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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study serves as baseline investigation into tuberculosis (TB) patient population characteristics and the compliance of clinics in rural settings to the national TB guidelines in terms of diagnosing the disease. A total of 62 TB positive patients’ files were reviewed. Patients were diagnosed using: smear microscopy (41.9%); chest radiography (37.1%); Xpert MTB/RIF (9.7%); symptoms (3.2%); abdomen sonar (1.6%); and, no record (6.5%). Lack of complete compliance was identified, including large dependencies on chest X-ray as the first line of diagnosis and inadequate diagnosis of extra-pulmonary TB. These findings could assist identifying health system gaps for provincial and national control programs. [ABSTRACT FROM PUBLISHER]

Background: African buffaloes are the maintenance host for Mycobacterium bovis in the endemically infected Kruger National Park (KNP). The infection is primarily spread between buffaloes via the respiratory route, but it is not known whether shedding of M. bovis in nasal and oral excretions may lead to contamination of ground and surface water and facilitate the transmission to other animal species. A study to investigate the possibility of water contamination with M. bovis was conducted in association with a BCG vaccination trial in African buffalo. Groups of vaccinated and nonvaccinated buffaloes were kept together with known infected in-contact buffalo cows to allow natural M. bovis transmission under semi-free ranging conditions. In the absence of horizontal transmission vaccinated and control buffaloes were experimentally challenged with M. bovis. Hence, all study buffaloes in the vaccination trial could be considered potential shedders and provided a suitable setting for investigating questions relating to the tenacity of M. bovis shed in water. Results: Serial water samples were collected from the drinking troughs of the buffaloes once per season over an eleven-month period and cultured for presence of mycobacteria. All water samples were found to be negative for M. bovis, but 16 non-tuberculous Mycobacterium spp. isolates were cultured. The non-tuberculous Mycobacterium species were further characterised using 5'-16S rDNA PCR-sequencing, resulting in the identification of M. terrae, M. vaccae (or vanbaalenii), M. engbaekii, M. thermoresistibile as well as at least two species which have not yet been classified. Conclusion: The absence of detectable levels of Mycobacterium bovis in the trough water suggests that diseased buffalo do not commonly shed the organism in high quantities in nasal and oral discharges. Surface water may therefore not be likely to play an important role in the transmission of bovine tuberculosis from buffalo living in free-ranging ecosystems. The study buffalo were, however, frequently exposed to different species of non-tuberculous, environmental mycobacteria, with an unknown effect on the buffaloes' immune response to mycobacteria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• BTB prevalence influences the performance of the TB ELISA in African buffaloes • NTM prevalence did not influence TB ELISA performance, despite high diversity • The TB ELISA is not suitable as a stand-alone test for BTB diagnosis in 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. [ABSTRACT FROM AUTHOR]

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.

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.

Background: Unpasteurised fresh and souring dairy products form an essential component of household diets throughout many rural communities in southern Africa. The presence of milk-borne zoonotic pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis and zoonotic tuberculosis in humans, constitute a public health threat, especially in remote areas with poor disease surveillance in livestock and highly compromised human health due to HIV/AIDS., Methods: In this study we used culture to determine the longevity of M. bovis in experimentally inoculated fresh and naturally souring milk obtained from communal cattle in the KwaZulu-Natal province of South Africa. The effect of bacterial load and storage temperature on the survival of M. bovis was evaluated by spiking mixtures of fresh milk and starter soured milk (aMasi) culture with three concentrations of bacteria (10(2), 10(4), 10(7) colony forming units/ml), followed by incubation under controlled laboratory conditions that mimicked ambient indoor (20°C) and outdoor (33°C) temperatures and periodic sampling and testing over time (0-56 days)., Results: M. bovis cultured from samples of the fresh and souring milk was identified by PCR analysis. At the highest spiking concentration (10(7) cfu/ml), M. bovis survived for at least 2 weeks at 20°C; but, at all concentrations in the 33°C treatment, M. bovis was absent by three days after inoculation. Logistic regression analysis was used to assess the effects of bacterial concentration and time since inoculation, as well as determine the potential half-life of M. bovis in raw souring milk. Given the most favourable tested conditions for bacterial survival (20°C), approximately 25% of mycobacteria were alive after one day of storage (95% CI: 9-53%), giving an estimated half-life of M. bovis in raw souring milk of approximately 12 hours (95% CI: 7-27 hours)., Conclusions: This study demonstrates that M. bovis may survive in fresh and souring milk for periods of time that represent a risk of exposure to people consuming these products, as well as domestic or wild animal populations that have reported opportunities to consume homemade unpasteurised dairy products. The temperature at which the milk is soured and stored substantially affects the survival time of M. bovis.

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.

Mycobacterium tuberculosis complex species cause tuberculosis disease in animals and humans. Although they share 99.9% similarity at the nucleotide level, several host-adapted ecotypes of the tubercule bacilli have been identified. In the wildlife setting, probably the most well-known member of this complex is Mycobacterium bovis, the causative agent of bovine tuberculosis. The recently described oryx bacillus is an extremely rare slow-growing member of the antelope clade of the M. tuberculosis complex and is closely related to the dassie bacillus, Mycobacterium africanum and Mycobacterium microti. The antelope clade is a group of strains apparently host adapted to antelopes, as most described infections were associated with deer and antelope, most specifically the Arabian oryx (Oryx leucoryx). In this study, oryx bacillus was isolated from a free-ranging adult female African buffalo (Syncerus caffer), in good physical condition, which tested strongly positive on three consecutive comparative intradermal tuberculin tests. Upon necropsy, a single pulmonary granuloma and an active retropharyngeal lymph node was found. Comprehensive molecular genetic assays were performed, which confirmed that the causative microorganism was not M. bovis but oryx bacillus. Oryx bacillus has never been reported in Southern Africa and has never been found to infect African buffalo. The identification of this microorganism in buffalo is an important observation in view of the large and ever-increasing epidemic of the closely related M. tuberculosis complex species M. bovis in some African buffalo populations in South Africa.

