"Spotting" Mycobacterium bovis infection in leopards (Panthera pardus) - novel application of diagnostic tools.

Background: Mycobacterium bovis (M. bovis) is the causative agent of animal tuberculosis (TB) which poses a threat to many of South Africa's most iconic wildlife species, including leopards (Panthera pardus). Due to limited tests for wildlife, the development of accurate ante-mortem tests for TB diagnosis in African big cat populations is urgently required. The aim of this study was to evaluate currently available immunological assays for their ability to detect M. bovis infection in leopards. Methods: Leopard whole blood (n=19) was stimulated using the QuantiFERON Gold Plus In-Tube System (QFT) to evaluate cytokine gene expression and protein production, along with serological assays. The GeneXpert® MTB/RIF Ultra (GXU®) qPCR assay, mycobacterial culture, and speciation by genomic regions of difference PCR, was used to confirm M. bovis infection in leopards. Results: Mycobacterium bovis infection was confirmed in six leopards and individuals that were tuberculin skin test (TST) negative were used for comparison. The GXU® assay was positive using all available tissue homogenates (n=5) from M. bovis culture positive animals. Mycobacterium bovis culture-confirmed leopards had greater antigen-specific responses, in the QFT interferon gamma release assay, CXCL9 and CXCL10 gene expression assays, compared to TST-negative individuals. One M. bovis culture-confirmed leopard had detectable antibodies using the DPP® Vet TB assay. Conclusion: Preliminary results demonstrated that immunoassays and TST may be potential tools to identify M. bovis-infected leopards. The GXU® assay provided rapid direct detection of infected leopards. Further studies should aim to improve TB diagnosis in wild felids, which will facilitate disease surveillance and screening.

The novel HLA-A*26:236 allele identified by sequence-based typing.

The new allele A*26:236 differs from A*26:01:01:01 at position 340 (G>T) of exon 2.

Whole genome sequencing improves the discrimination between Mycobacterium bovis strains on the southern border of Kruger National Park, South Africa.

Background: Mycobacterium bovis forms part of the Mycobacterium tuberculosis complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the molecular epidemiology of M. bovis. Advances in whole genome sequencing (WGS) have increased resolution to enable detection of genomic variants to the level of single nucleotide polymorphisms. This is especially relevant to One Health research on tuberculosis which benefits by being able to use WGS to identify epidemiologically linked cases, especially recent transmission. The use of WGS in molecular epidemiology has been extensively used in humans and cattle but is limited in wildlife. This approach appears to overcome the limitations of conventional genotyping methods due to lack of genetic diversity in M. bovis. Methods: This pilot study investigated the spoligotype and WGS of M. bovis isolates (n = 7) from wildlife in Marloth Park (MP) and compared these with WGS data from other South African M. bovis isolates. In addition, the greater resolution of WGS was used to explore the phylogenetic relatedness of M. bovis isolates in neighbouring wildlife populations. Results: The phylogenetic analyses showed the closest relatives to the seven isolates from MP were isolates from wildlife in Kruger National Park (KNP), which shares a border with MP. However, WGS data indicated that the KNP and MP isolates formed two distinct clades, even though they had similar spoligotypes and identical in silico genetic regions of difference profiles. Conclusions: Mycobacterium bovis isolates from MP were hypothesized to be directly linked to KNP wildlife, based on spoligotyping. However, WGS indicated more complex epidemiology. The presence of two distinct clades which were genetically distinct (SNP distance of 19-47) and suggested multiple transmission events. Therefore, WGS provided new insight into the molecular epidemiology of the M. bovis isolates from MP and their relationship to isolates from KNP. This approach will facilitate greater understanding of M. bovis transmission at wildlife-livestock-human interfaces and advances One Health research on tuberculosis, especially across different host species.

Viral dynamics and immune responses to foot-and-mouth disease virus in African buffalo (Syncerus caffer).

