Experimental Evidence of the Diarrheal Activity of Sarcocystis sp. in Sika Deer (Cervus nippon).

Recently, the wild deer population has been increasing in Japan, causing serious feeding-related damage to the agricultural and forestry industries. In conjunction with the government's promotion of hunting for population control, the effective utilization of resources and promotion of the game meat industry as a sixth sector of industrialization are desired by local governments. However, several cases in which patients showed intestinal symptoms such as diarrhea due to the consumption of sika deer meat infected with protozoan Sarcocystis spp. have been reported, and the pathogenic microorganisms found in wild deer should be investigated. In this study, Sarcocystis sp. parasitized Kyushu sika deer (Cervus nippon nippon) in Nagasaki Prefecture, Japan, was examined for its enterotoxicity. A phylogenetic analysis based on the sequence of the 18S rRNA gene and cox1 showed that the species was highly homologous to Sarcocystis japonica and/or Sarcocystis sp. HM050622. We attempted to confirm the diarrhea-evoking toxicity of Sarcocystis sp. in sika deer meat, which has been previously reported in human case reports. A mouse ileal loop assay showed that Sarcocystis sp. in sika deer meat induced significant fluid accumulation in the loop at doses of ∼5 × 106 bradyzoites. Western blotting showed that these Sarcocystis parasites possess actin-depolymerizing factor, a diarrhea-evoking factor, similar to Sarcocystis fayeri, which exists in horsemeat. However, the pathogenic conditions of the ileal loop were different from those of similar experiments with S. fayeri. This study suggests that S. japonica parasitizing C. n. nippon may cause diarrhea via a different mechanism from that of S. fayeri.

The Relationship Between Epidemiological Factors and the Parasite Load of Sarcocystis in Yezo-Deer (Cervus nippon yesoensis) in Hokkaido, Japan.

Several cases of gastrointestinal symptoms including diarrhea and vomiting due to the consumption of Sarcocystis-infected venison have been reported in Japan. However, the control of case incidence is difficult, as epidemiological information concerning Sarcocystis in venison in Japan is insufficient. We examined the prevalence and parasite load of Sarcocystis in 89 samples of Yezo-deer (Cervus nippon yesoensis) venison in Hokkaido by quantifying the copy numbers of the 18S rRNA gene of Sarcocystis, followed by a statistical analysis that considered the sampling area, age, and sex to clarify the parameters related to the parasite load. The copy numbers per gram of venison in samples ranged from 4.8 to 8.8 log. Wilcoxon rank-sum test, the one-way factorial analysis of variance (ANOVA), Steel-Dwass test, and a two-way factorial ANOVA showed significant differences in the copy numbers among sampling areas, not by age or sex, suggesting that the load of Sarcocystis in wild deer depended on the sampling area in Hokkaido. Notably, more than 80% of Hokkaido venison has a higher gene copy number than the meat that caused Sarcocystis fayeri-food poisoning. This information is expected to contribute to the establishment of hygiene standards for safe venison consumption and the control of gastrointestinal symptom cases due to consumption of Sarcocystis-infected venison.

Mite allergen avoidance decreases allergic symptoms in children in Ishinomaki city of Japan after natural disasters.

OBJECTIVE: We investigated the prevalence of asthma, rhinitis, and atopic dermatitis in children, evaluated the mite allergen levels in their bedding after the Great East Japan Earthquake, and assessed changes in allergic symptoms in children and their families after allergen avoidance practices. METHODS: We performed a survey for the International Study of Asthma and Allergies in Childhood (ISAAC) comprising 1109 children, aged 7-8 years, living in Ishinomaki, Japan. We collected responses from 464 children, and in 2016, measured the level of Dermatophagoides group 1 (Der 1) in the bedding of 202 of these children. The intervention group of children attended at least one allergen avoidance seminar. The levels of Der 1 in their bedding were measured, along with changes in allergic symptoms, in 17 children in 2017 and 14 children in 2018. The nonintervention group comprised children who did not attend an allergen avoidance seminar at any time. RESULTS: Of the 464 children who participated in the ISAAC, 50 (10.8%) reported having asthma, 179 (38.8%) allergic rhinitis, and 126 (27.3%) atopic dermatitis. The average level of Der 1 measured in the bedding of the 202 children in 2016 was 295.8 ng/m2. The levels of Der 1 in the intervention group-but not in the nonintervention group-significantly decreased in 2017 and 2018. The symptoms of asthma, allergic rhinitis, and atopic dermatitis in the children of intervention group and their families decreased after allergen avoidance practices. CONCLUSIONS: Allergen avoidance practices relieved allergic symptoms in school children after the Great East Japan Earthquake.

