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Foodborne disease outbreaks caused by raw olive flounders ( Paralichthys olivaceus) parasitized with Kudoa septempunctata have been reported in Japan. Origins of olive flounders consumed in Japan vary, being either domestic or imported, and aquaculture-raised or natural. Although it is unknown whether different sources are associated with different outcomes, it is desirable to identify whether this is the case by determining whether unique K. septempunctata strains occur and if so, whether some are associated with foodborne illness. We here developed an intraspecific genotyping method, using the sequence variation of mitochondrial genes. We collected olive flounder samples from foodborne disease outbreaks, domestic fish farms or quarantine offices and investigated whether K. septempunctata genotype is associated with pathogenicity or geographic origin. The 104 samples were classified into three genotypes, ST1, ST2 and ST3. Frequency of symptomatic cases differed by genotypes, but the association was not statistically significant. Whereas K. septempunctata detected from aquaculture-raised and natural fish from Japan were either ST1 or ST2, those from fish inspected at quarantine from Korea to Japan were ST3. Our method can be applied to phylogeographic analysis of K. septempunctata and contribute to containing the foodborne disease. The genotype database is hosted in the Pub MLST website (). [ABSTRACT FROM AUTHOR]

Species of the genusFusariumare well-known plant pathogens and mycotoxigenic fusaria are associated with health hazards to humans and animals. There is a need to understand the mechanisms of mycotoxin production byFusariumspecies and to predict which produce mycotoxins. In this study, theFusariumphylogenetic tree was first inferred among trichothecene producers and related species. We reconstructed the maximum likelihood (ML) tree based on the combined data from nucleotide sequences of rDNA cluster regions, the β-tubulin gene (β-tub) and the elongation factor 1α gene (EF-1α). Second, based on this tree topology, the ancestral states of the producing potential of type A and B trichothecenes (TriA and TriB), zearalenone (ZEN), moniliformin (MON), beauvericin (BEA) and enniatins (ENN) were reconstructed using the maximum parsimony (MP) method based on the observed production by extant species as reported in the literature. Finally, the species having the potential to produce each of these six mycotoxins was predicted on the basis of the parsimonious analysis. The ML tree indicated that theFusariumspecies analysed in this study could be divided into two major clades. Clade I was divided into four distinct subclades: I-a, I-b, I-c and I-d. Furthermore, the parsimony reconstruction suggested that the potential for producing MON and ZEN was gained or lost only once, and that the producing potential for TriA and TriB, BEA and ENN was repeatedly gained and lost during the evolutionary history of theFusariumspecies analysed in this study. Interestingly, the results showed the possibility that several species, about which reports were scarce with regard to mycotoxin production, have the potential to produce one or more of the six evaluated in this study. The phylogenetic information therefore helps one to predict the mycotoxin-producing potential byFusariumspecies, and these “phylotoxigenic relationships” may be useful for predicting the pathogenicity of fungi. [ABSTRACT FROM PUBLISHER]

To identify a rapid method for extracting a large amount of DNA from fungi associated with food hygiene, extraction methods were compared using fungal pellets formed rapidly in liquid media. Combinations of physical and chemical methods or commercial kits were evaluated with 3 species of yeast, 10 species of ascomycetous molds, and 4 species of zygomycetous molds. Bead grinding was the physical method, followed by chemical methods involving sodium dodecyl sulfate (SDS, cetyl trimethyl ammonium bromide (CTAB), and benzyl chloride and two commercial kits. Quantity was calculated by UV absorbance at 260 nm, quality was determined by the ratio of UV absorbance at 260 and 280 nm, and gene amplifications and electrophoresis profiles of whole genomes were analyzed. Bead grinding with the SDS method was the most effective for DNA extraction for yeasts and ascomycetous molds, and bead grinding with the CTAB method was most effective with zygomycetous molds. For both groups of molds, bead grinding with the CTAB method was the best approach for DNA extraction. Because this combination also is relatively effective for yeasts, it can be used to extract a large amount of DNA from a wide range of fungi. The DNA extraction methods are useful for developing gene indexes to identify, fungi with molecular techniques, such as DNA fingerprinting. [ABSTRACT FROM AUTHOR]

