with immunosuppressive drugs including corticosteroids will also negate the value of the
tuberculin test through diminution of inflammatory capacity (Dillehay Huerkamp 1990).
Furthermore, false negative reactions may result from incorrect injection and subjectivity inthe interpretation of the skin test.
False positive reactions may result from previous exposure to environmental MOTT,,
experimental injection of Freund`s complete adjuvant (see below) because it contains cellwall components of tubercle bacilli, trauma due to improper injection of the tuberculin, or
nonspecific reactions. Regarding the latter, contaminants like phenolic components may cause(transient) allergic reactions, which can appear already 30 minutes after injection and may
result in a false positive TST.

Tuberculous NHP can become anergic to mycobacterial antigens and appear negative in a
TST during certain stages of the disease (e.g end stage). Therefore, tuberculosis should beconsidered and further tests performed when a TST negative NHP displays unexplained
weight loss, non-healing wounds or any signs of distress or unexplained deviation from9

normal behaviour. Additional tests may include: culture swabs of non-healing wounds, chest
radiographs, selective culturing of a gastric or broncho-alveolar lavage.

Nonhuman primates display positive TST upon experimental injection of Complete Freunds
Adjuvant (CFA), because it contains cell wall components of tubercle bacilli. Ideally, othernon-mycobacterium-based adjuvants should be selected so that the diagnostic power of the
TST is not compromised. If the use of CFA is unavoidable (e. g. use of porcine zona pellucida
contraceptive vaccine), the individual animal has to be tuberculin tested the week before the
CFA is injected, or it has to be isolated from the colony. Because CFA application will induce
a positive TST, other indirect measures like weight monitoring, chest X-ray or bacterial
screening of biofluids might indicate TB infection. If tuberculosis is diagnosed in any otheranimal in the holding room housing a CFA exposed NHP, the nonhuman primate(s) that
previously received CFA should be regarded as potentially infectious and monitored
carefully. Because of the economic risk and possible ethical implication when a case of TB
remains unidentified, some dedicated primate facilities imply a stricter regime and do notallow animals to be in contact to their regular colony facilities at any time after the injectionof CFA.

It should be noted that some NHP species such as the orangutan (Pongo (P.) pygmaeus) havebeen reported to react false positive in the TST (Calle et al. 1989, Wells et al., 1990). In
Calle’s study, 60 % (12/20) of the orang-utans responded positively to at least one tuberculinthat was injected intradermally. Most animals reacted positive to MOT followed by M. avium
PPD and M. bovis PPD. Since none of the 20 tested individuals had evidence of tuberculosis,
as revealed by physical examination, hematologic and serum biochemical tests, thoracic
radiography, mycobacteriological examination of gastric lavage samples and ELISA, it ispostulated that immunological response was associated with the presence of non-tuberculous
10mycobacteria isolates. Eleven of twelve individuals were positive for mycobacteria other thantuberculosis (MOTT) in gastric lavage samples, and nine of those reacted positively in at least
one TST. It is therefore strongly recommended to carefully interpret single test results inspecies that have not been tested as yet, or those with high levels of false positive results. A
positive reaction is not necessarily a positive TB case, but in any way a challenge for the
responsible veterinarian or official. It is open to question, how, for example, orangutans
infected with MOTT react in the interferon-gamma test.
Given all these issues, a positive TST has to be confirmed by more definitive diagnostic
methods like thoracic radiographs, broncho-alveolar or gastric lavage, serology, and, finally,
isolation and culture of the bacteria.

3.2. Interferon-gamma test
Whole blood interferon gamma release assays (IGRAs) should be considered as analternative or as a complementary tool to the skin test. Both tests assess the cellular mediatedimmunity towards mycobacteria. IGRAs detect cellular reactivity to antigens through themeasurement of interferon-gamma (IFNγ) production by live lymphocytes contained in awhole blood sample. IFNγ is an important cytokine involved in the cell-mediated immune
response to mycobacteria. The assays may discriminate between M. bovis and M. avium
infection by exposing blood samples to avian and bovine PPD. Instead of PPDs, single
antigens (specific of mycobacteria groups or complex) may be used in the test to provide
more specificity.

