Prevalence and antimicrobial resistance of Enterococcus spp. isolated from animal feed in Japan.

The rising prevalence of antimicrobial resistance (AMR) of bacteria is a global health problem at the human, animal, and environmental interfaces, which necessitates the "One Health" approach. AMR of bacteria in animal feed are a potential cause of the prevalence in livestock; however, the role remains unclear. To date, there is limited research on AMR of bacteria in animal feed in Japan. In this study, a total of 57 complete feed samples and 275 feed ingredient samples were collected between 2018 and 2020. Enterococcus spp. were present in 82.5% of complete feed (47/57 samples), 76.5% of soybean meal (62/81), 49.6% of fish meal (55/111), 33.3% of poultry meal (22/66), and 47.1% of meat and bone meal (8/17) samples. Of 295 isolates, E. faecium (33.2% of total isolates) was the dominant Enterococcus spp., followed by E. faecalis (14.2%), E. hirae (6.4%), E. durans (2.7%), E. casseliflavus (2.4%), and E. gallinarum (1.0%). Of 134 isolates which were tested for antimicrobial susceptibility, resistance to kanamycin was the highest (26.1%), followed by erythromycin (24.6%), tetracycline (6.0%), lincomycin (2.2%), tylosin (1.5%), gentamicin (0.8%), and ciprofloxacin (0.8%). All Enterococcus spp. exhibited susceptibility to ampicillin, vancomycin, and chloramphenicol. Of 33 erythromycin-resistant isolates, only two showed a high minimum inhibitory concentration value (>128 µg/mL) and possessed ermB. These results revealed that overall resistance to antimicrobials is relatively low; however, animal feed is a source of Enterococcus spp. It is essential to elucidate the causative factors related to the prevalence of AMR in animal feed.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from pigs in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of infection in hospitalized patients and can be prevalent in humans and various animal species. In European countries, MRSA isolates belonging to clonal complex 398 have been detected at high rates in pigs. However, the prevalence of MRSA in pigs and farm environments in Japan remains unclear. MRSA isolates were obtained from pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust. We conducted whole-genome sequencing (WGS) and analyzed the molecular epidemiological relationship between these MRSA isolates using core genome multilocus sequence typing (cgMLST). The prevalence rates of MRSA among pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust were 5.2 %, 3.4 %, 28.8 %, and 0.06 %, respectively. ST 398 isolates that classified as ST398/t034 were isolated from pigs from all sources. The results of cgMLST showed that ST398/t034 isolates originating from domestic pigs clustered into the same cluster as the isolates from imported breeding pigs. However, some clusters only included isolates of domestic pig origin. Most MRSA isolates in this study carried resistance genes for aminoglycosides, ß-lactams, macrolides, tetracyclines, and zinc. None of the MRSA isolates in this study harbored Panton-Valentine leukocidin toxin genes. Molecular epidemiological analysis suggested a relationship between isolates from slaughter pigs and imported breeding pigs and the presence of MRSA isolates of domestic origin. However, more data are needed for elucidation of the origin of these MRSA variants in the pig industry in Japan.

Nationwide Monitoring of Antimicrobial-Resistant Escherichia coli and Enterococcus spp. Isolated From Diseased and Healthy Dogs and Cats in Japan.

The Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) was established for nationwide monitoring of antimicrobial-resistant bacteria isolated from animals. Here, antimicrobial resistance of Escherichia coli and Enterococcus spp. isolates from diseased and healthy dogs and cats was investigated. Isolates were collected from diseased dogs and cats and from healthy dogs and cats in 2018 to 2020. Minimum inhibitory concentrations were determined for 1873 E. coli and 1383 Enterococcus spp. isolates. E. coli isolates were most commonly resistant to nalidixic acid [diseased dog (DD), 62.1%; diseased cat (DC), 59.9%; healthy dog (HD), 23.5%; healthy cat (HC, 24.0%] and ampicillin (DD, 54.4%; DC, 64.1%; HD, 28.4%; HC, 25.2%), followed by ciprofloxacin (DD, 45.0%; DC, 44.0%; HD, 12.9%; HC, 10.4%). Enterococcus spp. isolates were most resistant to tetracycline (DD, 66.9%; DC, 67.8%; HD, 47.0%; HC, 52.0%), followed by erythromycin (DD, 43.2%; DC, 46.6%; HD, 27.8%; HC, 34.0%) and ciprofloxacin (DD, 27.9%; DC, 43.7%; HD, 9.7%; HC 12.9%). Only a few E. coli isolates were resistant to colistin and none were resistant to meropenem. Also, none of the Enterococcus spp. isolates we have tested were resistant to vancomycin. The significantly higher resistance rates of E. coli and Enterococcus spp. isolates from diseased, as opposed to healthy, dogs and cats against most of the tested antimicrobials indicates that the use of antimicrobials could select resistant E. coli and Enterococcus spp.

