BACKGROUND
the majority of the disease-causing species of the genus mycobacterium belong to two groups, the m. tuberculosis complex  and the m. avium-intracellulare complex .
mtc comprises of m. tuberculosis, m. bovis, m africanum and the attenuated m. bovis bacille calmette-guerin .
with the exception of bcg, these species are pathogenic and can cause tuberculosis  in humans and animals  <cit> .
disease caused by mtc pathogens is characterised by tubercle formation in the infected tissues.
these bacteria are considered obligate intracellular pathogens and the most efficient mode of transmission is via respiratory route  <cit> .
in contrast, environmental mycobacteria have the capacity to survive and multiply under a wide range of environmental conditions  <cit>  and to interact with a variety of environmental reservoirs, including protozoa and insects  <cit> .
the majority of environmental mycobacteria are considered non-pathogenic.
however, members of mac are capable of causing nontuberculous infections and disease in a wide range of animal species  <cit> .
mac comprises of two species, m. avium and m. intracellulare, and although these species are considered opportunistic pathogens, their role in human and animal diseases and their pathogenic potential have become increasingly recognised, especially in immunocompromised persons with hiv- <dig> infection  <cit> .
non-human primates  are susceptible to infections caused by members of mtc  <cit>  and mac  <cit> , including m. intracellulare  <cit> , indicating the existence of potential zoonotic transmission between nhp and visitors to zoos and game parks as well as animal handlers and laboratory workers using biological products from nhp.
the tuberculin skin test  is the traditional in vivo screening test for detecting tb in nhp  <cit> .
this test utilizes ppds, which are crude antigen preparations, usually made from m. tuberculosis, m. bovis or m. avium.
as ppd contains cross-reactive antigens  <cit> , a positive tst could indicate tuberculous and non-tuberculous disease, prior vaccination with bcg, or immune sensitisation with non-pathogenic environmental mycobacteria.
in addition, bcg vaccine vectors are increasingly being used in nhp studies  <cit> , making it important to distinguish such vaccinations from natural pathogenic mycobacterial infections.
the 6-kda early secretory antigenic target  and culture filtrate protein  <dig>  are well defined mycobacterial proteins  <cit> .
both are low molecular-weight proteins which are secreted in the culture filtrate by m. tuberculosis and other pathogenic mycobacteria, including m. bovis  <cit> , following short-term axenic culture.
these proteins are absent from the genomes of all bcg substrains and those of several non-pathogenic environmental mycobacteria including m. avium avium and m. avium paratuberculosis  <cit> .
both esat- <dig> and cfp- <dig> induce strong t-cell ifn-γ responses  <cit> , prompting their proposed use as diagnostic markers for m. tuberculosis and m. bovis infections  <cit> .
however, the presence of esat- <dig> and cfp- <dig> orthologues in other mycobacterial species, including non-pathogenic species such as m. smegmatis and m. gastri  <cit>  and environmental species like m. flavescens  <cit>  has raised some concerns regarding the potential use of these antigens as diagnostic markers  <cit> .
paradoxically, the presence of esat- <dig> and cfp- <dig> in other mycobacterial species does not seem to confound the detection of m. tuberculosis- and m. bovis-associated specific responses in clinical and epidemiological practice  <cit> , although these studies were mostly performed in low-incidence settings and patients may not have had significant previous contact with non-tuberculous mycobacteria.
a study by arend et al  <cit>  showed that when esat- <dig> and/or cfp- <dig> enzyme-linked immunosorbent assay  and enzyme-linked immunospot assay  were used to measure interferon-gamma production, most m. kansasii- or m. marinum-infected patients and several persons exposed to environmental mycobacteria responded to esat- <dig> and/or cfp- <dig>  ifn-γ production by t cells from leprosy patients, tb patients and unexposed controls in response to the m. leprae homologue of cfp- <dig> also showed significant crossreactivity with cfp- <dig> of m. tuberculosis  <cit> .
in the present study, we evaluated esat- <dig> and cfp- <dig> for use in the interferon-gamma  elispot assay for diagnosis of mycobacterial infections in baboons held in a research facility in south africa after the death of one baboon due to tb.
methods
baboons
ninety wild-caught chacma baboons  were involved in this study.
of these,  <dig> baboons had been newly introduced into the colony from the wild, while the other  <dig>  including the one that died of m. tuberculosis infection, had been kept in the colony for over  <dig> years.
of the  <dig> baboons,  <dig> had been sensitised to bcg by experimental inoculation,  <dig> to  <dig> years prior to the current study.
ethical approval for the experimentation on the baboons was obtained from the animal ethics committee of the university of cape town.
