[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.

Antiviral Effect of Ephedrine Alkaloids-Free Ephedra Herb Extract against SARS-CoV-2 In Vitro.

We report for the first time that ephedrine alkaloids-free Ephedra Herb extract (EFE) directly inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and that the addition of EFE to the culture medium before viral infection reduces virus titers in the culture supernatant of SARS-CoV-2, including those of variant strains, by more than 99%, 24 h after infection. The addition of Ephedra Herb macromolecule condensed-tannin, which is the main active ingredient responsible for the anticancer, pain suppression, and anti-influenza effects of EFE, similarly suppressed virus production in the culture supernatant by 99% before infection and by more than 90% after infection. Since EFE does not have the side effects caused by ephedrine alkaloids, such as hypertension, palpitations, and insomnia, our results showed the potential of EFE as a safe therapeutic agent against coronavirus disease 2019.

[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.

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%).

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.

Effect of the Photocatalyst under Visible Light Irradiation in SARS-CoV-2 Stability on an Abiotic Surface.

There is a worldwide attempt to develop prevention strategies against SARS-CoV-2 transmission. Here we examined the effectiveness of tungsten trioxide (WO3)-based visible light-responsive photocatalyst on the inactivation of SARS-CoV-2 under different temperatures and exposure durations. The viral titer on the photocatalyst-coated glass slides decreased from 5.93 ± 0.38 logTCID50 /mL to 3.05 ±. 25 logTCID50/mL after exposure to 3,000 lux of the visible light irradiation for 6h at 20℃. On the other hand, lighting without the photocatalyst, or the photocatalyst-coat without lighting retained viral stability. Immunoblotting and electron microscopic analyses showed the reduced amounts of spike protein on the viral surface after the photocatalyst treatment. Our data suggest a possible implication of the photocatalyst on the decontamination of SARS-CoV-2 in indoor environments, thereby preventing indirect viral spread.

Development of ciclesonide analogues that block SARS-CoV-2 RNA replication.

Ciclesonide is an inhaled corticosteroid used to treat asthma and is currently undergoing clinical trials for treatment of coronavirus disease 2019 (COVID-19). An active metabolite of ciclesonide, Cic2, was recently reported to repress severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomic RNA replication. Herein, we designed and synthesized a few types of ciclesonide analogues. Cic4 (bearing an azide group) and Cic6 (bearing a chloro group) potently decreased SARS-CoV-2 viral replication and had low cytotoxicity compared with Cic2 (bearing a hydroxy group). These compounds are promising as novel therapeutic agents for COVID-19 that show significant antiviral activity.

Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters.

In this study, we aimed to investigate the standard method used for quantification of norovirus in oysters in Japan for the provisional adaptation of the method as an alternative to ISO 15216-1:2017, to conduct a Japan baseline survey of norovirus in oysters. For this purpose, the method provided by the Japan Committee for Standardization of Virus Detection in Food was subjected to an interlaboratory study to determine the performance characteristics of the standard method used in Japan. As a result, the theoretical limit of quantification for norovirus GI and GII in oysters by the standard method used in Japan was expected to be 1.92 and 1.85 log10 copies/g, respectively. The repeatability standard deviations (Sr) were 0.26 and 0.30 log10 copies/g for GI and GII, respectively, and the reproducibility standard deviations (SR) were 0.47 and 0.44 log10 copies/g for GI and GII, respectively. Through the interlaboratory study, we specified several critical points to obtain scientifically reliable results by using the standard method used in Japan. Especially, necessity for application of using process control virus was the most crucial point that needed to be improved. In addition, there are many participating laboratories that could not handle dilution of standard and quantify or detect the viruses in the test samples. To ensure scientifically reliable test result, capacity building of laboratories and implementation of proficiency testing should be considered for future tasks in combination with an application of process control materials in the method. On the assumption that the problems revealed in this study will be solved, the standard method used in Japan would be suitable for use in Japan baseline survey of norovirus in oysters, which will contribute to the international action against norovirus in oysters, led by the EU.

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.

GII.17 norovirus infections in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan during two decades.

