The evaluation of a BCG vaccine against bovine tuberculosis in African buffaloes (Syncerus caffer)

To assist in the evaluation of BCG vaccination in African buffaloes (Syncerus caffer), an infection model for Mycobacterium bovis was established, using an intratonsilar route of inoculation. Two groups of 11 buffaloes each, aged approximately 18 months, were infected with either 3,2 x 102 cfu (low dose) or 3 x 104 (high dose) of virulent buffalo strain M. bovis. A control group of six buffaloes received saline via the same route. The infection status was monitored using the intradermal tuberculin test, an ELISA and a modified interferon-gamma assay. All buffaloes were euthanased 22 weeks post infection and the development of lesions in the left retropharyngeal lymph node was evaluated by macroscopic examination, mycobacterial culture and histopathology. It was found that the high dose caused macroscopic lesions in 9 out of 11 buffaloes that were comparable to that observed in buffaloes with natural disease. Mycobacterium bovis was isolated from all animals in the high dose and from 6 out of 11 buffaloes in the low dose group. The efficacy of a live BCG-Pasteur vaccine was tested in a group of buffalo calves captured in the northern districts of the Kruger National Park from herds with known negative tuberculosis status. Primary and booster vaccinations with BCG (1173P2) were administered to 15 calves, while another 15 were left unvaccinated as control animals. All the buffalo calves were challenged with the high dose of live M. bovis (as determined in the Infection Model) via intratonsilar inoculation. Laboratory tests were able to distinguish between infected and non-infected animals from an early stage. All buffaloes were euthanased 34 weeks after infection and the development of lesions in the lymph nodes of the head, thorax, carcass and abdomen was evaluated by macroscopic examination, mycobacterial culture and histopathology. The lungs were carefully palpated to detect the presence of tuberculous granulomas. Macroscopic lesions in the lymph nodes were found in 10 out of 14 control buffaloes and 7 out of 15 vaccinated animals. The lesions were comparable to that observed in buffaloes with natural infection. The lesion scores of individual animals were generally much higher in the BCG vaccine study than what was experienced with the Infection Model. Mycobacterium bovis was isolated from 12 out of 14 control animals and from 12 out of 15 vaccinated buffaloes. Although fewer vaccinated animals developed tuberculous lesions, the differences between the two groups were not statistically significant and it can be concluded that under the prevailing conditions the BCG vaccine was unable to protect buffalo calves against the establishment of M. bovis infection.Dissertation (MSc (Tropical Diseases))--University of Pretoria, 2004.Veterinary Tropical Diseasesunrestricte

Wildlife on the move : a hidden tuberculosis threat to conservation areas and game farms through introduction of untested animals

In South Africa, African buffaloes (Syncerus caffer) are one of the wildlife maintenance hosts for bovine tuberculosis (BTB) and play a key role in the spread of the disease to other wildlife species and potentially back to cattle. We report a trace-back investigation following the diagnosis of BTB in a previously BTB-free provincial game reserve, founded in the early 1990s in the North West Province of South Africa (SA). Using the intradermal tuberculin and interferon gamma tests, we detected Mycobacterium bovis infection in captured African buffaloes intended for sale. Detection of M. bovis was confirmed by culture and PCR. Molecular typing of M. bovis isolates from three African buffaloes revealed spoligotype SB0140 and a variable number of tandem repeat genotypes which had been previously isolated from wildlife in the KwaZulu-Natal Province of SA. Diagnosis of BTB in a previously uninfected buffalo population provides evidence that the disease can be introduced into an ecosystem through the translocation of untested plains game species. We further illustrate how BTB can remain unnoticed for considerable periods of time in free-ranging wildlife populations and emphasize the need for validated diagnostic tests for application in suitable and practical monitoring programs. This is especially important for species with maintenance host potential and those in high demand at game auctions.Department of Science and Technology and the National Research Foundation of South Africa.http://www.jwildlifedis.org/hb2016Veterinary Tropical Disease

Efficacy and Safety of BCG Vaccine for Control of Tuberculosis in Domestic Livestock and Wildlife

