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Environmental and climatic factors, as well as host demographics and behaviour, significantly influence the exposure of herbivorous mammalian hosts to pathogens such asBacillus anthracis, the causative agent of anthrax. Until the early 1990s in Kruger National Park (KNP), kudu (Tragelaphus strepsiceros) was the host species most affected by anthrax, with outbreaks occurring predominantly in the dry season, particularly during drought cycles. However, the most affected host species has shifted to impala (Aepyceros melampus), with more frequent anthrax outbreaks during the wet season. This study investigates the roles of environmental variation and other host species in this shift. Temporal trends in environmental variables such as precipitation, soil moisture, temperature, and normalised difference vegetation index (NDVI) were analyzed in relation to anthrax occurrence (presence/ absence and counts). Additionally, correlations between host species’ densities and anthrax mortalities over time were examined. Anthrax cases in 1990 were concentrated in the central and northern regions of KNP(excluding Pafuri), primarily affected kudus; while subsequent mortalities affected mostly impala and were restricted to the far north, in Pafuri. Significant correlations were found between kudu anthrax mortality and a decrease in NDVI, average temperature, SPI-6 and SPI-12 (Standardised Precipitation Index in various time intervals. Conversely, anthrax occurrence in impalas was associated with a decline in SPI-3, and temperature rise, with increased mortality during the rainy season. Elephant density correlated negatively with kudu mortality, but a positive correlation with both impala mortality and impala density. The study concludes that environmental variables and species’ densities may alter the diversity and frequency of hosts exposed toB.anthracis. Climate extremes and alterations therein may exacerbate anthrax severity by modifying species susceptibility and their probability of exposure over time. 

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. 

With emerging infectious disease outbreaks in human, domestic and wild animal populations on the rise, improvements in pathogen characterization and surveillance are paramount for the protection of human and animal health, as well as the conservation of ecologically and economically important wildlife. Genomics offers a range of suitable tools to meet these goals, with metagenomic sequencing facilitating the characterization of whole microbial communities associated with emerging and endemic disease outbreaks. Here, we use metagenomic sequencing in a case-control study to identify microbes in lung tissue associated with newly observed pneumonia-related fatalities in 34 white-tailed deer (Odocoileus virginianus) in Wisconsin, USA. We identified 20 bacterial species that occurred in more than a single individual. Of these, onlyClostridium novyiwas found to substantially differ (in number of detections) between case and control sample groups; however, this difference was not statistically significant. We also detected several bacterial species associated with pneumonia and/or other diseases in ruminants (Mycoplasma ovipneumoniae,Trueperella pyogenes,Pasteurella multocida,Anaplasma phagocytophilum,Fusobacterium necrophorum); however, these species did not substantially differ between case and control sample groups. On average, we detected a larger number of bacterial species in case samples than controls, supporting the potential role of polymicrobial infections in this system. Importantly, we did not detect DNA of viruses or fungi, suggesting that they are not significantly associated with pneumonia in this system. Together, these results highlight the utility of metagenomic sequencing for identifying disease-associated microbes. This preliminary list of microbes will help inform future research on pneumonia-associated fatalities of white-tailed deer. 

An important part of infectious disease management is predicting factors that influence disease outbreaks, such asR, the number of secondary infections arising from an infected individual. EstimatingRis particularly challenging for environmentally transmitted pathogens given time lags between cases and subsequent infections. Here, we calculatedRforBacillus anthracisinfections arising from anthrax carcass sites in Etosha National Park, Namibia. Combining host behavioural data, pathogen concentrations and simulation models, we show thatRis spatially and temporally variable, driven by spore concentrations at death, host visitation rates and early preference for foraging at infectious sites. While spores were detected up to a decade after death, most secondary infections occurred within 2 years. Transmission simulations under scenarios combining site infectiousness and host exposure risk under different environmental conditions led to dramatically different outbreak dynamics, from pathogen extinction (R< 1) to explosive outbreaks (R> 10). These transmission heterogeneities may explain variation in anthrax outbreak dynamics observed globally, and more generally, the critical importance of environmental variation underlying host–pathogen interactions. Notably, our approach allowed us to estimate the lethal dose of a highly virulent pathogen non-invasively from observational studies and epidemiological data, useful when experiments on wildlife are undesirable or impractical. 

