Am. J. Trop. Med. Hyg., 72(3), 2005, pp. 284–288 Copyright © 2005 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT: ECHINOCOCCUS MULTILOCULARIS CONFIRMED ON KUNASHIRI ISLAND, 15 KILOMETERS FROM THE EASTERN PART OF HOKKAIDO, JAPAN MASAO SATOH,* KAZUHIRO NAKAYA,* MINORU NAKAO, NING XIAO, HIROSHI YAMASAKI, YASUHITO SAKO, YUKAKO NAITOH, SEIJI KONDO, MARI KOBAYASHI, NORIYUKI OHTAISHI, AND AKIRA ITO* Department of Allergy and Immunology, Saitama Medical School, Saitama, Japan; Animal Laboratory for Medical Research and Department of Parasitology, Asahikawa Medical College, Asahikawa, Japan; Laboratory of Ecology and Genetics, Graduated School of Environmental Earth Science and Animal Production System, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan; Marine Wildlife Center of Japan, Sapporo, Japan
Abstract. There are two foci of alveolar echinococcosis (AE) caused by Echinococcus multilocularis in Japan. The first focus is on Rebun Island where AE patients were found from 1937, and the second is in eastern Hokkaido where patients have been found since the 1960s. The origin of the second focus is unknown. To further investigate AE in eastern Hokkaido, wild rodents (Muridae) were captured and examined for infection on Kunashiri Island, which is located 15 km off the northeastern coast of Hokkaido. Metacestodes of E. multilocularis were isolated from two of 31 voles, all of which were identified to be Clethrionomys rufocanus. Mitochondrial DNA sequencing data of recovered cestodes showed total identity with the cestode reported from Hokkaido. These results suggest that E. multilocularis may have been introduced to Hokkaido from Kunashiri Island during or after 1965.
Alveolar echinococcosis (AE),1 which is caused by ingestion of eggs of the fox tapeworm Echinococcus multilocularis, is the most potentially lethal parasitic infection of the nontropical areas in the northern hemisphere because of its tendency to invade and proliferate in the liver and the difficulty in treatment.1–3 Known endemic areas in Far East Asia include China3–5 and Hokkaido, Japan.6 However, there is no previous information available on the distribution of E. multilocularis or the endemic areas of AE in eastern Siberia, including State of Primorskii and Sakhalin (Figure 1). It has not been previously determined how E. multilocularis reached Hokkaido. A map of northeastern Hokkaido, Japan is shown in Figure 1. It has been suggested that E. multilocularis was introduced into some of the Chishima (Kurile) Islands with foxes from St. Lawrence Island through the Komandorski Islands.7,8 However, there is only a single report of the occurrence of E. multilocularis on Simushir Island (a in Figure 1) in the Chishima (Kurile) Islands.9 Historically, there have been two foci of AE recorded in Hokkaido, Japan. The first is on Rebun Island (b in Figure 1), which has an area of 83 km2 and is located 45 km off the northwestern coast of Hokkaido. It has been documented that E. multilocularis was accidentally introduced onto Rebun Island with 12 pairs of red foxes (Vulpes vulpes) from Simushir Island in the Chishima (Kurile) Islands between 1924 and 1926 for the purpose of control of voles and production of fox fur.6,8 The first human case of AE in Hokkaido was reported in a 28-year-old woman from Rebun Island in 1937. To date, 131 human AE cases have been reported on this small island.8 However, field studies conducted in 1948 showed few infected final hosts (dogs and cats) and no metacestodes in rodents.8 Therefore, transmission of the parasite was already considerably reduced at that time due to the earlier eradication of foxes, mainly through illegal hunting for selling fox fur.3,10,11 It is now illegal to introduce dogs and cats onto this island and the parasite has been eradicated from it since the 1960s.3,10–12
The other focus is in the eastern part of Hokkaido Island. In contrast, there are no critical historical data to explain the rapid spread of AE that has been reported since 1965 in the northeastern part of Hokkaido.6,12 To investigate the origin of AE in eastern Hokkaido, we conducted a field survey of wild rodents on Kunashiri Island from July 10 to July 27, 2003. This island is very close to eastern Hokkaido; however, there are no previous reports of E. multilocularis on this island. A total of 31 wild rodents (voles), including Clethrionomys rufocanus, were captured (20 at a location 44°17⬘N, 146°11⬘E,
FIGURE 1. Map of Hokkaido, Japan with surrounding areas. a ⳱ Simushir Island; b ⳱ Rebun Island; c ⳱ Kunashiri Island; d ⳱ Etorofu Island; e ⳱ Shikotan Island; f ⳱ Habomai Island.
