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Equine coronavirus (ECoV) causes pyrexia, anorexia, lethargy, and sometimes diarrhoea. Infected horses excrete the virus in their faeces, and ECoV is also detected in nasal samples from febrile horses. However, details about ECoV infection sites in the intestinal and respiratory tracts are lacking. To identify the ECoV infection sites in the intestinal and respiratory tracts, we performed an experimental infection study and analysed intestinal and respiratory samples collected from four infected horses at 3, 5, 7, and 14 days post-inoculation (dpi) by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and in situ hybridization (ISH). Two horses became febrile, but the other two did not. None of the horses had diarrhoea or respiratory signs, and severe cases were not observed in this study. None of the horses showed obvious abnormalities in their intestinal or respiratory tracts. Real-time RT-PCR and ISH showed that ECoV RNA was present throughout the intestinal tract, and ECoV-positive cells were mainly detected on the surface of the intestine. In one horse showing viremia at 3 dpi, ECoV RNA was detected in the lung by real-time RT-PCR, but not by ISH. This suggests that the lung cells themselves were not infected with ECoV and that real-time RT-PCR detected viremia in the lung. The other three horses were positive for ECoV RNA in nasal swabs but were negative in the trachea and lung by real-time RT-PCR and ISH. This study suggests that ECoV broadly infects the intestinal tract and is less likely to infect the respiratory tract.

Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were >100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the use of sensitive molecular assays is important for the efficient detection of T. equigenitalis in clinical samples.

Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.

Objectives Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have become a cause for great concern. Although some studies have reported the prevalence of ESBL-producing bacteria and ESBL-encoding genes in horses worldwide, the genetic structure surrounding the ESBL gene has not been analysed in detail. In the present study, we isolated two ESBL-producing Escherichia coli strains from diseased racehorses in Japan and demonstrated the mechanisms underlying the acquisition of their antimicrobial resistance (AMR) genes. Methods Two ESBL-producing E. coli strains (E148 and E189) were isolated from the heart and liver of horses with endocarditis and sepsis in 2014 and 2016, respectively, in Japan. Complete genomic sequences of the two strains were analysed using a PacBio RSII sequencer. Antimicrobial susceptibility testing was performed by the agar dilution method. Results The two isolates possessed a chromosomal AMR gene cluster containing blaCTX-M-1 that was similar to the pEQ1 plasmid found in E. coli isolated from a racehorse in the Czech Republic. In one of the two strains, tandem duplication of the 16-kb region containing blaCTX-M-1 and a class 1 integron, which occurred via IS26-mediated recombination, increased minimum inhibitory concentrations (MICs) associated with the duplicated AMR genes. Conclusion Chromosomal blaCTX-M-1 possibly derived from the pEQ1 or pEQ1-like plasmid was found in Japanese equine E. coli isolates. In Japanese strains, many AMR genes containing blaCTX-M-1 and the class 1 integron are highly accumulated in one region on the chromosome, and the AMR of E. coli was enhanced via the IS26-mediated duplication of the AMR gene cluster.

Mosquitoes and EDTA-treated blood samples from febrile racehorses were investigated for Getah virus infection from 2016 to 2019 at the Miho Training Center, where several outbreaks of Getah virus have occurred. We collected 5557 mosquitoes and 331 blood samples from febrile horses in this study. The most frequently captured mosquito species was Culex tritaeniorhynchus (51.9%), followed by Aedes vexans nipponii (14.2%) and Anopheles sinensis (11.2%). Getah virus was detected in mosquitoes (Aedes vexans nipponii) in 2016 (strain 16-0810-26) but not in 2017-2019. Six of 74 febrile horses in 2016 and one of 69 in 2019 tested positive for Getah virus; none of the horses tested positive in 2017 or 2018. Phylogenetic and sequence analysis showed that strain 16-0810-26 was closely related to strains that had been isolated from horses and a pig around the training center in 2014-2016 but have not been detected in samples collected at the training center since 2017. In contrast, the strain isolated from the infected horse in 2019 (19-I-703) was genetically distinct from the strains isolated from horses and a pig in 2014-2016 and was more closely related to a strain isolated in 1978 at the training center. The source of strain 19-I-703 is unclear, but the virus was not detected in other horses sampled in 2019. In summary, we found that the distribution of mosquito species present at the training center had not changed significantly since 1979, and although a small outbreak of Getah virus infection occurred among horses at the training center in 2016, limited Getah virus activity was detected in mosquitoes and horses at the training center from 2017 to 2019.

