The prevalence of vulvovaginal symptoms in Japanese women of reproductive and perimenopausal ages: A cross-sectional study across generations.

AIM: This cross-sectional study evaluated vaginal health and hygiene practices among reproductive and perimenopausal women in Japan using an online-based questionnaire. METHODS: The questionnaire included 11 well-structured questions concerning vulvovaginal symptoms and hygiene care practices. Participants' responses were anonymized and analyzed descriptively. The relationships of age, family income, occupation, and childbearing with women's concerns regarding vaginal or vulvar problems were analyzed by chi-square tests. RESULTS: About 80% of women in their 20s to 50s in Japan reported experiencing vulvovaginal symptoms. Women in their 40s had significantly fewer symptoms than women in their 20s (p = 0.04), and women in their 50s had significantly fewer symptoms than all other age groups (20s, 30s, and 40s) (p < 0.001). Among symptomatic women, 77.5% did not discuss their symptoms with anyone else and only 10% visited doctors. About 12.5% of women reported taking special care of their vagina or vulva regularly, whereas 38.2% expressed a desire to try some form of care but had not yet done so. Of the women who did not take special care of their vagina or vulva, 46.2% lacked knowledge about proper care, 42.2% did not want to spend money on care, 30.5% did not want to discuss care with others, and 21.3% were psychologically reluctant. CONCLUSION: Determination of the prevalence of vulvovaginal symptoms among Japanese women can enhance understanding of this prevalent condition and its impact on women's health. These findings may help formulate effective public health interventions and promote better hygienic practices, thus improving the well-being of women in Japan.

A giant calculus within urethral diverticulum causing urinary retention in an uncommunicative woman.

Introduction: There is no prior case report of calculus within a female urethral diverticulum causing urinary retention. We present such a case successfully treated by transurethral lithotripsy. Case presentation: A 34-year-old bedridden and uncommunicative woman with spinocerebellar degeneration presented with fever for 5 days. She was admitted to the hospital for a urinary tract infection with a 3-cm calculus in the lower urinary tract. At the time of admission, acute urinary retention occurred. A bladder catheter was placed, and antibiotics were administered; both improved the urinary tract infection. Subsequently, transurethral lithotripsy was performed and revealed that the giant calculus was incarcerated within the urethral diverticulum. The bladder catheter was removed postoperatively, and urinary retention did not recur. No calculus reformation or urinary tract infections were observed for 6 months after discharge. Conclusion: A giant calculus within a urethral diverticulum may cause acute urinary retention in an uncommunicative patient.

Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic Escherichia coli Serogroups O157, O26, and O111 Based on a De Novo Look-Up Table Constructed by Regression Analysis.

Multilocus variable-number tandem-repeat analysis (MLVA) is a widely accepted molecular typing tool for enterohemorrhagic Escherichia coli (EHEC). However, ensuring the accuracy of MLVA data among multiple laboratories remains difficult. We developed a method of constructing adjusted look-up tables, which are necessary for MLVA profiling, at each laboratory using a regression analysis based on electrophoresis data from 24 in-house reference strains. On performing MLVA against 51 EHEC O157 isolates, the repeat numbers of 46 isolates were determined accurately using the look-up table with a 99% prediction interval, an outcome superior to that when using a 95% prediction interval. For the remaining five isolates, although the electrophoresis size fell outside the look-up table, we were able to predict the repeat number accurately by extrapolation or the nearest values of the look-up table. Our approach provides more accurate results than a nonadjusted conventional look-up table for calibrating MLVA profiles.

Effect of TiB Orientation on Near-Threshold Fatigue Crack Propagation in TiB-Reinforced Ti-3Al-2.5V Matrix Composites Treated with Heat Extrusion.

TiB-reinforced Ti-3Al-2.5V matrix composites, in which TiB whiskers are oriented parallel to the direction of heat extrusion, were fabricated via mechanical alloying and hot isostatic pressing (HIP). To investigate the near-threshold fatigue crack propagation in TiB-reinforced Ti-3Al-2.5V matrix composites, stress intensity factor K-decreasing tests were conducted for disk-shaped compact specimens having two different orientations of TiB whiskers at force ratios from 0.1 to 0.8 under ambient conditions. The crack growth rates, da/dN, for the composites incorporating TiB whiskers oriented perpendicular to the direction of crack growth were constantly lower than those obtained in the case where the orientation was parallel at the same stress intensity range ΔK, while the threshold stress intensity range, ΔKth, was higher. This effect can be explained by the increase in the degree of roughness-induced crack closure resulting from the perpendicular TiB, because fatigue cracks preferentially propagated across the boundaries between the matrix and the TiB in certain regions. In contrast, the effective threshold stress intensity range, ΔKeff,th, for composites was unaffected by the TiB orientation at low force ratios.

