Bacteriological Survey of Insect Products in Japan.

A microbiological study was conducted on 41 insect product samples (29 raw frozen [21 domestic and 8 imported], 10 powdered, and 2 processed), which were commercially available in Japan. The total aerobic count for raw frozen insects was 5.61 log cfu/g (range: 2.52-8.40), whereas the powdered insect count was 2.89 log cfu/g (range: 1.00-4.57). The bacterial count was significantly higher in raw frozen insects (p < 0.05). The coliform count for the raw frozen insects ranged from <1 to 6.90 log cfu/g, and that for the powdered insects ranged from <1 to 1.00 log cfu/g. The number of samples with values above the detection limit was significantly higher in raw frozen insects (p < 0.05). The detection frequencies of aerobic spores (<1-4.63 log cfu/g), anaerobic spores (<0-4.40 log cfu/g), and Bacillus cereus (<1.7-3.83 log cfu/g) showed no sample type-related significant difference. Listeria spp. was isolated from four samples of raw frozen insects, one of which was Listeria monocytogenes. We did not detect any of the following: Salmonella spp., Shiga toxin-producing E. coli (STEC), Campylobacter jejuni/coli, or pathogenic Yersinia. We isolated insect products retailed in Japan harboring food poisoning bacteria, including L. monocytogenes and B. cereus. In particular, raw frozen products displayed high levels of hygienic indicator bacteria.

Analysis of Alternative Methods of Environmental Monitoring for Listeria in Food Production Facilities.

Validated alternative test methodologies may be used in place of culture-based methods recommended for environmental monitoring programs (EMPs) for Listeria in food production facilities. In order to help guide decisions on which testing method to use to simplify Listeria EMP implementation in food production facilities, alternative methods were compared to the culture-based method in actual EMPs for Listeria. Seventy-two samples collected from two facilities of souzai production businesses that use meat and meat products as ingredients, one facility of processed meat product production business, and one facility of processed meat product and souzai production business were applied to EMPs for Listeria using the culture-based method, 3MTM Molecular Detection System (MDS), and InSite L. mono Glo (InSite). The kappa coefficient in MDS was 0.65 for Listeria monocytogenes and 0.74 for Listeria spp., both of which were deemed substantial compared with the culture-based method. The kappa coefficient in InSite was -0.01 for L. monocytogenes and 0.50 for Listeria spp., which indicated poor and moderate reproducibility, respectively. When the medium of InSite was smeared on agar medium, 7 of the 19 samples tested positive only for Listeria spp. (negative for L. monocytogenes) but L. monocytogenes was cultured, indicating that the sensitivity of detecting L. monocytogenes via fluorescence may be low. MDS was considered a useful alternative for both L. monocytogenes and Listeria spp. as targets, and InSite was not possible as a substitute for detecting L. monocytogenes; however, it is considered a helpful alternative method for detecting Listeria spp. EMPs for Listeria often target Listeria spp. as an indicator of L. monocytogenes. The alternative methods studied in this study are rapid, simple, and useful in EMPs for Listeria. However, the data in this study were a comparatively small sample set and impacted by variability, so more robust comparisons are desirable in the future.

Seroprevalence of Orientia tsutsugamushi and Rickettsia typhi in water buffaloes (Bubalus bubalis) from Southern Thailand.

Background and Aim: Scrub typhus and murine typhus are globally distributed zoonoses caused by the intracellular Gram-negative bacteria Orientia tsutsugamushi and Rickettsia typhi, respectively. Numerous studies have been undertaken on rickettsial illnesses in humans and animals, including arthropod vectors, in Thailand. However, the reports on the seroprevalence of antibodies to O. tsutsugamushi and R. typhi in buffaloes is extremely rare. Thus, this study aimed to estimate the seroprevalence of both rickettsial infections in water buffaloes (Bubalus bubalis) in Phatthalung Province, southern Thailand. Materials and Methods: From February to March 2023, a total of 156 serum samples were collected from 156 water buffaloes on 29 farms in Phatthalung province. The sera were screened for antibodies against O. tsutsugamushi and R. typhi using an indirect immunofluorescence assay. Results: The seroprevalence of antibodies against O. tsutsugamushi and R. typhi in individual water buffaloes was 4.49% (95% confidence interval [CI]: 2.19%-8.97%) and 3.85% (95% CI: 1.77%-8.14%), respectively, whereas 31% (9/29) of the herds had buffaloes with antibodies. The number of buffaloes with scrub typhus infection and ectoparasite infestation was statistically significant (p < 0.05; odds ratio = 6.25 [95% CI: 1.19-33.33]). Intriguingly, the prevalence of scrub typhus antibodies in buffaloes that were not infested with ectoparasites was much higher than those that were. Conclusion: This is the first report of O. tsutsugamushi and R. typhi antibodies in water buffalo sera in Southern Thailand. Two serum samples showed a high antibody titer against O. tsutsugamushi. Seroprevalence mainly occurred in non-ectoparasite-infested buffaloes, especially for O. tsutsugamushi antibodies. At the herd level, one-third of the studied farms showed seroprevalence. Additional research on the occurrence of these pathogens in vectors and in other animal reservoirs is necessary.

