A coprological survey of the potential definitive hosts of Echinococcus multilocularis in Aomori Prefecture.

*Corresponding author: Mailing address: Department of Parasitology, National Institute of Infectious Diseases, Toyama 123-1, Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-52851111, Fax: +81-3-5285-1173, E-mail: morisima@nih.go.jp Alveolar echinococcosis (AE) is a zoonotic helminth disease classified as Category IV by the Infectious Diseases Control Law of Japan, which requires notification of both human and canine cases of the disease. The known domestic distribution of the causal agent, Echinococcus multilocularis, is exclusively restricted within Hokkaido; thus, human AE infections occur predominantly in the inhabitants of this region. On the other hand, there have also been AE patients reported from prefectures other than Hokkaido. Although many of these cases are thought to have had contact with either Hokkaido or foreign endemic countries, some infections were probably acquired autochthonously. It is noteworthy that half of the autochthonous cases were recorded in Aomori Prefecture (1), which is situated on the northern tip of the mainland of Japan and faces Hokkaido across the Tsugaru Strait (Fig. 1). Furthermore, slaughter pigs infected with metacestodes of E. multilocularis have recently been reported in this area (2). These data imply the spread of the parasite from Hokkaido to Aomori Prefecture, but adult E. multilocularis has not yet been detected in necropsy surveys on wild red foxes, though it must be kept in mind that relatively few animals were studied (3,4). Necropsy diagnosis of Echinococcus in the small intestines of the definitive hosts is laborious, time-consuming and even biohazardous when it is performed on a fresh carcass, but nevertheless serves as a gold standard owing to its reliability. However, postmortem examination is not suitable for areas like Aomori Prefecture, where animal carcasses are sparse or unavailable. According to game bag records, for instance, the mean annual number of red foxes captured in this prefecture during 1999 2001 was less than 100 (5) (Full data are available at http://www.sizenken.biodic.go.jp/ wildbird/flash/toukei/07toukei.html). Although domestic dogs can also become the definitive hosts of the parasite, they cannot be included in necropsy surveys for ethical reasons. Therefore, we attempted a survey with antemortem diagnostic approaches to increase the target population. The survey was undertaken on potential definitive hosts in Aomori Prefecture from December 2003 to February 2005. Forty-three fecal samples of wild red foxes, which included 17 rectal feces of shot individuals and 26 naturally excreted feces in fields, were collected by local hunters. With regard to the collection of the latter samples, local hunters were instructed in the criteria of vulpine feces (6) in order to avoid sample contamination with other carnivores. In addition, 134 fecal samples of hunting dogs were collected due to their management in fields in which they may acquire the infection by preying on rodent intermediate hosts. All samples were stored at –20°C until used. Microscopic egg detection was conducted with both a modified Wisconsin procedure (7) using a sucrose solution whose specific gravity was 1.27 and a formalin-ethyl acetate sedimentation technique (8). Because the eggs of Echinococcus morphologically resemble those of other taeniid tapeworms (9), they were subjected to PCR as described below. Echinococcus coproantigen was screened using an ELISA kit from Dr. Bommeli AG (CHEKITEchinotest; Liebefeld-Bern, Switzerland) following manufacturer’s instructions. The results of the test were expressed as percentages of likelihood for infection using the following equation: relative positivity (%) = {(OD of test sample–OD of negative control)/(OD of positive control–OD of negative control)}×100. A relative positivity value of less than 30% was regarded as a negative and a value greater than 40% was regarded as a positive. Values between 30 -40% were considered to be indeterminate. The test has a sensitivity rate of 90.9% and a specificity rate of 98.8% when applied to a group with a prevalence of 8% (data from the manufacturer). To confirm the results of the coproantigen test, and to distinguish E. multilocularis from other taeniid tapeworms, a specific nested PCR test (10) was carried out. The extraction of DNA from fecal samples and egg materials was performed using a commercial kit (QIAamp DNA Stool Mini Kit; Qiagen GmbH, Hilden, Germany) and the protocol described by Fig. 1. Location of Aomori Prefecture, Japan. N