The giant river shrimp ( Macrobrachium rosenbergii), a native species of Thailand, is either exported for commercial purposes or supplied to meet the local requirements in Thailand. Limited pharmacokinetic information of the major antibiotic, oxytetracycline (OTC), is available for this freshwater shrimp. The purpose of the present study was to investigate the muscle tissue kinetics of OTC in M. rosenbergii following either intramuscular (i.m.) or oral (p.o.) administration at two dosages of 11 and 22 mg/kg body weight (b.w.). The concentration of OTC in shrimp tissues was measured using high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. Muscle tissue concentrations were below the detection limit (LOD, 0.1 μg/g) after 96 and 120 h, following i.m. and p.o. administration, respectively. Peak muscle concentrations (Cmax) were 3.47 and 1.73 μg/g after i.m. and p.o. administration at a single dose of 11 mg/kg b.w. whereas they were 6.03 and 2.51 μg/g at a single dose of 22 mg/kg b.w., respectively. A noncompartment model was developed to describe the pharmacokinetics of OTC in the giant freshwater shrimp. The terminal half-lives of OTC were 28.68 and 28.09 h after i.m. and p.o. administration at a single dose of 11 mg/kg b.w., but 29.95 and 27.03 h at a single dose of 22 mg/kg b.w., respectively. The relative bioavailability was 82.32 and 64.67% following i.m. and p.o. administration, respectively. Based on the pharmacokinetic data, i.m. and p.o. administration with OTC at a dose of 11 mg/kg b.w. would be appropriate for use in giant freshwater shrimp farming. To avoid the OTC residue in shrimp muscle, it should take at least seven half-lives (8 days) to wash out the drug from the muscle of M. rosenbergii. [ABSTRACT FROM AUTHOR]

No information is available about the effects of Japanese radish sprout (JRS) on diabetes. To clarify the effects, the influence of JRS on carbohydrate and lipid metabolisms was investigated in normal and streptozotocin-induced diabetic rats. The rats were fed a diet containing 0%, 2.5% or 5% of JRS ad libitum for 21 days. Compared with the corresponding control groups, the JRS-fed normal rats showed lower plasma levels of total cholesterol (TC), triglycerides (TG), phospholipids (PL), fructosamine, glucose and insulin and higher plasma levels of low-density lipoprotein-cholesterol, whereas the JRS-fed diabetic rats showed lower plasma levels of fructosamine, glucose and insulin without changes in the plasma lipid parameters. JRS also decreased the hepatic TC, TG and PL levels in the normal rats and the TG level in the diabetic rats. These results showed that JRS had a hypoglycemic activity in both the normal and diabetic rats and partly improved lipid metabolism in the normal rats. JRS has the potential to alleviate hyperglycemia in cases where diabetes is present and to serve in the primary prevention of diabetes mellitus. [ABSTRACT FROM AUTHOR]

Abstract Background Species of the Fusarium genus are important fungi which is associated with health hazards in human and animals. The taxonomy of this genus has been a subject of controversy for many years. Although many researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, their phylogenetic relationships remain unclear only few comprehensive phylogenetic analyses of the Fusarium genus and a lack of suitable nucleotides and amino acid substitution rates. A previous stugy with whole genome comparison among Fusairum species revealed the possibility that each gene in Fusarium genomes has a unique evolutionary history, and such gene may bring difficulty to the reconstruction of phylogenetic tree of Fusarium. There is a need not only to check substitution rates of genes but also to perform the exact evaluation of each gene-evolution. Results We performed phylogenetic analyses based on the nucleotide sequences of the rDNA cluster region (rDNA cluster), and the β-tubulin gene (β-tub), the elongation factor 1α gene (EF-1α), and the aminoadipate reductase gene (lys2). Although incongruence of the tree topologies between lys2 and the other genes was detected, all genes supported the classification of Fusarium species into 7 major clades, I to VII. To obtain a reliable phylogeny for Fusarium species, we excluded the lys2 sequences from our dataset, and re-constructed a maximum likelihood (ML) tree based on the combined data of the rDNA cluster, β-tub, and EF-1α. Our ML tree indicated some interesting relationships in the higher and lower taxa of Fusarium species and related genera. Moreover, we observed a novel evolutionary history of lys2. We suggest that the unique tree topologies of lys2 are not due to an analytical artefact, but due to differences in the evolutionary history of genomes caused by positive selection of particular lineages. Conclusion This study showed the reliable species tree of the higher and lower taxonomy in the lineage of the Fusarium genus. Our ML tree clearly indicated 7 major clades within the Fusarium genus. Furthermore, this study reported differences in the evolutionary histories among multiple genes within this genus for the first time.

