| Cryptosporidisisis one of the important zoonosis with main clinic sign of diarrhea and worldwild distribution, and it endanger to human health and animal production. it was listed as B etiology by control and prevention centre of USA and one of the two kinds of important parasitic disease need to be prevented and controled in China in 2003. Recently, many achievments in epidemiology, pathogenic molecular biology , diagnosis and integrated control technology have been obtained, but the dispute on its taxonomy of the new found Cryptosporidium produced lacking of the marked difference in morphology between interspecies and unified standard for using to classify, which rsult in the difficulties to prevent and control it. Nowadays, its pathogenesis still wasn't clear, but no any medicine can be used to treat it effectively, hence medicine screening become one of the research domestic and abroad.In the thesis, Cryptosporidium originated from wild animals at X Zoo in Anhui province was investigated, the positive wild animals were screened preliminarily, and the oocysts of Cryptosporidium were isolated from the positive animals. It was identified by the techniques of morphology and molecular biology, and its CaM gene was cloned by RT-PCR. The aim was to be investigaed the infection of Cryptosporidium to provide the reference for preventing and controling it, it also will play foundation on the further study of metabolism mechanism.Firstly, 232 samples of fecal from X Zoo in Anhui provice were determined by floating saturated solution of sugar and acid-fast staining, the sorts of Cryptosporidium from camel and macaque were identified based on the characterization of oocyst in appearance, structure, size, Ovoid shape index. The primary identification result indicated that Cryptosporidium which infected camel was C. andersoni, macaque-derived Cryptosporidium was C. hominis. The infection rate of herbivbores, primates, meat omnivorous animals and birds was 4.88%(2/41),13.64%(6/44),9.09%(4/44) and 6.80%(7/103) among the 232 fecal samples by detection of the Sheather's sugar floatation technique;the infection rate of herbivbores, primates, meat omnivorous animals and birds was 7.32%(3/41), 2.27%(1/44), 0(0/44) and 0.97%(1/103) by detection of acid-fast staining. The total infection rate respectively was 8.19%(19/232)and 2.16%(5/232)among with the combination of acid-fast staining and he Sheather's sugar floatation technique, T test showed that there was markedly different between the two detection methods (P<0.01).Secondly, PCR was applied to identify the isolated Cryptosporidium. The oocysts was purified by discontinous sucrose gradients, the oocysts wall was break into pieces by shaking for 20min, then 2%DTT was added up into the suspension and mixed, the suspension was iced and mellted for three times, the genemic DNA was extracted. The two pair of primer was desiged besed on the repeorted sequence of 18S rRNA and HSP70 gene to amplify the aim genes, the PCR products were sequenced, and the obtained sequence were analysed with DNAStar software to construct cladogram . The result showed that there respectively was fragment of 540bp and 448bp according to the expected, the sequence analysis indicated that the isolated Cryptosporidium from camel is C.andersoni.Finally, RT-PCR method was applied to amplify calmodulin gene of Cryptosporidium. Ther total RNA was extracted, and reverse transcripted into cDNA, then the primer was designed according to the reported sequence to be used for amplification of CaM gene, and the obtained products was sequenced and analysed with blast of GenBank . The result showed that there was fragment of 297bp according to the expected.To sum up, the isolated Cryptosporidium from wild animals was C.andersoni and C. homonis, and the CaM gene also be cloned. The Cryptosporidium infected with wild animal was ensured will help for prevent and control it, and study on function of CaM gene will help for understanding the metabolism mechanism of Cryptosporidium.
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