Xylene causes oxidative stress and pronounced translation repression in Saccharomyces cerevisiae.

Organic solvent-resistant microorganisms are strongly desired for efficient fermentative production of hydrophobic substances in water-organic solvent two-phase systems. To improve organic solvent-resistance of microorganisms, a better understanding of the effects of organic solvents on microbial cells and cellular responses to organic solvents is essential. So far, various bacteria have been studied for their response mechanisms against organic solvents and improvement of their resistance to organic solvents. On the other hand, limited information is available on the effects of organic solvents on eukaryotic microorganisms. We herein examined the physiological effects of xylene, one of representative organic solvents, on the budding yeast Saccharomyces cerevisiae. We found that xylene induced fragmentation of mitochondria and the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, followed by the transcriptional activation of its target genes, GPX2 and TRX2, in yeast cells treated with xylene. These findings indicate that xylene caused oxidative stress in yeast cells. However, treatment with 0.03% (v/v) or more of xylene severely repressed the translation activity of yeast cells. Therefore, the expected protein synthesis of Yap1-target genes was not observed despite the transcriptional activation in cells treated with 0.03% (v/v) xylene. This is the first report on the inhibitory effects of xylene on bulk translation activity and provides novel insights into the toxicity of xylene.

New host records of three Kudoa spp. (K. yasunagai, K. thalassomi, and K. igami) with notable variation in the number of shell valves and polar capsules in spores.

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

Development of a Real-Time PCR Assay for Detection of Kudoa iwatai (Myxosporea: Multivalvulida) in Japanese Seabass ( Lateolabrax japonicus).

Kudoa iwatai, a myxosporean parasite, has low host fish specificity, and consumers encounter commercial marine fish or marketed marine fish infected with this parasite in Japan. Although the presence of this parasite infection in fish samples is traditionally determined by the microscopic morphological examination of extracted spores, this method lacks sensitivity and specificity. In this study, we developed a real-time PCR assay for the detection of K. iwatai 18S rDNA to achieve the rapid and specific identification of K. iwatai in foreign substance inspection. We also evaluated the usefulness of real-time PCR for Japanese seabass ( Lateolabrax japonicus) with or without K. iwatai cysts. Our real-time PCR assay was able to reliably detect the target plasmid DNA over a 7-log range (from 4.0 × 101 to 4.0 × 107 copies per reaction) and displayed a linear relationship, with a correlation of determination value of 0.9993 and slope of -3.3651. Moreover, the mean value of the intra-assay coefficient of variation was 0.89% in triplicate assays, and the detection limit of this method was 2.5 copies of K. iwatai 18S rDNA per reaction. The sensitivity of the real-time PCR was the same or higher than that of an established conventional PCR when DNA extracts from eight Japanese seabass with or without K. iwatai were used as templates. The specificity of the real-time PCR was comparable with that of conventional PCR by using DNA extracts from fish samples infected with nine Kudoa species. Together, these results indicate that our real-time PCR assay is highly sensitive, reproducible, and specific for detecting K. iwatai 18S rDNA in foreign substance inspection. We believe that this highly sensitive real-time PCR may also be useful for understanding the gastrointestinal diseases associated with K. iwatai and for studying the yet unknown life cycle of K. iwatai.

Production of a novel monoclonal antibody applicable for an immunochromatographic assay for Kudoa septempunctata spores contaminating the raw olive flounder (Paralichthys olivaceus).

Kudoa septempunctata, a myxosporean parasite of the olive flounder (Paralichthys olivaceus), causes foodborne gastroenteritis after ingestion of contaminated raw flounder. Available methods to detect K. septempunctata require expensive equipment, well-trained personnel, and lengthy procedures. Here we generated a novel monoclonal antibody (MAb 15G11) against K. septempunctata and used it to produce a prototype immunochromatographic assay (prototype Kudoa-ICA). Within 15min, the prototype Kudoa-ICA detected ≥1.0×105spores/mL in a spore suspension and ≥2.0×104spores/g of P. olivaceus muscle. The prototype Kudoa-ICA weakly cross-reacted with spores of K. lateolabracis and K. iwatai. cDNA sequence, expression, and western blot analyses revealed that MAb 15G11 detected an approximately 24-kDa protein encoded by a 573bp mRNA. The cDNA nucleotide and predicted amino acid sequences were not significantly similar to any sequence in the GeneBank database. Immunoelectron microscopy revealed that MAb 15G11 reacted with the sporoplasmic cells and mainly with the capsulogenic cells of the K. septempunctata spore. Although the Kudoa-ICA was weakly cross-reactive with two other Kudoa species, it detected >1.0×106spores/g of K. septempunctata in P. olivaceus muscle, which is the criterion used to indicate a violation of the Food Hygiene Law of Japan. We conclude that MAb 15G11 may be suitable for use in an immunochromatographic assay for screening P. olivaceus muscle contaminated with K. septempunctata at food distribution sites such as food wholesalers, grocery stores, and restaurants.

