Usefulness of PCR-restriction fragment length polymorphism analysis of the internal transcribed spacer region of rDNA for identification of Anisakis simplex complex.

Anisakiasis is a disease caused by infection with an anisakid larva, primarily Anisakis simplex, through the ingestion of raw marine fish, known worldwide as “sashimi”, or of a wide variety of undercooked marine fish. In Japan, anisakiasis is quite prevalent, with more than 1,000 new cases estimated to be diagnosed annually (1). At the end of 1999, “The Food Sanitation Law Enforcement Regulation” was amended, and anisakid larvae were newly added to the causative agents of food poisoning (2). Recent molecular analyses of the internal transcribed spacer (ITS) regions of rDNA, spanning the ITS-1, ITS-2 and the 5.8S subunit, demonstrated that the morphological species A. simplex is a complex of three or more sibling species, including A. simplex sensu strict, A. pegreffii and A. simplex C; the latter species has wider intraspecific genetic diversity than the other two (3). The marked differences in their respective ecological niches and their low proportion of hosts shared (3) may lead to different transmission pathways for the parasites as well as potential pathogenicity to humans. However, until now, the identification of the anisakid larvae recovered from gastric anisakiasis patients in Japan has been based exclusively on their morphological features. In the present study, a clinical isolate of anisakid larva obtained by an endoscopic operation from a 65-year-old Japanese female with epigastralgia (Fig. 1) was analyzed by both sequencing and PCR-restriction fragment length polymorphism (RFLP) mapping of the ITS regions of the rDNA. The clinical isolate was fixed in 70% ethanol and cut into two parts. The anterior half of the larva where the esophagus and ventriculus are located was cleared by immersion in the lactophenol solution for microscopic observations and was confirmed to be A. simplex complex by its morphological features. The remaining half of the larva was simultaneously