Tracing cross species transmission of Mycobacterium bovis at the wildlife/livestock interface in South Africa.

BACKGROUND: Bovine tuberculosis (bTB) affects cattle and wildlife in South Africa with the African buffalo (Syncerus caffer) as the principal maintenance host. The presence of a wildlife maintenance host at the wildlife/livestock interface acting as spill-over host makes it much more challenging to control and eradicate bTB in cattle. Spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) genotyping methods were performed to investigate the genetic diversity of Mycobacterium bovis (M. bovis) isolates from cattle and wildlife, their distribution and transmission at the wildlife/livestock interface in northern Kwa-Zulu Natal (KZN), South Africa. RESULTS: SB0130 was identified as the dominant spoligotype pattern at this wildlife/livestock interface, while VNTR typing revealed a total of 29 VNTR profiles (strains) in the KZN province signifying high genetic variability. The detection of 5 VNTR profiles shared between cattle and buffalo suggests M. bovis transmission between species. MIRU-VNTR confirmed co-infection in one cow with three strains of M. bovis that differed at a single locus, with 2 being shared with buffalo, implying pathogen introduction from most probably unrelated wildlife sources. CONCLUSION: Our findings highlight inter and intra species transmission of bTB at the wildlife/livestock interface and the need for the implementation of adequate bTB control measures to mitigate the spread of the pathogen responsible for economic losses and a public health threat.

Non-tuberculous Mycobacterium species causing mycobacteriosis in farmed aquatic animals of South Africa.

BACKGROUND: Mycobacteriosis caused by non-tuberculous mycobacteria (NTM), is among the most chronic diseases of aquatic animals. In addition, fish mycobacteriosis has substantial economic consequences especially in the aquaculture and fisheries industry as infections may significantly decrease production and trade. Some fish NTM pathogens are highly virulent and zoonotic; as such, infection of aquaria with these pathogens is a public health concern. In this study, we report isolation of nine different NTM species from sixteen aquatic animals including different fish species, frogs and a crocodile. Given the clinical significance of Mycobacterium marinum and its close relation to Mycobacterium tuberculosis, as well as the significance of ESAT 6 and CFP-10 secretion in mycobacterial virulence, we analysed the esxA and esxB nucleotide sequences of M. marinum isolates identified in this study as well as other mycobacteria in the public databases. RESULTS: Mycobacterium shimoidei, Mycobacterium marinum, Mycobacterium chelonae, Mycobacterium septicum /M. peregrinum and Mycobacterium porcinum were isolated from gold fish, Guppy, exotic fish species in South Africa, koi and undefined fish, Knysna seahorse, as well Natal ghost frogs respectively, presenting tuberculosis like granuloma. Other NTM species were isolated from the studied aquatic animals without any visible lesions, and these include Mycobacterium sp. N845 T, Mycobacterium fortuitum, a member of the Mycobacterium avium complex, and Mycobacterium szulgai. Phylogenetic analysis of mycobacteria, based on esxA and esxB genes, separated slow growing from rapidly growing mycobacteria as well as pathogenic from non-pathogenic mycobacteria in some cases. CONCLUSIONS: Isolation of the different NTM species from samples presenting granuloma suggests the significance of these NTM species in causing mycobacteriosis in these aquatic animals. The study also revealed the potential of esxA and esxB sequences as markers for phylogenetic classification of mycobacteria. Observations regarding use of esxA and esxB sequences for prediction of potential pathogenicity of mycobacteria warrants further investigation of these two genes in a study employing NTM species with well-defined pathogenicity.

Mycobacterium komaniense sp. nov., a rapidly growing non-tuberculous Mycobacterium species detected in South Africa.

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020T (=CIP 110823T=ATCC BAA-2758).

Spillover of Mycobacterium bovis from wildlife to livestock, South Africa.

During August 2012-February 2013, bovine tuberculosis was detected in communal livestock bordering the Greater Kruger National Park Complex (GKNPC) in South Africa. Using spacer oligonucleotide and variable number tandem repeat typing, we identified the Mycobacterium bovis strain endemic in GKNPC wildlife. Our findings indicate bovine tuberculosis spillover from GKNPC wildlife to neighboring livestock.

Species diversity of non-tuberculous mycobacteria isolated from humans, livestock and wildlife in the Serengeti ecosystem, Tanzania.

