Distribution and molecular characterization of South African Bacillus Anthracis strains and their associated bacteriophages

Chapter 1: Introduction and Literature Review; Chapter 2: A retrospective study of anthrax on the Ghaap plateau, Northern Cape Province of South Africa, with special reference to the 2007 - 2008 outbreaks; Chapter 3: Insights gained from sample diagnostics during anthrax outbreaks in the Kruger National Park, South Africa; Chapter 4: Through the lens: a microscopic and molecular evaluation of archival blood smears from the 2010 anthrax outbreaks in Kruger National Park, South Africa; Chapter 5: A distribution snapshot of anthrax in South Africa: multiple locus variable number of tandem repeats analyses of Bacillus anthracis isolates from epizootics spanning 4 decades across southern Africa; Chapter 6: Isolation and Whole Genome Analysis of a Lytic Bacteriophage Infected Bacillus anthracis Isolate from Pafuri, South Africa; Chapter 7: Characterisation of temperate bacteriophages infecting Bacillus cereus sensu stricto group in the anthrax endemic regions of South Africa; Chapter 8: General discussion, conclusions and recommendations.Thesis (PhD)--University of Pretoria, 2016.German Research Foundation (DFG)National Research Foundation (NRF) of South Africa.Veterinary Tropical DiseasesPhDUnrestricte

Genomic sequence data and single nucleotide polymorphism genotyping of Bacillus anthracis strains isolated from animal anthrax outbreaks in Northern Cape Province, South Africa

This report presents genomic data on sequence reads and draft genomes of Bacillus anthracis isolates from anthrax outbreaks in animals in an endemic region of South Africa as well as genotyping of the strains using canonical single nucleotide polymorphisms (canSNPs). It is derived from an article entitle “Phylogenomic structure of B. anthracis strains in the Northern Cape Province, South Africa revealed novel single nucleotide polymorphisms”. Whole genome sequencing (WGS) of twenty-three B. anthracis strains isolated during 1998 and 2009 anthrax outbreaks in the Northern Cape Province (NCP), as well as a strain from Botswana (6102_6B) and one from Namibia-South Africa transfrontier conservation area (Sendlingsdrift, 6461_SP2) were obtained using both the HiSeq 2500 and MiSeq Illumina platforms. Mismatch amplification mutation assay (melt-MAMA) qPCR were used to identify the canSNP genotypes within the global population of B. anthracis. DNA sequencing data is available at NCBI Sequence Read Archive and GenBank database under accession N0. PRJNA580142 and PRJNA510736 respectively. A phylogenetic tree and CanSNP typing profiles of the isolates are presented within this article.The Institute of Tropical Medicine (ITM), Belgium, National Research Foundation and AgriSETA (Agriculture Sector Education Training Authority) in South Africa.http://www.elsevier.com/locate/dibam2020Veterinary Tropical Disease

Development of a genus-specific brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer from Di erent specimen types

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was e cient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.This work was supported by the Belgian Directorate General for Development Co-operation Framework Agreement (FA4 DGD/ITM 2017-2021) (Grant No. NRF: CSRP170522231749) awarded to the Department of Veterinary TropicalDiseases, aswell asRedMeatResearch andDevelopment, SouthAfrica (GrantNo.VET2018,0023).The Belgian Directorate General for Development Co-operation Framework Agreement (FA4 DGD/ITM 2017-2021) and Red Meat Research and Development, South Africa.http://www.mdpi.com/journal/vetsciam2021Veterinary Tropical Disease

A retrospective study of anthrax on the Ghaap Plateau, Northern Cape province of South Africa, with special reference to the 2007–2008 outbreaks

Anthrax is a zoonotic disease caused by the gram-positive, endospore-forming and soil-borne bacterium Bacillus anthracis. When in spore form, the organism can survive in dormancy in the environment for decades. It is a controlled disease of livestock and wild ungulates in South Africa. In South Africa, the two enzootic regions are the Kruger National Park and the Ghaap Plateau in the Northern Cape province. Farms on the Plateau span thousands of hectares comprising of wildlife – livestock mixed use farming. In 2007–2008, anthrax outbreaks in the province led to government officials intervening to aid farmers with control measures aimed at preventing further losses. Because of the ability of the organism to persist in the environment for prolonged periods, an environmental risk or isolation survey was carried out in 2012 to determine the efficacy of control measures employed during the 2007–2008, anthrax outbreaks. No B. anthracis could be isolated from the old carcass sites, even when bone fragments from the carcasses were still clearly evident. This is an indication that the control measures and protocols were apparently successful in stemming the continuity of spore deposits at previously positive carcass sites.The National Research Foundation (NRF)http://www.ojvr.org/am2018Veterinary Tropical Disease

