Species composition and infection levels of Anisakis (Nematoda: Anisakidae) in the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Northwest Pacific.

Parasites can be used as biological tags to assess stock structures in various marine fish species. In the present study, the species composition and infection levels of parasitic nematodes of the genus Anisakis in the skipjack tuna Katsuwonus pelamis were examined in the Northwest Pacific and adjacent seas. A total of 867 third-stage larvae of Anisakis were collected from 112 skipjack tunas captured around Japan and in other subtropical localities. All larvae were identified as A. berlandi, A. pegreffii, A. simplex (s.s.), A. typica, and A. physeteris (s.l.) by the direct sequencing of the mitochondrial cox2 gene and real-time PCR assays targeting the nuclear ITS region. Anisakis species composition differed among northeastern Japan, the Sea of Japan, and other areas (central Japan, the Nansei Islands, and subtropical region), which is largely concordant with previous stock discrimination of skipjack tuna. Molecular phylogenetic analysis resulted in two intraspecific genetic groups in A. simplex (s.s.), one of which occurred almost exclusively in northeastern Japan. This could be a useful indicator for stock discrimination. Skipjack tunas from northeastern Japan were also characterized by a remarkable variety in the intensity of A. simplex (s.s.), suggesting the commingling of individuals with different migration patterns. This idea might be further justified by the geographic distribution of two genetically distinct groups of A. physeteris (s.l.).

[Prevalence of Cereulide-Producing Bacillus cereus in Pasteurized Milk].

To recognize the risk of Bacillus cereus in pasteurized milk, we investigated the prevalence of B. cereus and the rate of the production of cereulide from B. cereus isolates. B. cereus was found in 66 out of 101 (65.3%) domestically pasteurized milk samples in Japan. The ces gene was identified in 3 out of 90 B. cereus isolates that were isolated from three samples (one product) among the 101 samples. The ces gene positive isolate, the reference strain F4810/72 and a B. cereus isolate collected in a food poisoning incident were shown the productivity of cereulide using an LC-MS/MS analysis. The LC-MS/MS analysis was confirmed the ability of identification and quantification of cereulide produced in the milk samples. In this study, it was shown that B. cereus strains are prevalent in pasteurized milk, some of these strains produce cereulide, and confirmed usefulness of LC-MS/MS analysis to detect cereulide in milk.

Detection of Toxoplasma gondii and Sarcocystis sp. in the meat of common minke whale (Balaenoptera acutorostrata): A case of suspected food poisoning in Japan.

A case of suspected food poisoning related to the consumption of raw meat from a common minke whale (Balaenoptera acutorostrata) was reported in Tokyo, Japan, in June 2020. Microscopic analysis revealed tissue cysts of Toxoplasma gondii and sarcocysts of Sarcocystis sp. in whale meat. The SAG2 and ITS1 region sequences of T. gondii were detected in the DNA extracted from the meat. Genotyping of the multilocus nested PCR-RFLP using the genetic markers SAG1, SAG2 (5'- SAG2, 3'-SAG2, and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that the genotype of T. gondii was type II, with a type I pattern for the L358 locus. In the phylogenetic analyses of the six loci (GRA6, GRA7, SAG1, HP2, UPRT1, and UPRT7), these sequences clustered into haplogroup 2. Moreover, the sequences of the virulence-related genes ROP5 and ROP18 of T. gondii isolated from whale meat were similar to those of the type II ME49 reference strain. Sequence analyses of the mtDNA cox1 gene, 18S rRNA gene, and ITS1 region indicated the highest similarity of sarcocyst isolated from whale meat to Sarcocystis species that infect birds or carnivores as intermediate hosts; however, the species could not be identified. To our knowledge, this is the first report of T. gondii and Sarcocystis spp. being detected in same whale meat ingested by patients involved in a suspected food poisoning case in Japan.

Anisakid larvae in the skipjack tuna Katsuwonus pelamis captured in Japanese waters: Two-year monitoring of infection levels after the outbreak of human anisakiasis in 2018.