With increases in consumer demand for fried foods in Japan over the last several decades, the consumption of frying oil has also steadily increased. Fryers used in restaurants to cook large quantities of food are typically cleaned using neutral kitchen detergents at the end of the day after removing the oil from the tank. However, significant amounts of debris can remain in the fryer after cleaning, possibly accelerating oil deterioration and thus reducing the quality of the fried foods. In this study, debris obtained from fryer tanks used in actual restaurants was assessed using scanning electron microscopy-energy dispersive X-ray spectroscopy together with Fourier transform infrared spectroscopy, and were determined to comprise polymerized oil and carbonized organic matter. Experiments using artificially prepared debris confirmed that these materials increased the acid value (AV) of frying oil. Trials in two restaurants serving similar amounts of fried chicken, French fries and doughnuts examined the effects of cleaning the fryer with either an alkaline detergent or a neutral kitchen detergent on debris removal and oil life. The alkaline detergent was found to completely remove debris while the neutral detergent left significant amounts of debris. After cleaning, the fryers were operated with new oil as usual and the deterioration of this oil was monitored by assessing the color difference, AV, carbonyl value and peroxide value. These indices increased 1.3 to 2.0 times faster in the case that the neutral kitchen detergent was used, suggesting that cleaning fryer tanks with an alkaline detergent could contribute to extending the lifetime of frying oil, reducing food losses and thus achieving sustainable development goals.

Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food. A membrane-based yeast two-hybrid system using split-ubiquitin was successfully used to detect practically important concentration ranges of caffeine and aflatoxins using separated variable regions of an antibody. Moreover, enterohemorrhagic Escherichia coli O157 was detected using a specific single-chain antibody, in which Zymolyase was added to partially destroy the cell wall. The incorporation of secreted Cypridina luciferase reporter further simplified the signal detection procedures without cell lysis. The methodology is more cost-effective and faster than using mammalian cells. The ability to detect various targets renders Patrol Yeast a valuable tool for ensuring food and beverage safety and addressing other environmental and technological issues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Spices have been known to be highly contaminated commodities with mycotoxins. The Codex Alimentarius reports that nutmeg is particularly contaminated with aflatoxins (AFs) and ochratoxin A (OTA). To eliminate contaminated commodities, visual sorting and bright greenish-yellow fluorescence (BGYF) sorting are used as low-cost technologies in production engineering. In Indonesia, nutmeg is mainly sorted by visual sorting and classified into three grades according to the Indonesian national standards, with importers further defining their own brand as imported products. In this study, we evaluate the efficacy of BGYF sorting as a further selection method to reduce AFs and OTA using the importer's own brand. Further, the level of these mycotoxins and the relationship between fungal flora and mycotoxin contamination were examined. These results showed that BGYF sorting effectively reduces AFs as well as OTA. In addition, BGYF-positive groups were infected by Aspergillus sections Flavi, Nigri, and Circumdati.

