Molecular phylogenetic and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification of isolates from horses identified as Enterobacter cloacae by biochemical identification.

Enterobacter cloacae is an opportunistic pathogen of horses. Thirty isolates obtained from horses and their environments and identified as Enterobacter cloacae by biochemical methods were reidentified by taxonomic identification based on multilocus sequence analysis (MLSA) and by a commercial identification system based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MLSA identified the 30 equine isolates as E. ludwigii (9/30), E. asburiae (1/30), or E. cloacae (1/30); 19 isolates were not identified. The MALDI-TOF MS system could not clearly distinguish isolates to the species level, and the limited numbers of reference spectra for Enterobacter species might have contributed to the poor identification.

A novel selective medium for the isolation of Burkholderia mallei from equine specimens.

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 < 0.05). CONCLUSIONS: We believe that BM agar can be used to efficiently isolate B. mallei from mixed samples such as those typically collected from horses and other contaminated environments.

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.

Development of a loop-mediated isothermal amplification method for detecting virulent Rhodococcus equi.

Rhodococcus equi is the most important causative bacterium of severe pneumonia in foals. We report herein the development of a specific loop-mediated isothermal amplification (LAMP) assay, which targets a gene encoding vapA for detecting virulent R. equi The detection limit of the LAMP assay was 10(4) colony forming units (CFU)/mL, which was equal to 10 CFU/reaction. The clinical efficacy of the LAMP assay was compared with those of 2 published PCR-based methods: nested PCR and quantitative real-time (q)PCR. Agreements between bacterial culture, which is the gold standard for detection of R. equi, and each of the 3 molecular tests were measured by calculating a kappa coefficient. The kappa coefficients of the LAMP (0.760), nested PCR (0.583), and qPCR (0.888) indicated substantial agreement, moderate agreement, and almost perfect agreement, respectively. Although the clinical efficacy of LAMP was not the best among the 3 methods tested, LAMP could be more easily introduced into less well-equipped clinics because it does not require special equipment (such as a thermocycler) for gene amplification. Veterinary practitioners could diagnose R. equi pneumonia more quickly by using LAMP and could use the results to select an appropriate initial treatment.

Aneurysm of the cranial mesenteric artery as a site of carriage of Salmonella enterica subsp. enterica serovar Abortusequi in the horse.

Salmonella enterica subsp. enterica serovar Abortusequi is a pathogen restricted to horses. Our investigation targeted 4 draft horses (9-10 months old) kept on a Japanese farm that had suffered an outbreak of S. Abortusequi abortion. The 4 horses were suspected to be carriers of the bacterium owing to their high agglutination titers (≥1:2,560) in tube agglutination testing. The owners' on-farm observations confirmed that the horses had no apparent abnormalities, and S. Abortusequi was not isolated from their blood, rectal swabs, or sternal bone marrow fluid at antemortem investigation. However, at autopsy, all horses displayed the following: suppurative aneurysm of the cranial mesenteric artery with heavy infection with Strongylus vulgaris larvae; heavy intestinal parasitic infection with Gasterophilus intestinalis, Parascaris equorum, Anoplocephala perfoliata, and S. vulgaris; and enlargement of the systemic lymph nodes. In each case, large numbers of S. Abortusequi were isolated from the anterior mesenteric artery thrombus. The thrombus isolates harbored a single virulence plasmid, and the pulsed-field gel electrophoresis profiles of the isolates were identical not only to each other but also to those of Japanese enzootic strains of S. Abortusequi. These results reveal that parasitic aneurysms of the cranial mesenteric artery should be considered an important possible site of carriage of S. Abortusequi in horses. The results also suggest high clonality of the isolated serovar in the horse population in Japan.

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

UNLABELLED: 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.

Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis.

In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.

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.

Development of loop-mediated isothermal amplification methods for detecting Taylorella equigenitalis and Taylorella asinigenitalis.

Taylorella equigenitalis is a causative bacterium of contagious equine metritis (CEM), and Taylorella asinigenitalis is species belonging to genus Taylorella. The authors developed two loop-mediated isothermal amplification (LAMP) methods, Te-LAMP and Ta-LAMP, for detecting T. equigenitalis and T. asinigenitalis, respectively. Using experimentally spiked samples, Te-LAMP was as sensitive as a published semi-nested PCR method, and Ta-LAMP was more sensitive than conventional PCR. Multiplex LAMP worked well without nonspecific reactions, and the analytical sensitivities of multiplex LAMP in the spiked samples were almost equivalent to those of Te-LAMP and Ta-LAMP. Therefore, the LAMP methods are considered useful tools to detect T. equigenitalis and/or T. asinigenitalis, and preventive measures will be rapidly implemented if the occurrence of CEM is confirmed by the LAMP methods.