Foot-and-mouth disease (FMD) is one of the most important livestock diseases restricting international trade. While African buffalo (Syncerus caffer) act as the main wildlife reservoir, viral and immune response dynamics during FMD virus acute infection have not been described before in this species. We used experimental needle inoculation and contact infections with three Southern African Territories serotypes to assess clinical, virological and immunological dynamics for thirty days post infection. Clinical FMD in the needle inoculated buffalo was mild and characterised by pyrexia. Despite the absence of generalised vesicles, all contact animals were readily infected with their respective serotypes within the first two to nine days after being mixed with needle challenged buffalo. Irrespective of the route of infection or serotype, there were positive associations between the viral loads in blood and the induction of host innate pro-inflammatory cytokines and acute phase proteins. Viral loads in blood and tonsil swabs were tightly correlated during the acute phase of the infection, however, viraemia significantly declined after a peak at four days post-infection (dpi), which correlated with the presence of detectable neutralising antibodies. In contrast, infectious virus was isolated in the tonsil swabs until the last sampling point (30 dpi) in most animals. The pattern of virus detection in serum and tonsil swabs was similar for all three serotypes in the direct challenged and contact challenged animals. We have demonstrated for the first time that African buffalo are indeed systemically affected by FMD virus and clinical FMD in buffalo is characterized by a transient pyrexia. Despite the lack of FMD lesions, infection of African buffalo was characterised by high viral loads in blood and oropharynx, rapid and strong host innate and adaptive immune responses and high transmissibility.

Adaptation and Diagnostic Potential of a Commercial Cat Interferon Gamma Release Assay for the Detection of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis (M. bovis) infection in wildlife, including lions (Panthera leo), has implications for individual and population health. Tools for the detection of infected lions are needed for diagnosis and disease surveillance. This study aimed to evaluate the Mabtech Cat interferon gamma (IFN-γ) ELISABasic kit for detection of native lion IFN-γ in whole blood samples stimulated using the QuantiFERON® TB Gold Plus (QFT) platform as a potential diagnostic assay. The ELISA was able to detect lion IFN-γ in mitogen-stimulated samples, with good parallelism, linearity, and a working range of 15.6-500 pg/mL. Minimal matrix interference was observed in the recovery of domestic cat rIFN-γ in lion plasma. Both intra- and inter-assay reproducibility had a coefficient of variation less than 10%, while the limit of detection and quantification were 7.8 pg/mL and 31.2 pg/mL, respectively. The diagnostic performance of the QFT Mabtech Cat interferon gamma release assay (IGRA) was determined using mycobacterial antigen-stimulated samples from M. bovis culture-confirmed infected (n = 8) and uninfected (n = 4) lions. A lion-specific cut-off value (33 pg/mL) was calculated, and the sensitivity and specificity were determined to be 87.5% and 100%, respectively. Although additional samples should be tested, the QFT Mabtech Cat IGRA could identify M. bovis-infected African lions.

Detection of African Swine Fever Virus in Ornithodoros Tick Species Associated with Indigenous and Extralimital Warthog Populations in South Africa.

We investigated the possibility that sylvatic circulation of African swine fever virus (ASFV) in warthogs and Ornithodoros ticks had extended beyond the historically affected northern part of South Africa that was declared a controlled area in 1935 to prevent the spread of infection to the rest of the country. We recently reported finding antibody to the virus in extralimital warthogs in the south of the country, and now describe the detection of infected ticks outside the controlled area. A total of 5078 ticks was collected at 45 locations in 7/9 provinces during 2019-2021 and assayed as 711 pools for virus content by qPCR, while 221 pools were also analysed for tick phylogenetics. Viral nucleic acid was detected in 50 tick pools representing all four members of the Ornithodoros (Ornithodoros) moubata complex known to occur in South Africa: O. (O.) waterbergensis and O. (O.) phacochoerus species yielded ASFV genotypes XX, XXI, XXII at 4 locations and O. (O.) moubata yielded ASFV genotype I at two locations inside the controlled area. Outside the controlled area, O. (O.) moubata and O. (O.) compactus ticks yielded ASFV genotype I at 7 locations, while genotype III ASFV was identified in O. (O.) compactus ticks at a single location. Two of the three species of the O. (O.) savignyi complex ticks known to be present in the country, O. (O.) kalahariensis and O. (O.) noorsveldensis, were collected at single locations and found negative for virus. The only member of the Pavlovskyella subgenus of Ornithodoros ticks known to occur in South Africa, O. (P.) zumpti, was collected from warthog burrows for the first time, in Addo National Park in the Eastern Cape Province where ASFV had never been recorded, and it tested negative for the viral nucleic acid. While it is confirmed that there is sylvatic circulation of ASFV outside the controlled area in South Africa, there is a need for more extensive surveillance and for vector competence studies with various species of Ornithodoros ticks.