Association between Temporary Housing Habitation after the 2011 Japan Earthquake and Mite Allergen Sensitization and Asthma Development.

INTRODUCTION: We previously reported an increased prevalence of asthma in adults who lived in temporary housing after the 2011 Great East Japan Earthquake. The goal of the current study was to investigate changes in asthma prevalence and mite-specific immunoglobulin E (IgE) titers in temporary housing residents during 2014-2019. METHODS: By using the Global Initiative for Asthma guidelines, we diagnosed asthma in Ishinomaki city temporary housing residents aged 15 years or older. We then analyzed serum antigen-specific IgE levels to Dermatophagoides farinae (Der f), Dermatophagoides pteronyssinus (Der p), and Aspergillus fumigatus. RESULTS: The prevalence of asthma exceeded 20% across all age-groups throughout the study period. The proportion of study participants with a "positive" antigen-specific IgE titer (i.e., ≥0.35 IUA/mL) was higher in asthmatics than in nonasthmatics for Der f and Der p but not for Aspergillus fumigatus. Among residents ≥50 years old who were diagnosed with asthma, the percentage with a Der f-specific IgE titer ≥0.10 IUA/mL was higher than the proportion with ≥0.35 IUA/mL. Among study participants, asthma onset occurred before the earthquake, during residence in shelters or temporary housing, and (starting in 2016) after moving out of temporary housing. The Der p-specific IgE level was positively correlated with the duration of temporary housing (p < 0.05, r = 0.41) and inversely correlated with the time elapsed since moving out of temporary housing (p < 0.05, r = -0.35). CONCLUSION: Mite allergen sensitization was found in both asthmatic and nonasthmatic temporary housing residents after the 2011 Japan earthquake and tsunami; asthma developed even after subjects moved out of temporary housing.

Establishment of a Novel Molecular Detection Method for Sarcocystis in Venison.

Recently, horse meat (basashi) contaminated with Sarcocystis spp. caused food poisoning in Japan. An official detection method provided by the Ministry of Health, Labour and Welfare (MHLW), Japan, was designed to detect Sarcocystis fayeri to diagnose and control outbreaks of basashi food poisoning. In 2011, Sarcocystis-contaminated venison also caused food poisoning. However, the official MHLW detection method was not adequate for detecting Sarcocystis spp. in venison. In this study, we established a novel PCR-based detection method that amplifies 18S rRNA gene based on the conserved region of the sequence in 32 species of Sarcocystis for screening and quantification. Fifty venison samples from three areas in Hokkaido were examined by the MHLW method and the novel detection method. All samples were Sarcocystis spp.-positive. A sequence analysis indicated the presence of a species of Sarcocystis specific to sika deer (Cervus nippon), and not to horses. Another primer pair was designed for a quantitative real-time PCR assay to determine the copy number of the Sarcocystis-18S rRNA gene in parasitized venison. The melting curve analysis revealed high specificity of this assay. The calculated curve demonstrated that this quantitative PCR assay showed R2 value of 0.993 with 10-106 copies. Using this quantitative real-time PCR assay, the gene copy numbers were determined in 50 venison samples. The copy numbers of each sample ranged from 104 to 107 per gram. The copy numbers differed according to the area in Hokkaido. This indicates that the density of Sarcocystis spp. that infect Sika deer in Hokkaido is affected by the area. The novel screening and quantitative PCR method for Sarcocystis in venison was useful for collecting epidemiological information on Sarcocystis in wild Japanese sika deer, which will contribute to improve the safety of venison products in Japan.

Presence of Sarcocystis sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory and its phylogenetic relationship with Sarcocystis nipponi.

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.

[Behavior of Staphylococcus aureus and Staphylococcal Enterotoxins A and Q in Scrambled Eggs].

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 ≥107/10 g, reaching 109/10 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.

