Investigation of Bacillus anthracis spore survival in soils from Kruger National Park in South Africa and Etosha National Park in Namibia

Abstract

Bacillus anthracis is a soil-borne pathogen and the aetiological agent of anthrax, which is a disease that primarily affects ungulates in enzootic regions. These enzootic regions occur where suitable soil types promote the survival of B. anthracis endospores despite several environmental stresses. This study focuses on the enzootic regions of Kruger National Park (KNP) in South Africa and Etosha National Park (ENP) in Namibia where the disease is unmanaged, and some pathogen dynamics have been well documented over time. In KNP and ENP, Pafuri and Okaukuejo are described as the high-incidence anthrax regions, respectively, while Skukuza (KNP) and Otjovasandu (ENP) are described as the low incidence regions. The phylogenetic distribution of B. anthracis strains over time differs between the parks. All isolates in ENP belong to the A-subclade while in KNP, there are isolates from both the Aand B-subclades. In KNP, the B-subclade dominated before the 1990s, but the A-subclade dominated thereafter. The main aim of this study was to investigate how the different soil types from these highand low-incidence areas affect the survival of different B. anthracis strains in a laboratory experiment. A fully insulated terra-simulator with sensors which measured the soil moisture, ambient temperature, humidity, and light was designed and programmed to mimic the environmental conditions of Pafuri in real time. A pilot study using the Sterne strain was conducted initially to evaluate the experimental procedure, decontamination methods and the terra-simulator itself. Thereafter, two strains each from KNP and ENP were selected (one KNP A-, one B- and two ENP A-clade strains) as well as the Sterne strain and was inoculated into the four different soil types. The inoculated soils were incubated in the terra-simulator and monitored for 6 months. Spore counts were done at monthly intervals using classical microbiological techniques for the isolation of B. anthracis from soil and reported as CFU/g. In addition, soil analyses were conducted on the soil samples which looked at the soil pH, exchangeable cations, soil density and soil organic matter. The spores were also prepared for electron microscopy to assess the differences in the nap layer of the exosporium (outermost layer of the endospore) for the strains used in this study. Overall, Pafuri soil had the best spore survival for all strains except for Sterne, followed by Otjovasandu, Okaukuejo and Skukuza. The A-strains survived better than the B-strain in all soil types while the Kruger B-strain showed a better survivability only in Pafuri. Okaukuejo and Otjovasandu supported the spore survival of the A-strains equally well, especially for the ENP endemic strains. The Sterne strain showed an overall poor environmental survivability. By 6 months, there was very little decline in the spore counts between the sampling time points, thus, the spore counts appeared to stabilise, with the exception of Sterne which continued to decline. Our findings suggested that strain type, time, relative humidity, and temperature were significant determinants of spore survival across the different soil types. Furthermore, soil characteristics such as pH, the availability of macro and micronutrients, soil organic matter, texture and moisture also influenced the spore survival across the various soil types. We identified differences in the filament lengths of the nap layers where the ENP Astrains had the longest filaments followed by the KNP A-strains and then the B-strain. We also identified appendages on B. anthracis spores which had only been previously described in pathogenic B. cereus strains. In conclusion, our findings showed that the A-strains survived better than the B-strain and that the B-strain survives best in its endemic soil type, Pafuri. The Sterne strain showed significant environmental lability when compared to the fully virulent strains. Spore survival is dependent on a number of factors including strain type and environmental determinants such as temperature, relative humidity and soil characteristics. These environmental determinants are factors that are constantly changing due to a culmination of climactic events which highlights the importance of consistent disease surveillance for this pathogen. In addition, there is a need for improved predictive modelling systems which considers the changing climate and its effects on the soil characteristics and spore survival.