tuberculin skin test
baboons were anaesthetised with ketamine hydrochloride  and injected intradermally with  <dig> μl  of bovine tuberculin ppd  in the upper palpebrum of the eyelid.
reaction to tuberculin was checked by visual observation at  <dig>   <dig> and  <dig> hours and interpreted as tst positive or negative.
the tst positivity reactions comprised of erythema of palpebrum alone, various degrees of erythema with minimum swelling or slight swelling of palpebrum without erythema, and obvious swelling of palpebrum with drooping of the eyelid or swelling and/or necrosis of the palpebrum with eyelid closed.
bruise-extravasation of blood associated with injection of tuberculin or no detectable reaction on the palpebrum were interpreted as tst negative.
ifn-γ elispot assay
peripheral blood mononuclear cells  were isolated from heparinised blood by standard ficoll-gradient centrifugation.
freshly isolated or cryo-preserved pbmc were used in a standard ifn-γ elispot assay as previously described  <cit> .
briefly, elispot plates  were coated overnight at 4°c with purified anti-human ifn-γ monoclonal antibody .
pbmc were incubated for  <dig> h at 37°c in triplicate at  <dig>  cells per well with ppd , esat- <dig> , or cfp- <dig>  as the stimulant in a total volume of  <dig>  ml.
triplicate wells with cells and pha  and culture medium alone, served as positive and background stimulation controls respectively.
the average of triplicate counts of ifn-γ spot forming cells  was calculated for each stimulant and normalised to  <dig> pbmc to give ifn-γ sfc/ <dig> pbmc.
the results were reported as net ifn-γ sfc/ <dig> pbmc after subtracting the background sfc/ <dig> pbmc obtained in the absence of any stimulant .
to determine if a response was positive, a cut-off value was established for ppd, esat- <dig> and cfp- <dig> using pbmc from  <dig> tst negative healthy baboons whose background response or response to esat- <dig> or cfp- <dig> was < <dig> ifn-γ sfc/ <dig> pbmc.
the cut-off value was defined as the mean net ifn-γ sfc/ <dig> pbmc after subtracting the value of background reaction plus  <dig> standard deviations of the mean or twice the highest value of background reaction, whichever was greater.
the mean net responses plus three standard deviations for ppd, esat- <dig> and cfp- <dig> were  <dig> +  <dig>   <dig> +  <dig> and  <dig> +  <dig> ifn-γ sfc/ <dig> pbmc respectively while the highest value of background reactions were  <dig>   <dig> and  <dig>  thus, the cut-off values for ppd, esat- <dig> and cfp- <dig> were determined as  <dig>   <dig> and  <dig> ifn-γ sfc/ <dig> pbmc, respectively.
the mean background stimulation in the absence of any stimulant and in response to the pha positive controls were  <dig> ±  <dig> and  <dig> ±  <dig> sfc/ <dig> pbmc respectively.
euthanasia and necropsy
baboons with a positive tst were euthanased for necropsy using sodium pentobarbital .
in addition, baboons that were caged adjacent to those which were found to have tubercles at necropsy  were also euthanased for necropsy.
all the tst-positive baboons had been kept in the colony for over  <dig> years while  <dig> of  <dig> contact baboons were less than  <dig> months in the colony.
before euthanasia, blood for pbmc isolation was collected from these animals.
also, as part of ongoing research, cryo-preserved pbmc were available from some of these animals.
organs in the thoracic and abdominal cavities were examined macroscopically for the presence of tubercles, adhesions or enlargement and/or caseation of lymph nodes.
samples for histology, mycobacteria-specific staining and culture were taken from the lung tissue and/or bronchial lymph nodes .
histology, zn test and m. tuberculosis immunoperoxidase staining
tissues were fixed in 10% buffered formalin and sent to a veterinary pathologist  for histological examination, ziehl-neelsen  and m. tuberculosis immunoperoxidase  staining.
mycobacteria culture and identification
fresh tissues from the lung and/or bln collected at necropsy were cultured for mycobacterial culture, using standard methods in the tb research laboratory of the stellenbosch university.
standard precautions were followed to limit cross-contamination.
all cultures were genotyped by the is <dig> restriction fragment length polymorphism  method  <cit> .
isolates were also typed according to the mycobacterium tuberculosis complex typing method developed in our laboratory  <cit> .
mycobacterium tuberculosis complex negative samples were further subjected to a 5'-16s rrna gene pcr-sequencing assay, which is able to identify and speciate mycobacterium spp.
<cit> .
used in combination these methods would be able to identify superinfections between mycobacterial species of mtc and mac.