Mycoplasma species are often isolated from horses with respiratory symptoms; however, the pathogenicity of Mycoplasma is still unclear. In autumn of 2018, we encountered an increase in cases with respiratory symptoms, mainly coughing, in a group of Thoroughbred racehorses in Japan. We examined tracheal wash samples obtained from 40 of those cases. Bacteria and viruses that commonly cause respiratory symptoms were investigated, and anaerobes were detected in only 5 cases and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was detected in only 1 case of 40 cases with loop-mediated isothermal amplification assay. S. zooepidemicus and Streptococcus pneumoniae were isolated at a bacterial count of higher than 1.0 × 104 CFU/ml from 5 and 2 cases of 28 cases cultured, respectively. None of the viruses investigated was detected in 40 cases. Mycoplasma equirhinis (M. equirhinis) was isolated from 40.0% (16/40) of the cases, which was higher than previously reported isolation rates. The rate of M. equirhinis isolation in the cases from 2018 was significantly higher than the isolation rates in the other horses: clinical cases with respiratory symptoms in 2019-2020 (13.6%, 3/22) and healthy horses (13.5%, 5/37) in Japan. In this study, the isolation rate of M. equirhinis from horse group with cough symptoms in 2018 was high and no other common etiological agents were detected. The pathogenesis of M. equirhinis is still unclear, however, M. equirhinis might have been associated with respiratory symptoms in the Thoroughbred horse cases in 2018.

Bacterial placentitis in horses commonly results in abortion, premature birth or compromised neonatal foal health. Although mycobacterial infections are generally uncommon in horses, 10 equine abortion cases caused by Mycobacterium avium subsp. hominissuis (MAH) infections occurred between 2018 and 2019 in Japan. They occurred on seven Thoroughbred horse farms in the Hidaka district of Hokkaido, but direct contact among the mares on different farms was not recorded. Most cases were characterized by extensive pathological lesions of the placenta, which are not typical in cases of common pathogenic bacteria such as Streptococcus zooepidemicus and Escherichia coli. All abortions featured white–yellow exudates on the surface of the placenta. Mycobacterial granuloma formations were histologically found in the placenta and fetal organs, and acid‐fast bacteria were isolated from the placenta, fetal samples (heart, lung, liver, kidney, spleen and stomach contents) or uterine lavage fluid. The greatest number of bacteria was isolated from necrotic lesions on the placenta, which could be an important site for bacterial isolation in mycobacterial equine abortions. The isolates were identified as MAH based on internal genome sequences. In variable number tandem repeat analysis, all patterns of the strains were identical. Single nucleotide polymorphism analysis of the core genome grouped all strains in the II‐a/SC3 subcluster. Both results reveal that these strains share the same genetic background, suggesting that the horses had been infected by the same unknown contagious source., This article reports 10 equine abortions cases caused by Mycobacterial avium subsp. hominissuis infections. We confirmed the isolated strains shared the same genetic background, suggesting that the horses had been infected by the same contagious source.

We present the orientation order of push-pull chromophores consisting of phenyl vinylene thiophene vinylene (FTC) bridges between the N,-N-bis-(2-hydroxyethyl)-amine electron donor and phenyl-trifl...

Cold cracking sensibility is a severe problem in the welding fabrication of large-scale steel structures. It is well known that diffusible hydrogen content in the weld metal strongly influences thi...

Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the use of sensitive molecular assays is important for the efficient detection of T. equigenitalis in clinical samples.