Characterization of Sarcocystis fayeri's actin-depolymerizing factor as a toxin that causes diarrhea.

Raw horsemeat has the potential to induce food poisoning which often presents with diarrheal symptoms. A sample of horsemeat was found to be infected with Sarcocystis fayeri, and a 15-kDa protein isolated from the cysts of S. fayeri was found to clearly show its diarrhea-inducing activity. A nested polymerase chain reaction was used to clone the cDNA of the 15-kDa protein. The deduced amino acid sequence showed homology to actin-depolymerizing factor (ADF). A recombinant 15-kDa protein depolymerized prepolymerized actins in a test tube. The 15-kDa protein possessed conserved amino acid sequences of ADF of Toxoplasma gondii and Eimeria tenella. These characteristics indicate that the 15-kDa protein of S. fayeri belongs to the ADF/cofilin protein family. The recombinant 15-kDa protein evoked fluid accumulation in the looped ileum, resulting in diarrhea, but it did not kill the cultured fibroblast cells, macrophages or intestinal mucosal cells. In addition, the culture supernatant of the macrophages treated with the recombinant 15-kDa protein killed the fibroblast L929 cells. This fact indicates that ADF of S. fayeri induced cytotoxic substances, such as tumor necrosis factor-α, according to the published reports. Although further experiments are needed now to elucidate the enterotoxic mechanism of S. fayeri's ADF, our findings may offer new insight into research on parasites and parasite-instigated food poisoning.

Chronic toxicity of 1,3,5-triazine herbicides in the postembryonic development of the western clawed frog Silurana tropicalis.

Seven 1,3,5- triazine (s-triazine) herbicides (ametryn, prometryn, dimethametryn, simazine, atrazine, propazine, and cyanazine) were tested using an amphibian (Silurana tropicalis) metamorphosis assay focusing on morphometric, gravimetric, and thyroid-histological endpoints. Premetamorphic tadpoles were exposed to each s-triazine at 2 concentrations between 1/1000 and 1/10 of the 96-h acute toxicity values, until all tadpoles in the control group reached either the late prometamorphosic stages or the initial stage of metamorphic climax. All s-triazines tested induced significant retardation in growth and development at the higher concentrations (0.2-1.0mg/L), and some of them induced similar effects even at the lower concentrations (0.02-0.1mg/L) while each showing a linear dose-response. Total size of the thyroid glands tended to be reduced corresponding to the delayed development, but without showing histomorphological lesions typical of anti-thyroid chemicals. These consistent results suggest that the s-triazines can act as a chemical stressor inhibiting tadpole growth and development, possibly without disrupting the thyroid axis. In addition, tadpoles exhibiting spinal curvatures appeared in either one or both of the lower and higher concentration groups for each s-triazine tested. The incidence rate in the s-triazine exposure groups where tadpoles with scoliosis were observed ranged from 3.3% to 63.3%, some of which were significantly higher than that in the respective control groups (0-6.7%). It is speculated that the s-triazines may promote to occur axial malformations in developing tadpoles.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan.

Staphylococcal food poisoning (SFP), one of the commonest food-borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin-encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.

An experimental verification of fungal overgrowth in temporary houses at the site of the Great East Japan Earthquake.

Fungal contamination in the indoor air of prefabricated temporary houses at the site of the Great East Japan Earthquake revealed extremely high levels compared to those found in conventional residences. We experimentally investigated fungal growth levels on different interior materials to support fungal overgrowth in prefabricated temporary houses. Three species each of allergenic fungi and invasive fungi observed in temporary housing were selected for inoculation tests with various interior materials. The experiments with fungal inoculation were conducted in conformance with standards for industrial products described in the Japanese" JIS Z 2911:2018 Methods of test for fungus resistance" with small modifications. After incubation, visual and stereomicroscopic assessments were performed to determine fungal growth levels. The viability of the fungi varied according to the interior material type. Our findings demonstrate the importance of antifungal measures in indoor environments and the need for additional research on the growth levels of fungal species on various interior materials.

Specific properties of enteropathogenic Escherichia coli isolates from diarrheal patients and comparison to strains from foods and fecal specimens from cattle, swine, and healthy carriers in Osaka City, Japan.

