Contagious equine metritis eradicated from Japan.

Contagious equine metritis (CEM), a contagious venereal disease of horses, invaded Japan in 1980 and spread in the Thoroughbred population of the Hidaka-Iburi district of Hokkaido. To eradicate CEM, we ran a program aimed at detecting Taylorella equigenitalis, the causal agent, in carrier horses by using the PCR test, followed by culling or treatment. In 2001, the first year of the program, 12,356 Thoroughbred racing stallions and mares were tested and 11 carriers were found. Four, two, one, and one carrier mares were detected in 2002, 2003, 2004, and 2005, respectively, by application of the program at the same scale as in 2001. No PCR-positive horses were found from 2006 to 2010. These results strongly suggest that CEM was eradicated from Japan by 2010.

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.

Changes in Serum Antibody Levels after Vaccination for Strangles and after Intranasal Challenge with Streptococcus equi subsp. equi in Horses.

In this study, to evaluate the influence of strangles vaccination on serological test results, we investigated the changes in strangles serum antibody levels in horses after vaccination and subsequent intranasal challenge with S. equi. The horses were vaccinated for strangles with either a component vaccine (Group C) or a live vaccine (Group L). We measured changes in strangles serum antibody levels weekly for 20 weeks after vaccinating horses twice for strangles over a 3-week interval, and for 7 weeks after intranasal challenge with S. equi in the same horses. Serum antibody responses to the proline-glutamic acid-proline-lysine (PEPK) antigen with five repetitions (PEPK-5R) were higher at all times (up to 2.4-fold) following vaccination in Group C than in Group L, and the value peaked at 2.9-fold above the initial value after the second vaccination in Group C horses. However, the value was lower than that in horses infected with S. equi, and it gradually decreased, reaching the initial (week 0) value by the 15th week. Serum antibody responses to PEPK-5R after challenge with S. equi increased in both groups of horses, but the value tended to be lower than that reported for unvaccinated horses. In addition, the average value in Group C was 2.6-fold higher than that of Group L. These results suggest the serum antibody responses of horses infected with S. equi varies according to the type of vaccine with which they have been vaccinated. Although the serological diagnostic test for strangles in which PEPK-5R is used as an antigen is effective for the investigation of serum antibodies to strangles in vaccinated horses, the present data suggest it is necessary to consider the vaccination history when interpreting the results.

Antimicrobial resistance and genetic characteristics of Salmonella Typhimurium isolated from horses in Hokkaido, Japan.

In this study, we examined the antimicrobial susceptibility of 16 Salmonella Typhimurium isolates obtained from horses, and applied several genetic methods, namely polymerase chain reaction (PCR) for detecting class 1 integrons, multiplex PCR for detecting multidrug resistant S. Typhimurium definitive phage type 104 (MR-DT104), and fluorescent amplified-fragment length polymorphism (FAFLP). Seven isolates with an ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline (ACSSuT) type resistance pattern, harbored two class 1 integrons with sizes of 1.2 and 1.0 kb, and were identified as DT104 by bacteriophage typing. These isolates also showed a typical MR-DT104 amplification pattern, which was positive for flo(st), spvC, invA and int, in multiplex PCR. In the FAFLP analysis, the equine DT104 isolates and the previously reported ACSSuT-type resistant bovine isolates, which were also isolated in Hokkaido were included in the same genetic cluster. Our results retrospectively indicate that MR-DT104 infection has existed in horses in Japan at least since 1996, and it was suggested that there is a highly epidemiological relationship between the equine MR-DT104 isolates and certain multidrug resistant bovine isolates in the same area.

Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR).

Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was constructed by insertion of rpoB fragments from Rhodococcus equi into CEM-1P, which is a recombinant plasmid that includes a T. equigenitalis-specific sequence region. In CEM-PCR, the size of the PCR product from CEM-POS was clearly different from the true positive PCR product. In addition, CEM-POS retained high stability under convenient storage conditions of 4 degrees C. These results suggest CEM-POS to be a useful tool as a reaction control in routine CEM-PCR examinations.

Evaluation of serum amyloid A and surfactant protein D in sera for identification of the clinical condition of horses with bacterial pneumonia.