RESULTS
tuberculin skin test
following the death of one baboon  due to pulmonary tb diagnosed at necropsy, the remaining  <dig> baboons in the colony were screened for mycobacterial infection using bovine ppd tuberculin.
initially, only eight baboons tested positive, including three  with known prior vaccination with bcg or recombinant bcg.
one additional baboon  tested positive three months after initially testing negative, resulting in a total of nine tuberculin reactors .
the test reaction ranged from severe  to slight reactions .
reactions on eyelids of baboon b <dig> and b <dig> were moderate .
this table gives a summary of the major findings of the study.
nd: not determined; bln: bronchial lymph node; +: positive outcome; *: initially tst negative; Ψ: known prior vaccination with bcg or recombinant bcg; #: cut-off values for ppd, esat- <dig> & cfp- <dig> =  <dig>   <dig>  &  <dig> sfc/ <dig> respectively .
necropsy
the nine tst reactors and an additional eight contact baboons were euthanased for necropsy and further investigations.
of the nine tst positive baboons, four had classical tubercles in the lungs including at least one caseous bln, one had a non-classical tubercle-like lesion  with an enlarged but not caseous bln and four had no macroscopic lesions .
paradoxically, one of eight tst negative baboons  had a single caseous bln with no visible tubercles.
the severity of lesions varied widely, ranging from numerous small tubercles in the lungs and one or more caseous bln  to just a single caseous bln .
these macroscopic lesions were restricted to the pulmonary system.
histology, zn test and m. tuberculosis immunoperoxidase staining
as shown in the table  <dig>  necrotic granulomas and areas of necrosis were found in the lung and bln tissues of three of nine tst positive baboons while other lesions such as lymphocytic infiltration and granulomatous inflammation, suggestive of mycobacterial infection, were found in four other tst reactors.
also, five of seven histologic lesions were confirmed by zn and mtip staining.
for the tst negative baboons, only mild histological lesions were found in four of eight baboons and one  was positive by zn and mtip staining.
mycobacterial culture and identification
of the  <dig> baboons' specimens that were cultured, eight yielded mycobacterial growth.
m. tuberculosis was identified in five and m. intracellulare in three of these cultures by pcr-based methods and 16s rrna sequencing.
m. tuberculosis was cultured from four of the nine tst reactors and one of eight tst negative baboons while m. intracellulare was cultured from one tst reactor and two tst negative baboons .
it was unlikely that the culture of m. intracellulare represented laboratory cross-contamination as this species is rarely identified in sputum specimens routinely cultured in our laboratory.
no superinfection was detected in any of these samples.
ifn-γ elispot assay
pbmc were isolated from some baboons at the time of tst or just before euthanasia for determination of ifn-γ elispot response to bovine ppd, esat- <dig> and cfp- <dig>  as shown in figure  <dig> and table  <dig>  ppd elispot test was positive for all tst positive reactors and negative for all tst non-reactors, indicating agreement between these two tests.
however, two of the tuberculin reactors  were negative for both esat- <dig> and cfp- <dig> elispot test.
the mycobacterial culture in these animals was also negative.
the esat- <dig> and cfp- <dig> elispot test was positive for b <dig>  another tst reactor which had previously been vaccinated with bcg, although no mycobacteria was isolated from tissues from this animal.
the esat- <dig> and cfp- <dig> elispot test was also positive for b <dig>  which was initially tst negative and mycobacteria culture positive for m. intracellulare.
for the tst non-reactors, esat- <dig> and cfp- <dig> elispot test was positive for four baboons, two  from which m. intracellulare was isolated.
however, no mycobacteria was isolated from the other two baboons , which were also positive by esat- <dig> and cfp- <dig> elispot.
in an attempt to understand the kinetics of immune responses to m. tuberculosis and m. intracellulare, we performed retrospective measurements of t-cell ifn-γ responses in an elispot assay using cryo-preserved pbmc, previously obtained from baboons b <dig> and b <dig>  as depicted in figure  <dig>  the responses to esat- <dig> for b <dig> could be detected as early as  <dig> weeks before euthanasia but the ppd  responses were detectable only  <dig> weeks later.
thus, the tst was initially negative at  <dig> weeks before euthanasia although the elispot assay value was above the positive cut-off value for esat- <dig> .
this delay in induction of a ppd response was not observed for b <dig> that died of tb.
discussion
the tst, which is a delayed-type hypersensitivity  reaction, is partially t cell-mediated  <cit>  and ifn-γ plays a major role  <cit> .
the major drawback of this test is the inability to distinguish active pathogenic infection from healthy carrier status, or from non-pathogenic exposure to environmental mycobacteria or bcg.