A clinical case of equine fungal placentitis with reference to hormone profiles and ultrasonography.

Fungal placentitis is an infectious disease inducing abortion in pregnant mares. In the present report, we describe a field case of abortion caused by fungal placentitis with consecutive examinations. The progesterone level and combined thickness of the uterus and placenta (CTUP) were abnormal before the onset of clinical signs. Additionally, the estradiol level started to change before the appearance of clinical signs. Abnormal serum amyloid A values and an abnormal fetal heart rate were observed after the onset of clinical signs. The present report demonstrates that the progesterone level and CTUP may be adequate as early diagnostic markers of fungal placentitis and bacterial infection. Endocrinological evaluation based on cutoff values or serial measurements were also useful for early diagnosis.

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.

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.

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 × 10(4) CFU/ml) of S. zooepidemicus.

Dominant obligate anaerobes revealed in lower respiratory tract infection in horses by 16S rRNA gene sequencing.

Obligate anaerobes are important etiological agents in pneumonia or pleuropneumonia in horses, because they are isolated more commonly from ill horses that have died or been euthanized than from those that survive. We performed bacterial identification and antimicrobial susceptibility testing for obligate anaerobes to establish effective antimicrobial therapy. We used 16S rRNA gene sequencing to identify 58 obligate anaerobes and compared the results with those from a phenotypic identification kit. The identification results of 16S rRNA gene sequencing were more reliable than those of the commercial kit. We concluded that genera Bacteroides and Prevotella-especially B. fragilis and P. heparinolytica-are dominant anaerobes in lower respiratory tract infection in horses; these organisms were susceptible to metronidazole, imipenem and clindamycin.

Time-related Pathological Changes in Horses Experimentally Inoculated with Equine Influenza A Virus.

To investigate the pathology of equine influenza, necropsy of 7 horses experimentally infected with equine influenza A virus (EIV) subtype H3N8 was conducted on post-infection days (PID) 2, 3, 7, and 14. Histopathologically, rhinitis or tracheitis including epithelial degeneration or necrosis with loss of ciliated epithelia and a reduction in goblet cell numbers, was observed in the respiratory tracts on PIDs 2 and 3. Epithelial hyperplasia or squamous metaplasia and suppurative bronchopneumonia with proliferation of type II pneumocytes were observed on PIDs 7 and 14. Viral antigen was detected immunohistochemically in the epithelia of the nasal mucosa, trachea, and bronchi on PIDs 2 and 3. The sodA gene of Streptococcus equi subsp. zooepidemicus, a suspected cause of suppurative bronchopneumonia, was detected in paraffin-embedded lung tissue sections, but only on PIDs 7 and 14. These findings suggest that damage caused to ciliated epithelia and goblet cells by EIV infection results in secondary bacterial bronchopneumonia due to a reduction in mucociliary clearance.

Prevalence of equine herpesvirus type 1 strains of neuropathogenic genotype in a major breeding area of Japan.

A single non-synonymous nucleotide substitution of guanine (G) for adenine (A) at position 2254 in the viral DNA polymerase gene (encoded by open reading frame [ORF] 30) of equine herpesvirus type 1 (EHV-1) has been significantly associated with neuropathogenic potential in strains of this virus. To estimate the prevalence of EHV-1 strains with the neuropathogenic genotype (ORF30 G(2254)) in the Hidaka district--a major horse breeding area in Japan--we analyzed the ORF30 genomic region in cases of EHV-1 infection in this area during the years 2001-2010. Of the 113 cases analyzed, 3 (2.7%) were induced by ORF30 G(2254) strains. This prevalence is lower than those observed in the U.S.A. (10.8-19.4%), Argentina (7.4%), France (24%), and Germany (10.6%).

Mortierella wolfii keratomycosis in a horse.

PURPOSE: To describe a case of superficial keratomycosis caused by Mortierella wolfii (M. wolfii) in a horse. METHODS: A thoroughbred filly was presented with painful right eye of 2 days' duration. A superficial corneal ulcer was observed ventrally together with multifocal punctuate opacities axially. Samples were collected by swabbing and scraping the ulcerated lesion and submitted for microbiologic and cytologic examination. RESULTS: Microscopic evaluation of debrided corneal tissue revealed the presence of nonseptate fungal hyphae, and culture of a corneal swab yielded fungal growth. Medical treatment with topical antifungal, antibiotic and autogenous serum and systemic anti-inflammatory resolved the problem within 2 weeks. CONCLUSIONS: Cytologic evaluation of a corneal scraping was useful to make a clinical diagnosis of keratomycosis. Based on the mycological characteristics, the fungus isolated from the corneal lesion was identified as M. wolfii. To the authors' knowledge, this is the first case report of equine keratomycosis associated with this fungus, although the organism is known to infect various organs of cattle.

