【作者】 袁万哲；

【导师】 何孔旺； 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 产肠毒素性大肠杆菌(ETEC)是引起仔猪腹泻的主要病原之一，该菌的致病性与其具有粘附素和产肠毒素密切相关。迄今，在猪源ETEC中发现的粘附素抗原有K88(F4)、K99(F5)、987P(F6)、F41、F42、F165、F17、F18等，而以K88、K99、987P和F41最为流行。粘附素是一种具有良好免疫原性的蛋白质抗原，用带有粘附素的菌体或纯化的粘附素抗原免疫动物体均可以产生高效价的相应抗体。鉴于此，作者选取K88~+、K99~+、987P~+和F41~+株流行粘附素菌株，制备了仔猪大肠杆菌病四价亚单位疫苗，围绕新制品的研制本研究对其实验室试验进行了一些有益的探索。 1 制苗株部分生物学特性的研究 本试验主要对四株制苗株的血凝性、传代稳定性及毒力进行了研究。甘露糖抵抗血凝试验表明，K88~+株不能凝集绵羊红细胞，但能凝集鸡、猪、豚鼠、兔红细胞，而以鸡红细胞凝集价最高；K99~+株不能凝集兔红细胞，但能凝集鸡、猪、豚鼠、绵羊红细胞，而以绵羊红细胞凝集价最高；F41~+株能凝集所有五种红细胞，而以绵羊红细胞凝集价最高；987P~+株不能凝集这五种红细胞。传代结果表明，K88~+使用限定代次可达22代，K99~+、987P~+和F41~+株至少可达42代。毒力试验结果表明，K88~+、K99~+、987P~+和F41~+株对小鼠的最小致死量依次为10×10~8CFU／mL、10×10~8CFU／mL、5×10~8CFU／mL和10×10~8CFU／mL，对仔猪的最小腹泻量依次为150×10~8CFU／mL、300×10~8CFU／mL、150×10~8CFU／mL和300×10~8CFU／mL。 2 定量产肠毒素性大肠杆菌粘附素抗原反向间接血凝试验的建立 利用粘附素单因子血清IgG致敏绵羊红细胞，建立了定量ETEC粘附素抗原的反向间接血凝试验。该试验所用K88、K99、987P和F41单因子血清IgG的最佳浓度依次为30ug／mL、56ug／mL、28ug／mL和100ug／mL。经交叉试验、抑制试验和敏感性试验证实，该试验具有很高的特异性和敏感性，可用于生物制品中粘附素含量的定量。 3 检测产肠毒素性大肠杆菌粘附素抗体间接ELISA的建立 利用纯化粘附素为包被抗原，建立了检测ETEC粘附素抗体的间接ELISA。初步确定了各种反应条件：K88、K99、987P、F41粘附素抗原最适包被浓度依次为1：400、1：80、1：40和1：40，相应粘附素抗体最佳稀释度依次为1：400、1：200、1：200和1：200。经交叉试验、阻断试验和重复试验证实，本方法具中文摘要有良好的特异性和重复性，可用于粘附素杭体的检测.4疚苗生产与免疚效力试脸 将四株粘附素杭原株分别接种于适宜培养基，培养物经加热提取粘附素杭原后，加入油佐剂制成四价亚单位疫苗.经成品检验合格后分别免疫接种小鼠和怀孕母猪，同时监测怀孕母猪杭体.最后一次免疚后15d，分别用同源ETEC确定的攻毒剂1攻击.结果:经二次免疫后，K88、K99、987P、F41对小鼠的免疫保护率与一次免疚没有显著差异(P>0.05);对仔猪，一次免疫跟二次免疫均可显著地降低腹泻指数(P<0.05)，二次免疫与一次免疫没有显著差异(P>0.05).怀孕母猪免疫一周后(产前23d)杭体开始上升，第二周(产前16d)达到高峰.二次免疚后抗体迅速回升，第四周(产前Zd)达到最高峰，产后Zd杭体大幅度下跌，几近免疫前水平.二免母猪所产仔猪发病率明显低于一免母猪(P< 0.05).攻毒保护试验和杭体消长规律的结果表明，制备的仔猪大肠杆菌病“8一K99一987P一F41四价亚单位疫苗具有良好的免疫原性，能有效地预防仔猪大肠杆菌病的发生.更多还原

【Abstract】 Enterotoxigenic Escherichia coli(ETEC) is one of the major pathogens which cause piglet diarrhea.The pathogenesis of ETEC is closely related to adhesions and enterotoxins.So far,the discovered adhesins of porcine ETEC have K88(F4), K99(F5), 987P(F6),F41,F42,F165,F17,F18 and so on.Of those,K88,K99,987P and F41 are the most prevalent. Studies have shown adhesins to be immunogenic protein,and antibodies against adhesions can be produced by immunization using whole bacteria with adhesin or purified adhesin.Therefore,the tetravalent subunit vaccine against piglet colibacillosis was prepared with four different ETEC which contained K88,K99,987P and F41 adhesins,respectively.The study around the laboratory experiments of the vaccine had been gone on.1 Studies on some biological properties of vaccine strains K88+,K99+,987P+ and F41+ strains were studies with respect to their somebiological characteristics,including hemagglutination,passage stability and virulence. The mannose resistant hemagglutination test showed:K88+ strain could agglutinate erythrocytes of chicken,pig,guinea pig and rabbit,but not of sheep.For all sources of erythrocytes examined,chicken erythrocytes had the highest agglutination