Study On Identification Of Cryptosporidium And In Vitro Culture Of Cryptosporidium Andersoni

ObjectiveCryptosporidium is one of the most important pathogens causing diarrhea in human.In infants and immunodeficiencys,Cryptosporidium caused severe damage,and the treatments for cryptosporidiosis are less effective.It is urgent to establish diagnostic technique and screening effective drugs for the treatment of cryptosporidiosis.In this study, we established PCR-RFLP technique and gene sequencing technique to identify Cryptosporidium species,and economic and high performance method of oocyst genomic DNA purification for PCR assay,and C.andersoni in vitro culture system for screening anti-Cryptosporidium drugs.MethodsNest-PCR was used to amplify the SSU rRNA sequence of Cryptosporidium oocysts isolated from calf,mouse and rat.The RFLP technique and gene sequencing technique were used to identify Cryptosporidium species.Lysis solutions from imported and domestic genomic DNA purification kits,and 2% Trition X-100 and 5%guanidinium isothiocyanate were added into the oocysts,followed by different combining forms of freeze-thawing,proteinase K and sonication.Real-time PCR was used to determine the copies of Cryptosporidium oocyst wall protein(COWP) gene.HCT-8 cells were infected by C.andersoni oocysts.Real-time PCR was used to determine the enhancement effect of fetal calf serum(FCS),glucose(GLU),ascorbic acid(ASC),folic acid(FOLIC),calcium pantothenate(CAPAN) and insulin(INSULIN) while added into RPMI-1640 medium.Giemsa staining was used to observe the growth of C.andersoni,and transmission electron microscope(TEM) was used to observe the developmental stages of C.andersoni.MTT assay was used to evaluate the toxicity of nitazoxanid(NTZ) and ginkgoic acids (GAs) to HCT-8 cells.C.andersoni parasites growing in HCT-8 cells were treated with different concentrations of NTZ and GAs,and the copies of COWP gene were determined by Real-time PCR.The morphological characteristics of C.andersoni treated with NTZ and GAs were observed by TEM.ResultsCryptosporidium isolated from calf,mouse and rat were identified as C.andersoni,C. mouse genotype and C.rat genotype respectively,by RFLP and gene sequencing technique.The extracting effect of the imported commercial DNA extraction kit was the best,and the copies of COWP gene were(6.450-9.861 )×10~6 in the extraction.The copies of COWP gene were(2.377-3.689)×10~6 and(1.272-21.29)×10~5 in the extraction extracted by domestic commercial DNA kit and 5%guanidine thiocyanate,respectively.2%Triton X-100 had the poor effect on extraction,and the copies of COWP gene only about (2.060-866.7)×10~3.The method of freeze-thawing & proteinase K & sonication had the best effect on genomic DNA extraction among the four methods,and the effect of freeze-thawing & sonication and proteinase K & sonication were better,and effect of freeze-thawing & proteinase K was the worst.10%FCS was the best concentration of FCS for C.andersoni in vitro culture.The additional supplements including glucose,ascorbic acid and insulin had the enhancement effects on C.andersoni development,while folic acid had no significantly effect on C. andersoni development and calcium pantothenate had inhibited effect.When added 50 mmol/L glucose,50μg/ml ascorbic acid,0.3 U/ml insulin into 10%FCS RPMI-1640 medium,the proliferation of C.andersoni had a great significantly increase,and the gene copies of C.andersoni were 8-fold compared with that in 10%FCS RPMI-1640 medium. Giemsa staining had the similar result as above.Trophozoite,macrogamout,microgamout, typeⅠmeront,typeⅡmeront and sporozoite in the development of C.andersoni were observed by TEM.The proliferation of C.andersoni was significantly inhibited by 1.25μg/ml NTZ and 0.31μg/ml GAs.Treated with 20μg/ml NTZ and 5μg/ml GAs respectively,the numbers of C.andersoni were only 0.29%and 0.66%compared with the untreated.For TEM assay results,most of the host cells were destroyed when infected with C.andersoni,however the internal structures of C.andersoni were damaged and the structure of host cells remained normal while treated with NTZ and GAs.ConclusionsBoth PCR-RFLP technique and gene sequencing technique can be used for identifying Cryptosporidium species.RFLP technique is suitable for clinical diagnosis and epidemiological study.Gene sequencing technique can be used for identifying new species. The method of freeze-thawing & proteinase K & sonication can reach the maximum extraction effect of genomic DNA.The effect of the domestic commercial DNA extraction kit and 5%guanidine thiocyanate was similar to that of the imported commercial DNA extraction kit.C.andersoni can grow in HCT-8 cells,the different additional supplements in culture medium have the different enhancement effects on the development of C.andersoni.Similar to NTZ,GAs has significantly effect on the proliferation of C.andersoni in vitro, and GAs is a potential drug for the treatment of cryptosporidiosis.