Diarrheagenic Escherichia coli strains harboring the astA gene which encodes for the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) have been implicated in several foodborne outbreaks in Japan even in the absence of any other specific virulence marker of each pathovar. The polymerase chain reaction (PCR) assay is a critical tool used for detection and many astA specific primer sets have been developed though their specificity and sensitivity for astA detection directly from food samples have not been evaluated. Herein, four distinct PCR primer sets and three enzymes were evaluated in enriched food cultures to optimize astA detection. The Yamamoto & Echeverria PCR method yielded clear, easily interpretable results with high intensity of PCR product or no products. Combining this primer set with the Quick Taq HS DyeMix enzyme resulted in astA-specific amplicons without non-specific products from food cultures, indicating the superiority of this system in detecting astA in food samples. Furthermore, this primer set demonstrated the highest consistency with the E. coli harboring astA-isolation results. Subsequently, this system exhibited high specificity and sensitivity with a ≤5 log CFU/mL detection limit. These findings suggest that combining the Yamamoto & Echeverria primer set and the Quick Taq HS DyeMix offers an effective tool for detecting astA in food samples. We anticipate this PCR assay will enhance the detection and subsequent isolation of E. coli strains harboring astA from food products.

Escherichia albertii is an emerging enteropathogen and its distribution in various foods and environmental samples has been reported in many regions around the world. In this study, we aimed to identify effective isolation and detection methods for E. albertii in various foods and environmental water samples. E. albertii-specific polymerase chain reaction (PCR) was positive in chicken, oyster, river water, and wastewater samples, and E. albertii was isolated from these PCR-positive samples except the wastewater sample. E. albertii was not isolated from any of the samples without screening PCR; therefore, PCR is useful for the detection and isolation of E. albertii in foods and environmental water samples. The effect of two-step enrichment with four kinds of selective enrichment broth was compared with cycle threshold (Ct) values of the E. albertii-specific real-time PCR assay and the isolation results. The Ct values in three out of five samples were lower in the second enriched culture than those of the first enriched culture, and E. albertii was isolated from enriched cultures showed Ct values <25. These results suggest that the population of E. albertii in these three samples increased in the second enriched culture compared with the first enriched culture, and isolating E. albertii from an enriched culture showing Ct values <25 is an efficient method. Genetic analysis was performed to E. albertii isolates from food, environmental water, and human fecal samples, and all the isolates possessed eae, and isolates from chicken, pork, and river water samples showed the same EAOg type as E. albertii isolated from human fecal samples. Therefore, it was suggested that a continuous attention should be paid to E. albertii in food and environment.

Escherichia albertii is an emerging foodborne pathogen that causes diarrhea. E. albertii has been isolated from various foods, including pork and chicken meat, and environmental waters, such as river water. Although many food poisoning cases have been reported, there have been insufficient analyses of bacterial population behaviors in food and environmental water. In this study, we inoculated 2–5 log CFU of E. albertii into 25 g of pork, chicken meat, Japanese rock oyster, Pacific oyster, and 300 mL of well water and seawater at 4°C, 10°C, 20°C, and 30°C, and analyzed the bacterial population behavior in food and environmental water. After 3 days at 4°C, the population of E. albertii strain EA21 and EA24 in foods maintained approximately 4 log CFU/25 g. After 3 days at 10°C, the population of E. albertii strains in pork and oysters maintained approximately 4 log CFU/25 g, and that in chicken meat increased to approximately 5–6 log CFU/25 g. After 2 days at 20°C, E. albertii strains grew to approximately 6–7 log CFU/25 g in pork and chicken meat, and E. albertii strain EA21 but not EA24 grew to 4.5 log CFU/25 g in Japanese rock oyster, E. albertii strain EA21 but not EA24 slightly grew to 3.1 log CFU/25 g in Pacific oyster. After 1 day at 30°C, E. albertii strains grew to approximately 7–8 log CFU/25 g in chicken meat and pork, grew to approximately 4–6 log CFU/25 g in Japanese rock oyster, and 6–7 log CFU/25 g in Pacific oyster. These results suggest that E. albertii survives without growth below 4°C and grew rapidly at 20°C and 30°C in foods, especially in meat. E. albertii strains did not grow in well water and seawater at 4°C, 10°C, 20°C, and 30°C. The population of E. albertii strains in well water and seawater decreased faster at 30°C than at 4°C, 10°C, and 20°C, suggesting that E. albertii has low viability at 30°C in environmental water.

