Yuko Kumeda

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.

Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus Section Flavi strains.

ABSTRACT For genetic identification of Aspergillus SectionFlavi isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from Aspergillus flavus, A. parasiticus, A. tamarii, and A. nomius and subsequent minislab vinyl polymer gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1, and N-1) generated by pairwise reannealing among four reference fragments. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11A. flavus/A. oryzae panels were identical and typed as F-2 and 4 of 5 A. nomius panels were typed as N-2 or N-3. The other strain, A. nomius IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. WhenPenicillium or non-Section Flavi Aspergilluswas subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section Flavi and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64–97, in Introduction to Food- and Airborne Fungi, 6th ed., 2000).

Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

ABSTRACT Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

Development and Evaluation of Immunochromatographic Assay for Simple and Rapid Detection of Campylobacter jejuni and Campylobacter coli in Human Stool Specimens

ABSTRACT An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA, they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 × 104 to 8.2 × 105 CFU/ml of cell suspension, and those for the C. coli strains ranged from 1.4 × 105 to 4.6 × 106 CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens, suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent, and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens.

BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

ABSTRACT 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.

High prevalence of B and G aflatoxin‐producing fungi in sugarcane field soil in Japan: heteroduplex panel analysis identifies a new genotype within Aspergillus Section Flavi and Aspergillus nomius

Heteroduplex panel analysis (HPA) was previously developed for genetic identification of Aspergillus Section Flavi strains, utilizing polymerase chain reaction-amplified fragments of the internal transcribed spacer (ITS) regions of the rRNA gene. Application of HPA to a field study demonstrated that a new type of FP-1 strains belonging to Section Flavi is predominantly distributed throughout sugarcane field soil in the southernmost islands of Japan, and such a trend may also be the case in Vietnam. All of the 71 tested isolates of type FP-1 were able to produce aflatoxins B and G. The morphological observations of the type FP-1 isolates showed that a major part of them had broad interfaces with Aspergillus parasiticus and the remainder with Aspergillus flavus. Phylogenetic analysis based on the ITS sequences indicated that type FP-1 formed an independent clade positioned between A. parasiticus and A. flavus, and was more closely related to the former species. This is also the first report on the distribution of Aspergillus nomius in sugarcane field soil and/or sugarcane stems in Japan and Vietnam.

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10(3)CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.

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.

Antibiotic residue monitoring results for pork, chicken, and beef samples in Vietnam in 2012-2013.

A monitoring plan of residual antibiotics in food of animal origin was conducted in Vietnam from 2012 to 2013. Meat samples were collected from slaughterhouses and retail stores in Ho Chi Minh City and Nha Trang. A total of 28 antibiotics were analyzed using a LC-MS/MS screening method. Sulfonamides, fluoroquinolones, and tilmicosin were detected in some of the samples. Sulfaclozine and fluoroquinolones were mainly detected in chicken samples, and sulfamethazine was mainly detected in pork samples. High levels of sulfonamide residues, ranging between 2500 and 2700 μg/kg sulfaclozine and between 1300 and 3600 μg/kg sulfamethazine, were present in two chicken and three pork samples, respectively. Tilmicosin was detected at ranges of 150-450 μg/kg in 10 chicken samples. Positive percentages were 17.3, 8.8, and 7.4% for chicken, pork, and beef, respectively, for an average of 11.9%. The results suggest an appropriate withdrawal period after drug administration had not been observed in some livestock.

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)

ABSTRACT 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 × 101 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.