Investigation of Brucella melitensis in Sable Antelope (Hippotragus niger) in South Africa.

In this study, Brucella melitensis infection in sable antelope (Hippotragus niger) was investigated on two wildlife ranches in South Africa over a 12-year period in order to determine the origin of the outbreaks and the role of livestock in maintaining the disease. Retrospective data were obtained from farm records and interviews as well as samples tested from different disease scenarios and clinical settings. On one ranch, 10 of 74 sable tested seropositive for B. melitensis in 2004 but were certified clear of infection after no further brucellosis cases were detected following repeated serological tests and culling over a five-year period. Recrudescence occurred in 2013 (7 of 187 brucellosis positives) and in 2014 (one positive), with persistent, latent infection being the most reasonable explanation. In a second case study, linked to the first one through a common vendor, 15 of 80 sable tested positive in 2016, some five years after the acquisition of the animals from a putative source. Brucella melitensis biovar 1 and/or 3 were isolated from each outbreak on both ranches. Both outbreaks resulted in substantial losses for the owners, arising from testing and culling and significant resource expenditure by the state. The study identified the diagnostic challenges for identifying and resolving disease outbreaks in wildlife, the persistence of B. melitensis in sable, the risks associated with animal movements, and the need for a wildlife-sensitive disease control scheme. Although the actual source of infection could not be identified, the investigation points away from local livestock as a source of ongoing infection while the persistent infection is consistent with the disease circulating within small, ranched populations and being spread through the keeping and trading of high-value animals. The implications of the study findings to disease control in wildlife are discussed.

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.

Rift Valley Fever: Does Wildlife Play a Role?

Rift Valley fever (RVF) virus (RVFV) is an emerging vector-borne pathogen that causes sporadic epizootics and epidemics with multi-year, apparently quiescent, inter-epidemic periods. The epidemiology and ecology of the virus during these inter-epidemic periods is poorly understood. There is evidence for low-level circulation of the virus in livestock and wild ruminants; however, as of yet there is no evidence to identify a specific mammalian reservoir host. Using a systematic approach, this review synthesizes results from serosurveys, attempts at viral detection, and experimental infection of wildlife. These data demonstrate there is a gap in research conducted on RVF in wild ruminants. Specifically, there is very little published data on the pathogenicity of an RVFV infection in various wildlife species, validation of diagnostic assays for exposure to RVFV and understanding of epizootic or endemic disease dynamics in wild ruminants. We recommend that future research on RVFV incorporate a more systematic approach to understand the low-level cycling of the virus during inter-epidemic periods in both wild and domestic ruminant species.

Differential Persistence of Foot-and-Mouth Disease Virus in African Buffalo Is Related to Virus Virulence.

UNLABELLED: Foot-and-mouth disease (FMD) virus (FMDV) circulates as multiple serotypes and strains in many regions of endemicity. In particular, the three Southern African Territories (SAT) serotypes are maintained effectively in their wildlife reservoir, the African buffalo, and individuals may harbor 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 convincingly been demonstrated for only this species. Following coinfection of naive African buffaloes with isolates of three SAT serotypes from field buffaloes, palatine tonsil swabs were the sample of choice for recovering infectious FMDV up to 400 days postinfection (dpi). Postmortem examination identified infectious virus for up to 185 dpi and viral genomes for up to 400 dpi in lymphoid tissues of the head and neck, focused mainly in germinal centers. Interestingly, viral persistence in vivo was not homogenous, and the SAT-1 isolate persisted longer than the SAT-2 and SAT-3 isolates. Coinfection 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 that FMDV persistence occurs in the germinal centers of lymphoid tissue but that the duration of persistence is related to virus replication and cell-killing capacity. IMPORTANCE: Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in domestic livestock and wildlife species. African buffaloes (Syncerus caffer) are the primary carrier hosts of FMDV in African savannah ecosystems, where the disease is endemic. We have shown that the virus persists for up to 400 days in buffaloes and that there is competition between viruses during mixed infections. There was similar competition in cell culture: viruses that killed cells quickly persisted more efficiently in passaged cell cultures. These results may provide a mechanism for the dominance of particular viruses in an ecosystem.

