Identification Of Cows Source Cryptosporidium And Cloning Of Cryptosporidium Wall Protein Gene

Cryptosporidiosis is a global zoonotic parasitic disease caused by Cryptosporidium, which can infected people and animals.Cryptosporidium is one of the important pathogen for diarrhea in humans and animals.It is also one of the main cause of death of the human AIDS patients.In recent years, dispite scholars have a more extensive and in-depth research on cryptosporidiosis,Hefei region Cryptosporidium species is not clear.The subject has taking a survey of infective condition of Cryptosporidium for a diary in Anhui province. We appraisal of oocysts by adopted molecular biology technology methods and morphology methods for identification of Cryptosporidium. The total RNA of Cryptosporidium oocysts was extracted.Make a reverse transcription for Cryptosporidium cDNA.COWP gene was amplified by PCR in order to provide information for the screening of the development of Cryptosporidium antigen vaccine candidate molecules.Firstly,285fecal from Hefei diary were checked by saturated sucrose flotation technique and modified acid-fast staining. Oocysts from the5positive fecal samples were elliptical or ovoid detected by using floating saturated solution.The infection rate was1.8%. Oocysts from the6positive fecal samples were elliptical or ovoid detected by using modified acid-fast staining.The infection rate was2.1%.Bsaed on some characteristic feature such as morphology,size,strucrure,ovoid index(length/width), identifying the type of camels and the macaques source Cryptosporidium.The oocysts captured by floating saturated solution of sucrose was oval-shaped or oval with the size of the average7.37μm×6.13μm and oval index of1.20. The oocysts captured by modified acid-fast staining was oval or ovoid, red roses, surrounded by darkly stained, the central pale staining with the average size of7.58μm×6.20μm and the oval index of1.22. The oocysts captured by The oocysts captured by floating saturated solution of sucrose and modified acid-fast staining are Cryptosporidium andersoni by preliminary identification.Secondly,5positive fecals infected by Cryptosporidium oocysts were collected. Cryptosporidium oocysts are separated. According to the sequence of18SrRNA gene and HSP70gene from Cryptosporidium, the primers were designed and synthesized.PCR reactions and nested PCR reaction were taken place with the complate of Cryptosporidium genome. The nested PCR products were cloned and sequenced. The sequencing of Cryptosporidium parasite18SrRNA gene and HSP70gene sequence homology analysis and phylogenetic tree construction, in order to determine the types of Cryptosporidium obtained. The PCR amplified from the Cryptosporidium18SrRNA gene and HSP70gene sequences of540bp and448bp, respectively, by nested PCR amplified from the Cryptosporidium insects18S rRNA gene and HSP70gene sequences are250bp and325bp, respectively (GenBank accession numbers were JQ031803JQ031804, JQ031805JQ031806, JQ031807JQ031808, JQ031809, JQ031810, JQ031811, JQ031812), with the expected fragment size is basically the same nested PCR products sequence of Cryptosporidium sequence homology analysis found that the five isolates of18SrRNA gene Angle hidden Cryptosporidium andersoni DQ989573the highest sequence identity, both in the phylogenetic tree for the same branch five isolates of HSP70gene sequences Cryptosporidium andersoni AY954892and DQ989576sequence identity and phylogenetic tree for the same branch, to determine the isolation of the5cows Cryptosporidium isolates are Cryptosporidium andersoni.At last,positive fecal with Cryptosporidium oocyts were collected.Total RNA of Cryptosporidium andersoni was extracted. cDNA was amplified by reverse transcription.COWP gene was amplified by PCR.The PCR production was cloned and sequenced,and then taken a comparison with sequeces of COWP genes from GeneBank.A499bp gene fragment was amplified by reverse transcription and PCR with the complete of Cryptosporidium andersoni.The sequence homology is100%after cloning, sequencing and sequence analysis of the amplified COWP sequence in GeneBank accession number DQ989570.In conclusion,18S rRNA and HSP70gene fragments of Hefei cows Cryptosporidium isolates were obtained in this study.It provided the basis for the clinical diagnosis of cryptosporidiosis,the molecular biology and species identification.The amplifying of COWP gene of cows Cryptosporidium andersoni was layed the foundation for developing Cryptosporidium vaccine in the future.