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.

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.

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.

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 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.

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.

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.

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.

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 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.

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.

Reactive lymphoid hyperplasia with giant follicles associated with a posttherapeutic state of hematological malignancies. A report of eight cases.

AIMS AND BACKGROUND: To further clarify the clinicopathological, molecular genetic and karyotypic findings of reactive lymph node hyperplasia with giant follicles (RLHGF) associated with a posttherapeutic state of hematological malignancies, we studied eight such cases. METHODS: Using formalin-fixed, paraffin-embedded sections, histological, immunohistochemical, in situ hybridization (ISH), and polymerase chain reaction (PCR) were performed. RESULTS: Six patients had a history of malignant lymphoma (diffuse large B-cell lymphoma [DLBCL] = 4, marginal zone B-cell lymphoma = 2), and two had acute myeloid leukemia (AML). Six patients initially presented with lymphadenopathy of the head and neck area and the remaining one presented with swelling of the tonsil. All seven cases demonstrating analyzable metaphases showed a normal karyotype. Histologically, all eight lesions were characterized by numerous enlarged, bizarre-shaped coalescing lymphoid follicles with follicular lysis. Immunohistochemical and flow cytometry study demonstrated the reactive nature of the B cells in all eight lesions. However, three of our eight cases demonstrated immunoglobulin heavy-chain (IgH) gene rearrangement on PCR study. Different clonal bands were detected in the initial lymphomatous tissue and RLHGF in one of the studied cases. There was no development of B-cell lymphoma or recurrence of B-cell lymphoma in any of the three lesions demonstrating IgH rearrangement. There were no human herpes virus type-8+ or human immunodeficiency virus type-1+ cells in any of the eight lesions. ISH demonstrated Epstein-Barr virus (EBV)-encoded small RNA (EBER)+ cells in only two lesions. PCR analyses demonstrated that there was no Toxoplasma gondii DNA in any of the eight lesions. CONCLUSIONS: As suggested in RLHGF posttransplant, RLHGF arising after therapy for hematological malignancies is also a consequence of chronic stimulation in the setting of immune deregulation rather than various infectious agents. It is important for pathologists and clinicians to be aware of this type of lesion in diagnostic practice.

Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR.

We developed a new quantification method for the measles virus (MeV) nucleoprotein (N) gene using real-time reverse transcriptase PCR. This method allowed us to quantify 10(1)-10(7) copies per reaction (corresponding to 5x10(-1)-5x10(5) copies microl(-1)) of the MeV N gene. We also quantified the MeV N gene from the throat swabs of 22 patients with measles as well as the MeV genotypes A, D3, D5, D9 and H1 in viral suspensions derived from MeV-infected cells. As a result, 3.9x10(3)-5.2x10(6) copies ml(-1) and 7.4x10(7)-2.0x10(8) copies ml(-1) of the MeV genomes (N gene) were detected in the throat swabs and viral suspensions, respectively. No other viruses (enteroviruses, respiratory syncytial virus, human metapneumovirus or mumps virus) were detected in the assay. The results suggest that this method is applicable to the detection and quantification of some genotypes of MeV.

Prevalence of hepatitis E virus (HEV) infection in wild boars (Sus scrofa leucomystax) and pigs in Gunma Prefecture, Japan.

The prevalence of hepatitis E virus (HEV) infection in wild boars and pigs in Gunma Prefecture, Japan, was serologically and genetically examined. The positive detection rates of anti-HEV IgG and HEV RNA in the wild boars were 4.5% (4/89) and 1.1% (1/89), whereas those in the pigs were 74.6% (126/169) and 1.8% (3/169), respectively. The positive rates of anti-HEV IgG and HEV RNA on the 17 pig farms in the present study ranged from 20% to 100%, respectively. One male wild boar approximately 5 years of age was positive for HEV RNA but was negative for anti-HEV IgG. Three pigs from 2 farms were positive for HEV RNA; 2 of these pigs were negative for HEV IgG, and the other was positive. A phylogenetic analysis revealed that all of the HEV ORF1 genes detected in the present study belonged to genotype III. In Gunma Prefecture, HEV is highly prevalent and widespread, and uncooked wild boar and pig meat may have the potential to transmit HEV to humans.

