Genetic analysis based on next generation sequencing of Streptococcus equi subsp. equi isolated from horses imported into Japan.

Strangles is a globally widespread, commonly diagnosed and important infectious disease of equids caused by Streptococcus equi subsp. equi. We performed whole genome sequencing of 19 S. equi isolates collected from imported horses at the Japanese border. Of these isolates, 15 isolates were obtained from clinical cases and 4 were from subclinical cases. The 19 isolates were grouped into 3 Bayesian analysis of population structure (BAPS) groups by the core genome single nucleotide polymorphism analysis corresponding to exporting country, SeM typing, or exporter of the horses. The 19 isolates possessed same pathogenic genes regardless of clinical status in imported horses and no antimicrobial resistance genes. The disease status of the horses may rather reflect the prior exposure of animals with sub-clinical infection to S. equi.

Pharmacokinetics/pharmacodynamics cut-off determination for fosfomycin using Monte Carlo simulation in healthy horses.

Fosfomycin (FOM) is an approved veterinary medicinal product for large animals in Japan, but Clinical breakpoint (CBP) for antimicrobial susceptibility test (AST) is not defined for animals. This study aimed at conducting a pharmacokinetics/pharmacodynamics (PK/PD) analysis to determine the PK/PD cutoff for the CBP in horses. Drug concentrations following single intravenous administration (IV) of 20 mg/kg body weight (BW) FOM in nine horses were measured using liquid chromatography/mass spectrometry. The data were modelled using a nonlinear mixed-effects model, followed by Monte Carlo simulations. A 90% probability of target attainment for a PK/PD target of the ratio of Area Under the free plasma concentration-time curve divided by the minimal inhibitory concentration (MIC) >24 hr was set as PK/PD cut-off. The PK/PD cutoff for FOM 20 mg/kg BW q12 hr IV was estimated with the MIC value of ≤16.0 mg/L, and this regimen was considered effective against E. coli (MIC90; 16.0 mg/L) in healthy horses based on the MIC90 values of the wild population. Owing to the relevance of FOM to human health, veterinarians should use q 12 hr FOM 20 mg /kg against E. coli infections with an MIC <16 µg/mL, as suggested by our PK/PD cutoff after AST.

A real-time PCR assay for the quantification of Mycoplasma equirhinis in tracheal wash samples from Thoroughbred horses.

Mycoplasma equirhinis is the predominant equine Mycoplasma sp. isolated from clinically normal horses and is suspected to be associated with inflammatory airway disease in which cough is the primary sign. Quantitative evaluation of bacterial counts is useful in assessing the association between the bacteria in samples and observed clinical signs, but this evaluation has been difficult with conventional culture methods of M. equirhinis given the need for pre-enrichment using liquid cultures. We established a quantitative real-time PCR (qPCR) assay for the quantification of M. equirhinis, targeting the hypothetical protein FJM08_00025. We confirmed its high species-specificity for M. equirhinis and a limit of detection of 2.9 copies/reaction. We quantified M. equirhinis in tracheal wash samples from 20 clinically normal horses and 22 coughing horses. The copy numbers detected by qPCR in 18 of the 22 samples from clinically affected horses were within the range detected in the 20 clinically normal horses (0-84 copies/reaction). The remaining 4 samples had considerably higher copy numbers (734-1,620,000 copies/reaction), suggesting the likely involvement of M. equirhinis infection. Quantitative evaluation of M. equirhinis over time using our qPCR assay may allow a more accurate assessment of M. equirhinis infection in coughing horses compared to culture methods.

Bacterial and fungal isolates from 107 cases of ulcerative keratitis in Japanese Thoroughbred racehorses (2017-2021).

