Changes in the Phenotypes of Salmonella spp. in Japanese Broiler Flocks.

Salmonella infections represent a leading cause of foodborne illnesses; resistance to third-generation cephalosporins (TGCs), which are a first-choice antimicrobial for treating human Salmonella enteritis, has become a serious public health concern worldwide. Because the consumption of undercooked chicken meat products is a major cause of foodborne salmonellosis in Japan, we conducted three surveys at different periods between 2017 and 2022, with the cooperation of four abattoirs (two in Eastern and two in Western Japan). The first survey was conducted at abattoir A, which is located in Eastern Japan. Salmonella was detected in 84.4% of broiler flocks tested (27/32); among them, all the TGC-resistant isolates obtained from one farm (farm FA) were identified as S. Infantis. Salmonella was recovered from 62.5% of breast meat samples (20/32), with one case suggesting cross-contamination. The second survey was conducted at three other abattoirs to examine the prevalence of TGC-resistant Salmonella, in both Western (abattoirs B and C) and Eastern (abattoir D) Japan. Salmonella was detected in 90.6% of broiler flocks examined (29/32). TGC-resistant S. Infantis was isolated from 2 flocks until 2018 and not thereafter. Subsequently, isolates were identified as TGC-susceptible S. Schwarzengrund in both regions. The third survey was performed at abattoir A to elucidate whether there were changes in the phenotypes. Of the 11 broiler flocks introduced from farm FA, 10 were positive for Salmonella (90.9%); all the isolates were S. Schwarzengrund susceptible to TGC. This study shows that TGC-susceptible S. Schwarzengrund has replaced the resistant phenotypes among broiler flocks in both Eastern and Western Japan. Although chicken meat products could be cross-contaminated with Salmonella during the slaughtering process, reducing the prevalence of Salmonella in broiler flocks remains important to decrease Salmonella enteritis in humans.

[Prevalence and Characteristics of Campylobacter and Salmonella in Meat Derived from Spent Hens].

Meat derived from spent hens as well as broilers is destined for human consumption. There are many reports on the prevalence and antimicrobial resistance of Campylobacter and Salmonella in broiler meat, but few in spent hen meat. Therefore, we investigated the prevalence and antimicrobial resistance of these genera in spent hen meat collected at chicken processing plants. Campylobacter and Salmonella were isolated from 47 (92.2%) and 18 (35.5%), respectively, of breast meat derived from 51 spent hen flocks. Campylobacter jejuni accounted for 87.5% of Campylobacter isolates. The highest resistant rate in C. jejuni isolates was found for ampicillin (45.3%), followed by tetracycline (14.3%) and ciprofloxacin (14.3%). There was no Campylobacter isolate resistant to erythromycin, which is recommended as a first-choice antimicrobial for humans when Campylobacter enteritis is strongly suspected. Of Salmonella isolates, the first and second most frequent serovars were Salmonella Corvallis (30.4%) and S. Braenderup (21.7%), respectively. Of Salmonella isolates, 30.4% were resistant to streptomycin. There was no Salmonella isolate resistant to ciprofloxacin, which is one of the recommended antimicrobials for humans against Salmonella enteritis. This study shows that one third of spent hen meat is contaminated with Campylobacter or Salmonella, and administration of erythromycin or cefotaxime is an effective option for patients with Campylobacter- or Salmonella- enteritis, respectively, caused by consumption of spent hen meat.

Antimicrobial resistance profiles of Campylobacter jejuni and Salmonella spp. isolated from enteritis patients in Japan.

Understanding the antimicrobial resistance of Campylobacter jejuni and Salmonella spp. isolated from patients with enteritis will aid in therapeutic decision-making. This study aimed to characterize C. jejuni and Salmonella spp. isolates from patients with enteritis. For C. jejuni, the resistance rates against ampicillin, tetracycline, and ciprofloxacin were 17.2%, 23.8%, and 46.4%, respectively. All the C. jejuni isolates were susceptible to erythromycin, which is recommended as a first-choice antimicrobial if Campylobacter enteritis is strongly suspected. C. jejuni was classified into 64 sequence types (STs), and the five major STs were ST22, ST354, ST21, ST918, and ST50. The ciprofloxacin-resistance rate of ST22 was 85.7%. For Salmonella, the resistance rates against ampicillin, cefotaxime, streptomycin, kanamycin, tetracycline, and nalidixic acid were 14.7%, 2.0%, 57.8%, 10.8%, 16.7%, and 11.8%, respectively. All the Salmonella spp. isolates were susceptible to ciprofloxacin. Therefore, fluoroquinolones are the recommended antimicrobials against Salmonella enteritis. S. Thompson, S. Enteritidis, and S. Schwarzengrund were the three most prevalent serotypes. The two cefotaxime-resistant isolates were serotyped as S. Typhimurium and were found to harbor blaCMY-2. The results of this study would help select antimicrobials for treating patients with Campylobacter and Salmonella enteritis.

