An Animal Model For Cryptosporidium In Mice And The Detection Of Cryptosporidium By PCR And Differentiation By RFLP

This research work was conducted with the aims of establish an animal model for Cryptosporidium in mice and study anticryptosporidial activity of nitazoxanide; detect Cryptosporidium by routine way and a univeral prime species-specific PCR method and identificate two kinds of Cryptosporidium by Nested PCR-RFLP.The first trial is establishing a Cryptosporidium infective model in immunosuppressed mice. The optimal conditions for Cryptosporidium infection were determined by comparison between different mice strain (BALB/c and KM) ,parasite strains (C.parvum and C. andersoni), age of mice (sulking. 5-6 weeks. 10-12 weeks), DEX dosages( 0,2. 5,5, 7. 5,10,12. 5mg/L), inoculation dosages of CSO (75,1. 5×10~2, 1. 5× 10~3, 1.5×10~4 )and conservation time of CSO(1,2, 6,8months). The experimental results showed: (1) Both BALB/c and KM strain mice can be infected by C. parvum but C. andersoni can not; (2)5～6 weeks old KM mice survived longer than BALB/c mice with higher output of CSO. (3) KM strain mice immunosuppressed with 7. 5～10mg/L DEX in drinking water had the higher oocysts output and fewer dead than other groups. (4) There was no significant difference infection rate between groups inoculated with different number CSO and groups inoculated CSO with conserved different time. Trial were divided into control, groups of 50mg/kg /d, 100 mg/kg /d and 200 mg/kg /d dose of the drug to study the anticryptosporidial activities of nitazoxanide in an immunosuppressed mice model. The experimental results showed the treatment with 200mg/kg bw /day was significant inhibition of oocyst excretion.To detect the two cryptosporidiums strain (C. andersoni and C. parvum) we use three routine way and a Univeral prime species-specific PCR method. The experimental results showed:CSO can be easily detected out by saturated sucrose flotation technique and saturated sodium chloride flotation technique and the Modified Acid-fast staining mothed, the laboratory equipment are requested simply and conveniently. but the saturated sucrose flotation method can not be delayed, the CSO must be counted at once, or their will be destroyed, but this technique is more sensitive than the saturated sodium chloride flotation technique , because the latter may be get more impurity in sample. The Modified Acid-fast staining mothod of examining stool for oocysts request stain technique well ,and hence false negative results are common. But none these techniques allows discrimination regading the species of origin of the oocysts. To detect the two kinds of Cryptosporidiums infected in domestic animals in the same system and at the same time , DNA was extracted from Cryptosporidium oocysts and amplified by PCR, using a pairs of specific Univeral prime which based on the sequence of the 18S rRNA gene, and analyzed by the Danman and Genetxy softwares. This method is simply and conveniently, and it s more sensitive than the other routine way.The third trial used nested-PCR and RFLP. Based on www. ncbi. n1m. nih. gov GenBank . two pairs of primer (the primary forward primer 5'-GGGTTGTATTTATTAGATAAAGAAC-3'and the primary reverse prime 5-TCACTCCACCAACTAAGAACGG-3';the seconf forward primer5'-GAAACGGCTACCACA TCTAAGG-3' and the secondary reverse primer 5-GAAGGAGTAAGGAACAACC-3' )were designed and amplified by Nested-PCR ,The primary fragments of about 1100bp and the secondary fragments of about 660bp DNA of 18s rRNA gene respectively were amplified and sequenced the latter. A phylogenetic analysis...