Genetic diversity in Neospora caninum
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The apicomplexan parasite Neospora caninum has been identified as an important cause of disease in the dairy industry because it causes abortion and neonatal mortality in livestock such as cattle, sheep and goats and also causing neuromuscular disorders and death in dogs. Biological diversity within the species N. caninum isolates has not been studied in detail. Limited differences were reported using several genetic approaches such as sequencing of ribosomal DNA. Mini- and microsatellite DNA markers have recently been reported to represent a valuable tool to study genetic diversity within this parasite. This is the topic of this thesis. In the present study, mini- and microsatellite DNA markers were used successfully to genotype different strains of N. caninum which were isolated from bovine and canine hosts and from different geographical regions of the world. Several mini- and microsatellites were identified that detected differences among different isolates of this parasite. A prototype multiplex PCR assay was developed for strain typing of N. caninum based on the length polymorphisms detected amongst three different repetitive markers two- minisatellites and one microsatellite. This method is simple, rapid, highly informative and sensitive for the study of N. caninum diversity. Discrimination amongst isolates can be improved by the incorporation of more loci into a multiplex PCR and so additional DNA markers were sought. The study of genomic DNA resulted in the identification of two additional minisatellites and one microsatellite. A second generation multiplex PCR assay was developed for multilocus strain typing of N. caninum based on length polymorphisms of the six satellites. This multiplex included three microsatellites and three minisatellites. This assay provides a higher degree of resolution and provides a valuable tool to study genetic variation among different isolates of this parasite. Various uses of multiplex PCR were investigated in the study of N. caninum. Isolation of N. caninum from a litter of pups was attempted; the bitch was previously identified as seropositive to N. caninum. Multiplex PCR of DNA from the serum of the bitch showed a genotype for N. caninum not previously reported in Australia. Toxoplasma gondii was isolated from the brain of a dog for the first time in Australia. The identity of the parasite was confirmed by PCR, Western blotting, electron microscopy and cat bioassay. Genotyping of the isolate (TgDgAu1) was determined by 10 PCR-RFLP markers and revealed it to be a Type II strain. Westerb blotting demonstrated the presence of IgM antibodies to T. gondii suggesting the bitch was probably infected during pregnancy and the T. gondii was transmitted to the pups congenitally therefore, this represents the first description of a natural case of congenitally transmission of T. gondii in the dog. Unfortunately N. caninum was not isolated during these studies.
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