Jpn. J. Infect. Dis., 61, 315-317, 2008

Short Communication Serological and Molecular Survey of Rickettsial Infection in Cattle and Sika Deer in a Pastureland in Hidaka District, , Jilintai, Nobutaka Seino, Kotaro Matsumoto, Daisuke Hayakawa1, Masatsugu Suzuki2, Hirosi Hata3, Seiji Kondo3, Naoaki Yokoyama and Hisashi Inokuma* Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555; 1The United Graduate School of Veterinary Science and 2Faculty of Applied Biology Science, Gifu University, Gifu 501-1193; and 3Field Science Center of Northern Biosphere, Hokkaido University, Sapporo 060-8111, Japan (Received February 12, 2008. Accepted May 9, 2008) SUMMARY: Rickettsial infection of cattle and sika deer from a pastureland in Hidaka District, Hokkaido, Japan was examined by serological and molecular methods. Serum samples from 8 of 83 (9.6%) cattle reacted with helvetica antigens in an IFA test, with titers ranging from 1:160 to 1:640, while serum samples from 15 of 22 (68.2%) deer were positive for R. helvetica, with titers ranging from 1:80 to 1:640. In a genus- specific nested PCR based on gltA, no cattle were positive for Rickettsia, while 14 of 22 (63.3%) samples obtained from deer tested positive. Sequence analysis revealed that positive samples from sika deer showed 100% nucleotide sequence identity with the known sequence of Rickettsia asiatica.

Rickettsiae belong to the order and are obli- However, little is known about rickettsial infection in grazing gate intracellular, Gram-negative . Several species cattle in Japan. Thus, in the present study, we attempted to with worldwide distribution cause disease in humans and other detect Rickettsia using both serological and molecular tech- animals. This genus is subdivided into three groups on the niques in samples taken from sika deer and grazing cattle in basis of phenotypic criteria (1): the group (TG), the an area frequented by wild sika deer. ancestral group, and the spotted fever group (SFG). In Japan, A cattle herd (n = 83) on a pasture in the Hidaka District at was discovered to be the causative agent the Shizunai Livestock Farm at the Field Science Center of of (JSF) (2). JSF patients have mainly the Northern Biosphere, Hokkaido University, was examined been identified in western Japan since 1984, when the first in July 2007. A total of 22 sika deer (Cervus nippon yesoensis) case was reported in Prefecture (3). Recently, that appeared in this pasture were hunted from May 2006 to Rickettsia helvetica, another SFG rickettsia, has been isolated January 2007. Blood samples from the jugular vein of each from in Japan (1,4). Although R. helvetica was previ- animal were collected in both plain and EDTA tubes for ously only known to exist in European countries, R. helvetica serum and DNA extraction, respectively. The tubes were kept has now been found to be widespread in Japan, from Hokkaido, at 4°C until transfer to Obihiro University of Agriculture and a northern island, to Kyusyu, a southern island (5). Further- Veterinary Medicine. The sera were separated from plain tubes more, other Rickettsia spp., including Rickettsia tamurae and kept at –20°C for indirect immunofluorescence analysis (6), Rickettsia asiatica (7), and a species closely related to (IFA). A QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, “Candidatus Rickettsia tarasevichiae”, have also been de- Germany) was used to extract DNA from the EDTA blood tected from ticks in Japan (5,8). Cross-reactivities observed samples. DNA samples extracted from blood were stored at among these closely related rickettsial species render sero- –20°C in 200 μl of TE buffer until further use. logical studies complex (9,10). Despite the wide variety of The sera were evaluated using IFA. Antigens of R. helvetica Rickettsia spp. in Japan, little epidemiological information is for the IFA test were prepared by culturing Rickettsia available, including information about vectors and reservoir with L929 cells. Detection of antibodies was carried out as animals. described previously (10). Sera from dogs positive for R. As wild deer are often infested with numerous ticks (11), helvetica were used as positive controls (10). Sera from they can be important reservoir animals for -borne patho- healthy newborn calves kept at the Obihiro University of gens. Recently, the partial nucleotide sequences of R. helvetica Agriculture and Veterinary Medicine were used as negative genes were detected in peripheral blood samples of sika deer controls. Sera were screened at a 1:20 dilution in phosphate- (Cervus nippon yesoensis) in Hokkaido, Japan, which sug- buffered saline (pH 7.4) with 0.5% Tween 20 (PBST). An gests that sika deer could be a reservoir animal for R. helvetica. optimized dilution (1:200) of fluorescein isothiocyanate- The number of sika deer has been increasing in Japan, and labeled goat-anti-bovine IgG conjugate (ICN Pharmaceuti- these animals frequently approach areas where humans and cals Inc., Costa Mesa, Calif., USA) in PBST was used as the domestic animals live. Cattle are at great risk of rickettsial second antibody for the cattle samples. The same second infection because they live in pastures shared with sika deer, antibody was also used for the examination of the samples and cattle suffer frequent tick infestations while grazing. obtained from sika deer (12). Reactive antibodies were then detected using a fluorescence light microscope. The antibody *Corresponding author: Mailing address: Obihiro University of levels in the test samples were determined by comparison Agriculture & Veterinary Medicine, Inada, Obihiro 080-8555, with the appropriate positive and negative controls. Samples Japan. Tel/Fax: +81-155-49-5370, E-mail: inokuma@obihiro. that reacted with any antigens at the screening dilution were ac.jp then titrated using serial twofold dilutions in order to deter-