[Quantitative Survey of Campylobacter on Chicken Livers in Japan].

Chicken liver is a potential source of campylobacteriosis in humans. Therefore, we determined the number of Campylobacter in chicken liver. In total, 33 vacuum-packed liver products were obtained from retail stores, and found that 27 of the 33 products (81.8%) were contaminated with Campylobacter. Moreover, Campylobacter was isolated from 138 of 149 livers (92.6%) collected from the 27 Campylobacter-positive products. The mean Campylobacter count was 2.3 log10 CFU/g, while Campylobacter count in 22 of the 138 contaminated livers (15.9%) was >3.0 log10 CFU/g. Furthermore, gastrointestinal tract, liver, and bile samples were collected from 35 broilers at chicken processing plants. We isolated Campylobacter from the gastrointestinal tract of 27 broilers (77.1%). Of these 27 broilers, liver of 24 broilers (88.9%) was Campylobacter-positive, with a mean Campylobacter count of 2.8 log10 CFU/g. Of these 24 broilers, bile of 13 broilers (54.2%) was contaminated with Campylobacter (mean Campylobacter count, 3.5 log10 CFU/mL). Among them, bile of 2 broilers had a Campylobacter count of >8.3 log10 CFU/mL. Collectively, these results indicate that livers derived from broilers colonized with Campylobacter are contaminated with Campylobacter at the time of evisceration. Therefore, to prevent foodborne campylobacteriosis in humans, chicken livers should be thoroughly heated before consumption.

Prevalence and antimicrobial resistance of Campylobacter and Salmonella in layer flocks in Honshu, Japan.

Campylobacter and non-typhoidal Salmonella are the major causes of bacterial gastrointestinal infections in humans. Although antimicrobial therapy is typically not recommended in many cases of these infections, it may be life-saving in patients with severe symptoms. Since chicken eggs and meat derived from layers are destined for human consumption, we investigated the prevalence and antimicrobial resistance of these two bacterial genera in 82 layer flocks at chicken processing plants in Honshu, Japan. Campylobacter was isolated from 77 flocks (93.9%). Resistance to ampicillin, tetracycline, and ciprofloxacin was documented in 42.3 (30/71), 16.9 (12/71), and 14.1% (10/71) of Campylobacter jejuni, respectively. Multilocus-sequence typing identified ST4389 and ST5262 as the most frequent C. jejuni sequence types. In C. coli, resistance to ampicillin, tetracycline, and ciprofloxacin was found in 20.0 (7/35), 20.0 (7/35), and 25.7% (9/35), respectively. The most frequent sequence type in C. coli was ST8292. Erythromycin resistance was not observed among Campylobacter species. Salmonella was isolated from 14 flocks (17.1%). The two most frequent serovars were Salmonella Corvallis and S. Braenderup. Neither S. Enteritidis nor S. Infantis were isolated. Streptomycin resistance was observed in six isolates (26.1%), and all isolates were susceptible to cefotaxime and ciprofloxacin. Thus, chicken eggs and meat derived from layers are possible sources of these bacterial infections in humans. The antimicrobial susceptibility of these isolates was maintained, reflecting restrictions on the use of antimicrobial agents on layers.

Quantitative detection and genetic characterization of thermotolerant Campylobacter spp. in fresh chicken meats at retail in Japan.

