Hidekazu Niwa

Ulcerative keratitis in thoroughbred racehorses in Japan from 1997 to 2008

OBJECTIVE To describe the number of cases, etiologies, healing times, and results of microbial culture and susceptibility testing of corneal ulcers in thoroughbred racehorses in Japan. PROCEDURE Retrospective study of the medical records of racehorses belonging to the Japan Racing Association (JRA) from 1997 to 2008. RESULTS A total of 2846 horses suffered ulcerative keratitis. These horses accounted for 90.5% of all the corneal problems and 54.9% of the entire number of horses with ocular diseases. Running in races was the cause in 64.3% of the cases. The mean healing time of the ulcers developed in races was 5.5 + or - 9.6 days, which was shorter than that of the ulcers unrelated to racing (8.6 + or - 11.7 days). In horses presented for examination by the next day after the race, healing was even more rapid (4.1 + or - 7.5 days) than in horses presented later (12.4 + or - 14.7 days). Microbial culture was performed on 74 samples of 58 horses. Forty-four bacterial and four fungal organisms were isolated from 35 samples of 29 horses. Pseudomonas aeruginosa (10) and Streptococcus equi subspecies zooepidemicus (5) were the bacteria most frequently isolated. There was no tendency of increasing antibiotic resistance of these organisms. CONCLUSIONS Ulcerative keratitis is the most frequent corneal and ocular disease of the racehorse in Japan. Careful observation of the eyes after racing is necessary as early diagnosis and treatment of corneal ulcers speeds healing. Many antibiotic have efficacy against the bacterial isolates, however, potent antibiotics should be reserved for the most severe corneal ulcers.

Equine keratomycosis in Japan.

OBJECTIVE To describe the incidence, clinical progress, visual outcome, and laboratory findings of equine keratomycosis in Japan. PROCEDURE  Retrospective study of the medical records of horses clinically and mycologically diagnosed with keratomycosis at the Equine Hospitals of the Japan Racing Association from 2005 to 2011. RESULTS The diagnosis of keratomycosis was confirmed in eight horses (40.0% of the 20 horses with infectious keratitis from which fungi and/or bacteria were isolated). Fungi recovered from corneal swabs were identified as Aspergillus flavus (4), Aspergillus niger (1), Fusarium solani (1), and Mortierella wolfii (2). All horses were treated medically with topical antifungals, and one horse was also treated surgically. The median of treatment period was 40 days. Two horses were rendered blind in the affected eye and the others retained vision. CONCLUSIONS Equine keratomycosis comprises a considerable portion of infectious keratitis in Japan, and the causative fungi that we isolated had been isolated previously from horses with keratomycosis in other regions with the exception of M. wolfii. Culture and cytological examination of corneal lesions should be immediately performed on eyes with signs of keratitis, particularly on those not improving with antibacterial medication, as early initiation of aggressive antifungal treatment tended to result in better outcome and shorter treatment period.

Development of a Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus equi subsp. zooepidemicus and Analysis of Its Use with Three Simple Methods of Extracting DNA from Equine Respiratory Tract Specimens

ABSTRACT Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 104 CFU/ml) of S. zooepidemicus.

No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8)

BackgroundSince equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission.MethodsThree pairs of animals consisting of a dog inoculated with CIV (108.3 egg infectious dose50/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia.ResultsAll three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses.ConclusionsThese findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.

Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification

Abstract A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of equine coronavirus (ECoV). This assay was conducted at 60°C for 40min. Specificity of the RT-LAMP assay was confirmed using several equine intestinal and respiratory pathogens in addition to ECoV. The novel assay failed to cross-react with the other pathogens tested, suggesting it is highly specific for ECoV. Using artificially synthesized ECoV RNA, the 50% detection limit of the RT-LAMP assay was 101.8 copies/reaction. This is a 50-fold greater sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) assays, but a 4-fold lower sensitivity than quantitative RT-PCR (qRT-PCR) assays. Eighty-two fecal samples collected during ECoV outbreaks were analyzed. ECoV was detected in 59 samples using the RT-LAMP assay, and in 30 and 65 samples using RT-PCR or qRT-PCR assays, respectively. Although the RT-LAMP assay is less sensitive than qRT-PCR techniques, it can be performed without the need for expensive equipment. Thus, the RT-LAMP assay might be suitable for large-scale surveillance and diagnosis of ECoV infection in laboratories with limited resources.

Methicillin-resistant Staphylococcus aureus ulcerative keratitis in a Thoroughbred racehorse.

ABSTRACT 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.

Postoperative Clostridium difficile infection with PCR ribotype 078 strain identified at necropsy in five Thoroughbred racehorses

Clostridium difficile is an important cause of acute enterocolitis in horses. We describe five cases of C difficile infection occurring postoperatively in Thoroughbred racehorses. Following diarrhoea or colic accompanied by a marked increase in packed cell volume (to ≥60 per cent) and leucopenia (≤4000 cells/μl) within two to four days after surgery in all five horses, four of them died or were euthanased because of colitis or severe diarrhoea. In these four horses, necrotising entero-typhlo-colitis was revealed by postmortem examination, and C difficile was recovered from the contents of the small and/or large intestine. The remaining horse was euthanased because of marked decline in general condition and the presence of a lung abscess, from which C difficile was isolated. The horse had had severe postoperative diarrhoea before the onset of respiratory disorder; laboratory tests for C difficile were not performed on the faeces. All C difficile isolates were toxin-A-positive, toxin-B-positive and actin-specific ADP-ribosyltransferase (CDT)-positive. The isolates were indistinguishable by pulsed field gel electrophoresis analysis, PCR ribotyping, and slpA sequence typing, and the slpA sequences and PCR ribotype patterns were identical to those of known PCR type 078. This case sequence might have been healthcare-associated infection, although there was about a four-month interval between each disease onset.

Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis

ABSTRACT Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes 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 isolated Myoviridae or Podoviridae phages, 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 by P. aeruginosa in 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. IMPORTANCE Antibiotic-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 by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad range of P. aeruginosa strains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models of P. aeruginosa keratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Use of loop-mediated isothermal amplification to detect six groups of pathogens causing secondary lower respiratory bacterial infections in horses.

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.

Meticillin-resistant Staphylococcus aureus colonisation and infection in Thoroughbred racehorses and veterinarians in Japan

Meticillin-resistant Staphylococcus aureus (MRSA) infections have been confirmed in hospitalised Thoroughbred racehorses at the hospitals of two training centres in Japan since 2009. To investigate the source of infection, the authors examined the rate of nasal MRSA colonisation in 600 healthy Thoroughbred racehorses, 53 veterinarians and 16 office staff at the racehorse hospitals of the two training centres. MRSA was not isolated from healthy Thoroughbred racehorses or hospital office staff. However, MRSA was isolated from 16 veterinarians (30.1 per cent), and the colonisation rate was significantly higher in veterinarians than in the office staff of the same hospitals. Also, 10 of the 16 MRSA strains (62.5 per cent) isolated from veterinarians were classified as type II by staphylococcal cassette chromosome mec (SCCmec) typing and ST5 by multilocus sequence typing. Pulsed-field gel electrophoresis analysis demonstrated that these 10 MRSA strains of SCCmec type II and ST5 were genetically identical or very similar to 9 MRSA strains isolated from infected horses hospitalised at these hospitals between 2009 and 2013. These results indicate that SCCmec type II and ST5 MRSA strains were probably transmitted between veterinarians and infected horses.