Eco-epidemiological and pathological features of wildlife mortality events related to cyanobacterial bio-intoxication in the Kruger National Park, South Africa.

Over the past decade, several clustered, multispecies, wildlife mortality events occurred in the vicinity of two man-made earthen dams in the southern and south central regions of the Kruger National Park, South Africa. On field investigation, heavy cyanobacterial blooms were visible in these impoundments and analysis of water samples showed the dominance of Microcystis spp. (probably Microcystis aeruginosa). Macroscopic lesions seen at necropsy and histopathological lesions were compatible with a diagnosis of cyanobacterial intoxication. Laboratory toxicity tests and assays also confirmed the presence of significant levels of microcystins in water from the two dams. These outbreaks occurred during the dry autumn and early winter seasons when water levels in these dams were dropping, and a common feature was that all the affected dams were supporting a large number of hippopotamuses (Hippopotamus amphibius). It is hypothesised that hippopotamus' urine and faeces, together with agitation of the sediments, significantly contributed to internal loading of phosphates and nitrogen - leading to eutrophication of the water in these impoundments and subsequent cyanobacterial blooms. A major cause for concern was that a number of white rhinoceros (Ceratotherium simum) were amongst the victims of these bio-intoxication events. This publication discusses the eco-epidemiology and pathology of these clustered mortalities, as well as the management options considered and eventually used to address the problem.

Context-dependent survival, fecundity and predicted population-level consequences of brucellosis in African buffalo.

Chronic infections may have negative impacts on wildlife populations, yet their effects are difficult to detect in the absence of long-term population monitoring. Brucella abortus, the bacteria responsible for bovine brucellosis, causes chronic infections and abortions in wild and domestic ungulates, but its impact on population dynamics is not well understood. We report infection patterns and fitness correlates of bovine brucellosis in African buffalo based on (1) 7 years of cross-sectional disease surveys and (2) a 4-year longitudinal study in Kruger National Park (KNP), South Africa. We then used a matrix population model to translate these observed patterns into predicted population-level effects. Annual brucellosis seroprevalence ranged from 8·7% (95% CI = 1·8-15·6) to 47·6% (95% CI = 35·1-60·1) increased with age until adulthood (>6) and varied by location within KNP. Animals were on average in worse condition after testing positive for brucellosis (F = -5·074, P < 0·0001), and infection was associated with a 2·0 (95% CI = 1·1-3·7) fold increase in mortality (χ(2)  = 2·039, P = 0·036). Buffalo in low body condition were associated with lower reproductive success (F = 2·683, P = 0·034), but there was no association between brucellosis and pregnancy or being observed with a calf. For the range of body condition scores observed in the population, the model-predicted growth rate was λ = 1·11 (95% CI = 1·02-1·21) in herds without brucellosis and λ = 1·00 (95% CI = 0·85-1·16) when brucellosis seroprevalence was 30%. Our results suggest that brucellosis infection can potentially result in reduced population growth rates, but because these effects varied with demographic and environmental conditions, they may remain unseen without intensive, longitudinal monitoring.

Evaluation of the sensitivity and specificity of an enzyme-linked immunosorbent assay for diagnosing brucellosis in African buffalo (Syncerus caffer).

Brucellosis is a disease of veterinary and public health importance worldwide. In sub-Saharan Africa, where the bacterium Brucella abortus has been identified in several free-ranging wildlife species, successful disease control may be dependent on accurate detection in wildlife reservoirs, including African buffalo (Syncerus caffer). We estimated the sensitivity and specificity of a commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX Brucellosis Serum Ab test, IDEXX Laboratories, Westbrook, Maine, USA) for B. abortus based on a data set of 571 serum samples from 258 buffalo in the Kruger National Park, South Africa. We defined a pseudogold standard test result as those buffalo that were consistently positive or negative on two additional serologic tests, namely, the rose bengal test (RBT) and the complement fixation test (CFT). The ELISA's cutoff value was selected using receiver operating characteristics analysis, the pseudogold standard, and a threshold criterion that maximizes the total sensitivity and specificity. Then, we estimated the sensitivity and specificity of all three tests using Bayesian inference and latent class analysis. The ELISA had an estimated sensitivity of 0.928 (95% Bayesian posterior credibility interval [95% BCI] = 0.869-0.974) and specificity of 0.870 (95% BCI = 0.836-0.900). Compared with the ELISA, the RBT had a higher estimated sensitivity of 0.986 (95% BCI = 0.928-0.999), and both the RBT and CFT had higher specificities, estimated to be 0.992 (95% BCI = 0.971-0.996) and 0.998 (95% BCI = 0.992-0.999), respectively. Therefore, no single serologic test perfectly detected the antibody. However, after adjustment of cutoff values for South African conditions, the IDEXX Brucellosis Serum Ab Test may be a valuable additional screening test for brucellosis in Kruger National Park's African buffalo.