Citreoviridin (CTV) is a mycotoxin produced by various fungi, including Penicillium citreonigrum. One of the toxicities reportedly associated with CTV is neurotoxicity. CTV is also suspected to be associated with acute cardiac beriberi (also known as "Shoshin-kakke") and Keshan disease, which can have adverse effects on the heart, so the in vivo and in vitro toxicity of CTV on the heart or cardiomyocytes in experimental animal models have been reported. However, the toxicity of CTV for the human heart, especially its electrophysiological effect, remains poorly understood. Therefore, to investigate the electrophysiological effect of CTV on the human cardiomyocytes, we conducted a multi-electrode array (MEA) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The MEA revealed that 30 μmol/L of CTV stopped the beating of hiPSC-CMs, and the field potential duration and first peak amplitude were shortened at 10 μmol/L. Before the hiPSC-CMs stopped beating, the length of the inter-spike interval varied two- to four-fold. These results demonstrated that CTV induced an electrophysiological disturbance on human cardiomyocytes. This is first paper to elucidate the electrophysiological effect of CTV on human heart directly and may aid in analyzing the risk associated with CTV to ensure food safety.

Exposure to sterigmatocystin (STC) raises concerns on developmental neurological disorders. The present study investigated the effects of maternal oral STC exposure on postnatal hippocampal neurogenesis of offspring in rats. Dams were exposed to STC (1.7, 5.0, and 15.0 ppm in diet) from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without STC exposure until adulthood on postnatal day (PND) 77, in accordance with OECD chemical testing guideline Test No. 426. On PND 21, 15.0-ppm STC decreased type-3 neural progenitor cell numbers in the subgranular zone (SGZ) due to suppressed proliferation. Increased γ-H2AX-immunoreactive ( + ) cell numbers in the SGZ and Ercc1 upregulation and Brip1 downregulation in the dentate gyrus suggested induction of DNA double-strand breaks in SGZ cells. Upregulation of Apex1 and Ogg1 and downregulation of antioxidant genes downstream of NRF2-Keap1 signaling suggested induction of oxidative DNA damage. Increased p21 WAF1/CIP1+ SGZ cell numbers and suppressed cholinergic signaling through CHRNB2-containing receptors in GABAergic interneurons suggested potential neurogenesis suppression mechanisms. Multiple mechanisms involving N-methyl-d-aspartate (NMDA) receptor-mediated glutamatergic signaling and various GABAergic interneuron subpopulations, including CHRNA7-expressing somatostatin + interneurons activated by BDNF-TrkB signaling, may be involved in ameliorating the neurogenesis. Upregulation of Arc, Ptgs2, and genes encoding NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors suggested synaptic plasticity facilitation. On PND 77, ARC + granule cells decreased, and Nos2 was upregulated following 15.0 ppm STC exposure, suggesting oxidative stress-mediated synaptic plasticity suppression. Inverse pattern in gene expression changes in vesicular glutamate transporter isoforms, Slc17a7 and Slc17a6, from weaning might also be responsible for the synaptic plasticity suppression. The no-observed-adverse-effect level of maternal oral STC exposure for offspring neurogenesis was determined to be 5.0 ppm, translating to 0.34-0.85 mg/kg body weight/day., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1β, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function., Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-021-00487-y., Competing Interests: Conflict of interestThe authors have no conflicts of interest to disclose., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

To understand fungal contamination in the indoor environment of the disaster region, a field survey was performed to measure the number of fungal counts and identify isolates in the indoor air of prefabricated temporary housing, privately independent-housing, and rented apartments flooded by the East Japan Great Earthquake disaster tsunami. As a result, the period with the highest detected fungal count was from the rainy season to summer in independent-housing and rented apartments. Moreover, in the temporary housing, the fungal number increased further in winter as indicated by the maximum fungal-number throughout the measurement period. The detection frequency of Aspergillus species was relatively higher in the indoor air of temporary housing than in typical housing in the non-disaster area. Since Aspergillus is known as an allergenic genus, it requires careful attention to the health risk for residents. The extremely high level of fungal condensation in indoor air possibly occurred due to high relative humidity and loss of heat insulation in the building attics. It is suggested that this problem commonly happened in the cold region including the entire disaster region of the East Japan Great Earthquake.

