Parasitol Res (2004) 92: 352–353 DOI 10.1007/s00436-003-1048-6
SH O RT CO MM U N IC A T IO N
Ning Xiao Æ Tiao-Ying Li Æ Jia-Min Qiu Minoru Nakao Æ Xing-Wang Chen Æ Kazuhiro Nakaya Hiroshi Yamasaki Æ Peter M. Schantz Æ Philip S. Craig Akira Ito
The Tibetan hare Lepus oiostolus : a novel intermediate host for Echinococcus multilocularis Received: 23 September 2003 / Accepted: 5 November 2003 / Published online: 14 January 2004 Ó Springer-Verlag 2004
Abstract Multicystic hydatids have been found in the livers of hares (Lepus oiostolus) examined from the Qinghai-Tibet plateau of China. In this study, the causative species was definitively identified as Echinococcus multilocularis by mitochondrial DNA sequencing. This is the first confirmation of larval E. multilocularis from hares. The hydatids contained protoscolices, suggesting that the hare may contribute to the transmission of E. multilocularis on the Tibetan plateau.
The canine intestinal tapeworm Echinococcus multilocularis (Cestoda: Taeniidae) is the causative pathogen of human alveolar echinococcosis, a potentially fatal liver metacestode infection. Its transmission cycle occurs in foxes and small mammals and is completed by means of the predator–prey relationship between these species. According to Rausch (1995), larval E. multilocularis have been recorded from various small mammals belonging to eight families: Arvicolidae (voles and lemmings), Muridae (rats and mice), Soricidae (shrews), N. Xiao Æ M. Nakao Æ K. Nakaya Æ H. Yamasaki Æ A. Ito (&) Department of Parasitology, Asahikawa Medical College, Midorigaoka-Higashi 2-1, 078-8510 Asahikawa, Japan E-mail:
[email protected] Tel.: +81-166-682420 Fax: +81-166-682429 T.-Y. Li Æ J.-M. Qiu Æ X.-W. Chen Sichuan Institute of Parasitic Diseases, Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, China P. M. Schantz Division of Parasitic Diseases, NCID, Centers for Disease Control and Prevention, Atlanta, Georgia, USA P. S. Craig Cestode Zoonoses Research Group, Bioscience Research Institute and School of Environment and Life Sciences, University of Salford, Salford, Greater Manchester, UK
Talpidae (moles), Sciuridae (squirrels), Cricetidae (gerbils), Dipodidae (jerboas) and Ochotonidae (pikas). A number of arvicolid rodents appear to be highly susceptible with production of protoscolices by the larval cestode stage and, consequently, these most commonly serve as the intermediate hosts. Medium-sized mammals of the family Leporidae (rabbits and hares) have been regarded as unsuitable hosts for larval E. multilocularis (Ohbayashi et al. 1971). The Tibetan hare, Lepus oiostolus, predominantly inhabits the grasslands of Tibet and adjacent highlands (Nowak 1991). Recently, Qiu et al. (1995) reported finding ‘‘multicystic’’ hydatids in 5 (6.7%) of 75 hares from the eastern Tibet plateau of western Sichuan. More recent surveys, from 1997 to 2002, found that 4 (7.0%) of 57 hares harbored multicystic hydatids in their livers (He et al. 2000; unpublished data). These were morphologically identified as larval E. multilocularis; however, the species identification requires confirmation because both Echinococcus granulosus and E. multilocularis are highly endemic to the survey area. Previous reports showed that hares serve exceptionally as intermediate hosts for E. granulosus (Kontrimavichus 1959; Schantz and Lord 1972). Moreover, the multicystic hydatid of E. granulosus developing in unsuitable hosts has a superficial similarity to larval E. multilocularis (Vogel 1957). Therefore, in this study, a multicystic hydatid from a Tibetan hare was subjected to mitochondrial DNA sequencing for definitive identification of the causative species. The hydatid tissue from one Tibetan hare captured in 2002 was available for this study. Multicystic lesions were present in both liver and lungs of the hare. As shown in Fig. 1, the lesions consisted of numerous small vesicles without severe inflammatory reaction, suggesting that active multivesiculation had occurred. The vesicles had well-developed germinal layers and the primordia of brood capsules. The mature vesicles contained protoscolices and calcareous corpuscles, morphologically compatible with those of larval E. multilocularis. Genomic DNA was purified from the
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small mammals such as voles (e.g. Microtus fuscus) and pikas (e.g. Ochotona curzoniae) appear to be involved as intermediate hosts in the transmission cycle of E. multilocularis. Based on our findings, we propose that a medium-sized mammal, the Tibetan hare, could be potentially involved in this cycle as a novel intermediate host for E. multilocularis. Our histological observations showed that 5 (55.6%) of 9 naturally infected Tibetan hares possessed protoscolices in their multicystic lesions, suggesting that L. oiostolus could contribute to the transmission of E. multilocularis. Confirmation of this could be made after dietary analysis of foxes and dogs, as well as experimental infection of canids using parasites derived from Tibetan hares. Acknowledgements This study was supported by the U.S. National Institute of Health (1R01 TW01565-01; Principal Investigator, Philip S. Craig) and by a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society of Promotion of Science to A. I. (1255702414, 14256001).
References
Fig. 1 Echinococcal lesion in the liver of Lepus oiostolus. A Low magnification (PAS staining). B High magnification (HE staining). Abbreviations are as follows: PS protoscolex, GL germinal layer, LL laminated layer, BC brood capsule
hydatid tissue by using a spin column kit (DNeasy tissue kit; Qiagen, Hilden, Germany) and then used as a template for polymerase chain reaction (PCR). As reported previously (Xiao et al. 2003), partial fragments of cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) genes were amplified by PCR. The PCR amplicons were directly sequenced by using a dye terminator cycle sequencing kit (DYEnamic ET terminator; Amersham Biosciences, Tokyo, Japan) and an ABI PRISM 377 sequencer (Perkin Elmer Applied Biosystems, Foster City, Calif.). The amplified sequences of cox1 (815 bp) and cytb (568 bp) genes were completely identical with those of E. multilocularis (Nakao et al. 2002). These findings confirm that the hare was infected with larval E. multilocularis. In the Qinghai-Tibet plateau region of China, the transmission dynamics of Echinococcus spp. depend upon the ecosystem of high altitude steppe and the human activities associated with stock-farming (Qiu et al. 2000). In this environment, domestic and wild canids (dog, Tibetan fox and red fox) may serve as definitive hosts for E. granulosus or E. multilocularis. Livestock, primarily sheep and yaks may act as the intermediate hosts of E. granulosus (Xiao et al. 2003). In contrast,
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