Dissertation submitted in partial compliance with the requirements for a Master’s Degree in Technology: Chiropractic, Durban University of Technology, 2012. / Background: Animal chiropractic, an internationally sanctioned profession, assists veterinarians with a complementary approach to animal healthcare therapy. Animal chiropractic in South Africa appears to be within its novel stages of development and no clear parameters define its present position. As a result of ambiguity and present concern dictated by veterinary regulation, animal chiropractic has not developed along well defined parameters, and it is thought that this study will contribute to achieving some clarity in this regard. The impetus for this study originated as a result of an increased awareness within the complementary and alternative medicine (CAM) field of these CAM therapies in the management of animal healthcare, and a growing interest in their application. Objective: The purpose of the study is to identify the current status of animal chiropractic in South Africa and to explore ways in which the integration of animal chiropractic into the animal healthcare setting of South Africa might be achieved. Method: This study is an interpretive investigation set in a post-positivistic paradigm and used a grounded theory approach. Data was collected from twelve semi-structured interviews (digitally voice recorded) with relevant stakeholders who were knowledgeable within their respective fields (animal chiropractic; veterinary health science; their respective governing bodies; and owners of animals which had received treatment from animal chiropractic). Questions addressed participants’ perceptions and experiences of animal chiropractic with regard to the role it plays, current interprofessional interactions and developmental issues facing the profession. Qualitative analysis of the data was done using NVIVO 9 software (NVivo 9, developed in Australia, copyright 2011 QSR International Pty Ltd.). The purpose of the data collection was to obtain knowledge presently available within the proposed field in order to build a credible theory which might explain the current status of animal chiropractic in South Africa and the way forward to professional integration with mainstream animal healthcare practice. iv Results: Applying a process of grounded theory methodology revealed that certain key prerequisites were needed for integration of animal chiropractic with mainstream animal healthcare to take place. Firstly, animal chiropractic practitioners had to be seen to have reached a certain level of competence, which could be achieved through a recognised educational programme and by following practising standards at the same (or similar) level as veterinary practitioners. Secondly, acceptance by the public and mainstream practitioners is vital, and requires that the need for animal chiropractic is well motivated, that the role of animal chiropractic is better defined, and that collaboration with mainstream professionals is implemented. Thirdly, animal chiropractic needs careful regulation in order for it to be controlled appropriately. This will require a suitable professional body to govern it, legislation to endorse it and guidelines to direct its actions. Lastly, resources must be available, such as enough animal chiropractors interested in entering the field, sufficient qualified instructors (i.e. experienced animal chiropractors) to provide tuition, adequate amenities, and sufficient time available for the profession to develop and the integration process to take place. Conclusion: It would appear that, with CAVM therapies becoming popular, more people are becoming interested in animal chiropractic. The study suggests that, if the animal chiropractic profession makes provision for achieving the prerequisites of competence, acceptance, regulation and resources in terms of its future development, it might be possible to achieve professional integration with mainstream animal healthcare within the next ten years.

The molecular evolution of feline calicivirus (FCV) was studied in cell culture and in persistently infected cats. Sequence analysis of the 5' hypervariable region of the FCV capsid (5'HVR; located at the 5'end of variable region E), a region known to contain linear neutralising B cell epitopes, showed FCV existed as a quasispecies which evolved at the nucleotide and amino acid level during persistent infection. Quasispecies heterogeneity tended to decrease during the course of persistence. Sequential isolates from a cat showed marked antigenic variation during the course of persistent infection. Sequential passage of FCV in cell culture was also associated with sequence evolution of the 5'HVR. However, these isolates showed no change in antigenicity suggesting that individual substitutions observed in viruses from cats, but not in viruses from cell culture, may be responsible for changes in antigenicity. Alternatively, the observed antigenic changes may be associated with mutations elsewhere in the genome. In order to identify regions of 'the FCV capsid protein containing linear B-cell epitopes, two approachesw ere used.F irstly, an expressionl ibrary containingr andom, short (100- 300bp) fragments of an FCV capsid gene was constructed. This library was screened using polyclonal antisera from a cat that had been challenged experimentally with FCV to identify immunoreactive clones containing B-cell epitopes. Initial screening identified five clones that reacted positively to feline antisera in immunoblots. FCV derived sequencefr om thesec lones all mappedt o the 5'HVR suggestingt his region containst he immunodominant linear epitopes of the capsid. The second approach used to identify B-cell epitopes was to map more accurately the epitope of a neutralising monoclonal antibody (IG9) which had already been shown to lie in a 37 amino acid region of the 5'HVR (Milton et al. (1992), Journal of General Virology 73,2435-2439). Replication of plaque purified IG9-sensitive parent virus in sub-neutralising concentrations of IG9 led to the generation of a neutralisation resistant escape mutant. Sequence analysis of this mutant and the parent virus revealed a single non-synonymous nucleotide substitution within the 5'HVR suggesting this residue is critical to the correct formation of the IG9 epitope. A method to type FCVs based on sequence analysis of the 5'HVR was established. Most isolates appeared relatively homogenous. However, some isolates, both from vaccines and vaccine failures, appeared to contain more than one FCV. Comparison of 5'HVR sequencesfr om different isolatess howed that most isolates were either 0-5.3% different (related isolates) or 20.7-42.7% different (unrelated isolates). The majority of the relatedi solatess hareda n epidemiologicall ink, implying they representedis olatest hat originated from a common source. Comparisons of sequences obtained from vaccine failures and vaccine virus fell into two similar categories; those with closely related sequences(0 .0-5.3%) implying a role for the vaccinei n diseasea nd those with divergent sequences(2 1.3-38.7%)i mplying field virus causedt he disease. These results were compared with those obtained by using a serotyping method based on virus neutralisation (VN) which exploits differences in antigenicity between most FCVs gene (Dawson et al. (1993), Veterinary Record 132,346-350). VN and sequence analysis gave the same typing result in 65-73% of individual cases. Based on these results and the difficulty of interpreting VNs, we suggest that molecular based sequence analysis may be more suitable to the epidemiological investigation of FCV related disease particularly in the case of vaccine reactions.