Recently, a whole blood stimulation assay has become commercially available as the socalled PRIMAGAM® test licensed for use in cynomolgus and rhesus macaques. The assays
may discriminate between M. tuberculosis complex and M. bovis infection by including avian
and bovine PPD separately in the test. The amount of interferon produced in response to11

stimulation with different antigens can be used to differentiate a reaction due to M.
bovis/tuberculosis or atypical mycobacteria (Lerche et al. 2008). It is a quantifiable diagnostic
test with good sensitivity and specificity compared with disease status determined bypathologic examination (Garcia et al. 2004 a, b Vervenne et al. 2004). Recent results suggestthat the IFNγ response to tuberculin antigen may not be reliable in cynomolgus macaques andthat another cut-off should be used to read the test (Garcia et al 2004a).
Compared to the TST-test the PRIMAGAM test has a low sensitivity and high specificity(Garcia et al. 2004b, Vervenne et al. 2004, Lin et al. 2008). A combination of both tests may
increase the overall sensitivity and specificity.
Whole blood with anticoagulant, preferably lithium-heparin, is needed for testing. As thetest relies on (re)stimulation of primed T-lymphocytes, the first compulsory step is to keep
blood cells viable for in vitro culture. Thus, samples must be available for testing not later
than 8-10 hours after sampling and meanwhile should be kept at ambient temperature.
Particular care must be paid to homogeneous and full mixing of blood and anticoagulant(heparin) at collection.
IGRA is an in vitro test, and thus discards some effects of in vivo tuberculin injections (e.
g. sensitisation/desensitization). Over the past few years, comparable assays for humans(Quantiferon®, Quantiferon Gold®, T-SPOT-TB®) and cattle (Bovigam®) gave reliable andinformative results on the status of TB infection. It is a quantitative diagnostic test with goodsensitivity and specificity values. There is only sparse information about the use of
Quantiferon Gold® test in nonhuman primates. A comparison of this test with the TST-test
showed that the Quantiferon Gold® test might be a highly sensitive tool for the diagnosis of
mycobacterial infection in rhesus macaques but it was not possible to detect specific infection
with M. tubucolusis with this test (Parsons et al. 2010).
However, three important caveats should be known about IGRAs used in NHP. First,
there are several different interpretation guidelines, based on different calculation methods
12with various cut-off thresholds. In humans, IGRAs are known to have a high variability (>80%) between individuals. Cut-off values, therefore, might be influenced by the species as wellas the individual knowledge.l Experienced technicians are required for the completion of
IGRAs, and raw data (optic density) shall be communicated and documented properly. It isessential to have an equalized interpretation scheme. The test implies the use of a mitogen as apositive control. An aliquot of the sample is exposed to this mitogen, which in turn elicits anunspecific application and IFNγ production in lymphocytes. However, this mitogen is usually
not provided within the test and should be selected by the lab. Different mitogens can be used
(ConA, PHA, SEB, Pokeweed etc.). The level of unspecific stimulation is species dependantand needs to be assessed when an IGRA gets established. For instance, a human IGRA
(Quantiferon®) could be considered to be used for some species of NHP (apes) but contains
PHA as a mitogen, which is unfortunately known to be a weak stimulant in NHPs. Lab
technicians who perform IGRA must be aware of species/mitogen differences. Unfortunately,
yet, the majority of NHP species lacks such information. It is therefore recommend to use
two or more different mitogens to test NHP species with paucity of information about themost suitable mitogen.
Third caveat is the detection of IFNγ itself. IGRAs rely on ELISA or ELISPOT that aredesign to reveal IFNγ molecules for certain species. Through all the NHP taxonomy, IFNγ
varies in composition and structure, and there is no broad ELISA that is able to detect
interferon of all species. For instance, ELISA embedded in the PRIMAGAM® test is a“monkey INFγ” ELISA, but unable to detect IFNγ in most New World monkeys.
Therefore the parallel use of TST and PRIMAGAM® test is advisable for an optimal coveragein a diagnostic regimen. TST might influence IGRA results when performed a few days
before blood is sampled. As observed in other species (cattle, human), a “booster effect”
could occur with the previous injection of tuberculin strengthening the immune response
obtained on subsequent IGRA. Said to increase sensitivity of the test, questions may arise on13

the influence on its specificity, as the boost effect may be created only by tuberculin
sensitization. Therefore, the use of a booster procedure cannot be recommended at that stage.
Further studies are needed to answer those questions.

3.3. Serological tests: antibody detection
Recent work suggests that the serological detection of antibodies against Mycobacteriumspecific antigens might be a useful tool for the development of an immunodiagnostic method
for tuberculosis (Kanaujia et al. 2003). There is a strong association between tuberculosis in
NHPs and an immune response against early secretory antigenic target-6 (ESAT-6), a protein
especially secreted by the metabolically active virulent bacilli belonging to the tuberculosis
complex. Some indirect ELISA tests are designed and marketed to detect ESAT-6. Other
laboratories are proposing ELISA targeting on single antigens like the 10-kDa culture filtrate
protein (CFP-10) or broader antigenic compounds from culture filtrates. Purified single
antigens are known to increase specificity. However, ESAT-6 has been reported to be present
in mycobacteria out of the tuberculosis complex, which might cause cross-reactions that
potentially produce false positive results.

