Genetic diversity and relationships among native Japanese horse breeds, the Japanese Thoroughbred and horses outside of Japan using genome-wide SNP data.

Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni-are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.

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

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

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

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

Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles.

The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.

Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles.

The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.

Effects of orientation, intermittent rest and vehicle cleaning during transport on development of transport-related respiratory disease in horses.

The effects of various factors on the inflammatory and stress response in horses during transportation were examined in Experiments 1 and 2, carried out in April and August, respectively. In Experiment 1, three groups (G1-G3) of four Thoroughbreds were used, and in Experiment 2, two groups (G4, G5). G1 animals were loaded into lorries with their heads facing forwards (FF) and given periods of short rest (SR) (30 min for every 4 h driven). G2 horses were loaded facing rearwards (FR) and given SR. G3 horses were FF and given periods of long rest (LR) (2 h rest for every 4 h driven). G4 horses were transported FF with hay suspended in front of them and no cleaning of faeces or urine during rest stops. G5 horses were FF and given pellets instead of hay, and the lorry was cleaned and washed at each rest stop. G4 and G5 horses were rested for 1 h after every 5 h of driving. All groups were driven a distance of 1500 km, and the total journey time was 37 h for G1 and G2, 49 h for G3, and 40 h for G4 and G5. In Experiment 1, indicators of inflammatory and stress responses tended to be highest in G1, intermediate in G2 and lowest in G3. In Experiment 2 they tended to be higher in G4 than in G5. The results suggested that increasing the rest time and cleaning the interior of the vehicle during rest stops reduced transportation stress and respiratory insults, factors that may lead to respiratory disease.

Morphology of the nerve endings in laryngeal mucosa of the horse.

To discuss the significance of laryngeal sensation on various disorders of the horse, we studied the morphological and topographical characteristics of sensory structures in the laryngeal mucosa using immunohistochemistry and immunoelectron microscopy. Various sensory structures, i.e. glomerular endings, taste buds and intraepithelial free nerve endings, were found in the laryngeal mucosa by immunohistochemistry for protein gene product 9.5 (PGP 9.5) and neurofilament 200kD (NF200). Glomerular nerve endings were distributed mainly in the epiglottic mucosa; some endings were also found in the arytenoid region arising from thick nerve fibres running through the subepithelial connective tissue. Some terminals directly contacted the epithelial cells. Taste buds were distributed in the epithelium of the epiglottis and aryepiglottic fold. In the whole mount preparation, the taste buds were supplied by the terminal branching of the thick nerve fibres. In some cases, the taste buds were arranged around the opening of the duct of the epiglottic glands. The intraepithelial free nerve endings were found to be immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP). These nerve endings were surrounded by the polygonal stratified epithelial cells in the supraglottic region, and by the ciliated cells in the subglottic region. The density of the intraepithelial free nerve endings was highest in the corniculate process of the arytenoid region and lowest in the vocal cord mucosa. The densities of CGRP- and SP-immunoreactive nerve endings in the arytenoid region were (mean +/- s.d.) 30.6+/-12.0 and 10.0+/-4.9 per unit epithelial length (1 mm), respectively and in the vocal fold mucosa, 1.1+/-0.9 and 0.8+/-0.7, respectively. Approximately one half of the CGRP immunoreactive nerve endings were immunoreactive for SP, and most SP-immunoreactive nerve endings were also immunoreactive for CGRP. Well-developed subepithelial plexus with numerous intraepithelial fibres were observed in flat or round mucosal projections that existed on the corniculate process of the arytenoid region. In conclusion, the laryngeal mucosa of the horse seems to have morphology- and/or location-dependent sensory mechanisms against various endo-and exogenious stimuli.

Freestanding and multiunit immediate loading of the expandable implant: an up-to-40-month prospective survival study.

