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[PubMed] [Google Scholar] 14. rectal heat throughout the study period. We attempted to detect rickettsial DNA from peripheral blood and aspiration samples from kidney and spleen by nested PCR, but all samples examined were unfavorable. The control doggie lacked detectable titers to antigen on day 14, while positive antibodies to were detected in all four experimentally infected dogs, with titers of 1 1:160 to 1 1:80. In the epidemiologic survey, 24 (1.8%) of the 1,363 dogs examined throughout Japan had antibodies against and are obligate intracellular, Gram-negative bacteria. Several species cause disease in humans and other animals and are distributed worldwide. This genus comprises the spotted fever group (SFG) rickettsiae and the typhus group (TG) rickettsiae (18). In Japan, and are confirmed vectors of (9). Isolation of from wild mice indicated that mice are a mammalian reservoir for the pathogen (22). Dogs have also been thought to be a mammalian reservoir for have been detected in canine serum (6, 8). A recent epidemiologic study revealed that antibodies against were detected in 20 of 1 1,207 dogs in Japan (21). Dogs are often uncovered to a large number of tick species, depending on the distribution of these arthropod vectors in the environment (19). They most likely have an increased risk of tick bites compared to humans, due at least in part to their activity in woodland and bush areas. However, no previous studies have resolved whether dogs that are naturally infected with readily exhibit detectable clinical indicators or whether dogs are important sources of infection. The present study evaluated pathogenesis and the reservoir potential of dogs through experimental inoculation and an epidemiologic survey. MATERIALS AND METHODS Experimental inoculation of in dogs. Five 6-month-old male beagle dogs weighing between 8 and 11 kg were provided by a commercial breeder. They DS18561882 were housed indoors and managed in a biosafety level P3 animal care facility, as dictated by the Animal Care and Use Committee regulations of Obihiro University or college of Agriculture and Veterinary Medicine (permission Rabbit Polyclonal to Notch 2 (Cleaved-Asp1733) number 20-86). During a 2-week acclimatization period, we monitored clinical signs, food consumption, and rectal temperatures on a daily basis. During that DS18561882 time 2 milliliters of blood was obtained from the cephalic vein of each doggie. Complete blood counts were performed using EDTA-anticoagulated blood. All hematological parameters fell within normal ranges (1). Extracted serum was subjected to an indirect immunofluorescence assay (IFA) to confirm the absence of reactive antibodies to strain Aoki (day 0). Each inoculum dose contained approximately 2 107 infected cells suspended in minimum essential medium (MEM) with 1% fetal bovine serum (FBS). The control doggie was inoculated with the same amount of uninfected L929 cells suspended at the same concentration. Clinical indicators and food consumption were monitored, and rectal temperatures were recorded twice a day. Blood from your cephalic vein was collected in EDTA tubes every 2 days from day 0 until day 14. Each blood sample was subjected to hematological analysis and PCR. Peripheral blood smears were examined for neutrophil counts and evidence of DS18561882 platelet aggregation. Sera were collected and IFA was conducted on day 14. Kidney and spleen samples were aspirated for PCR use on day 14. IFA. Detection of antibodies against was carried out using IFA as explained previously (21). Serum samples were screened at a 1:20 dilution in phosphate-buffered saline (pH 7.2) with 0.5% Tween 20 (PBST). Fluorescein isothiocyanate (FITC)-labeled rabbit anti-canine IgG (Fc) conjugates (Rockland Inc., Gilbertsville, PA) or FITC-labeled rabbit anti-feline IgG (Fc) conjugates (Rockland Inc.) were used as secondary antibodies for the IFA. Reactive antibodies were then detected using a fluorescence light microscope. Samples that reacted with the antigen at the screening dilution were then.