Absence of 2899CPanthera leo) with Polymyopathy.

Polyphasic skeletal muscle degeneration, necrosis and mineralization of skeletal muscle was diagnosed in eight juvenile free-ranging lions (Panthera leo), from five different litters in the Greater Kruger National Park area that were unable to walk properly. A detailed investigation was not possible in free-ranging lions, so the cause could not be determined. The cases resembled hypokalemic polymyopathy in domestic cats with muscle weakness. A candidate-gene approach previously identified a nonsense mutation in the gene coding for the enzyme lysine-deficient 4 protein kinase (WNK4) associated with the disease in Burmese and Tonkinese cats. In this study, we sequenced all 19 exons of the gene in one case, and two control samples, to identify possible mutations that may be associated with polymyopathy in free-ranging lions. Here, no mutations were detected in any of the exons sequenced. Our findings indicate that the WNK4 gene is not a major contributor to the condition in these lions. Further studies into the pathogenesis of this condition are needed to inform conservation policies for this vulnerable, iconic African species.

Diagnosis of Mycobacterium bovis infection in free-ranging common hippopotamus (Hippopotamus amphibius).

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB-endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB-endemic ecosystems and evaluate serological and other diagnostic tools in this species.

Extension of Sylvatic Circulation of African Swine Fever Virus in Extralimital Warthogs in South Africa.

Sylvatic circulation of African swine fever virus (ASFV) in warthogs and Ornithodoros ticks that live in warthog burrows historically occurred in northern South Africa. Outbreaks of the disease in domestic pigs originated in this region. A controlled area was declared in the north in 1935 and regulations were implemented to prevent transfer of potentially infected suids or products to the rest of the country. However, over the past six decades, warthogs have been widely translocated to the south where the extralimital animals have flourished to become an invasive species. Since 2016, there have been outbreaks of ASF in pigs outside the controlled area that cannot be linked to transfer of infected animals or products from the north. An investigation in 2008-2012 revealed that the presence of Ornithodoros ticks and ASFV in warthog burrows extended marginally across the boundary of the controlled area. We found serological evidence of ASFV circulation in extralimital warthogs further south in the central part of the country.

Endemic persistence of a highly contagious pathogen: Foot-and-mouth disease in its wildlife host.

Extremely contagious pathogens are a global biosecurity threat because of their high burden of morbidity and mortality, as well as their capacity for fast-moving epidemics that are difficult to quell. Understanding the mechanisms enabling persistence of highly transmissible pathogens in host populations is thus a central problem in disease ecology. Through a combination of experimental and theoretical approaches, we investigated how highly contagious foot-and-mouth disease viruses persist in the African buffalo, which serves as their wildlife reservoir. We found that viral persistence through transmission among acutely infected hosts alone is unlikely. However, the inclusion of occasional transmission from persistently infected carriers reliably rescues the most infectious viral strain from fade-out. Additional mechanisms such as antigenic shift, loss of immunity, or spillover among host populations may be required for persistence of less transmissible strains.

First report of cystic echinococcosis in rhinos: A fertile infection of Echinococcus equinus in a Southern white rhinoceros (Ceratotherium simum simum) of Kruger National Park, South Africa.