Infectious ulcerative keratitis is a common disease in racehorses. To improve treatment outcomes, this study aimed to assess the antimicrobial susceptibilities of bacterial and fungal isolates obtained from the cornea of Japanese Thoroughbred racehorses with equine infectious ulcerative keratitis. Bacterial and fungal cultures were performed for 166 corneal swabs from 107 cases. A disc diffusion test and minimum inhibitory concentration test were also performed to assess antimicrobial susceptibility of the bacterial and fungal isolates, respectively. Bacterial and/or fungal isolates were obtained from 85.0% (91/107) of the cases. Staphylococcus was primarily isolated from bacterial isolates, including methicillin-resistant Staphylococcus aureus (MRSA), Aerococcus, Streptococcus, Acinetobacter, and Pseudomonas. Aspergillus was primarily isolated from filamentous fungi, and Debaryomyces species was primarily identified in yeast-like fungi. Ofloxacin resistance was observed in 100% (12/12), 15.9% (7/44), and 25.0% (3/12) of MRSA, Staphylococcus, and Streptococcus isolates, respectively. The prevalence of quinolone-resistant Staphylococci and Streptococci has increased in the past two decades. All Aspergillus isolates were susceptible to voriconazole, whereas other filamentous fungi, including Fusarium, were less susceptible to voriconazole. Further studies are required to determine effective treatments for antimicrobial-resistant isolates.

Incidence of surgical site infection after internal fixation of the first phalangeal bone and the third metacarpal/metatarsal bone fractures in Thoroughbred racehorses.

Surgical site infection (SSI) is one of the major complications of equine fracture surgery. The purpose of this study was to investigate the incidence of and risk factors for SSI after internal fixation of the first phalangeal bone (P1) and the third metacarpal/metatarsal bone (MC3/MT3) fractures in Thoroughbred racehorses. Between 2011 and 2020, 451 cases underwent surgery with screws or a locking compression plate (LCP) for sagittal fractures of P1 or condylar fractures of MC3/MT3. Overall, 2.9% (13/451) of the cases developed an SSI. The incidence was significantly higher in plate fixation (21.4%) than in screw fixation (2.3%). There was no significant association with other variables, such as sex, age, number of screws, experience of surgeon, or prophylactic antimicrobials. The median duration of hospitalization for screw fixation was 14 days without an SSI and 20 days with an SSI, and those for plate fixation were 26 and 25-88 days, respectively, indicating that the development of SSI prolongs the duration of hospitalization. On the other hand, there were no significant differences in discharge and race resumption rates between cases with and without an SSI. These data indicate that the incidence of SSI in this study was low and that it was higher following plate fixation than screw fixation.

Clostridioides difficile infection in thoroughbred horses in Japan from 2010 to 2021.

We encountered 34 Clostridioides difficile (C. difficile) infection (CDI) cases among Thoroughbred horses in Japan from 2010 to 2021. Among them, 79.4% (27/34) either died or were euthanised. The risk factors associated with CDI and mortality among Japanese Thoroughbred horses remain unclear. We used genetic methods to examine C. difficile strains and their relationships with prognosis. Twenty-two (64.7%) cases were hospitalised at the onset of colitis. Outcomes were balanced for hospitalisation rates at the onset of colitis. The mortality rates of cases treated with metronidazole (65.0%) were significantly lower than untreated cases (100%). The predominant genotype of C. difficile isolate was polymerase chain reaction ribotype (RT) 078, isolated from 12 cases (35.3%), followed by RT014 (six cases, 17.6%). Binary toxin (C. difficile transferase [CDT])-positive strains, including all RT078 strains, were isolated from 16 horses. Mortality rates in RT078 strain (75.0%) or CDT-positive strain (83.3%) cases were comparable to that in cases of other types. Sufficient infection control is needed to prevent CDI in Thoroughbred horses. A timely and prompt CDI diagnosis leading to metronidazole treatment would improve CDI outcomes.

Assessment of tetanus revaccination regimens in horses not vaccinated in the previous year.

A two-dose revaccination against tetanus is recommended for horses over 2 years old in Japan with no history of vaccination in the previous year. Here, the need for two-dose revaccination was evaluated in terms of antibody titers for each vaccine type, namely monovalent or multivalent. There was no difference in antibody titers between one- and two-dose regimens for up to 1 year, except at 8 weeks with the multivalent vaccine, and all horses had sufficient antibody titers for 1 year of tetanus prophylaxis. These results suggest that one-dose revaccination, regardless of the vaccine type, is as effective as two-dose in preventing tetanus for at least 1 year in horses not vaccinated in the previous year.