Norovirus (NoV) is a major cause of viral gastroenteritis, and GII.4 has been the predominant genotype worldwide since the mid-1990s. During the 2014 to 2015 winter, a rare genotype, NoV GII.17, emerged and became prevalent mainly in East Asia. Over the past two decades, NoV molecular surveillance in Osaka City, Japan, has revealed that NoV GII.17 was detected for the first time in February 2001 and that NoV GII.17-associated outbreaks remarkably increased during the 2014 to 2015 season, with higher incidence recorded in January to March 2015. Genetic analysis indicated that 28 GII.17 outbreak strains were closely related to the novel GII.P17-GII.17 variants represented by the Kawasaki308/2015/JP strain, similar to that in other regions. Statistical analysis showed that NoV GII.17 infections were more common in adults than GII.3 and GII.4 infections, suggesting that the affected adults most likely did not have antibodies against NoV GII.17 and the novel GII.17 variant had recently appeared. Regarding transmission, food was one of the most important factors involved in the spread of NoV GII.17 among adults; 61% of GII.17 outbreaks were foodborne, with oysters being the most common vehicle. Interplay between pathogens, hosts, and environmental factors was considered to be important in the 2014 to 2015 NoV GII.17 epidemic.

Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome.

We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5' untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

Effect of High Pressure Processing on a Wide Variety of Human Noroviruses Naturally Present in Aqua-Cultured Japanese Oysters.

The contamination of oysters with human norovirus (HuNoV) poses a human health risk, as oysters are often consumed raw. In this study, the effect of high pressure processing (HPP) on a wide variety of HuNoVs naturally present in aqua-cultured Japanese oysters was determined through a polymerase chain reaction-based method with enzymatic pretreatment, to distinguish between infectious HuNoV. Among five batches, genogroup I. genotype 1 (GI.1), GI.2, GI.3, and GI.8 HuNoV were detected from only one oyster not treated with HPP in the fifth batch, while genogroup II. genotype 1 to 4 (GII.1 to 4), GII.6, GII.8., GII.9, GII.13, GII.16, GII.17, and GII.22 HuNoV were detected from oysters not treated with HPP in all tested batches as determined by next-generation sequencing analysis. Neither GI nor GII HuNoV was detected in the oysters of any of the batches after HPP treatment. To our knowledge, this is the first study to investigate the effect of HPP on a wide variety of HuNoVs naturally present in aqua-cultured oysters.

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.

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.

Effect of High-Pressure Processing on Human Noroviruses in Laboratory-Contaminated Oysters by Bio-Accumulation.

The contamination of oysters with human noroviruses poses a human health risk, since oysters are often consumed raw. In this study, human norovirus genogroup II was allowed to bio-accumulate in oysters, and then the effect of high-pressure processing (HPP) on human noroviruses in oysters was determined through a polymerase chain reaction (PCR)-based method with enzymatic pretreatment to distinguish infectious noroviruses. As a result, oysters could be artificially contaminated to a detectable level of norovirus genome by the reverse transcription-PCR. Concentrations of norovirus genome in laboratory-contaminated oysters were log normally distributed, as determined by the real-time PCR, suggesting that artificial contamination by bio-accumulation was successful. In two independent HPP trials, a 1.87 log10 and 1.99 log10 reduction of norovirus GII.17 genome concentration was observed after HPP at 400 MPa for 5 min at 25°C. These data suggest that HPP is a promising process of inactivation of infectious human noroviruses in oysters. To our knowledge, this is the first report to investigate the effect of HPP on laboratory-contaminated noroviruses in oysters.

Next-Generation Sequencing Analysis of the Diversity of Human Noroviruses in Japanese Oysters.

To obtain detailed information on the diversity of infectious norovirus in oysters (Crossostrea gigas), oysters obtained from fish producers at six different sites (sites A, B, C, D, E, and F) in Japan were analyzed once a month during the period spanning October 2015-February 2016. To avoid false-positive polymerase chain reaction (PCR) results derived from noninfectious virus particles, samples were pretreated with RNase before reverse transcription-PCR (RT-PCR). RT-PCR products were subjected to next-generation sequencing to identify norovirus genotypes in oysters. As a result, all GI genotypes were detected in the investigational period. The detection rate and proportion of norovirus GI genotypes differed depending on the sampling site and month. GII.3, GII.4, GII.13, GII.16, and GII.17 were detected in this study. Both the detection rate and proportion of norovirus GII genotypes differed depending on the sampling site and month. In total, the detection rate and proportion of GII.3 were highest from October to December among all detected genotypes. In January, the detection rates of GII.4 and GII.17 reached the same level as that of GII.3. The proportion of GII.17 was relatively lower from October to December, whereas it was the highest in January. To our knowledge, this is the first investigation on noroviruses in oysters in Japan, based on a method that can distinguish their infectivity.