Blowflies as vectors of Bacillus anthracis in the Kruger National Park

Anthrax, caused by Bacillus anthracis, is endemic in the Kruger National Park (KNP). The epidemiology of B. anthracis is dependent on various factors including vectors. The aims of this study were to examine non-biting blowflies for the presence of B. anthracis externally and internally after feeding on an anthrax-infected carcass and to determine the role of flies in disseminating B. anthracis onto the surrounding vegetation. During an anthrax outbreak in 2014 in the endemic Pafuri region, blowflies associated with two 2–3-day-old anthrax-positive carcasses (kudu and impala) as well as surrounding vegetation were collected and investigated for the presence of B. anthracis spores. The non-biting blowflies (n = 57) caught included Chrysomya albiceps, Ch. marginalis and Lucilia spp. Bacillus anthracis spores were isolated from 65.5% and 25.0% of blowflies collected from the kudu and impala carcasses, respectively. Chrysomya albiceps and Ch. marginalis have the potential to disseminate B. anthracis to vegetation from infected carcasses and may play a role in the epidemiology of anthrax in the KNP. No B. anthracis spores were initially isolated from leaves of the surrounding vegetation using selective media. However, 170 and 500 spores were subsequently isolated from Abutilon angulatum and Acacia sp. leaves, respectively, when using sheep blood agar. Conservation implications: The results obtained in this study have no direct conservation implications and only assist in the understanding of the spread of the disease

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho

INTRODUCTION : Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa. Accurate identification of virulent B. anthracis is essential but challenging due to its close relationship with other members of B. cereus group. This study characterized B. anthracis and Bacillus species that were recovered from animals and the environment where animals died of anthrax symptoms in southern Africa using a polyphasic approach. METHODOLOGY : For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, BiologOmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, polymerase chain reaction (PCR) detection of protective antigen (pag) and capsule (cap) regions, and real-time PCR using hybridization probes targeting chromosomal, pag, and capC genes. RESULTS : The Bacillus isolates were non-hemolytic, non-motile, and susceptible to penicillin, which is typical of B. anthracis, but resistant to gamma phage, unlike typical B. anthracis. The Biolog system and 16S rRNA gene sequence analysis identified most of the Bacillus isolates as B. endophyticus (7 of 10). Conventional PCR revealed that most of the Bacillus isolates contained capBCA gene regions. This highlights the limitation of the specificity of conventional PCR and the fact that the real-time PCR is more specific and reliable for anthrax diagnosis. CONCLUSIONS : Real-time PCR, 16S rRNA sequencing, and confirmatory microbiology tests including phage resistance distinguished Bacillus isolates from B. anthracis in this study. Identification of B. anthracis should be done using a polyphasic approach.The National Research Foundation (NRF) and NRF-THRIP.http://www.jidc.orgam2017Veterinary Tropical Disease

Immunological evidence of variation in exposure and immune response to Bacillus anthracis in herbivores of Kruger and Etosha national parks

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.NSF Division of Environmental Biology.https://www.frontiersin.org/journals/immunologydm2022Veterinary Tropical Disease

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

Environmental factors are common forces driving infectious disease dynamics. We compared interannual and seasonal patterns of anthrax infections in two multihost 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 interannual variation in case numbers, and large outbreaks seemed to follow a roughly 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 implies 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 multihost 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.The National Science Foundation of South Africa.https://onlinelibrary.wiley.com/r/ecmam2023Veterinary Tropical Disease

