J Med Primatol doi:10.1111/j.1600-0684.2008.00303.x

ORIGINAL ARTICLE

Guidelines for the prevention and control of tuberculosis
in non-human primates: recommendations of the European
Primate Veterinary Association Working Group on
Tuberculosis
Moshe Bushmitz1, Alex Lecu2, Frank Verreck3, Ellen Preussing4, Susanne Rensing5 & KerstinMätz-Rensing6 of the EPV-Tuberculosis Working Group on Non-human Primate Health
1 BFC, Mazor, Israel
2 Parc Zoologique de Paris, Paris, France
3 Biomedical Primate Research Centre, Rijswijk, the Netherlands
4 Novartis Pharma AG, Basel, Switzerland
5 Covance, Münster, Germany
6 German Primate Centre, Göttingen, Germany

Keywords
Diagnosis – handling – non-human primate –recommendation – tuberculosis
Correspondence
Dr Kerstin Mätz-Rensing, Department of
Infectious Pathology, German Primate
Centre, Kellnerweg 4, D-37077 Göttingen,Germany.
Tel.: +49 551 3851 386;
fax: +49 551 3851 442;
e-mail: kmaetz@gwdg.de
Accepted June 14, 2008.

Abstract
Background Effective tuberculosis (TB) control requires accurate diagnostic
methods but the tuberculin skin test has serious limitations. Both false-negative and false-positive reactions are common, resulting in the spread of
the infection and devastating TB outbreaks. Results of questionnaire surveys concerning TB testing practices in primate housing facilities showed
great differences in testing practices. Although there was some uniformity
regarding the sites of application, the amounts of tuberculin used and thetime intervals for retesting, a great deal of variety was revealed considering
the types of tuberculin preparations, the interpretation of tests and thesusceptibility of animals.
Conclusion Here, we summarize the most common practices as regards TB
control and prevention for non-human primates, and attempt to establish a
uniform guideline based upon our experience with primate husbandry andcare programmes as well as recent developments in the literature. The present guideline represents a consensus recommendation intending to harmonize the existing protocols.

Introduction
Simian tuberculosis (TB) is one of the most important
bacterial diseases of non-human primates because of
its ubiquitous and insidious nature and its ability to
spread rapidly. It is caused by two aerobic facultative
intracellular bacillius species, Mycobacterium tuberculosis and M. bovis [1]. Tuberculosis in non-human primates is a disease most often contracted from humans.
Although it usually becomes manifest as a respiratory
disease, tuberculosis can disseminate to almost any
organ and it should be regarded as a systemic infection
J Med Primatol 38 (2009) 59–69 ª 2008 The Authors
Journal compilation ª 2008 Blackwell Munksgaard[1, 2, 10]. Outbreaks of tuberculosis have beenreported in non-human primate colonies almost as
long as primates have been used as experimental
animal models or kept in zoological gardens. Despite
the signiﬁcant reduction in the incidence of tuberculosis among captive non-human primates since 1970,
tuberculosis remains a serious threat to the health of
non-human primates and their caretakers. Outbreaksof tuberculosis still occur in established colonies andcan have economic consequences because of the loss ofanimals, disruption of research and costs associatedwith disease control. Infected animals with active
59