Ciguatera fish poisoning (CFP) is one of the most frequently encountered seafood poisoning syndromes; it is caused by the consumption of marine finfish contaminated with ciguatoxins (CTXs). The majority of CFP cases result from eating fish flesh, but a traditional belief exists among people that the head and viscera are more toxic and should be avoided. Unlike the viscera, scientific data to support the legendary high toxicity of the head is scarce. We prepared tissue samples from the fillet, head, and eyes taken from five yellow-edged lyretail ( Variola louti ) individuals sourced from Okinawa, Japan, and analyzed the CTXs by LC-MS/MS. Three CTXs, namely, CTX1B, 52- epi -54-deoxyCTX1B, and 54-deoxyCTX1B, were confirmed in similar proportions. The toxins were distributed nearly evenly in the flesh, prepared separately from the fillet and head. Within the same individual specimen, the flesh in the fillet and the flesh from the head, tested separately, had the same level and composition of toxins. We, therefore, conclude that flesh samples for LC-MS/MS analysis can be taken from any part of the body. However, the tissue surrounding the eyeball displayed CTX levels two to four times higher than those of the flesh. The present study is the first to provide scientific data demonstrating the high toxicity of the eyes.

Boiled noodles are considered to be one of the most perishable foods due to their high moisture content and high water activity. Thus, the hygiene control measures based on HACCP manuals has been recommended in the noodle manufacturing industry. However, there were several cases in which post-packaged products manufactured at the Boiled noodles factory of small-to-medium size company detected a viable cell count higher than their voluntary standards. To identify the source of microbial contamination, an investigation based on the Plan-Do-Check-Act (PDCA) cycle was conducted. The results showed that the bacteria causing the contamination were environmental bacteria. Secondary contamination occurred during the cooling process after sterilization. Airborne environmental bacteria and oxygen may have been introduced into the rinsing and cooling water tank by the strong water flow during the rinsing and cooling process, inducing growth of microorganisms in the cooling water and contaminating the final product. This is a new finding, as such occurrence was not listed in the HACCP manual and should be contributed to plan HACCP system.

Ciguatera poisoning (CP) is one of the most abundant seafood poisonings in the world. CP frequently occurred in the tropical and subtropical Indo-Pacific Ocean and the Caribbean Sea. In Japan, CP cases have been reported annually, from the subtropical regions, including Okinawa Prefecture and Amami Islands, Kagoshima Prefecture. The principal toxins, named ciguatoxins (CTXs), are bio-synthesized by benthic dinoflagellate of genera Gambierdiscus and Fukuyoa. They are bio-transferred herbivorous animals to carnivorous fishes via the food chain.The Ogasawara Islands comprise more than 30 islands, Mukojima Islands, Chichijima (Bonin) Islands, Hahajima Islands, Iwo Islands, Nishinoshima, Minamitorishima, and Okinotorishima, which locate in the tropical to subtropical regions. The Mukojima Islands, Chichijima Islands, and Hahajima Islands locate approximately the same latitude as Okinawa. The distance from Tokyo is approximately 1,000 km for Chichijima, 1,700 km for Okinotorishima (the southernmost tip of Japan), and 1,900 km for Minamitorishima (the easternmost tip of Japan). These islands exist in a wide range of waters, latitudes from 20°25' to 27°44' North and longitudes from 136°04' to 153° 59' East. We collected 65 specimens of a grouper, Variola louti, the most frequent species implicated in CP in Japan, from the waters around the Chichijima, Mukojima, and Hahajima islands. The fish flesh specimens were analyzed CTXs using the liquid chromatography-tandem mass spectrometer (LC-MS/MS). While the peak whose retention time is almost identical to that of CTX1B was detected in all specimens on our routine protocol, no 52-epi-54-deoxyCTX1B nor 54-deoxyCTX1B was detected. The peak retention time was quite different from that of CTX1B when re-analyzing by changing the analytical column. Thus, the CTXs in the specimens in the waters of these islands seemed to be undetectable levels.

