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Equine multinodular pulmonary fibrosis (EMPF) is a recently described form of interstitial pneumonia associated with equine herpesvirus type 5 (EHV-5). This disease has been reported in North and South America, Europe and Oceania but not, to our knowledge, in horses in Japan. We diagnosed EMPF in two Thoroughbred horses in Japan on the basis of gross and histopathological findings. In both cases, significant gross lesions, restricted to the lungs, consisted of numerous firm and coalescing nodules widely distributed throughout the lung. The nodules were3 cm in diameter and pale white to tan in colour. Microscopically, they showed severe interstitial fibrosis and infiltration of macrophages, neutrophils, lymphocytes and a few eosinophils. The residual alveoli were lined by cuboidal epithelial cells (type II pneumocytes) and filled with many macrophages, which rarely displayed oval eosinophilic to amphophilic intranuclear inclusion bodies. Polymerase chain reaction and sequence analyses identified the glycoprotein H gene of EHV-5, and in-situ hybridization detected EHV-5 in the alveolar macrophages in the lesions. In one case, electron microscopy revealed herpesvirus-like particles and EHV-5 was isolated from pulmonary lesions.

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.

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

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.

application/pdf, 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.

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.

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.

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.

Pneumocystis pneumonia is an opportunistic respiratory infection that occurs in immunocompromised animals. In horses, pneumocystic pneumonia is observed mostly in foals and often progresses rapidly. Here, we report pneumocystic pneumonia in a Thoroughbred racehorse. A 3-year-old Thoroughbred racehorse colt had marked respiratory symptoms for 3 weeks and was unresponsive to antibiotic treatment. At necropsy, firm, tan, patchy lesions were scattered diffusely in the lungs. Microscopically, alveolar septa thickened by proliferation of collagen fibers and infiltration of inflammatory cells were observed. In the alveolar spaces, many brown-black yeast-like organisms similar to cystic forms of Pneumocystis carinii were recognized by staining with Gomori's methenamine silver. Bronchoalveolar lavage fluid (BALF) obtained before necropsy included macrophages engulfing the fungus bodies. Amplified products were obtained from BALF and lung tissue samples by Pneumocystis-specific nested PCR. Phylogenetic analysis based on the 18S rRNA gene sequence revealed that the P. carinii organism from BALF was related to the Pneumocystis spp. detected in other animals and was especially close to P. carinii derived from ferrets. This is a rare case of pneumocystic pneumonia in a colt with chronic pulmonary lesions.

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.

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-de- hydrogenase (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.

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 104colony 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.

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.

spread of pathogens to other patients or surfaces. Therefore cleaning and disinfection are paramount for keeping the environment and fomites safe for animals andhumans in ahospital and field environments. Studies in humans and veterinary settings have shown that contaminated environment and fomites (stethoscopes, thermometers, etc.) are routinely found, thus posing a risk for disease transmission. Furthermore routine cleaning and disinfection has been shown to be effective in decreasing the bioburden on environmental surfaces, thus decreasing the chances of disease transmission. Practices such as improved education and training, checklists to assure that all surfaces and equipment/devises are treated and assessment of environmental cleanliness with feedback to the environmental service worker have been shown in human medicine to improve frequency of adequate cleaning by 71 to 77%. Practices like these, with the added environmental surveillance cultures for Salmonella spp.were instituted by the author with success in the past in both the field and hospital environments. Even though studies have shown that environmental cleaning and disinfection reduces pathogen loads, thesemeasures should be complementedbyother interventions that strengthen and complement it. For example monitoringof Salmonella shedderswill enhanced infection control practices byperformingamore thorough cleaninganddisinfection of high risk areas. Moreover biosecurity education of personnel involved with animal care would increase engagement and compliance, thus strengthening infectious control measures.