Pharmacokinetic/pharmacodynamic analysis of cephalothin after intramuscular administration in Thoroughbred horses.

A pharmacokinetic/pharmacodynamic (PK/PD) approach was used to determine a dosage regimen of cephalothin (CET) after intramuscular (IM) administration in horses. CET plasma concentrations were measured in eight horses after a single IM administration of 11 mg/kg bwt of CET. The data were modeled using a nonlinear mixed-effect model, and the probability of target attainment (PTA) of the PK/PD target was calculated for 5,000 horses generated by Monte Carlo simulations. IM administrations of CET at 11 mg/kg bwt q 8 hr and q 6 hr achieved a PTA of 90% against the MIC90 of S. zooepidemicus and S. aureus, respectively, and were considered to be effective dosage regimens. The total dose for the IM administration recommended in this study was lower than that for intravenous (IV) administration in previous studies.

Concentration of cephalothin in body fluids and tissue samples of Thoroughbred horses.

Cephalothin (CET) concentrations in body fluids (plasma, synovial fluid, pleural fluid, peritoneal fluid, and aqueous humor) and tissue samples (bone, lung, jejunum, hoof, and subcutaneous tissue) were investigated to consider the treatment of infectious diseases in horses. CET 22 mg/kg body weight was intravenously administered to 12 horses. Samples were collected from four different horses at 1, 3, and 5 hr after administration. The CET concentration in body fluids other than aqueous humor was maintained above the MIC90 values of Streptococcus zooepidemicus and Staphylococcus aureus until 5 hr, but it was not maintained above that of S. aureus in bone. CET (22 mg/kg twice a day) is effective for septic arthritis, pleuritis, and peritonitis caused by gram-positive bacteria but ineffective for osteomyelitis.

Outbreak of methicillin-resistant Staphylococcus aureus sequence type 1, spa type t1784, in an equine hospital in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) has often been isolated from livestock and companion animals, including horses. Seven cases of MRSA infection in Thoroughbred racehorses were observed in an equine hospital in Japan in 2020. In this study, MRSA isolates from these seven horses and nine veterinarians in the equine hospital were studied to examine their genetic relatedness and evaluate the possibility of MRSA transmission. The MRSA isolates were subjected to whole-genome sequencing for multi-locus sequence typing, S. aureus protein A (spa) typing, staphylococcal cassette chromosome typing, and antimicrobial resistance gene detection. Minimum inhibitory concentrations of antibiotics were assessed to determine the antimicrobial susceptibility phenotype of the isolates. Phylogenetic trees based on single nucleotide polymorphisms were constructed to identify genetically close isolates. All isolates from horses and veterinarians belonged to sequence type (ST) 1, spa type t1784, with a point mutation in gyrA and double point mutations in grlA, which is known to cause fluoroquinolone resistance. All ST1-t1784 isolates were genetically closely related based on the phylogenetic tree. Our results suggested an outbreak and horse-veterinarian transmission of ST1-t1784 strains in an equine hospital.

Simultaneous Daily Fecal Microbiota Transplantation Fails to Prevent Metronidazole-Induced Dysbiosis of Equine Gut Microbiota.

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.

Comparison of seven nucleic acid amplification tests for detection of Taylorella equigenitalis.

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.

Prevalence of equine proliferative enteropathy in Hidaka district, Hokkaido, over five seasons.

Equine proliferative enteropathy (EPE) is an equine infectious disease that can lead to severe weight loss and hyperplasia of the intestinal mucosa due to infection with Lawsonia intracellularis. In this study, we investigated the prevalence of EPE in a major Thoroughbred breeding area: Hidaka district, Hokkaido, Japan. Of the 252 symptomatic horses that we tested, 192 EPE cases (76.2%), including 8 fatal cases, were confirmed from April 2015 to March 2020 by etiological and/or serological investigation. Most of the EPE cases were observed in foals (88.5%), with fewer cases in yearlings (7.3%) and adults (4.2%). Asymptomatic infection was observed in 62.9% of the horses kept with affected horses. These results suggest that EPE is an enzootic disease in Hidaka district.

