【作者】 程学慧；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 本研究针对导致仔猪腹泻的主要产肠毒素大肠杆菌K88，建立了K88及其三种变异体K88ab、K88ac和K88ad的PCR鉴定方法，进而对武汉及其周边地区规模化猪场进行了ETEC K88及其变异体的流行病学调查，确定了K88ac是武汉及周边地区腹泻仔猪粪样中ETEC K88变异体最常见的一类；通过对ETEC K88ac培养条件、黏附素抗原提取、黏附素诱导蛋鸡免疫应答和卵黄抗体的加工特性的研究，建立了抗ETEC K88ac黏附素卵黄抗体的制备技术；通过体外和体内试验研究了抗ETEC K88ac黏附素卵黄抗体预防仔猪腹泻的机理和应用效果。结果如下： 1．以ETEC K88ab、K88ac和K88ad标准菌株为试验材料，建立了ETEC K88及其三种变异体K88ab、K88ac和K88ad的PCR鉴定方法，并对武汉及其周边地区猪场进行了ETEC K88及其变异体的流行性调查。结果表明：K88ab、K88ac和K88ad用设计的引物，经PCR均扩增出特异性目的大小的DNA片段；对291株已经血清学鉴定未发现携带K88粘附素的大肠杆菌用PCR方法进行检验，发现有一株大肠杆菌是ETEC K88，其变异体为K88ac。对武汉及周边地区采集227份哺乳仔猪腹泻粪样进行了PCR检测，发现有23份含有K88菌，占总数的10.1％，且均为K88ac变异体。因此，PCR方法鉴定ETEC K88及其变异体具有快速、特异性强、敏感性和准确性高的优点，适合于ETEC K88及其变异体大规模鉴定和流行病学调查；K88ac是武汉及周边地区腹泻仔猪粪样中ETEC K88变异体最常见的一类。 2．比较了振荡培养和静止培养两种方式对ETEC K88ac菌体产量和黏附素表达效果的影响。研究了ETEC K88ac黏附素蛋白提取纯化方法和ETEC K88黏附素诱导蛋鸡的免疫应答。确立了卵黄抗体粉适宜加工方式，并对其稳定性和储藏性能进行了初步探讨。结果表明：振荡培养条件下ETEC K88ac的生长量比静止培养多1倍左右，MRHA效价分别为2~6和2~5；60℃加热—磁力搅拌法及等电点沉淀法可以获得较高产量纯化的黏附素蛋白，平均每升菌液可收获1.5毫克，在SDS—PAGE中呈一条带，分子量约为28KDa；用纯化的ETEC K88ac黏附素蛋白免疫蛋鸡在二免1周后卵黄中的抗体明显升高，4周达到高峰(1：2560)，并持续到13周后才开始缓慢下降。因此，TSB液体培养基振荡培养有利于K88ac的繁殖及其黏附素的生长，60℃加热—磁力搅拌法及等电点连续沉淀法可以获得较高产量纯化的K88ac黏附素蛋白，适用于实验室及小规模黏附素生产。K88ac黏附素可以诱导蛋鸡长时间，高水平卵黄抗体的产生。喷雾干燥(140℃进气，65℃出气)对卵黄抗体活性无影响，加工前后抗体滴度均为1：1280；卵黄抗体粉在90℃干热条件下处理5分钟后抗体效价下降50％，而90℃湿热条件下处理5分钟抗体效价无明显变化，但处理15分钟后下降75％；经过1小时的干热或湿热处理，抗体均基本失活；4℃和常温下放置半年对卵预防仔猪腹泻外源抗体一卵黄抗体研究黄抗体粉活性无影响;因此，喷雾干燥是加工卵黄抗体粉的适宜加工方式，喷雾千燥卵黄抗体粉具有良好的加工和储藏性能。3.应用细菌体外粘附试验研究了卵黄抗体预防仔猪ETEC感染性腹泻的机制，并通过饲养试验研究了卵黄抗体预防断奶仔猪腹泻的效果。结果表明:在体外试验中，无抗体的对照组平均每个上皮细胞勃附16.4个K88aC;抗体效价分别为1:640和1:1280的卵黄抗体较对照组均极显著抑制了K88ac对肠上皮细胞的勃附作用 (p<0.01)，平均每个上皮细胞分别仅勃附8.5和2.4个细菌，二者之间也存在极显著差异(p<0 .01);抗体效价为l:320的抗体组相对对照组有一定抑制细菌粘附作用，但差异不显著(p=0.15)。90头30士2日龄断奶仔猪随机分成‘4组，每组分3栏饲养，公母各半，采用单因子设计，对照组日粮采用玉米一豆粕典型日粮，在对照组日粮中分别添加0.5%、1%、2%的喷雾千燥卵黄粉形成试验一组、试验二组和三组的试验日粮。添加卵黄抗体粉虽然对仔猪的平均日增重和采食量均没有显著影响 (p>0.05)，但添加1%卵黄抗体粉显著降低了料肉比和腹泻率(p<0.05)，同时对仔猪的后期生长有一定促进作用。更多还原

【Abstract】 It has been well recognized that ETEC was the major pathogenic Escherichia coli that caused piglet diarrheal in pig farm .A polymerse chain reaction (PCR) technology was developed to detect ETEC K88 and its variants (ab, ac, ad). The presence of the K88 and its variants (ab, ac and ad) in some pig farms around Wuhan were investigated by PCR methology, and the K88ac was confirmed the predominant variants in Wuhan area. Through the research of ETEC K88ac cultivation, fimbriae extraction, immune reponse and egg-yolk processing character. An appropriated way of producing anti-K88ac fimbriae egg-yolk antibodies was established. The active mechanism and efficiency of egg yolk antibodies was evaluated by in vivo and in vitro experiments. The results were as follows:1. A polymerse chain reaction (PCR) technology to detect ETEC K88 and its variants (ab, ac, ad) was developed to investigate the presence of the K88 and its variants (ab, ac and ad) in some pig farms around Wuhan. K88ab, K88ac and K88ad by PCR with special primers showed target bands. One of 291 E. coli, which were not