A 15-year-old Dutch Warmblood gelding suddenly developed incoordination and hindlimb stumbling. The horse had a history of eyelid lymphoma. Necropsy revealed yellow-white or dark reddish-brown masses adhering to the outer surface of the spinal dura mater from the first cervical vertebra to the seventh thoracic vertebra. The spinal cord close to the first cervical vertebra and the seventh thoracic vertebra was markedly compressed by the masses filling the epidural space. The masses were also observed in the larynx, eyelids, and adipose-rich tissues, including the joints and orbits. They appeared similar in shape. The mandibular, retropharyngeal, axillary, superficial inguinal, deep inguinal, and lateral iliac lymph nodes were solid and enlarged. Histologically, the masses were composed of small or medium-sized lymphocyte-like tumor cells, but atypical cells and mitotic figures were rare. There were moderate infiltrations of macrophages and multinucleated giant cells, which were occasionally ingesting the surrounding tumor cells. Immunohistochemically, the tumor cells were classified as T-cell-derived cells. Throughout the spinal cord, enlargement or loss of nerve axons, dilation of periaxonal spaces, and macrophage infiltration into periaxonal spaces were observed, mainly in the ventral funiculus. Spinal cord compression by the tumor mass was suggested as a cause of the locomotive dysfunction. This is the first report of equine lymphoma with ataxia located from the proximal cervical to middle thoracic dura mater and in joint cavities.

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.

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.

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 μg/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. (Am J Vet Res 2010;71:1062–1066)

Streptococcus zooepidemicus is a dominant pneumococcal pathogen in adult horses and comprises a wide diversity of strain genotypes. Minocycline has been used as a second-line drug for lower respiratory bacterial infection in the facilities of Japan Racing Association. The aim of this study was to reveal the relationship between genotypes of equine S. zooepidemicus and tetracycline resistance genes. Sixty-two strains of S. zooepidemicus were isolated from ill horses between 2000 and 2014, and 20 (32.3%) strains possessed one of tetracycline resistance genes, tet(M), tet(O), or tet(W). Nine of the 20 strains harboring the resistance genes were isolated in 2010–2014, of which eight had the tet(O). Interestingly, the presence of tet(O) was strongly correlated with high minimum inhibitory concentration of minocycline. Genetic diversities were widely observed in S. zooepidemicus by multilocus sequence typing, and the resistance genes were distributed across many genotypes. However, all five ST-62 strains had tet(O), and both of two ST-61 strains had tet(W), suggesting that certain genotypes are associated with particular resistance genes. These data could help us to understand the relationship between the genotypes and tetracycline resistance genes in equine S. zooepidemicus.

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.

TA and BALF obtained from 44 Thoroughbred racehorses that developed pneumonia after long-distance transportation were examined cytologically and bacteriologically. The percentages of neutrophils in both TA and BALF collected from the same horses at the same time were almost the same and were very high indicating inflammation. Various bacteria, including obligative anaerobius which are categorized as opportunistic pathogens, were isolated from BALF, and the bacteria isolated mainly in the later stages of the clinical course tended to be resistant to cephalotine and gentamicin, which are used as the first choice in the racehorse hospital of JRA where this study was performed.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.

The pathology of aortic-iliac thrombosis(AIT) in two adult Thoroughbred horses from the same breeding farm is reported. No. 1 showed sudden falls with moaning and profuse sweating at mating and during hard exercise repeatedly for more than two years. The symptoms subsided within several tens of minutes. No. 2 showed intermittent lameness of the left hind leg progressively for more than three years. In both horses, thromboembolism was observed in the abdominal aorta, terminal quadrifurcation, and branchial arteries bilaterally. Arterial intima without adherent thrombi had formed intimal plaques frequently. Other noticeable changes were parasitic aneurysm and Hemomelasma ilei. From these findings of the two cases it is suggested that AIT is one of the pathological conditions caused by Strongylus vulgaris infection.

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.

Two enzyme-linked immunosorbent assays (ELISA) with recombinant protein as antigens were evaluated by comparison with the indirect fluorescent antibody tests (IFAT) for the detection of specific antibodies to Babesia caballi and Babesia equi, respectively in 380 sera from experimentally infected, uninfected, and field horses. The high concordances of 92.4% (351/380) and 98.2% (373/380) between ELISA and IFAT for B. caballi and B. equi, respectively suggest that ELISA, especially for B. equi infection, could be alternative to the corresponding IFAT for serodiagnoses of equine piroplasmosis, although some improvements are required in ELISA for B. caballi.