Despite being a parasitic disease known since ancient times, some epidemiological aspects of cystic echinococcosis (CE) remain unclear. Many studies describe its prevalence and genotyping in populations of domestic animals and livestock, but data regarding wildlife are often scarce and incomplete. The available literature suggests that CE has never been reported in African rhinos. Considering the fragile conservation status of these species due to continued poaching, this study tries to clarify some neglected epidemiological aspects. In February 2020, an adult female of the Southern white rhinoceros, Ceratotherium simum simum (Burchell, 1817), was killed by poachers. The subsequent necropsy performed by the state veterinary team revealed the presence of seven cysts within the pulmonary tissue (four cysts in the right medio-caudal lobe and three cysts in the left medio-caudal lobe) with a diameter of between 1.5 and 2.3 cm. Given the state of decomposition of the carcass, only two of these were suitable for microscopic examination. Specimens were examined under 10x and 40x microscopic magnification for the confirmation of fertility of the cysts, based on the presence of numerous protoscoleces in different stages of maturation. A histopathological examination was also performed to describe the relationship between parasite and host tissue reaction. Cyst samples were subjected to PCR. The primers successfully amplified the expected fragments of the cox-1 and the nad-1 gene from the isolated genomic DNA, revealing high sequence identity with published sequences of Echinococcus equinus Williams & Sweatman, 1963 isolate G4 and E. equinus isolate SLG5-G4.

Shedding of Mycobacterium bovis in respiratory secretions of free-ranging wild dogs (Lycaon pictus): Implications for intraspecies transmission.

It has recently been discovered that Mycobacterium bovis (M. bovis) causes disease in the endangered African wild dog (Lycaon pictus) in areas endemic for bovine tuberculosis (bTB), including the Kruger National Park (KNP). However, information on M. bovis infection dynamics within this species is limited and requires investigation as M. bovis can cause conservation consequences due to movement restrictions, crucial for genetic management. This study had two aims: firstly, to investigate mycobacterial shedding in free-ranging wild dogs from KNP by culturing oropharyngeal swab (OS) and bronchoalveolar lavage (BAL) samples. Secondly, to determine the relationship between ante-mortem culture and interferon gamma release assay (IGRA) results as well as agreement between OS culture and BAL culture results. Mycobacterial culture revealed that 6 of 173 (3.5%) OS samples and 1 of 32 (3.1%) BAL samples (from 7 different wild dogs) were M. bovis culture positive, suggesting that wild dogs can shed M. bovis through respiratory secretions. However, the possibility of contamination by ingestion of infected prey cannot be excluded in wild dogs with positive OS culture results. Furthermore, the test outcomes between IGRA and culture (OS and BAL) differed substantially. Samples from 172 wild dogs were available for IGRA screening and 134 had positive results (detectable M. bovis immune sensitization). Seven wild dogs had culture-positive results, which included one additional wild dog that did not have an IGRA result (total 173 wild dogs). Out of these 7 M. bovis culture-positive wild dogs, 3 were IGRA positive initially, however, after repeat sampling and testing, 5 out of 7 were IGRA positive. These findings suggest that intraspecies transmission of M. bovis may be possible among wild dogs. Although the risk of intraspecies transmission is currently unknown, this knowledge is important for assessing the risk of M. bovis transmission from infected wild dogs to uninfected populations during translocations.

ANTIBODY PREVALENCE TO AFRICAN SWINE FEVER VIRUS, MYCOBACTERIUM BOVIS, FOOT-AND-MOUTH DISEASE VIRUS, RIFT VALLEY FEVER VIRUS, INFLUENZA A VIRUS, AND BRUCELLA AND LEPTOSPIRA SPP. IN FREE-RANGING WARTHOG (PHACOCHOERUS AFRICANUS) POPULATIONS IN SOUTH AFRICA.

