鸭疫里氏杆菌分离株抗原性分析及乳胶凝集试验的建立
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摘要
鸭疫里氏杆菌（Riemerellaanatipestifer,RA）病是由鸭疫里氏杆菌引起家鸭、火鸡、鹅和其它多种禽类的一种接触性传染病，又名称鸭传染性浆膜炎；感染鸭表现主要临床症状是精神沉郁、共济失调、角弓反张和淡绿色下痢等；病理变化主要有纤维素性心包炎、肝周炎、气囊炎、干酪性输卵管炎、脑膜炎、结膜炎、关节炎和败血症等。该病的发病率和死亡率很高，血清型众多，而各个血清型之间缺乏免疫保护，给该病的防治工作带来困难，已对养鸭业造成了巨大的经济损失。
从黑龙江、山东和广东疑似RA感染的鸭场分离到4株细菌，经菌落形态观察、染色镜检、生化鉴定确定为RA，分别命名为HLG1、SD0901、SD0902和GD0901；利用平板凝集试验对RA进行血清型鉴定，其中HLG1、SD0901和GD0901是血清1型菌株。药敏试验表明，4株分离菌株对嗯诺沙星、氧氟沙星和先锋霉素V相对敏感，但对卡那霉素和庆大霉素均耐药。PCR扩增了RA的外膜蛋白A（OmpA）基因，克隆测序结果显示，4个RA分离株的ompA基因全长均为1164bp，编码387个氨基酸，在核苷酸水平上的同源性为89%~100%。将HLG1分离株5×108CFU，颈部皮下接种SPF鸭，病死的SPF鸭的心、肝、脾、肺、脑和肾具有典型的病变，并在人工感染鸭的组织中分离到RA细菌。根据RADSM15868株（登录号为NC014738）全基因组序列设计了3对引物，PCR成功的
扩增了HLG1株编码的脂蛋白（Riean0791）、外膜脂蛋白（Riean1110）和外膜脂蛋白（Riean1852）的目的基因，克隆至pMD18-T载体中测序结果表明，Riean0791、Riean1110和Riean1852基因的ORF分别为1404bp、1437bp和1374bp。分别将Riean0791、Riean1110和Riean1852基因克隆到pPRO-EX-Htb载体构建原核表达质粒，转化至大肠杆菌BL21（DE3）plysS感受态细胞后，经IPTG诱导和SDS-PAGE分析结果表明，重组蛋白Riean0791、Riean1110和Riean1852的分子量分别约为55ku、56ku和54ku，均以包涵体形式的表达。Westernblot结果证实，重组蛋白Riean0791、Riean1110和Riean1852均与RA阳性血清发生特异性反应，具有很好的抗原性。纯化的重组蛋白Riean0791、Riean1110和Riean1852作为免疫原对SPF鸭进行了免疫试验证实，两次免疫后，Riean0791、Riean1110和Riean1852免疫组的SPF鸭均产生了较高水平的抗体；对HLG1株的免疫保护率分别为5/6、0/6和2/6。提取HLG1株外膜蛋白，利用制备的Riean0791、Riean1110和Riean1852的血清Westernblot证实，Riean0791、Riean1110和Riean1852蛋白主要位于细菌的膜蛋白中。以纯化的Riean0791蛋白为致敏抗原，初步建立了检测RA血清中的特异性抗体的乳胶凝集试验。对乳胶凝集试验的条件进行优化的结果表明，最佳羧化乳胶浓度为1%，最佳偶联蛋白初始量为200μg，最佳偶联时间为6h。以最佳条件制备的乳胶凝集试剂可与RA阳性鸭血清出现
肉眼可见的较强的凝集反应；与PBS、BBS、鸭阴性血清、鸭大肠杆菌、鸭多杀性巴氏杆菌、鸭肠炎病毒和鸭肝炎病毒等阳性血清均无凝集反应，建立的乳胶凝集试验具有较强的特异性、敏感性和重复性。该建立的RA的乳胶凝集试验与OmpA间接酶联免疫吸附试验的符合率为91.04%。本研究中RA的分离鉴定和建立的乳胶凝集试验，为临床鸭群RA的检测提供了一种快速的诊断方法，也可为更好地预防和控制RA提供技术保障。
Riemerella anatipestifer disease, also called duck infectious serositis, is a contagious disease of many types of poultry, such as domestic ducks, turkeys, geese and other many kinds of birds, caused by Riemerella anatipestifer(RA). Infected domestic ducks show the main clinical symptoms which were depression, ataxia, opisthotonos, dysentery. The disease is accompanied with pathological changes, such as fibropericarditis, perihepatitis, salpingitis, meningitis, conjunctivitis, arthritis, septicemia and so on. RA causes high morbidity and mortality with various serotypes, but the lack of cross-protection between different serotypes causes difficulties to the prevention and control of the disease, RA causes important economic losses to the duck industry.