Comparative Genomic Analysis of Third-Generation-Cephalosporin-Resistant Escherichia coli Harboring the blaCMY-2-Positive IncI1 Group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in Japan.

The off-label use of third-generation cephalosporins (3GCs) during in ovo vaccination or vaccination of newly hatched chicks has been a common practice worldwide. CMY-2-producing Escherichia coli strains have been disseminated in broiler chicken production. The objective of this study was to determine the epidemiological linkage of blaCMY-2-positive plasmids among broilers both within and outside Japan, because the grandparent stock and parent stock were imported into Japan. We examined the whole-genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002 to 2014. The predominant 3GC resistance gene was blaCMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-Iγ (n = 21; 24.1%), IncI (n = 12; 13.8%), IncB/O/K/Z (n = 28; 32.2%), and IncC (n = 22; 25.3%). Those plasmids were subjected to gene clustering, network analyses, and plasmid multilocus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single-nucleotide variant (SNV)-based phylogenetic analysis. MLST and SNV-based phylogenetic analysis revealed high diversity of E. coli isolates. The sequence type 429 (ST429) cluster harboring blaCMY-2-positive IncB/O/K/Z was closely related to isolates from broilers in Germany harboring blaCMY-2-positive IncB/O/K/Z. pST55-IncI, pST12-IncI1-Iγ, and pST3-IncC were prevalent in western Japan. pST12-IncI1-Iγ and pST3-IncC were closely related to plasmids detected in E. coli isolates from chickens in North America, whereas 26 IncB/O/K/Z types were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria inside and outside Japan.

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.

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

Immunoreactivity and morphological changes of bursal follicles in chickens infected with vaccine or wild-type strains of the infectious bursal disease virus.

Infectious bursal disease (IBD) is characterized by immunosuppression due to the depletion of lymphocytes in the atrophied bursa of Fabricius (BF). We have sometimes encountered contradictory findings: chickens infected with the vaccine IBD virus (IBDV) strain have sometimes exhibited a highly atrophied BF, but not immunosuppression. In this study, chickens administered vaccine or wild-type strains of IBDV were later vaccinated with the B1 strain of the Newcastle disease virus (NDV). Bursal changes were examined histologically with a focus on the bursal follicle. The immunoreactivity to NDV was also evaluated with the hemagglutination inhibition test. In gross examination, we observed a few chickens with a severely atrophied BF in vaccine strain-administered groups (vaccine groups), and the level of severity was the same as that in the wild-type strain-administered group (wild-type group). However, these chickens retained humoral antibody responses to NDV and were revealed to possess a higher number of bursal follicles than those of the wild-type group. These results indicated that macroscopic evaluation dose not accurately reflect the immunoreactivity and degree of bursal damage in IBDV-administered chickens. We also found non-immunosuppressed chickens in the wild-type group. These non-immunosuppressed chickens retained a significantly higher number of normal follicles and total follicles according to our statistical analysis. Furthermore, a high correlation coefficient between the NDV-HI titer and the number of normal follicles was found in the wild-type group. These results implied that the retained number of normal follicles is important for the immunoreactivity of chickens infected with IBDV.

Detection of hepatocellular carcinoma with ferucarbotran (resovist)-enhanced breath-hold MR imaging: feasibility of 10 minute-delayed images.

PURPOSE: We evaluated the optimal timing for breath-hold MR imaging with bolus-injectable superparamagnetic iron oxide (SPIO) for detecting hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Twenty patients with 62 HCCs (52 hypervascular, 10 non-hypervascular) underwent MR imaging that included unenhanced and SPIO-enhanced T1-weighted gradient echo (GRE) and T2-weighted fast spin echo (FSE) sequences, perfusion study, and SPIO-enhanced T2*-weighted GRE sequences. We obtained SPIO-enhanced T2*-weighted sequences 10 and 30 min after injecting SPIO and made 2 image sets, comprising 10- or 30-min delayed T2*-weighted images. Three observers performed alternative free response receiver operating characteristic (AFROC) analysis, and quantitative evaluation was performed. RESULTS: Only Observers 2 and 3 recognized a significant difference in the area under the AFROC curve (Az) value in the 10-min delayed images; no significant difference was observed in the 30-min delayed images. There was no significant difference in the sensitivity of individual observers between 10- and 30-min delayed images. The contrast-to-noise (C/N) ratio of the 30-min delayed images was significantly higher than that of the 10-min delayed images. The C/N ratio of hypervascular HCCs in the 30-min delayed images was significantly higher than in the 10-min delayed images, but that of non-hypervascular HCCs showed no significant difference. CONCLUSION: In most cases, 10-min delayed SPIO-enhanced T2*-weighted images are sufficient to detect HCCs.