High prevalence of Mycoplasma equirhinis in Thoroughbred horses with respiratory symptoms in autumn 2018.

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.

Construction and Application of an In-House Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Database Specific to Bacteria From Horses.

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.

Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome.

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.

Antifungal susceptibility of dermatophytes from racehorses in Japan.

BACKGROUND: Luliconazole (LCZ) is an imidazole antifungal medication that exhibits excellent activity against dermatophytes. As a topical cream and lotion (approved for human use), LCZ has demonstrated a broad spectrum of activity against human dermatophytoses. OBJECTIVES: This is the first study to investigate the in vitro susceptibility of clinical isolates from horse dermatophytoses to LCZ. ANIMALS: No animals were used in this study. METHODS AND MATERIALS: In the present study, the in vitro susceptibilities of clinical isolates of dermatophytes to LCZ, clotrimazole (CTZ), miconazole (MCZ) and terbinafine (TRF) were investigated using the Clinical & Laboratory Standards Institute M38-A2 test. RESULTS: The minimum inhibitory concentrations (MICs) for all 16 clinical isolates of Trichophyton equinum, Microsporum equinum/canis and M. gypseum for LCZ were <0.03 mg/L. The MICs of all isolates were <0.03-0.5 mg/L for CTZ, 0.03-16 mg/L for MCZ and <0.03-1 mg/L for TRF. CONCLUSIONS: LCZ demonstrated a broad spectrum of activity against clinical isolates from horse dermatophytoses. We consider that LCZ will become the primary antifungal agent for treating horse dermatophytosis.

The Clonal Population of Trichophyton equinum from Dermatophytoses of Japanese Racehorses.

Trichophyton equinum is a zoophilic dermatophyte that is frequently isolated from horse dermatophytosis and rare infections in humans. In the present study, molecular and physiological testing were performed on T. equinum isolates from dermatophytoses of Japanese racehorses to assess genotype and phenotype patterns of these strains. Comparative nucleotide sequence analysis showed that internal transcribed spacer (ITS) region sequences amplified from all Japanese isolates were 99.5% identical to T. equinum reference strains. ITS sequences amplified among the isolates were 100% (BT2) showed that isolates were 100% identical and harbored a "T" single nucleotide polymorphism at position 18. The sequences of ß-tubulin (BT2) showed that isolates were 100% identical to T. equinum reference strains. The MAT1-2 allele was detected by PCR in all seven isolates, whereas none of the isolates contained the MAT1-1 allele. All isolates grew only on Trichophyton Agar 5 and did not grow on Trichophyton Agar 1 and 4, indicating nicotinic acid requirement. These results suggest that Japanese T. equinum isolates are derived from a clonal population.

First isolation of Trichophyton bullosum from a horse with dermatophytosis in Japan.

Trichophyton bullosum is a zoophilic dermatophyte that has been rarely isolated from horses and humans in Africa and Europe. This is the first reported isolation of T. bullosum from a horse with dermatophytosis in Japan. The isolate from a skin lesion formed a cream-colored and waxy colony that was slightly elevated in the center. Sequencing of the internal transcribe spacer region of the isolate revealed that it was 100% identical to that of T. bullosum.

Comparison of the proteomes in sera between healthy Thoroughbreds and Thoroughbreds with respiratory disease associated with transport using mass spectrometry-based proteomics.

In the past decade, mass spectrometry has become an important technology for protein identification. Recent developments in mass spectrometry allow a large number of identifications in samples; therefore, mass-spectrometry-based techniques have been applied to the discovery of biomarkers. Here, we conducted a proteomic study to compare the proteomes in sera between healthy Thoroughbreds and Thoroughbreds with respiratory disease associated with transport (RDT). We found that four proteins, apolipoprotein F, lipopolysaccharide binding protein, lysozyme and protein S100-A8, were upregulated, while keratin 1 was downregulated in the RDT group. It is assumed that inflammation and immune response are involved in the changes of these proteins. The findings suggested that these proteins are potentially useful for elucidating the mechanism of development of RDT.