BACKGROUND: Non-tuberculous mycobacteria (NTM), which are ubiquitous micro-organisms occurring in humans, animals and the environment, sometimes receive public health and veterinary attention as opportunistic disease-causing agents. In Tanzania, there is limited information regarding the diversity of NTM species, particularly at the human-livestock-wildlife interface such as the Serengeti ecosystem, where potential for cross species infection or transmission may exist. METHODS: Mycobacterial DNA was extracted from cultured isolates obtained from sputum samples of 472 suspect TB patients and 606 tissues from wildlife species and indigenous cattle. Multiplex PCR was used to differentiate NTM from Mycobacterium tuberculosis complex (MTBC) members. NTM were further identified to species level by nucleotide sequencing of the 16S rRNA gene. RESULTS: A total of fifty five (55) NTM isolates representing 16 mycobacterial species and 5 isolates belonging to the MTBC were detected. Overall, Mycobacterium intracellulare which was isolated from human, cattle and wildlife, was the most frequently isolated species (20 isolates, 36.4%) followed by M. lentiflavum (11 isolates, 20%), M. fortuitum (4 isolates, 7.3%) and M. chelonae-abscessus group (3 isolates, 5.5%). In terms of hosts, 36 isolates were from cattle and 12 from humans, the balance being found in various wildlife species. CONCLUSION: This study reveals a diversity of NTM species in the Serengeti ecosystem, some of which have potential for causing disease in animals and humans. The isolation of NTM from tuberculosis-like lesions in the absence of MTBC calls for further research to elucidate their actual role in causing disease. We are also suggesting a one health approach in identifying risk factors for and possible transmission mechanisms of the NTM in the agro-pastoral communities in the Serengeti ecosystem.

Vaccines to control tuberculosis in cattle.

The age-old cattle disease has resisted rigorous control, but the BCG vaccine may do better.

Prevalence and risk factors for infection of bovine tuberculosis in indigenous cattle in the Serengeti ecosystem, Tanzania.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic debilitating disease and is a cause of morbidity and mortality in livestock, wildlife and humans. This study estimated the prevalence and risk factors associated with bovine tuberculosis transmission in indigenous cattle at the human-animal interface in the Serengeti ecosystem of Tanzania. RESULTS: A total of 1,103 indigenous cattle from 32 herds were investigated for the presence of bTB using the Single Intradermal Comparative Tuberculin Test. Epidemiological data on herd structure, management and grazing system were also collected.The apparent individual animal prevalence of tuberculin reactors was 2.4% (95% confidence interval (CI), 1.7 - 3.5%), whereas the true prevalence was 0.6% CI, 0.6 - 0.7% as indicated by a reaction to avian tuberculin purified protein derivatives (PPD) which is more than 4 mm greater than the reaction to avian tuberculin PPD. The results showed that 10.6% (117/1,103) showed non-specific reactions (atypical mycobacterium). The herd prevalence of 50% (16/32) was found. Tuberculin skin test results were found to be significantly associated with age, location, size of the household and animal tested. Of 108 respondents, 70 (64.8%) individuals had not heard about bovine tuberculosis at all. Thirty five percent (38/108) of respondents at least were aware of bTB. About 60% (23/38) of respondents who were aware of bTB had some knowledge on how bTB is spread. Eighty one percent (87/108) of respondents were not aware of the presence of bTB in wildlife. There is regular contact between cattle and wild animals due to sharing of grazing land and water sources, with 99% (107/108) of households grazing cattle in communal pastures. CONCLUSION: The study has demonstrated a high reported interaction of livestock with wildlife and poor knowledge of most cattle owners concerning bTB and its transmission pathways among people, livestock and wildlife. Although the overall proportion of animals with bTB is relatively low, herd prevalence is 50% and prevalence within herds varied considerably. Thus there is a possibility of cross transmission of bTB at wildlife-livestock interface areas that necessitates use of genetic strain typing methods to characterize them accurately.

Optimization and Application of Real-Time qPCR Assays in Detection and Identification of Chlamydiales in Products of Domestic Ruminant Abortion.

