Investigating The Infective Condition Of Cryptosporidium In Some Area Of Anhui Province And Study On Cryptosporidium Genotypes

Cryptosporidium is one of the important prozotoan parasite,which can infected people and animals.It was found in experimental mice gland in 1907.Cryptosporidisis is a worldwild distribution anthropozoonosis which with the main clinical signs of diarrhea.The disease has severely endanger to human health and animal production and has been recognized as one of the cause of deadly in association with the acquired immunodeficiency syndrome(AIDS).The control and prevention centre of USA has classified the disease as B etiology.The disease was also classified as one of the two kinds of important parasitic disease that our country took precautions against especially in 2003. In recent years,Cryptosporidium has been studying and acquired many important achievements in many respects such as aetiology,morphology,epidemiology,pathogenic molecular biology,diagnosis and integrated control technology etc.;But as the morphologic features among differential genus not notable,Besides lack of unified criteria in the clasiification of protozoan parasite at home and abroad at the present stage.With the development of molecular biology technology,Many reports about new genotypes of Cryptosporidium are coming in all the time.These factors all causes the precise kinds of Cryptosporidium is still unknown.We confronted greatly difficult in prevention and control the disease.The subject has taking an survey of infective condition of Cryptosporidium for four dairy farm in Anhui province,preliminary clear about the infective condition of Cryptosporidium in Anhui province.We seperated oocysts from positive cows,In addition to,We appraisal of oocysts by adopted molecular biology technology methods and morphology methods,trying to inspect which type of Cryptosporidium in Anhui province in a way of molecular biology technology,Supplying theory principles to preventive and control which disease of anthropozoonosis of Cryptosporidiosis.Firstly,the 502 fecal specimen gathered form cows was floation with satturated white sugar,We have not found Cryptosporidium oocysts in D dairy farm.Bsaed on some characteristic feature such as morphology,size,strucrure,ovoid index,appraisal oocyst which acquired is C.andersoni.Using test of significance,The Cryptosporidium of infection rate of B dairy is very significantly higher than C dairy(P＜0.01);The divergence infection rate of A dairy and B dairy also significant(P＜0.01);The range of infective rate for four dairy farms are 0%-14.79%,the average rate is 5.18%.Counted and analyied infection of cow in different age,The Cryptosporidium of infection rate between calf and youth cattle divergencet is significant(0.01＜p＜0.05),The Cryptosporidium of infection rate between calf and cattle divergence is also significant(p＜0.01).The infection rate between youth and cattle had no significant difference.Counted and analyied infection of cow in different age,each age cow could infect cryptosporidium.but different age cow with different situation.Secondly,We comparaed some kinds of methods of purified Cryptosporidium oocysts.These methods included:discontinous sucrose gradients,aether-saturated Zinc sulfate,sheather's floation.The result indicates that the best purified method is discontinous sucrose gradients which recovered the most oocysts and free of debris.Oocysts recovered by sheather's floation were the second.But as the adhesive of sucrose,the recovered oocysts contained a quantity of debris,it may inhibits the PCR reaction.The oocysts coefficient of recovery and purified effect for aether-saturated Zinc sulfate is situated between sucrose gradients and sheather's floation.Thirdly,We adopted discontinous sucrose gradients to purified oocysts.Adding splitting liquid(2%DTT,1×TE) to purified oocysts,put these oocysts to-70℃refrigerator and room temperature repeatedly three times.Designed the Ccryptosporidium specific primer according to the 18SrRNA gene of Crytposporidium,amplified the 540 bp fragment. Nested-PCR reaction based on the PCR products,amplified the 250 bp fragment.Designed the Cryptosporidium specificity primer according to the HSP70 gene of Crytposporidium, amplified the 448 bp fragment.Nested-PCR reaction based on the PCR products,amplified the 325 bp fragment.At last,we raclaimed Nested-PCR products by using reagent box of PCR.Applied for shanghai sangon to sequenced these products.Then analysis the sequence and draws phylogentic trees using DNAStar,MEGA3.1.To make the results as far as possible accurate,We used two algorithm include Neighbour-jointing(NJ) and maximum parsimony(MP).The analysis of software demonstrated that the two phylogentie trees similar,Further proved that oocysts of Cryptosporidium which separated from four dairy farms in Anhui province is C.andersoni in a way of molecular biology technologyTo sum up the above arguments,the results acquires from the subject supplied theory basis for preventive and treatment of Cryptosporidium of Anhui province.Establishing the identified methods by molecular biology and reaserch on genotypes supplied some significance reference for study on Cryprosporidium and pathogens...