Diagnosis of tuberculosis in non-human primatestuberculosis may show overt signs of disease for weeks
or months [3, 11, 21]. The tuberculous monkey is ahealth hazard especially to other monkeys in thegroup, but (re)transmission of the infection to man has
been reported as well. For this reason, an effective preventive tuberculosis screening programme is essential
to reduce the risk of infection with this classical anthropozoonosis.
Guidelines for the prevention and control oftuberculosis in non-human primates
Before acquiring new animals in a facility, efﬁcient
preventive measures and procedures have to be inplace, so that the spread of TB from the new animals
to the colony is prevented. Speciﬁc examination of animals including TB testing at the supplier facility andcareful retracing of the source/breeder will help in preventing the introduction of mycobacterially infectedindividuals into the colony. Preventive measures also
require the protection of non-human primates as wellas the personnel in contact with these primates, possibly harbouring Mycobacterium spp. Efﬁcient quarantine procedures, husbandry practices, and monitoringprocedures, and protection of personnel and nonhuman primates are useful for preventing of TB outbreaks in a colony. A quarantine procedure at the timeof the animal’s arrival in a country or facility is mandatory for controlled animal health assessments. The
basic components of quarantine include isolation ofimported animals, veterinary health observations,
physical examinations, diagnostic testing for important
zoonotic diseases and protection of the personnel
involved. In general, the quarantine programme musttake into account the zoonotic potential of non-humanprimates and depends on the information available onindividual animals undergoing the quarantine procedures. The life history and available documentation on
the health status of an animal may inﬂuence the duration and extensiveness of the quarantine process. As arule, the less the information available on an animal
and for animals originating from an infected source,
the longer and more stringent the quarantine procedures. For all aspects of this guideline the legal
requirements which could be differing from country tocountrym, should be followed.
Quarantine and testing
As it takes a minimum of 3 weeks for infected animals to develop a delayed hypersensitivity reaction
to tuberculin testing, a series of consecutive testing is
recommended during any quarantine period to60

Bushmitz et al.

increase the possibility of detecting TB positive animals upon importation. The duration of quarantine
has to be a minimum of 42 days (6 weeks) withthree tuberculin skin tests (TSTs) incorporated into
the routine at 2-week intervals and starting 1 week
after arrival. Whenever feasible, it is recommended
to extend the quarantine to a length of 3 months(13 weeks). In case of a positive TB test, the respective animal(s) will be isolated and/or killed, while forall other animals the quarantine procedure will berestarted at that point.
Performing pre-transport TB testing on animals
from controlled breeding or supplier facilities withgood documentation is desirable, as this testing can be
performed in a controlled manner under veterinarycare. This pre-transport testing will reduce the risk ofimporting animals with TB and may allow for reduced
testingrequirements
duringthepost-importquarantine.
Because of the enormous economic interests andpossible ethical impact, some dedicated primate centres
routinely comply with a 3-month (13 weeks) quarantine period, with four skin tests at four weekly intervals, regardless of the origin of the animals. Moreover,two PRIMAGAM tests are incorporated at the startand the end of the quarantine period for an optimal
diagnostic coverage.
Animals from an approved source or the so-called
good-quality animals are those which are captive bredand conditioned in their country of origin andexported for research. Animals that are wild-caught
and kept in captivity for a variable amount of time
before their export to another country bear a higher
risk of disease. In cases of pre-export tuberculosis testing, three negative tests at 2-week intervals, with thelast test not more than 10 days prior to shipment are
required. The supplier should provide health certiﬁcates including dates of all previously performed TB
tests and the parameters tested [puriﬁed protein derivates (PPD), mammalian old tuberculin (MOT), etc.].
These documents should accompany all primate shipments (from suppliers to research institutions or zoos,
between institutions, etc.). Animals should not be
accepted from suppliers who have reportable diseases
or signiﬁcant losses in animal numbers. All animals
have to be physically examined by a veterinarian after
arrival. Very valuable animals diagnosed with TB may
be treated; others are to be qualiﬁed as non-acceptable
and should be killed or returned to the supplier.
Quarantine groups should be established in consideration of the species, disease status (if known), sourceand date of arrival in quarantine. Juvenile macaques
are the most susceptible to TB. Animals should be
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Bushmitz et al.

