间接血凝方法检测副猪嗜血杆菌抗体

将副猪嗜血杆菌(Hps)4、5、13型分离菌株超声产物致敏经戊二醛和鞣酸处理的绵羊红细胞,建立了检测Hps抗体的间接血凝试验方法。对15种血清型Hps阳性血清进行检测,结果均为阳性,抗体效价达1∶2⁵~1∶2¹⁰,敏感性明显高于琼脂扩散试验。对其它16种病毒和细菌的阳性血清进行检测,结果除胸膜肺炎放线杆菌有凝集外,其余均为阴性。对620份临床血清进行检测,阳性率为67.58%。结果表明,该方法敏感性较高、特异性强,可用于Hps抗体水平的检测和流行病学调查。     

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.     

副猪嗜血杆菌耐药性研究进展

作者主要对国内外副猪嗜血杆菌耐药性的研究进展进行综述,基于国内外副猪嗜血杆菌的最新耐药性研究报道,揭示了当前副猪嗜血杆菌的耐药性趋势,并根据其试验结果证明了前人提出的治疗方案确实、可靠,指出了目前治疗副猪嗜血杆菌病的有效药物,为养猪生产中综合防制副猪嗜血杆菌病提供了参考和依据。     

副猪嗜血杆菌病流行病学及致病因子的研究进展

副猪嗜血杆菌（Haemophilus parasuis,Hps）是猪上呼吸道的一种常在菌,常引起猪的多发性浆膜炎、关节炎、脑膜炎,由该菌引起的病又称格拉泽氏病。随着世界养猪业的发展,该病已成为全球范围内影响养猪业的一种重要细菌性疾病。为了更全面的了解副猪嗜血杆菌病的研究现状,笔者对该病的病原学、流行病学、致病因子的研究进展作一综述。     

高致病性猪繁殖与呼吸综合征、猪圆环病毒病和副猪嗜血杆菌病混合感染的诊断与防制

目前在集约化养殖场的猪群中经常出现病毒病和细菌病的混合感染,严重影响中国养猪业的发展。2011年9月,广东某个千头母猪场的猪群大量发病,表现为高热、咳嗽、食欲下降、皮肤发绀、关节肿大等临床症状。通过流行病学调查、剖检、细菌分离鉴定、药敏试验、PCR和RT-PCR等实验室检测最终确诊为高致病性猪繁殖与呼吸综合征、猪圆环病毒病和副猪嗜血杆菌病的混合感染,在此基础上提出和实施了该病的防控方案。     

锦州地区副猪嗜血杆菌分离鉴定与药敏试验

从锦州市某猪场7份疑似副猪嗜血杆菌病料中分离到4株革兰氏阴性细小杆菌,进行了细菌培养特性试验、纯化后镜检、V因子需要试验、卫星现象检查、生化试验、PCR鉴定、药物敏感试验、细菌的致病性试验。结果表明,分离的菌株为副猪嗜血杆菌,4株分离菌株对林可霉素、环丙沙星、强力霉素、头孢噻肟、氧氟沙星和恩诺沙星均较敏感;对卡那霉素、复方新诺明、克林霉素、金霉素、四环素均为耐药;对链霉素呈现不同程度的敏感性。     

副猪嗜血杆菌血清型和外膜蛋白P5基因型多样性的研究

副猪嗜血杆菌(Haemophilus parasuis,Hps)的血清型及基因型具有复杂多样性。本研究用间接血凝试验对某一猪场分离的Hps进行血清分型鉴定,并对部分菌株的外膜蛋白P5基因进行克隆测序和进化发育分析。试验结果显示,在21头病死猪不同部位共分离了42株Hps,其中 Hps血清5型17株(40.7%)、血清14型12株(28.5%)、血清4型1株(2.3%)和未定型12株(28.5%)。本试验中有5头猪同时感染2种不同血清型的Hps。21株Hps临床分离株分属5个不同基因序列型(STA～STE),其中STA有12株(12/21)为优势基因型;相同血清型的Hps分离株具有不同的STs型,来自同一头猪的Hps血清型和STs型也不相同。以上结果表明,同一猪群感染的Hps血清型和基因型存在明显的多样性,同一头猪感染Hps的血清型和基因型也存在多样性。本研究为进一步揭示猪群感染Hps的复杂性,研究该病的感染与流行机制提供了有价值的参考。     

应用对流免疫电泳法鉴定胸膜肺炎嗜血杆菌血清型

本文建立的对流免疫技术可用于胸膜肺炎嗜血杆菌(H·T)的血清型鉴定。应用0.05M,PH8.0的巴比妥缓冲液,以自行制备的已知血清型兔免疫血清检测H·P菌浸出抗原,通电20—45分钟获得结果。本法比琼脂扩散法敏感8—12倍,克服2—ME试管凝集试验不能检测自身凝集菌株的缺点,不存在种间、血清型间交叉反应,是一种快速、敏感和特异的方法。     

副猪嗜血杆菌血清5型TbpA蛋白的表达及其免疫原性分析

为原核表达副猪嗜血杆菌血清5型TbpA蛋白,本研究利用特异性引物扩增tbpA基因,并将其克隆到pMD 18-T载体中进行序列测定.再将其亚克隆于pET-28a中构建重组表达质粒pET-tbpA.将其转化受体菌E.coli BL21 (DE3),经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约106 ku,并以包涵体形式存在.表达的重组蛋白经纯化后,免疫6周龄昆明小鼠制备免疫血清,ELISA分析表明制备的血清抗体效价在1∶3 200以上,表明TbpA具有良好的免疫原性.     

Cloning and Bioinformatics Analysis of espP Gene of Haemophilus parasuis

In order to evaluate whether Haemophilus parasuis espP gene could use as a vaccine candidate antigen or not,the espP gene was amplified by PCR using the specific primers that designed according to corresponding sequence getting form GenBank (accession No.:HAPS_1381) and then sent to sequence.After translating the DNA sequence into amino acid sequence,the signal peptide,hydrophilic region,linear B cell epitopes and 3D structure of Hps espP protein were predicted using multiple bioinformatics analysis.The results showed that the full length of Hps espP gene was 2 343 bp,encoding 781 aa.The first 23 aa were predicted as signal peptide.There were 12 hydrophilic regions and 12 B cell epitopes in the Hps espP protein.The 3D structure of C-terminal of the protein was consisted of a monomeric β-barrel containing 12 β-strands and a central pore.The study laid the foundation for further development of molecular vaccines based on espP protein against Hps infection.