*These authors contributed equally to this work.
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8 at 43°55⬘N, 145°39⬘E, 2 at 44°02⬘N, 145°50⬘E, and 1 at 44°25⬘N, 146°30⬘E). A detailed report on the fauna at this field survey will be published elsewhere. Thirteen livers and one spleen were collected from 13 rodent specimens and preserved in 100% ethanol for parasitologic examinations. These 14 specimens were examined for parasites at Asahikawa Medical College as a joint project. Eight of the 13 hepatic specimens had no observable cysts on their surfaces. Five others showed macroscopic cyst-like lesions that were dehydrated and embedded in paraffin wax. Sections (5 m thickness) were stained with hematoxylin and eosin or periodic acid/Schiff. They were confirmed to be E. multilocularis (n ⳱ 2), Capillaria hepatica (n ⳱ 2), and Cladothyridium sp. (n ⳱ 1). Among the two infected specimens of C. rufocanus, one was infected with both E. multilocularis and eggs of C. hepatica (Figure 2), whereas the other only with E. multilocularis in both the liver and spleen. Echinococcus multilocularis and Cladothyridium sp. were confirmed by mitochondrial DNA (mtDNA) sequencing analysis. The mtDNA samples were prepared using the DNeasy Tissue kit (Qiagen, Hilden, Germany). For E. multilocularis, a partial nucleotide se-
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quence of the cytochrome b gene was determined according to a previously published method.13 For Cladothyridium sp., the 16S ribosomal RNA gene (rrnL) was amplified using primers designed based on the nucleotide sequence of rrnL from E. multilocularis. The polymerase chain reaction protocols consisted of 30 cycles of denaturation (30 seconds at 94°C), annealing (30 seconds at 56°C), and extension (90 seconds at 72°C) plus one cycle of 5 minutes at 72°C. DNA sequencing for both genes was performed using the Genetic Analyzer (ABI PRISM model 310; Applied Biosystems, Foster City, CA). The nucleotide sequences (568 basepairs) of the cytochrome b gene from all three E. multilocularis specimens from Kunashiri Island were identical to that of E. multilocularis from Hokkaido (accession no. AB018440) (Figure 3). The partial nucleotide sequences of rrnL (336 basepairs) from Cladothyridium sp. (accession number AB126579) and two related cestode species are aligned and shown in Figure 4. The homologies of rrnL from Cladothyridium sp. with those from Taenia pisiformis (accession number AB020394) and T. taeniaeformis (accession number AB020395) were 81.2% and
FIGURE 2. Histopathologic section of the liver of a vole infected with a metacestode of Echinococcus multilocularis and eggs of Capillaria hepatica, stained with hematoxylin and eosin. LL ⳱ laminated layer; GL ⳱ germinal layer; VL ⳱ echinococcal vesicular lumen; BP ⳱ primodium of brood capsule and protoscolex; Ch ⳱ egg of C. hepatica (scale bar ⳱ 100 m).
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FIGURE 3. Comparison of partial nucleotide sequences of cytochrome b genes from isolates of Echinococcus multilocularis from Kunashiri Island and Hokkaido. The partial sequences of 568 basepairs (positions 402 to 969 of the cytochrome b gene) are shown. Asterisks indicate identical nucleotides. The accession number for the Hokkaido isolate is AB018440.