housed together by shipment and not mixed with different shipments or species. If groups are housed
together, the start of the quarantine is deﬁned as theday when the last animal enters the quarantine room.
At no time should different species be housed
together during quarantine. To minimize the risk ofcontamination, movement of animals should be
avoided and is acceptable only with prior approval of
the attending veterinarian. Any movement of animals
during quarantine should be strictly recorded to allow
tracing in case of disease. Other factors that have to
be considered when establishing quarantine groups
include the age of animals and social history. The
quarantine facility should contain a minimal numberof animals per room and have good and efﬁcient environmental conditions.
Loss of weight is an important indicator for tuberculosis. Therefore, weight of the animals should be carefully monitored periodically and animals that have lost
more than 10% of their body weight from the start of
the quarantine procedure or young animals which do
not gain weight after 42 days should be re-examined
and carefully evaluated.
Prior to release of animals from quarantine, allinformation pertinent to the group should be reviewed,
including shipping documents, applicable permits,
health certiﬁcates and records from the supplier (originator), tuberculin test records for each animal, individual health records containing the results of all
surveillance and diagnostic procedures and all prophylactic measures performed during quarantine. The animals should be subjected to a ﬁnal physicalexamination, preferably including a thoracic radiograph. Release from quarantine should be possible
only with the permission of the attending veterinarian
after all physical examination records have beenchecked.
Unexpected deaths in quarantine should be handledwith caution. Animals should carefully be doublebagged, weighed and submitted for necropsy to acompetent pathology section. The veterinarian and/or
veterinary pathologist should be notiﬁed immediately.
The remaining animals of the group should be kept
totally isolated in the quarantine until the cause ofdeath is identiﬁed.
Husbandry practices
The animal husbandry and sanitation practices applied
to non-human primates are designed to prevent the
spread of pathogens including tubercle bacilli. Optionally, tuberculocidal detergent disinfectants can be usedin facilities housing non-human primates. Cleaning
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Diagnosis of tuberculosis in non-human primatesequipment must be kept in one room unless it will beeffectively disinfected between rooms. Sanitation
schedules and practices must be in compliance with allapplicable regulations, policies and guidelines.
Non-human primate holdings and procedure rooms
must be under negative pressure relative to adjacentcorridors. Husbandry practices must minimize the production of aerosols in animal rooms, e.g. sanitizing
room surfaces and sanitizing animal cages and lifter
pans or trays. Other procedures, including research
procedures, must be carried out in a manner to prevent the generation of aerosols that potentially containpathogens. High-pressure washing of cages and room
surfaces can be performed only after the non-humanprimates have been removed from the room and withproper protection of personnel.
Monitoring procedures
TST, the primary tool used to detect tuberculosis in
nonhuman primates
Antemortem diagnosis of tuberculosis is based upon
the intradermal TST using MOT or PPD. They are
injected intradermally into an eyelid near its edge orinto the abdominal skin, or both. The test relies on thedevelopment of a delayed hypersensitivity response
against a mycobacterial antigen. In non-human primates this delayed-type hypersensitivity develops aspart of the adaptive immune cascade within 3–4 weeks
post-infection. The protein fraction of the tuberculin isrecognized by sensitized T lymphocytes causing releaseof lymphokines, local oedema and local cellular inﬁltration. The amplitude of the hypersensitivity response
and therefore the accuracy of the TST reading maycorrelate with the number of (replicating) tubercule
bacilli and depend on various factors including theamount of circulating, primed, antigen-speciﬁc T cells
and the amount of speciﬁc antigen in the tuberculin
preparation that is used for the screening. MOT is apoorly deﬁned preparation composed of various mycobacterial antigens that are known to be highly crossreactive. In comparison, the PPD are not only a
precipitated fraction of culture ﬁltrates, but also comprise a mixture of mycobacterial antigens. MOT is lesspuriﬁed, but it holds more tuberculin units than PPD.
In non-human primates, MOT has a greater reactivity
than PPD and is therefore preferred to PPD as thereagent to use in a TST to identify infected animals.
The TST is limited in its efﬁcacy as animals withearly or advanced infection may give false-negative
reactions because of a latency period upon infection or
immunosuppression in case of progressive TB disease,61