Campylobacter jejuni and C. coli are one of the leading causes of gastrointestinal illnesses, and which are considered to be transmitted to humans mainly from chicken meats. Considering the less availability of quantitative contamination data in the retail chicken meats in Japan, 510 fresh chicken meats retailed at five distinct regions in Japan between June 2019 and March 2021 were examined. The quantitative testing resulted that 45.7% of the samples (254/510) were positive at mean ± standard deviation of 1.15 ± 1.03 logCFU/g, whereas 43 samples (8.4%) exceeded 3.0 logCFU/g. Seasonal comparison revealed increased bacterial counts in fall compared with spring and summer. As for the chicken slaughter age, those slaughtered at >75 days old were less contaminated than those at <75 days old. Genome sequencing analyses of 111 representative C. jejuni isolates resulted in the detection of three antimicrobial resistance genes (gyrA substitution T86I, tetO and blaOXA-61) at 25.2, 27.9 and 42.3%, respectively. In silico MLST analysis revealed the predominance of sequence types (ST)-21 clonal complex (CC), followed by ST-45CC and ST-464CC. The single nucleotide polymorphism (SNP)-based phylogenetic tree largely classified the sequenced C. jejuni isolates into two clusters (I and II), where all C. jejuni from highly contaminated samples (STs-21CC, -22CC and -45CC) belonged to cluster I, independent of both season and slaughter age. To our knowledge, this is the first example to study the current status of Campylobacter contamination levels in fresh chicken meats retailed in Japan. Our data would be contributable to future quantitative microbial risk assessment, to establish effective control measures for campylobacteriosis.

First isolation of ST398 methicillin-resistant Staphylococcus aureus carrying staphylococcal cassette chromosome mec type IVd from pig ears in Japan.

The emergence and increasing prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) are a global concern. To investigate the prevalence and characteristics of sequence type 398 (ST398) MRSA in pig ears, 102 pig's ears were collected from 102 animals shipped from 51 farms at an abattoir. Eight ST398 MRSA isolates were isolated from the ears of eight pigs shipped from seven farms. Of the eight ST398 isolates, seven had the staphylococcal cassette chromosome mec (SCCmec) type IVd and these were obtained from seven pigs shipped from six farms. Single nucleotide polymorphisms ranging from 13 to 26 were observed in the core-genome regions in the seven SCCmec type IVd isolates. We believe that this is the first report on the isolation of ST398 MRSA SCCmec type IVd in Japan.

Characterization of Campylobacter jejuni in large-billed crows (Corvus macrorhynchos) in Tochigi prefecture, Japan.

As free-living crows are a potential source of Campylobacter infections in broilers and cattle, we characterized Campylobacter spp. isolated from crows using multilocus sequence typing and antimicrobial susceptibility testing. We obtained 82 samples from 27 birds captured at seven different times using a trap set in Tochigi prefecture, Japan. Campylobacter jejuni was isolated from 55 (67.1%) of the 82 samples and classified into 29 sequence types, of which 21 were novel. Tetracycline and streptomycin resistance rates were 18.2% and 3.6%, respectively. These results show that most types of C. jejuni infecting crows differ from those isolated from humans, broilers, and cattle. Thus, the importance of free-living crows as reservoirs of Campylobacter infections in broilers and cattle may be limited.

Campylobacter spp. prevalence and fluoroquinolone resistance in chicken layer farms.

Chicken is a major source of human campylobacteriosis. Chicken meat originates not only from broilers but also from spent layers; however, few reports have documented the prevalence and antimicrobial resistance of Campylobacter spp. in layers in Japan. Therefore, we investigated the prevalence and antimicrobial susceptibility of Campylobacter spp. in 47 layer farms in Japan. Fecal samples were collected from the youngest and oldest flocks on the farm, and Campylobacter spp. was isolated from 46/47 (97.9%) farms. Among the C. jejuni isolates, the resistance rates to ampicillin, tetracycline, and ciprofloxacin were 29.6%, 22.2%, and 19.8%, respectively. The ciprofloxacin resistance rate (7.3%) in C. jejuni isolated from old flocks was significantly (P<0.01) lower than that in young flocks (32.5%).

Bacterial Distribution and Community Structure in Beef Cattle Liver and Bile at Slaughter.

ABSTRACT: In this study, the distribution of hygienic indicator bacteria in cattle livers and bile was examined at slaughterhouses. One hundred twenty-seven cattle livers with gallbladders were carefully eviscerated from carcasses at 10 slaughterhouses. Microbiological examination revealed that nine bile samples (7.1% prevalence) and 19 liver parenchyma samples (15.0% prevalence) were positive for Enterobacteriaceae (EB) with means ± standard deviations of 3.68 ± 4.63 log CFU/mL and 1.59 ± 2.47 log CFU/g, respectively; thus, bacterial contamination was apparent even at the postevisceration stage. Subsequently, 70 cattle livers were obtained at the postprocessing and storage stage from 7 of the 10 slaughterhouses. Microbiological analysis revealed significantly higher levels of EB in the liver parenchyma (3.00 ± 3.89 log CFU/g, P = 0.011) than those at the postevisceration stage, suggesting that bacterial dissemination and/or replication occurred in the liver parenchyma during processing and storage. According to 16S rRNA ion semiconductor sequencing analysis of representative samples from 12 cattle, Proteobacteria, Firmicutes, and Actinobacteria were dominant in both the parenchyma and bile in which EB and Escherichia coli were predominant among livers with higher EB levels. These results suggest that bile plays a role as a vehicle for bacterial transmission to the liver parenchyma. This study is the first to evaluate bacterial distribution and community structure in the liver and biliary microecosystem of cattle at slaughter. Our data support the use of EB testing of bile to screen cattle livers contaminated with high levels of fecal indicator bacteria.