The warthog (Phacochoerus africanus) can be used as a model for investigating disease transmission at the human, wildlife, and livestock interface. An omnivore and scavenger, a warthog moves freely between natural ecotypes, farmland, and human communities and is susceptible to diseases of zoonotic, agricultural, and conservation concern. A retrospective study using 100 individual serum samples collected from May 1999 to August 2016 was performed to determine antibody prevalence to seven pathogens in warthogs from five locations in northeastern South Africa. Higher prevalence of antibodies to African swine fever virus and Mycobacterium bovis were detected in warthogs from the Greater Kruger National Park ecosystem in comparison to lower prevalence of antibodies to M. bovis and no antibodies to African swine fever virus in warthogs from uMhkuze Game Reserve. Low prevalence of antibodies to foot-and-mouth disease virus, Rift Valley fever virus, and influenza A virus was detected in all locations, and no antibodies against Brucella and Leptospira spp. were detected. No statistically significant difference in antibody prevalence was found between sexes for any disease. At the univariate analysis, M. bovis seropositivity was significantly different among age categories, with 49% (35/71) of adults found positive versus 29% (4/14) of juveniles and 9% (1/11) of sub-adults (Fisher's exact test, P=0.020), and between the sampling locations (Fisher's exact test, P=0.001). The multivariate model results indicated that juvenile warthogs had lower odds of testing positive to M. bovis antibodies than adults (juveniles' odds ratio [OR]=0.17, 95% confidence interval [CI]: 0.02-1.0), although this result was not statistically significant at the 5% level (P=0.052). For warthogs sampled at Satara Buffalo Camp, the odds (OR=0.22, 95% CI: 0.035-0.96) of being M. bovis antibody positive were significantly lower (P=0.043) than for warthogs sampled at Skukuza. Of particular interest in this study was the detection of warthogs seropositive for influenza A virus.

Correction: Pervasive within-host recombination and epistasis as major determinants of the molecular evolution of the foot-and-mouth disease virus capsid.

[This corrects the article DOI: 10.1371/journal.ppat.1008235.].

Pervasive within-host recombination and epistasis as major determinants of the molecular evolution of the foot-and-mouth disease virus capsid.

Although recombination is known to occur in foot-and-mouth disease virus (FMDV), it is considered only a minor determinant of virus sequence diversity. Analysis at phylogenetic scales shows inter-serotypic recombination events are rare, whereby recombination occurs almost exclusively in non-structural proteins. In this study we have estimated recombination rates within a natural host in an experimental setting. African buffaloes were inoculated with a SAT-1 FMDV strain containing two major viral sub-populations differing in their capsid sequence. This population structure enabled the detection of extensive within-host recombination in the genomic region coding for structural proteins and allowed recombination rates between the two sub-populations to be estimated. Quite surprisingly, the effective recombination rate in VP1 during the acute infection phase turns out to be about 0.1 per base per year, i.e. comparable to the mutation/substitution rate. Using a high-resolution map of effective within-host recombination in the capsid-coding region, we identified a linkage disequilibrium pattern in VP1 that is consistent with a mosaic structure with two main genetic blocks. Positive epistatic interactions between co-evolved variants appear to be present both within and between blocks. These interactions are due to intra-host selection both at the RNA and protein level. Overall our findings show that during FMDV co-infections by closely related strains, capsid-coding genes recombine within the host at a much higher rate than expected, despite the presence of strong constraints dictated by the capsid structure. Although these intra-host results are not immediately translatable to a phylogenetic setting, recombination and epistasis must play a major and so far underappreciated role in the molecular evolution of the virus at all scales.

Severe infection caused by nymphs of Armillifer armillatus (Pentastomida, Porocephalidae) in a leopard, Panthera pardus, in the Kruger National Park, South Africa.

The necropsy of an adult male leopard, Panthera pardus, shot in the Kruger National Park, revealed the presence of large numbers of Armillifer armillatus nymphs in the intestine, liver, spleen, mesentery, peritoneal fold, mediastinum and lungs. The animal had been observed to be blind in the right eye and severely debilitated. The infection with A. armillatus clearly contributed to its emaciation and anaemia. Armillifer armillatus is a parasite of snakes, using mammals that form part of the snakes' prey as intermediate hosts. It is also one of the pentastomids with the highest zoonotic potential in Africa. It is unclear if the leopard's partial blindness and injuries of its extremities forced it to forego larger prey items for easier prey, such as snakes, and this in turn led to exposure to this unusual parasite, or if he had simply developed a preference for snakes. The incidental finding of A. armillatus in a large carnivore emphasises the importance of necropsies in expanding our knowledge on wildlife diseases.

Cytokine gene expression assay as a diagnostic tool for detection of Mycobacterium bovis infection in warthogs (Phacochoerus africanus).

