Preparation Of Egg Yolk Antibodies On Cryptosporidium Andersoni And Immuno-protection And Cross Protection Analysis

100 fecal samples in one cattle farm in Zhengzhou were surveyed with both saturated sucrose solution floatation and modified Ziehl-Neelsen staining (acid-fast stain) technique. Two types of oocyst were found.one is of 7.25μm×6.33μm with shape index of 1.22,and another is of 3.32μm×3.16μm with shape index of 1.05. They were identified firstly as Cryptosporidium parvum and C. andersoni respectively according to their morphological characteristics. Nested PCR-RFLP by analysis of the 18SrRNA gene was used to detect Cryptosporidium from cow feces.The fragments of 18SrRNA gene was amplified by Nested PCR from the two Cryptosporidium isolates respectively.RFLP restriction maps of two isolates formed after PCR products were digested by SspⅠwere identital to RFLP restriction maps of 450bp,and further showed the two isolates were all C. andersoni.The oocysts of C.andersoni strain were collected by saturated sucrose solution floatation,and then were purified by concentration,absolute-rest precipitation,vacuum filtration,following by amelioration discontinuous sucrose gradients. Purified oocysts were freeze-thawed and sonicated to prepare particulate antigen and. treated according to C.WEIR to prepare soluble antigen,mixed with Freund complete or incomplete adjuvant to immunize the five-month-old laying hens according to the immunity procedure.using the gradually augment the dosage of antigen got the best result. Immunity procedure of particulate antigen group:the dosage of antigen in the 1st time and the 2nd time all is 1×107 oocysts per hen,1.5×107 oocysts per hen in the 3rd time and the 4th time,2.5×107 oocysts per hen in the 5th time and the 6th time,and15～20 days arfter the next immunity injection. Immunity procedure of soluble antigen group:the dosage of antigen in the 1st time and the 2nd time all is 40μg per hen,80μg per hen in the 3rd time and the 4th time,120μg per hen in the 5th time and the 6th time,and15～20 days arfter the next immunity injection. The titers of yolk antibodies of two groups gradually raise.The titer of soluble antigen group rise to 1:12800 on 15d after the 6th times,the extent of peak titer (1:12800～1:102400)lasted 12 weeks . The titer of particulate antigen group rise to 1:12800 on 45d after the 6th times,the extent of peak titer (1:12800～1:51200)lasted 10 weeks. It was found that the growth-decline trends of yolk-antibody and serum-antibody were generally similar,but the yolk-antibody titers inclined to decreased markedly.The egg yolk antibodies were examined with indirect ELISA assay that has been set up,and testified it was against Cryptosporidium.The egg yolk antibodies were purified with water dilution method , and passed through SDS-PAGE electrophoresis:the result shown that the purified egg yolk show heavy chains were approximately 68 kDa and light chains was about26 kDa.Molecular weights of bands identified by IgY antibody were 18Kda-34KDa through Western-blotting analysis. The IgY was stable under 65℃. However,at 70℃for 15 min,the titers of IgY lost approximately 20%, at 75℃for 15 min,the titers of IgY lost approximately 80%, at 80℃for 15 min,the IgY-activity lost completely.The IgY-activity showed broad stability between the pH 4 and 10. However, at less than pH 3 and more than pH 11 , the IgY-activity declined evidently.It was an aim to study the treatment effect of anti-C.andersoni egg yolk in naturally infected cows by C.andersoni. The result suggested anti-C.andersoni egg yolk could reduce the oocyts ,decline the OPG value of peak at the same time,compared to negative yolk control group.In this study we also had research on cross-protection at different anti-Cryptosporidium egg yoik on the same Cryptosporidium.The result showed anti-C.andersoni egg yolk and anti-C.baileyi egg yolk had favor effect in protecting chicken infected by C.baileyi .They all decreased the infection intensity,the time of oocysts sheded decreased 2～3 days. Middle dosage of Anti-C.baileyi egg yolk had the same effect as the large dosage and the middle dosage of anti- C.andersoni egg yolk,but better than the small dosage of anti- C.andersoni egg yolk.