Studies On DNA Polymorphisms In Mitochondrial, Apicoplast Genes Of Eimeria And Mitochondrial Genomes

Coccidiosis is the most serious parastic disease in chicken. It is widely distributed around the world. Coccidiosis is likely to exist in every chicken farm. Seven Eimeria species are recognized in the world and all the species have pathogenicity. Coccidiosis causes economic lossess of billions of dollars annually in China. The accurate identification and studying genetic variation in chicken Eimeria has important implications for the prevention and control of chicken coccidiosis.In the first part of this study, the objectives were to investigate nucleotide variation in sequences of two mitochondrial genes and to study phylogenetic relationships. A total of 17 Eimeria strains belonging to 6 species from different geographical origins in China were examined. The partial sequences of two mitochondrial DNA genes, namely cytochrome c oxidase subunitâ…¢(cox3) and cytochrome b gene (cytb), were amplified separately by PCR and sequenced in order to find sequence differences. The results showed that no lenth difference was found in two gene sequences. Intra-specific and inter-specific of pcox3 sequence variation were 0-0.3% and 3.1-25.8%, respectively. There had no difference in pcytb within a species, whereas inter-specific differences was 1.5-19.5%. After analyzing polymorphisms of the two genes, the cox3 and cytb genes could be used as ideal genetic markers. The molecular phylogeny using the combined sequences of cox3 and cytb gene was reconstructed. It showed that E. necatrix was closer to E. tellena, then grouped with E. praecox, E. brunetti and E. acervulina, and E. maxima was the most distinct species.In the second part of this study, partial apicoplast sequences of 14 Eimeria strains were amplified and sequenced. Apicoplast is a unique organelle similar to plant plastid organelles. Based on E. tenella apicoplast sequence and Toxoplasma gondii apicoplast sequence, a pair of primers was designed and used to amplify partial apicoplast sequences (pApico). Intra-specific variation in pApico was 0-0.3% and inter-specific sequence difference was 3.1-12.6%, with 178 positions of nucleotide variations. The results showed that intra-specific nucleotide variations was low, whereas inter-specific nucleotide differences were significant.In the third part of this study, the complete mitochondrial genome of E. brunette was sequenced. Using obtained E. brunetti cox3 seuqnecs to design primers, the entire mitochondrial genome of approximately 6.0kb in size was amqlified by long PCR. The PCR product was cloned and sequenced. The mitochondrial genome of E. brunetti was 6155 bp in length. This genome had the identical gene organization and contents to that of Leucocytozoon caulleryi and Plasmodium juxtanucleare 3 protein genes were identified, cox3, cox1 and cytb. The content of A + T in mtDNA was 65.43%.The present study provided mitochondrial and apicoplast sequences for Eimeria, which has implications for studying molecular taxonomy, phylogeny, and population genetic structure of Eimeria, as well as for effective prevention and treatment of coccidiosis.