Surveillance of Getah virus in mosquitoes and racehorses from 2016 to 2019 at a training center in Ibaraki Prefecture, Japan, a site of several previous Getah virus outbreaks.

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.

Distribution of equine coronavirus RNA in the intestinal and respiratory tracts of experimentally infected horses.

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.

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.

Capillaria hepatica (Calodium hepaticum) infection in a horse: a case report.

BACKGROUND: Capillaria hepatica is a zoonotic parasite in humans and animals and has a worldwide distribution. However, infections in mammals apart from rodents, which are natural hosts of the parasite, have rarely been reported. This report describes the first known case of C. hepatica infection in a horse in Japan. CASE PRESENTATION: A 3-year-old filly without clinical signs was presented at a slaughterhouse in Japan. Gross examination revealed white to tan nodules 0.5 to 1.5 cm in diameter in the parenchyma of the liver. Histologically, the nodules had mature fibrous capsules and consisted of multifocal to coalescing granulomatous inflammations with numerous nematode eggs. The eggs were barrel shaped with an opercular plug on each end and double-layered shells; these findings are consistent with the features of C. hepatica eggs. CONCLUSIONS: To our knowledge, this is the first case of C. hepatica infection in a horse in Japan. The pathological findings confirmed the presence of this pathogen in this part of the world, and they highlight the importance of this nematode in the differential diagnosis of hepatic granulomatous lesions in horses.

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

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.

Experimental inoculation of equine coronavirus into Japanese draft horses.

Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.

Rupture of the gastrocnemius muscle in neonatal thoroughbred foals: a report of three cases.

Rupture of the gastrocnemius muscle is occasionally found in neonatal foals associated with dystocia and assisted delivery. In this report, 3 cases of gastrocnemius muscle disruption in newborn Thoroughbred foals (6, 5 and 2 days old) are reported. In all cases, the foals were presented with inability to rise unassisted postpartum, a dropped tarsus and swelling in the caudal aspect of the thigh accompanied by a hematoma. Ultrasonography, radiography, computerized tomography (CT) and subsequent autopsy were performed to confirm the clinical and pathological features of these cases.

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.

Selenium deposition in an atypically disintegrated hoof wall in a Thoroughbred racehorse with alkali disease: Proof by energy-dispersive X-ray fluorescence analysis.

From summer 2018 to summer 2019, several Thoroughbred racehorses held at the Miho Training Centre of the Japan Racing Association inadvertently ingested excessive amounts of sodium selenite, resulting in typical chronic selenium (Se) poisoning - the so-called alkali disease. The typical abnormality was a hoof wall disorder with a circumferentially deep ring and/or transverse hoof wall cracks parallel to the coronet on all feet and appearing after excessive ingestion. One affected Thoroughbred male was unique in that all the hooves had a rough surface with a very fragile hoof wall, but no wall rings or transverse cracking. This horse was euthanized because of dysstasia due to the permanent foot pain associated with hoof wall deformities in the front feet. To detect Se deposition in the hooves, we used energy-dispersive X-ray fluorescence (EDXRF) analysis to measure the Se signal intensity of each lesion. Characteristic Se-kα signals were emitted from the areas of histologically damaged hoof wall at 33.76 ± 11.78 (mean ± SD) counts per second (cps)/mm2. In contrast, the signal from the uninjured proximal hoof wall was 1.43 ± 0.14 cps/mm2 and that from the uninjured distal hoof wall was 1.51 ± 0.23 cps/mm2. The much greater Se deposition in the injured hoof walls suggests that their disintegration was caused by alkali disease. These results indicate that atypical hoof wall abnormalities due to alkali disease can be diagnosed by EDXRF analysis.

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.

Histopathological and Virological Findings of a Penile Papilloma in a Japanese Stallion with Equus Caballus Papillomavirus 2 (EcPV2).

Equus caballus papillomavirus 2 (EcPV2) is known to cause genital neoplasms in horses. However, reports on EcPV2 in Japan and Asia are limited. Herein, we present the histopathological and virological findings of the first reported case of an EcPV2-associated penile mass in Japan. The patient was a 22-year-old stallion with a history of breeding in Japan and abroad. Histopathological examination contained RNA in situ hybridization targeting the E6/E7 region and an immunohistochemical approach, and whole-genome sequencing was conducted within the viral examination. Proliferating epidermal cells were observed, and EcPV2 E6/E7 mRNA was detected within the epidermis, which was interpreted as viral papilloma. The detected EcPV2 virus was genetically close to foreign strains and different from the strain previously reported from a Japanese mare. This suggests that various types of EcPV2 might already exist among horses in Japan. Although the mass reported herein was not malignant based on histopathological findings and the absence of recurrence, its presence on the penis would be an obstacle to breeding. These results provide a better understanding of the pathogenesis and diversity of EcPV2.

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.

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.

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.

Immunohistochemical Characteristics of Spindle Cell Ameloblastic Carcinoma in a Horse.

A 2-year-old male Thoroughbred horse presented with a mass in the maxilla. The focally ulcerated mass, approximately 8 cm in diameter, covered the upper left intermediate and corner incisor teeth (nos. 602 and 603 according to the modified Triadan system) and radiographic examination revealed displacement and lysis of the incisors. Histologically, the tumour was composed of a dense proliferation of spindle-shaped cells and neoplastic odontogenic epithelial cells arranged in island, follicular, plexiform or sheetlike patterns. The spindle-shaped cells were immunopositive for cytokeratins AE1/AE3, 5/6, 14 and 19. The Ki-67 index was 32.6% in the spindle cell component. Based on the histological and immunohistochemical findings, the tumour was diagnosed as spindle cell ameloblastic carcinoma.

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.

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.

Bone marrow oedema-type signal in the proximal phalanx of Thoroughbred racehorses.

This study focused on 8 Thoroughbred racehorses showing bone marrow oedema-type signal in the proximal sagittal groove of the proximal phalanx, with the aim of understanding its clinical significance. Standing magnetic resonance imaging played an important role in assessing osseous abnormalities that were not radiographically identifiable. Further, a histopathological result from one of the cases showed there was oedema surrounding adipose tissues with increase in density of trabecular scaffolding. This may indicate presence of osseous injury within the area of decreased elasticity due to subchondral bone modeling. This study suggests that detection of osseous abnormality based on bone marrow oedema-type signal, and application of appropriate care following injury would contribute to prevent deterioration of stress-related fractures of the proximal phalanx.

Comparison of two agar gel immunodiffusion protocols for diagnosing equine infectious anemia.

This study compared agar gel immunodiffusion (AGID) protocols for diagnosing equine infectious anemia. Two commercial testing kits were used: one following the Japanese Act on Domestic Animal Infectious Diseases Control and one following the World Organisation for Animal Health (OIE) manual. From 651 samples tested, both protocols gave identical results for 647 samples (23 samples tested positive; 624 tested negative). Non-specific reactions were observed in 21 samples testing negative by the Japanese protocol, but none were observed with the OIE protocol. The kappa coefficient value was 0.962, indicating almost perfect agreement between the two protocols. This study found no difference in diagnostic agreement between the two protocols, but the OIE protocol produced non-specific reactions less frequently than the Japanese protocol.