Specific diagnostic tools for protozoan infection of ruminants
Neospora caninum, Toxoplasma gondii and Sarcocystis spp. are closely related intracellular protozoan parasites causing neosporosis, toxoplasmosis and sarcocystosis, respectively. Toxoplasma and Neospora are major causes of abortion in livestock worldwide leading to substantial economic losses. Toxoplasma is a well-known infectious parasite of sheep and a wide range of warm-blooded animals, including humans. Neospora predominantly causes disease in cattle, although infections in other warm-blooded animals have been found to cause disease. Sarcocystis infect a wide range of intermediate and definitive mammalian hosts. Although abortion is a major problem for livestock operations and animal welfare worldwide, the identification of a specific cause is particularly difficult and achieved in less than 50% of the cases, even in well-established diagnostic laboratories. Neospora, Toxoplasma, and Sarcocystis spp. share many common morphological and biological similarities making differentiation through immunological methods between these protozoan parasites challenging. Aetiological diagnostics tests are required to confirm the presence of association of disease with specific parasites, and to determine the cause of abortion to adopt the most relevant disease control strategy. As such, it is necessary to have access to specific diagnostic tools to confirm or rule out the presence of Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. as the cause of abortion in mammalian species. The primary aim of this PhD was to develop a genus-specific PCR assay for the detection of N. caninum, T. gondii and Sarcocystis spp. using fixed and fresh tissue material. Suitable target regions for the production of genus-specific PCR primers were identified using the 18S rRNA gene and ITS1 regions. Primers were tested for sensitivity and specificity using DNA from various protozoan parasites. For the 18S rRNA gene, general PCR primers were developed to amplify DNA from Neospora, Toxoplasma and Sarcocystis spp. 18S group-specific primers were developed to enable detection of T. gondii and N. caninum from Sarcocystis spp. Several 18S Sarcocystis specific group primers were developed to enable differentiation of a variety of Sarcocystis spp. Species-specific primers were developed using the ITS1 region to enable diagnosis of Neospora from Toxoplasma. PCR assays were standardized, and primers were validated using DNA extracted from fixed and fresh tissues to enable the diagnosis of different protozoan species. The second aim was to develop genus-specific antibodies (polyclonal and monoclonal) raised against recombinant proteins of N. caninum, T. gondii and Sarcocystis spp. to enable specific diagnosis. For this aim, suitable target genes for the production of recombinant proteins for Neospora, Toxoplasma and Sarcocystis spp. were identified. Recombinant proteins were expressed using E. coli and analysed for cross-reactivity. Three recombinant proteins for Neospora, three for Toxoplasma and one for Sarcocystis were successfully expressed. Of those, two recombinant proteins for Neospora (rNcSRS2 and rNcSAG1) and one recombinant protein for Toxoplasma (rTgSRS2) were used for subsequent antibody production. For the development of polyclonal antisera, rabbit pre-immune sera were tested to choose the best candidate for immunisation using the recombinant proteins. Polyclonal sera were tested using immunohistochemistry for functionality and specificity. The polyclonal antibodies were validated using a range of ruminant clinical cases suspected of protozoal infections. Based on the recombinant protein expression the best candidates were taken forward for the development of a genus-specific monoclonal antibody. For this study, three rabbits were chosen for immunisation using recombinant proteins, and three polyclonal rabbit sera (anti- Neospora NcSAG1, anti- Neospora-NcSRS2, anti- Toxoplasma TgSRS2) were generated. Each polyclonal sera was shown to be specific, and results showed that the sera can be used in immunohistochemical detection of the parasite on formalin fixed paraffin embedded samples. For monoclonal antibody production, mice were immunised with recombinant proteins NcSRS2 and TgSRS2. Hybridoma clones were generated, and clones that showed reactivity and specificity using ELISA and immunohistochemistry were selected to produce monoclonal antibodies. The study achieved the successful production of Neospora monoclonal antibody ME7.1.B12.C9. No Toxoplasma specific monoclonal antibody was produced. This study shows that the genus-specific PCR assays and genus-specific antibodies can be used for the identification of Neospora, Toxoplasma and Sarcocystis in formalin fixed paraffin embedded samples.