the majority of tb outbreaks in nhp are associated with human contact or tb infected animals and carcasses  <cit>  and rarely give rise to latent tb infection  <cit> .
thus, mtc infections in nhp are considered progressive and almost always fatal.
however, infection with mac is usually chronic  <cit>  and sometimes refractory to tst  <cit> .
in order to eliminate their zoonotic potential, early and reliable detection of potential pathogenic mycobacterial infections, including members of mac in the nonhuman primate colonies is crucial.
both esat- <dig> and cfp- <dig> have been proposed as diagnostic markers for pathogenic infection caused by m. tuberculosis and m. bovis in human and animals  <cit>  because they are well defined antigens  <cit> , absent from bcg vaccine strains and some non-pathogenic environmental mycobacteria  <cit> , and they associate in m. tuberculosis-exposed people with a higher risk of developing active tuberculosis  <cit> .
in our study, the ifn-γ elispot response to esat- <dig>  cfp <dig> and ppd were compared to the tst.
there was concordance between the tst and ppd elispot tests.
however the results for esat- <dig> and cfp- <dig> elispot and tst were discordant in two of seven tst-positive and four of seven tst-negative animals.
one of the two tst-positive but esat- <dig> and cfp- <dig> negative baboon  could be a result of previous exposure to bcg via inoculation with a recombinant bcg vaccine 2– <dig> years previously, but the other baboon  possibly represents environmental sensitisation as this animal had been caught from the wild less than  <dig> months before the test and had no known exposure to bcg.
this outcome demonstrates that esat- <dig> and cfp- <dig> elispot test is capable of distinguishing bcg vaccination and environmental exposure by non-pathogenic mycobacterial infection  from tb disease.
however, esat- <dig> and cfp- <dig> elispot test for another tst-positive baboon  that had previously been vaccinated with bcg, was not conclusive as no mycobacteria was isolated from tissues from this animal.
it was noteworthy that one m. intracellulare infection  was detected by both tst and esat- <dig> and cfp- <dig> elispot tests.
since the methods used to identify and speciate the mycobacteria excluded the possibility of superinfection with m. tuberculosis, this result suggests a cross-reactivity between m. tuberculosis-derived esat- <dig> and cfp- <dig> antigens and m. intracellulare.
paradoxically, tst failed to detect two other m. intracellulare-infected baboons , possibly because the stage of infection in these animals were too early for the ppd-specific t responses to have developed.
our data on the kinetics of immune responses induced by m. tuberculosis and m. intracellulare show that induction of ppd responses occurs much later than that of esat- <dig> in case of m. intracellulare infection.
thus, it is possible for tst to miss early m. intracellulare infection, as we observed in this study.
however, the isolation of m. intracellulare from two other tst-negative but esat- <dig> and cfp- <dig> elispot-positive baboons  casts some doubt regarding the specificity of this elipot test.
this needs to be resolved by further investigation using larger sample sizes.
although limited by the small sample size and an assumed exposure in the in-contact baboons, the findings in this study indicate that the esat- <dig> and cfp- <dig> elispot assays were highly effective in detecting m. tuberculosis and m. intracellulare infections .
further, these results show the ability of this method to distinguish bcg vaccination from infection with these mycobacteria.
in addition, our results show that the tst failed to detect two infections with m. intracellulare and one infection with m. tuberculosis.
we further show that t-cell responses to esat- <dig> and cfp- <dig> develop much earlier than those of ppd, especially in m. intracellulare infection and hypothesise that this could possibly facilitate earlier diagnosis of mycobacterial infection by elispot test.
CONCLUSIONS
this study demonstrated that the traditional tst can manifest significant false-negatives in screening for tuberculosis in nhp while the esat- <dig> and cfp- <dig> elispot assays are highly effective in detecting m. tuberculosis and m. intracellulare infections, thus facilitating an improvement in identifying and controlling a potential zoonotic transmission hazard, especially where nhp are infected with m. intracellulare.
the study also presents the first evidence of cross-reactivity between m. tuberculosis esat- <dig> and cfp- <dig> and m. intracellulare antigens, where animals infected with m. intracellulare react to esat- <dig> and cfp- <dig> of m. tuberculosis.
whilst this study may support the use of an esat- <dig> or cfp- <dig> ifn-γ elispot assay to screen for tuberculosis in nhp facilities, a positive result may arise from infection by mycobacteria other than m. tuberculosis.
competing interests
the author declare that they have no competing interests.
authors' contributions
gkc participated in collection and subsequent delivery of animals' samples to the laboratory, processing pbmc and performing of the elispot tests, helped in tst and autopsies, compiled data and drafted primarily the manuscript.
rmw performed cultures, mycobacteria speciation and helped drafting the manuscript.
ncgvp helped in mycobacteria speciation and drafting the manuscript.
wab helped in elispot assay and compiling of data.
rjw participated partially in necropsies and helped in drafting the manuscript.
egs contributed in elispot assay analysis and facilitating histopathology.
a-lw was the principal investigator and was responsible for the study design.
all the authors read and approved the final manuscript.

