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1. Immunological Evidence of Variation in Exposure and Immune Response to Bacillus anthracis in Herbivores of Kruger and Etosha National Parks

   https://doi.org/10.3389/fimmu.2022.814031  

   Ochai, Sunday O.; Crafford, Jan E.; Hassim, Ayesha; Byaruhanga, Charles; Huang, Yen-Hua; Hartmann, Axel; Dekker, Edgar H.; van Schalkwyk, O. Louis; Kamath, Pauline L.; Turner, Wendy C.; et al (February 2022, Frontiers in Immunology)

   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. 

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2. Roles of host and environment in shift of primary anthrax host species in Kruger National Park

   https://doi.org/10.1371/journal.pone.0314103  

   Ochai, Sunday O; Snyman, Lourens; Dolfi, Amelie C; Ramoelo, Abel; Reilly, Brian K; Botha, Judith M; Dekker, Edgar H; van_Schalkwyk, O Louis; Kamath, Pauline L; Archer, Emma; et al (December 2024, PLOS ONE)

   Serrano, Emmanuel (Ed.)

   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. 

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3. Environmental drivers of biseasonal anthrax outbreak dynamics in two multihost savanna systems

   https://doi.org/10.1002/ecm.1526  

   Huang, Yen‐Hua; Kausrud, Kyrre; Hassim, Ayesha; Ochai, Sunday O.; Schalkwyk, O. Louis; Dekker, Edgar H.; Buyantuev, Alexander; Cloete, Claudine C.; Kilian, J. Werner; Mfune, John K.; et al (April 2022, Ecological Monographs)

   Environmental factors are common forces driving infectious disease dynamics. We compared inter-annual and seasonal patterns of anthrax infections in two multi-host 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 inter-annual variation in case numbers, and large outbreaks seemed to follow roughly a 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 may imply 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 multi-host 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. 

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4. Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax

   https://doi.org/10.1371/journal.pntd.0012122  

   Ochai, Sunday Ochonu; Hassim, Ayesha; Dekker, Edgar H; Magome, Thuto; Lekota, Kgaugelo Edward; Makgabo, S Marcus; de_Klerk-Loris, Lin-Mari; van_Schalkwyk, Louis O; Kamath, Pauline L; Turner, Wendy C; et al (November 2024, PLOS Neglected Tropical Diseases)

   Chang, Yung-Fu (Ed.)

   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. 

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5. Development of conjugated secondary antibodies for wildlife disease surveillance

   https://doi.org/10.3389/fimmu.2023.1221071  

   Ochai, Sunday O.; Crafford, Jan E.; Kamath, Pauline L.; Turner, Wendy C.; van Heerden, Henriette (July 2023, Frontiers in Immunology)

   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. 

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