Sarcocystis cruzi is a member of the genus Sarcocystis, infecting bovine animals such as cattle and bison as intermediate hosts, and canids such as dogs and raccoon dogs as definitive hosts. Acute sarcocystosis of S. cruzi causes occasional symptoms in cattle, including weight loss, reduced milk production, abortions, and death, and similar to other Sarcocystis species can potentially cause food poisoning in humans when raw or undercooked infected cattle meat is consumed. Despite these issues, genetic information on S. cruzi is scarce, and there is no specific quantitative method for the detection and quantification of the parasite in infected cattle. In this study, we aimed to develop a method based on high-throughput sequencing of S. cruzi genome and transcriptome that specifically and quantitatively detects the S. cruzi acetyl-CoA synthetase gene (ScACS). Cardiac muscles were collected from slaughterhouses in Saitama Prefecture to obtain sarcocysts from which DNA and RNA were extracted for the high-throughput sequencing. Using the sequences, we developed a specific quantitative PCR assay which could distinguish S. cruzi ACS from that of Toxoplasma gondii by taking advantage of the differences in their exon/intron organizations and validated the assay with the microscopic counting of the S. cruzi bradyzoites. Thus, this assay will be useful for future studies of S. cruzi pathogenesis in cattle and for the surveillance of infected animals, thereby easing public health concerns.

Histamine is produced from histidine using histidine decarboxylase of histamine-producing bacteria. However, associated histamine food poisoning demands microbiological controls. Furthermore, studies reported that histamine production by histamine-producing bacteria is affected by temperature. Therefore, to prevent histamine food poisoning, it is desirable to store foods below 4℃. However, it is challenging to maintain the storage temperature of food substances in refrigerators constantly below 4℃. Thus, we investigated histamine production capacity using seven histamine-producing bacterial strains under storage at 10℃, a more reasonable cold storage condition. Subsequently, we examined the variation of histamine production in buffers, the correlation between bacterial density and histamine production quantities, and the growth rate in broths. Results showed that similar levels of histamine were produced in buffers even after 5 days of storage under certain conditions in which histamine-producing bacteria did not grow. Moreover, bacterial density was proportional to histamine production, and the coefficient of determination was more than 0.97, and the bacterial density required to produce 200 μg/mL of histamine during storage at 10℃ was calculated to be 4×107-4×108 CFU/mL. When the initial bacterial density was 102-103 CFU/mL, psychrophilic bacteria required 2 or 3 days and mesophilic bacteria required more than 4 days to grow above 107 CFU/mL. The above results suggest that understanding the capacity of histamine-producing bacteria to produce histamine and its growth rate in foods is important for the prevention of histamine food poisoning.

ISO/IEC 17025 : 2017 requires laboratories to verify the performance of methods before introducing them. ISO 16140-3 : 2021 employs LOD50 (the level of detection for which 50% of tests give a positive result) as an index for evaluating the performance of microbial qualitative methods. While fecal coliforms and Staphylococcus aureus qualitative methods are widely implemented in Japan, their LOD50 values have not been established, making performance evaluation of these methods impossible at the time of introduction. As a result, we looked into estimating the LOD50 values of fecal coliforms and S. aureus qualitative methods to evaluate the index of method performance. The fecal coliforms method had an LOD50 value of 19–31 CFU/mL and the S. aureus method had an LOD50 value of 29–49 CFU/mL by laboratory personnels using samples without food matrix. We estimated LOD50 of six food samples using each method in a single laboratory. The LOD50 values of all foods calculated using the fecal coliforms method were in the range of 14–27 CFU/g and those calculated using the S. aureus method were in the range of 25–48 CFU/g, indicating that these methods did not have low levels of sensitivity detection compared with skill variation. LOD50 values for food samples with the highest or lowest LOD50 in each method were compared among five laboratories. Because the estimated LOD50 values of both the methods were within four times of each laboratory result, which is the acceptable limit specified in ISO 16140-3 : 2021, they were assumed to be suitable values. This study is expected to be utilized as a benchmark in the verification of method performance in each laboratory.