Diagnosis of tuberculosis in non-human primates

respectively. Concomitant diseases like measles or fungal infections may also result in a false-negative TSTreaction because of immunosuppression [17]. Vaccination for polio, measles or yellow fever may have thesame effect [15, 19]. Therapy with isoniazid or treatment with immunosuppressive drugs including corticosteroids will also negate the value of the tuberculin
test. Furthermore, false-negative reactions may result
from incorrect injection, subjectivity in the interpretation of the skin test, or a suboptimal concentration of
antigen in the applied preparation.
False-positive reactions may result from previous(experimental) exposure to complete Freund’s adjuvant (CFA), trauma because of improper installationof the tuberculin test substance, or non-speciﬁc reactions [5, 14, 16]. As regards non-speciﬁc reactions,contaminants like phenolic components may lead to(transient) allergic reactions, which can present
within 30 minutes after injection. Furthermore, crossreactivity after exposure to atypical or saphrophytic
mycobacteria like M. gordoneae may result in a
false-positive TST [18].
Methods
Using a sterile 25–27 gauge needle for each non-humanprimate, inject 0.1 ml of MOT (2 500 IU), intradermally into one eyelid near the margin or into theabdominal skin or both; 0.05 ml can be used in small
non-human primates, e.g. marmosets. Usually, the eyelid is preferred as it is relatively easy to observe. An
injection of the solvent into the second eye lid or theabdominal skin can be considered as a control. If theabdomen is used, the hair should be shaved off without traumatizing the skin and the injection site should
be marked with ink or edding to make the readingeasy. The abdominal skin test is most commonly used
when retesting non-human primates suspected of TB
infection after a ﬁrst TST. The advantage of using theabdomen is that any indurations can be measured anda saline control injection can be used. In marmosets
and tamarins, this site is recommended as injection
into the eyelid may be difﬁcult.
Reading TST
Observe the animals for reactions at 24, 48 and72 hours after injection under optimal lighting conditions. Animals which are suspected to be anergic
should be observed after 2–8 hours and everyday as‘ﬂash’ reactions can quickly recede. The initial readings
may be made by a trained technician. Any reactions orsuspected reactions are to be observed and interpretedby the attending veterinarian. The following grading
systems should be used:
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Bushmitz et al.

Eyelid injections
When using the following grading system, the actual
results should be recorded for every animal:
Reaction: scoring of changes
0 no reaction
1 bruise: extravasation of blood in the eyelid associated with the injection of tuberculin: negative
2 varying degrees of erythema of the palpebrum withminimal swelling: negative
3 moderate swelling with or without erythema: positive
4 obvious swelling of the palpebrum with drooping
and varying degrees of erythema: positive
5 marked swelling with necrosis and closed eyelid:
positive
Abdominal injections
Reaction: scoring of changes
0 no reaction
1 moderate swelling, height of induration 3–5 mm:
negative
2 moderate swelling, height of induration 5–10 mm:
questionable
3 obvious swelling, height of induration <10 mm:
positive
Cave: a case with two questionable test results
should be regarded as positive!
Finally, again, the visual scoring of the skin test is largely subjective promoting confusion between negativeand positive results. Therefore, alternative diagnostic
approaches for antemortem TB testing are needed.
Frequency of TST
Before importation
Three negative tests at 2 week intervals are required.
The last test should be performed 10 days before shipment. The supplier should provide data on tests conducted before importation (Fig. 1).
In quarantine
The minimal duration of quarantine is 42 days. During
this time period, three tests are required. The ﬁrst test
should be performed 1 week after arrival to ensure theanimals a short acclimation period (Fig. 1). In casethere were any reports of TB infections in the origin/source of the monkey within the past year, a prolonged quarantine period of 60 days including two
additional tests with bovine PPD is advisable. As
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Bushmitz et al.

Diagnosis of tuberculosis in non-human primatesFig. 1 Interpretation of Tuberculin Skin Test.