Aflatoxins (AFs) are known to be oncogenic mycotoxins. This study investigated the mitigation effects of lactic acid bacteria (LAB) isolated from four types of vegetable, cucumber, Chinese cabbage, Japanese radish and eggplant, which are used to make Japanese traditional fermented pickles, on AFs. Using aflatoxin M 1 (AFM 1 ) binding assay for screening, four representative strains were selected (one from each vegetable) from total 94 LAB strains, based on the highest binding ratio. The ranges of the binding ratio of these representative strains to aflatoxin B 1 (AFB 1 ), aflatoxin B 2 , aflatoxin G 1 , aflatoxin G 2 and AFM 1 were 57.5%-87.9% for the LAB strain derived from cucumber, 18.9%-43.9% for the LAB strain derived from Chinese cabbage, 26.4%-41.7% for the LAB strain derived from Japanese radish, and 15.0%-42.6% for the LAB strain derived from eggplant. The strains isolated from cucumber, Chinese cabbage, Japanese radish and eggplant were identified as Lactococcus lactis subsp. lactis, Weissella cibaria, Leuconostoc mesenteroides and Leu. mesenteroides, respectively. An in vitro binding assay of the four strains under acidic conditions showed that the number of living bacteria decreased, while the binding ratio increased in some strains, suggesting that the LAB maintained their capacity to bind aflatoxins even in an environment that imitated the stomach. An in vivo experiment using L. lactis subsp. lactis derived from cucumber revealed that the bacteria significantly inhibited the absorption of AFB 1 into blood. These results showed that the LAB used for Japanese vegetable pickles was an effective binding agent of AFs and suggested that they might play a role in mitigating AF absorption.

Entoloma sarcopum is widely known as an edible mushroom but appears morphologically similar to the poisonous mushrooms E. rhodopolium sensu lato (s. l.) and E. sinuatum s. l. Many cases of food poisoning caused by eating these poisonous mushrooms occur each year in Japan. Therefore, they were recently reclassified based on both morphological and molecular characteristics as sensu stricto species. In this study, we analyzed the nucleotide sequences of the rRNA gene (rDNA) cluster region, mainly including the internal transcribed spacer regions and mitochondrial cytochrome oxidase 1 (CO1) gene, in E. sarcopum and its related species, to evaluate performances of these genes as genetic markers for identification and molecular phylogenetic analysis. We found that the CO1 gene contained lineage-specific insertion/deletion sequences, and our CO1 tree yielded phylogenetic information that was not supported by analysis of the rDNA cluster region sequence. Our results suggested that the CO1 gene is a better genetic marker than the rDNA cluster region, which is the most widely used marker for fungal identification and classification, for discrimination between edible and poisonous mushrooms among Japanese E. sarcopum and related species. Our study thus reports a new genetic marker that is useful for detection of Japanese poisonous mushrooms, Entoloma.

The first study reporting the morphological characterization of Sarcocystis sybillensis was performed in 1983; however, without any molecular analysis. Sarcocystis nipponi has been recently described as a species synonymic to S. sybillensis. We reconfirmed the presence of S. sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory; and performed its molecular and phylogenetic characterization. The morphological characteristics of the sarcocysts were consistent with those of S. nipponi and S. sybillensis described in the first report. However, the nucleotide sequence of 18S rRNA gene of S. sybillensis showed only 91.9% identity to that of S. nipponi, suggesting low homology among the concerned Sarcocystis spp. Accordingly, S. sybillensis was found to occupy a clade distinct from that of S. nipponi in a phylogenetic tree of Sarcocystis. Therefore, the present study provides essential information on 18S rRNA-based molecular characterization of S. sybillensis and disproves the existing notion of morphology-based species synonymity of S. sibillensis and S. nipponi. These results also suggest that S. sybillensis belongs to type 2 Sarcocystis.