In a comprehensive approach, ESAT-6 is one of several antigens used in the PrimaTB STATPAK® assay, a new lateral flow test that has been developed as a TB specific serodiagnostic.
The test was evaluated in comparison with the intradermal palpebral tuberculin test on NHPsof two different species of macaques and on green monkeys. Some of the animals wereexperimentally infected with M. tuberculosis, and serologic evaluation demonstrated highdiagnostic sensitivity (90 %) and specificity (99 %) (Lyashchenko et al. 2007).
This commercial test is handy and easy to use and works with serum, plasma or possibly anyother antibody containing body fluid. It may, therefore, be an attractive option for institutes
with limited capacity. This serodiagnostic test employs a selective array of recombinant M.
14

tuberculosis proteins covering several immunodominant mycobacterial antigens for immune
detection. A combination of PrimaTB STAT-PAK® assay and the TST seems to be a sensitiveand reliable diagnostic approach for the detection of TB in NHPs, as it includes both ways of
immune-reaction towards tuberculosis infection. Yet, scientific validation of the test wasassessed for three species of Old World monkeys only. Several studies, unpublished or inpress, report its use in free ranging and captive primate species with lower sensitivity andspecificity than in Macaca species. However, as antibody detection in culture positive NHP
species, other than the three published species, was already noticed, serological tests remain
an interesting ancillary tool for the TB screening in monkey species.
As for IGRAs, an anamnestic rise in antibody titres has been observed in some species (cattle,
elephant) on serum samples collected between 1 and 3 weeks after tuberculin injection. Even
if not yet reported in NHP, it is likely to occur in primate species as well and, in absence ofany validation of this effect, serologic evaluation should be separated from TST by at least 2
months.

4 Direct examination
4.1. Culture: the Gold standard
The presence of acid-fast-bacilli (AFB) on a sputum smear or other specimen may indicate
TB disease. Acid-fast microscopy is easy and quick, but has its limitations. Some acid-fastitems are not M. tuberculosis (Nocardia, some coccidian, sperm heads). Ziehl-Neelsen is thestain of choice. A positive result has to be confirmed by culture,
Bacterial culture is required on all initial samples to confirm the diagnosis of a positive
suspicion. A positive culture for M. tuberculosis confirms the diagnosis of TB infection.
Culture examinations should be completed on all suspected samples regardless of AFB smear
results. Culture should last at least 8 weeks before a final conclusion can be made. Samples tobe cultivated for mycobacteria from clinical materials using Löwenstein-Jensen agar or other15

suitable cultivation media (liquid, semi liquid) include•

gastric lavage with acid fast cytology and culture of gastric mucus•

tracheal or broncho-alveolar lavage with acid fast cytology and culture of tracheal
mucus

•

fecal examination with acid fast staining and culture•biopsy of altered or unaltered organs (lymph nodes) with acid fast stain and culture•bio-fluids such as urine, pleural or abdominal effusion, cerebrospinal fluid, milk etc.

Although they belong to the group of “slow-growing mycobacteria”, culture methods arehighly standardized for M. tuberculosis and M. bovis with significant reduction of culture
delay. However, for other mycobacteria from the TB complex, such as M. pinnipedii orDassie Bacillus (M. tuberculosis complex sp.), a waiting period of at least 8 weeks is strongly
recommended before declaring the culture negative.
Finding NTMs out of gastric lavage or stools is common. In the case of NTM discovery on asuspected or TST positive animal, the conclusion of false positivity due to cross reaction
should not be taken too quick, as NTM and TB complex mycobacteria infection can occur
simultaneously.

4.2. Molecular biological diagnostic
Polymerase chain reaction (PCR) can be used to detect mycobacterial DNA in any biologicalsamples and intrinsically has the advantage of being much quicker than the conventional
culture methods of diagnosis (one day versus several weeks). Detection of infection byscreening faeces or sputum for mycobacterial DNA may therefore be considered as a rapid
and alternative diagnostic tool. Commercial laboratories often consider PCR on pure culture,
only, and therefore may refuse to run PCR on non-purified sample material. Moreover,sensitivity of PCR will be likely lower compared to pure culturewhen applied to biopsies or16

stools (presence of inhibitors etc.).
Beyond PCR, other DNA fingerprinting methods are available such as spoligotyping or otherstrain identification process. Their main interest is to identify strains of mycobacteria, andthen track epidemiological circulation and origin of the infection. These methods are mainly
applied on pure culture, but some laboratories accept to perform it on samples, provided thatthe number of bacilli inside is high enough.