stated above, some dedicated primate centres (e.g. Biomedical Primate Research Centre (BPRC), Rijswijk)
follow a 3-month (13 weeks) quarantine procedureregardless of the origin/history of the animals.
Post-quarantine
The following interval for TST of species or groups
of non-human primates is recommended during thepost-quarantine housing period for institutes with frequent animal transfer and where humans work inclose contact with animals: macaques and vervets
quarterly; baboons, prosimians and New World monkeys semiannually; and great apes annually. A socalled closed institute holding monkeys with limited
exposure to humans may have a routine of TB testing
every 12 months, depending on the policy of theinstitute.
Because of a number of variables, the facility veterinarian may opt for TST at less frequent time intervals.
When non-human primates are shipped from a facility
with less frequent TB testing than that advised in theserecommendations, the veterinarian, to whose institutethe animals are shipped, must be notiﬁed about thedeviation in the procedure before the animals aretransferred.
It is important that the tuberculin test is accurately
recorded for each animal. An animal’s clinical record
should also include information about the building
and/or room in which it was kept, its social housing
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partners, and any movement (when it applies) during
quarantine. A digital image and exact measurement ofa suspect skin reaction is a useful tool to compare test
results in repeated tests.
Tuberculous non-human primates can become anergic to mycobacterial antigens and appear negative ina TST. At present, it is unclear to what extent a condition of TB-associated immunosuppression or anergy
affects other immunodiagnostics. Therefore, TB
should be considered and further tests performed if
an animal displays unexplained weight loss, non-healing wounds or any signs of distress or unexplained
deviation from normal behaviour. Additional tests
may include culture swabs of non-healing wounds,
chest radiographs, and selective culturing of a gastriclavage.
Non-human primates display a positive TST upon
experimental injection of CFA, because it contains cellwall components of tubercle bacilli. Ideally other, nonmycobacterium-based adjuvants should be selected
such that the diagnostic power of the TST is not compromised. If the use of CFA is unavoidable, the individual animal has to be tuberculin-tested the week
before the CFA is injected or it has to be isolated fromthe colony. Because CFA application will induce a
positive TST, other indirect measures like weight monitoring, chest X-ray or bacterial culturing of bioﬂuid
might indicate TB infection. If TB is diagnosed inany other animal in the holding room housing a
CFA-exposed non-human primate, the non-human63Diagnosis of tuberculosis in non-human primatesprimate(s) that previously received CFA should be
regarded as potentially infectious and monitored carefully. Because of the economic risk and possible ethicalimplications of a case of TB remaining unidentiﬁed,
some dedicated primate facilities insists on a strict
regime and do not allow animals to be in contact with
their regular colony facilities at all after the injectionof CFA.

Bushmitz et al.

l

l

l

l

l

gastric lavage with acid-fast cytology and culture ofgastric mucus;
tracheal or bronchoalveolar wash with acid-fastcytology and culture of tracheal mucus
faecal examination with acid-fast staining andculturebiopsy of altered organs with acid fast stain andculturelaparoscopy.

Chest radiographs
A posterior–anterior chest radiograph can be used todetect chest abnormalities. Lesions may appear anywhere in the lungs and may differ in size, shape,density and cavitations. These abnormalities may
suggest TB, but cannot be used for deﬁnitive differential diagnosis. Therefore, chest radiographs may be
used as an additional test procedure only for screening TB. Moreover, considering the limited sensitivity,
this additional test, however, is very valuable for theidentiﬁcation of animals that have a negative TSTreaction because of immunosuppression associatedwith fulminant disease. Chest radiographs can be difﬁcult to interpret and ideally are presented to an
experienced radiologist or lung physician for interpretation. Because of the rareness of calciﬁed tubercles, TB may present with a weak radiologicalcontrast in non-human primates compared with otheranimals. There are no pathognomonic lesions, but
enlargement of the bronchial lymph nodes may bean early sign of pulmonary mycobacteriosis. Larger
tubercles or caviations may be appreciated radiographically. Abdominal radiography may aid in the identiﬁcation or conﬁrmation of splenomegaly or mesentericlymphadenopathy. Computed tomography scanninggave detailed real-time imaging of disease progression[12].
Diagnostic microbiology
The presence of acid-fast bacilli (AFB) on a sputum
smear or other specimen may indicate TB. Acid-fast
microscopy is easy and quick, but it has a limitation inthat some AFBs are not M. tuberculosis. Therefore, abacterial culture is required on all initial samples to
conﬁrm the diagnosis. A positive culture of M. tuberculosis conﬁrms a positive acid-fast staining andthereby the diagnosis of TB infection. Culture examinations should be completed on all specimens regardlessof AFB smear results.
lCulture of mycobacteria from clinical materials
using Löwenstein–Jensen agar or other suitable cultivation media;
64

