miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells.

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210-3p, and miR-224-5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210-3p or miR-224-5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210-3p, and miR-224-5p in sEVs was compared. The amount of miR-33a-5p and miR-210-3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224-5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210-3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.

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.

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.

Application of Next-Generation Sequencing to Evaluate the Profile of Noroviruses in Pre- and Post-Depurated Oysters.

The development of procedures for the efficient removal or inactivation of noroviruses from contaminated oysters is of great interest in oyster production. However, there is a critical limitation for evaluating the depuration efficacy of presently available procedures, as no suitable cell culture system currently exists to cultivate noroviruses. Thus, we applied a next-generation sequencing (NGS) technique to characterize norovirus genotypes in pre- and post-depurated oysters. As a result, we revealed the diversity of noroviruses in pre- and post-depurated oysters. Although the applied depuration procedure could reduce the number of bacterial agents to the level recommended by the Japanese Ministry of Health, Labour and Welfare, no significant changes were observed in the detection rate and the proportion of norovirus group (G) I and GII genotypes. To our knowledge, this is the first report to evaluate the profile of noroviruses in pre- and post-depurated oysters, specifically with respect to norovirus removal, using NGS; the findings imply that the removal of noroviruses from oysters through depuration is not presently sufficient. Further studies are needed to develop a more suitable depuration procedure for removing and/or inactivating noroviruses from contaminated oysters.

Application of next-generation sequencing to investigation of norovirus diversity in shellfish collected from two coastal sites in Japan from 2013 to 2014.

A better understanding of the role played by shellfish regarding the manner of pathogen contamination, persistence, and selection may help considering epidemiology of noroviruses. Thus, norovirus genotype profiles in shellfish (Crassostrea gigas and Mitilus galloprovincialis) were investigated by using Next-generation sequencing (NGS) technology. In genogroup I (GI), 7 genotypes (abbreviated as GI.2 to GI.7, and GI.9) were detected from C. gigas, whereas 9 genotypes (GI.1 to GI.9) were detected from M. galloprovincialis. The genotype with the highest proportion found in both C. gigas and M. galloprovincialis was GI.4, and the second highest was GI.3. In genogroup II (GII), 17 genotypes (GII.1 to GII.9, GII.11 to GII.17, GII.21 and GI.22) were detected from C. gigas, whereas 16 genotypes (GII.1 to GII.8, GII.11 to GII.17, GII.21 and GI.22) were detected from M. galloprovincialis. The genotype with the highest proportion in both C. gigas and M. galloprovincialis was GII.4, the next highest differed between C. gigas and M. galloprovincialis. To our knowledge, this study may be the first trial to utilize the latest technology in this field, and reveal the diversity of norovirus genotypes present in shellfish.

Contamination of poultry products with Listeria monocytogenes at poultry processing plants.

This study aimed to confirm that poultry products packed at poultry processing plants have already been contaminated with Listeria monocytogenes and that poultry products contaminated with L. monocytogenes are derived from broiler flocks infected with L. monocytogenes. L. monocytogenes was isolated from 16.8% (58/345) of chicken breast products and 2.3% (8/345) of chicken liver products. In contrast, L. monocytogenes was isolated from the pooled cecal content sample from only 1 (4%) of 25 flocks and was never isolated from any pooled dropping samples collected from 25 farms. The results of our study indicate that cecal content does not seem to be an important source of L. monocytogenes in poultry products.

Prevalence and antimicrobial susceptibility of foodborne bacteria in wild boars (Sus scrofa) and wild deer (Cervus nippon) in Japan.

This study aimed to evaluate the role of wild boars and deer as reservoirs of foodborne bacteria. We investigated the prevalence and antimicrobial susceptibility of Campylobacter spp., Salmonella spp., Shiga toxin-producing Escherichia coli (STEC) O157 and O26, and Listeria monocytogenes isolated from wild boars and deer in Japan, from July through December 2010. Campylobacter spp. and Salmonella spp. were isolated from 43.8% (95% confidence interval [CI]: 35.0-52.6) and 7.4% (95% CI: 2.8-12.1) of rectal content samples of wild boars, respectively, but not from wild deer. The most common Campylobacter species was C. lanienae and C. hyointestinalis. The nine Salmonella serovars isolated were S. enterica subsp. enterica serovar Agona (three isolates), S. Narashino (two), S. Enteritidis (one), S. Havana (one), S. Infantis (one), and S. Thompson (one). Five (16%) and 6 (29%) isolates of C. lanienae and C. hyointestinalis, respectively, were resistant to enrofloxacin. STEC O157 and O26 and L. monocytogenes were isolated from 2.3% (95% CI: 0-5.0), 0.8% (95% CI: 0-2.3), and 6.1% (95% CI: 1.7-10.5) of the rectal content samples of wild deer, respectively, but not from wild boars. This first nationwide survey of the prevalence of foodborne bacteria in wild boars and wild deer in Japan suggests that consumption of meat from these animals is associated with the risk of causing infection with these bacteria in humans. Moreover, these animals are potential vehicles for distribution of antimicrobial-resistant bacteria into their habitat. The prevalence and antimicrobial susceptibility of such foodborne bacteria in these wild animals should be monitored periodically.