Detection of hepatitis E virus RNA from pig bile collected at a slaughterhouse in Japan.

Hepatitis E virus (HEV) is a zoonotic pathogen that circulates mainly between pigs and humans. In Japan, the number of confirmed HEV cases has increased over the past decade, with the majority reported as domestic HEV infections. HEV-infected pork products may be associated with this increase, but there is limited information on HEV in pork in Japanese markets. From February to March 2020, gallbladders were collected from 200 slaughtered pigs shipped from 14 farms and were surveyed to detect HEV RNA in bile using reverse transcription quantitative polymerase chain reaction. The samples were then sequenced and genotyped. Twenty pigs were positive for HEV ribonucleic acid, and seven samples had Ct values of less than 30. Among these 20 pigs, virus strains from 14 pigs were determined as genotype 3. This report indicated that HEV-contaminated pork liver was shipped to consumer markets and demonstrated the importance of detection of HEV in meat ready for shipment.

Antimicrobial Resistance in Salmonella Isolated from Food Workers and Chicken Products in Japan.

Salmonella is an enteric bacterial pathogen that causes foodborne illness in humans. Third-generation cephalosporin (TGC) resistance in Salmonella remains a global concern. Food workers may represent a reservoir of Salmonella, thus potentially contaminating food products. Therefore, we aimed to investigate the prevalence of Salmonella in food workers and characterize the isolates by serotyping and antimicrobial susceptibility testing. Salmonella was isolated from 583 (0.079%) of 740,635 stool samples collected from food workers between January and December 2018, and then serotyped into 76 Salmonella enterica serovars and 22 untypeable Salmonella strains. High rates of antimicrobial resistance were observed for streptomycin (51.1%), tetracycline (33.1%), and kanamycin (18.4%). Although isolates were susceptible to ciprofloxacin, 12 (2.1%) strains (one S. Infantis, one S. Manhattan, two S. Bareilly, two S. Blockley, two S. Heidelberg, two S. Minnesota, one S. Goldcoast, and one untypeable Salmonella strain) were resistant to the TGC cefotaxime, all of which harbored ß-lactamase genes (blaCMY-2, blaCTX-M-15, blaCTX-M-55, and blaTEM-52B). Moreover, 1.3% (4/309) of Salmonella strains (three S. Infantis and one S. Manhattan strains) isolated from chicken products were resistant to cefotaxime and harbored blaCMY-2 or blaTEM-52B. Thus, food workers may acquire TGC-resistant Salmonella after the ingestion of contaminated chicken products and further contaminate food products.

Development and Evaluation of Fluorescence Immunochromatography for Rapid and Sensitive Detection of Thermophilic Campylobacter.

Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are leading causes of foodborne gastroenteritis in Japan. Epidemiological surveillance has provided evidence that poultry meat is one of the main reservoirs for human campylobacteriosis, and therefore, improvement in process hygiene at slaughter is required to reduce the number of human infections. This study thus aimed to develop fluorescent immunochromatography strips for rapid and sensitive detection of thermophilic Campylobacter on poultry carcasses at slaughter. To establish the required detection levels, we first determined the numbers of C. jejuni and C. coli on poultry carcasses at one large-scale poultry slaughterhouse in Japan, resulting in the detection of Campylobacter at 1.97 ± 0.24 log CFU/25 g of neck skin during the post-chilling process by using ISO 10272-2:2017. Our developed Campylobacter fluorescence immunochromatography (FIC) assay exhibited a 50% limit of detection of 3.51 log CFU or 4.34 log CFU for C. jejuni NCTC 11168 or C. coli JCM 2529, respectively. Inclusive and exclusive tests resulted in good agreement. The practical usefulness of this test toward poultry carcasses should be evaluated in future studies, perhaps concentration of the target microorganisms prior to the testing might be helpful to further enhance sensitivity. Nevertheless, our data suggest the potential of FIC for rapid and sensitive detection of thermophilic Campylobacter for monitoring the process hygiene of poultry carcasses at slaughter.