Domestic ruminant abortions due to infectious agents represent an important cause of economic losses in the agricultural industry. This study aimed to optimise and apply qPCR assays for detection of Chlamydiales in domestic ruminant abortion cases. Primers and probes for detection of the order Chlamydiales, Chlamydia abortus, Chlamydia pecorum, Parachlamydia acanthamoeba and Waddlia chondrophila were taken from the literature to create one singleplex and two duplex assays and the assays were optimised. Placentitis and pneumonia are pathological lesions associated with Chlamydiales infection. In a previous study, twenty-five clinical cases had pathological lesions of placentitis or pneumonia. These cases were investigated further by application of the qPCR assays in this study. Chlamydiales were detected in 16 cases. C. abortus, P. acanthamoeba and W. chondrophila were detected in bovine; and C. pecorum and W. chondrophila in ovine and caprine cases. Chlamydiales were detected in three previously inconclusive cases. Identification was improved from genus to species level (C. pecorum). Four cases remained inconclusive. In conclusion, detection of Chlamydiales and differentiation to species level was improved. This study reports the first detection of P. acanthamoeba and W. chondrophila in abortion cases in South Africa, indicating a potentially significant role in abortions in this country.

Approaches to increase recovery of bacterial and fungal abortion agents in domestic ruminants.

Abortions in domestic ruminants cause significant economic losses to farmers. Determining the cause of an abortion is important for control efforts, but it can be challenging. All available diagnostic methods in the bacteriology laboratory should be employed in every case due to the many limiting factors (autolysis, lack of history, range of samples) that complicate the investigation process. The purpose of this study was to determine whether the recovery of diagnostically significant isolates from domestic ruminant abortion cases could be increased through the use of a combination of the existing aerobic culture and Brucella selective method with methods that are commonly recommended in the literature reporting abortion investigations. These methods are examination of wet preparations and impression smears stained by the modified Ziehl-Neelsen method, anaerobic, microaerophilic, Leptospira, Mycoplasma and fungal culture. Samples of placenta and aborted foetuses from 135 routine clinical abortion cases of cattle (n = 88), sheep (n = 25) and goats (n = 22) were analysed by the new combination of methods. In 46 cases, bacteria were identified as aetiological agents and in one case a fungus. Isolation of Brucella species increased to 7.4% over two years compared with the previous 10 years (7.3%), as well as Campylobacter jejuni (n = 2) and Rhizopus species (n = 1). Salmonella species (5.9%) and Trueperella pyogenes (4.4%) were also isolated more often. In conclusion, the approach was effective in removing test selection bias in the bacteriology laboratory. The importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by increased isolation of Brucella abortus and isolation of C. jejuni. The combination of methods that yielded the most clinically relevant isolates was aerobic, microaerophilic, Brucella and fungal cultures.

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda.

BACKGROUND: Bovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health. MATERIALS AND METHODS: A cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence. FINDINGS: Of the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle. CONCLUSIONS: This study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.

African 2, a clonal complex of Mycobacterium bovis epidemiologically important in East Africa.

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Zoonotic Tuberculosis - The Changing Landscape.

Despite slow reductions in the annual burden of active human tuberculosis (TB) cases, zoonotic TB (zTB) remains a poorly monitored and an important unaddressed global problem. There is a higher incidence in some regions and countries, especially where close association exists between growing numbers of cattle (the major source of Mycobacterium bovis) and people, many suffering from poverty, and where dairy products are consumed unpasteurised. More attention needs to be focused on possible increased zTB incidence resulting from growth in dairy production globally and increased demand in low income countries in particular. Evidence of new zoonotic mycobacterial strains in South Asia and Africa (e.g. M. orygis), warrants urgent assessment of prevalence, potential drivers and risk in order to develop appropriate interventions. Control of M. bovis infection in cattle through detect and cull policies remain the mainstay of reducing zTB risk, whilst in certain circumstances animal vaccination is proving beneficial. New point of care diagnostics will help to detect animal infections and human cases. Given the high burden of human tuberculosis (caused by M. tuberculosis) in endemic areas, animals are affected by reverse zoonosis, including multi-drug resistant strains. This, may create drug resistant reservoirs of infection in animals. Like COVID-19, zTB is evolving in an ever-changing global landscape.

Novel Mycobacterium tuberculosis complex pathogen, M. mungi.

Seven outbreaks involving increasing numbers of banded mongoose troops and high death rates have been documented. We identified a Mycobacterium tuberculosis complex pathogen, M. mungi sp. nov., as the causative agent among banded mongooses that live near humans in Chobe District, Botswana. Host spectrum and transmission dynamics remain unknown.

Wildlife-cattle interactions emerge as drivers of bovine tuberculosis in traditionally farmed cattle.