Pansteatitis of unknown etiology associated with large-scale Nile crocodile (Crocodylus niloticus) mortality in Kruger National Park, South Africa: pathologic findings.

Annual mortality events in Nile crocodiles (Crocodylus niloticus) in the Olifants River Gorge in Kruger National Park, South Africa, were experienced between 2008 and 2012, during which at least 216 crocodiles died. Live crocodiles were lethargic. Necropsy examination of 56 affected crocodiles showed dark yellow-brown firm nodules in both somatic fat and the abdominal fat body. In all of the 11 crocodiles submitted for histology, degenerative, necrotic, and inflammatory changes supported a diagnosis of steatitis in both fat types. Crocodiles are apex predators in this anthropogenically changed aquatic ecosystem that is used by humans upstream and downstream from the park for domestic, agricultural, fishing, and recreational purposes. This pathologic review of pansteatitis in crocodiles in the Olifants River system was part of a broad multidisciplinary research program. To date, no definitive causative agent has been identified. Epidemiologic evidence suggests that this event may have been a one-time event with long-standing repercussions on the health of the crocodiles. Pathologic findings are rarely documented in wild crocodilians. This study also reports on other conditions, including the presence of coccidian oocysts, capillarid and filaroid nematodes, digenetic trematodes, and pentastomes.

Detection of antibodies to tuberculosis antigens in free-ranging lions (Panthera leo) infected with Mycobacterium bovis in Kruger National Park, South Africa.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become established in Kruger National Park, South Africa, in the cape buffalo (Syncerus caffer) population and in other species. TB in prey species has resulted in infection and morbidity in the resident lion (Panthera leo) prides. The only validated live animal test currently available for lions is the intradermal tuberculin test. Because this test requires capture twice, 72 hr apart, of free-ranging lions to read results, it is logistically difficult to administer in a large ecosystem. Therefore, development of a rapid animal-side screening assay would be ideal in providing information for wildlife managers, veterinarians, and researchers working with free-living lion prides. This study reports preliminary descriptive results from an ongoing project evaluating two serologic tests for M. bovis (ElephantTB Stat-Pak and dual path platform VetTB). Disease status was determined by postmortem culture and presence of pathologic lesions in 14 free-ranging lions. Seropositivity was found to be associated with M. bovis infection. Extended field studies are underway to validate these rapid animal-side immunoassays for antemortem screening tests for TB in lions.

The African buffalo: a villain for inter-species spread of infectious diseases in southern Africa.

The African buffalo (Syncerus caffer) is a large wild bovid which until recently ranged across all but the driest parts of sub-Saharan Africa, and their local range being limited to about 20 km from surface water. They are of high ecological value due to their important role as bulk feeders in the grazing hierarchy. They also have high economic value, because they are one of the sought after 'Big Five' in the eco-tourism industry. In Africa, buffaloes have been recognised for some time as an important role player in the maintenance and transmission of a variety of economically important livestock diseases at the wildlife and/or livestock interface. These include African strains of foot-and-mouth disease (FMD), Corridor disease (theileriosis), bovine tuberculosis and bovine brucellosis. For a number of other diseases of veterinary importance, African buffaloes may also serve as amplifier or incidental host, whereby infection with the causative pathogens may cause severe clinical signs such as death or abortion as in the case of anthrax and Rift Valley fever, or remain mild or subclinical for example heartwater. The long term health implications of most of those infections on the buffalo at a population level is usually limited, and they do not pose a threat on the population's survival. Because of their ability to harbour and transmit important diseases to livestock, their sustainable future in ecotourism, trade and transfrontier conservation projects become complex and costly and reliable diagnostic tools are required to monitor these infections in buffalo populations.