Mycobacterium bovis infection has been described in many wildlife species across Africa. However, diagnostic tests are lacking for many of these, including warthogs (Phacochoerus africanus). Most literature on suids has focused on using serological tools, with few studies investigating the use of cell-mediated immune response (CMI) assays. A recent study showed that warthogs develop measurable CMI responses, which suggests that cytokine gene expression assays (GEAs) may be valuable for detecting M. bovis-infection, as shown in numerous African wildlife species. Therefore, the aim of the study was to develop GEAs capable of distinguishing between M. bovis-infected and uninfected warthogs. Whole blood was stimulated using the QuantiFERON-TB Gold (In-Tube) system, using ESAT-6 and CFP-10 peptides, before determining the relative gene expression of five reference (B2M, H3F3A, LDHA, PPIA and YWHAZ) and five target (CXCL9, CXCL10, CXCL11, IFNG and TNFA) genes through qPCR. The reference gene H3F3A was the most stably expressed, while all target genes were significantly upregulated in M. bovis-infected warthogs with the greatest upregulation observed for CXCL10. Consequently, the CXCL10 GEA shows promise as an ante-mortem diagnostic tool for the detection of M. bovis-infected warthogs.

Mycobacterium bovis Infection in African Wild Dogs, Kruger National Park, South Africa.

We screened African wild dogs (Lycaon pictus) in Kruger National Park, South Africa, for Mycobacterium bovis infection using an interferon-gamma release assay. We detected M. bovis sensitization in 20 of 21 packs; overall apparent infection prevalence was 83%. These animals experience high infection pressure, which may affect long-term survival and conservation strategies.

Persistent Infection of African Buffalo (Syncerus caffer) with Foot-and-Mouth Disease Virus: Limited Viral Evolution and No Evidence of Antibody Neutralization Escape.

African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centers of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centers, probang samples (oropharyngeal scrapings), and tonsil swabs to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1 coding region of the SAT1 virus inoculum clustered around 2 subpopulations differing by 22 single-nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions, depending on the day postinfection (dpi). There was a significant difference between the populations of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral populations in samples from individual animals, germinal centers, and epithelial tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to postmortem showed less within-sample variability of VP1 than that of tissue sample sequences analyzed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralize the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study, there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo.IMPORTANCE Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven-hoofed animals and is recognized as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV, and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low-frequency sequence variants over time, there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.

MYCOBACTERIUM BOVIS IN FREE-RANGING LIONS (PANTHERA LEO) - EVALUATION OF SEROLOGICAL AND TUBERCULIN SKIN TESTS FOR DETECTION OF INFECTION AND DISEASE.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis infection, causes morbidity and mortality in free-ranging lions in bTB-endemic areas of South Africa. However, the only currently used diagnostic test is the tuberculin skin test (TST). This test is logistically challenging to perform because it requires immobilization of lions twice in a 72-hr period. Blood-based diagnostic tests, such as serological assays, have been previously reported for M. bovis detection in lion populations, and have the advantage of only requiring a single immobilization. In addition, serological assays can be used for retrospective testing. Therefore, the aim of this study was to test free-ranging lions with the STAT-PAKt (Chembio Diagnostics Systems, Medford, NY 11763, USA) and DPPt VetTB (Chembio Diagnostics Systems) serological assays and compare those results with the tuberculin skin test. The serological assays were also used to determine prevalence in bTB-endemic and uninfected lion populations. The results showed that the serological assays could distinguish between M. bovis culture-positive and -negative lions. In addition, antigen-specific humoral responses were present in lions that had clinical signs of bTB disease or were shedding M. bovis antemortem. Although the seroprevalence of M. bovis infection in Kruger National Park lions was similar to that obtained from antemortem mycobacterial culture (4.8 and 3.3%, respectively), it was less than that estimated by the TST (72%). These findings support the hypothesis that assays based on cell-mediated immune responses are more sensitive than serology is in detecting M. bovis infection in lions. However, serological assays can have a role in bTB disease detection in lions and are especially useful for retrospective studies.