The epidemiology of Neospora caninum
A seroepidemiological study was undertaken in a pedigree dairy herd that had a history of abortions due to neosporosis. The infection in this closed herd was thought to have arisen from a point-source infection, after which sporadic abortions have occurred. All cattle were bled twice, once in the winter and again the following summer and antibodies to N. caninum measured using an ELISA. The overall seroprevalence of Neospora was found to be 18 %. Three data sets; age-prevalence data, dam-daughter pair analysis and family tree data showed vertical transmission to be an important route of transmission of neosporosis in this herd. Analysis of anti- Neospora antibody titres with respect to the stage in the breeding cycle of cows appeared to show no association on a herd level. Data was collected on the number of Artificial Insemination (AI) services per successful pregnancy which showed a significantly greater number of Al services in Neospora-seropositive cattle compared with Neospora-seronegative cattle. This is the first study to assess the effect of neosporosis on cattle fertility in a quantitative manner and suggests that a wider study is justified. N. caninum shares many similarities with T gondii and has widely been assumed also to have a world-wide distribution. Two regions of Africa, Ghana in West Africa and Tanzania in East Africa, were studied in a cross-sectional survey of neosporosis in cattle indigenous to these areas. A prevalence of 8.1 % and 2% was found in two different areas in cattle native to Tanzania. Despite sampling a significant number of cattle in all three ecological zones of Ghana and of several different breeds, no Neospora-seropositive cattle were found. Possible reasons for the apparent absence of N. caninum in West Africa are discussed. To determine the overall genetic diversity in laboratory isolates of N. caninum, RAPD and AFLP methods were used. Genetic diversity was found to be low amongst Neospora laboratory isolates, relative to T. gondii, but demonstrated that genetic heterogeneity does exist within the species. Both RAPD and AFLP data were subjected to pair-wise similarity and cluster analysis and showed that there was no clustering with respect to host or geographical origin. The genetic similarity between cattle and dog isolates suggests that these hosts are epidemiologically related. In order to exploit the genetic heterogeneity in N. caninum to analyse a wider range of clinical field samples, several methods were attempted to devise PCR-based sequence-specific typing approaches that could be used on infected bovine tissue. Microsatellite markers were identified in N. caninum DNA sequences, however none of the microsatellite regions gave rise to detectable size differences, although they remain to be tested on a wider range of field samples. Laboratory isolates of N. caninum were also analysed for polymorphisms with two conserved minisatellite probes, 33.6 and 33.15, but although hybridisation occurred to digested parasite DNA, identical fingerprints were obtained for each isolate. In a final attempt to identify sequence-specific polymorphic markers, intron regions from two genes, actin and tubulin, were amplified and sequenced in both laboratory and field isolates. This approach revealed a number of single nucleotide polymorphisms (SNPs) that were able to differentiate between some isolates of N. caninum and might serve as useful molecular markers. SNPs were found more frequently in the clinical field samples, suggesting that the diversity of N. caninum is greater than that represented by current laboratory isolates. Further genotyping of field samples will enable the genetic population structure of N. caninum to be determined to facilitate molecular epidemiological studies.