Ticks transmit more types of microorganisms and infect more people with disease than any other arthropod in the U.S. Lyme disease is responsible for a resurgence of concern about tick, although Rocky Mountain spotted fever, Colorado tick fever and relapsing fever remain a continuous threat to the human population on a regional basis. The focus of our proposal is on the tick's salivary glands that facilitate disease transmission and produce t he antihemostasis, antiinflammatory and immunosuppressive agents, which control the bite site so the parasite can feed. Our overall objective is to characterize the primary salivary components that mediate the feeding-site environment. Understanding heir roles should provide fundamental insights into the disease transmission process and how the host's responses to the parasite are neutralized or even used to the tick's advantage. Until recently, progress towards characterization of organic salivary components was only modest, and based upon responses of in vitro assays to saliva or gland extracts. The primary rate- limiting steps has been that of tissue (salivary gland) abundance. Advances in molecular genetics technology now provide the opportunity to overcome this limitation. We have identified a form of calreticulin in two medically important tick species (feeding Amblyomma americanum & Dermacentor variabilis females), based upon complete sequencing of a cDNA, (69% deduced amino acid homology with rabbit skeletal muscle calreticulin) and positive immunoblotting of gland extracts and saliva with goat anti-rabbit calreticulin. Calreticulin is a highly conserved Ca2+ binding protein within non-muscle cell endoplasmic reticulum and smooth muscle sarcoplasmic reticulum. That the protein within non-muscle cell endoplasmic reticulum and smooth muscle sarcoplasmic reticulum. That the protein is likely to be synthesized for secretion is supported by sequence data as the ER retention signal (KDEL) is apparently not present; and, it is found in the saliva. Light and ultrastructural level immunolocalization of the protein and in situ hybridization studies of glands during morphogenesis will help correlate protein secretion with host-related responses, and determine where the protein is synthesized and located within the cell. Hemostasis (platelet aggregation) and vascular (smooth muscle contraction) assays will provide valuable information about the role of the gland protein as a secreted product. Ca2+-dependent and independent processes will be investigated. Characterization of calreticulin using these methods will present a significant advancement in describing when and where its is secreted, while beginning the process of defining its physiological significance in the feeding process. This understanding will be critical in understanding the parasite-host relationship and possibly disease transmission by ixodid ticks.
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