Between the laminar epidermis and the laminar dermis of laminar region (LR) in equine foot, it can be observed the basement membrane zone (BMZ), which is composed of a basement membrane and its accompaniments like the hemidesmosome and anchoring fibril. Alteration in the BMZ in equine laminitis is possibly related with not only development but also recovery outcome and recurrence of this disease. However, there is little known about the structure of the BMZ during the recovery phase of this disease. To assess the condition of the BMZ of LR affected by chronic laminitis, the tissue was examined in three cases at two weeks, four weeks and three months after the onset of laminitis, using pathological, immunohistochemical and electron microscopic techniques. Histologically in all laminitis cases, there was a regenerated laminar epidermis with proliferating keratinocytes between the Stratum medium and the dermis, but it included the undeveloped secondary epidermal laminae (ud-SELs) structure in one side of the primary epidermal laminae, especially in the part of the deep area of LR. Immunohistochemical results were positive for the anti-type IV collagen, anti-type VII collagen and anti-laminin 5 antibodies in the most BMZs. However, partial BMZs adjacent to the ud-SELs were negative for the anti-type VII collagen and anti-laminin 5 antibodies. Ultrastructurally, in the BMZ of the ud-SEL, the lamina densa and the lamina lucida were present. In contrast, the anchoring fibrils and the hemidesmosomes were either absent, or present at lower than normal levels. In conclusion, the present study indicated that the part of regenerated LR in chronic laminitis was not able to fully restore to construct the BMZ for a long time, especially in the unilateral side of laminar epidermis. It might be related with recurrence of this disease.

To demonstrate the participation of the 95-kb virulence plasmid in the pathogenicity of S. Abortusequi in horses, the prevalence of the plasmid in S. Abortusequi field isolates was examined, and experimental inoculation of mice and horses was carried out using strains with and without the plasmid. Single, large plasmids were detected in 226 of the 242 field isolates (93.4%), and spvC was detected by PCR testing from all the isolates with the plasmid. Mouse virulence tests revealed that the LD50 of 11 of 13 isolates harboring the virulence plasmid was less than 104 CFU, whereas the LD50 of 12 isolates not harboring the virulence plasmid was, without exception, over 106 CFU. Experimentally, 6 horses inoculated with S. Abortusequi strains 17N1 or Sal-107 harboring the 95-kb virulence plasmid were infected, and showed similar clinical symptoms, even though Sal-107 is avirulent in IVCS mice. On the other hand, the 17N3 plasmid-cured strain did not infect horses. We conclude that the 95-kb virulence plasmid of S. Abortusequi plays a role as a pathogenic factor in mice, but is also important in horses.

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.

Seven horses were exercised five days a week for 12 months at different gaits on a treadmill, and eight horses were pastured freely in a paddock without any training programs. They were 60 days of age at the beginning of the experiment. We compared the number of C cells in the thyroid, ultrastructure of chief cells in the parathyroid and radiography of the third metatarsal bone of the training group with the control group. In the training group, the number of C cells was not increased and chief cells were not in a phase of secretion. In addition, the bone mineral content and the cortical area did not show high values. We considered that an additional exercise used in this study had suppressive effects on the thyroid and parathyroid. Our results also suggest an additional and/or intense exercise in early stage of life (foal to weanling) may have an adverse effect on bone growth and metabolism.

Six live horses with various stages of acute to chronic superficial digital flexor (SDF) tendinitis were examined using magnetic resonance imaging (MRI). In each case, MRI findings were compared to the corresponding ultrasonographic (USD) and histologic findings, to establish the usefulness of MRI. In the acute cases, lesions characterized by hemorrhage were well defined as high signal intensity on MRI and hypoechoic regions on USD. Chronic tendon fibrosis was slightly hyperechoic and difficult to distinguish from the normal tendon tissue around the original injury by using USD. In contrast, MRI visualized the chronic lesion as a low intensity signal, which could be distinguished from the black background of the normal SDF tendon tissue. This study clearly demonstrated MRI was the better imaging modality for the objective detection of chronic scar tissue in live horses. These findings, from living horses, suggest an advantage of MRI in the clinical application to diagnose tendinitis in cases where there is chronic scar tissue that is difficult to discern on USD.

A rare case of pulmonary edema due to cardiac valve obstruction has been encountered in a racehorse. It is suspected that the pulmonary edema was caused by high blood pressure in the lungs due to mitral insufficiency resulting from bacterial endocarditis. If pansystolic heart murmur and rough bronchial sounds are detected during auscultation of a horse exhibiting flu-like symptoms, pulmonary edema and mitral insufficiency should be suspected.