77.1%, respectively. It has been reported that Cladothyridium sp. is found in C. rufocanus bedfordiae and cycles between rodents and birds, including Milvus sp., in the eastern part of Hokkaido.14 Kunashiri Island is only 15 km from the eastern coast of Hokkaido and the Sea of Okhotsk and Pacific Ocean off the northeastern part of Hokkaido are often frozen during the winter. Thus, E. multilocularis might have been introduced from Kunashiri Island to eastern Hokkaido through migration of infected foxes during the winter. Although there was no formal communication between Hokkaido and Kunashiri Island between 1945 and 1991, the local capital (Nemuro City) on the eastern coast (Figure 1) has been known to be endemic for E. multilocularis. The wild vole C. rufocanus bedfordiae is the dominant species infected with E. multilocularis.3,14,15 It would be interesting to conduct field surveys on these four northern islands (Etorofu, Kunashiri, Shikotan, and Habomai). It is evident from this preliminary work that wild rodents on Kunashiri Island are highly infected with E. mul-
tilocularis. This island has a rich mammalian fauna, including red fox (V. vulpes) and brown bear (Ursus arctos). These four northern islands have been inhabited by Russian residents for the past 60 years, and they may have been exposed to eggs of E. multilocularis. A diagnostic survey of these human residents is necessary to evaluate the possibility of AE infection especially on the islands of Etorofu, Kunashiri, and Shikotan, where relatively large populations reside and are involved in the marine product industry. Received February 25, 2004. Accepted for publication June 8, 2004. Acknowledgments: Masao Satoh, Yukako Naitoh, Seiji Kondo, and Mari Kobayashi visited Kunashiri Island through the program of Japanese and Russian specialists exchange without visa hosted by the Marine Wildlife Center of Japan (Incorporated Non Profit Organization). We thank Dr. P.M. Schantz (Centers for Disease Control and Prevention, Atlanta, GA) for improving this manuscript. Financial support: This work was supported by the Nippon Foundation, the Pro Nature Foundation-Japan, the Japan Fund for Global Environment, the Sonpo Japan Environment Foundation, and the
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FIGURE 4. Alignment of partial nucleotide sequences of 16S ribosomal RNA genes from Cladothyridium sp. and two related cestodes. Multiple alignment of sequences was achieved using the CLUSTAL W program available over the World Wide Web (http://www.ddbj.nig.ac.jp). Asterisks indicate identical nucleotides. Dashes are introduced to maximize alignment. The accession numbers are AB126579, AB020394, and AB020395 for Cladothyridium sp., Taenia pisiformis, and T. taeniaeformis, respectively.
Keidanren Nature Conservation Fund, and by a grant-in-aid for the Scientific Research sponsored by the Japanese Society for Promotion of Science (14256001) to Akira Ito. Authors’ addresses: Masao Satoh, Department of Allergy and Immunology, Saitama Medical School, Morohongo 38, Moroyama, Irumagun, Saitama, 350-0495 Japan, Telephone: 81-492-76-1173, Fax: 81492-94-2274, E-mail:
[email protected]. Kazuhiro Nakaya, Animal Laboratory for Medical Research, Asahikawa Medical College, Asahikawa, Japan. Minoru Nakao, Ning Xiao, Hiroshi Yamasaki, Yasuhito Sako, and Akira Ito, Department of Parasitology, Asahikawa Medical College, Midorigaoka-Higashi 2-1-1-1, Asahikawa, 078-8510, Japan. Yukako Naitoh, Laboratory of Ecology and Genetics, Graduate School of Environmental Earth Science, Hokkaido University, Nishi 5, Kita 10, Kita-ku, Sapporo, 060-0810, Japan. Seiji Kondo, Animal Production System, Graduate School of Agriculture, Hokkaido University, Nishi 9, Kita 9, Kita-ku, Sapporo, 0608589, Japan. Mari Kobayashi and Noriyuki Ohtaishi, Marine Wildlife Center of Japan, 1-35-103, Nishi 6, Kita 21, Kita-ku, Sapporo, 0010021, Japan.
