Diagnosis and Pathobiology of Emerging Enterohepatic Helicobacter spp. in Mice

描述（由申请人提供）：我们很高兴有机会响应NCRR RFA-RR-10-005“实验动物中新发病原体的病理生物学”。在过去的15年中，8种新型小鼠肠肝幽门螺杆菌（EHS）已被正式命名，另外9种新物种（基于16S rRNA序列）已在实验室培养和鉴定。我们已经收集了，我们相信，是世界上最大的EHS培养集合，并准备表征这些小鼠菌株，既方便他们的诊断，也确定他们的致病潜力。肝嗜血杆菌是EHS的原型，引起持续的肠道感染，在世界各地的学术小鼠群体中普遍存在。肝芽胞杆菌感染可导致肝癌，在几种肝癌发生模型中被认为是一种强肿瘤启动子，并在免疫功能低下的小鼠中诱导炎症性肠病。EHS的存在混淆了许多研究，可以污染肿瘤细胞系，在免疫缺陷小鼠和免疫能力小鼠的特定品系中引起显著的发病率和死亡率，并且最近与小鼠群体的生殖失败有关。我们在2007年进行的一项研究表明，在全球学术环境中使用的小鼠中，85%感染了一种或多种EHS。然而，我们和其他人已经发现，大多数EHS不容易培养，没有特定的分子或血清学检测来诊断，也不知道这些H. spp是否具有致病性以及在什么条件下具有致病性。因此，我们打算开发敏感和特异性的分子检测方法来诊断小鼠EHS。我们期望开发一种微型微阵列芯片，用于研究界检测EHS并防止其在已建立的H. spp自由菌落中的定植。由于IL10-/-在我们的实验室和其他使用肝芽胞杆菌解剖IBD发病机制方面的效用，我们将采用该模型筛选新的H. spp的致病潜力，并确定新测序的H. spp中毒力基因的假定作用。这些结果将与我们用于预测肝芽胞杆菌毒力潜力的体外测试进行比较和验证。该项目的具体目标是：1)提供新型肠肝幽门螺杆菌（EHS）的形态学、遗传学和生化特征，并建立小鼠分离的EHS特异性检测方法；2)在IL10-/-小鼠模型中明确小鼠EHS体外毒力因子与体内致病潜能的关系，确定宿主免疫反应如何影响疾病进展；3)鉴定从啮齿动物和临床疾病患者分离的3种肠道幽门螺杆菌（Helicobacter spp.）以及肝炎嗜血杆菌（H. hepatcus）基因组序列中的毒力决定因素，并确定这些基因是否可在C57BL IL10-/-小鼠中诱导IBD。为了实现这些目标，我们组建了一个科学家团队，他们在过去十年中成功地合作研究了小鼠肝芽胞杆菌胃肠道疾病的发病机制。

DESCRIPTION (provided by applicant): We are excited about the opportunity to respond to the NCRR RFA-RR-10-005 "Pathobiology of Emerging Pathogens in Laboratory Animals". During the past 15 years, 8 novel enterohepatic Helicobacter spp. (EHS) in mice have been formally named, and 9 additional clusters (based on 16S rRNA sequences) of novel species have been cultured and identified in our lab. We have collected, what we believe, is the largest EHS culture collection available worldwide and are poised to characterize these murine strains, both to facilitate their diagnosis and also to establish their pathogenic potential. H. hepaticus, the prototype EHS, causes a persistent enteric infection and is prevalent in academic mouse colonies throughout the world. H. hepaticus infection causes liver cancer, is known to be a strong tumor promoter in several models of hepatocarcinogenesis, and induces inflammatory bowel disease in immunocompromised mice. The presence of EHS has confounded a number of studies, can contaminate tumor cell lines, causes significant morbidity and mortality in immunodeficient mice and selected strains of immune-competent mice, and has been recently associated with reproductive failures in mouse colonies. A 2007 study conducted by us indicated that 85% of mice used in academic settings worldwide are infected with one or more EHS. However the majority of EHS, we and others have identified, are not easily cultured, do not have specific molecular or serological assays for diagnosis, nor is it known whether and under what conditions these H. spp. have pathogenic potential. We therefore intend to develop sensitive and specific molecular assays to diagnose EHS in mice. We anticipate the development of a mini-microarray chip for use by the research community to detect EHS and prevent their colonization in established H. spp. free colonies. Because of the utility of the IL10-/- in our lab and others in using H. hepaticus to dissect etiopathogenesis of IBD, we will employ this model in screening novel H. spp. for pathogenic potential as well as determine the putative role of virulence genes in newly sequenced H. spp. These results will be compared and validated with in vitro tests we have used to predict virulence potential in H. hepaticus. The specific aims of the project are: 1) To provide morphologic, genetic, and biochemical characterization of novel enterohepatic Helicobacter species (EHS) and develop species specific assays to diagnose EHS isolated from mice; 2) To define the relationship of in vitro virulence factors of murine EHS to in vivo pathogenic potential in the IL10-/- mouse model and to determine how the hosts' immune responses influence disease progression; and 3) To identify and characterize virulence determinants in the genome sequences of 3 enteric Helicobacter spp. isolated from rodents and humans with clinical disease, as well as those of H. hepaticus and determine if these genes are operable in inducing IBD in C57BL IL10-/- mice. To accomplish these aims we have assembled a team of scientists who have successfully collaborated together for the last decade to study the etiopathogenesis of H. hepaticus gastrointestinal disease in mice.