Sarcocystis causes various veterinary and human infections. A few agents are available for treating Sarcocystis infections, but their mechanisms of action are not known. Phytohormonal herbicides are antiparasitic agents that reduce the viability of organisms belonging to the phylum, Apicomplexa. Some of these herbicides prevent Apicomplexan parasites from infecting cells and organisms, yet the effects of such drugs on Sarcocystis are unclear. We evaluated the activity of phytohormonal herbicides against Sarcocystis bradyzoites using mortality and morphological changes with methylene blue staining as an indicator. Antiparasitic activity of 17 agrochemicals that act on plant plastids-classified into the following six herbicide groups: acetyl-CoA carboxylase (ACC) inhibitors, acetolactate synthase (ALS) inhibitors, photosynthesis inhibitors, protoporphyrinogen oxidase (protox) inhibitors, carotenoid biosynthesis pathway inhibitors, very-long-chain fatty acid (VLCFA) biosynthesis inhibitors, and auxin-like herbicides-were examined. The in vitro system enables the screening of drugs against Sarcocystis species, for which a well-established culturing method is not yet available. Nine herbicides, including ALS inhibitors, ACC inhibitors, and auxin-like herbicides, caused mortality of Sarcocystis cruzi bradyzoites by more than 90%. Further, all ACC inhibitors caused the formation of vacuolar structures in bradyzoites, and two ALS inhibitors caused bradyzoites rounding.

A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis.

Between May 1999 and March 2001, a total of 166 raw meat specimens collected from retail markets in Saitama Prefecture was examined for the presence of Salmonella and Listeria sp. Salmonella was isolated from 9.5% of domestic chicken specimens and 13.6% of imported chicken specimens. According to the most-probablenumber (MPN) method, numbers of contaminating Salmonella cells were less than 10 cfu/g. Main Salmonella serotypes were S. Infantis for domestic chicken and S. Enteritidis for imported chicken. Listeria sp. were isolated from all the kinds of meat examined. Isolation rates among domestic meats were 22.7% for beef, 40.0% for pork, and 42.9% for chicken. Rates among imported meats were 34.5% for beef, 30.0% for pork, and 64.4% for chicken. According to the MPN method, contaminating cell numbers were less than 10 cfu/g for all kinds of domestic meat but more than 10 cfu/g for all kinds of imported meat. Some specimens of both imported pork and imported chicken showed more than 100 cfu/g. L. monocytogenes were isolated from all kinds of meat. The most prevalent serotype was 1/2a, accounting for 38.2% of all isolates of the organism.

By the examination of Campylobacter species, 47 Campylobacter jejuni strains were isolated from 107 cattle cecal specimens obtained in a Saitama slaughterhouse brought from 20 farms in 5 prefectures. On the basis of the PCR-based randomly amplified polymorphic DNA (RAPD) method, the isolates were divided into 27 types. This method is recommended for discriminating strains of C. jejuni isolated from cattle for the purposes of epidemiological study.

An examination was conducted to determine whether the isolation ratio of two serotypes of Salmonella inoculated simultaneously and then incubated varies with the enrichment broth and isolating medium used. Some serotypes were found to have a higher isolation ratio when a certain broth or medium was used. The isolation ratio varied with the serotype combinations. When S. Oranienburg and S. Chester were simultaneously inoculated and cultured in SC. S. Oranienburg was isolated in TSA at a higher ratio. When S. Oranienburg and S. Chester were simultaneously inoculated in sterile physiological saline solution and isolated in SS agar, S. Oranienburg was detected at a ratio lower by one order. When S. Infantis and S. Chester were simultaneously inoculated and cultured in TT or RV, the isolation ratio of S. Infantis lowered. When S. Enteritidis and S. Chester were simultaneously inoculated and cultured in RV, the isolation ratio of S. Enteritidis decreased.