 the majority of the disease-causing species of the genus mycobacterium belong to two groups, the m. tuberculosis complex and the m. avium-intracellulare complex. mtc comprises of m. tuberculosis, m. bovis, m africanum and the attenuated m. bovis bacille calmette-guerin. with the exception of bcg, these species are pathogenic and can cause tuberculosis in humans and animals. disease caused by mtc pathogens is characterised by tubercle formation in the infected tissues. these bacteria are considered obligate intracellular pathogens and the most efficient mode of transmission is via respiratory route. in contrast, environmental mycobacteria have the capacity to survive and multiply under a wide range of environmental conditions and to interact with a variety of environmental reservoirs, including protozoa and insects. the majority of environmental mycobacteria are considered non-pathogenic. however, members of mac are capable of causing nontuberculous infections and disease in a wide range of animal species. mac comprises of two species, m. avium and m. intracellulare, and although these species are considered opportunistic pathogens, their role in human and animal diseases and their pathogenic potential have become increasingly recognised, especially in immunocompromised persons with hiv- infection. non-human primates are susceptible to infections caused by members of mtc and mac, including m. intracellulare, indicating the existence of potential zoonotic transmission between nhp and visitors to zoos and game parks as well as animal handlers and laboratory workers using biological products from nhp. the tuberculin skin test is the traditional in vivo screening test for detecting tb in nhp. this test utilizes ppds, which are crude antigen preparations, usually made from m. tuberculosis, m. bovis or m. avium. as ppd contains cross-reactive antigens, a positive tst could indicate tuberculous and non-tuberculous disease, prior vaccination with bcg, or immune sensitisation with non-pathogenic environmental mycobacteria. in addition, bcg vaccine vectors are increasingly being used in nhp studies, making it important to distinguish such vaccinations from natural pathogenic mycobacterial infections. the 6-kda early secretory antigenic target and culture filtrate protein are well defined mycobacterial proteins. both are low molecular-weight proteins which are secreted in the culture filtrate by m. tuberculosis and other pathogenic mycobacteria, including m. bovis, following short-term axenic culture. these proteins are absent from the genomes of all bcg substrains and those of several non-pathogenic environmental mycobacteria including m. avium avium and m. avium paratuberculosis. both esat- and cfp- induce strong t-cell ifn-γ responses, prompting their proposed use as diagnostic markers for m. tuberculosis and m. bovis infections. however, the presence of esat- and cfp- orthologues in other mycobacterial species, including non-pathogenic species such as m. smegmatis and m. gastri and environmental species like m. flavescens has raised some concerns regarding the potential use of these antigens as diagnostic markers. paradoxically, the presence of esat- and cfp- in other mycobacterial species does not seem to confound the detection of m. tuberculosis- and m. bovis-associated specific responses in clinical and epidemiological practice, although these studies were mostly performed in low-incidence settings and patients may not have had significant previous contact with non-tuberculous mycobacteria. a study by arend et al showed that when esat- and/or cfp- enzyme-linked immunosorbent assay and enzyme-linked immunospot assay were used to measure interferon-gamma production, most m. kansasii- or m. marinum-infected patients and several persons exposed to environmental mycobacteria responded to esat- and/or cfp- ifn-γ production by t cells from leprosy patients, tb patients and unexposed controls in response to the m. leprae homologue of cfp- also showed significant crossreactivity with cfp- of m. tuberculosis. in the present study, we evaluated esat- and cfp- for use in the interferon-gamma elispot assay for diagnosis of mycobacterial infections in baboons held in a research facility in south africa after the death of one baboon due to tb. methods baboons ninety wild-caught chacma baboons were involved in this study. of these, baboons had been newly introduced into the colony from the wild, while the other including the one that died of m. tuberculosis infection, had been kept in the colony for over years. of the baboons, had been sensitised to bcg by experimental inoculation, to years prior to the current study. ethical approval for the experimentation on the baboons was obtained from the animal ethics committee of the university of cape town. tuberculin skin test baboons were anaesthetised with ketamine hydrochloride and injected intradermally with μl of bovine tuberculin ppd in the upper palpebrum of the eyelid. reaction to tuberculin was checked by visual observation at and hours and interpreted as tst positive or negative. the tst positivity reactions comprised of erythema of palpebrum alone, various degrees of erythema with minimum swelling or slight swelling of palpebrum without erythema, and obvious swelling of palpebrum with drooping of the eyelid or swelling and/or necrosis of the palpebrum with eyelid closed. bruise-extravasation of blood associated with injection of tuberculin or no detectable