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M.Comm. / The animal health industry in South Africa is static and products are of a generic nature. Most of the international players are represented in South Africa. The fact that there are so many players in the market leads to severe competition. In this study the possible application of relationship marketing in the animal health industry was investigated. Sanvet, one of the leading companies in the industry was used for the study. The company was established in September 1994 through an amalgamation of the animal health divisions of Agrihold and Premier Pharmaceuticals. In relationship marketing the focus moves from a short-term transactional approach to long-term relationship marketing. Relationship marketing is the integration of marketing, quality and customer service. The provision of quality customer service involves an understanding of what the customer buys and determines how additional value can be added to the product or service being offered. Quality is the link between what a customer expects and the customers perception of what is being offered. The four P's model is very limited and in relationship marketing three additional elements are being used. The elements are people, processes and the provision of customer service. Customer service creates a clearly differentiated and superior value proposition, and is the central focus of all the other elements. Through the acknowledgement of the contribution of people to getting and keeping customers, within the overall marketing mix, the company's competitive performance will be substantially enhanced. People can be categorised into the following groups based on their role in the company: contractors, modifiers, influencers and isolators. Although the human element is very important in customer service, no amount of energy from personnel can counter poor performance due to unsatisfactory processes. In relationship marketing not only the relationship with the target markets is being addressed but also the relationship with the intemal markets, referral markets, supplier markets, employee markets and influence markets. The employees of the company are the core of an internal marketing plan. Employees are seen as internal customers and jobs as internal products. The ideal form of marketing is to get customers to do the marketing on your behalf. Referral markets within the industry must be identified and marketing activities directed at them. The traditional adversarial relationship between suppliers and their customer must change to a new form of relationship based on co-operation. The competition between companies in their effort to attract suitably motivated and trained employees is increasing. Suitable employees are becoming very scarce resources. Companies must focus their marketing activities to ensure that they and the company are first choice for a potential employee. Financial markets, regulatory markets, the government and shareholders form the influence markets and marketing activities should be addressed to these markets to ensure a long-term relationship. This study was limited to investigating the relationship with the internal markets, referral markets and supplier markets. It was found that relationship marketing can be applied in the animal health industry. The recommendations in this study can be used to draw up a complete relationship marketing strategy for Sanvet.

This study reports the development of chromosome-specific libraries by chromosome microdissection and microcloning and its utility in developing high density linkage map for particular chromosomes. Chromosome-specific painting probes were prepared for bovine (Bos taurus) autosomes 11 and 23 using two different translocation cell lines. Chromosome painting probe for swine chromosome 6 was developed using chromosomes from primary swine fibroblast cultures. The purity and specificity of the painting probes was verified by fluorescent in situ hybridization (FISH) on bovine and swine metaphase chromosomes. Bovine painting probes were used on sheep (Ovis aries) and goat (Capra hircus) metaphases to identify their corresponding homologs. BTA 11 and SSA 6-specific DNA fragments were cloned in Lambda Zap Express vector to develop high titer chromosome-specific libraries. BTA 11 library was screened for microsatellite-containing clones using (AC)$\sb{12}$ oligos. Primer pairs developed for 17 microsatellites yielded successful amplifications with bovine genomic DNA. Three markers were binned on Illinois Reference/Resource family (IRRF) and 14 were mapped on the USDA-MARC resource family. Two point analysis was done on MARC population to generate a preliminary linkage map for BTA 11. A bovine YAC library was screened with BTA 11-specific microsatellite primers. Four YACs were identified and physically mapped by FISH. Two YAC clones that were mapped to BTA 11 by linkage and by FISH helped to orient and anchor the linkage map on bovine chromosome 11. BTA 11-specific DNA was subjected to subtractive hybridization using bovine Cot4 DNA and the subtracted product was used to screen a bovine cosmid library. Positive clones were pooled and mapped to bovine metaphases by FISH. All the clones showed very strong hybridization on the centromeres of all autosomes in the bovine chromosome complement. However, they failed to hybridize to the sex chromosome centromeres suggesting that the sequences are specific to autosomal centromeres. The same probes failed to hybridize to sheep and goat metaphases suggesting species specificity of these probes. An answer to the exact function of these DNA sequences need to be investigated.