REFERENCES 1. Pawlowski ZS, Eckert J, Vuitton DA, Amman RW, Kern P, Craig PS. Dar KF, De Rosa F, Filice C, Gottstein B, Grinn F, Macpherson CNL, Sato N, Todorov T, Uchino J, von Sinner W, Wen H, 2001. Echinococcosis in humans: clinical aspects, diagnosis and treatment. Eckert J, Gemmell MA, Meslin FX, Pawlowski ZS, eds. WHO/OIE Manual on Echinococcosis in
2. 3. 4.
5.
6.
7. 8.
9.
Humans and Animals: A Public Health Problem of Global Concern. Paris: Office International des Epizooties, 20–71. McManus DP, Zhang W, Li J, Bartley PB, 2003. Echinococcosis. Lancet 362: 1295–1304. Ito A, Romig T, Takahashi K, 2003. Perspective on control option for Echinococcus multilocularis with particular reference to Japan. Parasitology 127: S159–S172. Vuitton AD, Zhou H, Bresson-Hadni S, Wang Q, Piarroux M, Raoul F, Giraudoux P, 2003. Epidemiology of alveolar echinococcosis with particular reference to China and Europe. Parasitology 127: S87–S107. Schantz PM, Wang H, Qiu J, Liu FJ, Saito E, Emshoff A, Ito A, Roberts JM, Delker C, 2003. Echinococcosis on the Tibetan Plateau: prevalence and risk factors for cystic and alveolar echinococcosis in Tibetan populations in Qinghai Province, China. Parasitology 127: S109–S120. Ito A, Urbani C, Qiu JM, Vuitton DA, Qiu DC, Heath DD, Craig PS, Feng Z, Schantz PM, 2003. Control of echinococcosis and cysticercosis: a public health challenge to international cooperation in China. Acta Trop 86: 3–17. Rausch R, Yamashita J, 1957. Occurrence of Echinococcus multilocularis Leuckart, 1863, in Japan. Proc Helminthol Soc (Wash) 24: 128–133. Yamashita J, 1973. Echinococcus and echinococcosis. Morishita K, Komiya Y, Matsubayashi H, eds. Progress of Medical Parasitology in Japan. Tokyo: Meguro Parasitological Museum, 64– 123. Ishino E, 1941. Discussion about the development of alveolar type of echinococcosis. J Anim Health Assoc 9: 115–128.
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10. Minagawa T, 1999. The reconsideration of natural history of echinococcosis at Rebun Island. Hokkaido Med J 74: 113– 134. 11. Doi R, Nakao M, Nihei N, Kutsumi H, 2000. Epidemiology of alveolar hydatid disease (AHD) and estimation of infected period of AHD in Rebun Island, Hokkaido. Jpn J Public Health 47: 145–152. 12. Minagawa T, 1997. Survey of echinococcosis in Hokkaido and measure against it. Hokkaido Med J 72: 569–581. 13. Xiao N, Qiu J, Nakao M, Nakaya K, Yamasaki H, Sako Y, Ma-
muti W, Schantz PM, Craig PS, Ito A, 2003. Identification of Echinococcus species from a yak in the Qinghai-Tibet plateau region of China. Am J Trop Med Hyg 69: 445–446. 14. Kamiya H, Ohbayashi M, Sugawara K, Hattori K, 1977. An epidemiological survey of multilocular echinococcosis in small mammals of eastern Hokkaido, Japan. Jpn J Parasitol 26: 148– 156. 15. Saitoh T, Takahashi K, 1998. The role of vole populations in prevalence of the parasite (Echinococcus multilocularis) in foxes. Res Popul Ecol 40: 97–105.