Environmental Monitoring of Food Manufacturing Facilities for Listeria: A Case Study.

Environmental monitoring programs (EMPs) for food production facilities are useful for verifying general sanitation controls and are recommended as verification measures to ensure that the Hazard Analysis Critical Control Point plan is working effectively. In this study, EMPs for Listeria were conducted at three food production facilities to assess the efficacy of sanitation control and establish effective sanitation control methods. In Facility A, L. monocytogenes was detected in the clean area although in Zone 3, non-food-contact surfaces. To prevent contamination from dirty areas, the cleaning practices in the preparation room were investigated. Normal cleaning combined with disinfection with carbonated hypochlorite water (chlorine concentration, 150 ppm) proved effective. At Facility B, a salad product and its ingredients (pastrami and salami) were positive for L. monocytogenes serotype 3b. The bacterial count was <10/g in all samples. However, when inoculated with L. monocytogenes isolates, the growth of approximately 2 log cfu/g was observed on pastrami after 48 h of incubation at 10°C. The ingredients were commercially purchased blocks that were sliced in a slicer at Facility B and used as salad toppings. Because both unopened blocks were negative for L. monocytogenes, contamination of the slicer was suspected. Sampling of the slicer revealed that contamination by L. monocytogenes serotype 3b was more extensive after use than before use. Therefore, the slicer was disassembled, cleaned, and disinfected thoroughly. In Facility C, L. monocytogenes serotype 4b (4e) was detected in all the dirty, semiclean, and clean areas. The strain was also isolated from the wheels of a smoking cart transported across the zones. Therefore, efforts were made to frequently clean and disinfect the cart. EMPs revealed the presence of Listeria in each facility and allowed remedial measures to be undertaken. Continued monitoring and Plan-Do-Check-Act cycles were considered desirable.

Food safety, livestock health, and productivity of a farm fatting Japanese black cattle following implementation of a certificated Hazard Analysis and Critical Control Point (HACCP) system.

This study examined the safety and productivity data analysis of a beef cattle farm with Japanese black cattle over a 4-year period following the implementation of a Hazard Analysis and Critical Control Point (HACCP) system in 2018. The critical control point was "the selection of shipping cows" and no deviations from critical limits were observed, indicating the beef were safe. In addition, cattle treated for bovine respiratory disease (BRD) less than 6 months after the introduction of cattle decreased, while the average Beef Marbling Standard and quality of meat (i.e., grades A5, B5, A4, and B4) in beef carcass trade standard scores increased. These results suggest that the HACCP system had a positive effect on the health and meat quality of the cattle.

Survival of Campylobacter jejuni, Salmonella, and Listeria monocytogenes and Temperature Change in Low-Temperature-Longtime-Cooked Chicken Meat.

ABSTRACT: Low-temperature and longtime (LT-LT) cooking, also known as sous vide cooking, is the process in which meat is sealed in a bag and cooked in hot water at a relatively low temperature of around 60°C. This cooking method has increased in popularity, and low-temperature cookers for home use are now commercially available. However, after LT-LT cooking, if any foodborne bacteria remain, they could cause infection and foodborne illnesses. Therefore, in the present study, the aim was to determine the appropriate LT-LT cooking methods for chicken by assessing temperature changes and studying the bacteria in LT-LT-cooked chicken meat. At set cooking temperatures of 60 and 65°C, the temperatures were measured at the surface and in the centers of single- and double-layer samples of 300 g of chicken breast meat. The times required to reach 50°C were 5 to 14 min at the surface, 25 min in the center of the single-layer sample, and 33 to 35 min in the center of the double-layer sample. The time taken to reach 50°C was fastest in the surface of single-layer chicken meat, followed by the center of single-layer and double-layer chicken meat (P < 0.05). When the meat was LT-LT cooked at 60 and 65°C for 60 min, color changes in the meat and heating of the meat were observed all the way to the interior. Campylobacter jejuni, Salmonella O7, and Listeria monocytogenes were inoculated into chicken breasts, which were then cooked at set temperatures of 60 and 65°C for 15, 30, 60, 90, and 120 min. C. jejuni survived for up to 30 min of cooking, Salmonella O7 survived for up to 60 min of cooking at 60°C and 30 min at 65°C, and L. monocytogenes survived for up to 90 min of cooking at 60°C and 60 min at 65°C. Thus, to prevent infection and illness caused by the three tested bacteria species, LT-LT cooking for 120 min at 60°C and 90 min at 65°C is recommended.