In the present study, the concentrations of serum amyloid A and surfactant protein D in sera were measured to evaluate them for identification of the clinical condition of horses with bacterial pneumonia. The study utilized 185 clinically healthy control thoroughbreds and 9 thoroughbreds for experimental infectious study with S. zooepidemicus. Blood samples were collected from the 185 healthy control thoroughbreds. The 9 thoroughbreds were experimentally infected S. zooepidemicus using an endoscopic injection to a lung lobe and were then observed of clinical conditions. Blood samples were collected before inoculation and on the 1-15th, 22nd, and 29th days after inoculation (follow-up group). The levels of SAA and SP-D in the healthy control thoroughbreds were very low. In the follow-up group, the levels of SAA and SP-D changed in parallel with the horses' clinical condition. The pyrexia observed after bacterial inoculation faded by the 11th day, and the changes in SAA and SP-D occurred simultaneous to disappearance of the clinical signs. Measurement of SAA and SP-D proved useful for monitoring the clinical condition of the horses with bacterial pneumonia. Changes in the SP-D value were preceded by changes in the SAA value. Since the changes in SP-D occurred approximately simultaneous to the changes in the horses' clinical signs, we believe that they reflect the condition of the alveolar membranes. We conclude that measurement of SAA and SP-D in sera is useful for identification of the clinical condition of horses with bacterial pneumonia.

Morphological survey of bovine Setaria in the abdominal cavities of cattle in Aomori and Kumamoto Prefectures, Japan.

An epidemiological survey of bovine Setaria collected from the abdominal cavities was performed morphologically on the cattle in Aomori and Kumamoto Prefectures, Japan, between August 2005 and July 2006. Fifty Setaria worms were collected from the cattle in Aomori Prefecture and 847 from those in Kumamoto Prefecture. Of these worms, 35 were identified as Setaria digitata, 14 as S. marshalli, and one as S. labiatopapillosa in Aomori Prefecture, while 816 were identified as S. digitata and 31 as S. marshalli in Kumamoto Prefecture.

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis.

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.

Evaluation of enzyme-linked immunosorbent assays with recombinant antigens for the serodiagnosis of equine Babesia infections.

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.

A nucleotide mutation associated with fluoroquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants.

In this study, the quinolone resistance-determining region (QRDR) in gyrA and gyrB of in vitro fluoroquinolone-resistant Rhodococcus equi mutants was sequenced. These mutants were selected from four R. equi strains on blood agar plates containing ciprofloxacin or enrofloxacin. Each mutant became 8- to 64 or greater-fold resistant to fluoroquinolones compared with their parent strains. From the results of sequence analysis of QRDR in gyrA and gyrB, a nucleotide mutation of codon GAC for GGC in gyrA was detected in all mutants, but no mutation was observed in gyrB. This mutation leads to amino acid substitution of Asp for Gly in putative GyrA in R. equi. The position of this substitution corresponds to position 87 of GyrA in Escherichia coli. Our results suggest that the mutation of QRDR in gyrA, which was observed in in vitro fluoroquinolone-resistant R. equi mutants in this study, is closely associated with fluoroquinolone resistance.

Variation in the N-terminal region of an M-like protein of Streptococcus equi and evaluation of its potential as a tool in epidemiologic studies.

OBJECTIVE: To develop a method for typing Streptococcus equi on the basis of the DNA sequence of the genes that produce an M-like protein and to compare isolates among the United States, Japan, and other countries. SAMPLE POPULATION: S equi strains CF32, Hidaka/95/2, and NCTC9682 as well as 82 other isolates from the United States, Japan, and other countries obtained during 1975 to 2001. PROCEDURE: DNA sequences of the structural genes ( SeM and SzPSe) that produce M-like proteins were determined for 3 representative strains to find a variable region. Variability in this region of SeM was then determined for the other isolates. Amino acid sequences were deduced and analyzed phylogenetically by use of the neighbor-joining method. RESULTS: Sequence diversity was detected in the N-terminal region of SeM but not in SzPSe of the 3 representative strains. Base substitutions in the variable region of SeM varied in a nonsynonymous manner, resulting in variation in the amino acid sequence. Eighty-five isolates were categorized as 32 types of SeM on the basis of differences in the deduced amino acid sequences. CONCLUSIONS AND CLINICAL RELEVANCE: This study documented a region in the N-terminal portion of SeM that varies in a nonsynonymous manner. This information should be useful in molecular epidemiologic studies of S equi.

