Insights gained from sample diagnostics during anthrax outbreaks in the Kruger National Park, South Africa

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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.

Abstract

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. Vaccination of livestock is a common practice in endemic areas; however, the vaccination of wildlife can be costly and logistically difficult. In South Africa, the two endemic regions are the Kruger National Park and the Ghaap Plateau in the Northern Cape Province. Farms on the Plateau span thousands of hectares. The Ghaap region is comprised of wildlife – livestock mixed use farming. In 2008, an anthrax outbreak in the province decimated the stock numbers in the region and government officials stepped in to aid farmers in control measures against further losses. Due to the ability of the organism to persist in the environment for prolonged periods, an environmental risk/isolation survey was carried out in 2012 to determine the efficacy of control measures employed during the 2008 anthrax outbreak. 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 successful in stemming the continuity of spore deposits at previously positive carcass sites.

Introduction

The Northern Cape Province (NCP) in South Africa is situated at 30° S, 22° E. It is the largest province in South Africa spanning 372 889 km2. A dolomitic escarpment elevates the mid-eastern border of the province, extending 275 km to the south west, known as the Ghaap Plateau (Smit, 1978, Partridge et al., 2010). The province is divided into two ecological areas, namely the Savannah biome makes up the north eastern half of the province while the south western half hosts the rare and more arid Nama Karoo biome (Fourie and Roberts, 1973). The province contains a number of state, provincial and privately owned wildlife conservancy areas. The NCP also services the Kgalagadi Transfrontier Park bordering Botswana and the Richtersveld Tranfrontier Park bordering Namibia (http://www.sanparks.co.za/conservation). Due to this predominating savannah in the eastern part of the province, the majority of the remaining land is utilized for extensive farming of sheep, cattle and mixed farming which includes wild game (www.southafrica.info/about/geography/northern–cape.htm).
The NCP has alkali, phosphorus deficient soils which leads to pica in grazers and browsers in the form of osteophagia and geophagia (Theiler, 1912, Boyazoglu, 1973, De Vos and Turnbull, 2004). This animal behavioural characteristic has resulted in a variety of infections with similar pathologies of which ‘lamsiekte’ (botulism, Clostridium botulinum), ‘miltsiekte’ (anthrax, Bacillus anthracis) and ‘stijfsiekte’ (Three Day Sickness, rhabdovirus) are amongst the most common (Theiler, 1912; Viljoen, 1928). Both anthrax and botulism are caused by soil-born, spore-forming and toxin-producing bacteria. Carcasses are typically observed to have an opisthotonic form with oedema or protruding of the tongue for both diseases (Theiler, 1912; Edmonds, 1922; Van der Lugt, 1995). Anthrax and botulism have been observed to occur on the same farm, even simultaneously at times and has been a cause for misdiagnosis in the past by Theiler (1912), (Theiler, 1927, Viljoen et al., 1928, Kriek and Odendaal, 1994).
Anthrax is an acute or peracute zoonotic disease predominantly affecting livestock and wild ungulates with episodic spill over to humans and carnivores. It is characterized by oedema, sudden death syndrome, black eschars and haemorrhaging from the orifices (Turnbull, 2008), which also differentiates it from botulism (Viljoen et al., 1928). The disease is caused by the Gram-positive, aerobic, endospore-forming bacterium Bacillus anthracis. Vegetative B.
anthracis cells have a distinct encapsulated, square ended “box-shaped” appearance on Giemsa stained blood smears (Hugh-Jones and de Vos, 2002, Turnbull, 2008), which is a means of distinguishing them from other Gram-positive rod shaped bacteria microscopically (Theiler, 1912). The use of selective media and morphological selection are typically employed for bacteriologic isolation of B. anthracis followed by testing for sensitivity to Gamma phage and penicillin as well as verification of virulence factors as added confirmation for the bacterium and differentiation from closely related Bacillus cereus sensu lato group organisms (Knisely, 1966, Turnbull, 1999).