Food safety, livestock health, and productivity of a dairy farm following implementation of a certificated Hazard Analysis and Critical Control Points system.

This study examined the safety and productivity data analysis of a dairy farm over a 3-year period following the implementation of a Hazard Analysis and Critical Control Point (HACCP) system in 2018. The CCP was "the selection of milking cows" and the critical limit was "the withholding period has passed". No deviation from the critical limit was observed, the safety of the milk is ensured. In addition, the average daily milk yield per cow increased, while the average number of somatic cells/ml decreased. The number of cows with newly diagnosed mastitis increased, and the product excluded. These results suggest that the HACCP system had a positive effect on milk yield per cow and led to a decrease in somatic cells.

Detailed Structure and Pathophysiological Roles of the IgA-Albumin Complex in Multiple Myeloma.

Immunoglobulin A (IgA)-albumin complexes may be associated with pathophysiology of multiple myeloma, although the etiology is not clear. Detailed structural analyses of these protein-protein complexes may contribute to our understanding of the pathophysiology of this disease. We analyzed the structure of the IgA-albumin complex using various electrophoresis, mass spectrometry, and in silico techniques. The data based on the electrophoresis and mass spectrometry showed that IgA in the sera of patients was dimeric, linked via the J chain. Only dimeric IgA can bind to albumin molecules leading to IgA-albumin complexes, although both monomeric and dimeric forms of IgA were present in the sera. Molecular interaction analyses in silico implied that dimeric IgA and albumin interacted not only via disulfide bond formation, but also via noncovalent bonds. Disulfide bonds were predicted between Cys34 of albumin and Cys311 of IgA, resulting in an oxidized form of albumin. Furthermore, complex formation prolongs the half-life of IgA molecules in the IgA-albumin complex, leading to excessive glycation of IgA molecules and affects the accumulation of IgA in serum. These findings may demonstrate why complications such as hyperviscosity syndrome occur more often in patients with IgA dimer producing multiple myeloma.

Epidemiological Aspects of Escherichia albertii Outbreaks in Japan and Genetic Characteristics of the Causative Pathogen.

Zoonotic pathogen Escherichia albertii has been identified as the cause of several human disease outbreaks; however, factors such as the general symptoms and incubation period of E. albertii infection have yet to be defined. Therefore, we aimed to determine the unique aspects of E. albertii outbreaks in Japan and to examine the genetic characteristics of the causative pathogen. We studied all known E. albertii outbreaks that occurred in Japan up until 2015, which consisted of five confirmed outbreaks and one putative outbreak (Outbreaks 1-6). Outbreaks were re-examined based on personal communications between researchers in prefectural and municipal public health institutes, and through examination of any published study conducted at the time. Draft genome sequences of outbreak-associated E. albertii isolates were also generated. The most common symptom displayed by patients across the six episodes was watery diarrhea (>80%), followed by abdominal pain (50-84%) and fever (37.0-39.5°C) (26-44%). The estimated average incubation period of E. albertii infection was 12-24 h. We assumed that most of the outbreaks were foodborne or waterborne, with restaurant foods, restaurant water, and boxed lunches being the suspected transmission vehicles. Three of the six outbreak-associated E. albertii isolates possessed intact ETT2 regions, while the remaining isolates contained disrupted ETT2-encoding genes. Virulence gene screening revealed that more than half (44/70) of the tested genes were present in all 5 strains examined, and that each of the strains contained more than 1 gene from 14 out of the 21 groups of virulence genes examined in this study. The five E. albertii strains were classified into four of the five known phylogroups. Therefore, we determined that multiple E. albertii genotypes in Japan have the potential to cause outbreaks of diarrhea, abdominal pain, and/or fever following infection of a human host.

Molecular Evolution of the VP1 Gene in Human Norovirus GII.4 Variants in 1974-2015.