BCG vaccination failed to protect yearling African buffaloes (Syncerus caffer) against experimental intratonsilar challenge with Mycobacterium bovis.

Vaccination has been discussed as a practical option to control bovine tuberculosis in countries where a wildlife reservoir of the disease is present. African buffaloes (Syncerus caffer) are the main wildlife reservoir of Mycobacterium bovis in certain South African game parks and vaccination is not only the most promising but the only ethically acceptable control measure currently available. The use of Bacillus Calmette-Guérin vaccine (Pasteur strain) to vaccinate fourteen African buffalo yearlings and their reactions to subsequent intratonsilar challenge with a field strain of M. bovis are described. The BCG vaccine was administered twice intramuscularly, six weeks apart. All vaccinates and thirteen control buffaloes were euthanized and necropsies performed 9 months after the challenge. Standard sets of lymph nodes from the head, the thoracic cavity and abdomen were cultured and examined histopathologically. No significant reduction in number of lesions or severity of disease was noted, concluding that the BCG vaccine did not induce sufficient protection able to limit the shedding of organisms. The age of the buffaloes, route of vaccination and prior exposure to environmental mycobacteria are among the possible reasons for vaccination failure.

Assessment of microcystis bloom toxicity associated with wildlife mortality in the Kruger National Park, South Africa.

Based on previous necropsy results, Microcystis blooms in constructed water impoundments in the Kruger National Park (KNP) have been identified as a cause of wildlife mortality. In response to wildlife mortality during 2007, water samples, containing algal bloom material, were collected during February 2007 and July 2007 from four dams (Nhlanganzwani, Mpanamana, Makhohlola, and Sunset) in the southeastern part of the KNP as part of the follow-up investigation. The toxicity of the Microcystis blooms was determined using the enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition (PPI) assay, mouse bioassay, and African sharptooth catfish (Clarias gariepinus) primary hepatocytes. Both the ELISA and PPI assays indicated that the water sample collected during February 2007 from the Nhlanganzwani Dam, and samples collected from the Nhlanganzwani and Sunset dams in June 2007, were toxic. These dams, exhibiting the toxic Microcystis blooms, were also associated with the wildlife mortality. Mice injected intraperitoneally with water samples from Nhlanganzwani Dam (February 2007) induced hepatotoxicity and mortality within 1 hr. Primary hepatocytes from the sharptooth catfish exposed to samples from these dams gave similar results. This laboratory investigation and results strongly incriminate the toxic Microcystis blooms as the cause of the wildlife mortality. Eutrophication and bloom formation appear to have been the consequence of the high numbers of hippopotami (Hippopotamus amphibius) in specific dams.

Clinical demodicosis in African buffalo (Syncerus caffer) in the Kruger National Park.

We investigated the relationship between prevalence and severity of clinical signs of Demodex cafferi infection in free-ranging African buffalo (Syncerus caffer) and other factors such as age, sex, pregnancy status, and concomitant infections with bovine tuberculosis (BTB), Rift Valley fever (RVF), and brucellosis (BA). Approximately half of 203 buffalo examined in this study had clinical signs of demodicosis (cutaneous nodules); younger age classes had the highest prevalence and severity of lesions (chi(2)=21.4, df=6, P=0.0015). Nodules were generally limited to the head and neck region, but in severe cases were present over the entire animal. We found no significant association between clinical severity of the Demodex infection and gender, pregnancy status, or infection with BTB, RVF, or BA.

Identification of toxigenic Microcystis strains after incidents of wild animal mortalities in the Kruger National Park, South Africa.