Mycoplasma species are often isolated from horses with respiratory symptoms; however, the pathogenicity of Mycoplasma is still unclear. In autumn of 2018, we encountered an increase in cases with respiratory symptoms, mainly coughing, in a group of Thoroughbred racehorses in Japan. We examined tracheal wash samples obtained from 40 of those cases. Bacteria and viruses that commonly cause respiratory symptoms were investigated, and anaerobes were detected in only 5 cases and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was detected in only 1 case of 40 cases with loop-mediated isothermal amplification assay. S. zooepidemicus and Streptococcus pneumoniae were isolated at a bacterial count of higher than 1.0 × 104 CFU/ml from 5 and 2 cases of 28 cases cultured, respectively. None of the viruses investigated was detected in 40 cases. Mycoplasma equirhinis (M. equirhinis) was isolated from 40.0% (16/40) of the cases, which was higher than previously reported isolation rates. The rate of M. equirhinis isolation in the cases from 2018 was significantly higher than the isolation rates in the other horses: clinical cases with respiratory symptoms in 2019–2020 (13.6%, 3/22) and healthy horses (13.5%, 5/37) in Japan. In this study, the isolation rate of M. equirhinis from horse group with cough symptoms in 2018 was high and no other common etiological agents were detected. The pathogenesis of M. equirhinis is still unclear, however, M. equirhinis might have been associated with respiratory symptoms in the Thoroughbred horse cases in 2018.

Obesity is a risk factor for gastrointestinal malignancies and tumors. However, which factors either protect or predispose the gastrointestinal organs to high-fat diet (HFD)-induced neoplasia remains unclear. Here, we demonstrate that HFD impacts the stomach to a greater extent as compared to the colorectum, resulting in leptin receptor (LepR) signaling-mediated neoplasia in the tissues. HFD activated leptin signaling, which in turn, accelerates the pathogenesis in the gastric mucosa more than that in the colorectum along with ectopic TFF3 expression. Moreover, in the stomach, higher levels of phosphorylated epidermal growth factor receptor (EGFR) in addition to the activation of STAT3 and Akt were observed as compared to the colorectum. The mice with LepR deletion in the gastrointestinal epithelium exhibited a suppressed induction of leptin, TFF3, and phosphorylated EGFR in the stomach, whereas the levels in the colorectum were insignificant. In co-transfected COS-7 cells with LepR and EGFR plasmid DNA, leptin transactivated EGFR to accelerate TFF3 induction along with activation of STAT3, ERK1/2, Akt, and PI3K p85/p55. Furthermore, TFF3 could bind to EGFR but did not transactivate LepR. Leptin-induced TFF3 induction was markedly suppressed by inhibitors of PI3K (LY294002) and EGFR (Erlotinib). Together, these results suggest a novel role of LepR-mediated signaling in transactivating EGFR that leads to TFF3 expression via the PI3K-Akt pathway. Therefore, this study sheds light on the identification of potentially new therapeutic targets for the treatment of pre-cancerous symptoms in stomach and colorectum.

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have become a cause for great concern. Although some studies have reported the prevalence of ESBL-producing bacteria and ESBL-encoding genes in horses worldwide, the genetic structure surrounding the ESBL gene has not been analysed in detail. In the present study, we isolated two ESBL-producing Escherichia coli strains from diseased racehorses in Japan and demonstrated the mechanisms underlying the acquisition of their antimicrobial resistance (AMR) genes.Two ESBL-producing E. coli strains (E148 and E189) were isolated from the heart and liver of horses with endocarditis and sepsis in 2014 and 2016, respectively, in Japan. Complete genomic sequences of the two strains were analysed using a PacBio RSII sequencer. Antimicrobial susceptibility testing was performed by the agar dilution method.The two isolates possessed a chromosomal AMR gene cluster containing blaChromosomal bla

