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.