Diagnosis and Pathobiology of Emerging Enterohepatic Helicobacter spp. in Mice

描述(申请人提供)：我们很高兴有机会对NCRR RFA-RR-10-005“实验动物中新出现的病原体的病理生物学”做出回应。在过去的15年中，发现了8个新的肠肝型螺旋杆菌。小鼠的EHS已被正式命名，并在我们实验室培养和鉴定了另外9个新物种的簇(基于16S rRNA序列)。我们已经收集了，我们相信，是世界上可用的最大的EHS培养样本，并准备对这些小鼠菌株进行特征鉴定，以方便它们的诊断和建立它们的致病潜力。肝型血吸虫是EHS的原型，会引起持续性的肠道感染，并在世界各地的学术小鼠群体中流行。肝吸虫感染会导致肝癌，已知在几种肝癌发生模型中是强烈的肿瘤促进剂，并在免疫功能低下的小鼠中诱导炎症性肠道疾病。EHS的存在扰乱了许多研究，可以污染肿瘤细胞系，在免疫缺陷小鼠和选定的免疫能力小鼠品系中导致显著的发病率和死亡率，最近还与小鼠群体中的繁殖失败有关。我们在2007年进行的一项研究表明，全世界在学术环境中使用的小鼠中有85%感染了一种或多种EHS。然而，我们和其他人已经确认，大多数EHS不容易培养，没有专门的分子或血清学检测来诊断，也不知道这些H.spp是否以及在什么条件下。具有致病潜能。因此，我们打算开发灵敏和特异的分子检测方法来诊断小鼠的EHS。我们预计将开发一种微型微阵列芯片，供研究界使用，以检测EHS并防止它们在已建立的H.spp中定植。自由殖民地。由于本实验室和其他实验室的IL10-/-在利用肝包虫研究IBD的发病机制方面的应用，我们将利用该模型来筛选新的H.spp。以及确定毒力基因在新测序的H.spp中的可能作用。这些结果将与我们用来预测肝片吸虫毒力潜力的体外试验进行比较和验证。该项目的具体目标是：1)提供新的肠肝螺杆菌(EHS)的形态、遗传和生化特征，并建立从小鼠分离的EHS的物种特异性诊断方法；2)在IL10-/-小鼠模型中确定EHS的体外毒力因子与体内致病潜能的关系，并确定宿主的免疫反应如何影响疾病进展；以及3)鉴定和表征3株肠道螺杆菌基因组序列中的毒力决定因素。从啮齿动物和有临床疾病的人以及肝炎病毒中分离出这些基因，并确定这些基因是否可用于在C57BLIL10-/-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炎性胃肠道疾病的病因。

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.