PCR
Polymerase chain reaction (PCR) can be used to detect
mycobacterial DNA in any biological samples andintrinsically has the advantage of being much quicker
than the conventional culture methods of diagnosis.
Detection of infection by screening faeces or sputumby PCR for mycobacterial DNA may therefore be considered as a rapid and alternative diagnostic tool fortuberculosis.
PRIMAGAM-IFN-c test
In vitro immune stimulation assays have been reported
to distinguish between TB-infected and TB-noninfected macaques. Recently, a whole-blood stimulation assay has become commercially available as theso-called PRIMAGAM (Product number 63301,
Prionics, Va Vista, Nebras ka, USA) test. This test
detects cellular immune reactivity to PPD antigen
through the measurement of interferon-gamma (IFNc) production in whole-blood samples [4]. IFN-c is an
important cytokine involved in the cell-mediatedimmune response to mycobacteria. The assays may discriminate between M. tuberculosis complex and M. bovis infection by including avian and bovine PPD
separately in the test. It is a quantiﬁable diagnostic testwith good sensitivity and speciﬁcity compared with disease status determined by pathological examination [6,7, 20]. Recent results suggest that the IFN-c response
to tuberculin antigen may not be reliable in cynomolgus macaques and that another cut-off should be used
to read the test [6]. Moreover, a more formal validation of this test kit would wait extensive testing in larger cohorts of naturally TB-exposed cohorts ofmonkeys. Thus, in general, the parallel use of TST and
PRIMAGAM test is advisable for an optimal coverage in a diagnostic regimen.
The PRIMAGAM-IFN-c test is an additional test
which can be used for gorillas, chimpanzees, orangutans, gibbons, colobids, baboons, mandrills, macaques, vervets, guenons, squirrel monkeys, langurs andmarmosets. As both tests can suffer from intermittent
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Bushmitz et al.

positive and negative reactions on repeated testing, theparallel use of skin testing while performing a PRIMAGAM assay is recommended for maximal overallsensitivity in TB screening programmes, particularly
also for cynomolgus monkeys.
PrimaTB STAT-PAK assay
Recent studies suggest that the serological detection of
antibody against Mycobacterium-speciﬁc early secretory
antigenic target-6 (ESAT-6) by enzyme-linked immunosorbent assay may be a useful tool for the developmentof an immunodiagnostic tool for TB [9]. There is astrong association between TB in non-human primatesand an immune response against ESAT-6, a protein
secreted by the metabolically active, virulent tubercle
bacilli.
In a comprehensive approach, ESAT-6 is one of several antigens used in the PrimaTB STAT-PAK assay– as a new lateral ﬂow test that has been developed asa TB-speciﬁc serodiagnosticum. This test was evaluatedin comparison with the intradermal palpebral tuberculin test on non-human primates of three different species. Some of the animals were experimentally infectedwith M. tuberculosis. Serological evaluation demonstrated high diagnostic sensitivity (90%) and speciﬁcity(99%) [13].
This commercial test is very easy to use, and canbe used to analyse serum, plasma or possibly anyother antibody-containing body ﬂuid. It may therefore be an attractive option for institutes with more
limited capacity or facilities. This serodiagnostic test
employs a selective array of recombinant M. tuberculosis proteins covering several immunodominant
mycobacterial antigens for immune detection. A combination of PrimaTB STAT-PAK assay and theskin test seems to be a sensitive and reliable diagnostic approach for detection of TB in non-humanprimates.
Shipment of material to the laboratory
If specimens have to be shipped they should be packed
in compliance with regulations covering the transportation of aetiological agents. See Guideline: Diagnostic
Specimens, Category B, assigned to UN identiﬁcation
number 3373. A triple packaging system must be used(see Appendix7.1.).
General remark: Intradermal testing alone is not sufﬁcient to adequately diagnose tuberculosis. Limitationsof the TST, particularly its inability to identify latent
TB infections reliably makes it unsuitable for use as asingle, stand-alone test for TB surveillance. The use of
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Diagnosis of tuberculosis in non-human primatesa combination of intradermal testing and PRIMAGAM-IFN-c test or PrimaTB STAT-PAK assay isadvisable. In doubtful cases, a range of additional test
methods like chest radiographs and microbiology
should be applied and the results evaluated critically.
The use of several tests in combination will help toincrease the overall sensitivity and speciﬁcity of screening and surveillance programmes for non-humanprimates.
Handling of tuberculous non-human primates
In case of a suspicious TB test
The animal has to be taken into quarantine.
Repeated testing according to the quarantine procedures is necessary. The use of alternative sites (othereyelid or abdomen) and possibly more potent tuberculin preparation (see above) are advisable to compare the results of the skin test. Other diagnostic
tools like chest X-rays, bacterial culturing and/or
immune assays like the PRIMAGAM test or PrimaTB STAT-PAK may be considered. All animals
in the group of the TB suspected animal should be
regarded as possibly infected and therefore be tested
up to ﬁve times. The quarantine period should be
extended to 90 days (Fig. 2).
In case of positive tuberculin test
Immediate killing
When a clinical diagnosis of TB is made in a nonhuman primate, it has to be killed immediately and thecarcass has to be taken to the pathology section for
necropsy. The cage and room, where the tuberculous
non-human primate(s) was/were held, have to be sanitized and the remaining non-human primate(s) shouldbe placed under a whole period of quarantine or be
killed. In case an animal tests positive for TB during
quarantine, the quarantine period should be extended
to 90 days. During this time ﬁve TST should be
performed (Fig. 2).
Quarantine sanctions
Notiﬁcation to all workers, to government authorities
and a clear indication of the infected room areessential.
l
Access to the room is limited to assigned personnel
only,lProtective clothing (Tyvek overall, DuPont,
France, N95 respirator masks, shoe covers, headbonnet, vinyl or rubber gloves and eye protection)
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Diagnosis of tuberculosis in non-human primates