Distribution of influenza virus sialoreceptors on upper and lower respiratory tract in horses and dogs.

It is strongly suspected that equine influenza virus (EIV) is the origin of canine influenza virus (CIV, H3N8), which was first isolated in U.S.A. in 2004, on the basis of phylogenetic analyses. Although the distribution of influenza virus sialoreceptors seems to be associated with this interspecies transmission, there have been scant data of comparison about distributions of sialoreceptors on the whole respiratory tract between horses and dogs. We examined the histological distribution of influenza virus sialoreceptors on the upper and lower respiratory tract in detail in both animals using double lectin staining with Maackia amurensis (specific for SAα2,3Gal) and Sambucus sieboldiana (specific for SAα2,6Gal). SAα2,3Gal was observed on the surface of ciliated epithelial cells in the nasal mucosa, trachea and bronchus in both animals. The results may indicate that dogs are susceptible to EIV without alteration of receptor binding specificity.

Pharmacokinetics and tissue distribution of minocycline hydrochloride in horses.

OBJECTIVE: To determine the pharmacokinetics and tissue distribution of minocycline in horses. ANIMALS: 5 healthy Thoroughbred mares for the pharmacokinetic experiment and 6 healthy Thoroughbred mares for the tissue distribution experiment. PROCEDURES: Each mare was given 2.2 mg of minocycline hydrochloride/kg, IV. Blood samples were collected once before minocycline administration (0 hours) and 10 times within 48 hours after administration in the pharmacokinetics study, and 24 tissue samples were obtained at 0.5 and 3 hours in the distribution study. RESULTS: No adverse effects were observed in any of the mares after minocycline administration. The mean+/-SD elimination half-life was 7.70+/-1.91 hours. The total body clearance was 0.16+/-0.04 L/h/kg, and the volume of distribution at steady state was 1.53+/-0.09 L/kg. The percentage of plasma protein binding was 68.1+/-2.6%. Plasma concentration of free minocycline was 0.12 microg/mL at 12 hours. Minocycline was not detected in brain tissue, CSF or aqueous humor at 0.5 hours; however, it was found in all tissues, except in the aqueous humor, at 3 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Clearance of minocycline in healthy mares was greater than that reported for humans. For effective treatment of infections with common equine pathogens, it will be necessary to administer minocycline at a dosage of 2.2 mg/kg, IV, every 12 hours. This drug could be useful for infections in many tissues, including the CNS. The pharmacokinetic and tissue distribution data should aid in the appropriate use of minocycline in horses.

Histopathological characteristics of an ossifying fibroma formed in the maxilla of a racehorse.

A 1-year-old male thoroughbred racehorse experienced swelling of the left upper lip. The swelling was attributable to enlargement around the incisive bone of the interdental space posterior to the third incisor in the left maxilla. Even after two operations to reduce the bulk of the mass, it continued to increase in size. Dyspnea caused by stenosis of the nasal cavity forced us to perform euthanasia, and a pathological examination was conducted. Macroscopic examination of a section of the mass revealed the formation of multiple areas of solid fibrous tissue, and trabeculae within the incisive bone which had displaced the cortical bone. On histology, the mass was composed of trabecular bone-like structures due to the proliferation and aggregation of fibroblasts. Therefore, we diagnosed it as an ossifying fibroma. Equine ossifying fibroma is characterized by development in the mandible, but was formed in the maxilla in this case. Equine ossifying fibroma has not been reported previously in Japan. This is the first case of equine ossifying fibroma identified in Japan.

Antibody responses induced by experimental West Nile virus infection with or without previous immunization with inactivated Japanese encephalitis vaccine in horses.

A group of horses immunized with inactivated Japanese encephalitis (JE) vaccine (JE-Immune Group) and a group of non-immunized horses (Non-Immune Group) were infected with West Nile virus (WNV). After WNV infection, neutralizing (Nt) antibody (Ab) titers to WNV were higher than those to JE virus (JEV) in the Non-Immune Group, but the NtAb titers to JEV were higher than those to WNV during most of the post-challenge observation period in the JE-Immune Group. Immunoglobulin M (IgM) Abs to WNV tested positive in the Non-Immune Group but negative in the JE-Immune Group, except for in one horse. These results suggest that diagnosis of WNV infection in JE-immunized horses requires serological tests for NtAb and IgM titers to both WNV and JEV.