Prevalence, antibiotic resistance, and extended-spectrum and AmpC ß-lactamase productivity of Salmonella isolates from raw meat and seafood samples in Ho Chi Minh City, Vietnam.

Salmonellosis is a type of foodborne disease caused by Salmonella enterica and is a frequent cause of childhood diarrhea in Vietnam. Of particular concern is the dissemination of multidrug-resistant Salmonella, as extended-spectrum ß-lactamase (ESBL)-positive isolates were recently detected in children in Vietnam. In the present study, the prevalence and antibiotic resistance of Salmonella isolates obtained from 409 raw meat and seafood samples collected between October 2012 and March 2015 from slaughterhouses, wholesale fish market, and retail markets in Ho Chi Minh City, Vietnam were examined. A high rate of Salmonella contamination was detected in the pork (69.7%), poultry (65.3%), beef (58.3%), shrimp (49.1%), and farmed freshwater fish samples (36.6%). A total of 53 Salmonella serovars were found, of which S. Rissen, S. Weltevreden, S. London, S. Anatum, S. Typhimurium, and S. Corvallis were the most prevalent. In addition, 4 monophasic S. Typhimurium strains were identified using a PCR method for the detection of a specific IS200 fragment within the fliB-fliA intergenic region. The Salmonella isolates had a high prevalence (62.2%) of resistance to antimicrobial agents, particularly tetracycline (53.3%), ampicillin (43.8%), chloramphenicol (37.5%), and trimethoprim/sulfamethoxazole (31.3%). Isolates with resistance to three or more classes of antimicrobials were found (41.1%). Especially, isolates such as S. monophasic Typhimurium, S. Schwarzengrund, S. Indiana, S. Newport, S. Saintpaul and S. Bovismorbificans exhibited resistance to 6 classes of antimicrobials (3.3%). All 7 S. Indiana strains were resistant to between 4 and 6 classes of antimicrobials, including ciprofloxacin, which is commonly used for the treatment of human Salmonella infections. Two fish isolates were confirmed to be CTX-M-55 ESBL-producing Salmonella serovars Bovismorbificans and Newport, and five CMY-2 AmpC-producing Salmonella isolates of serovars Braenderup (4) and Typhimurium (1) were detected in poultry samples. The findings from this study, which is the first report of ESBL- and AmpC-producing Salmonella isolates from food in Vietnam, indicate that multidrug-resistant Salmonella are widely disseminated not only in meats, but also in seafood, within the food distribution system of Vietnam. The presence of these multidrug-resistant strains is a public health concern and suggests that the use of antimicrobial agents in both humans and animals in Vietnam should be tightly controlled.

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

Dissemination of Extended-Spectrum ß-Lactamase- and AmpC ß-Lactamase-Producing Escherichia coli within the Food Distribution System of Ho Chi Minh City, Vietnam.

To investigate the dissemination of ESBL/pAmpC-producing E. coli within the food distribution system of Ho Chi Minh City (HCMC), Vietnam, the prevalence of ESBL/pAmpC-producing E. coli strains in chicken meat, pork, beef, and fish/shrimp samples obtained from slaughterhouses, a wholesale market, and supermarkets was examined. Among the total of 330 collected food samples, ESBL/pAmpC-producing E. coli was detected in 150 samples (45.5%). The highest prevalence of these isolates was in chicken meat (76/82, 92.7%), followed by pork (32/92, 34.8%), beef (18/74, 34.3%), and fish/shrimp (24/82, 29.3%). A total of 342 strains of ESBL/pAmpC-producing E. coli were isolated from 150 positive food samples. The most prevalent genes responsible for ESBL or pAmpC activity belonged to the CTX-M-9 (110/342, 31.2%), CTX-M-1 (102/342, 29.8%), and CIT (118/342, 34.5%) groups. To our knowledge, this is the first report of the high occurrence of pAmpC (37.1%) in animal-based food in Vietnam. Among the 342 total ESBL/pAmpC-producing E. coli isolates, 276 (80.7%) were resistant to at least 6 antibiotic agents. Notably, high percentages of resistance to ciprofloxacin and fosfomycin were found in isolates from chicken (80.5% and 50.8%, resp.). These findings demonstrate that animal-based food products in HCMC represent a major reservoir of ESBL/pAmpC-producing E. coli.

Prevalence and Antimicrobial Susceptibility of Enterobacteriaceae Isolated from Retail Pepper in Vietnam.