Bushmitz et al.

Fig. 2 Frequency of Tuberculin Skin Test.

is worn in the room and is not removed except for
autoclaving,lno other non-human primates are placed in theroom; no animals are removed from the room,lthe non-human primates in the room are tuberculin-tested every 2 weeks until ﬁve tests have been
performed with no reactions. The ﬁrst of these tests
is administered 2 weeks after the test that identiﬁed
the tuberculous non-human primate.
All previously negative contact personnel should betuberculin-tested again and personnel with access tothe primates should be kept to a minimum.
If all animals are negative the quarantine may beterminated after the 90-day period except that nonhuman primates are not placed in or removed fromthe room until a tuberculin test is administered
4 weeks after the last of the ﬁve tests with noreactions. A diligent effort should be made to locate
all non-human primates that were housed in the roomin which the tuberculous non-human primate washoused over the last 60 days. These non-humanprimates should be tuberculin tested on the sameschedule as the non-human primates currently housedin the quarantined room.
Delayed killing
Killing of a tuberculous non-human primate can bedelayed if the animal is of great value for a research
project and can be isolated to minimize the spreadof tubercle bacilli to other non-human primates orhumans. The room in which such a non-human primate is held at the time of clinical diagnosis has tobe placed under quarantine. Biosafety level 3 prac66

tices and facilities are recommended for animal studies using non-human primates that are naturally or
experimentally infected with M. tuberculosis and M.
bovis. Therefore, animals that are tuberculin-positive
should be killed if this level of containment is notavailable. Multidrug treatment may be considered,but only if appropriate isolation and containment
facilities can be provided, if such containment andisolation is ethically justiﬁed, and if accurate
detection of infection is possible. Every single animal
that could have been exposed (via direct contact, air
supply, etc.) has to be handled according to quarantine conditions. All previously negative contact personnel should be tuberculin-tested again and thepersonnel with access to the primates should be kept
to a minimum.
Treatment of tuberculous non-human primates
Valuable non-human primates of those species that are
not highly susceptible to tuberculosis or belonging toendangered species may be treated to free them oftubercle bacilli, e.g. chimpanzees. A multiple drug regimen based upon the most current information must be
used for treatment. Effective drug combinations include
isoniazid and streptomycin, isoniazid and p-aminosalicyclic acid, or isoniazid, ethambutol and rifampin [8,22, 23]. In all cases of successful treatment, the organism had to be isolated and antibiotic sensitivity determined beforehand. Therapy must be maintained for atleast 6 months. The room, in which such a non-humanprimate was kept when the clinical diagnosis was
made, has to be placed under quarantine. An animal
under therapy has to be kept under quarantine
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Bushmitz et al.