For exhaustive detection of diarrheagenic Escherichia coli, we previously developed a colony-hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR. To assess the role of domestic animals as the source of atypical enteropathogenic E. coli (aEPEC), a total of 679 samples (333 from foods, fecal samples from 227 domestic animals, and 119 from healthy people) were examined. Combining 48 strains previously isolated from patients and carriers, 159 aEPEC strains were classified by phylogroup, virulence profile, and intimin typing. Phylogroup B1 was significantly more prevalent among aEPEC from patients (50%) and bovine samples (79%) than from healthy carriers (16%) and swine strains (23%), respectively. Intimin type ß1 was predominant in phylogroup B1; B1-ß1 strains comprised 26% of bovine strains and 25% of patient strains. The virulence profile groups Ia and Ib were also observed more frequently among bovine strains than among porcine strains. Similarly, virulence group Ia was detected more frequently among patient strains than strains of healthy carriers. A total of 85 strains belonged to virulence group I, and 63 of these strains (74%) belonged to phylogroup B1. The present study suggests that the etiologically important aEPEC in diarrheal patients could be distinguished from aEPEC strains indigenous to humans based on type, such as B1, Ia, and ß1/γ1, which are shared with bovine strains, while the aEPEC strains in healthy humans are different, and some of these were also present in porcine samples.

Fatal pneumonia caused by Penicillium digitatum: a case report.

BACKGROUND: Penicillium species are among the most common fungi present in the environment and are usually considered non-pathogenic to humans. However, in immunocompromised hosts they can be virulent pathogens and can cause death. Penicillium digitatum is a plant pathogen that commonly causes a postharvest fungal disease of citrus called green mould; it very rarely causes systemic mycosis in humans. Here, we report a case of fatal pneumonia due to P. digitatum infection, as confirmed by repeated examination of cultured sputum. CASE PRESENTATION: A cavity was found in the left upper lung on routine chest X-ray in a 78-year-old undernourished male who had been diagnosed at age 66 with bronchial asthma and pulmonary emphysema. No increased sputum production was present. The presence of antigen-specific precipitating antibodies to Aspergillus flavus and P. digitatum was confirmed in the patient's serum and also later pleural fluid by using Ouchterlony double immunodiffusion testing with A. flavus and P. digitatum antigens. The patient was treated over a period of months with itraconazole, micafungin, voriconazole, amphotericin B, and antibacterials. However, the cavity enlarged, the pleural effusion increased, and the patient began producing purulent sputum. He died from progressive renal failure. From sputum culture only one fungus was isolated repeatedly on potato-dextrose agar in large quantities. This fungus was confirmed to be P. digitatum by molecular identification. Partial sequences of the beta-tubulin gene were determined by using the primers Bt2a and Bt2b for PCR amplification and sequencing and underwent a BLAST search at the National Centre for Biotechnology Information, these results confirmed that the isolated fungus was P. digitatum. CONCLUSION: To our knowledge, this is the first report of pulmonary infection with P. digitatum. Our patient had pulmonary emphysema and was elderly, and undernourished. These factors might have facilitated the infection. In his case, antimycotics were ineffective in treating the lung involvement. Although human infection with P. digitatum is considered rare, it appears that this organism can be very virulent and resistant to antimycotics.

Characterization of the ribosomal RNA gene of Kudoa neothunni (Myxosporea: Multivalvulida) in tunas (Thunnus spp.) and Kudoa scomberi n. sp. in a chub mackerel (Scomber japonicus).

