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.

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.

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.).

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.

Molecular identification and characterization of Sarcocystis spp. in horsemeat and beef marketed in Japan.

Human Sarcocystis infections are known to be caused by the ingestion of raw or undercooked beef or pork containing mature sarcocysts of Sarcocystis hominis or S. suihominis, respectively. In addition, several cases of parasitic food poisoning in Japan have recently been reported after consumption of raw horsemeat containing sarcocysts of S. fayeri. In this study, the presence of sarcocysts in 28 horsemeat and 121 beef samples collected in Tokyo was investigated. Sarcocysts of S. fayeri were found in 16 horsemeat samples. Sarcocysts of S. hominis were not detected in beef samples, while sarcocysts of S. cruzi were detected in 60 beef samples. In addition, S. hirsuta and S. bovini were isolated only from New Zealand beef samples. Bradyzoites in sarcocysts collected from 62/73 sarcocyst-positive refrigerated horsemeat and beef samples were determined to be viable. Molecular analysis of S. fayeri 18S rRNA gene sequences revealed that intraspecific variation among eight individual bradyzoites from a single sarcocyst was as high as 9.8%. In contrast, mitochondrial cytochrome c oxidase subunit 1 (mtDNA cox1) gene sequences from the six fragments of a single sarcocyst were 100% identical. Sarcocysts of S. bovini isolated from beef also exhibited intraspecific variation in 18S rRNA gene sequences and had to be cloned before sequencing, while mtDNA cox1 gene sequences were obtained by direct sequencing. Therefore, we conclude that molecular analysis of the mtDNA cox1 gene is the most useful for identification of Sarcocystis species. This study provides the first published partial sequence of the S. fayeri mtDNA cox1 gene.

Detection rate of diarrhoea-causing Kudoa hexapunctata in Pacific bluefin tuna Thunnus orientalis from Japanese waters.

Diffuse outbreaks of food poisoning with unknown aetiologies leading to diarrhoea and vomiting within a short time after ingesting flatfish (Paralichthys olivaceus), tuna (Thunnus spp.), or amberjack (Seriola dumerili) have occurred nationwide in Japan, including the Tokyo metropolitan area. In this study, we surveyed the detection rates of kudoid parasites in 12 tuna samples that caused clinical diarrhoea from 2009 to 2012; we assessed 104 samples of whole juvenile Pacific bluefin tuna (PBT, Thunnus orientalis) and 153 block samples of other tuna distributed in the Tokyo Metropolitan Central Wholesale Market. The survey revealed that more than 70% of clinical diarrhoea cases due to tuna ingestion occurred between June and September, and Kudoa hexapunctata were detected in 9 of 12 tuna samples associated with clinical diarrhoea cases. The numbers of spores and 18S ribosomal DNA (rDNA) copies per gram of fish in 8 of 9 samples were more than 1×10(6) spores and 1×10(9) copies, respectively. Market research revealed that the K. hexapunctata-positive rate in juvenile PBT from Japanese waters was 64.4% (67/104) but that in adult PBT was 10.4% (7/67). The numbers of K. hexapunctata 18S rDNA copies in 64.5% (20/31) samples and 72.7% (16/22) of <5kg fish samples collected between May and July were more than 1×10(9)copies/g. On the other hand, kudoid parasites were not detected from 73 tuna samples except for a single sample of Thunnus albacares. Cell monolayer permeability assays performed to examine the toxicity of K. hexapunctata against Caco-2 cells revealed that the transepithelial electrical resistance (TER) in 5×10(7)K. hexapunctata spores decreased by 80% within 2-4h. In conclusion, K. hexapunctata was commonly detected in juvenile PBT from Japanese waters and are a likely cause of the diarrhoea outbreaks.

Soluble interleukin-2 receptor level on day 7 as a predictor of graft-versus-host disease after HLA-haploidentical stem cell transplantation using reduced-intensity conditioning.