Antimicrobial administration can lead to imbalances of gastrointestinal microbiota, called dysbiosis. Dysbiosis sometimes results in diarrhea and enteritis in horses. Fecal microbiota transplantation (FMT) is used to treat affected horses, but whether it is effective as a prophylactic approach for dysbiosis in horses receiving antimicrobials remains unknown. The aim of this study was to assess the efficacy of simultaneous FMT against metronidazole-induced dysbiosis in horses. Changes in the ratios of bacterial families, determined by metagenomic analysis, were similar between the metronidazole-treated group and the simultaneous metronidazole- and FMT-treated group, notably in the Clostridiaceae, Ruminococcaceae, and Enterobacteriaceae. Differences in fecal bacterial compositions were due mainly to metronidazole administration (P = .0003), but not to FMT (P = .3136). Simultaneous FMT at 500 g of donor feces in 1 L of suspension once a day did not inhibit metronidazole-induced dysbiosis. The results show that the FMT protocol needs to be improved to prevent metronidazole-induced gut dysbiosis in horses.

Actinobacillus species are known to be pathogenic to horses. To clarify etiological agents of actinobacillosis in Japanese adult horses, 27 isolates from Japanese Thoroughbred racehorses putatively identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as Actinobacillus were further identified by PCR of the A. equuli toxin gene, by CAMP test, and by 16S rRNA sequencing analysis. Actinobacillus equuli subsp. haemolyticus was isolated most frequently (16/27) and was related to respiratory infections. Actinobacillus equuli subsp. equuli (4/27) was isolated from chronic cases or concomitant with other bacterial infections. The remainder were A. pleuropneumoniae, unclassified Actinobacillus species and Pasteurella caballi. Actinobacillus equuli including subsp. haemolyticus and subsp. equuli were the species most frequently isolated from equine actinobacillosis in Japan.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi , establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such ‘carrier’ animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

OBJECTIVE To determine plasma pharmacokinetics of metronidazole and imipenem following administration of a single dose PO (metronidazole, 15 mg/kg) or IV (imipenem, 10 mg/kg) in healthy Thoroughbreds and simulate pleural fluid concentrations following multiple dose administration every 8 hours. ANIMALS 4 healthy Thoroughbreds. PROCEDURES Metronidazole and imipenem were administered, and samples of plasma and pleural fluid were collected at predetermined time points. Minimum concentrations of metronidazole and imipenem that inhibited growth of 90% of isolates (MIC90), including 22 clinical Bacteroides isolates from horses with pleuropneumonia, were calculated. For the computer simulation, the target ratio for area under the pleural fluid concentration-versus-time curve during 24 hours to the MIC90 for metronidazole was > 70, and the target percentage of time per day that the pleural fluid concentration of imipenem exceeded the MIC90 was > 50%. RESULTS Mean ± SD pleural fluid concentrations of metronidazole and imipenem were 12.7 ± 3.3 μg/mL and 12.1 ± 0.9 μg/mL, respectively, 1 hour after administration and 4.9 ± 0.85 μg/mL and 0.3 ± 0.08 μg/mL, respectively, 8 hours after administration. For both antimicrobials, concentrations in the pleural fluid and plasma were similar. The ratio for area under the pleural fluid concentration-versus-time curve during 24 hours to the MIC90 for metronidazole was 84.9, and the percentage of time per day the pleural fluid concentration of imipenem exceeded the MIC90 was 70.9%. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that administration of metronidazole (15 mg/kg, PO, q 8 h) or imipenem (10 mg/kg, IV, q 8 h) resulted in their accumulation in the pleural fluid in healthy horses and concentrations were likely to be effective for the treatment of pneumonia and pleuropneumonia caused by Bacteroides spp.

Mycoplasma felis causes conjunctivitis in cats and respiratory diseases in horses. We report here the complete genome assembly of equine Mycoplasma felis strain Myco-2, which was isolated from an ill horse in Japan.

Here, we describe the complete genome assembly of Mycobacterium avium subsp. hominissuis strain JP-H-1, collected from an equine abortion case in Japan. JP-H-1 has a 5,491,452-bp circular chromosome and 3 plasmids.