deteced K88 by slide agglutination, were checked out to be K88 (K88ac) by PCR. 23(10.1%) K88 were found in 227 feces from diarrheic piglets in some pig farms of Wuhan area, and all contained genes for K88ac. The result suggested that the PCR methodology be a quick, sensitive and specific for the detection and epidemiological investigation of ETEC K88 and its variants (ab, ac, ad). K88ac is the predominant K88 variant associate with diarrhea in piglets of Wuhan area.2. The effect of shaking cultivation and resting cultivation on the production of ETEC K88ac and its fimbriae was compared. An appropriated method of separating-purifying K88ac fimbrial was developed and the immune response of ETEC K88ac fimbriae to hens was studied. A feasible processing of egg yolk antibodies powders was established and its stability and storage performance were also studied. The result suggested that the output of ETEC K88ac with shaking cultivation be about once more than that of resting cultivation. Fimbrial MRHA titer of shaking cultivation and resting cultivation were 26 and 25 respectively. The extraction of ETEC K88ac fimbriae, separated and purified by 60 ℃-heating-magnetism-stir and isoelectric point deposition was 1.5mg/L. The purified fimbriae showed one band in SDS-PAGE with a molecular mass of about 28 KDa. Titerof egg-yolk antibodies rose quickly in a week after the second immune and achieved the top at the 4th week (1:2560), then fell slowly at the 13th week. The results confirmed that shaking cultivation of K88ac with TSB, separating-purifying fimbriae 60℃-heating-magnetism-stir and isoelectric point deposition was a suitable way for the extration of ETEC K88ac fimbriae in the laboratory or on the small scale. Activity of egg-yolk antibodies was not inflenced by spray drying. liter of egg-yolk antibodies did not change with the treatment of 90℃ wet-hot for 5 minutes, but declined 75% for 15 minutes and 50% with the treatment of 90℃ hot-air for 5 minutes; 90℃ wet-hot or hot-air treatment for 1 hour would inactive the antibodies. The antibodies would not be destroyed under the 4℃ and normal atmospheric temperature in half a year. The results suggested that the spray drying be a suitable processing for egg yolk antibodies powders. The egg yolk antibodies powders had good processing stability and storage performance.3. The active mechanism and efficiency of egg yolk antibodies was evaluated by in vivo and vitro experiments. The results suggested that the average numbers of adherent bacterial cells per enteroepithelial cell for control without antibodies was 16.4, the egg-yolk anti-K88ac fimbrial antibodies with liter 1:640 (8.5 bacterial cells per epithelial cell) and 1:1280 (2.4 bacterial cells per epithelial cell) could significantly inhabited the adhesion of ETEC K88ac to piglet epithelial cell (p<0.01) ; Antibodies with titer 1:320 had no significant effect on the adhesion of ETEC K88ac (p>0.05) .90 28-32-days-old piglets更多还原