In the present study, we analyzed the kinetics of serum soluble interleukin-2 receptor (sIL-2R) using data from 77 patients undergoing HLA-haploidentical transplantation using reduced-intensity conditioning (RIC), who were at an advanced stage or at high risk for relapse, to clarify the usefulness of sIL-2R as a biomarker of acute graft-versus-host disease (GVHD). Anti-T-lymphocyte globulin and methylprednisolone were used as GVHD prophylaxis. While the median sIL-2R in 38 patients not developing GVHD was suppressed at levels <740 U/ml, sIL-2R in 25 patients developing severe GVHD peaked on day 11 (1,663 U/ml), and thereafter decreased to <1,000 U/ml after day 30. The occurrence of GVHD was not limited to times of high sIL-2R level, but occurred at any time point on the sIL-2R curve. Most patients developing GVHD, however, experienced a higher sIL-2R level early in their transplant course. The combination of RIC and glucocorticoids sufficiently suppressed sIL-2R levels after HLA-haploidentical transplantation. In a multivariate analysis to identify factors associated with GVHD, day 7 sIL-2R >810 U/ml was the only factor significantly associated with the occurrence of severe GVHD (p = 0.0101).

Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters.

The third-stage (L3) larvae of Anisakis, which are the etiological agents of human anisakiasis, have been categorized morphologically into Anisakis Type I larvae and Anisakis Type II larvae. Genetic analysis has allowed easy identification of these larvae: Anisakis Type I larvae include the species Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis simplex C, Anisakis typica, Anisakis ziphidarum, and Anisakis nascettii, whereas Anisakis Type II larvae include the species Anisakis physeteris, Anisakis brevispiculata, and Anisakis paggiae. Since human consumption of raw fish and squid is common in Japan, we investigated Anisakis L3 larvae in 44 specimens of Beryx splendens from Japanese waters. A total of 730 Anisakis L3 larvae collected from B. splendens were divided morphologically into 4 types: Type I, Type II, and 2 other types that were similar to Anisakis Type III and Type IV described by Shiraki (1974). Anisakis Type II, Type III, and Type IV larvae all had a short ventriculus, but their tails were morphologically different. In addition, data from genetic analysis indicated that Anisakis Type II, Type III, and Type IV larvae could be identified as A. physeteris, A. brevispiculata, and A. paggiae, respectively. Therefore, A. physeteris, A. brevispiculata, and A. paggiae can be readily differentiated not only by genetic analysis but also by morphological characteristics of L3 larvae.

Risk factors for human Anisakis infection and association between the geographic origins of Scomber japonicus and anisakid nematodes.

It has been reported that nearly all cases of anisakiasis in Japan are caused by Anisakis simplex sensu stricto. To elucidate this further, we investigated the presence of Anisakis type I larvae and Pseudoterranova decipiens in 218 Scomber japonicus fish collected from the seas of Japan. Anisakis type I larvae were detected in 74.3% (162/218) of the fish, and 99.8% of the Anisakis type I larvae comprised A. simplex sensu stricto and Anisakis pegreffii. Molecular identification techniques were used for 7.5% (360/4806) of the Anisakis type I larvae. The larvae found in the fish of the Pacific stock (the Pacific coast of Japan) and the Tsushima Warm Current stock (the East China Sea and the Sea of Japan) were primarily A. simplex sensu stricto and A. pegreffii, respectively. In addition, for the first time in Japan, Anisakis simplex C and Anisakis ziphidarum were detected in the fish of the Pacific stock. The average number of A. pegreffii and A. simplex sensu stricto larvae per fish was 47 and 6, respectively. However, the average number (0.61 larvae) of A. simplex sensu stricto in the muscle per fish was 12 times the average number (0.05 larvae) of A. pegreffii. When fish on the purchased day were compared with those held at 4 degrees C and 20 degrees C for 20h, the penetration rates (ratio of the number of larvae detected in the muscle to the total number of larvae detected) of A. pegreffii and A. simplex sensu stricto were as high as 1.8% and 5.8%, respectively. In conclusion, we suggest that anisakiasis in Japan is mainly caused by A. simplex sensu stricto because it penetrates the muscle of the fish at a higher rate than A. pegreffii.

A survey of amoebic infections and differentiation of an Entamoeba histolytica-like variant (JSK2004) in nonhuman primates by a multiplex polymerase chain reaction.