Simple and rapid PCR method for identification of streptococcal species relevant to animal infections based on 23S rDNA sequence.

A PCR identification system targeting 23S rDNA sequences for the identification of eight streptococcal species relevant to animal infections (Streptococcus agalactiae, S. bovis, S. canis, S. dysgalactiae, S. equi, S. porcinus, S. suis and S. uberis) was developed. This system consists of two PCR reactions, A and B, in which seven and eight primers, respectively, are used simultaneously, and was designed so that each amplification product indicates a species by its size. A total of 111 cultures, including the type strain of eight species, could be successfully identified and differentiated as individual species, except for the cross reactivity between S. bovis and S. equinus. The developed PCR system can complete the identification procedure for eight streptococcal species through two tube reactions per isolate, and, therefore, might provide a rapid, simple and accurate diagnostic tool for veterinary laboratories.

Nonulcerative keratouveitis as a manifestation of Leptospiral infection in a horse.

A 2-year-old Thoroughbred filly presented with ocular pain and epiphora of the left eye. The pupil was miotic and the cornea edematous near the ventro-temporal limbus, but did not retain any fluorescein. The topical antibiotics and atropine and diclofenac, and systemic flunixin meglumine and antibiotic therapy did not resolve the condition. A pink and fleshy infiltrate developed near the limbus indicating nonulcerative keratouveitis. The anterior uveitis deteriorated as manifested by the presence of dyscoria, hypopyon, and organized fibrin in the anterior chamber. Ocular signs were improved by topical and subconjunctival corticosteroids, but repeatedly deteriorated as the frequency of medication was reduced. The horse was seropositive to three serovars of Leptospira interrogans. The animal was diagnosed as blind on day 91 by the absence of pupillary light and menace reflexes, and donated for histopathologic diagnosis. The corneal opacity was histologically fibrotic and infiltrated predominantly by lymphocytes with Descemet's membrane partially disrupted by macrophages. The choroid was infiltrated by lymphocytes, eosinophils and basophils, and was positive to IgG and C3. There were filamentous or spiral structures positive to Warthin-Starry stain in the renal cortex. There was also polymerase chain reaction amplification of the leptospiral gene in the kidney. From these findings nonulcerative keratouveitis was believed to be caused by systemic infection with Leptospira.

Pneumonia in horses induced by intrapulmonary inoculation of Streptococcus equi subsp. zooepidemicus.

To evaluate the possibility that Streptococcus equi subsp. zooepidemicus (S.z) the causative bacterial agent of equine shipping fever pneumonia (ESFP), as well as to investigate its pathogenesis, 10 horses (seven Thoroughbreds and three Anglo-Arab species, ranging from 2-4 years in age) were experimentally inoculated, via an endoscope, into bronchus of the lung lobe with a dose of 30 ml of 1-7 x 10(8) CFU/ml of S.z. After inoculation, autopsy and pathological examinations were sequentially conducted 30 min, 1, 2, 3, 4, 17, 20 hr and 2 weeks later. Pneumonia induced by the intrapulmonary inoculation of S.z was characterized by small purulent pneumonic foci in the inoculated areas. With the lapse of time, these foci developed into serous hemorrhagic pneumonia, hemorrhagic purulent pneumonia, and then purulent, coagulation necrotic pneumonia. These pathomorphological characteristics of experimental pneumonia closely resemble those naturally occurring ESFP. There is strong evidence that S.z. is implicated as a causal factor in ESFP. S.z. grew in the mucus, exudate, and pulmonary effusions. Further, the bacteria showed resistance against phagocytosis by pulmonary alveolar macrophages (PAM) and neutrophils. Inhibition of PAM and neutrophil function is considered to be important in the development of pneumonia. With the progression of the disease, the neutrophils often adhered to the endothelial surface of the alveolar capillary lumen and played a role in generating coagulation necrosis of lung tissues.

First case report of Sarcocystis neurona-induced equine protozoal myeloencephalitis in Japan.

Equine protozoal myeloencephalitis developed in a three-year-old male Thoroughbred racehorse imported from the United States. The animal showed astasia five days after the onset of ataxia. Histopathologically, focal nonpurulent myelitis accompanied by hemorrhage and perivascular infiltration was observed in the fourth and fifth cervical spinal cord. Immunohistochemically, shizonts were occasionally observed and were positive for anti-Sarcocystis neurona (S. neurona) antiserum. S. neurona-specific antibodies were detected in the serum and cerebrospinal fluid by Western blot. This is the first equine protozoal myeloencephalitis case in Japan.