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.

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.

We tested the hypothesis that frequency of intense exercise during training would affect the likelihood of bucked shins complex developing in young horses. The relative prevalence of bucked shins were examined in two groups of Anglo-Arabian horses that trainined by different training programs. One group (6 horses) exercised once per week at maximal speed, the other group (7 horses) exercised twice per week at maximal speed. They ran on similar track surface conditions on the same track. Although the group exercised twice per week at maximal speed had a prevalence, there was no significant difference in the incidence of bucked shins between the two groups. It suggest that the number of maximum gallop than the duration of intense exercise. Pathomorphogenetically, there was suggestion that focal radiolucent areas, indicating focal remodeling regions consisting of a focal conglomerate of irregular-sized osteons, at the site of the middle layer in the cortical bone beneath periosteal bony proliferation might play a role as a precursor lesion for the occurrence of otitis of the third metacarpal bone (Mclll).

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons. The nucleotide sequence encoding equine CGA was determined using RT-PCR and rapid amplification of complementary DNA (cDNA) ends (RACE) techniques. A total 1,828 bp of the nucleotide sequence reveals that equine CGA is a 448-residue protein preceded by an 18-residue signal peptide. Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH2-terminal 1-77 amino acids regions (94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively) and COOH-terminal 314-430 amino acids regions (90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation (52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively). Northern blot analysis and RT-PCR elucidated the tissue distribution of equine CGA mRNA. Its expression was confirmed not only in the adrenal medullary cells but also in other organs (cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland). Further, in adrenal chromaffin cells and pituitary cells of the anterior-intermediate lobe, the expression was confirmed by in situ hybridization with anti-sense CGA cRNA probe.

The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acids. Further, the expression of Cu/Zn-SOD and Mn-SOD genes were confirmed in the equine tissues by RT-PCR and in situ hybridization.

A case of a racehorse that suddenly developed acute laminitis after participation in a race and succeeding transport was studied. The horse had shown higher γ-GT levels since the preliminary examination two months prior to the crisis until the onset of the disease. However, the horse had been trained on a regular alimentary schedule until the race, because it was free from an abnormal level of biochemical items other than γ-GT and also showed no clinical signs. These pathological characteristics included the palmarly displacement of the distal phalanx (P.III) maintaining parallelism between the hoof wall and the P.III, conspicuous proliferation of a wide range of laminar layers, marked swelling of axillary and cubital lymph nodes, and mild mononuclear cell infiltration to the portal area. Accordingly, this case was considered to be sinker laminitis induced by stress attributable to race participation and transportation, possibly with mild hepatic anomaly laid at the root of the disease.

To clarify the effects of ammonia on the respiratory tract during trailer transports, horses in stalls were exposed to either 2-17 ppm (horse No. 1), which is a level similar to the interior vehicle environment, or 40-130 ppm (horse No. 2) of gaseous ammonia for 40 hr. Clinically, coughing and hypersecretion of nasal discharge, but no significant hematological changes, were observed in the exposed horses. Morphologically, swelling of the tip of cilia in the tracheal epithelium was observed in horse No.1. In contrast, marked changes in the form of lost cilia and degenerative cytoplasmic change of the tracheal epithelium were observed in horse No.2. These histological changes suggest a direct effect of ammonia gas on the respiratory tract.

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%).

A 4-year-old female thoroughbred race horse died of acute peritonitis caused by necrotizing granulomatous duodenitis. Yersinia enterocolitica was immunohistochemically demonstrated in macrophages in granulomas developed in the duodenum, lung, liver and abdominal lymph nodes. The yeast-like fungi were found in the cytoplasmic vacuoles of macrophages in the lung that infiltrated into the granulomas and surrounding alveoli with congestive edema. The yeast-like fungi were positively stained by Gomori-Grocott chromic acid methenamine silver stain and immuno-histochemically stained with anti-histoplasma antibody. In this case, it was considered that granulomas formed in the duodenum, lung, liver and abdominal lymph nodes were primarily caused by Yersinia enterocolitica due to idiopathic weakening of the immune system. Yeast-like fungi immunohistochemically identified as histoplasmas secondarily infected the lung. This is the first case regarded as equine histoplasmosis capsulati in Japan.

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.