Jpn. J. Infect. Dis., 63, 353-354, 2010

Short Communication Molecular Survey of Rickettsial Agents in Feral Raccoons (Procyon lotor) in ,

Mariko Sashika1,2,GoAbe3, Kotaro Matsumoto1, and Hisashi Inokuma1* 1Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555; 2United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193; and 3Wildlife Management Research Center, Hyogo 669-3842, Japan (Received April 9, 2010. Accepted July 26, 2010)

SUMMARY: Rickettsial infection in feral raccoons (Procyon lotor) in Hokkaido, Japan was analyzed by molecular methods. Genus-specific nested polymerase chain reaction (PCR) analysis based on the Rickettsia citrate synthase (gltA) gene showed that 13 of 699 raccoons (1.9z) examined were positive for Rickettsia. Twelve of the 13 partial gltA sequence amplicons were successfully analyzed. The nucleo- tide sequence of one amplicon was identical to both Rickettsia heilongjiangensis and R. japonica, one was identical to R. felis,andtheresttoR. helvetica. This is the first report on the detection of rickettsial agents in peripheral blood of raccoons.

The raccoon is a medium-sized carnivore native to extracted with a QIAamp DNA Mini Kit (Qiagen North America; however, a large number of raccoons GmbH,Hilden,Germany).DNAsampleswerestored have been imported from North America as pets since at －209Cin200ml of Tris-EDTA (TE) buffer until fur- the 1970s (1). Subsequently, large numbers of raccoons ther use. have naturalized in many parts of Japan due to the in- Nested PCR was performed with genus-specific tentional release or escape of pet raccoons (2). Feral rac- primers for the rickettsial citrate synthase (gltA)gene coons cause heavy damage to crops and native (9), and the primer pair RpCS.877p and RpCS.1273r ecosystems in Japan. Furthermore, raccoons may have was used for the first amplification. The first round of introduced nonindigenous pathogens into Japan, such PCR was carried out in a 25-ml reaction mixture (5 mlof as rabies, raccoon ascarid, and rickettsiosis (3), which DNA template) under the following conditions: 35 cy- have been reported in the United States. Because of cles of denaturation at 959C for 30 s, annealing at 549C these problems, the Hokkaido Government initiated a for 30 s, and extension at 729C for 90 s. The resulting feral raccoon management program in 1999. PCR products were then used as a template for the sec- Spotted fever group (SFG) Rickettsia is a significant ond amplification with primers RpCS.896f and emerging infectious disease whose principal clinical fea- RpCS.1258n. Cycling conditions for the second round tures are fever and rash. In Japan, the first case of rick- of PCR were the same as the first round, except that an- ettsiosis caused by Rickettsia japonica was reported in nealing was carried out at 569C for 30 s. DNA extracted Prefecture in 1984 (4). Other SFG Rickettsia from the Rickettsia AT-1 strain was used as a positive spp. have recently been detected in Japan, including control, and distilled water was used for the blank con- Rickettsia helvetica (5), R. tamurae (6), R. asiatica (7), trol. PCR products were purified with the Qiaquick and R. tarasevichiae (8); however, little epidemiologic PCR purification kit (Qiagen) and sequenced as de- data is available (i.e., vectors and reservoir animals). scribed previously (9). Sequence homology searches of We therefore aimed to characterize rickettsial pathogens the PCR products were performed with the Basic Local by a molecular analysis of peripheral blood samples ob- Alignment Search Tool (BLAST; National Center for tained from feral raccoons in Japan. Another objective Biotechnology Information). of this study was to clarify the epidemiologic role of rac- According to genus-specific nested PCR analysis, 13 coons for these pathogens in Japan. of 699 (1.9z) raccoons were positive for Rickettsia.In A total of 699 raccoons were captured between May 12 samples, approximately 322 bp of the gltA gene, ex- and October 2007 and between March and October 2008 cluding the primer region, was successfully sequenced. as part of raccoon population control programs im- These sequences have been deposited in GenBank plemented by the Hokkaido Government and the Minis- under accession numbers HM049647 to HM049658. try of the Environment in west-central Hokkaido, the One amplicon showed 100z nucleotide identity with northernmost of the main islands of Japan. Blood sam- both R. japonica (AY743327) and R. heilongjiangensis ples were collected from 699 raccoons, and DNA was (AY285776), and another was identical to R. felis (U33922). The remaining amplicons were identical to the gltA gene of R. helvetica (AM418450). *Corresponding author: Mailing address: Obihiro Uni- To distinguish between R. japonica and R. heilong- versity of Agriculture and Veterinary Medicine, Inada, jiangensis, species-specific PCR for the R. japonica Obihiro 080-8555, Japan. Tel & Fax: ＋81-155-49-5370, E- 17-kDa antigen gene was performed with the primers mail: inokuma＠obihiro.ac.jp Rj5 and Rj10 (10). Given the negative result, a conclu-

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