Molecular characterisation of southern African Bacillus anthracis strains

With important diseases including zoonotic diseases, it is necessary to find accurate and reliable techniques in the diagnosis of the causal agent. Bacillus anthracis the causal agent of anthrax has received a great deal of attention due to its negative association with biological warfare. Microbiological tests have routinely been used to confirm diagnoses of B. anthracis in suspected anthrax cases and to distinguish it from B. cereus and B. thuringiensis that also belong to the B. cereus group, along with B. anthracis. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is the current, rapid, molecular assay of choice in typing B. anthracis strains. It relies on commonly practiced PCR based methods to target regions which differ in tandem repeat unit. Various MLVA panels, of which the first consisted of 8 VNTR markers, followed by the MLVA15 and MLVA25 panels have been used to differentiate anthrax strains and to evaluate the diversity of B. anthracis from different geographical areas. In this study, we investigated the use of 31 VNTR markers (combination of MLVA15 and 25 panels) to type B. anthracis isolates from southern Africa using both the capillary and agarose electrophoresis methods to determine the comparative value of each. The samples included B. anthracis sensu lato isolates from southern Africa (n=112), a clinical B. cereus isolate and 34F2 Sterne vaccine strain. This study indicated that the resolution using agarose gel electrophoresis does not allow the accurate separation of 6 VNTR loci with tandem repeat consisting of 6 bp or less, but that the remaining 25 VNTR loci are sufficient to type B. anthracis strains for the purpose of epidemiological study. Agarose electrophoresis is also the most cost effective and appropriate technique for the average African / developing country laboratory. Despite the fact that the 31 MLVA panel using capillary electrophoresis is not cost effective, it is a rapid and accurate method for B. anthracis typing. A comparison of the discriminative power of the four MLVA systems, using 8, 15, 25, and 31-markers clearly showed the superiority of the 31-marker MLVA. However cluster analysis of 113 B. cereus/ B. antrhacis sensu lato isolates from southern Africa indicated that the MLVA25 and MLVA31 panels are very similar and the latter only differentiated an additional 7 genotypes. As MLVA is known to reveal the genetic relationships within B. anthracis, we used the MLVA31 panel to also investigate whether it will differentiate isolates amongst the B. cereus group. The study revealed that MLVA alone may not be sufficient in resolving isolates from the B. cereus group, but is an effective tool in determining genetic and geographical distance and identifies isolates with anomalies that can be definitively identified with further study. Lastly, as blood smears are the most common method to diagnose anthrax and often the only available sample, 14 stained blood smear slides were evaluated as a source of DNA for the fingerprinting of anthrax using MLVA. This preliminary study indicated that typing of 31VNTR loci was only successful from freshly made stained blood smear slides. With stored, stained blood smears slide the quantity and quality of DNA from the slide was insufficient and had to be amplified using GenomiPhi and only amplified VNTR loci with fragment size smaller than 300 bp. This study identified factors that influenced fingerprinting / typing to be primarily the small amount of target DNA (anthrax spores) from a blood smear slide and the conditions of collection and storage. Results from this thesis, highlighted the use of MLVA for typing of B. anthracis and also identified areas that need further investigation.Dissertation (MSc)--University of Pretoria, 2011.Veterinary Tropical DiseasesMScUnrestricte

A retrospective study of anthrax on the Ghaap Plateau, Northern Cape province of South Africa, with special reference to the 2007–2008 outbreaks

Anthrax is a zoonotic disease caused by the gram-positive, endospore-forming and soil-borne bacterium Bacillus anthracis. When in spore form, the organism can survive in dormancy in the environment for decades. It is a controlled disease of livestock and wild ungulates in South Africa. In South Africa, the two enzootic regions are the Kruger National Park and the Ghaap Plateau in the Northern Cape province. Farms on the Plateau span thousands of hectares comprising of wildlife – livestock mixed use farming. In 2007–2008, anthrax outbreaks in the province led to government officials intervening to aid farmers with control measures aimed at preventing further losses. Because of the ability of the organism to persist in the environment for prolonged periods, an environmental risk or isolation survey was carried out in 2012 to determine the efficacy of control measures employed during the 2007–2008, anthrax outbreaks. No B. anthracis could be isolated from the old carcass sites, even when bone fragments from the carcasses were still clearly evident. This is an indication that the control measures and protocols were apparently successful in stemming the continuity of spore deposits at previously positive carcass sites