titers. K99+ strain could do erythrocytes of chicken,pig,guinea pig and sheep,but not of rabbit.The sheep erythrocytes could receive the highest agglutination titers,which was the same to F41+.F41+ strain could do those all erythrocytes, but 987P+ not.Passage results showed K88 strain could passage to 22nd passage,K99+,987P+ and F41+ strains could do to 42nd passage at least. Virulence detection indicated that the minimal lethal dose to mice of K88+, K99+, 987P+ and F41+ was 10 108CFU/mL,10 108CFU/mL,5 108CFU/mL and10 108CFU/mL,respectively. The minimal diarrhea dose to piglet of those was150 108CFU/mL,300 108CFU/mL,150 108CFU/mL and 300 108CFU/mL,respectively.2 Development of titrating ETEC adhesin contents with reverse indirect hemagglutination testReverse indirect hemagglutination test was developed by using monospecific anti-adhesins IgG sensitizing sheep red cells to quantitate adhesin antigens of ETEC. The optimal concentration of monospecific anti-K88,anti-K99,anti-987Pand anti-F41 IgG was 1:400,1:80,1:40 and 1:80 respectively. It was proved that the experiment was well specific and sensitive by cross experiment,mhibition test and sensitivity experiment.lt could be used for titrating contents of adhesin antigens in vaccines.3 Development of an indirect ELISA for detecting antibodies to adhesins of ETECAn indirect ELISA for detecting antibodies to adhesins of ETEC was established by using purified adhesins for coating antigens.Conditions of reaction were as follows:The optimal coating concentration of K88,K99,987P and F41 antigens was 1:400,1:80,1:40 and 1:40 respectively. The optimal dilution of corresponding serum samples was 1:400,1:200,1:200 and 1:200 respectively.lt was proved that the method was well specific and repeatable by cross experiment,blocking test and repeated experiment. It could be used for detecting adhesin antibodies.4 Vaccine production and immunization efficacy experimentK88-ETEC,K99-ETEC,987P-ETEC and F41-ETEC strains were inoculated on appropriate medium repectively and bacteria fluids were collected.The tetravalent subunit vaccine containing purified K88,K99,987P and F41 adhesin antigens which were extracted by thermoextract method from four different ETEC and employing white oil as adjuvant was prepared. The vaccine was inoculated to mice and pregnant sows,at the same time antibodies produced from pregnant sows were detected after the product inspection.Challenging with the homogeneous strains at the determinate dose on days 15 after the vaccines was inoculated secondarily,the results were:after the second immunization,there was not a significant difference in immune protection rate between the one-time-immunization group and the two-time-immunization one to mice.To piglets,in comparison with control group,there was a significant difference,but not a significant difference be更多还原