Equine pyoderma associated with malnutrition and unhygienic conditions due to neglect in a herd.

Twelve horses kept at a riding club suffered from pyoderma. All the horses displayed crusting, scaling and alopecia. The lesions were distributed in the chest, back, rump and limbs. Some of the horse patients also showed epilation with an attached crust similar to a 'paintbrush lesion' of dermatophilosis, but normal skin flora or opportunistic pathogenic bacteria were only isolated from the lesions. Some patients clearly showed weight loss, anemia and low levels of serum protein and cholesterol. General condition and skin lesions of the patients were improved gradually with improvement of feed and environment after being moved to new stables. Malnutrition under conditions of poor hygiene and poor management due to neglect might be associated with these equine cases of pyoderma in the herd.

Comparison of Salmonella enterica serovar Abortusequi isolates of equine origin by pulsed-field gel electrophoresis and fluorescent amplified-fragment length polymorphism fingerprinting.

Equine paratyphoid is caused by Salmonella enterica serovar Abortusequi, and manifests mainly as abortion in the mare. We compared S. Abortusequi strains isolated in Japan and other countries using pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment length polymorphism (FAFLP) analysis. PFGE analysis of S. Abortusequi strains gave 21-27 fragments ranging in size from 33 to 602kb. Although two PFGE profiles were observed among the 20 S. Abortusequi isolates in Japan, the restriction fragments originating from the chromosome were common between the two profiles. The similarity index of the two profiles was 90.9%, while those between Japanese and five other S. Abortusequi strains were 29.8-37.5%. On the other hand, FAFLP analysis of S. Abortusequi strains generated 64-67 amplified fragments ranging in size from 100 to 400bp. One polymorphic fragment was observed among the 20 S. Abortusequi isolates in Japan. These data indicate the close relation of this agent in Japan. S. Abortusequi strains sharing a common ancestry might have been conserved in Japan.

Evaluation of the field application of PCR in the eradication of contagious equine metritis from Japan.

The effectiveness of the polymerase chain reaction (PCR) as a field application test for the eradication of contagious equine metritis (CEM) was evaluated. Seven-thousands five-hundred and thirty-four genital swabs were collected from 4,026 Thoroughbred broodmares and stallions in Japan to test "high risk" horses as well as for general surveillance testing from 1998 to 2001. Bacterial isolation as well as PCR testing of original specimens and cultured specimens was performed for detection of Taylorella equigenitalis from genital swabs. As a result, T. equigenitalis was detected in 12 mares and 1 stallion by PCR, although the bacteria were isolated from only 2 of the PCR-positive mares. CEM-infected and carrier horses were treated by a combination of chemotherapy and surgery. Subsequent follow-up testing over a 3-year period did not detect T. equigenitalis. It was demonstrated that PCR testing was more sensitive than isolation as a method for the detection of T. equigenitalis from genital swabs of horses in the field. It was therefore suggested that a combination of PCR testing and treatment were useful measures in the eradication of CEM from Japan.

Distribution of mecA-harboring staphylococci in healthy mares.

The prevalence of staphylococci that harbor the mecA gene responsible for methicillin resistance was examined in healthy breeding mares. Staphylococci often cause diseases of horses such as metritis, keratitis, and abscess. Methicillin-resistant staphylococci would make antibiotic treatments ineffective, so it may be significant to know the distribution of mecA-harboring staphylococci in mares. Isolation of mecA-harboring staphylococci was achieved from nares and pasterns of 100 mares in Hokkaido, Japan. From 13% of the mares, mecA-harboring staphylococci, including 15 isolates of Staphylococcus sciuri and 3 of Staphylococcus lentus, were isolated. Isolates of S. sciuri were found to be genetically polyclonal by pulsed-field gel electrophoresis. These isolates produced no PCase and showed low or no resistance to beta-lactam and other classes of antibiotics. Distribution of staphylococcal species and levels of antibiotic resistance were found to be different between isolates from the present mares and those previously reported from riding-horses. Antibiotic pressure may lead to these differences. In addition, it appears that mecA-harboring S. sciuri may be native to horses.