Effectiveness of ear skin swabs for monitoring methicillin-resistant Staphylococcus aureus ST398 in pigs at abattoirs.

Monitoring the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs could be useful for managing transmission risk to humans. To optimize sampling for LA-MRSA monitoring, we compared the sensitivity of MRSA isolation from skin swabs taken behind the ear and nasal swabs collected from 276 pigs and investigated the prevalence of MRSA in their carcasses. MRSA was isolated from 40 behind the ear skin swabs (14.5%), which was statistically higher than the number isolated from nasal swabs (23 samples, 8.3%). MRSA prevalence in the carcasses was 0.4%. All MRSA isolates were sequence type 398 lineage. Sampling of both the skin behind the ear and nasal mucosa in a pig is recommended to investigate the prevalence of LA-MRSA in pigs.

[Prevalence and Characterization of Campylobacter in Bile from Bovine Gallbladders].

Campylobacter is one of the most important causes of food-borne infectious diseases. Antibiotics are rarely needed to treat campylobacteriosis, but occasionally used in severe or prolonged cases. Consumption of contaminated bovine liver is a source of campylobacteriosis. Bovine liver can be contaminated with Campylobacter on the surface and inside by the bile at slaughterhouses. Therefore, we investigated the current prevalence and characteristics of Campylobacter in bovine bile at a slaughterhouse. Campylobacter was isolated from 35.7% (55/154) of bile samples. C. jejuni and C. fetus were the two most frequent species. High antimicrobial resistant rates in C. jejuni were observed against tetracycline (63.0%) and ciprofloxacin (44.4%). Multi-locus sequence typing divided C. jejuni isolates (27 isolates) into 12 sequence types (STs) in which ST806 was the most frequent ST and accounted for 37.0%. All C. fetus were identified as C. fetus subsp. fetus which can cause systemic infections. High antimicrobial resistant rates in C. fetus were observed against ciprofloxacin (66.6%), streptomycin (58.3%) and tetracycline (33.3%). All the C. fetus isolates were divided into two STs, ST3 (16 isolates) and ST6 (8 isolates). Of the 16 ST3 isolates, 15 (93.8%) were resistant to both streptomycin and ciprofloxacin. Our data shows high prevalence of Campylobacter in bovine bile and their high rates of antimicrobial resistance. Preventing bile contamination of bovine liver at slaughterhouses is thus considered to be one of control measures to reduce the risk of Campylobacter infections.

Isolation of ST398 methicillin-resistant Staphylococcus aureus from pigs at abattoirs in Tohoku region, Japan.

Here, we investigated the presence of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in nasal swabs of 420 slaughtered pigs from 84 farms at three abattoirs in Tohoku, Japan. MRSA were isolated from 13 (3.1%) samples from 9 (10.7%) farms at two abattoirs. All isolates were classified as ST398 and were resistant to ampicillin and tetracycline. Ten and three isolates were classified as Staphylococcal Cassette Chromosome mec (SCCmec) types V and IVa, respectively. All type V isolates possessed czrC. The minimum inhibitory concentrations (MICs) of zinc chloride against types IVa and V were 1 and 4 mM, respectively. This study shows the presence of ST398 MRSA in pigs in this region. Antimicrobials and zinc compounds in feed and drugs might select SCCmec type V ST398 MRSA.

Prevalence and Phylogenetic Analysis of Hepatitis E Virus among Pigs in Japan.

The number of reported cases of human hepatitis E virus (HEV) infection has increased since 2012. Pigs are considered an important source of viruses causing human HEV infection. It is possible that the prevalence of HEV among pigs at slaughter age (approximately 6 months old) has increased in the last decade. Therefore, we investigated the current prevalence of HEV among pigs in Japan. Although HEV RNA was detected in rectal content samples from pigs aged from one to 5 months, no HEV RNA was detected in any samples from 6-month-old pigs. The highest viral shedding prevalence (33%) was detected among 3-month-old pigs. This study shows that there has been no change in the prevalence of HEV among pigs at the slaughter age, in the prevalence of HEV by age group on pig farms, or in the phylogenetic classification of HEV isolates in the last decade. Therefore, factors downstream of the pork production stage may be contributing to the increased number of human HEV infection cases.

Imperfection of Commercial Inactivated Salmonella Vaccine Against Salmonella Infantis During Induced Molting in Chickens and Proposed Evaluation Method.