Image quality using dynamic MR imaging of the temporomandibular joint with true-FISP sequence.

We evaluated the quality of dynamic magnetic resonance (MR) imaging of the temporomandibular joint (TMJ) in 20 normal volunteers with 40 TMJs. To confirm TMJ, we obtained static proton density weighted images (PDWI) before performing dynamic MR imaging with true-fast imaging in a steady-state precession (true-FISP) sequence. Four sequences of the first 10 volunteers were examined to determine the optimal sequence. The 4 sequences included the integrated parallel acquisition technique (iPAT) and/or fat saturation technique. The optimal sequence was then determined and performed in all 20 volunteers. The quality of imaging was evaluated, especially with respect to the conspicuity of the articular disk, mandibular condyle, articular eminence and lateral pterygoid muscle. One of 3 confidence levels was assigned for this evaluation. Neither iPAT nor fat saturation obtained the best quality imaging. Detection rates in the 20 volunteers were 83% for the articular disk, 95% for the mandibular condyle, 96% for the articular eminence and 7.5% for the lateral pterygoid muscle. We recommend dynamic MR imaging of the TMJ with the true-FISP sequence using neither iPAT nor fat saturation. Nevertheless, dynamic MR imaging was inferior to static imaging in detecting the articular disk and still requires improvement.

Gd-EOB-DTPA enhanced MRI for hepatocellular carcinoma: quantitative evaluation of tumor enhancement in hepatobiliary phase.

OBJECTIVE: The purpose of this study was to evaluate the utility of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) for the quantitative evaluation of hepatocellular carcinoma (HCC) and dysplastic nodules in the hepatobiliary phase. MATERIAL AND METHODS: The subjects comprised 12 patients with 27 lesions (22 HCCs and 5 dysplastic nodules). Chemical-shift-selective fat-suppressed T1-weighted sequences were obtained before and 10, 20, and 40 min after the injection of Gd-EOB-DTPA. Quantitative analyses were performed with the enhancement ratio of the lesion and the contrast-to-noise (C/N) ratio. RESULTS: The enhancement ratios of the HCCs were 44.0+/-36.5, 44.7+/-46.8, and 47.7+/-52.8 (%) at 10, 20, and 40 min, respectively, after the injection of Gd-EOB-DTPA. The enhancement ratios of the dysplastic nodules were 36.2+/-34.3, 44.3+/-37.3, and 40.1+/-46.8 (%). The C/N ratios of the HCCs were 0.2+/-6.6 for the precontrast image, and -9.2+/-12.6, -9.9+/-14.8, and -12.7+/-15.7 at 10, 20, and 40 min, respectively, after the injection of Gd-EOB-DTPA. The C/N ratios of the dysplastic nodules were 1.4+/-8.0, -13.7+/-11.1, -13.3+/-7.6, and -13.1+/-10.4. No significant differences were found between the HCCs and the dysplastic nodules in the enhancement ratio and the C/N ratio. Only two HCCs showed a positive C/N ratio value, and these HCCs were pathologically confirmed to be a well differentiated and a moderately differentiated carcinoma, respectively. CONCLUSION: HCCs and some of the dysplastic nodules showed hypointensity in the hepatobiliary phase in Gd-EOB-DTPA-enhanced MRI. No specific enhancement was observed, regardless of tumor differentiation.

Perfusion study of hypervascular hepatocellular carcinoma with SPIO.

PURPOSE: We evaluated whether a perfusion study with Resovist is useful to assess blood flow in tumors in patients with hypervascular hepatocellular carcinoma (HCC). SUBJECTS AND METHODS: The subjects were 28 patients; the lesions consisted of 68 hypervascular HCC, 14 post-treatment nodules, and 7 hepatocellular hypovascular nodules. After rapid intravenous injection of Resovist, 7-phase imaging was performed using the single-shot echo-planar method. Diagnostic accuracy and tumor vascularity were evaluated by 3 radiologists using the alternative free response receiver operating characteristic method. Sensitivity, Az values, and positive predictive values were calculated. To assess interobserver variability, we evaluated the kappa static to measure the degree of agreement. RESULTS: The 3 observers indicated no significant difference in Az value related to the presence or absence of a perfusion study, and only one remarked a significant difference in sensitivity. However, kappa values were better in the presence than in the absence of a perfusion study. Blood flow assessment was poor in less than 1 cm. The 3 observers showed a positive predictive value of 90% or more. CONCLUSION: A perfusion study may facilitate the diagnosis of hypervascular HCC, improving the diagnostic accuracy.