Bovine tuberculosis (bTB) in cattle is recognized to be associated with several risk factors that include herd size, cattle movement, ownership of other domestic animals, confinement of cattle in enclosures at night, water sources, communal grazing area and proximity to wildlife, especially bTB maintenance hosts. A questionnaire survey was used to investigate the risk factors associated with Mycobacterium bovis (M. bovis) infection and transmission in traditionally farmed cattle at the wildlife/livestock interface in uMkhanyakude district, northern Kwa-Zulu Natal (KZN), South Africa. The questionnaire comprised of semi-structured questions that were used to gather data on livestock management practices and knowledge about bTB from 71 respondents from households that owned either bTB infected cattle herds or uninfected herds. Multiple correspondence analysis (MCA) was used to explore the association between the risk factors for M. bovis transmission and the bTB herd status. Bovine TB positive herds were associated with a herd size of n > 15, movement of cattle to areas adjacent to the game parks for grazing, cattle grazing inside the game parks as well as cattle sharing water and pasture with wildlife. The multivariable logistic regression model identified movement of animals to areas adjacent to the game parks and cattle sharing water with wildlife as highly significant risk factors for bTB infection in cattle. The findings of this study emphasized the need for the implementation of bTB control strategies in both cattle and wildlife populations for the successful control of the disease.

Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes.

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattle-specific serological assay for BTB (TB ELISA). Buffaloes (n = 997) were tested with the tuberculin skin test and TB ELISA; a subset (n = 119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes.

Accuracy of three diagnostic tests for determining Mycobacterium bovis infection status in live-sampled wild meerkats (Suricata suricatta).

Accurate diagnosis of Mycobacterium bovis infection (bovine tuberculosis [bTB]) in live animals is notoriously problematic. The aim of the present study was to evaluate the diagnostic potential of 2 new serologic tests (multiantigen print immunoassay [MAPIA] and lateral flow immunoassay rapid test [RT]) in comparison with mycobacterial culture of tracheal washes for determining M. bovis infection status in a free-ranging population of wild meerkats (Suricata suricatta). During a longitudinal study lasting 2.5 years, 240 individually identifiable meerkats were each sampled up to 8 times under anesthesia every 3 months. Diagnostic accuracy was determined through Bayesian and maximum likelihood estimations of sensitivity, specificity, and likelihood ratios for each diagnostic test when used independently and in parallel to classify the disease status of individual meerkats in the absence of a gold standard. Culture of tracheal washes was highly specific (0.99; 95% confidence interval [CI] = 0.77, 1.00) but of low sensitivity (0.36; 95% CI = 0.24, 0.50) for diagnosing M. bovis-infected individuals. The longitudinal nature of the study with repeated sampling of the same individual animals served simultaneously to improve chances of detecting infection and increase confidence in a negative result in individual animals repeatedly testing negative. Although MAPIA and RT were individually of limited diagnostic use, interpreting the results of these 2 tests in parallel produced estimates of sensitivity (0.83; 95% CI = 0.67, 0.93) and specificity (0.73; 95% CI = 0.62, 0.82) high enough to usefully inform decision making when determining exposure to bTB in wild meerkats and potentially other species in which bTB poses a diagnostic challenge.

Detection of native interferon-γ in nyala (Tragelaphus angasii): Towards diagnosing tuberculosis.

Mycobacterium bovis is the main cause of tuberculosis in wildlife. In South Africa, African buffaloes (Syncerus caffer) are a wildlife maintenance host while a number of other species are considered spillover hosts. Nyala (Tragelaphus angasii), a large antelope species from Southern Africa, is frequently traded and can be infected with M. bovis. Interferon gamma (IFN-γ) release assays that detect cell-mediated immune (CMI) responses to M. bovis infection have shown promise in elephants, rhinoceroses and buffaloes. The BOVIGAM® assay is a commercial IFN-γ release assay designed to detect tuberculosis in cattle and has been validated in buffaloes. We tested the suitability of the BOVIGAM® assay to detect native IFN-γ release in nyala. Blood samples collected from 17 nyalas were stimulated with different mitogens and IFN-γ release measured. We found that incubating whole blood with phorbol 12-myristate 13-acetate and calcium ionophore (PMA/CaI) resulted in the highest levels of IFN-y release. Samples stimulated with tuberculin purified protein derivatives of M. bovis (PPDb) and M. avium (PPDa) did not show significant IFN-γ production. An intradermal tuberculin test (IDT) and culture of tissues from 15 of the 17 culled nyala were also performed, which supported the findings of the BOVIGAM® assay, suggesting the potential value of this assay for the diagnosis of tuberculosis in nyala.