Comparison of the prevalence of shiga toxin-producing Escherichia coli strains O157 and O26 between beef and dairy cattle in Japan.

With the aim of comparing the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157 and O26 between beef and dairy cattle, we collected rectal content samples from 250 beef cattle on 25 beef farms and 250 dairy cows on 25 dairy farms from July through September 2011. STEC O157 was isolated from 16 beef cattle on 7 beef farms, while no STEC O157 was isolated from any dairy farms. This result suggests that the prevalence of STEC O157 is higher in beef cattle than in dairy cattle. STEC O26 was isolated from 1 animal each from beef and dairy cattle herds, and therefore, it was not possible to compare statistically the prevalence of STEC O26 in beef and dairy cattle.

Prevalence of Campylobacter spp., Salmonella spp., Listeria monocytogenes, and hepatitis E virus in swine livers collected at an abattoir.

We investigated the prevalence of Campylobacter spp., Salmonella spp., Listeria monocytogenes, and hepatitis E virus (HEV) in swine liver. We collected swine livers from 110 pigs at an abattoir from September 2011 to March 2012 [corrected] . Pathogens were detected in the liver samples of 19 (17.3%) pigs. Campylobacter spp. were isolated from the liver samples of 14 (12.7%) pigs. In 10 of the 14 Campylobacter-positive pigs, bacteria were present in the internal regions of the liver. Salmonella spp. and L. monocytogenes were detected in the liver samples of 5 (4.5%) pigs and 1 (1%) pig, respectively. No HEV was detected in the swine liver samples tested. Regarding antimicrobial resistance in Campylobacter and Salmonella isolates, all isolates, except 1 Campylobacter jejuni isolate, were resistant to 1 or more antimicrobial agent. Campylobacter spp. resistant to erythromycin and/or enrofloxacin were isolated from the liver samples of 9 (8%) pigs. These results suggest that the consuming swine liver without proper heat treatment may increase the risk of foodborne illnesses.

Prevalence and characterization of foodborne pathogens in dairy cattle in the eastern part of Japan.

To investigate the prevalence and characterization of foodborne pathogens [Campylobacter spp., Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Salmonella spp.] in dairy cows, rectal content grab samples were collected from 250 dairy cows reared on 25 dairy farms in eastern Japan from December 2010 through February 2011. Campylobacter jejuni was isolated from 106 (42%) cows on 23 (92%) farms, STEC O157 from three cows on one farm, L. monocytogenes from three cows on another three farms and Salmonella enterica subsp. enterica serovar Typhimurium from eight cows on another farm. STEC O26 was not isolated from any of the dairy farms investigated. The results suggest that C. jejuni is widespread in dairy farms in eastern Japan.

Prevalence and antimicrobial resistance of Campylobacter isolates from beef cattle and pigs in Japan.

Rectal contents of beef cattle and pigs were collected between October 2010 and February 2011 in Japan. Campylobacter jejuni were isolated from 36.0% (90/250) of beef cattle from 88.0% (22/25) of beef farms. C. coli were isolated from 3.6% (9/250) of beef cattle from 16.0% (4/25) of beef farms and from 42.4% (106/250) of pigs from all 25 pig farms. As to enrofloxacin, 40.0% (36/90) of C. jejuni isolates and 66.7% (6/9) of C. coli isolates from beef cattle and 44.3% (47/106) of C. coli isolates from pigs were resistant. Additionally, 15.1% (16/106) of C. coli isolates from pigs were resistant to erythromycin and enrofloxacin. The high prevalence of Campylobacter carriers and significant antimicrobial resistance of the isolates were found.

Antimicrobial resistance in Shiga toxin-producing Escherichia coli O157 and O26 isolates from beef cattle.

This study was conducted to determine the prevalence of antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) O157 (n = 241) and O26 (n = 11) isolated from beef cattle and to characterize their antimicrobial resistance profiles. Resistance to dihydrostreptomycin was detected most frequently (STEC O157, 9.5%; STEC O26, 54.5%), followed by resistance to oxytetracycline (7.9%; 45.5%) and ampicillin (5.4%; 36.4%). Resistance to one or more antimicrobial agents was detected in 13.3% (32/241) of the STEC O157 isolates and 54.5% (6/11) of the STEC O26 isolates. The antimicrobial resistance rate in the STEC O26 isolates was significantly higher than that in the STEC O157 isolates (P = 0.002, Fisher's exact test). The antimicrobial resistance rate in the STEC O157 isolates possessing both stx(1) and stx(2) genes was 26.3% (15/57), while that in the isolates possessing stx(2c) gene alone was 3.9% (3/77). These findings suggest that the antimicrobial resistance in STEC O157 is associated with serogroups and the Shiga toxin genotype.