Kudoa neothunni is the first described Kudoa species having six shell valves and polar capsules, previously assigned to the genus Hexacapsula Arai and Matsumoto, 1953. Since its genetic analyses remain to be conducted, the present study characterizes the ribosomal RNA gene (rDNA) using two isolates from a yellowfin tuna (Thunnus albacares) with post-harvest myoliquefaction and a northern bluefin tuna (Thunnus thynnus) without tissue degradation. Spores of the two isolates localized in the myofiber of trunk muscles, forming pseudocysts, and showed typical morphology of K. neothunni with six equal-sized shell valves radially arranged in apical view: spores (n = 15) measuring 9.5-11.4 µm in width, 7.3-8.6 µm in suture width, 8.9-10.9 µm in thickness, and 7.3-7.7 µm in length; and polar capsules measuring 3.6-4.1 µm by 1.8-2.3 µm. In lateral view, the spores were pyramidal in shape without apical protrusions. Their 18S and 5.8S rDNA sequences were essentially identical, but variations in the ITS1 (62.4 % similarity across 757-bp length), ITS2 (66.9 % similarity across 599-bp length), and 28S (99.0 % similarity across 2,245-bp length) rDNA regions existed between the two isolates. On phylogenetic trees based on the 18S or 28S rDNA sequence, K. neothunni formed a clade with Kudoa spp. with more than four shell valves and polar capsules, particularly K. grammatorcyni and K. scomberomori. Semiquadrate spores of a kudoid species with four shell valves and polar capsules were detected from minute cysts (0.30-0.75 mm by 0.20-0.40 mm) embedded in the trunk muscle of a chub mackerel (Scomber japonicus) fished in the Sea of Japan. Morphologically, it resembled K. caudata described from a chub mackerel fished in the southeastern Pacific Ocean off Peru; however, it lacked filamentous projections on the shell valves of spores. Additionally, it morphologically resembled K. thunni described from a yellowfin tuna also fished in the Pacific Ocean; spores (n = 30) measuring 8.2-10.5 µm in width, 7.0-8.8 µm in thickness, and 6.1-6.8 µm in length; and polar capsule measuring 2.5-3.4 µm by 1.3-2.0 µm. The similarities of the 18S and 28S rDNA sequences between these two species were 98.5 % and 96.3 %, respectively. Simultaneously, the dimensions of cysts in the trunk muscle formed by K. thunni are clearly larger than those of the present species from a chub mackerel: 1.3-2.0 mm by 1.1-1.4 mm (n = 14) vs. 0.30-0.75 mm by 0.20-0.40 mm (n = 7), respectively. Thus, Kudoa scomberi n. sp. is proposed for this multivalvulid species found in the chub mackerel.

Application of an amphibian (Silurana tropicalis) metamorphosis assay to the testing of the chronic toxicity of three rice paddy herbicides: simetryn, mefenacet, and thiobencarb.

We examined the chronic toxicity of three rice paddy herbicides (simetryn, mefenacet, and thiobencarb) using an amphibian (Silurana tropicalis) metamorphosis assay (a 28-day semistatic test under an individual-separated exposure system). Each herbicide was tested at two concentrations (1/100 and 1/10 of the 96-h LC50 value reported previously) with morphometric, gravimetric, and thyroid-histological endpoints. Simetryn caused significant retardation in growth and development at both test concentrations (0.04 and 0.40mg/L), as indicated by significantly shorter total body lengths and hind limb lengths, smaller wet body masses, and delayed developmental stages compared to those observed in the control tadpoles. However, no clear histopathology was observed in the thyroid glands of the tadpoles exposed to simetryn. These results suggest that simetryn can act as a chemical stressor retarding tadpole growth and development without disrupting thyroid functions, even at 1/100 of the 96-h LC50 value. In addition, scoliosis near the tail base was observed in the tadpoles exposed to 0.40mg/L of simetryn at a significantly high incidence (7/30=23.3%). Therefore, simetryn can also act as a teratogen inducing axial malformations at 1/10 of the 96-h LC50 value. During the 28 days of exposure, neither mefenacet (0.03 and 0.30mg/L) nor thiobencarb (0.008 and 0.080mg/L) induced any abnormalities, although the test concentrations measured immediately before the solution renewals decreased to nearly 50 percent of the nominal concentrations since day 14. Because the concentrations tested for simetryn are likely to occur in paddy water, wild anuran tadpoles in paddy water may therefore be adversely impacted by simetryn.

Kudoa septempunctata invasion increases the permeability of human intestinal epithelial monolayer.

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins.

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ß-pore-forming toxins such as aerolysin, C. perfringens ε-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ß-strands, two of which span its entire length. Domain II and domain III have three short ß-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the ß-strands. The sequence of amino acids composing the α-helix and preceding ß-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ß-barrel-made pores. These structural features imply that CPE is a ß-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ß-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.

Identification of Kudoa septempunctata as the causative agent of novel food poisoning outbreaks in Japan by consumption of Paralichthys olivaceus in raw fish.