reaction on the palpebrum were interpreted as tst negative. ifn-γ elispot assay peripheral blood mononuclear cells were isolated from heparinised blood by standard ficoll-gradient centrifugation. freshly isolated or cryo-preserved pbmc were used in a standard ifn-γ elispot assay as previously described. briefly, elispot plates were coated overnight at 4°c with purified anti-human ifn-γ monoclonal antibody. pbmc were incubated for h at 37°c in triplicate at cells per well with ppd, esat-, or cfp- as the stimulant in a total volume of ml. triplicate wells with cells and pha and culture medium alone, served as positive and background stimulation controls respectively. the average of triplicate counts of ifn-γ spot forming cells was calculated for each stimulant and normalised to pbmc to give ifn-γ sfc/ pbmc. the results were reported as net ifn-γ sfc/ pbmc after subtracting the background sfc/ pbmc obtained in the absence of any stimulant. to determine if a response was positive, a cut-off value was established for ppd, esat- and cfp- using pbmc from tst negative healthy baboons whose background response or response to esat- or cfp- was < ifn-γ sfc/ pbmc. the cut-off value was defined as the mean net ifn-γ sfc/ pbmc after subtracting the value of background reaction plus standard deviations of the mean or twice the highest value of background reaction, whichever was greater. the mean net responses plus three standard deviations for ppd, esat- and cfp- were + + and + ifn-γ sfc/ pbmc respectively while the highest value of background reactions were and thus, the cut-off values for ppd, esat- and cfp- were determined as and ifn-γ sfc/ pbmc, respectively. the mean background stimulation in the absence of any stimulant and in response to the pha positive controls were ± and ± sfc/ pbmc respectively. euthanasia and necropsy baboons with a positive tst were euthanased for necropsy using sodium pentobarbital. in addition, baboons that were caged adjacent to those which were found to have tubercles at necropsy were also euthanased for necropsy. all the tst-positive baboons had been kept in the colony for over years while of contact baboons were less than months in the colony. before euthanasia, blood for pbmc isolation was collected from these animals. also, as part of ongoing research, cryo-preserved pbmc were available from some of these animals. organs in the thoracic and abdominal cavities were examined macroscopically for the presence of tubercles, adhesions or enlargement and/or caseation of lymph nodes. samples for histology, mycobacteria-specific staining and culture were taken from the lung tissue and/or bronchial lymph nodes. histology, zn test and m. tuberculosis immunoperoxidase staining tissues were fixed in 10% buffered formalin and sent to a veterinary pathologist for histological examination, ziehl-neelsen and m. tuberculosis immunoperoxidase staining. mycobacteria culture and identification fresh tissues from the lung and/or bln collected at necropsy were cultured for mycobacterial culture, using standard methods in the tb research laboratory of the stellenbosch university. standard precautions were followed to limit cross-contamination. all cultures were genotyped by the is restriction fragment length polymorphism method. isolates were also typed according to the mycobacterium tuberculosis complex typing method developed in our laboratory. mycobacterium tuberculosis complex negative samples were further subjected to a 5'-16s rrna gene pcr-sequencing assay, which is able to identify and speciate mycobacterium spp.. used in combination these methods would be able to identify superinfections between mycobacterial species of mtc and mac.  tuberculin skin test following the death of one baboon due to pulmonary tb diagnosed at necropsy, the remaining baboons in the colony were screened for mycobacterial infection using bovine ppd tuberculin. initially, only eight baboons tested positive, including three with known prior vaccination with bcg or recombinant bcg. one additional baboon tested positive three months after initially testing negative, resulting in a total of nine tuberculin reactors. the test reaction ranged from severe to slight reactions. reactions on eyelids of baboon b and b were moderate. this table gives a summary of the major findings of the study. nd: not determined; bln: bronchial lymph node; +: positive outcome; *: initially tst negative; Ψ: known prior vaccination with bcg or recombinant bcg; #: cut-off values for ppd, esat- & cfp- = & sfc/ respectively. necropsy the nine tst reactors and an additional eight contact baboons were euthanased for necropsy and further investigations. of the nine tst positive baboons, four had classical tubercles in the lungs including at least one caseous bln, one had a non-classical tubercle-like lesion with an enlarged but not caseous bln and four had no macroscopic lesions. paradoxically, one of eight tst negative baboons had a single caseous bln with no visible tubercles. the severity of lesions varied widely, ranging from numerous small tubercles in the lungs and one or more caseous bln to just a single caseous bln. these macroscopic lesions were restricted to the pulmonary system. histology, zn test and m. tuberculosis immunoperoxidase staining as shown in the table necrotic granulomas and areas of necrosis were found in the lung and bln tissues of three of nine tst positive baboons while other lesions such as lymphocytic infiltration