| To unserstand the prevalence of Cryptosporidium spp. in water buffalo and beef catlle in Henan province, an investigatioin was conducted to the beef cattle in Nanle county and the water buffalo in Jigong Mountain in Xinyang city. The fecal samples were examined using the Sheather's floatation technique and the modified acid-fast staining method. The results showed that the infection rate of water buffalo was 16.73% (43/257), and two distinct Cryptosporidium spp. were discovered. Likewise, the infection rate of beef cattle was 4.32% (9/208), and only one species was found.Moreover, the genetical characterization of Cryptosporidium isolates was also studied. The partial fragment of 18S rRNA gene was successfully amplified from the nine beef cattle isolates, then the PCR products were ingested by Ssp I and Vsp I. The results showed that the nine Cryptosporidium isolates all belonged to C. andersoni. Subsequently, the phelogenetic analysis further supported the results done by PCR-RFLP method.The oocysts of C. andersoni 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 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. |
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