An efficient system for genetic modification and large scale cloning of cattle is of importance for agriculture, biotechnology and human medicine. Two approaches were investigated here to produce a transgenic bovine. First, the use of embryonic stem (ES) cells in production of chimeras and second, the use of nuclear transfer procedures using genetically modified somatic cells as nuclear donors. Embryonic stem (ES) cells could be useful for generating transgenic cattle or cells for xenotransplantation. The in vitro production of tissue for xenotransplantation requires the development of an unlimited source of pluripotent cells. Furthermore, these cells should be capable of being genetically modified to aid in directing differentiation, reducing graft rejection, improving graft survival and for producing therapeutic proteins in vivo. A novel method was developed, using nuclear transplantation, to produce transgenic ES cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, were able to differentiate into derivatives from the three embryonic germ layers; ectoderm, mesoderm and endoderm in adult animals. Six out of seven calves born were found to be chimeric for at least one tissue (86%). This work demonstrates that somatic cells can be genetically modified and then dedifferentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy. In order to produce a whole transgenic bovine in one generation, fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected and the cells were fused to enucleated mature oocytes. Out of 130 embryos transferred to 53 recipient cows, seven healthy, identical, transgenic calves were generated. Furthermore, the lifespan of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40 day fetal clone. With the ability to extend the lifespan of these primary cultured cells, this system should be very useful for inducing complex genetic modifications in cattle.

Host control of Trypanosoma brucei infections relies on adequate B cell mediated responses including anti-VSG antibody responses. Trypanotolerant animals, namely; Cape buffalo maintain anti-trypanosome specific antibody responses throughout infection. Studies in mice, however; show a failure to maintain adequate antibody responses to trypanosomes as well as a failure to generate subsequent specific responses to antigens. T. brucei infections in mice result in the loss of mature conventional B cell subsets presumed to be important in host control of the parasites including marginal zone B cells and follicular B cells. Mature cell subset losses are coupled with plasma cell expansion early during infection. Mature B cell pools are not replenished as there is a loss of transitional cells due in part to higher levels of apoptosis and a failure to replenish these cells from bone marrow. B1 B cells appear to constitute the majority of plasma cells and resist infection induced losses to a greater extant than B2 B cells.

The interaction between Ixodid ticks and their mammalian hosts is a complex relationship. While the mammalian host tries to avoid the completion of the feeding process, the tick has devised strategies to counteract these attempts. Tick saliva contains a vast array of pharmacological activities that presumably aid the tick to evade host responses, including anti-complement, oxidative and innate and adaptive immune responses. The characterization of these activities has gained momentum in the last several years. One of the best-studied activities present in tick saliva corresponds to the antigen known as Salp15, which inhibits TCR-mediated CD4+ T cell activation and IL-2 production. This study identifies CD4 as the specific receptor for Salp15 on T cells. A direct association occurs between CD4 and the C-terminal amino acids of Salp15, which allows Salp15 to act at the very beginning of TCR ligation-induced signaling cascades. Salp15 prevents the activation of Lck upon TCR engagement and the formation of lipid rafts and actin polymerization. Salp15 also affects tyrosine phosphorylation of several early signal components downstream of Lck, including Zap70, Lat, and PLCγ1. This study also demonstrates that the peptide that mediates the interaction of Salp15 with CD4, P11, is able to recapitulate the immunosuppressive activity of the whole saliva protein. These results clarify the molecular mechanisms of action of Salp15 on T cells and suggest that binding to CD4 is sufficient to elicit its immunosuppressive activity. The differentiation of Th17 cells requires the concerted action of IL-6 and TGFβ. However, the exact contribution of each cytokine has not been elucidated. This study also provides evidence indicating that the role of TGFβ during the differentiation of CD4+ T cells is exclusively the repression of IL-2 production, which has been shown to counteract the generation of these effector cells. The inhibition of IL-2 during the differentiation of CD4+ T cells mediated by Salp15 and the specific IP 3 receptor inhibitor 2-APB resulted in Th17 differentiation in vitro. Furthermore, the treatment of PLP139-151-immunized mice with Salp15 also resulted in increased differentiation of Th17 cells in the central nervous system and augmented pathology. Our results demonstrate that the role played by TFGβ is circumscribed to the inhibition of IL-2 during the differentiation of CD4+ T cells. Finally, this study shows that Salp15 inhibits the interaction between HIV-1 gp120 and CD4. Furthermore, Salp15 prevents the formation of syncitia of HL2/3 (a stable HeLa cell line expressing the envelop protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interaction with the glycoprotein. A phage display library screen provided the interacting residues in the C1 domain of gp120. These results provide a potential basis to define exposed gp120 epitopes for the generation of neutralizing vaccines against HIV.

