PDF(Pubmed)
Abstract：
Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA1,628-1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA1,628-1,899 was used as antigen coating on ELISA plates to capture FHA1,628-1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.
摘要：
肝弯曲菌是轻度肝病（SLD）的病因。SLD会在产蛋高峰及其附近引起产蛋鸡的大量生产损失和死亡率。我们以前开发了一种酶联免疫吸附测定(ELISA)，SLD-ELISA1，使用肝梭菌总蛋白和用空肠弯曲杆菌蛋白预吸收的血清检测鸟类血清中的肝梭菌特异性抗体。使用SLD-ELISA1实现的高特异性表明在感染的鸟类的血清中存在肝梭菌特异性抗体。然而,SLD-ELISA1中使用的一些试剂制备耗时且难以进行质量控制。这种理解导致寻找可以以重组形式用作免疫测定设计中的抗体捕获抗原的肝念珠菌特异性免疫原性蛋白。在这项研究中,结合生物信息学分析的免疫蛋白质组学方法，西方印迹,并使用LCMS/MS蛋白谱分析，和丝状血凝素粘附素（FHA）的片段，FHA1,628-1,899具有肝梭菌特异性抗原性。重组FHA1,628-1,899用作ELISA板上的抗原包被,以捕获感染鸟类血清中的FHA1,628-1,899特异性抗体。SLD-ELISA2，基于纯化的重组FHA片段，比SLD-ELISA1更具用户友好性和标准化，用于筛选对母鸡肝梭菌暴露的抗体反应。这项研究是首次报道在免疫测定中使用弯曲杆菌属的FHA，这也为研究FHA在肝梭菌发病机制中的作用及其作为候选疫苗的有效性开辟了未来的研究方向。