To investigate the microbial quality of retail pepper in Vietnam, the enumeration and detection of Enterobacteriaceae and the screening of cefotaxime (CTX)-resistant coliforms were performed by using 84 commercial samples. Although Enterobacteriaceae were isolated from 78 samples, the number of Enterobacteriaceae was lower than 1.0 log CFU/g in 46 samples. For the detection of Enterobacteriaceae with the International Organization for Standardization methods, Salmonella spp., Escherichia coli, Klebsiella pneumoniae, Cronobacter sakazakii, and Enterobacter cloacae complex were isolated from 5, 12, 36, 19, and 30 samples, respectively. During screening of CTX-resistant coliforms, K. pneumoniae, C. sakazakii, and E. cloacae complex were isolated from 8, 1, and 21 samples, respectively. Seven K. pneumoniae and seven E. cloacae complex isolates obtained in the screening of CTX-resistant coliforms were resistant to at least one of the three third-generation cephalosporins (CTX, ceftazidime, and cefpodoxime). Moreover, one E. cloacae complex cluster IV and all K. pneumoniae isolates were positive for extended-spectrum ß-lactamase genes or plasmid-mediated AmpC ß-lactamase genes or both. Additionally, two extended-spectrum ß-lactamase-producing K. pneumoniae isolates and one AmpC ß-lactamase-producing E. cloacae complex cluster IV isolate were positive for the plasmid-mediated quinolone resistance determinants and also had amino acid alterations in the quinolone resistance-determining regions of GyrA and ParC. Furthermore, 10 E. cloacae complex isolates were positive for the plasmid-mediated fosfomycin resistance gene fosA. As pepper is often consumed without a heating process, the possible spread to humans of foodborne, opportunistic, and nosocomial infection pathogens or resistance genes from foods prepared or seasoned with pepper cannot be excluded. Therefore, it is necessary to handle pepper by using hygienic conditions during the cultivation, harvesting and processing steps.

BEC, a novel enterotoxin of Clostridium perfringens found in human clinical isolates from acute gastroenteritis outbreaks.

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

ELISA detection of Kudoa septempunctata in raw Paralichthys olivaceus (olive flounder) using a chicken anti-Kudoa antiserum.

Kudoa septempunctata is the causative agent of a foodborne disease associated with the consumption of raw Paralichthys olivaceus (olive flounder). Chickens were used to establish specific antibodies against K. septempunctata spores. A specific antiserum, CS#3, raised against sonicated spores, also recognized intact spores. The CS#3 antiserum showed high titers for sonicated and intact K. septempunctata spores and was suitable for both ELISA and immunohistochemical staining. Using homogenated raw olive flounder meat, the ELISA system detected more than 5.0×10(5) spores in 1 g of tissue, which was consistent with the number determined by microscopic examination. The preparation of rapid detection kits for K. septempunctata spores in P. olivaceus muscle tissue using immunochromatography with CS#3 antiserum should be useful for preventing the foodborne disease in the field.

A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis.

The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml(-1), respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.

Biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger RNP granules and attenuate translation activity in Saccharomyces cerevisiae.

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

Detection of Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder (Paralichthys olivaceus).

Kudoa septempunctata is a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata 18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctata in feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctata DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctata DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10(1) spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctata DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctata in laboratory investigations.

Development of a quantitative polymerase chain reaction assay for detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus).

Kudoa septempunctata is a newly identified myxosporean parasite that infects the trunk muscles of olive flounder (Paralichthys olivaceus) and a causative agent of the increasing number of foodborne gastroenteritis outbreaks with unknown etiology which have occurred in Japan over the last few years. Here, we developed a quantitative polymerase chain reaction (QPCR) assay for the detection of K. septempunctata 18S rDNA in olive flounder muscle tissue samples. Additionally, we compared the relative efficacy of four DNA extraction methods, including two commercial kits, and assessed intrafish variability in the distribution of K. septempunctata spores in flounder using this QPCR method in order to establish a more accurate quantitative measurement. Our QPCR assay displayed high sensitivity, specificity, and reproducibility, and had good correlation with a microscopic detection method. Our data also indicated that the DNeasy® Blood & Tissue Kit was more efficient method for the extraction of K. septempunctata DNA than the three other methods (heating, alkaline lysis, and FastDNA® SPIN Kit method). We believe that our method would be useful for investigating foodborne outbreaks caused by K. septempunctata and for the monitoring and quantification of this parasite in retail or aquacultured olive flounders to prevent such outbreaks.

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.

Isolation of VanA-type vancomycin-resistant Enterococcus strains from domestic poultry products with enrichment by incubation in buffered peptone water at 42 degrees C.

Eight VanA-type enterococcal strains were isolated from 8 of 171 domestic poultry products by using enrichment by incubation in buffered peptone water at 35 degrees C and 42 degrees C. The pulsed-field gel electrophoresis patterns of all six VanA-type Enterococcus faecalis isolates were nearly indistinguishable, indicating the presence of a specific clone in Japan.