In 2018, human anisakiasis caused by the ingestion of the skipjack tuna Katsuwonus pelamis occurred frequently in Japan. This may be attributable to a heavy infection of A. simplex (s.s.) in the host's muscle tissue. In this study, we investigate infection levels of anisakid L3 larvae in skipjack tuna captured in Japanese waters afterward (2019-2020) to contribute to predict and prevent the outbreak of human anisakiasis. A total of 476 larvae were detected from 78 out of 85 skipjack tuna captured at 14 stations of the Pacific and East China Sea. The present parasitological survey suggests that infection levels in 2019-2020 were low, comparing that in 2018; in total only seven larvae were found from the host's muscle tissue. The collected larvae were identified by molecular methods to Anisakis berlandi, A. pegreffii, A. simplex (s.s.), A. typica and Skrjabinisakis physeteris (s.l.). Not only larvae of A. simplex (s.s.) but also those of A. berlandi were found from the muscle tissue and thus the latter species may also be a causative agent of human anisakiasis. In addition, this study confirmed the geographic distribution pattern that A. simplex (s.s.) is abundant in the Pacific, while A. pegreffii is dominant in the East China Sea. Our results contribute to understanding the risk of food poisoning and stock delimitation of host animals.

Prevalence of Anisakis larvae in cultured mackerel Scomber japonicas in Japan and the relationship between the intensity of Anisakis infection in cultured mackerel and fish fatness.

Incidences of food poisoning caused by Anisakis have increased in Japan, and a significant number of anisakiasis cases in Tokyo attributed to the consumption of mackerel (Scomber japonicus) have been reported. There are two types of cultured mackerel in Japan: those cultured fully from controlled parent fish eggs and those cultured from wild juveniles collected from the sea. In this study, we aimed to investigate the prevalence of Anisakis larvae in cultured mackerel (184 fish) in 15 products and identified the species using molecular analysis to evaluate the risk of food poisoning. In total, 1567 Anisakis larvae were detected in 70 of 130 mackerel in 10 products; however, Anisakis larvae were not detected in 54 mackerel using artificially reared juveniles in 5 products. Moreover, 277 larvae were detected in fish muscle, and 98.6 % (273/277 larvae) were molecularly identified as Anisakis simplex sensu stricto (A. simplex). Conversely, 1043 Anisakis pegreffii larvae were identified genetically and/or morphologically but only 2 larvae were identified in the muscle. There was no significant relationship between the host coefficient of fatness and the infection intensity of Anisakis larvae in individual fish (Spearman's rank correlation coefficient test, P > 0.05). Based on the results of the analysis of the cytochrome c oxidase subunit2 (cox2) gene of A. simplex and A. pegreffii detected in this study, we attempted to estimate the catch area of the juveniles (Pacific stock and Tsushima Warm Current stock). The clusters on the phylogenetic tree of the cox2 gene of A. pegreffii from the mackerel presumed to be the two above mentioned geographic distributions were not separated and these geographic origins could not be estimated. This study revealed that mackerel cultured using wild juveniles are likely to be contaminated with Anisakis larvae, which can be detected not only in the visceral organs, but also in the muscle. Anisakis infection in cultured mackerel did not influence fish growth and evaluating the intensity of Anisakis based on the fatness level of the mackerel was complicated. To prevent anisakiasis caused by the consumption of mackerel cultured using wild juveniles, it is important to steadily control Anisakis through heating and freezing.

Current Status of Anisakiasis and Anisakis Larvae in Tokyo, Japan.