Disease monitoring in free-ranging wildlife is a challenge and often relies on passive surveillance. Alternatively, proactive surveillance that relies on the detection of specific antibodies could give more reliable and timely insight into disease presence and prevalence in a population, especially if the evidence of disease occurs below detection thresholds for passive surveillance. Primary binding assays, like the indirect ELISA for antibody detection in wildlife, are hampered by a lack of species-specific conjugates. In this study, we developed anti-kudu ( Tragelaphus strepsiceros ) and anti-impala ( Aepyceros melampus ) immunoglobulin-specific conjugates in chickens and compared them to the binding of commercially available protein-G and protein-AG conjugates, using an ELISA-based avidity index. The conjugates were evaluated for cross-reaction with sera from other wild herbivores to assess future use in ELISAs. The developed conjugates had a high avidity of >70% against kudu and impala sera. The commercial conjugates (protein-G and protein-AG) had significantly low relative avidity (<20%) against these species. Eighteen other wildlife species demonstrated cross-reactivity with a mean relative avidity of >50% with the impala and kudu conjugates and <40% with the commercial conjugates. These results demonstrate that species-specific conjugates are important tools for the development and validation of immunoassays in wildlife and for the surveillance of zoonotic agents along the livestock-wildlife-human interface. 

Datasets collected from monitored zebra anthrax carcass sites in Etosha National Park, Namibia, 2010-2021. One data file describes the anthrax mortality, soil characterization of the carcass sites and details on the types and timing of different sampling efforts at these sites. The other data file is counts of Bacillus anthracis spores in surface soils collected annually at these carcass sites. 

Bacillus anthracis, the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. While analyses of genetic data from across the globe have increased our understanding of this bacterium’s population genomic structure, the influence of selective pressures on this successful pathogen is not well understood. In this study, we investigate the effects of antimicrobial resistance, phage diversity, geography and isolation source in shaping population genomic structure. We also identify a suite of candidate genes potentially under selection, driving patterns of diversity across 356 globally extant B. anthracis genomes. We report ten antimicrobial resistance genes and 11 different prophage sequences, resulting in the first large-scale documentation of these genetic anomalies for this pathogen. Results of random forest classification suggest genomic structure may be driven by a combination of antimicrobial resistance, geography and isolation source, specific to the population cluster examined. We found strong evidence that a recombination event linked to a gene involved in protein synthesis may be responsible for phenotypic differences between comparatively disparate populations. We also offer a list of genes for further examination of B. anthracis evolution, based on high-impact single nucleotide polymorphisms (SNPs) and clustered mutations. The information presented here sheds new light on the factors driving genomic structure in this notorious pathogen and may act as a road map for future studies aimed at understanding functional differences in terms of B. anthracis biogeography, virulence and evolution. 

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. 

Disease outbreaks are a consequence of interactions among the three components of a host–parasite system: the infectious agent, the host and the environment. While virulence and transmission are widely investigated, most studies of parasite life-history trade-offs are conducted with theoretical models or tractable experimental systems where transmission is standardized and the environment controlled. Yet, biotic and abiotic environmental factors can strongly affect disease dynamics, and ultimately, host–parasite coevolution. Here, we review research on how environmental context alters virulence–transmission relationships, focusing on the off-host portion of the parasite life cycle, and how variation in parasite survival affects the evolution of virulence and transmission. We review three inter-related ‘approaches’ that have dominated the study of the evolution of virulence and transmission for different host–parasite systems: (i) evolutionary trade-off theory, (ii) parasite local adaptation and (iii) parasite phylodynamics. These approaches consider the role of the environment in virulence and transmission evolution from different angles, which entail different advantages and potential biases. We suggest improvements to how to investigate virulence–transmission relationships, through conceptual and methodological developments and taking environmental context into consideration. By combining developments in life-history evolution, phylogenetics, adaptive dynamics and comparative genomics, we can improve our understanding of virulence–transmission relationships across a diversity of host–parasite systems that have eluded experimental study of parasite life history.