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.