A pathogenic Entamoeba histolytica-like variant (JSK2004 strain) with genetic variations and a novel isoenzyme pattern isolated from a De Brazza's guenon in a Tokyo zoo in Japan has previously been documented. In this study, a multiplex polymerase chain reaction (PCR) assay that could distinguish the JSK2004-type E. histolytica-like variant (JSK04-Eh-V) from E. histolytica and Entamoeba dispar using three newly designed primer sets for amplifying each specific DNA fragment from their small-subunit ribosomal RNA genes was developed and established. Forty-seven primates (11 species) from the zoo were surveyed by multiplex PCR to assess the prevalence of JSK04-Eh-V infection, which was recognized in six individuals of four species, including an Abyssinian colobus monkey, a De Brazza's guenon (including the individual from whom JSK2004 was isolated), a white-faced saki, and a Geoffroy's spider monkey. In addition, the autopsied individuals of an Abyssinian colobus and Geoffroy's spider monkey that died of amoebic liver abscess were also evaluated. DNA samples were also analyzed for specific genotypes based on the nucleotide sequencing of two protein-coding (chitinase and serine-rich E. histolytica protein) genes and the protein-noncoding locus 1-2 that was used for fingerprinting of the E. histolytica strain. These studies indicated that the E. histolytica-like variant infection in this zoo was caused by the same type (i.e., JSK04-Eh-V). An axenic culture medium (yeast extract-iron-maltose-dihydroxyacetone-serum) was developed based on the yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium, which is designed for axenic culture of E. dispar. This new medium could be used for axenically culturing E. histolytica, JSK04-Eh-V, and E. dispar in a single medium.

Seroprevalence of Entamoeba histolytica infection in female outpatients at a sexually transmitted disease sentinel clinic in Tokyo, Japan.

From 2003 to 2006, we surveyed the seroprevalence of amoebic infection in female outpatients at a gynecologist's office, which was designated as a sexually transmitted disease sentinel clinic by the Tokyo Metropolitan Government, using an enzyme-linked immunosorbent assay (ELISA). The annual rate of anti-Entamoeba histolytica (HM-1:IMSScl6 strain; HM-1) antibody-positive cases as detected by ELISA increased during that period, and anti-Chlamydia trachomatis antibodies were detected in 60%, i.e., 24 of 40 anti-HM-1 antibody-positive individuals, suggesting sexual transmission of E. histolytica. We designed an ELISA with better sensitivity using the antigen extracted from the virulence-augmented E. histolytica strains (LHM-1 and LLA526 strains) by liver-passaging in hamsters. The average ratios of the S/N value (optical density [OD] of sample/OD of negative control) of ELISA with either the LHM-1 or LLA526 antigen and that of ELISA with the HM-1 antigen were significantly higher in intestinal amoebiasis cases with low S/N values than in amoebic liver abscess cases. In the present study of the seroprevalence of E. histolytica infection, the sera testing positive with low S/N values (<10) by ELISA with HM-1 antigen exhibited higher S/N values by ELISA using LHM-1 and LLA526 antigens. This modification of the antigen preparation for ELISA is expected to be effective in detecting anti-E. histolytica antibodies from such asymptomatic patients who have low antibody titers.

Profiles of a pathogenic Entamoeba histolytica-like variant with variations in the nucleotide sequence of the small subunit ribosomal RNA isolated from a primate (De Brazza's guenon).

A pathogenic Entamoeba histolytica-like variant (JSK2004) with genetic variations and a novel isoenzyme pattern was isolated from a De Brazza's guenon. A homology of 99.1% was found between the clones of E. histolytica (HM-1:IMSS) and JSK2004 in the 1,893 nucleotide bases of the small subunit rRNA (SSU-rRNA) gene. The DNA of the pathogenic amoeba species was also extracted from two sterile liver abscesses during the autopsies of an Abyssinian colobus and a Geoffroy's spider monkey occurring in the same institution in which JSK2004 was isolated, and the homology of the nucleotide sequences in the SSU-rRNA gene of the DNAs was identical to that of JSK2004.