Jpn. J. Med. Sci. Biol., 45, 81-86, 1992. Short Communication ISOLATION OF SPOTTED FEVER GROUP RICKETTSIA FROM APODEMUS SPECIOSUS IN AN ENDEMIC AREA IN JAPAN Seigo YAMAMOTO, Chiharu MORITA1 and Kimiyuki TSUCHIYA2 Miyazaki Pre fectural Institute for Public Health and Environment, Gakuen Kibanadai, Miyazaki-shi, Miyazaki 889-21, 1Department of Veterinary Science, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141, and 2Experimental Animal Center, Miyazaki Medical College, Kiyotake-cho, Miyazakigun, Miyazaki 889-16 (Received February 3, 1992. Accepted April 27, 1992) SUMMARY: A spotted fever group rickettsia was isolated from Apodemus mice captured near the site where a person had been infected in Miyazaki Prefecture. Antigenic characteristics of the isolate designated as strain TO-1 were compared with those of Rickettsia japonica (strain YH) and Rickettsia montana (ATCC VR611) by the indirect fluorescent antibody test with 14 serum specimens obtained from Apodemus speciosus, immune rat antisera against the strains TO-1 and YH, and three patients' sera. The titers of these sera measured with strain TO-1 were identical to those with strain YH, suggesting the antigenic similarity between these two organisms. It was also suggested that Apodemus speciosus is likely to be an important vertebrate host for a spotted fever group rickettsia in Japan. The existence of spotted fever group (SFG) rickettsiosis of humans in Japan was revealed in 1985 (1,2), and after this first recognition, patients have been reported from several areas, especially from the southern area, in Japan (3-8). In 81
Table I. Antibody titers of Apodemus speciosus sera YH: R ickettsia japonica, VR611: Rickettsia montana. 1) Titration was performed by the indirect IFA test and the titers are expressed as the reciprocal of the highest serum dilution that was positive. 1986, Uchida et al. (9) succeeded in isolating the causative rickettsia from a patient on Shikoku Island. Since the antigenic characteristics of SFG rickettsiae isolated from the blood of patients differed from those of the known SFG rickettsiae, the taxonomic name Rickettsia (R.) japonica, with strain YH as the type strain, was approved for the new isolates (10). In Japan, many people were affected in fields where Apodemus mice, especially Apodemus speciosus, are usually the dominant species of fauna (11). Recently, we reported a high incidence of antibodies to SFG rickettsiae in Apodemus mice in an endemic area (12). In this study, we attempted to isolate SFG rickettsia from Apodemus mice. 82
Table II. Antibody titer of immune rat sera 1) The same as Table I. This communication is the first report on isolation of a SFG rickettsia from rodent in Japan. The strains of SFG rickettsiae used were R. japonica, strain YH, and a strain of R. montana, ATCC VR-611. The isolate designated as strain TO-1 is described in the text. Vero-E6 cells provided by Dr. J. McCormick, Special Pathogens Branch, CDC, Atlanta, GA were used throughout the study. The antigenic characteristics of strain TO-1 were compared with those of R. japonica and R. montana by the indirect fluorescent antibody (IFA) test with Apodemus speciosus sera, immune rat sera, and patients' sera. Fourteen serum specimens of Apodemus speciosus were described previously (12): from one of Apodemus mice, strain TO-1 was isolated. Four-week old Wister rats (Shizuoka Experimental Animal Center, Hamamatsu-shi, Shizuoka) were inoculated subcutaneously with strain TO-1 or strain YH. Serum samples were collected 3, 4, and 6 weeks after inoculation. Sera from three patients, who showed symptoms of spotted fever like-illness in Miyazaki Prefecture, were used. The methods for the IFA test and preparation of antigens were described previously (6). In the IFA test, fluorescein isothiocyanate-conjugated anti-rat IgG goat serum (Cappel Lab., Cochranville, PA) and anti-human IgG and 1gM (ć- chain specific and Đ-chain specific) goat sera (Tago Inc., Burlingame, CA) were employed. 83
Table III. Antibody titers of sera from patients in Miyazaki Prefecture 1) The same as Table I. Each spleen of 12 out of 14 Apodemus speciosus captured near the site where a person had been infected (the patient is designated as B in Table III) was minced with a pestle in Eagle's minimum essential medium without antibiotics and phenol red (Nissui Pharmaceutical Co. Ltd., Tokyo) supplemented with 2% newborn calf serum. Each supernatant fluid after centrifugation of the homogenate at 2,000 rpm for 10 min was inoculated into Vero-E6 cell monolayers. One out of the 12 cultures inoculated with the spleen homogenate showed cytopathic effects (CPE) in 7 days after inoculation. After three serial passages in Vero-E6 cells, CPE appeared within 4 days after inoculation. About 80% of the cells contained the rickettsial particles showing specific fluorescence by the IFA test with a patient serum. The isolate was designated as strain TO-1. The antibody titers of the sera from Apodemus speciosus are shown in Table I. Although the geometric mean of the antibody titers measured with R. montana was lower than that with strain TO-1 or YH (P<0.01 by the paired comparison test), there was no 84
difference between the geometric means of the titers measured with strains TO-1 and YH. The antibody titers of the immune rat sera are shown in Table II. Although the antibody titers of rat sera to R. montana were lower than those to strain TO-1 or YH, no difference was demonstrated between the titers to strain TO-1 and those to strain YH. As seen in Table III, paired sera from three patients showed a significant rise in the antibody titers to strains TO-1 and YH, which were identical to each other. We reported that Apodemus speciosus seemed to be one of the important field vertebrate hosts of an SFG rickettsia from the results of comparative seroepidemiological surveys in an endemic area in Miyazaki Prefecture and in an nonendemic area in Kanagawa Prefecture (12). In this study, we successfully isolated an SFG rickettsia from Apodemus speciosus captured near the place where one of the patients had become infected. The isolate, strain TO-1, is likely to be antigenically similar to R. japonica, the isolate from humans in Japan. The results suggested also that Apodemus speciosus is likely to be an important vertebrate host of an SFG rickettsia in Japan. We thank Dr. T. Uchida, Department of Virology, School of Medicine, University of Tokushima, for his kind supply of the strain of R. japonica, and Dr. Y. Tuboi, Department of Virology and Rickettsiology, National Institute of Health, Tokyo, Japan, for providing VR611 of R. montana obtained from American Type Culture Collection. REFERENCES 1. Funato, T., Kitamura, Y, and Kawamura, A. (1988): Rickettsiosis of spotted fever group encountered in Muroto area of Shikoku, Japan -clinical and epidemiological features of 23 cases. J. Jpn. Assoc. Infect. Dis., 62,783-791 (in Japanese). 2. Mahara, F., Koga, K., Sawada, S., Taniguchi, T., Shigemi, F., Suto, T., Tsuboi, Y., Oya, A., Koyama, H., Uchiyama, T, and Uchida, T. (1985): The first report of the rickettsial infections of spotted fever group in Japan; three clinical cases. J. Jpn. Assoc. Infect. Dis., 59,1165-1172 (in Japanese). 3. Kaiho, I., Tokieda, M., Ohtawara, M., Uchiyama, T, and Uchida, T. (1988): Occurance of rickettsiosis of the spotted fever group in Chiba Prefecture in Japan. Jpn. J. Med. Sci. Biol., 41, 69-71. 85
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