Four bacteria were isolated from affected ducks in duck farms from Heilongjiang, Shandong, Guangdong province, respectively,and identified as Riemerella anatipestifer,designated HLGl, SD0901, SD0902and GD0901, according its morphology, serologic test and biochemical characteristics. The four isolates were high sensitive to enrofloxacin, ofloxacin and cephalosporin V, but resistive to kanamycin and gentamycin. The cloning and sequencing of ompA genes of the isolates showed that ompA gene was1164bp in length, which shared89%to100%similarity with other RA reference strains, Furthermore, SPF ducks were subcutaneously injected in cervical part at a dosage of5X108CFU of isolate HLGl and showed the clinical signals of RA infection and pathological changes in heart, liver, spleen, lung, brain and kidney.
The Riean0791, Riean1110and Riean1852genes of HLGl strain of Riemerella anatipestifer were amplified using3pairs of primers and cloned into pMD18-T vector. Sequencing showed that lengths of ORFs encoding Riean0791, Riean1100and Riean1852were1404bp,1437bp and1374bp, respectively. Riean0791, Riean1110and Riean1852were cloned into pPRO-EX-Htb to construct the recombinant plasmids pHTb-Riean0791, pHTb-Riean1110and pHTb-Riean1852. The recombinant plasmids were transformed into the E.coli BL21(DE3) plysS and induced by IPTG. SDS-PAGE showed that the rRiean0791, rRiean1110and rRiean1852were about55ku,56ku and54ku and in the form of inclusion body. Western blot analysis was indicated that rRiean0791, rRiean1110and rRiean1852were recognized by the positive serum of RA. SPF ducks were immunized with rRiean0791, rRiean1110and rRiean1852and produced high level of antibody titers by ELISA and immune protective rate were5/6,0/6and2/6, respectively. Western blot analysis showed that native Riean0791, Riean1110and Riean1852protein of HLGl strain was mainly in membrane protein of bacteria by positive duck serum of immunized by rRiean0791, rRiean1110and rRiean1852.
A latex agglutination test based on rRiean0791was constructed to detect specific antibodies of RA. The optimization conditions were that the concentration of carboxylated latex was1%, the quantity of rRiean0791was200μg and the time was6hour. LAT had good reaction with RA positive serum apparently, and showed no reaction with PBS, BBS, negative duck serum and other positive serum. The LAT had better specificity, sensitivity and repetitiveness. The agreement rate between RA latex agglutination test and indirect ELISA was91.04%. In this study, isolation and identification of RA from ducks and construction of latex agglutination test provides the method to diagnosis and monitoring SPF ducks and a rapid diagnostic method to prevent and control RA in field.
从黑龙江、山东和广东疑似RA感染的鸭场分离到4株细菌，经菌落形态观察、染色镜检、生化鉴定确定为RA，分别命名为HLG1、SD0901、SD0902和GD0901；利用平板凝集试验对RA进行血清型鉴定，其中HLG1、SD0901和GD0901是血清1型菌株。药敏试验表明，4株分离菌株对嗯诺沙星、氧氟沙星和先锋霉素V相对敏感，但对卡那霉素和庆大霉素均耐药。PCR扩增了RA的外膜蛋白A（OmpA）基因，克隆测序结果显示，4个RA分离株的ompA基因全长均为1164bp，编码387个氨基酸，在核苷酸水平上的同源性为89%~100%。将HLG1分离株5×108CFU，颈部皮下接种SPF鸭，病死的SPF鸭的心、肝、脾、肺、脑和肾具有典型的病变，并在人工感染鸭的组织中分离到RA细菌。根据RADSM15868株（登录号为NC014738）全基因组序列设计了3对引物，PCR成功的
扩增了HLG1株编码的脂蛋白（Riean0791）、外膜脂蛋白（Riean1110）和外膜脂蛋白（Riean1852）的目的基因，克隆至pMD18-T载体中测序结果表明，Riean0791、Riean1110和Riean1852基因的ORF分别为1404bp、1437bp和1374bp。分别将Riean0791、Riean1110和Riean1852基因克隆到pPRO-EX-Htb载体构建原核表达质粒，转化至大肠杆菌BL21（DE3）plysS感受态细胞后，经IPTG诱导和SDS-PAGE分析结果表明，重组蛋白Riean0791、Riean1110和Riean1852的分子量分别约为55ku、56ku和54ku，均以包涵体形式的表达。Westernblot结果证实，重组蛋白Riean0791、Riean1110和Riean1852均与RA阳性血清发生特异性反应，具有很好的抗原性。纯化的重组蛋白Riean0791、Riean1110和Riean1852作为免疫原对SPF鸭进行了免疫试验证实，两次免疫后，Riean0791、Riean1110和Riean1852免疫组的SPF鸭均产生了较高水平的抗体；对HLG1株的免疫保护率分别为5/6、0/6和2/6。提取HLG1株外膜蛋白，利用制备的Riean0791、Riean1110和Riean1852的血清Westernblot证实，Riean0791、Riean1110和Riean1852蛋白主要位于细菌的膜蛋白中。以纯化的Riean0791蛋白为致敏抗原，初步建立了检测RA血清中的特异性抗体的乳胶凝集试验。对乳胶凝集试验的条件进行优化的结果表明，最佳羧化乳胶浓度为1%，最佳偶联蛋白初始量为200μg，最佳偶联时间为6h。以最佳条件制备的乳胶凝集试剂可与RA阳性鸭血清出现