Bovine tuberculosis (TB) continues to be an intractable problem in many countries, particularly where “test and slaughter” policies cannot be implemented or where wildlife reservoirs of Mycobacterium bovis infection serve as a recurrent source of infection for domestic livestock. Alternative control measures are urgently required and vaccination is a promising option. Although the M. bovis bacille Calmette-Guérin (BCG) vaccine has been used in humans for nearly a century, its use in animals has been limited, principally as protection against TB has been incomplete and vaccination may result in animals reacting in the tuberculin skin test. Valuable insights have been gained over the past 25 years to optimise protection induced by BCG vaccine in animals and in the development of tests to differentiate infected from vaccinated animals (DIVA). This review examines factors affecting the efficacy of BCG vaccine in cattle, recent field trials, use of DIVA tests and the effectiveness of BCG vaccine in other domestic livestock as well as in wildlife. Oral delivery of BCG vaccine to wildlife reservoirs of infection such as European badgers, brushtail possums, wild boar, and deer has been shown to induce protection against TB and could prove to be a practical means to vaccinate these species at scale. Testing of BCG vaccine in a wide range of animal species has indicated that it is safe and vaccination has the potential to be a valuable tool to assist in the control of TB in both domestic livestock and wildlife

Bovine tuberculosis in African buffaloes : observations regarding Mycobacterium bovis shedding into water and exposure to environmental mycobacteria

Includes bibliographyBackground: 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.Peer Reviewe

Ancient diversity and geographical sub-structuring in African buffalo Theileria parva populations revealed through metagenetic analysis of antigen-encoding loci

An infection and treatment protocol involving infection with a mixture of three parasite isolates and simultaneous treatment with oxytetracycline is currently used to vaccinate cattle against Theileria parva. While vaccination results in high levels of protection in some regions, little or no protection is observed in areas where animals are challenged predominantly by parasites of buffalo origin. A previous study involving sequencing of two antigen-encoding genes from a series of parasite isolates indicated that this is associated with greater antigenic diversity in buffalo-derived T. parva. The current study set out to extend these analyses by applying high-throughput sequencing to ex vivo samples from naturally infected buffalo to determine the extent of diversity in a set of antigen-encoding genes. Samples from two populations of buffalo, one in Kenya and the other in South Africa, were examined to investigate the effect of geographical distance on the nature of sequence diversity. The results revealed a number of significant findings. First, there was a variable degree of nucleotide sequence diversity in all gene segments examined, with the percentage of polymorphic nucleotides ranging from 10% to 69%. Second, large numbers of allelic variants of each gene were found in individual animals, indicating multiple infection events. Third, despite the observed diversity in nucleotide sequences, several of the gene products had highly conserved amino acid sequences, and thus represent potential candidates for vaccine development. Fourth, although compelling evidence for population differentiation between the Kenyan and South African T. parva parasites was identified, analysis of molecular variance for each gene revealed that the majority of the underlying nucleotide sequence polymorphism was common to both areas, indicating that much of this aspect of genetic variation in the parasite population arose prior to geographic separation

Absence of 2899C<T mutation in the WNK4 gene in a free-ranging lion (Panthera 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 specieshttp://www.mdpi.com/journal/animalsCentre for Veterinary Wildlife StudiesParaclinical Science

BCG vaccination for bovine tuberculosis; conclusions from the Jerusalem One Health workshop.

The global burden of bovine tuberculosis (bTB) remains poorly characterized, with spill-over impacts on multiple species. The "One Health" concept is especially relevant given the bidirectional risk of cattle infecting humans with Mycobacterium bovis and humans infecting cattle with Mycobacterium tuberculosis. "Test and cull" is the traditional bTB control method, but the strategy may not be economically feasible or culturally acceptable where cattle are highly prized or their killing is a religious taboo; it is also less effective when there are wildlife reservoirs of infection. Vaccination with M. bovis bacille Calmette-Guerin (BCG) provides protection against bTB, but its use in animals has been limited. The Jerusalem One Health workshop considered key bTB knowledge gaps and innovative solutions. Knowledge gaps identified included (a) the poorly quantified prevalence of M. bovis infection and disease in cattle, domestic camelids and human populations in developing countries, (b) the absence of alternatives to a "test and cull" strategy in settings where the killing of infected animals is culturally or economically unacceptable, or where affected species are protected and (c) an understanding of the induction of mucosal immunity against bTB. We summarize discussions on the use of BCG vaccination in domestic animals and wildlife and list potential projects to address the knowledge gaps identified