conditions for the whole duration of therapy. In
general, treatment is only advisable for very valuable
animals considering costs and ethical issues involved
with containment.
Protection of non-human primates and personnel
Protection of personnel
Biosafety precautions must be taken when dealing witha suspected or diagnosed tuberculous non-human primate (a non-human primate suspicious of tuberculosis)
and when collecting and handling samples to be cultured
for tubercle bacilli. Access to quarantine is limited toauthorized personnel, who should have participated inseminars about the risks of handling non-human primates. They have to wear safety clothing. Serum samples of workers have to be stored as reference serum.
Protection of non-human primates
All humans working with primates should have negative
skin tests or be non-infectious. The skin test should be
repeated every 6 months. People with positive reaction(>10 mm) (some due to BCG vaccination) have toundergo chest X-ray or any alternative TB diagnostic
test like the QuantiFERON-TB Gold test. These complementary diagnostic tests may be used in an alternatingfashion, each of them being repeated every 12 months.
Tuberculin test (Teinte test or Goldmann Mantheux)positive personnel should be monitored medically, whenever they have respiratory symptoms that persist longer
than those usually seen with transient viral upper respiratory infections. Human tuberculin converters should
be referred to a physician for follow-up with radiographs, cultures, or other diagnostic procedures andtreatment. TB-positive people should not be allowed towork with primates. People whose jobs require contact
with animals should be assigned other duties until all
diagnostic tests are completed and they are considered tobe free of infection and not shedding tubercle bacilli.
Acknowledgments
The authors would like to thank Dr. Peter Heidt, Biomedical Primate Research Centre, Rijswijk and Prof.
Dr. Franz-Josef Kaup, German Primate Center,Göttingen, Germany for helpful discussions.
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Appendix
Guideline: Packing Diagnostic Specimens forTransport(Diagnostic Specimens, Category B, assigned to UN
identiﬁcation number 3373)

68

Bushmitz et al.

Triple packaging system
Primary PackinglPrimary receptacle(s) must be water tight, e.g.
screw cap seal with paraﬁlm or adhesive tape orsimilar.

lMultiple primary receptacles must be wrapped individually to prevent breakage.

l

When determining the volume of diagnostic specimens being shipped, include the viral transport
media.

Primary receptacle(s) must not contain more than
500 ml or 500 g.
The entire content of the primary receptacle is thediagnostic specimen.

lSecondary Packing
Use enough absorbent material in the secondary
container to absorb the entire contents of all primary receptacles in case of leakage or damage.
lSecondary packaging must meet the IATA packaging requirements for diagnostic specimens
including 1.2 meter (3.9 feet) drop test procedure.
Since infectious substance packaging surpasses therequirements for diagnostic specimen packaging
stated in the IATA Packing Instruction 602, it
can be used.
l
Infectious substance packaging must have therequired speciﬁcation markings on packaging.
Secondary packaging must be watertight. Follow the
packaging manufacturer or other authorized party’s
packing instructions included with the secondary
packaging.
Secondary packaging must be at least 100 mm (4inches) in the smallest overall external dimension, mustbe large enough for shipping documents, e.g. air
waybill.
l

Outer Packing
The outer packaging must not contain more than
4 l or 4 kg.
lBoth dry ice and wet ice must be placed outside thesecondary packaging.
l
Dry ice: packaging must permit the release of carbon dioxide gas and not allow a build-up of pressure that could rupture the packaging.
l
Wet ice: the packaging must be leak-proof.
lEach package and the air waybill must be marked
with the following exact wording:– UN 3373 Diagnostic Specimenl

J Med Primatol 38 (2009) 59–69 ª 2008 The Authors
Journal compilation ª 2008 Blackwell Munksgaard

Bushmitz et al.

– Packed in compliance with IATA Packing instruction 650l
An itemized list of contents must be enclosed
between the secondary packaging and the outer
packaging.
l
Place in a sealed plastic bag to protect frommoisture.

J Med Primatol 38 (2009) 59–69 ª 2008 The Authors
Journal compilation ª 2008 Blackwell Munksgaard

Diagnosis of tuberculosis in non-human primates

A shippers declaration for dangerous goods is
NOT required.
This document was compiled using the expertise of
the members of the working group and their personal
literature resources. For further reading, we refer tothe following articles and documents.

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