Plasmacytic hyperplasia in age-related Epstein-Barr virus-associated lymphoproliferative disorders: a report of two cases.

Age-related Epstein-Barr virus (EBV)+B-cell lymphoproliferative disorder (LPD) is a recently recognized entity that occurs in patients over 40 years of age without any known immunodeficiency. However, this entity is thought to be related to the immunological deterioration that is part of the aging process. Histologically, age-related EBV+B-cell LPDs are classified into the polymorphous subtype and large B-cell lymphoma subtypes. However, plasmacytic hyperplasia, which is thought to be the earliest recognizable EBV+PT-LPD, has not been reported among EBV+B-cell LPDs. We report here two cases of age-related EBV-associated LPD and demonstrate the histological evolution. Pathologically, the initial lymph node biopsy specimens from both cases showed the classical Hodgkin lymphoma-like polymorphous subtype. Hodgkin (H) and Reed-Sternberg (RS) cells were CD3-, CD20+, CD15-. CD30+, CD45RB+, and latent membrane antigen-1+. In-situ hybridization (ISH) study demonstrated that numerous H and RS cells contained EBV-encoded small RNA (EBER)+. Repeated lymph node biopsy specimens from each case contained a mixture of lymphoid cells with prominent plasma cell differentiation, including immunoblasts without atypia. A portion of B-immunoblasts were CD30+ and EBER+. As assessed by polymerase chain reaction (PCR) assay, only the initial biopsy specimen in Case 1 displayed a solitary faint immunoglobulin heavy chain (IgH) gene rearrangement, consistent with the presence of a small clonal B-cell population. However, PCR analyses for EBV-genomes demonstrated the same single clonal infection of EBV in the initial and recurrent lymph node lesions in the present two cases. These two cases demonstrated the presence of plasmacytic hyperplasia in age-related EBV+B-cell LPDs, and plasmacytic hyperplasia also appears to be the earliest lesion of age-related EBV+B-cell LPDs.

Salmonella prevalence in slaughtered buffaloes and pigs and antimicrobial susceptibility of isolates in Vientiane, Lao People's Democratic Republic.

This is the first report regarding isolation of Salmonella from cecum samples of buffaloes and pigs and characterization of the isolates in Laos. The organisms were isolated from 8% (4/50) of buffaloes and 76% (37/49) of pigs. In buffaloes, 3 animals harbored serotype 9,12: -:1,5, and 1 animal harbored both S. Derby and S. Javiana. In pigs, the most predominant serotypes were S. Derby (51%) followed by S. Anatum (45%), S. Weltevreden (15%) and S. Stanley (5%). The buffalo isolates were susceptible to the antimicrobials tested, whereas the pig isolates showed 10 resistance patterns to 1-5 antibiotics. Of the 59 pig isolates, the resistance rates to tetracycline, streptomycin, ampicillin, sulfamethoxazole-trimethoprim, chloramphenicol, amoxicillin-clavulanic acid and nalidixic acid were 24%, 22%, 14%, 5%, 2%, 2% and 2%, respectively. The results suggest that pigs and buffaloes harbor Salmonella, with a higher prevalence especially in pigs, and all the isolates showed sensitivity to cefotaxime, norfloxacin and ciprofloxacin.

Rotavirus associated acute encephalitis/encephalopathy and concurrent cerebellitis: report of two cases.

It is known that rotavirus gastroenteritis can accompany some neurological manifestations, including encephalitis/encephalopathy or seizures. However, the detailed pathogenesis involved has not been fully understood. To date, acute cerebellitis associated rotavirus gastroenteritis has not been previously reported, except for one case. Herein, we describe two cases of acute encephalitis/encephalopathy and concurrent cerebellitis, associated rotavirus gastroenteritis. Following vomiting and diarrhea, case 1 experienced convulsions and consciousness disturbance and case 2, transient loss of consciousness with eye deviation. After these symptoms subsided, cerebellar signs became evident and a brain MRI showed cerebellar involvement in both cases. Both cases showed speech disturbances, such as mutism, slow speech and dysarthria. In this report, we will discuss the possible pathogenesis of rotavirus associated acute encephalitis/encephalopathy and concurrent cerebellitis.