The eutrophic process potentially caused by a high urine and faecal load resulting from an unusually high hippopotamus (Hippopotamus amphibious) density in the Nhlanganzwane Dam, Kruger National Park, South Africa, triggered a chain of events characterised by an increase in the growth of primary producers (Microcystis aeruginosa). This increase in M. aeruginosa biomass was followed by bio-intoxication incidents in wild animals. In this study, we determine if a M. aeruginosa bloom with a total microcystin level of 23,718microgl(-1) have been responsible for mortalities of megaherbivores in the Nhlanganzwane Dam. We further use microcystin molecular markers derived from the mcy gene cluster to identify potentially toxigenic environmental Microcystis strains in the dam during the occurrence of animal intoxications. The estimated total microcystin-LR daily intake by an adult male white rhinoceros (Ceratotherium simum) from cyanobacterial-contaminated water of the dam during the toxic event was an order of magnitude higher (754.29microgkg(-1)bw) in comparison with the lowest observed adverse effecting level (LOAEL) value measured for pigs in a previous study by other authors. In this study the presence of toxic cyanobacterial strains was confirmed with the use of molecular markers that detected the presence of the mcy gene cluster responsible for the production of toxin by M. aeruginosa.

Pulmonary infection due to Mycobacterium goodii in a spotted hyena (Crocuta crocuta) from South Africa.

We report a case of pyogranulomatous pneumonia due to infection with Mycobacterium goodii in an adult female spotted hyena (Crocuta crocuta). The lungs of the animal showed consolidated, granulomatous lesions, and they were extensively and severely infiltrated. Polymerase chain reaction sequencing of isolated crude lung tissue DNA, and boiled lung culture samples, all confirmed that the causative organism was M. goodii, a recently described fast-growing organism closely related to the nonpathogenic mycobacterial species M. smegmatis. The current study illustrates that this organism can be pathogenic and cause extensive pulmonary disease.

Recombinant nucleocapsid-based ELISA for detection of IgG antibody to Rift Valley fever virus in African buffalo.

Wild ruminants are thought to serve as natural hosts for Rift Valley fever virus (RVFV) but the role of these animals as reservoirs for RVFV during inter-epidemic periods and as amplifiers during epidemics is not well understood. An indirect enzyme-linked immunoassay (I-ELISA) based on the recombinant nucleocapsid protein (rNp) of RVFV was validated for the detection of specific IgG antibodies in African buffalo. Data sets derived from testing buffalo sera from Kenya (n=405) and South Africa (n=618) were dichotomised according to the results of a virus neutralisation test. The assay characteristic performance was analysed using threshold values optimised by the two-graph receiver operating characteristics (TG-ROC) analysis, and by mean plus two, as well as by mean plus three standard deviations derived from I-ELISA PP values in uninfected animals. Among 1023 buffalo sera tested, 77 (7.5%) had detectable virus neutralising antibodies. The assay had high intra- and inter-plate repeatability in routine runs. At a cut-off optimised by the TG-ROC at 95% accuracy level, the diagnostic sensitivity of the I-ELISA was 98.7% and diagnostic specificity 99.36% while estimates for the Youden's index (J) and efficiency (Ef) were 0.98 and 99.31%. When cut-off values determined by traditional statistical approaches were used, the diagnostic sensitivity was 100% but estimates of J, Ef and other combined measures of diagnostic accuracy were lower compared to those based on cut-off value derived from the TG-ROC. Results of the study indicate that the I-ELISA based on the rNp would be useful for seroepidemiological studies of RVFV infections in African buffalo.

Molecular epidemiology of SAT3-type foot-and-mouth disease.

VP1 gene nucleotide sequences of 51 SAT3-type foot-and-mouth disease (FMD) viruses from seven southern and eastern African countries were used to infer a gene phylogeny. Results obtained by phylogenetic analysis of the homologous 405 nt region corresponding to the C-terminal 128 amino acids of 1D and adjacent 7 amino acids of 2A indicate that there are six distinct virus lineages evolving independently in different geographical localities in accordance with the FMD topotype concept. Topotypes I-IV occur in southern Africa, whilst topotypes V and VI are unique to East Africa. Viruses of different topotypes differ from each other at 20% or more of the nucleotide sites, specified in this study. Despite the limited geographical distribution of this serotype, the level of intratypic variation is intermediate between that of SAT1 and SAT2, both of which are widely distributed in sub-Saharan Africa. Within SAT3, 37.3% and 47.4% of sites were completely conserved on nucleotide and amino acid levels, respectively. The locality-specific grouping of viruses permits accurate determination of the sources of outbreaks, whilst the high levels of variation within the immunodominant 1D protein has implications for the control of the disease through vaccination.