Bovine tuberculosis (BTB), a chronic disease of mammals caused by Mycobacterium bovis, is a threat to South African wildlife. It has been reported that African buffaloes (Syncerus caffer) are reservoir hosts of BTB in South African wildlife populations. This study reports on the molecular identification and typing of 31 M. bovis isolates collected between 1993 and 2008, mainly from buffaloes but also from two lions and a bush pig, in the Hluhluwe-iMfolozi Park (HiP) in KwaZulu-Natal. To study the dynamics of BTB in the buffalo populations, 28 M. bovis isolates from the HiP and epidemiologically related parks were characterised using regions of difference deletion analysis for species identification and spoligotyping, variable number of tandem repeats (VNTR), polymorphic G-C-rich sequences and IS6110 restriction fragment length polymorphism (RFLP) genotyping methods. At least three distinct M. bovis genotypes were found amongst HiP samples. The combination of VNTR typing (using a 16-loci panel) and IS6110 RFLP revealed the presence of three additional genetic profiles in individual buffaloes, demonstrating that the highest level of discrimination was achieved by these typing methods. One of the observed spoligotypes (SB0130) was dominant and represented 75% of isolates from buffaloes. A novel M. bovis spoligotype (SB1474), which is reported for the first time in this study, was observed in 14.3% of isolates from buffaloes. Based on the observed genetic relationships, the findings suggest independent introductions from at least three unrelated sources. These findings improve the knowledge regarding the diversity of circulating M. bovis strains in the HiP.

We report a case of tuberculosis due to infection with Mycobacterium bovis in an elderly male black rhinoceros (Diceros bicornis minor) from the Limpopo Province in South Africa. The animal was euthanized due to very poor condition, old age, and dental attrition. Necropsy examination revealed two small nonencapsulated granulomas (approximately 40-mm diameter) in the dorsocaudal lobe of the left lung. Sequencing of isolated crude lung tissue PCR product and boiled lung culture samples confirmed that the causative organism was M. bovis. Genotyping revealed limited similarities with M. bovis strains isolated thus far from South African cattle or wildlife. The source of the infection could not be determined. This case illustrates that M. bovis could impact conservation of free-ranging rare and endangered species. Effective diagnostics are urgently needed for different animal species, such as white or black rhinoceroses, to certify with a reasonable degree of certainty that these animals are free of tuberculosis in natural habitats.

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.

The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl-Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs.

Between 1991 and 2001 a total of 12 cases of Mycobacterium tuberculosis infection in eight different species were recorded in the National Zoological Gardens of South Africa in Pretoria (Tshwane). The genetic relatedness between seven of the M. tuberculosis isolates was determined by IS6110 restriction fragment length polymorphism analysis. For the majority of isolates that were analyzed, a high degree of polymorphism suggested different sources of infection. Evidence of M. tuberculosis transmission between animals is reported in two chimpanzees (Pan troglodytes) housed together, from which samples were collected for analysis 29 mo apart.

In most countries, tuberculosis caused by Mycobacterium bovis is mainly a disease of domestic cattle and can be controlled successfully by means of a test-and-slaughter program. Once the infection spills over into a wild animal species with maintenance host potential, conventional measures are no longer sufficient to provide effective control. In South Africa, African buffaloes (Syncerus caffer) represent the most important maintenance host for M. bovis. Apart from transmitting the disease to predators and scavengers that feed on them, buffalo also serve as a source of infection to other wildlife species through environmental contamination. In several countries, it was shown that an infected wildlife reservoir that interacts with livestock causes frequent herd breakdowns and substantial economic losses to the agricultural sector. The outbreak of tuberculosis in free-ranging wildlife populations thus poses a huge challenge on long-term management and control strategies to prevent spillover into other wildlife, especially endangered, wildlife species and domestic livestock.

A survey to determine the bovine tuberculosis status of buffalo herds north of the Olifants River in the Kruger National Park was conducted, using a new diagnostic approach. Diagnosis of Mycobacterium bovis infection was accomplished using the gamma-interferon assay technique in 608 adult buffaloes out of a total of 29 discreet herds. The animals were immobilized in groups of 10-15, bled, individually marked and then revived and released on site. As soon as test results were available (after 26-36 h), the same buffalo herd was relocated by tracking the frequency of a radio-collar previously fitted to one adult cow per group during the initial operation. Bovine reactors were identified, darted and euthanased from the helicopter. Necropsy and culture findings of all culled buffaloes showed excellent correlation with the results of the ante-mortem gamma-interferon test. The survey revealed that over and above the two positive herds that had been identified during a previous survey carried out in 1996, there were three additional, but previously unidentified, infected herds in the region north of the Olifants River.

Expansion of ecotourism-based industries, changes in land-use practices, and escalating competition for resources have increased contact between free-ranging wildlife and humans. Although human presence in wildlife areas may provide an important economic benefit through ecotourism, exposure to human pathogens may represent a health risk for wildlife. This report is the first to document introduction of a primary human pathogen into free-ranging wildlife. We describe outbreaks of Mycobacterium tuberculosis, a human pathogen, in free-ranging banded mongooses (Mungos mungo) in Botswana and suricates (Suricata suricatta) in South Africa. Wildlife managers and scientists must address the potential threat that humans pose to the health of free-ranging wildlife.