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.File. Supplementary methods and figures. Supplementary Information containing further details on statistical methods, data analysis and evolutionary consequences.Writing – review & editing: Luca Ferretti, Eva Pe´rez-Martı´n, Franc¸ois Maree, Bryan Charleston, Paolo Ribeca.The Pirbright Institute receives grant aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom (projects BB/E/I/00007035, BB/E/I/ 00007036, BB/E/I/00007032, BBS/E/I/00007039 and grant BB/L011085/1 as part of the joint USDANSF- NIH-BBSRC Ecology and Evolution of Infectious Diseases program).http://www.plospathogens.orgam2020Microbiology and Plant Patholog

Experimental Mycobacterium bovis infection in three white rhinoceroses (Ceratotherium simum):Susceptibility, clinical and anatomical pathology

Tuberculosis caused by Mycobacterium bovis is endemic in the African buffalo (Syncerus caffer) population in the Kruger National Park and other conservation areas in South Africa. The disease has been diagnosed in a total of 21 free ranging or semi-free ranging wildlife species in the country with highly variable presentations in terms of clinical signs as well as severity and distribution of tuberculous lesions. Most species are spillover or dead-end hosts without significant role in the epidemiology of the disease. White rhinoceroses (Ceratotherium simum) are translocated from the Kruger National Park in substantial numbers every year and a clear understanding of their risk to manifest overt tuberculosis disease and to serve as source of infection to other species is required. We report the findings of experimental infection of three white rhinoceroses with a moderately low dose of a virulent field isolate of Mycobacterium bovis. None of the animals developed clinical signs or disseminated disease. The susceptibility of the white rhinoceros to bovine tuberculosis was confirmed by successful experimental infection based on the ante mortem isolation of M. bovis from the respiratory tract of one rhinoceros, the presence of acid-fast organisms and necrotizing granulomatous lesions in the tracheobronchial lymph nodes and the detection of M. bovis genetic material by PCR in the lungs of two animals

Differential persistence of foot-and-mouth disease virus in African buffalo is related to virus virulence

Foot-and-mouth disease virus (FMDV) circulates as multiple serotypes and strains in many endemic regions. In particular the three Southern African Territories (SAT) serotypes are maintained effectively in their wildlife reservoir, the African buffalo, and individuals may harbour multiple SAT-serotypes for extended periods in the pharyngeal region. However the exact site and mechanism for persistence remain unclear. FMD in buffaloes offers a unique opportunity to study FMDV-persistence, as transmission from carrier ruminants has only convincingly been demonstrated for this species. Following co-infection of naïve African buffaloes with three SAT-serotypes isolated from field buffaloes; palatine tonsil swabs were the sample of choice for recovering infectious FMDV up to 400 days post infection (dpi). Post-mortem examination identified infectious virus for up to 185 dpi and viral genome up to 400 dpi in lymphoid tissue of the head and neck, mainly focussed in germinal centres. Interestingly viral persistence in vivo was not homogenous and the SAT-1 isolate persisted for longer than SAT-2 and SAT-3. Co-infection and passage of these SAT isolates in goat and buffalo cell lines demonstrated a direct correlation between persistence and cell killing capacity. These data suggest FMDV persistence occurs in the germinal centres of lymphoid tissue but the duration of persistence is related to virus replication and cell killing capacity.NJ was funded as a Wellcome Trust Intermediate Clinical Fellow and funding is acknowledged from the Biotechnology and Biological Sciences Research Council (BBS/E/I/00001523 and BBS/E/I/00001717).http://jvi.asm.org2016-11-30hb2016Microbiology and Plant Patholog