本文選題：犢牛腹瀉 + 大腸桿菌　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：犢牛腹瀉是造成規(guī)模化奶牛場(chǎng)內(nèi)犢牛死亡的主要疾病之一,腸致病性大腸桿菌(EPEC)及產(chǎn)腸毒素大腸桿菌(ETEC)是引起初生犢牛腹瀉和敗血癥的主要病原,其中以K99、F41菌毛致病菌株的致病幾率最高。免疫預(yù)防是控制該病最有效的方法,但目前國(guó)內(nèi)外尚無(wú)牛用的商品化疫苗。為此本研究采用自本地區(qū)致病犢牛腹瀉病料分離并鑒定的O8(K99)、O78(F41)菌株,采用菌毛抗原和全菌體抗原分別制成油佐劑滅活苗,通過(guò)免疫小鼠攻毒保護(hù)試驗(yàn),間接ELISA方法測(cè)定免疫母牛初乳及所產(chǎn)犢牛的血清抗體,觀察記錄犢牛腹瀉自然發(fā)病率的情況確定其免疫效果,結(jié)果如下:1.將O8、O78菌株分別接種LB肉湯及改良Minca肉湯,經(jīng)擴(kuò)大培養(yǎng)后滅活,離心菌體在60℃水浴振蕩下提取菌毛,飽和硫酸銨鹽析法提取菌毛蛋白,加入ISA206雙相油佐劑分別制備了犢牛腹瀉大腸桿菌K99-F41菌毛油佐劑苗和全菌體疫苗。2.疫苗檢驗(yàn):對(duì)兩種菌苗的抗原含量進(jìn)行測(cè)定、物理性狀檢查,無(wú)菌檢驗(yàn),安全檢測(cè),免疫小鼠攻毒實(shí)驗(yàn),結(jié)果顯示,菌毛苗蛋白含量為0.5~1.0 mg/m L,全菌體苗細(xì)菌數(shù)為40~150億/m L。免疫小鼠攻毒實(shí)驗(yàn)結(jié)果中菌毛疫苗和全菌體苗的保護(hù)率分別是100%和93.3%,表明兩種疫苗達(dá)到無(wú)菌,安全,性狀穩(wěn)定,免疫原性良好指標(biāo)。3.利用O8、O78菌株所提取的K99-F41菌毛抗原作為包被抗原,通過(guò)棋盤(pán)滴定法建立了K99-F41菌毛疫苗免疫抗體的間接ELISA檢測(cè)方法,并測(cè)定陰性血清的OD450值計(jì)算出陰性臨界值,結(jié)果表明,抗原包被稀釋倍數(shù)為1:80,被檢血清及乳清稀釋倍數(shù)為1:100為最佳條件。4.將兩種疫苗分別按不同劑量給產(chǎn)前2-4周的懷孕母牛免疫接種,通過(guò)已建立的間接ELISA方法檢測(cè)免疫組母牛產(chǎn)后3 h、6 h、12 h、24 h、36 h、72 h的初乳及所產(chǎn)犢牛灌服初乳1 d、3 d、5 d、7 d血清中相應(yīng)大腸桿菌抗體滴度,來(lái)比較兩種大腸桿菌油佐劑疫苗不同注射劑量對(duì)妊娠母牛及所產(chǎn)犢牛免疫抗體滴度的差異。結(jié)果表明,全菌體疫苗劑量為5 m L組的母牛初乳中抗體滴度最高;菌毛疫苗5 m L組犢牛血清中的抗體滴度最高;兩組疫苗免疫劑量5 m L組的初乳及犢牛血清中相應(yīng)大腸桿菌抗體滴度均顯著高于3 m L組;未免疫母牛初乳及所產(chǎn)犢牛的大腸桿菌抗體顯著低于免疫組,初生犢牛腹瀉自然發(fā)病率明顯高于免疫組。
[Abstract]:Calf diarrhea is one of the major causes of calf death in a large scale dairy, and enteropathogenic Escherichia coli EPECand enterotoxigenic Escherichia coli ETEC) are the main pathogens causing diarrhea and septicemia in newborn calves. Among them, the pathogenic strain K 99 F 41 had the highest pathogenicity. Immunization prevention is the most effective method to control the disease, but there is no commercial vaccine for cattle at home and abroad. In this study, oil adjuvant inactivated vaccine (O8K99O78F41) isolated and identified from disease-causing calves in this area was made into oil adjuvant inactivated vaccine using fimbriae antigen and whole cell antigen respectively. Indirect Elisa method was used to detect the serum antibody of immune mother bovine colostrum and calves, and to observe and record the natural incidence of diarrhea in calves to determine the immune effect. The results were as follows: 1. Strain O8 O78 was inoculated with LB broth and modified Minca broth, and then inactivated after expanded culture. The fimbriae were extracted by centrifugal bacteria in water bath at 60 鈩，

本文編號(hào)：2030625