Mining the Genetic Diversity of the Pathogenic Protozoan, Neospora caninum

Publication Type:
Thesis
Issue Date:
2020
Full metadata record
๐˜•๐˜ฆ๐˜ฐ๐˜ด๐˜ฑ๐˜ฐ๐˜ณ๐˜ข ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ is a cyst-forming apicomplexan parasite, responsible for economic and reproductive losses to cattle industries worldwide, and represents a serious neurological disease in canines. Although discovered over three decades ago, progress towards treatment and control strategies against neosporosis, remains stagnant. Currently, common practices to combat the disease include passivity, or expensive culling of seropositive dams. However, vaccination represents a cost-effective and efficacious option, especially using live, attenuated isolates. Members of the Apicomplexa consist of populations that vary enormously in their disease-causing potential, where ๐˜ช๐˜ฏ ๐˜ท๐˜ช๐˜ท๐˜ฐ experiments have demonstrated pathogenic variability between ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ isolates. The underlying question therefore, is what is the genetic basis of virulence within the species, and consequently, how can such information be exploited in vaccine development? Thus far, conventional techniques have been employed to study the intraspecies genetic diversity associated with ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ, generally involving PCR-based approaches targeting repetitive elements. However, a direct causal relationship between such diversity and important parasite phenotypes such as virulence, is yet to be established. Alternatively, burgeoning next generation sequencing (NGS) technologies and ๐˜ช๐˜ฏ-๐˜ด๐˜ช๐˜ญ๐˜ช๐˜ค๐˜ฐ tools have provided new opportunities to perform genome-wide scans in such organisms. Hence the objective of this body of work was to compare the genomes and transcriptomes of two distinct ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ isolates, using NGS data and bioinformatics workflows, to identify sequence variants in coding and non-coding DNA. Annotation of variable regions would reveal potential virulence markers distinguishing isolates of this species. Challenges accompanying such research include the lack of optimisation and standardisation of NGS analysis tools for non-model organisms such as pathogenic Protozoa. This is compounded by the dubious accuracy of the ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ reference genome, as well as the disturbingly large number of proteins described as โ€˜hypotheticalโ€™ or โ€˜uncharacterisedโ€™. This body of research represents a thesis by compilation, consisting of four publications, and one chapter under review. Each chapter represents an independent study, which collectively address the research objective and current gaps in the literature. The results present polymorphic โ€œhotspotsโ€ in concentrated windows of the ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ genome, where there is a correlation between hypervariable regions within protein-coding genes, and non-coding regions. Furthermore, an ๐˜ช๐˜ฏ-๐˜ด๐˜ช๐˜ญ๐˜ช๐˜ค๐˜ฐ pipeline is developed to annotate uncharacterised proteins, subsequently identifying a subset of proteins potentially implicated in crucial parasite mechanisms, conducive to ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎโ€™s success. It is trusted that this thesis contributes vital knowledge pertaining to ๐˜•. ๐˜ค๐˜ข๐˜ฏ๐˜ช๐˜ฏ๐˜ถ๐˜ฎ intraspecies diversity, aiding in the quest to develop a vaccine against neosporosis.
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