Here, we describe the complete genome assembly of Mycobacterium avium subsp. hominissuis strain JP-H-1, collected from an equine abortion case in Japan. JP-H-1 has a 5,491,452-bp circular chromosome and 3 plasmids.

Objectives MRSA is a known pathogen that affects horses. We investigated an equine MRSA isolate for potential antimicrobial resistance genes, classified the staphylococcal cassette chromosome mec (SCCmec) and identified the strain-specific dissemination in the horse community based on WGS. Methods WGS, using short-read sequencing, and subsequent long-read sequencing by hybrid assembly, was conducted to obtain a complete genome sequence. Pairwise sequence alignment of relative SCCmec sequences and core-genome phylogenetic analysis were performed to highlight transmission routes of the SCCmec and MRSA strain-specific lineages. Results In 2018, we isolated the MRSA JRA307 strain from the pus of a wound on a racehorse and the complete genome sequence suggests that it is a clinically relevant pvl-negative ST1-t127 MRSA that harbours both mecA and mecC on SCCmec-307. SCCmec-307 exhibited marked sequence identity to the previously reported SCCmec–mecC in the Staphylococcus sciuri GVGS2 strain isolated from cattle. The JRA307 mecC gene was classified as a mecC allotype of S. sciuri rather than that of Staphylococcus aureus. Conclusions We demonstrated the complete genome sequence of equine isolate JRA307, which is a clinically relevant MRSA harbouring mecA and mecC on SCCmec-307. The finding of mecC MRSA suggests a possible SCCmec transmission between distinct staphylococcal species. To the best of our knowledge, this is the first report of mecC detection in Japan.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used for identification of bacterial species isolated from horses. However, because of insufficiencies in the reference database, some bacterial species isolated from horses are difficult to identify with MALDI-TOF MS, and enriching the databases is expected to enhance the accuracy of MALDI-TOF MS identification. Here we created an in-house database including 271 bacterial isolates from horses. Furthermore, we used an enhanced database (our in-house database plus a commercially provided database) to examine 91 newly obtained isolates that could not be identified with MALDI-TOF MS using the commercially provided database. The enhanced database could identify 15 of those 91 isolates to the species level; including streptococcus (3/19), Gram-positive rod (4/17), Gram-negative rod (8/17) isolates. The enhanced database increased the average identification score of the 91 isolates (1.64-1.76). The in-house database increased the number of isolates that MALDI-TOF MS could identify to the species level and contributed to more accurate identification of bacterial isolates from horses.

Actinobacillus species are known to be pathogenic to horses. To clarify etiological agents of actinobacillosis in Japanese adult horses, 27 isolates from Japanese Thoroughbred racehorses putatively identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as Actinobacillus were further identified by PCR of the A. equuli toxin gene, by CAMP test, and by 16S rRNA sequencing analysis. Actinobacillus equuli subsp. haemolyticus was isolated most frequently (16/27) and was related to respiratory infections. Actinobacillus equuli subsp. equuli (4/27) was isolated from chronic cases or concomitant with other bacterial infections. The remainder were A. pleuropneumoniae, unclassified Actinobacillus species and Pasteurella caballi. Actinobacillus equuli including subsp. haemolyticus and subsp. equuli were the species most frequently isolated from equine actinobacillosis in Japan.