肉眼可见的较强的凝集反应；与PBS、BBS、鸭阴性血清、鸭大肠杆菌、鸭多杀性巴氏杆菌、鸭肠炎病毒和鸭肝炎病毒等阳性血清均无凝集反应，建立的乳胶凝集试验具有较强的特异性、敏感性和重复性。该建立的RA的乳胶凝集试验与OmpA间接酶联免疫吸附试验的符合率为91.04%。本研究中RA的分离鉴定和建立的乳胶凝集试验，为临床鸭群RA的检测提供了一种快速的诊断方法，也可为更好地预防和控制RA提供技术保障。
Riemerella anatipestifer disease, also called duck infectious serositis, is a contagious disease of many types of poultry, such as domestic ducks, turkeys, geese and other many kinds of birds, caused by Riemerella anatipestifer(RA). Infected domestic ducks show the main clinical symptoms which were depression, ataxia, opisthotonos, dysentery. The disease is accompanied with pathological changes, such as fibropericarditis, perihepatitis, salpingitis, meningitis, conjunctivitis, arthritis, septicemia and so on. RA causes high morbidity and mortality with various serotypes, but the lack of cross-protection between different serotypes causes difficulties to the prevention and control of the disease, RA causes important economic losses to the duck industry.
Four bacteria were isolated from affected ducks in duck farms from Heilongjiang, Shandong, Guangdong province, respectively,and identified as Riemerella anatipestifer,designated HLGl, SD0901, SD0902and GD0901, according its morphology, serologic test and biochemical characteristics. The four isolates were high sensitive to enrofloxacin, ofloxacin and cephalosporin V, but resistive to kanamycin and gentamycin. The cloning and sequencing of ompA genes of the isolates showed that ompA gene was1164bp in length, which shared89%to100%similarity with other RA reference strains, Furthermore, SPF ducks were subcutaneously injected in cervical part at a dosage of5X108CFU of isolate HLGl and showed the clinical signals of RA infection and pathological changes in heart, liver, spleen, lung, brain and kidney.
The Riean0791, Riean1110and Riean1852genes of HLGl strain of Riemerella anatipestifer were amplified using3pairs of primers and cloned into pMD18-T vector. Sequencing showed that lengths of ORFs encoding Riean0791, Riean1100and Riean1852were1404bp,1437bp and1374bp, respectively. Riean0791, Riean1110and Riean1852were cloned into pPRO-EX-Htb to construct the recombinant plasmids pHTb-Riean0791, pHTb-Riean1110and pHTb-Riean1852. The recombinant plasmids were transformed into the E.coli BL21(DE3) plysS and induced by IPTG. SDS-PAGE showed that the rRiean0791, rRiean1110and rRiean1852were about55ku,56ku and54ku and in the form of inclusion body. Western blot analysis was indicated that rRiean0791, rRiean1110and rRiean1852were recognized by the positive serum of RA. SPF ducks were immunized with rRiean0791, rRiean1110and rRiean1852and produced high level of antibody titers by ELISA and immune protective rate were5/6,0/6and2/6, respectively. Western blot analysis showed that native Riean0791, Riean1110and Riean1852protein of HLGl strain was mainly in membrane protein of bacteria by positive duck serum of immunized by rRiean0791, rRiean1110and rRiean1852.
A latex agglutination test based on rRiean0791was constructed to detect specific antibodies of RA. The optimization conditions were that the concentration of carboxylated latex was1%, the quantity of rRiean0791was200μg and the time was6hour. LAT had good reaction with RA positive serum apparently, and showed no reaction with PBS, BBS, negative duck serum and other positive serum. The LAT had better specificity, sensitivity and repetitiveness. The agreement rate between RA latex agglutination test and indirect ELISA was91.04%. In this study, isolation and identification of RA from ducks and construction of latex agglutination test provides the method to diagnosis and monitoring SPF ducks and a rapid diagnostic method to prevent and control RA in field.
引文
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