and granulomatous inflammation, suggestive of mycobacterial infection, were found in four other tst reactors. also, five of seven histologic lesions were confirmed by zn and mtip staining. for the tst negative baboons, only mild histological lesions were found in four of eight baboons and one was positive by zn and mtip staining. mycobacterial culture and identification of the baboons' specimens that were cultured, eight yielded mycobacterial growth. m. tuberculosis was identified in five and m. intracellulare in three of these cultures by pcr-based methods and 16s rrna sequencing. m. tuberculosis was cultured from four of the nine tst reactors and one of eight tst negative baboons while m. intracellulare was cultured from one tst reactor and two tst negative baboons. it was unlikely that the culture of m. intracellulare represented laboratory cross-contamination as this species is rarely identified in sputum specimens routinely cultured in our laboratory. no superinfection was detected in any of these samples. ifn-γ elispot assay pbmc were isolated from some baboons at the time of tst or just before euthanasia for determination of ifn-γ elispot response to bovine ppd, esat- and cfp- as shown in figure and table ppd elispot test was positive for all tst positive reactors and negative for all tst non-reactors, indicating agreement between these two tests. however, two of the tuberculin reactors were negative for both esat- and cfp- elispot test. the mycobacterial culture in these animals was also negative. the esat- and cfp- elispot test was positive for b another tst reactor which had previously been vaccinated with bcg, although no mycobacteria was isolated from tissues from this animal. the esat- and cfp- elispot test was also positive for b which was initially tst negative and mycobacteria culture positive for m. intracellulare. for the tst non-reactors, esat- and cfp- elispot test was positive for four baboons, two from which m. intracellulare was isolated. however, no mycobacteria was isolated from the other two baboons, which were also positive by esat- and cfp- elispot. in an attempt to understand the kinetics of immune responses to m. tuberculosis and m. intracellulare, we performed retrospective measurements of t-cell ifn-γ responses in an elispot assay using cryo-preserved pbmc, previously obtained from baboons b and b as depicted in figure the responses to esat- for b could be detected as early as weeks before euthanasia but the ppd responses were detectable only weeks later. thus, the tst was initially negative at weeks before euthanasia although the elispot assay value was above the positive cut-off value for esat-. this delay in induction of a ppd response was not observed for b that died of tb. discussion the tst, which is a delayed-type hypersensitivity reaction, is partially t cell-mediated and ifn-γ plays a major role. the major drawback of this test is the inability to distinguish active pathogenic infection from healthy carrier status, or from non-pathogenic exposure to environmental mycobacteria or bcg. the majority of tb outbreaks in nhp are associated with human contact or tb infected animals and carcasses and rarely give rise to latent tb infection. thus, mtc infections in nhp are considered progressive and almost always fatal. however, infection with mac is usually chronic and sometimes refractory to tst. in order to eliminate their zoonotic potential, early and reliable detection of potential pathogenic mycobacterial infections, including members of mac in the nonhuman primate colonies is crucial. both esat- and cfp- have been proposed as diagnostic markers for pathogenic infection caused by m. tuberculosis and m. bovis in human and animals because they are well defined antigens, absent from bcg vaccine strains and some non-pathogenic environmental mycobacteria, and they associate in m. tuberculosis-exposed people with a higher risk of developing active tuberculosis. in our study, the ifn-γ elispot response to esat- cfp and ppd were compared to the tst. there was concordance between the tst and ppd elispot tests. however the results for esat- and cfp- elispot and tst were discordant in two of seven tst-positive and four of seven tst-negative animals. one of the two tst-positive but esat- and cfp- negative baboon could be a result of previous exposure to bcg via inoculation with a recombinant bcg vaccine 2– years previously, but the other baboon possibly represents environmental sensitisation as this animal had been caught from the wild less than months before the test and had no known exposure to bcg. this outcome demonstrates that esat- and cfp- elispot test is capable of distinguishing bcg vaccination and environmental exposure by non-pathogenic mycobacterial infection from tb disease. however, esat- and cfp- elispot test for another tst-positive baboon that had previously been vaccinated with bcg, was not conclusive as no mycobacteria was isolated from tissues from this animal. it was noteworthy that one m. intracellulare infection was detected by both tst and esat- and cfp- elispot tests. since the methods used to identify and speciate the mycobacteria excluded the possibility of superinfection with m. tuberculosis, this result suggests a cross-reactivity between m. tuberculosis-derived esat- and cfp- antigens and m. intracellulare. paradoxically, tst failed to detect two other m. intracellulare-infected baboons, possibly because the stage of infection in