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Molecular Identification of Cryptosporidium Isolates from Cattles and Antibodies Preparation for Prevention and Cure
Author: WangKun
Tutor: ZhangLongXian
School: Henan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Cryptosporidium Buffalo Beef cattle PCR-RFLP C. andersoni Yolk antibody Preparation
CLC: S858.23
Type: Master's thesis
Year: 2009
Downloads: 41
Quote: 0
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Abstract
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To understand the prevalence of Henan Province, buffalo and beef cattle Cryptosporidium, free-range buffalo of Nanyang Nanle beef cattle and Xinyang Jigongshan areas were investigated. Cryptosporidium oocysts in fecal samples detected with saturated sugar flotation method and modified acid-fast staining, buffalo Cryptosporidium infection rate was 16.73% (43/257), found that two kinds of morphology; beef cattle Cryptosporidium The infection rate was 4.32% (9/208), only to find a form of Cryptosporidium oocysts. Genetic Analysis of beef Cryptosporidium isolates using nested PCR amplification of the 18S rRNA gene sequence fragments of Cryptosporidium isolates from nine beef cattle source Ssp I and Vsp I restriction endonuclease PCR products digested digested the results of the restriction map of the nine isolates and C. andersoni. Subsequent phylogenetic analysis confirmed the results of the RFLP method. Collected above bovine Cryptosporidium andersoni improved Sheather's sucrose gradient centrifugation purified oocysts, filter filtration was purified through repeated freezing and thawing, and ultrasonic treatment prepared particulate antigen, soluble antigens were prepared, Freund's complete and non-complete adjuvant agent immune antigen was prepared to set the immunization program, the immunization open production of healthy hens. The immunohistochemical gradient of increasing the amount of antigen immune achieved satisfactory antibody levels. Particulate antigen group exemption, the amount of antigen were two free only -1,1 1 × 107 · 107 · only -1, three free the Four Frees antigen both only 1.5 × 107 · -1, five Free of six free amount of antigen are 2.5 × 107 · -1 each immunization interval 15 ~ 20d, free a total of six times. Soluble antigen group a Free only -1, 40μg · Free 40μg · only -1, three free 80μg · only -1 the Four Frees 80μg · only -1, five free 120μg · only -1, six free 120μg · only -1 each immunization interval 15 ~ 20d, free a total of six times. Two groups hen egg yolk antibody after immunization gradually increased six free 15d soluble yolk antibody levels in ELISA titer of 1:12800, peak area (1:12800 to 1:102400) lasts about 12 weeks; particulate group 6 Free 45 yolk antibody levels 1:12800, the peak area (1:12800 to 1:51200) for 10 weeks; yolk antibody its trend of serum antibodies to basically the same, but the level of serum antibodies was significantly higher than the corresponding yolk antibody levels. Indirect ELISA yolk antibody obtained a preliminary identification of Cryptosporidium genus-specific. This experiment - salting purified IgY antibody obtained was diluted with water, after SDS-PAGE analysis, purified IgY antibody, there are two main protein band, i.e., a heavy chain of about 68kD, the light chain of about 26 kD. Western-blotting analysis the the IgY recognition cattle Cryptosporidium andersoni the soluble protein molecular weight bands 26KDa and 68KDa. The IgY antibody on the activity below 65 ℃ no effect on the activity decreased by about 20% at 70 ° C for 15 min, 75 ° C for 5 min, activity dropped by about 80%, 80 ℃ 15 min Basic inactive. IgY activity in PH4 ~ 10 no significant change in the PH lt; 3 or gt; 11, the activity decreased, with good acid and alkali resistance.
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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Cow
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