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Animals
Eight-week old female BALB/c mice (n = 6) (South African Vaccine Producers, Sandringham, South Africa) were used to confirm the virulence of the B. anthracis challenge strain. Twenty-six healthy one-year naïve old Boer goats (females and emasculated males) were housed at Onderstepoort Biological Products (OBP), South Africa after screening for background anti-rPA83 cross-reactive antibodies by ELISA. Lethal challenge studies were conducted at a remote site in an endemic area of the Kruger National Park (KNP), South Africa. Animal experiments were performed according to the guidelines of the National Research Council of the USA (Clark et al. 1996) and approved by the animal use and care committees of the South African National Parks, OBP and University of Pretoria (Protocol numbers V041-10 and V065/12) respectively. Approval for Section 20 of the animal disease act 35 of 1984 was granted by the Directorate of Animal Health, Department of Agriculture, Forestry and Fisheries, South Africa (registration number 12/11/1/1/6).
Animals were vaccinated using the SLSV by subcutaneous injection in the inner thigh, as stipulated for goats (OBP). Challenge was performed with a virulent South African B. anthracis strain (Lekota et al. 2015) prepared as indicated in the methods section. Virulence of the spores was confirmed in BALB/c mice and naïve goats. Two groups of three mice received an intra-peritoneal challenge of ~500 and ~1000 spores respectively and monitored till death. Cause of death was confirmed by re-isolation of B. anthracis colonies from liver and spleen cultures made on sheep blood agar.
Two goats from each of the three negative control groups (NCG1-3) were challenged (subcutaneously in the thigh) with 36, 172 and 844 spores respectively (Table 4.1). Spore numbers in the respective challenge doses were estimated by counting colony forming units (cfu) prepared from redundant doses. The highest dose of 844 spores was subsequently used for the challenge of the SLSV vaccinated goats (SVG1 to 3) (Table 4.1). Death from anthrax was confirmed after microscopic demonstration of Gram-positive encapsulated rod-shaped bacilli in stained blood smears. All virulent challenge (mice and goats) were performed in the anthrax-endemic region of the Kruger National Park (KNP). Decontamination of challenge environment was carried out with 4 % formalin after incineration of animals and bedding materials. Following decontamination, soil amples were collected from the incineration and challenge sites to verify if free of B. anthracis spores contamination.
Experimental design
The immunogenicity and protectiveness of the SLSV were evaluated in four scenarios using five goats per group (SVG1 to 4) (Table 3.1). Two groups were vaccinated once and challenged after 6 (SVG1) and 62 (SVG2) weeks respectively. SVG3 was vaccinated twice at weeks 0 and 58 before lethal challenge 4 weeks later. The rationale was to evaluate the protection elicited by this vaccine one month (SVG1), one year (SVG2) after single vaccinations and one month after a double (year apart) vaccination (SVG3). A fourth group of goats (SVG4) was vaccinated at weeks 0 and 12 to evaluate the titre development in a shortened two vaccination schedule. However, ethical approval for direct lethal challenge was not obtained for this group due to unavailability of the challenge facility. Blood for serum harvest was collected as indicated in Table 3.1 and stored at -20 ˚C.
Following lethal challenge, the goats were monitored for a period of 14 days for signs of pyrexia (temperatures of ≥ 40 °C) and abnormal behavior. Goats were euthanized by sodium pentobarbitone overdose (Eutha-naze®, Bayer, Isando, South Africa, 400 mg/kg body mass) as soon as pyrexia and bacilli rods were detected in blood smears. Survivors were treated with procaine benzylpenicillin (Depocillin®, Intervet, Spartan, South Africa, 20 mg/kg body mass) and euthanized after confirmation of absence of bacilli rods in blood smears. This was a condition stipulated by the animal health authorities, as a precautionary measure against environmental dispersion of the challenge strain (even though animals protected by vaccination should have no detectable B. anthracis at this stage).
Serology
Sera were analysed with indirect ELISAs for specific antibodies against recombinant protective antigen (rPA83); recombinant bacillus collagen-like protein of anthracis (rBclA) (Steichen et al. 2003); formaldehyde inactivated spores (FIS) (Brossier, Levy & Mock 2002) and a vegetative antigen formulation derived from a capsule and toxin deficient strain (CDC 1014) as described in the methods section. Neutralizing antibody titres were assessed using an in vitro toxin neutralizing assay (TNA) as described in methods section.
CHAPTER ONE Introduction
CHAPTER TWO Literature Review
2.1 Introduction – History of anthrax
2.2 Routes of infection
2.3 Clinical signs
2.4 Host immune response to B. anthracis
2.5 Anthrax immunology: Evolving diagnostic tools
2.6 Anthrax veterinary vaccines
2.7 Experimental anthrax vaccines
2.8 References
CHAPTER THREE General Methods
3.1 Bacillus anthracis vaccine and challenge strain preparation
3.2. Preparation and purification of recombinant proteins
3.3. ELISA
3.4. Toxin neutralization assay
3.5. Lymphocyte proliferation assay
3.6 References
CHAPTER FOUR Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model
4.1. Introduction
4.2. Materials and methods
4.3. Results
4.4 Discussion
4.5. References
CHAPTER FIVE Immunogenicity of anthrax recombinant peptides and killed spores in goats and protective efficacy f immune sera in A/J mouse model
5.1. Introduction
5.2. Experimental Procedures
5.3. Results
5.4. Discussion
5.5. References
CHAPTER SIX Immunogenicity of the Sterne live spore vaccine versus non-living anthrax vaccine candidates in combination with simultaneous penicillin G treatment in goats
6.1. Introduction
6.2. Materials and Methods
6.3. Results
6.4. Discussion
6.5. References
CHAPTER SEVEN Immunogenicity of a DNA prime/recombinant protein boost and DNA prime/formaldehyde inactivated spores boost formulations in goats and protective efficacy of immune goat sera in an A/J mouse model
7.1. Introduction
7.2. Materials and Methods
7.3. Results
7.4. Discussion
7.5. References
CHAPTER EIGHT General Conclusions