5. CONCLUSION
Intradermal testing alone is not sufficient to adequately diagnose tuberculosis. Each of themethods listed include the risk of false positive or false negative reaction. Therefore, acombination of the TST test with an IFNγ assay or a lateral flow assay is advisible to increase
the sensitivity and specificity. In doubtful cases, a range of test methods relying on different
aspects of the pathogen (immunology, serology) should be applied and the results should be
evaluated critically. During evaluation, the veterinarian has to set established laboratory andspecies specific cut-off values for most of the available tests, and this is directly influencing
the predictive positive and negative values.

References

Calle, P., Thoen, C. and Roskop, M.: Tuberculin skin test responses, mycobacterolgical examinationsof gastric lavage, and serum enzyme linked immunosorbet assays in orangutans (Pongo pygmaeus).
J. of Zoo and Wildlife Med. 20: 307-314, 1989.

Garcia, M.A., Bouley, D.M., Larson, M.J., Lifland, B., Moorhead, R., Simkins, M.D., Borie,
D.C., Tolwani, R. and Otto, G.: Outbreak of Mycobacterium bovis in a conditioned colonyof rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Comp.
Med. 54:578-584, 2004a.
Garcia, M.A., Yee, J., Bouley, D.M., Moorhead, R. and Lerche, N.W.: Diagnosis of17

tuberculosis in macaques using whole blood in vitro interferon-gamma (Primagam) testing.
Comp. Med. 54:86-92,2004b.

Kanaujia, G.V., Garcia, M.A., Bouley, D.M., Peters, R. and Gennaro, M.L.: Detection ofearly secretory antigenic target-6 antibody for diagnosis of tuberculosis in non-humanprimates. Comp. Med. 53:602-606, 2003.
Lerche, N. W., Yee, J. L., Capuano, S. V. and Flynn, J. L. : New approaches to tuberculosis
surveillance of nonhuman primates. ILAR Journal 49, 170–178, 2008.

Lewinsohn, D.M., Tydeman, I.S., Frieder, M., Grotzke, J.E., Lines, R.A., Ahmed, S., Prongay, K.D.,
Primack, S.L., Colgin, L.M.A., Lewis, A.D. and Lewinsohn, D.A.: High resolution radiographic
and ﬁne immunologic deﬁnition of TB disease progression in the rhesus macque. Microbes Infect.
8: 2587-2598, 2006.

Lin, P.L., Yee, J., Klein E. and Lerche, N.W.: Immunological concepts in tuberculosis diagnostics fornonhuman primates: a review. J Med Primatol, 37 (1): 44-51, 2008.

Lyashchenko, K. P., Greenwald, R., Esfandiari, J., Greenwald, D., Nacy, C.A., Gibson, S., Didier, P.
J., Washington, M., Szczerba, P., Motzel, S. (2007): PrimaTB STAT-PAK assay, a novel, rapid
lateral-flow test for tuberculosis in nonhuman primates. Clinical and Vaccine Immunology 14:
1158–1164.

Panarella, M.L. and Bimes, R.S.: A naturally occurring outbreak of tuberculosis in a group ofimported cynomolgus monkeys (Macaca fascicularis). J. Am. Ass. Lab. Anim. Sci. 49 (2): 221225, 2010.

Parsons, S.D., de Villiers, C., Gey van Pittius, N.C, Warren, R.M. and van Helden, P.D.: Detection of
Mycobacterium kansasii infection in a rhesus macaque (Macaca mulatta) using a modified
QuantiFERON-TB Gold assay. Vet Immunol. Immunopathol., 136 (3-4), 330-334, 2010.

Roberts, J.A. and Andrews, K. : Nonhuman primates quarantine: its evolution and practice. ILAR
Journal49, 145-156, 2008.

Vervenne, R. A .W, Jones, S. L., Van Soolingen, D., Van Der Laan, T., Andersen, P., Heidt, P. J.,
Thomas, A. W. and , Langermans, J. A. M.: TB diagnosis in non-human primates: comparison of
two interferon-g assays and the skin test for identification of Mycobacterium tuberculosis infection.

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Vet. Immunol. and Immunopathol. 100: 61–71, 2004.

Wells, S. K., Sargent, E. L., and Andrews, M. E. : Tuberculosis and tuberculin testing in Orang-utangs(Pongo pygmaeus). Proc Am. Assoc. Zoo Vet., 119–125,1990.

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