BACKGROUND: Outbreaks of an unidentified food-borne illness associated with the consumption of raw fish have increased in Japan since 2003. Those affected with this illness develop diarrhea and emesis within 2-20 hours after a meal including raw fish. No known causative agents such as bacteria, viruses, bacterial toxins, or toxic chemicals have been detected in the foods that were ingested. Fortunately, this illness is self-limiting with good prognosis in all cases. METHODS: We conducted an epidemiological analysis of outbreaks that occurred during 2008 and 2010 and analysed a fish sample from one outbreak by metagenomic DNA sequencing, real-time polymerase chain reaction, and direct microscopic observations. The pathogenicity of a putative risk factor identified by these techniques was assessed using the suckling-mouse test and a house musk shrew emetic assay. RESULTS: The epidemiological analysis of outbreaks in 24 municipalities involving >1300 subjects implicated an olive flounder (Paralichthys olivaceus) as the causative food source. The presence of Kudoa septempunctata, a recently-described myxosporean species in P. olivaceus, was prevalent in the causative foods. K. septempunctata induced watery stools and an elevated fluid accumulation ratio in suckling mice, as well as vomiting in house musk shrews. CONCLUSIONS: These results identify K. septempunctata as the etiological agent of this novel food-borne illness outbreak associated with consumption of raw P. olivaceus. This is the first report, to our knowledge, demonstrating the human pathogenicity of Kudoa spores.

Three novel myxobolid species of genera Henneguya and Myxobolus (Myxosporea: Bivalvulida) from marine fish in Japan.

Myxosporean genera Henneguya and Myxobolus (Bivalvulida: Myxobolidae) are closely related in morphology and molecular phylogeny, speciose with approximately 1,000 nominal species. The majority of them are recorded from freshwater fish worldwide, and few are known from marine fish. In this study, three myxobolid spp. are described from marine fish around Japan. Two novel Henneguya spp., Henneguya ogawai sp. n. and Henneguya yokoyamai sp. n., are described from two black sea breams (Acanthopagrus schlegelii) fished in the Inland Sea (Setonaikai), Japan. Plasmodia of the former species were localized in the esophageal or intestinal wall, and those of the latter species were in the wall of the gall bladder and peritoneum. Spore development in plasmodia of these two species was synchronous. The spore body of H. ogawai sp. n. was 11.0 (8.9-12.2) µm in length, 6.9 (6.3-7.5) µm in width, 5.9 (5.2-6.6) µm in thickness, with a bifurcated caudal process of equal length, 10.0 (8.4-12.7) µm long; total spore length, 21.1 (19.2-23.4) µm. It contained two polar capsule, 4.3 (3.8-5.2) × 1.9 (1.4-2.3) µm. The spore body of H. yokoyamai sp. n. was 11.0 (10.1-13.7) µm in length, 7.1 (6.6-7.5) µm in width, and 5.6 (4.5-6.4) µm in thickness, with a bifurcated caudal process of equal length, 14.1 (10.8-17.0) µm long; total spore length, 25.0 (21.9-29.2) µm. It contained two polar capsules, 3.7 (3.1-4.2) × 2.0 (1.8-2.4) µm. A novel Myxobolus sp., Myxobolus machidai sp. n., is described from a spotted knifejaw (Oplegnathus punctatus) fished in the Sea of Japan, off Shimonoseki, Yamaguchi Prefecture, Japan. Plasmodia were embedded in the esophageal wall. Its round spore was small in size, 9.0 (8.1-9.4) µm in length, 7.8 (7.5-8.3) µm in width, and 5.5 (5.1-6.0) µm in thickness. It contained two polar capsules, 3.5 (3.2-3.8) × 2.3 (2.2-2.5) µm. Spore development in a plasmodium was asynchronous. Nucleotide sequencing of the small subunit ribosomal RNA gene (SSU rDNA) of these two novel Henneguya spp. revealed a close phylogenetic relationship with the marine clade of Henneguya spp.; however, they were distinct in morphology and SSU rDNA sequence from any known species. M. machidai sp. n. was grouped with freshwater Henneguya spp. in a phylogenetic tree based on the SSU rDNA, distant from a known marine clade of Myxobolus spp. reported mainly from the Mediterranean Sea. This is the first record of Henneguya-Myxobolus spp. from natural marine water in Japan.