Citreoviridin (CTV) is a mycotoxin that is produced by Aspergillus terreus , Eupenicillium ochrosalmoneum and Penicillium citreonigrum , and CTV has been detected in a wide range of cereal grains throughout the world. Furthermore, it is especially a serious problem in regions where rice is consumed as a staple food. Moreover, CTV is a well-known yellow rice toxin, and outbreaks of beriberi have occurred due to consumption of rice that is contaminated by CTV even in the recent years. Although CTV biosynthetic genes of A. terreus have been described, those of P. citreonigrum remain unclear, which is concerning since P. citreonigrum is the main cause of CTV contamination in rice. In the present study, we determined the draft genome of the P. citreonigrum strain IMI92228 and revealed the presence of all four genes that form a gene cluster and that are homologous to the CTV biosynthesis genes of A. terreus . The expression of these four homologous genes were highly correlated with CTV production, suggesting that they may play an important role in CTV biosynthesis in P. citreonigrum . We concluded that the gene cluster is a CTV biosynthesis cluster of P. citreonigrum . The findings will contribute to the understanding of the biosynthetic pathway of CTV and will ultimately lead to improvements in the CTV management of agricultural products.

To investigate the developmental exposure effect of diacetoxyscirpenol (DAS) on postnatal hippocampal neurogenesis, pregnant ICR mice were provided a diet containing DAS at 0, 0.6, 2.0, or 6.0 ppm from gestational day 6 to day 21 on weaning after delivery. Offspring were maintained through postnatal day (PND) 77 without DAS exposure. On PND 21, neural stem cells (NSCs) and all subpopulations of proliferating progenitor cells were suggested to decrease in number in the subgranular zone (SGZ) at ≥ 2.0 ppm. At 6.0 ppm, increases of SGZ cells showing TUNEL + , metallothionein-I/II + , γ-H2AX + or malondialdehyde + , and transcript downregulation of Ogg1, Parp1 and Kit without changing the level of double-stranded DNA break-related genes were observed in the dentate gyrus. This suggested induction of oxidative DNA damage of NSCs and early-stage progenitor cells, which led to their apoptosis. Cdkn2a, Rb1 and Trp53 downregulated transcripts, which suggested an increased vulnerability to DNA damage. Hilar PVALB + GABAergic interneurons decreased and Grin2a and Chrna7 were downregulated, which suggested suppression of type-2-progenitor cell differentiation. On PND 77, hilar RELN + interneurons increased at ≥ 2.0 ppm; at 6.0 ppm, RELN-related Itsn1 transcripts were upregulated and ARC + granule cells decreased. Increased RELN signals may ameliorate the response to the decreases of NSCs and ARC-mediated synaptic plasticity. These results suggest that DAS reversibly disrupts hippocampal neurogenesis by inducing oxidative cellular injury and suppressed differentiation of granule cell lineages. The no-observed-adverse-effect level of DAS for offspring neurogenesis was determined to be 0.6 ppm (0.09-0.29 mg/kg body weight/day)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Staphylococcal food poisoning (SFP) is caused by Staphylococcus aureus, and its typical symptom of vomiting is evoked by staphylococcal enterotoxins (SEs). SEs are classified as classical and new types. SEQ is a new-type enterotoxin predicted to have a high potential risk for SFP. To elucidate the correlation between the number of S. aureus cells and the production of SEs as well as classical and new-type enterotoxins in the food environment, the numbers of S. aureus strain cells carrying sea and seq genes and the production of SEA and SEQ protein were examined under 3 pHs values (pH 6.0, 7.0 and 8.0) and 2 NaCl concentrations (0.5 and 1.0%) conditions. The experiments were performed at 25℃, resembling the setting of scrambled eggs at room temperature after cooking. By 24 hr after incubation, the cell number in the scrambled egg was ≥10 7 /10 g, reaching 10 9 /10 g by 48 hr under all conditions. The productions of both SEA and SEQ were detected in the scrambled egg under all conditions by 48 h. SEQ was detected from 24 hr at all 3 pH values in the egg containing 1.0% NaCl, whereas in the egg containing 0.5% NaCl, it was detected from 24 hr at pH 6.0 and from 48 hr at other pHs. The SEQ production was consistently 100-1,000 times less than that of SEA. These results suggest that the new-type enterotoxin SEQ has the potential to evoke symptoms related to SFP following the consumption of egg products cooked under relative lower pH and water activity.

