Interspecific Competition Between Rickettsiae in Ticks

描述（由申请人提供）：尽管蜱传斑点热群立克次体的多样性和广泛分布，现场和实验室研究都表明，虽然蜱虫能够获得多种立克次体感染，但大多数蜱虫调查显示，在受感染的蜱虫中只存在一种立克次体。在之前的资助期间，我们已经表明，稳定的山立克次体感染阻断了蜱卵巢的维持和第二种新引入的立克次体的传播。在我们之后的研究提供了经验证据，表明细胞进入可能是蜱卵巢系统多次立克次体定植的限制步骤。在本应用中，我们将重点关注通过经卵巢传播调节立克次体维持的潜在机制，特别是蜱虫基因产物导致经卵巢干扰现象。特别是，我们将测试假设斑点热组立克次体在宿主细胞进入或通过竞争中相互竞争蜱卵巢生态位。本研究的目的是：(1)确定细胞进入或生长是否为防止卡丘氏r.p ocockii感染的andersonderentor蜱双重立克次体感染的限制步骤。对卵巢作为生态位的竞争将通过分子或通过对表达GFP或mCherry的立克次体感染细胞的目视检查来测量。在此目的下，我们还将研究立克次体致病性程度是否与andersoni中致病性立克次体对蜱卵巢维持作用的降低相关；(2)研究在感染了共生的孔雀或蒙塔那氏蜱的蜱中，免疫活性是否持续保持在较高水平，从而阻止卵巢中立克次体的重复感染。我们将测试假设，免疫反应持续在一个较高的水平在蜱构成感染立克次体，这有助于干扰现象的表型。利用qRT-PCR技术，我们将比较防御素、溶菌酶和库尼茨型蛋白酶抑制剂（DvKPI）在未感染蜱和孔雀r.p ockii或蒙塔那r.p ontanensis构成型感染蜱中的表达水平。然后，我们将对具有立克次体感染的蜱进行互惠挑战，并比较防御基因与未挑战的构成性感染蜱的表达水平。

DESCRIPTION (provided by applicant): Despite the diversity and wide distribution of tick-borne spotted fever group rickettsiae both field and laboratory studies have demonstrated that while ticks are capable of acquiring multiple rickettsial infections most often tick surveys revealed the presence of only a single rickettsial species in infected tick. During previous funding period, we have shown that stable Rickettsia montanensis infection blocks the tick ovarian maintenance and transmission of a second newly introduced rickettsiae. Studies following ours have offered empirical evidence that cell entry may be a limiting step to multiple rickettsial colonization of tick ovarial system. In this application we are focusing on underlying mechanisms that regulate rickettsial maintenance via transovarial transmission and in particular the tick gene products contributing to the transovarial interference phenomenon. In particular, we will test the hypothesis that spotted fever group rickettsiae outcompete each other for the tick ovarian niche during host cell entry or through competition. The aims of the proposed research are: (1) To determine if cell entry or growth is the limiting step preventing dual rickettsial infection in R. peacockii - infected Dermacentor andersoni ticks. Competition for the ovary as a niche will be measured molecularly or via visual inspection of cells infected with GFP- or mCherry- expressing rickettsiae. Under this aim we will also investigate whether the degree of rickettsial pathogenicity correlates with reduced role for tick ovarian maintenance of virulent R. rickettsii in D. andersoni; and (2) To investigate if immune activity persists at a heightened level in ticks with a symbiotic R. peacockii- or R. montanensis-infected ticks thereby prohibiting rickettsial superinfection in the ovaries. We will test the hypothesis that the immune response persists at a heightened level in ticks constitutively infected with rickettsiae, which contributes to the interference phenomenon phenotype. Using qRT-PCR, we will compare defensin, lysozyme and a kunitz-type protease inhibitor (DvKPI) expression levels in uninfected and R. peacockii or R. montanensis-constitutively infected ticks. We will then perform a reciprocal challenge of ticks with a constitutive rickettsia infection and compare the expression level of defense genes to unchallenged constitutively infected ticks. PUBLIC HEALTH RELEVANCE: Nine different tick-transmitted spotted fever group rickettsiae are recognized in the United States including the highly pathogenic Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF). RMSF is characterized by headache, fever, myalgia, nausea and vomiting early in the illness and if untreated it could progress to multi-organ failure and death. While the human RMSF cases more than doubled since 2000 overall the U.S. Rickettsia/tick surveys indicate that R. rickettsii is less prevalent in vector ticks and in most cases the positive ticks harbor nonpathogenic rickettsiae such as R. peacockii, R. montanensis and R. rhipicephali. The low prevalence of R. rickettsii in positive ticks has generated intriguing questions, thus leading us to investigate the consequences and epidemiological significance of multiple rickettsial infections in ticks. In particular, our research focused on underlying mechanisms that regulate rickettsial maintenance in ticks.