Background Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules). B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but inhibit the growth of other bacteria and fungi based on their antimicrobial profiles. We compared the growth of B. mallei on BM with Xie’s and PC agars, the two previously described selective agars for B. mallei. Results BM agar could sufficiently grow almost all of the tested B. mallei strains within 72 h: only one out of the 38 strains grew scantly after 72 h of incubation. BM agar was further tested with other Burkholderia species and various bacterial species commonly found in the nasal cavities and on the skin of horses. We have found that other Burkholderia species including B. pseudomallei and B. thailandensis can grow on BM agar, but non-Burkholderia species cannot. Furthermore, the specificities of the three selective agars were tested with or without spiking B. mallei culture into clinical specimens of non-sterile sites collected from healthy horses. The results showed that BM agar inhibited growths of fungi and other bacterial species better than PC and Xie’s agars. We have also found that growth of B. mallei on BM agar was equivalent to that on 5% horse blood agar and was significantly greater than those on the other two agars (P

The monoclonal antibody-based latex agglutination tests targeting a high molecular weight exopolysaccharide antigen of Burkholderia pseudomallei are commercially available. The tests are primarily used in routine diagnosis of melioidosis in major hospitals in Thailand and some endemic countries. Being a rapid test, this technique was employed as a presumptive test to identify colonies of B. pseudomallei among many others grown from soil specimens collected from southern Thailand. Cross-reactivity of these tests with other soil bacteria was a concern since it complicated the identification of B. pseudomallei. Here, we describe the cross-reactivity of two commercial latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates from soil.

The incidence of equine proliferative enteropathy, caused by Lawsonia intracellularis, is increasing around the world. To investigate the relationships of variable-number tandem repeat (VNTR) patterns with host species and clinical status in horses, m ulti l ocus V NTR a nalysis (MLVA) was applied to 98 L. intracellularis samples collected from horses, seven from pigs, seven from wildlife, one vaccine strain, and 17 public strains. The VNTR patterns were highly diverse: a total of 130 samples identified 99 distinct patterns, and the 98 horses were classified into 71 different patterns. A phylogenetic tree based on the MLVA showed three clusters: porcine, equine, and miscellaneous cluster. The equine cluster contained 46 horse samples, of which 42 (91.3%) were collected from two sampling areas. The MLVA could discriminate horse samples from pig, but the horse samples in the miscellaneous cluster could not be distinguished from wildlife samples. As for clinical data of the horses, the VNTR patterns were unrelated to horse age, clinical signs, and clinical outcomes. This study shows that VNTR patterns had no clear connection with equine clinical status, but the MLVA could be useful to investigate its epidemiological relationships, and interspecific transmission of L. intracellularis between horse and wildlife cannot be ruled out.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used for bacterial identification by analyzing the spectra of isolates and comparing them against a database of reference spectra; it is known for its rapidity and accuracy. Although MALDI-TOF MS is used for identification of bacterial isolates from animals, not all animal pathogens are identified correctly. In this study, we used a commercial MALDI-TOF MS identification system to examine 3724 bacterial isolates from horses and their environments. Isolates that could not be identified with MALDI-TOF MS were identified by 16S rRNA gene sequence taxonomic analysis. MALDI-TOF MS could identify 86.2% of the isolates from horses to the species level, showing that this method could be successfully applied for bacterial identification in horses. However, some species known to be equine pathogenic agents including Taylorella equigenitalis and Rhodococcus equi were difficult to identify with MALDI-TOF MS, which might be the result of an inadequate reference database. Some Prevotella, Staphylococcus, and Streptococcus isolates, which could not be identified with either MALDI-TOF MS or 16S rRNA gene sequencing analysis, formed clusters in the 16S rRNA phylogenic tree, and might be unknown species isolated from horses. Adding the spectra of isolates identified in this study to an in-house database might make MALDI-TOF MS a more useful tool for identifying equine isolates.

A rapid and sensitive diagnostic method is needed to help prevent the spread of equine influenza virus. The cobas Influenza A/B & RSV test for the cobas Liat system (Roche Diagnostics) is based on real-time reverse transcription polymerase chain reaction and is designed to broadly detect influenza A virus RNA within 20 minutes. It detected a broad range of equine influenza virus strains, and detected equine influenza virus RNA from nasal swabs of infected horses at the same level as real-time reverse transcription polymerase chain reaction, although it returned some invalid results (7.7%). This suggests that cobas Influenza A/B & RSV test is highly sensitive to equine influenza virus, but should be improved to reduce the rate of invalid results.

Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria,Pseudomonas aeruginosasometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolatedMyoviridaeorPodoviridaephages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused byP. aeruginosain a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis.IMPORTANCEAntibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused byPseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work hadin vitroeffectiveness for a broad range ofP. aeruginosastrains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models ofP. aeruginosakeratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

$^{195}$Pt-NMR measurements of Pt nanoparticles with a mean diameter of 4.0 nm were performed in a high magnetic field of approximately $\mu_0 H = 23.3$ T to investigate the low-temperature electronic state of the nanoparticles. The characteristic temperature $T^*$, below which the nuclear spin-lattice relaxation rate $1/T_1$ deviates from the relaxation rate of the bulk, shows a magnetic-field dependence. This dependence supports the theoretical prediction of the appearance of discrete energy levels., Comment: 6 pages, 2 figures, To appear in short notes of J. Phys. Soc. Jpn

Adrenomedullin (AM) exerts a potent anti-inflammatory effect. Intrarectal or consecutive intravenous administrations of AM reduce pathological manifestations in rodent colitis models. However, in clinical applications, a safer administration route that provides stronger alleviation of patient burden is preferred. We investigated whether subcutaneously administered AM is effective against dextran sulfate sodium (DSS)-induced colitis. C57BL/6J mice were administered 1% DSS in drinking water and received AM at 8, 40 or 80 nmol/kg subcutaneously once a day for 7 consecutive days. Subcutaneously administered AM significantly and dose-dependently ameliorated body weight loss, diarrhea, and histological severity of colonic inflammation in DSS-treated mice. The AM therapeutic effect was associated with the upregulation of the production of autocrine AM, and expression of cAMP, c-fos, KLF4, and downregulation of STAT3 and NF-κB p65 phosphorylation, as well as a decrease in proinflammatory cytokine expression in the colon. Subcutaneous AM treatment potently attenuated DSS-induced colitis, which suggests that AM administered subcutaneously in ulcerative colitis (UC) patients may decrease diseases burden and improve quality of life.

The monoclonal antibody-based latex agglutination tests targeting a high molecular weight exopolysaccharide antigen of Burkholderia pseudomallei are commercially available. The tests are primarily used in routine diagnosis of melioidosis in major hospitals in Thailand and some endemic countries. Being a rapid test, this technique was employed as a presumptive test to identify colonies of B. pseudomallei among many others grown from soil specimens collected from southern Thailand. Cross-reactivity of these tests with other soil bacteria was a concern since it complicated the identification of B. pseudomallei. Here, we describe the cross-reactivity of two commercial latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates from soil.

Microbial substitution occasionally occurs following the administration of antimicrobials to horses that have pneumonia or pleuropneumonia. Four specific loop-mediated isothermal amplification (LAMP) assays were developed to detect some equine respiratory pathogens, namely strains of the Bacteroides-Prevotella group, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Staphylococcus aureus. These four LAMP assays and two previously published LAMP assays targeting Escherichia coli or Pseudomonas aeruginosa were used on clinical respiratory specimens and a high accordance found between the results of the LAMP assays and bacterial culture. Use of these LAMP assays could enable rapid detection of pathogenic bacteria and swift administration of the appropriate antimicrobials.

We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a racehorse. A 5-year-old mare developed punctate keratitis after racing. The corneal ulcer continued to expand despite ophthalmic antimicrobial therapy. On day 6, a conjunctival graft surgery was performed. The mare was euthanized, following colitis and laminitis development on day 10. MRSA was isolated from the corneal swab taken at the time of euthanasia. Immunohistochemical analysis demonstrated gram-positive and anti-S. aureus monoclonal antibody-positive cocci infiltration of the corneal stroma; and a diagnosis of MRSA ulcerative keratitis was made. An ophthalmic antimicrobial against the isolated MRSA did not improve the ocular lesion. The MRSA strain was found to be staphylococcal cassette chromosome mec type II, a strain frequently isolated from humans in Japan.