Infection of a host is achieved through ingestion, inhalation or cutaneously. Once an infected animal has died, its carcass becomes a potential site of infection for the next host (Dragon et al., 2005). Anthrax is an OIE reportable disease and opening of carcasses is strictly prohibited (Turnbull, 2008). Sporulation is triggered by nutrient shortages and exposure to oxygen (Sterne, 1937b, Turnbull, 2008). Bacterial spore counts are higher where bloody discharge from the orifices and bodily fluids from the carcass soak the ground (Bellan et al., 2013). Sporulation also takes place when a carcass is opened by scavengers such as vultures (Gyps spp. / Trigonoceps occipitalis / Torgos tracheliotos), crows (Corvus spp.), jackal (Canis spp.) or hyena (Crocuta crocuta) (Hugh-Jones and de Vos, 2002, Turnbull, 2008). Blowflies are considered as mechanical vectors of anthrax in KNP because they feed on a carcass and then deposit B. anthracis laden regurgitate on vegetation around the carcass. This contaminated browse may then be a potential source of infection to susceptible kudus (Tragelaphus strepsiceros) and other browsers (Braack and De Vos, 1990, Hugh-Jones and de Vos, 2002, De Vos and Turnbull, 2004, Blackburn et al., 2014). Spores in the environment have been recovered by De Vos (1990) from bones during archaeological excavations at a site in KNP that were estimated to be 200 ± 50 years old by carbon-dating.
To reduce such spore inoculum in the environment incineration of anthrax carcasses and burial have been the preferred method and in accordance with WHO and OIE guidelines (Turnbull, 2008). Treating anthrax carcasses with 10% formalin would kill the bacteria, deter scavengers that would open the carcass and decrease spread by flies, but remains controversial due to the negative impact of formalin on the environment. Turnbull (2008) also proposed covering or wrapping carcasses in plastic or tarpaulins to keep the skin intact and reduce vegetative Bacilli through putrefaction and thus anthrax spores in the environment.
The two anthrax endemic areas in South Africa are the NCP and the Kruger National Park (KNP) (Smith et al., 2000, Bengis et al., 2002, Hugh-Jones and de Vos, 2002, De Vos and Turnbull, 2004). While mandatory vaccination schemes have virtually abolished anthrax in livestock in most of the country, these areas remain enzootic due to the predominance of wildlife conservancies and game farms (Gilfoyle, 2006; Turnbull, 2008). A large number of animals usually succumb to the disease before it is noticed due to the short incubation period and the large areas involved. Kudus are typical fatalities of anthrax, but in 2008 large numbers of antelope and equids were also affected in the NCP outbreak. The outbreak gained momentum, and by the end of March 2008, had resulted in the deaths of thousands of game and massive economic losses to the farmers of the Ghaap Plateau tallying to millions of South African rand (Visagie, 2008). It was the largest recorded outbreak in the region in recent history and the Department of Agriculture Fisheries and Forestry in South Africa mobilized national and provincial state veterinary services to aid in diagnosis, surveillance and control measures in order to stem the outbreak (Visagie, 2008, Nduli, 2009). The aims of this study were (i) to report the 2008 B. anthracis outbreak in the NCP, (ii) to determine the B. anthracis spore concentrations from bone and environmental samples during the 2008 NCP anthrax outbreak using bacteriological methods and (iii) to compare the spore concentrations from bone and soil collected at the same sites in 2012 to determine the spore endurance and/or efficacy of the control measures employed during the 2008 outbreak in reducing the inoculum in the environment.