these animals were too early for the ppd-specific t responses to have developed. our data on the kinetics of immune responses induced by m. tuberculosis and m. intracellulare show that induction of ppd responses occurs much later than that of esat- in case of m. intracellulare infection. thus, it is possible for tst to miss early m. intracellulare infection, as we observed in this study. however, the isolation of m. intracellulare from two other tst-negative but esat- and cfp- elispot-positive baboons casts some doubt regarding the specificity of this elipot test. this needs to be resolved by further investigation using larger sample sizes. although limited by the small sample size and an assumed exposure in the in-contact baboons, the findings in this study indicate that the esat- and cfp- elispot assays were highly effective in detecting m. tuberculosis and m. intracellulare infections. further, these results show the ability of this method to distinguish bcg vaccination from infection with these mycobacteria. in addition, our results show that the tst failed to detect two infections with m. intracellulare and one infection with m. tuberculosis. we further show that t-cell responses to esat- and cfp- develop much earlier than those of ppd, especially in m. intracellulare infection and hypothesise that this could possibly facilitate earlier diagnosis of mycobacterial infection by elispot test.  this study demonstrated that the traditional tst can manifest significant false-negatives in screening for tuberculosis in nhp while the esat- and cfp- elispot assays are highly effective in detecting m. tuberculosis and m. intracellulare infections, thus facilitating an improvement in identifying and controlling a potential zoonotic transmission hazard, especially where nhp are infected with m. intracellulare. the study also presents the first evidence of cross-reactivity between m. tuberculosis esat- and cfp- and m. intracellulare antigens, where animals infected with m. intracellulare react to esat- and cfp- of m. tuberculosis. whilst this study may support the use of an esat- or cfp- ifn-γ elispot assay to screen for tuberculosis in nhp facilities, a positive result may arise from infection by mycobacteria other than m. tuberculosis. competing interests the author declare that they have no competing interests. authors' contributions gkc participated in collection and subsequent delivery of animals' samples to the laboratory, processing pbmc and performing of the elispot tests, helped in tst and autopsies, compiled data and drafted primarily the manuscript. rmw performed cultures, mycobacteria speciation and helped drafting the manuscript. ncgvp helped in mycobacteria speciation and drafting the manuscript. wab helped in elispot assay and compiling of data. rjw participated partially in necropsies and helped in drafting the manuscript. egs contributed in elispot assay analysis and facilitating histopathology. a-lw was the principal investigator and was responsible for the study design. all the authors read and approved the final manuscript. 
both esat- and cfp- ifn-γ elispot assays were able to detect early m. tuberculosis but also m. intracellulare infection. although this indicates potential cross-reactivity with m. intracellulare antigens, the method was able to distinguish m. bovis bcg vaccination from m. tuberculosis infection. this assay performed better than the tst, which failed to detect one m. tuberculosis and two early m. intracellulare infections. there is some doubt, however, concerning specificity, as the assay scored positive three animals infected with m. intracellulare. both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates, indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. these results suggest that the ifn-γ elispot assay could improve the detection of m tuberculosis infections when screening nhp. screening of mycobacterial infections in nhp is traditionally done by tuberculin skin test, which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay assay were obtained from tst positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. in this study, we investigated the use of esat- and cfp- for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis.
 both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates, indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. screening of mycobacterial infections in nhp is traditionally done by tuberculin skin test, which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. in this study, we investigated the use of esat- and cfp- for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis. methods peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay assay were obtained from tst positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture.  both esat- and cfp- ifn-γ elispot assays were able to detect early m. tuberculosis but also m. intracellulare infection. although this indicates potential cross-reactivity with m. intracellulare antigens, the method was able to distinguish m. bovis bcg vaccination from m. tuberculosis infection. this assay performed better than the tst, which failed to detect one m. tuberculosis and two early m. intracellulare infections.  these results suggest that the ifn-γ elispot assay could improve the detection of m tuberculosis infections when screening nhp. there is some doubt, however, concerning specificity, as the assay scored positive three animals infected with m. intracellulare. 