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The diagnosis of anthrax, a zoonotic disease caused byBacillus anthraciscan be complicated by detection of closely related species. Conventional diagnosis of anthrax involves microscopy, culture identification of bacterial colonies and molecular detection. Genetic markers used are often virulence gene targets such asB. anthracisprotective antigen (pagA, also called BAPA, occurring on plasmid pXO1), lethal factor (lef, on pXO1), capsule-encodingcapB/C(located on pXO2) as well as chromosomal Ba-1. Combinations of genetic markers using real-time/quantitative polymerase chain reaction (qPCR) are used to confirmB.anthracisfrom culture but can also be used directly on diagnostic samples to avoid propagation and its associated biorisks and for faster identification. We investigated how the presence of closely related species could complicate anthrax diagnoses with and without culture to standardise the use of genetic markers using qPCR for accurate anthrax diagnosis. Using blood smears from 2012–2020 from wildlife mortalities (n = 1708) in Kruger National Park in South Africa where anthrax is endemic, we contrasted anthrax diagnostic results based on qPCR, microscopy, and culture. From smears, 113/1708 grew bacteria in culture, from which 506 isolates were obtained. Of these isolates, only 24.7% (125 isolates) were positive forB.anthracisbased on genetic markers or microscopy. However, among these, merely 4/125 (3.2%) were confirmedB.anthracisisolates (based on morphology, microscopy, and sensitivity testing to penicillin and gamma-phage) from the blood smear, likely due to poor survival of spores on stored smears. This study identifiedB.cereus sensu lato, which includedB.cereusandB.anthracis,Peribacillusspp., andPriestiaspp. clusters usinggyrBgene in selected bacterial isolates positive forpagAregion using BAPA probe. Using qPCR on blood smears, 52.1% (890 samples) tested positive forB.anthracisbased on one or a combination of genetic markers which included the 25 positive controls. Notably, the standardlefprimer set displayed the lowest specificity and accuracy. The Ba-1+BAPA+lefcombination showed 100% specificity, sensitivity, and accuracy. Various marker combinations, such as Ba-1+capB, BAPA+capB, Ba-1+BAPA+capB+lef, and BAPA+lef+capB, all demonstrated 100.0% specificity and 98.7% accuracy, while maintaining a sensitivity of 96.6%. Using Ba-1+BAPA+lef+capB, as well as Ba-1+BAPA+lefwith molecular diagnosis accurately detectsB.anthracisin the absence of bacterial culture. Systematically combining microscopy and molecular markers holds promise for notably reducing false positives. This significantly enhances the detection and surveillance of diseases like anthrax in southern Africa and beyond and reduces the need for propagation of the bacteria in culture. 

Exposure and immunity to generalist pathogens differ among host species and vary across spatial scales. Anthrax, caused by a multi-host bacterial pathogen, Bacillus anthracis , is enzootic in Kruger National Park (KNP), South Africa and Etosha National Park (ENP), Namibia. These parks share many of the same potential host species, yet the main anthrax host in one (greater kudu ( Tragelaphus strepsiceros ) in KNP and plains zebra ( Equus quagga ) in ENP) is only a minor host in the other. We investigated species and spatial patterns in anthrax mortalities, B. anthracis exposure, and the ability to neutralize the anthrax lethal toxin to determine if observed host mortality differences between locations could be attributed to population-level variation in pathogen exposure and/or immune response. Using serum collected from zebra and kudu in high and low incidence areas of each park (18- 20 samples/species/area), we estimated pathogen exposure from anti-protective antigen (PA) antibody response using enzyme-linked immunosorbent assay (ELISA) and lethal toxin neutralization with a toxin neutralization assay (TNA). Serological evidence of pathogen exposure followed mortality patterns within each system (kudus: 95% positive in KNP versus 40% in ENP; zebras: 83% positive in ENP versus 63% in KNP). Animals in the high-incidence area of KNP had higher anti-PA responses than those in the low-incidence area, but there were no significant differences in exposure by area within ENP. Toxin neutralizing ability was higher for host populations with lower exposure prevalence, i.e., higher in ENP kudus and KNP zebras than their conspecifics in the other park. These results indicate that host species differ in their exposure to and adaptive immunity against B. anthracis in the two parks. These patterns may be due to environmental differences such as vegetation, rainfall patterns, landscape or forage availability between these systems and their interplay with host behavior (foraging or other risky behaviors), resulting in differences in exposure frequency and dose, and hence immune response. 

Abstract Environmental factors are common forces driving infectious disease dynamics. We compared interannual and seasonal patterns of anthrax infections in two multihost systems in southern Africa: Etosha National Park, Namibia, and Kruger National Park, South Africa. Using several decades of mortality data from each system, we assessed possible transmission mechanisms behind anthrax dynamics, examining (1) within‐ and between‐species temporal case correlations and (2) associations between anthrax mortalities and environmental factors, specifically rainfall and the Normalized Difference Vegetation Index (NDVI), with empirical dynamic modeling. Anthrax cases in Kruger had wide interannual variation in case numbers, and large outbreaks seemed to follow a roughly decadal cycle. In contrast, outbreaks in Etosha were smaller in magnitude and occurred annually. In Etosha, the host species commonly affected remained consistent over several decades, although plains zebra (Equus quagga) became relatively more dominant. In Kruger, turnover of the main host species occurred after the 1990s, where the previously dominant host species, greater kudu (Tragelaphus strepsiceros), was replaced by impala (Aepyceros melampus). In both parks, anthrax infections showed two seasonal peaks, with each species having only one peak in a year. Zebra, springbok (Antidorcas marsupialis), wildebeest (Connochaetes taurinus), and impala cases peaked in wet seasons, while elephant (Loxodonta africana), kudu, and buffalo (Syncerus caffer) cases peaked in dry seasons. For common host species shared between the two parks, anthrax mortalities peaked in the same season in both systems. Among host species with cases peaking in the same season, anthrax mortalities were mostly synchronized, which implies similar transmission mechanisms or shared sources of exposure. Between seasons, outbreaks in one species may contribute to more cases in another species in the following season. Higher vegetation greenness was associated with more zebra and springbok anthrax mortalities in Etosha but fewer elephant cases in Kruger. These results suggest that host behavioral responses to changing environmental conditions may affect anthrax transmission risk, with differences in transmission mechanisms leading to multihost biseasonal outbreaks. This study reveals the dynamics and potential environmental drivers of anthrax in two savanna systems, providing a better understanding of factors driving biseasonal dynamics and outbreak variation among locations.