Aspergillus section Versicolores species, except Aspergillus sydowii, produce a carcinogenic mycotoxin sterigmatocystin (STC). Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP)-based PCR amplification. Using specific primer pairs based on the SNPs between A. sydowii and other strains of Aspergillus section Versicolores, we succeeded in amplifying the genomic DNA all target strains except A. sydowii. These results confirm that the SNP-based PCR amplification technique, using a high discrimination DNA polymerase, was a reliable and robust screening method for target fungal strains.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC 50 s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

A survey of the contamination of foods by sterigmatocystin (STC) was performed by an analytical method based on LC-MS/MS. STC was extracted from samples with acetonitrile/water (85/15, v/v) and then purified with immunoaffinity columns. The method was validated by a small-scale inter-laboratory study using spiked wheat samples. Mean recoveries of STC were 100.3% and 92.5% from two samples spiked at 0.5 and 5.0 µg/kg, respectively. A total of 583 samples were analysed between 2016 and 2018, and STC was detected in 19.9% of all samples at >0.05 μg/kg (limit of quantification). The foods that were contaminated by STC were wheat flour, Job's tears products, rye flour, rice, buckwheat flour, white sorghum, barley products, azuki bean and corn flour. STC was not found in beer or wine. The occurrence of STC in domestic wheat flour (44.4%), Job's tears products (41.7%) and rye flour (29.9%) accounted for the three highest values. The highest mean concentrations were obtained for Job's tears products (0.3 μg/kg) and rye flour (0.3 μg/kg). The maximum contamination level was present in a sample of rye flour (7.1 μg/kg). Although the contamination levels were low, these results indicate that STC frequently contaminates Japanese retail foods. A continuous survey is required to assess exposure to STC in Japan.

This study was conducted to give a preliminary estimation of deoxynivalenol (DON) dietary exposure in Japanese university students (n = 30, aged 22-25 years) using a biomarker approach and to examine the correlation between wheat food intake and DON exposure levels. Spot urine samples were collected from 30 students of Azabu University, Tokyo. Urine samples were treated with enzyme digestion (for total DON measurement) and without (for unconjugated DON analysis) before clean-up using an immuno-affinity column and analysis using an LC-MS method, with a 13 C 15 - DON internal standard used for accurate quantification. The limit of detection for this method is 0.5 ng/mL urine. The geometric mean (95% CI) of DON concentration was 2.03 (1.64 - 6.87) ng per mL urine. Ninety of the urine samples had detectable levels of urinary DON. The DON dietary intake exposure estimation suggested that one out of the 30 subjects had an intake of DON that exceeded Joint FAO/WHO Expert Committee on Food Additives (JECFA) provisional maximum tolerable daily intake (PMTDI) level. Mean ratio of free DON to total DON was determined to be 19%. Wheat intake assessed using a basic food frequent questionnaire method did not show a significant correlation with the urinary DON level., Competing Interests: Conflicts of interest: The authors declare they have no competing financial interests., (©2019 Food Safety Commission, Cabinet Office, Government of Japan.)

Citreoviridin (CTVD), a mycotoxin called yellow rice toxin, is reported to be related to acute cardiac beriberi; however, its toxicokinetics remain unclear. The present study elucidated the toxicokinetics through in vivo experiments in swine and predicted the human toxicokinetics by comparing the findings to those from in vitro experiments. In vivo experiments revealed the high bioavailability of CTVD (116.4%) in swine. An intestinal permeability study using Caco-2 cells to estimate the toxicokinetics in humans showed that CTVD has a high permeability coefficient. When CTVD was incubated with hepatic S9 fraction from swine and humans, hydroxylation and methylation, desaturation, and dihydroxylation derivatives were produced as the predominant metabolites. The levels of these products produced using human S9 were higher than those obtained swine S9, while CTVD glucuronide was produced slowly in human S9 in comparison to swine S9. Furthermore, the elimination of CTVD by human S9 was significantly more rapid in comparison to that by swine S9. These results suggest that CTVD is easily absorbed in swine and that it remains in the body where it is slowly metabolized. In contrast, the absorption of CTVD in humans would be the same as that in swine, although its elimination would be faster.