Invalidation of Diphyllobothrium hottai (Cestoda: Diphyllobothriidae) based on morphological and molecular phylogenetic analyses.

Diphyllobothrium hottai Yazaki, Fukumoto & Abe, 1988 was described based on the morphology of adult worms recovered from golden hamsters that had been experimentally infected with plerocercoids obtained from Japanese surf smelts (Hypomesus pretiosus japonicus) and olive rainbow smelts (Osmerus eperlanus mordax). Although D. hottai was considered to be distinct from Diphyllobothrium ditremum (Creplin, 1825), their taxonomic relationship requires further clarification. In our study, D. hottai and D. ditremum obtained from hamsters experimentally infected with plerocercoids isolated from Japanese surf smelts were compared using morphological and molecular methods. The criterion usually used to differentiate between D. hottai and D. ditremum is the difference in the angle between the long axis of the cirrus sac and that of the seminal vesicle. However, we found variation of the angle within the same individual and, one specimen showed both of the different angles that were supposedly unique to each of the species. Furthermore, phylogenetic analysis of the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes revealed that both species were genetically indistinguishable. Therefore, D. hottai is considered to be a junior synonym of D. ditremum.

Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan.

Anisakid nematodes are known to cause the zoonotic disease, anisakiasis, through the consumption of raw or undercooked fish. The parasites most frequently associated with the disease in humans are categorized as Anisakis type I, which comprise several species of the genus Anisakis. The larvae show primitive forms and lack the detailed morphological characteristics required for precise species identification. Thus, molecular characterization is necessary for determining the species of Anisakis type I larvae and acquiring important clinical and epidemiological information. In this study, we isolated Anisakis type I larvae from hairtail fish caught off the coasts of Taiwan and Japan. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was sequenced, and restriction fragment length polymorphism (RFLP) analyses using HinfI and HhaI was carried out for species identification. Most larvae isolated from hairtail caught in Taiwan were Anisakis typica (84%), while those isolated from hairtail caught in Japan were almost exclusively identified either as Anisakis simplex sensu stricto (65%) or Anisakis pegreffii (33%). This is the first report of A. typica in fish obtained from Taiwan. Our results shed the light on the epidemiology of Anisakis type I larvae, which is a potential cause of human anisakiasis in Taiwan and Japan.

Multiplex PCR for the identification of Anisakis simplex sensu stricto, Anisakis pegreffii and the other anisakid nematodes.

A multiplex PCR method was established for the rapid identification of Anisakis simplex sensu stricto, A. pegreffii, A. physeteris, Pseudoterranova decipiens, Contracaecum osculatum and Hysterothylacium aduncum. The sequence alignment of the internal transcribed spacer 1 region (ITS-1) between A. simplex s. str. and A. pegreffii showed a high degree of similarity, but only two C-T transitions were observed. To differentiate A. simplex s. str. from A. pegreffii, an intentional mismatch primer with an artificial mismatched base at the second base from the primer 3' end was constructed. This intentional mismatch primer, which produced a PCR band only from A. pegreffii DNA, was able to differentiate the two morphologically indistinguishable sibling species of A. simplex. Specific forward primers for other anisakid species were also designed based on the nucleotide sequences of the ITS region. The multiplex PCR using these primers yielded distinct PCR products for each of the anisakid nematodes. The multiplex PCR established in this study would be a useful tool for identifying anisakid nematodes rapidly and accurately.

Molecular identification of the etiological agent of the human anisakiasis in Japan.

Anisakis simplex complex presently comprises three sibling species, A. simplex sensu stricto, A. pegreffii and A. simplex C. A. simplex is a common parasite in fishes and cephalopods and capable of causing anisakiasis in humans. Therefore, identification of sibling species of A. simplex was important for human health. In this study, one hundred Anisakis type I larvae isolated from eighty five patients with anisakiasis in Hokkaido and Kyushu in Japan were analyzed by adapting the new molecular method that can identify the sibling species of A. simplex complex. Based on the restriction fragment length polymorphism (RFLP) pattern of ITS regions including 5.8 subunit rRNA gene, we identified two sibling species, A. simplex s. str. and A. pegreffii. However, the infection rate of A. simplex s. str. was significantly higher than that of A. pegreffii. Eighty four (98.8%) out of the eighty five patients were infected with A. simplex s. str. On the contrary, one patients (1.2%) in Kyushu infected with A. pegreffii. This study provided basic information about human infection with A. simplex complex. Furthermore, we suggested that A. simplex s. str. is the most important etiological agent in Japan.

Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetacean in Japanese waters.

Parasites morphologically consistent with Anisakis simplex sensu lato collected from the coast of Japan and Western North Pacific Ocean were examined by PCR-RFLP of the ITS region (ITS1, 5.8 subunit rRNA gene and ITS2) and mtDNA cox1. The RFLP patterns of rDNA generated by HinfI and HhaI showed that 100% of the larvae collected from Hokkaido and 94% of adults collected from Western North Pacific Ocean were identified as A. simplex sensu stricto. On the other hand, 97% of the larvae collected from Fukuoka prefecture were identified as A. pegreffii. A hybrid genotype was found in adults in Western North Pacific Ocean and larva in Fukuoka prefecture. These findings revealed that A. simplexs. str. is primarily distributed in the North Pacific Ocean and A. pegreffii is primarily distributed in the southern Sea of Japan. RFLP analysis of mtDNA cox1 showed different patterns between A. simplex s. str. and A. pegreffii after digestion with HinfI. This polymorphism obtained by RFLP analysis of mtDNA cox1 proved the usefulness as new genetic markers to distinguish two sibling species.

Scuticociliata infection in the weedy sea dragon Phyllopteryx taeniolatus.

The weedy sea dragon Phyllopteryx taeniolatus at an aquarium in Kanagawa Prefecture were found infected by protozoan ciliate, 2001. The infected fish in particular, showed sloughing of the epidermis. Fish with intense infections showed sloughing of the dorsal fin, depigmentation of skin, anal distension and accumulation of ascitic fluid in the body cavity. In biopsies, ciliates were detected only in fresh mounts of abdominal dropsy and in the mucus on the body surface. Histopathological studies revealed ciliates mainly infected the dermis, and induced extensive detachment of the epidermis from the skin. Based on the arrangement and shape of the buccal structure and the number of somatic ciliature, ciliates isolated from the fishes belonged to the order Scuticociliatida. We discuss the major factors that bring about the death of weedy sea dragons.