Risk Factors for Zoonotic Tuberculosis at the Wildlife-Livestock-Human Interface in South Africa.

A cross-sectional study was conducted to investigate the risk factors associated with zoonotic tuberculosis in humans and its transmission to people living at the wildlife-livestock-human interface. A questionnaire was administered to collect information on food consumption habits, food handling practices, and knowledge of zoonotic TB. Sputum samples were also collected from 150 individuals that belonged to households of cattle farmers with or without a bTB infected herd. In addition, 30 milk samples and 99 nasal swabs were randomly collected from cattle in bTB infected herds for isolation of Mycobacterium bovis (M. bovis). The sputum samples were screened for TB using the GeneXpert test and this was followed by mycobacterial culture and speciation using molecular techniques. No M. bovis was isolated from TB positive sputum samples and only one sample was confirmed as Mycobacterium tuberculosis (M. tuberculosis). M. bovis was isolated from 6.6% (n = 2/30) milk samples and 9% (n = 9/99) of nasal swabs. Ownership of a bTB infected herd and consumption of milk were recognized as highly significant risk factors associated with a history of TB in the household using multiple correspondence analysis (MCA) and logistic regression. The findings from this study have confirmed the potential for zoonotic TB transmission via both unpasteurized milk and aerosol thus, the role of M. bovis in human TB remains a concern for vulnerable communities.

First detection of Mycobacterium bovis infection in Giraffe (Giraffa camelopardalis) in the Greater Kruger National Park Complex: Role and implications.

Bovine tuberculosis (bovine TB) caused by Mycobacterium bovis has become endemic in some wildlife populations in South Africa. The disease has been reported in 21 wildlife species in the country. In this study, we report M. bovis infection in two female giraffes (Giraffa camelopardalis) from two different nature reserves within the Greater Kruger National Park Complex (GKNPC). Mycobacterium bovis was isolated from tissue lesions consistent with macroscopic appearance of tuberculosis (TB) and confirmed by polymerase chain reactions (PCRs), targeting the RD4 region of difference on the genome of the isolates. Spoligotyping and variable number of tandem repeat (VNTR) typing revealed infection of one giraffe with a strain (SB0294) previously not detected in South Africa, while a resident M. bovis strain (SB0121) was detected from the other giraffe. Our work is first to report M. bovis infection in free-ranging giraffes in South Africa. We have further demonstrated the existence of at least three genetically unrelated strains currently infecting wildlife species within the GKNPC. This finding suggests that the epidemiological situation of M. bovis within the GKNPC is not only driven by internal sources from its established endemic presence, but can be additionally fuelled by strains introduced from external sources. It further emphasizes that regular wildlife disease surveillance is an essential prerequisite for the timely identification of new pathogens or strains in ecospheres of high conservation value.

Mycobacterium fortuitum infection interference with Mycobacterium bovis diagnostics: natural infection cases and a pilot experimental infection.

Mycobacterium fortuitum and at least 1 unidentified species of soil mycobacteria were isolated from lymph nodes from 4 of 5 African buffalo (Syncerus caffer) that had been culled because of positive test results using the Bovigam assay. The buffalo were part of a group of 16 free-ranging buffalo captured in the far north of the Kruger National Park (South Africa) assumed to be free of bovine tuberculosis. No Mycobacterium bovis was isolated. To investigate the possible cause of the apparent false-positive diagnosis, the Mycobacterium isolates were inoculated into 4 experimental cattle and their immune responses monitored over a 13-week period, using the gamma interferon assay. The immune reactivity was predominantly directed toward avian tuberculin purified protein derivative (PPD) and lasted for approximately 8 weeks. During that period 3 of 4 cattle yielded positive test results on 1 or 2 occasions. The immune responsiveness was boosted when the inoculations were repeated after 15 weeks, which led to 2 subsequent positive reactions in the experimental animal that did not react previously. Including an additional stimulatory antigen, sensitin prepared from M. fortuitum in the gamma interferon assay, showed that it was able to elicit a detectable gamma interferon response in all 4 experimentally inoculated cattle when applied in parallel with bovine and avian tuberculin PPD for the stimulation of blood samples. The implications of occasional cross-reactive responses in natural cases of infection with environmental mycobacteria in the diagnosis of bovine tuberculosis in African buffalo and cattle in South Africa are discussed.