Diagnosis of anthrax and the roles of host and environment in the transmission of anthrax in Kruger National Park

Abstract

Over the years, various techniques have been employed to diagnose infectious zoonoses, such as anthrax caused by Bacillus anthracis. These methods encompass microscopic examination of blood smears, bacterial culture, molecular diagnostics targeting genetic markers of the pathogen, and serological tests to identify antibodies against pathogen antigens. The accuracy of these techniques largely depends on the specificity and sensitivity of the tests used. Disease monitoring in free-roaming wild animals is challenging, often relying on passive surveillance. However, proactive surveillance, which involves detecting specific antibodies, can provide more reliable and timely insights into disease presence and prevalence within populations, especially when disease signs are below passive surveillance detection thresholds. Nevertheless, primary binding assays, such as the indirect enzyme-linked immunosorbent assay (ELISA) used for detecting antibodies in wildlife, face challenges due to the absence of species-specific conjugates. Also, the diagnosis of anthrax, remains a matter of concern due to the challenges posed by the identification of closely related species that carry regions on plasmids (pXO1 and pXO2) and chromosome that highly resembling those found in B. anthracis. As mentioned, traditional methods for diagnosing anthrax include microscopy, identifying isolates through culture, and using genetic markers such as B. anthracis protective antigen (pagA also known as BAPA on pXO1), lethal factor (lef on pXO1), chromosomal (Ba-1), and capsule (capB on pXO2) genes for molecular detection. Because anthrax is not contagious, the exposure of herbivorous mammalian hosts to B. anthracis is greatly influenced by environmental and climatic factors, as well as host demographics and behaviour. In Kruger National Park (KNP), the most impacted host species used to be kudu (Tragelaphus strepsiceros) until the early 1990s, and outbreaks were more common during the dry season. However, there has been a shift in this pattern, and impala (Aepyceros melampus) is now the most affected species, with outbreaks occurring more frequently during the wet season. In this study, we first developed anti-kudu and anti-impala immunoglobulin-specific conjugates in chickens to compare their binding efficiency with that of commercially available protein-G and protein-AG conjugates. This was done using an ELISA-based avidity index to enhance the serological diagnosis of anthrax. Second, we investigated the complications posed by the presence of atypical B. cereus and other closely related species in diagnosing anthrax with genetic markers and qPCR. For this purpose, we analyzed blood smears from wildlife mortalities in Kruger National Park (KNP), South Africa, comparing the outcomes of anthrax diagnostics using qPCR, microscopy, and culture methods. Finally, we explored the transition of the primary anthrax host from kudu to impala within KNP. Our focus was on identifying potential links between environmental factors—such as precipitation, soil moisture, temperature, and the Normalized Difference Vegetation Index (NDVI)—and the patterns of anthrax mortality occurrences and frequencies. Additionally, we examined the variations in environmental factors and the population densities of various host species over time, aiming to identify any correlations between the densities of host species and the rates of anthrax mortalities. 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 additional wildlife species exhibited cross-reactivity, showing a mean relative avidity of over 50% with the developed impala and kudu conjugates, compared to less than 40% with the commercial conjugates. This study underscores the value of species-specific conjugates as crucial tools for developing and validating immunoassays in wildlife, and for monitoring zoonotic diseases across the livestock-wildlife-human interface. In our analysis of 1,706 blood smears from wildlife mortalities, 890 samples were positive for B. anthracis, detected either through genetic markers or microscopy. Specifically, 15.2% of these samples tested positive for the lef marker, and 12.6% for BAPA. The use of both BAPA and lef markers together identified 24.4% of samples as positive, which increased to 44.4% when combined with microscopy, indicating strong concordance between molecular and microscopic methods (p<0.0001). Out of 506 cultured isolates, 24.7% tested positive by either genetic markers or microscopy, but only 4 samples were definitively confirmed as B. anthracis through culture, microscopy, and sensitivity testing to penicillin and gamma-phage. The lef marker was found to have the lowest specificity and accuracy. Conversely, combinations such as Ba-1/capB, BAPA/capB, Ba-1/BAPA/capB/lef, and BAPA/lef/capB achieved 100% specificity and accuracy, with a sensitivity of 75%. The combination of BAPA/lef/Ba-1 also reached 100% in specificity, sensitivity, and accuracy. The findings emphasize the need to identify precise markers for B. anthracis in southern Africa to enhance anthrax diagnosis. The strategic use of both microscopy and multiple markers can significantly reduce false positives. The study also noted distinct trends in anthrax mortality over different years and regions, with a notable shift in the primary host species from kudu to impala. Furthermore, significant correlations were found between anthrax mortality in kudu and environmental factors such as NDVI, average temperature, and standardized precipitation indexes (SPI-6 and SPI-12). In contrast, impala mortality was associated with changes in SPI-3, temperature increases, and higher mortality rates during the rainy season. Interestingly, elephant density was negatively correlated with kudu mortality but positively correlated with impala mortality and density. These observations suggest that environmental conditions and the density of species play significant roles in determining the frequency and variety of hosts exposed to B. anthracis. The study concludes that over time, climate extremes could amplify the severity of anthrax outbreaks by affecting species susceptibility and exposure chances.