315 Table 1. Results of screening PCR and antibody titers against Rickettsia helvetica in 83 cattle and 22 sika deer examined Antibody tites Animal PCR No. of animals <40 40 80 160 320 640 Cattle + 0 ––––– – – 83 75* 0 0 4 3 1 Sika deer + 14 3 0 2 3 5 1 – 8 4 0 1 3 0 0 * No. of animals. mine the end titers. In this study, antibody titers of 1:40 or (13) and Ixodes persulcatus ticks (1) in Hokkaido. Although above were considered positive, as in a previous survey (10). several animals possessed positive antibodies against R. Nested polymerase chain reaction (PCR) amplification was helvetica, the DNA fragments detected in this study were all performed using the same oligonucleotide primer pairs for of R. asiatica. This finding suggests that sika deer in this area rickettsial gltA as discussed in our previous report (13). Briefly, suffer from rickettsiemia due to R. asiatica. The positive two genus-specific primer sets, RpCS.877p/RpCS.1273r and antibodies against R. helvetica observed in this study might RpCS.896f/PpCS.1258n, were used for the first and second have been due to a cross-reaction of antibodies against R. PCR analysis, respectively. Positive PCR products were asiatica (9,10). These results also suggest that sika deer might purified using a QIA PCR purification kit (QIAGEN) for be a reservoir animal for R. asiatica in Japan. In our previous direct sequence analysis with a Perkin-Elmer ABI Prism 3100 study, another strain of R. helvetica was detected in sika deer automated DNA sequencer. The sequence data of the PCR blood samples obtained on Nakanoshima Island in Lake Toya products were analyzed using the BLAST 2.0 program (Na- (13). This difference in detected species might reflect differ- tional Center for Biotechnology Information) for homology ences in vector tick species or geographical location. Further searching. To confirm the results obtained by the gltA PCR epidemiological studies will still be necessary to clarify the and sequencing analyses, another nested PCR to amplify role played by sika deer as reservoirs of different species of the rickettsial 17-kDa gene was performed using gltA PCR- Rickettsia in Japan. positive samples according to a previously reported method All of the examined cattle tested negative for Rickettsia in (14). the nested PCR in this study, although 8 cattle possessed Chi-square analysis was used to compare the rates of posi- antibodies against R. helvetica. One explanation for this nega- tive IFA and PCR reactions between cattle and deer. P-values tive result could be the use of acaricide in the cattle on this of less than 0.05 were considered to be significant. pasture. It is also possible that cattle are not a suitable res- The results are summarized in Table 1. The serum from ervoir animal for R. asiatica. Different animal species can a total of 8 of 83 (9.6%) cattle reacted with R. helvetica be expected to differ in terms of their susceptibility to SFG antigens in the IFA test, with titers ranging from 1:160 to rickettsia. More studies are needed to clarify the epidemio- 1:640, while serum samples from 15 of 22 (68.2%) deer were logical role of cattle as reservoirs of SFG rickettsia. positive for R. helvetica, with titers ranging from 1:80 to 1:640. The proportion of R. helvetica-positive sika deer was ACKNOWLEDGMENTS significantly higher than that of R. helvetica-positive cattle This work was supported in part by grant H18-Shinkou-Ippan-014 for (P < 0.0001). Although no cattle tested positive for Rickett- Research on Emerging and Re-emerging Infectious Diseases from the Min- sia by nested PCR, 14 of 22 (63.3%) deer samples were istry of Health, Labour and Welfare of Japan, Grants-in-Aid for Scientific positive. The difference in the positive reaction rates was Research from the Japan Society for the Promotion of Science (No. 18380185) significant (P < 0.0001). The nucleotide sequences of seven and the Akiyama Foundation. randomly selected positive nested PCR products were ana- lyzed. Approximately 342 bp of the rickettsial gltA sequences, REFERENCES excluding the primer region, were determined. All 7 samples 1. Fournier, P-E., Fujita, H., Takada, N., et al. (2002): Genetic identifica- were identical to each other, and showed 100% nucleotide tion of rickettsiae isolated from ticks in Japan. J. Clin. Microbiol., 40, sequence identity with the sequence of R. asiatica registered 2176-2181. 2. Mahara, F., Koga, K., Sawaga, S., et al. (1985): The first report of the in GenBank (accession no. AB114797). The nucleotide se- rickettsial infection of spotted fever group in Japan: three clinical cases. quences of the 17-kDa gene PCR products from these 3 Jpn. J. Assoc. Infect. 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