Human norovirus (HuNoV) is a leading cause of viral gastroenteritis worldwide, of which GII.4 is the most predominant genotype. Unlike other genotypes, GII.4 has created various variants that escaped from previously acquired immunity of the host and caused repeated epidemics. However, the molecular evolutionary differences among all GII.4 variants, including recently discovered strains, have not been elucidated. Thus, we conducted a series of bioinformatic analyses using numerous, globally collected, full-length GII.4 major capsid (VP1) gene sequences (466 strains) to compare the evolutionary patterns among GII.4 variants. The time-scaled phylogenetic tree constructed using the Bayesian Markov chain Monte Carlo (MCMC) method showed that the common ancestor of the GII.4 VP1 gene diverged from GII.20 in 1840. The GII.4 genotype emerged in 1932, and then formed seven clusters including 14 known variants after 1980. The evolutionary rate of GII.4 strains was estimated to be 7.68 × 10-3 substitutions/site/year. The evolutionary rates probably differed among variants as well as domains [protruding 1 (P1), shell, and P2 domains]. The Osaka 2007 variant strains probably contained more nucleotide substitutions than any other variant. Few conformational epitopes were located in the shell and P1 domains, although most were contained in the P2 domain, which, as previously established, is associated with attachment to host factors and antigenicity. We found that positive selection sites for the whole GII.4 genotype existed in the shell and P1 domains, while Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants were under positive selection in the P2 domain. Amino acid substitutions overlapped with putative epitopes or were located around the epitopes in the P2 domain. The effective population sizes of the present strains increased stepwise for Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants. These results suggest that HuNoV GII.4 rapidly evolved in a few decades, created various variants, and altered its evolutionary rate and antigenicity.

Molecular evolution of the capsid gene in human norovirus genogroup II.

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.

A food-borne outbreak of gastroenteritis due to genotype G1P[8] rotavirus among adolescents in Japan.

Six high school students in Tochigi prefecture, Japan, developed gastroenteritis after eating at a pork cutlet shop. Molecular epidemiologic analyses showed that the causative agent was genotype G1P[8] rotavirus (RV), this being detected in stool samples from both the patients and the asymptomatic food handlers. The detected RV strains were closely related genetically. The only uncooked food that all victims had eaten was raw sliced cabbage. These findings results suggest that uncooked foods contaminated with RV may be sources of infectious gastroenteritis in adolescents.

[Analysis of survival of enterohemorrhagic Escherichia coli in the steps during cooking on a Japanese barbecue].

Foodborne infections with enterohemorrhagic Escherichia coli (EHEC) related to food in each step of the cooking of a Japanese barbecue have been reported in Japan. We examined the survival of EHEC during various types of cooking on a Japanese barbecue. The number of EHEC in barbecue sauce remained stable during short-term storage at low temperature. In a series of experiments on survival of EHEC on beef during cooking on an electric griddle or a gas cooktop, the population was reduced by at least 1/1,100. Although these results suggested that EHEC are effectively killed by adequate cooking, the degree of reduction of EHEC varied among types of meat and was affected by uneven cooking. Furthermore, when the same cooking equipment was used to handle meats before and after cooking, 1/500 to 1/300,000 of EHEC population of contaminated uncooked meat cross-contaminated the cooked meat. Adequate cooking of beef, including internal organs, and use of separate cooking equipment for uncooked and cooked beef are important to avoid EHEC infection caused by Japanese barbecues.

Triplex real-time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus.

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT-PCR method was established. This triplex real-time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real-time PCR across a broad dynamic range of 10(2) -10(7) copies/assay using plasmid DNA standards. The limit of detection was 10(2) copies/assay. The quantitative value was comparable with that of monoplex real-time PCR of stool samples. Our triplex real-time PCR is useful for detection of NoV and SaV infections.

Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan.

We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p-distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10(-3) substitutions/site per year). The p-distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV-A emerged approximately10 years ago and spread to some countries with a high evolution rate.

Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008-2011.

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008-2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63×10⁻³ and 4.56×10⁻³ substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.

Salmonella prevalence in meat at retail markets in Pakse, Champasak Province, Laos, and antimicrobial susceptibility of isolates.

Non-typhoidal Salmonella is one of the most common causes of human gastroenteritis worldwide and most human outbreaks are associated with the consumption of contaminated food. However, there are no reports on Salmonella contamination in market meat in Laos. The objective of this study was to determine the prevalence of Salmonella in meat samples in Pakse, Champasak Province, Laos, as well as the antimicrobial susceptibility of isolates. The prevalence of Salmonella was 82% in beef, 93% in pork and 80% in buffalo meat. In total, 80 isolates and 11 serovars were found, including serovars Stanley (n=15), Anatum (n=14), Derby (n=11), Rissen (n=9) and Amsterdam (n=7). The drug susceptibility of 60 strains against 10 antimicrobial agents was tested. The 60 isolates examined were sensitive to ciprofloxacin (100% susceptible), norfloxacin (100%), cefotaxime (95%), nalidixic acid (90%) and chloramphenicol (88%), but were resistant to streptomycin (67% resistant), tetracycline (67%) and ampicillin (63%). Of the isolates, 73% were multidrug-resistant. These findings indicate a high Salmonella prevalence in market meat in Pakse. Therefore, programmes to control Salmonella contamination are needed.

Genetic analysis of non-hydrogen sulfide-producing Salmonella enterica serovar typhimurium and S. enterica serovar infantis isolates in Japan.

Whole-genome sequencing of non-H(2)S-producing Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 ß-lactamase. The lack of production of H(2)S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistance genes.