Theileria parva is a tick-borne intracellular protozoan parasite which infects cattle and African buffalo in Eastern and Southern Africa. Cattle may be immunised against T. parva by the infection and treatment method (ITM), which involves inoculation with live sporozoites and simultaneous treatment with oxytetracycline. One such ITM vaccine is the Muguga Cocktail, which is composed of a mixture of three parasite stocks: Muguga, Serengeti-transformed and Kiambu 5. Although the vaccine has been used with success in the field in several areas in Eastern Africa, there is evidence that vaccination using cattle-derived parasites does not always provide adequate protection against buffalo-derived T. parva. A number of T. parva antigens recognised by CD8+ T cells from cattle immunised by ITM have been identified in previous studies. A proportion of these antigens show a high degree of sequence polymorphism and allelic diversity is believed to be much greater in buffalo-derived T. parva than in cattle-derived parasites. The present study focussed on the development and application of a deep sequencing technique for characterising genotypically heterogeneous T. parva DNA samples. A panel of genes encoding CD8+ T cell antigens was used as the basis of a multi-locus sequence typing system (MLST) built upon Roche 454 amplicon sequencing technology. This system was validated using parasite stocks of known composition and then utilised to investigate genetic and antigenic diversity in vaccine stabilates and samples derived from African buffalo. The MLST profile obtained for the Muguga Cocktail stocks was compared to those of African buffalo in two geographically separated sites and was also compared with micro/mini-satellite DNA profiles of Muguga Cocktail stocks. The three components of the T. parva Muguga Cocktail vaccine were found to have limited genotypic and antigenic diversity using both methods. The composition of vaccine batches produced in a single production run (ILRI0801-ILRI0804) was shown to be relatively consistent. In contrast, the composition of the component stocks was shown to alter following passage through cattle and ticks. The deep multi-locus sequence profile and satellite DNA profile established in this study may be used as a reference for comparison with future vaccine batches. It is suggested that formulation of a new cocktail vaccine containing three parasite clones selected on the basis of genotypic and antigenic divergence may well provide protection comparable to that obtained with the Muguga Cocktail. The components of such a vaccine could readily be distinguished and the composition of vaccine batches monitored, thus allowing improved quality control and greater consistency of the vaccine. Genetic and antigenic diversity was found to be very high in parasite populations from African buffalo from the Kruger National Park, South Africa and the Ol Pejeta conservancy, Kenya. The estimated average genetic ‘distance’ between any two alleles in the Kruger National Park and within the Ol Pejeta conservancy was very similar for all six genes investigated. Many of the identified alleles were ‘private’ to either the buffalo from Ol Pejeta or the Kruger National Park and many of these alleles were present in several individuals in one location. Principal co-ordinate analysis and phylogenetic investigation of several antigen-encoding loci indicated that extant buffalo parasite populations are geographically sub-structured although some of the underlying diversity may reflect ‘ancient’ polymorphism in an ancestral population. A subset of the CD8+ T cell antigens examined exhibited extensive antigenic polymorphism while others were highly conserved at the amino acid level. These conserved genes may represent good candidates for the development of next generation vaccines, as strain specificity may be overcome if protective CD8+ T cell responses could be generated against these conserved antigens. This would enable the use of sub-unit vaccines in areas where cattle co-graze with buffalo. Theileria sp (buffalo) was identified in cell lines isolated from cattle, indicating that this parasite can transform bovine lymphocytes and may therefore be implicated in pathology in cattle. Phylogenetic analysis of T. parva and T. sp (buffalo) clones using the 5S subunit ribosomal RNA gene, Tp6, Tp7 and Tp8 showed a clear distinction between the two parasite species. These genes could thus be considered as candidates for an improved diagnostic test for T. parva in South Africa.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:630351 |
| Date | January 2014 |
| Creators | Hemmink, Johanneke Dinie |
| Contributors | Morrison, Ivan |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/9622 |
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