Semen cryopreservation has allowed the establishment of genome banks and the large scale propagation of species. The development of simple techniques to cryopreserve semen or alternatives to efficiently use cryopreserved semen from males of valuable genetics that have become infertile will permit continuous propagation of the genetics from these males and may serve as a model for preservation and propagation of endangered species. Sperm cryopreservation without cryoprotectants is a simple process, and offspring have been produced following intracytoplasmic sperm injection (ICSI); however the ability of frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI was unknown. In the series of experiments performed, bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants was used to activate intra- and interspecies oocyte following ICSI. Additionally, equine cumulus-oocyte complexes (COCs) glucose metabolism during in vitro maturation was evaluated. The first experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had their plasma membrane damaged; however the DNA was unaffected. The second experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had the ability to activate bovine oocytes following intracytoplasmic sperm injection; although at a lower rate compared to frozen-thawed sperm. The third experiment demonstrated that frozen-thawed stallion sperm refrozen without the addition of cryoprotectants was unable to activate equine oocytes. The exact reason for this failure could not be explained from the experiment; however COC metabolism during in vitro maturation impacts embryo activation/development and required further investigation. The fourth experiment demonstrated that equine COCs consume and metabolize glucose through glycolysis during in vitro maturation; however, results from this experiment were unable to explain the failure of refrozen stallion sperm to activate equine oocytes. To our knowledge, this is the first report of the use of bull or stallion frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI. Furthermore, this is also the first report of equine COCs glucose metabolism during in vitro maturation.

Optimal conditions for preparation and storage of haemolysin were determined using spirochaetes harvested from rabbit serum broth. Haemolysin was purified by fractionation on Whatman DEAE Cellulose and Sephadex G100, purity being assessed by SDS-PAGE. The molecular weight of the haemolysin was estimated by gel filtration to be 19,000 daltons although analysis on SDS-PAGE suggested that the molecular weight of haemolysin dissociated from the RNA-core carrier was much lower. The purified haemolysin was not antigenic. The unpurified haemolysin caused lysis of several species of erythrocyte and was cytotoxic towards a range of cell monolayers, embryo bovine lung fibroblasts being the most sensitive. The cytotoxic activity of the haemolysin was quantified using a 51Chromium release assay. Of the porcine inflammatory cells tested, lymphocytes were the most sensitive. After purification the preparation was haemolytic and leucotoxic but was less cytotoxic. Lower concentrations of haemolysin were produced by an avirulent strain of T. hyody4enteniae than from the virulent strain and it was not toxic for embryo bovine lung fibroblasts. No toxic effect of haemolysin from virulent strains of T. hyody4entetiae was demonstrated either by inoculation of high concentrations into ligated colonic loops in pigs or by intragastric inoculation of CFI mice. The treponemal haemolysin is similar to Streptolysin S in the requirement for a carrier molecule to demonstrate invino haemol. ysis, and with respect to molecular weight, lack of antigenicity, cytotoxic activity and the effect on lymphocytes. When T. hyodyzente4iaew as incubated with excised colonic tissue slices and inoculated into ligated colonic loops in pigs, spirochaetes associated with the mucus rather than attaching to the colonic epithelium. Within 2 hours, spirochaetes were observed in the bottom of crypts suggested that a chemotactic mechanism may be more important than attachment in enabling T. hyodyzenten£ae to establish in the colon. iii

The objectives of this study were to investigate concentration dependent effects of canine prostatic fluid (PF) on in vitro seminal parameters of cooled canine semen. Sperm motility parameters, plasma membrane integrity and stability, acrosome integrity and DNA fragmentation were measured after the addition of 0%, 10%, 25%, or 50% PF to extended semen of fertile dogs. Assessments were made at 0 h pre-cooling, at 24, and 48 h of cooled storage (4 °C), and after freezing and thawing followed by incubation (37 °C) at 0, 4 and 24 h. Our hypothesis was that lower dilutions of canine semen with PF in an egg yolk-Tris extender would improve plasma membrane stability and acrosome integrity, and preserve sperm kinetics and reduce DNA fragmentation in comparison with higher concentrations of PF in fertile dogs during cooling. Sperm motility parameters were assessed by computer assisted sperm analysis, and plasma membrane integrity by the hypo-osmotic swelling test. Flow cytometry was used after staining with YO-PRO-1/Ethidium Homodimer 1 (EthD-1) to evaluate membrane stability, fluorescent isothiocynate-PNA (Arachis Hypogaea)/propidium iodide to assess acrosome integrity, and sperm chromatin structure assay to assess DNA fragmentation. The data was analyzed using a mixed linear model (ANOVA) and in case of significant effects of time, treatment, or treatment*time interaction (P < 0.05), least square means were used for pairwise comparisons. Acrosome integrity and DNA fragmentation were not affected by treatment with PF. During the cooling period motility parameters were not influenced by PF treatment. A lower proportion of early apoptotic and higher proportion of early necrotic cells was seen during cooling with 50% PF (YO-PRO-1/EthD-1). Although lower concentrations of PF did not improve the evaluated spermatozoal parameters, they did not seem to compromise sperm motility and plasma membrane stability. The presence of 50% PF prior to cryopreservation decreased post thaw motility and produced a shift towards early necrotic cells after thawing. Therefore admixture with more than 10% PF should be avoided prior to cryopreservation of canine semen.

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