Human, antigen-specific polyclonal antibodies are in high demand for therapeutic and research applications. However, the supply of these antibodies currently comes from only human donors and cannot satisfy the demand. The application of pathogen-specific human polyclonal antibodies is limited because there are severe restrictions on the kinds of antigens and the immunization protocols that can be used in humans. In the attempt to resolve the issue of the supply of antigen-specific, human polyclonal antibodies, transgenic for human Ig (hIg) animals were created. The most sufficient and promising model was the human artificial chromosome (HAC) transgenic animal. This model was successful due to the most efficient transferring vector CS20, comprising the entire unrearranged human heavy (1.5MB) and lambda locus (1 MB). In order to produce a large quantity of polyclonal hIg, cloned HAC transgenic cattle were created. Highly human-specific and bovine non-cross reactive, polyclonal bovine anti-human antibody and sensitive solid phase ELISA were created to determine, quantify, and characterize hIg in the sera of HAC transgenic cattle. Using the assay, that have been developed in this study; it was found that the majority of the HAC cattle produce hIg. It was also found that both heavy and light chains of hIg are produced by HAC bovines. The heavy chain of hIg undergoes class switching to the IgG and its half-life is 30 days, which is longer than hIgG in humans (21 days) or bovine IgG in bovines (19 days). Highly human-specific and bovine non-cross reactive monoclonal antibodies for the characterization of hIg produced by HAC cattle were created to recognize hIg heavy chain classes/subclasses and light chains. Analysis of the human V lambda genes sequences derived from HAC transgenic cattle demonstrated that human genes undergo extensive rearrangement and somatic hypermutation following normal Ig patterns. This study has demonstrated that hIg produced by HAC cattle diversifies according to normal Ig patterns and undergoes class switching to IgG. The half-life of hIgG is sufficiently long for protection from pathogens of the homozygous Ig deficient HAC cattle, and for harvesting of human IgG. HAC transgenic cattle can be potential donors of human polyclonal Ig.

African trypanosomes are parasitic protozoa that cause fatal disease in humans and domestic livestock. Though domestic animals are susceptible to trypanosomiasis, certain wild animals have developed resistance mechanisms. The African cape buffalo can control the early stages of trypanosome infection by increasing plasm and erythrocyte levels of the reactive oxygen intermediate (ROI), hydrogen peroxide (H2O2). Cape buffalo are able to increase the amount of H2O2 produced by the purine catabolic enzyme xanthine oxidase by supplying more substrate in the blood microenvironment and by decreasing the plasma, and erythrocyte levels of catalase, a H2O2 degrading enzyme. My work has been to attempt to manipulate the ROI generating capabilities of mice to recreate this defense mechanism in a small laboratory animal model. Initial experiments revealed that Balb/c, C57Bl/6 and C3H/HeOu mice have all the purine catabolic enzymes necessary to generate trypanocidal amounts of H2O2. However, unlike the buffalo, all of these mouse strains are susceptible to infection by trypanosomes and their serum is not trypanocidal in vitro. Therefore, the ROI generating capabilities of Balb/c, gamma-interferon knockout Balb/c mice and OH catalase-reduced mutant mice were altered by feeding additives, either in a pellet or liquid diet base, to the mice to either inhibit or enhance ROI generation. Only one combination resulted in a slight but significant decrease in parasitemia: C3H catalase-reduced mice fed the catalase inhibitor 3-amino-triazole. Though parasitemia was not effected in any of the other mouse/diet combinations, the mice were effected in some experiments. Gamma-interferon knockout male mice died significantly earlier than female mice, with or without ROI alterations. Mice maintained on a liquid diet had a significantly reduced acceleration of trypanosome-induced inflammation, but this effect was abrogated when the glutathione precursor N-acetyl-cysteine was included in the diet. This indicates that the diets do have an effect on ROI generation in mice, though parasitemia remained largely unaffected. Thus trypanosomes are able to avoid or neutralize ROI in mice. However, they are susceptible to similar ROI levels in buffalo, suggesting a component in the mice that the trypanosomes are able to utilize to inhibit ROI-induced damage.