In the present study, we evaluated the continuance and efficacy of inactivated vaccine against Salmonella Infantis (SI) in chickens raised on a commercial farm. Chickens (88-days-old) were inoculated with 1 or 0.5 doses of commercially available trivalent inactivated Salmonella vaccine; anti-SI antibody titer was examined continuously for 11 mo thereafter. Molting was induced 11 mo after vaccination, and SI was administered orally. SI colony-forming units (CFUs) were measured in cecal feces, cecal contents, liver, and spleen samples. Anti-SI antibodies in the 1 dose vaccination group could be detected in at least 90% of cases until the end of testing. SI discharge was significantly reduced in birds treated with either dose of vaccine. However, SI CFUs were elevated in the induced molting group, regardless of vaccination dose, particularly in the cecal feces, cecal contents, and spleen. Thus, the vaccine provided remarkable protection against SI infection under ordinary rearing methods but not during induced molting. To achieve sufficient SI protective efficacy, we recommend inoculation with 1 dose of vaccine. Moreover, the efficacy of inactivated Salmonella vaccine is recommended to be evaluated by challenging chickens with live Salmonella in addition to Salmonella antibody titration.

Limitaciones de la vacuna comercial contra Salmonella inactivada contra Salmonella infantis durante la muda inducida en pollos y propuesta de un para evaluación. En el presente estudio, se evaluó la continuidad y la eficacia de la vacuna inactivada contra Salmonella Infantis (SI) en pollos criados en una granja comercial. Los pollos (de 88 días de vida) se inocularon con una o media dosis de una vacuna trivalente de Salmonella inactivada disponible comercialmente. El título de anticuerpos contra S. Infantis se examinó de forma continua durante once meses. La muda se indujo a los once meses después de la vacunación y S. Infantis se administró por vía oral. Se contaron las unidades formadoras de colonias (UFC) de S. Infantis de muestras de heces cecales, contenido cecal, hígado y bazo. Los anticuerpos contra S. Infantis en el grupo que recibió una dosis de la vacuna pudieron detectarse en al menos el 90% de los casos hasta el final de la prueba. La eliminación de descarga de S. Infantis se redujo significativamente en las aves tratadas con cualquiera de las dosis de vacuna. Sin embargo, las unidades formadoras de colonias de S. Infantis se elevaron en el grupo al que se le indujo la de muda, independientemente de la dosis de vacunación, particularmente en las heces del ciego, el contenido cecal y el bazo. Por lo tanto, la vacuna proporcionó una protección importante contra la infección por S. Infantis bajo los métodos de cría ordinarios, pero no durante la muda inducida. Para lograr suficiente eficacia protectora contra S. Infantis se recomienda la inoculación con 1 dosis de vacuna. Además, se recomienda evaluar la eficacia de la vacuna de Salmonella inactivada mediante desafío de los pollos con Salmonella viva además de la titulación de anticuerpos contra Salmonella.

Microbiological Quality Assessment of Game Meats at Retail in Japan.

In this study, we examined the prevalence of Shiga toxin-producing Escherichia coli and Salmonella spp. and the distribution of indicator bacteria in 248 samples of game meats (120 venison and 128 wild boar) retailed between November 2015 and March 2016 in Japan. No Salmonella spp. were detected in any of the samples, whereas Shiga toxin-producing Escherichia coli serotype OUT:H25 (stx2d+, eae-) was isolated from one deer meat sample, suggesting a possible source for human infection. Plate count assays indicated greater prevalence of coliforms and E. coli in wild boar meat than in venison, whereas their prevalence in processing facilities showed greater variation than in animal species. The 16S rRNA ion semiconductor sequencing analysis of 24 representative samples revealed that the abundances of Acinetobacter and Arthrobacter spp. significantly correlated with the prevalence of E. coli, and quantitative PCR analyses in combination with selective plate count assay verified these correlations. To our knowledge, this is the first report to characterize the diversity of microorganisms of game meats at retail in Japan, together with identification of dominant microbiota. Our data suggest the necessity of bottom-up hygienic assessment in areas of slaughtering and processing facilities to improve microbiological safety.

Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan.

To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years after manufacture, as compared with a fresh vial. No statistically significant non-linearity or non-parallelism of the regression lines was observed (p > 0.05). Statistical assessment of inter- and intra-laboratory variability revealed acceptable coefficients of variation of 3.2% and 2.4-3.1%, respectively. Based on these results, the potency of the potential reference standard was calculated at 40 units of antitoxin activity per 1-mL vial. Vials of this preparation were distributed for use as the first equine tetanus antitoxin reference standard for veterinary use in September 2015.