Materials and Methods

Observations, sample collection and control measures during 2008 anthrax outbreak

The first cases of the 2008 anthrax outbreak in NCP were reported in November 2007 at Doringbult farm (Figure 2.1) after heavy spring rainfall during mid-August to November 2007. Later that month, the skeletal remains of 1 female Kudu (NC/17) on Grootsalmonsfontein and 2 female kudus (NC/27 and NC/28) on the adjacent farm Dikbosch were recovered, all of which were gestating (personal communication EH Dekker; Nduli, 2009). The first isolated cases of anthrax from kudu were identified in the latter farm Dikbosch (Figure 2.1). In February 2008, 5 more kudu carcasses (both males and females) were discovered on Dikbosch and Kleinsalmonsfontein farms and by the following month anthrax cases included zebra, wildebeest, impala, sheep and kudu along the length of the Ghaap escarpment (Figure 2.1).
After the kudu at Doringbult was confirmed suspicious for anthrax from a Giemsa stained blood smear, more comprehensive samples were collected for diagnostics. KNP (Skukuza Veterinary Services) and Northern Cape Province Veterinary Services visited farms to aid farmers in the diagnosis and control of the outbreak. The condition of the carcasses were documented along with collection of blood smears and bone samples (mandibular, orbital, rib, vertebra, femur and/or pelvic bones if possible). Soil samples were collected from under the head, abdomen and tail of the carcass for diagnostic purposes. Environmental samples such as crow faeces and bone fragments were collected from carcass sites where evidence of scavenger activity was visible (Table 2.1). Louse flies (Hippobosca rufipes) were observed and collected from recently dead kudu carcasses at Clearwater and Klipfontein (Table 2.1, Figure 2.2A). Random soil samples were also collected from farms at the top of the plateau and pans at the base of the escarpment to determine spore counts in areas not contaminated by fresh carcasses (Table 2.1).
Table 2.1: Anthrax positive carcasses (based on Giemsa stained blood smears) and environmental samples collected from farms along the Ghaap Plateau in the Northen Cape Province, South Africa during outbreaks in 2008 indicating control measures implemented by farm owners and State Veterinary Services, as well as, the subsequent soil and bone sampling of these sites in 2012. Supplementary Data\Table 2.1.xlsx
Control measures included vaccination of livestock; treatment of the carcass sites which involved spraying either 10% chlorine or 10% formalin on the carcasses; burning of the carcasses and covering up each animal with black plastic/tarpaulin sails to increase bacterial vegetative cell death and limit blowfly and scavenger access to the carcass. Table 2.1 indicates the farms affected by the outbreak based on Giemsa stained blood smears, as well as, carcass condition and observable blowfly, louse fly and crow activity around carcasses.

List of Abbreviations 
List of Figures 
List of Tables 
Chapter 1: Intoduction and Literature Review 
1.1. Introduction
1.2. Literature Review
1.2.1. Taxonomy
1.2.2. Ecology
1.2.3. Routes of Infection
1.2.4. Mode of Action
1.2.5. Symptoms and Pathology
1.2.6. Diagnostics
1.2.7. Vaccine and Control
1.2.8. Distribution and Spread
1.2.9. Southern African Outbreaks
1.2.10. Molecular Characterization
1.2.11. Bacteriophages
1.3 References
Chapter 2: A retrospective study of anthrax on the Ghaap plateau, Northern Cape Province of South Africa, with special reference to the 2008 outbreak. 
2.1. Introduction
2.2. Materials and Methods
2.3. Results
2.4. Discussion
2.5. Conclusion
2.6. References
Chapter 3: Insights gained from sample diagnostics during anthrax outbreaks in the Kruger National Park, South Africa.
3.1. Introduction
3.2. Materials and Methods
3.3. Results
3.4. Discussion
3.5. Conclusions
3.6. References
Chapter 4: Through the lens: a microscopic and molecular evaluation of archival blood smears from the 2010 anthrax outbreak in Kruger National Park, South Africa
4.1. Introduction
4.2. Materials and Methods
4.3. Results
4.4. Discussion
4.5. Conclusion
4.6. References
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.
5.1. Introduction
5.2. Materials and Methods
5.3. Results
5.4. Discussion
5.5. Conclusions
5.6. References
Chapter 6: Through the lens: a microscopic and molecular evaluation of archival blood smears from the 2010 anthrax outbreak in Kruger National Park, South Africa
6.1. Introduction
6.2. Materials and methods
6.3. Results
6.4. Discussion
6.5. Conclusion
6.6. References
Chapter 7: Characterisation of temperate bacteriophages infecting Bacillus cereus sensu stricto group in the anthrax endemic regions of South Africa.
7.1. Introduction
7.2. Materials and Methods
7.3. Results
7.4. Discussion
7.5. Conclusions
7.6. References
Chapter 8: General Discussion, Recommendations and Conclusion
8.1. Anthrax on the Ghaap Plateau
8.2. Anthrax in the Kruger National Park
8.3. Bacillus anthracis in the environment
8.4. Conclusion
8.5. References
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