| Coccidiosis in chicken is one of the major reasons caused the losses of poultry industry. Which has a great hazard, with hiding the growing development and decling the performance at least. However, in Serious cases, the deaths of a large of chicken will occur, especially in the younger chicken group. For example, the Mortality rate can reach 80% in 15～50 days chicken. With the developing of intensive aquaculture husbandry, the enhancing of Resistance to chemotherapy, the produing of medical resistance, and the contamination of medicine, which all make coccidiosis becoming more and more serious. Therefore, there is an important significance in conducting epidemiological investigation for preventing and curing this disease.Based on described above, an epidemiological invstigation on chicken, including the prevalence of Eimeria sp. and the species identification were performed in Harbin areas in the present study. Moreover, to the Highly pathogenic E.tenella, the 3-1E gene, with a higher cross-protection among different birds, was selected to further cloned and expressed. So to make a solid foundation for discussing whether this recombinant protein can be as genetically engineered vaccine or not.A total of 218 chicken farms in Harbin areas were surveyed, after examining the fecal samples, 136 chicken farms were positive for Eimeria sp. infection, the overall prevalence was 62.39%. Thereinto, there were higher infection rate in Hulan and Xiangfang areas, with more than 80% of prevalence. According to the shape characterizaion of oocysts by microscopical identification, six Eimeria sp. were discovered in chicken in Harbin areas, namely E. tenella, E .mitis, E. necatrix, E. acervulina, E. praecox and E. maxima.Under natural infection condition, mixed infection of Eimeria sp. is often observed in chicken. In the present study, all the farms examined for positive had the mixed infection, with not less than five speices. Another founding was that E. tenella was examined in all the positive farms, with the most infection rate of 39.83%. The number of oocysts were calculated by microscopical observation under 100×. The average number of oocysts calculated in a total of five field was considered as the standard of infection intensity. The results showed that the infection intensity of Eimeria in Harbin areas was mild or moderate. Therefore,suitable measures are needed to prevent and cure this disease. Form the perspective of age group, the 0～30 days chicken of infection rate reach to 40.00%. However, the highest prevalence was 76.47% in 30～60 days chicken.By the single oocyst sepration technique, six Eimeria sp. were successfully seprated from Harbin areas, namely E. tenella, E. mitis, E. necatrix, E. acervulina, E. praecox, and E. brunetti.The ITS1 gene was successfully amplified from the pure oocyst by nested-PCR method, and then the speices were further molecularly identified using the sequences obtained by similarity analysis and phylogenetical analysis.The fragment of 3-1E gene exited in sporozoite was amplified from the E. tenella Harbin strain by RT-PCR technique. The result showed that there was a open reading frame (ORF), with a length of 513bp. This gene was further cloned and after identified accurately, which was sub-cloned to the vector of pET-32a (+). Then, Prokaryotic expression was conduted in the receptor bacteria of Rosetta. The SDS-PAGE electrophoresis analysis illustrated that the fusion protein mainly existed in the supernatant, indicating a soluble expression. The immunity reactivity of the recombinant protein was confirmed by Western-blot. |
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