Anisakiasis is a gastrointestinal disease caused by infection with anisakid nematodes. Anisakis larvae have been listed as distinct food poisoning agents in the manual of Food Poisoning Statistics, Japan since 2013. The reported numbers of food poisoning cases caused by Anisakis larvae are gradually increasing. A total of 94.0% of the causative larvae species were identified as Anisakis simplex sensu stricto (A. simplex), and 4.4% were identified as Anisakis pegreffii, among human-isolated anisakid nematodes examined in Tokyo Metropolitan Institute of Public Health, Japan from 2011 to 2018. Anisakis species infecting fishes in Japanese waters differ depending on their habitat and depth. A. simplex mainly infects fishes in the Pacific side of Japan, and A. pegreffii mainly infects fishes in the East China Sea and Sea of Japan sides. Regarding the causative foods of anisakiasis, cases by ingestion of mackerel (Scomber spp.) have been the most common in Japan, and cases caused by eating "marinated mackerel" accounted for 32.8% of the total in Tokyo from 2011 to 2017. However, the number of reports of food poisoning caused by skipjack tuna (Katsuwonus pelamis) was highest in May 2018 in Japan. A parasitological surveys of Anisakis third-stage larvae in skipjack tuna in Japanese waters were conducted in 2018 and 2019, and it was confirmed that more A. simplex infections of skipjack tuna may have occurred in 2018 than usual due to the meandering flow of the Black Current. Moreover, a portion of A. simplex larvae migrated from visceral organs to the ventral muscle in live skipjack tuna before capture, suggesting that an extensive cold chain after capture cannot prevent anisakiasis. In fish species that were reported to be high frequency of causative food of anisakiasis, it is necessary to freeze or at least remove the ventral muscle.

Probable association between Anisakis infection in the muscle of skipjack tuna (Katsuwonus pelamis) and human anisakiasis in Tokyo, Japan.

Anisakiasis is a gastrointestinal disease caused by parasitic anisakid nematodes, mainly Anisakis simplex sensu stricto (A. simplex). Anisakiasis is prevalent in Japan and approximately 40% of anisakiasis cases in Tokyo occur through the consumption of raw or marinated mackerel. However, in 2018, there was a sudden increase in the number of the food poisoning cases in Tokyo caused by consumption of skipjack tuna (Katsuwonus pelamis). Therefore, we investigated anisakiasis cases resulting from ingestion of skipjack tuna in Tokyo, and surveyed the presence of Anisakis larvae in skipjack tuna in 2018 and 2019. Nineteen samples from 15 patients (13 in 2018 and 2 in 2019) with anisakiasis surely caused by ingestion of skipjack tuna were all identified as A. simplex. The higher mean abundance of Anisakis simplex larvae in skipjack tuna muscle in May 2018 (1.30; 13 larvae/10 fishes) compared to that in the other periods was regarded as a contributing factor in the increase in anisakiasis cases by ingesting skipjack tuna in 2018. To verify whether Anisakis larvae migrate from the visceral organs to the muscle during the period from fishing on the boat until processing for sale, the number of Anisakis larvae in skipjack tuna caught from August to November 2018 was investigated by removing the visceral organs at three different timings, i.e., immediately after catching, after landing, and after transport to the laboratory. Anisakis larvae were detected in the muscle irrespective of the timings at which visceral organs were removed. All larvae from the muscle were detected only from the ventral part and were identified as A. simplex. We thus consider that avoiding raw consumption of the ventral muscle should be an effective measure to prevent anisakiasis.

Hybrid origin of gynogenetic clones and the introgression of their mitochondrial genome into sexual diploids through meiotic hybridogenesis in the loach, Misgurnus anguillicuadatus.

In a few Japanese populations of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae), clonal diploid lineages produce unreduced diploid eggs that normally undergo gynogenetic reproduction; however the origin of these clones remains elusive. Here, we show the presence of two diverse clades, A and B, within this loach species from sequence analyses of two nuclear genes RAG1 (recombination activating gene 1) and IRBP2 (interphotoreceptor retinoid-binding protein, 2) and then demonstrate heterozygous genotypes fixed at the two loci as the evidence of the hybrid nature of clonal lineages. All the clonal individuals were identified by clone-specific mitochondrial DNA haplotypes, microsatellite genotypes, and random amplified polymorphic DNA fingerprints; they commonly showed two alleles, one from clade A and another from clade B, whereas other wild-type diploids possessed alleles from either clade A or B. However, we also found wild-type diploids with clone-specific mitochondrial DNA and nuclear genes from clade B. One possible explanation is an introgression of a clone-specific mitochondrial genome from clonal to these wild-type loaches. These individuals likely arose by a cross between haploid sperm from bisexual B clade males and haploid eggs with clone-specific mtDNA and clade B nuclear genome, produced by meiotic hybridogenesis (elimination of unmatched A genome followed by meiosis after preferential pairing between two matched B genomes) in clone-origin triploid individual (ABB).