||Parasitological analyses of Eimeria infections in domestic animals and the development of molecular methods for species discrimination
The identification of Eimeria species which infect domestic animals has been exclusively based upon a morphological approach of observing oocyst appearances. However, the discrimination of Eimeria to the species level is difficult, unreliable and subjective due to overlapping morphological features compounded by intra-species variation. New user-friendly molecular methods for species-specific detection of Eimeria are required and can complement clinical and epidemiological applications. Chapter I describes nucleotide sequences of the ITS-1 region from the ribosomal RNA locus of bovine Eimeria species. The results of analysis conducted for 21 ITS-1 sequences to define the variation among 18 Eimeria field collections and analysis of the phylogenetic relationship of each sequence and Eimeria species are also described. The ITS-1 regions were found to show sufficient inter-species variations for the development of reliable PCR diagnostics to identify species. The PCR assays could detect and identify six species of bovine Eimeria in species-specific manner. Chapter II deals with the development of a SYBR Green-based real-time PCR assay for the diagnosis of field-isolated Eimeria species of chicken. Real-time PCR offers the advantage of avoiding post-PCR processing steps, saving enormous time and laboratory labor to accomplish diagnostic examinations. The sensitivity of real-time PCR was slightly superior to a conventional fecal examination which shows a detectable level over 100 oocysts per 1g feces. Eimeria brunetti was found in 21 farms examined by the PCR assay, 20 of which came from breeder and layer farms on this survey throughout Japan. Chapter III describes the characterization of a Japanese field strain of E. brunetti, defining its pathogenicity and sensitivity to drugs for the first time. The strain of E. brunetti was strong enough to cause clinical coccidiosis in pathogenicity. Many diagnostic samples have been found to contain E. brunetti, despite the presence of E. brunetti in Japan long being regarded as scarce. It suggests that E. brunetti may be common throughout Japan and prompts the reassessment of Eimeria species occurrence across the country. Morphological and molecular differentiation methods should be combined to gain objective results in epidemiological surveys and studies. It is desirable that a PCR-based technique will provide new epidemiological data to reveal potential problems such as the example of E. brunetti prevalence in Japan and thus it helps to formulate strategies for controlling the parasites as quickly as possible.
Hokkaido University（北海道大学）. 博士(獣医学)