STATEMENT OF PROBLEM: The subject of immediate loading of dental implants has received substantial attention, although it appears to conflict with the basic principles of dental implant therapies developed and established over the past 20 years. Clinicians would welcome the use of a reliable, immediate-loading implant system. PURPOSE: The purpose of this study was to evaluate the effectiveness of an expandable implant design for immediate and delayed loading and for freestanding and multiunit situations. MATERIAL AND METHODS: Two hundred eighty-six immediate-load Sargon implants were placed in 75 patients during a 40-month period. Of the 273 implants that survived, 81 were placed into fresh extraction sockets and immediately loaded, 162 were placed into healed sites and immediately loaded, and 30 were delay loaded. Some implants that had failed to remain stable after immediate loading became stable and osseointegrated after the load was removed and their expansion mechanisms were reactivated. RESULTS: The overall survival rate during the 40-month period was 96.0% in the maxilla and 94.8% in the mandible. Implants placed in fresh extraction sockets showed a 98.9% survival rate. Healed sites showed a 93.9% survival rate. Immediate loading of 52 fresh extraction socket implants in the maxilla showed a 100% survival rate during the evaluation period. CONCLUSION: Within the limitations of this study, it was shown that the feature of mechanical expandability may provide operators some control over implant stability during the vulnerable period after immediate loading of single, freestanding implants.

Bronchoalveolar lavage for the diagnosis and treatment of pneumonia associated with transport in Thoroughbred racehorses.

To evaluate a hypothese that use of bronchoalveolar lavage (BAL) for early treatment of pneumonia would improve their prognosis by reducing bacterial numbers and excessive numbers of neutrophils in the lung, initial experiences with BAL in the diagnosis and treatment of pneumonia were performed in 36 racehorses that became ill within 24 hr of long distance travel (1,200-1,600 km, 26-32 hr) by road. Comparisons were made of the outcomes of the 36 horses and those of 42 horses (81.0% recovered, 50.0% returned to racing) treated for transport associated pneumonia without BAL. The total amount of BAL fluid injected during hospitalization varied from 700 to 3,700 ml and the duration of antibiotic treatments ranged from 5 to 40 days. Clinical symptoms after lavages showed good results with no side effects. None of the horses required thoracic drainage. Horses treated with BAL required shorter period of antibiotic therapy, a greater percentage recovered (100%, 36/36) and a greater percentage returned to racing (77.8%, 28/36). Eight (22.2%) never raced because of lameness or other considerations.

Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport.

An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-equine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma technology, purified by the antibody purification reagent, and analysed by Western blotting analysis. The immunoreaction (two-site sandwich ELISA) with a mAb, peroxidase-labelled mAb and BALF sample was carried out simultaneously and analytical recovery and precision were assayed. Six mAb for SP-A and four mAb for SP-D were successfully cloned in limiting dilution to monoclonality. These mAb were reacted with equine SP-A or SP-D on Western blotting analysis. For SP-A, a combination of solid-phase TA08 and horseradish peroxidase (HRP)-conjugated WA28 was found to be more sensitive than other combinations, gave a good dose response and was capable of measuring 0.78 to 100 ng of protein/ml. For SP-D, a combination of solid-phase TD13 and HRP-conjugated WD19 was found to be more sensitive than other combinations, had a good dose response and was capable of measuring 0.78 to 200 ng of protein/ml. The assay was used to determine the effect of 41 hours of road transport on the concentrations of SP-A and SP-D in the BALF of 30 horses. The concentrations of SP-A and SP-D decreased by 55 per cent and 36 per cent, respectively, decreases similar to the decrease in phosphatidylglycerol concentration previously reported by the authors.

Use of sevoflurane for anesthetic management of horses during thoracotomy.

OBJECTIVE: To evaluate sevoflurane as an inhalation anesthetic for thoracotomy in horses. ANIMALS: 18 horses between 2 and 15 years old. PROCEDURE: 4 horses were used to develop surgical techniques and were euthanatized at the end of the procedure. The remaining 14 horses were selected, because they had an episode of bleeding from their lungs during strenuous exercise. General anesthesia was induced with xylazine (1.0 mg/kg of body weight, IV) followed by ketamine (2.0 mg/kg, IV). Anesthesia was maintained with sevoflurane in oxygen delivered via a circle anesthetic breathing circuit. Ventilation was controlled to maintain PaCO2 at approximately 45 mm Hg. Neuromuscular blocking drugs (succinylcholine or atracurium) were administered to eliminate spontaneous breathing efforts and to facilitate surgery. Cardiovascular performance was monitored and supported as indicated. RESULTS: 2 of the 14 horses not euthanatized died as a result of ventricular fibrillation. Mean (+/- SD) duration of anesthesia was 304.9 +/- 64.1 minutes for horses that survived and 216.7 +/- 85.5 minutes for horses that were euthanatized or died. Our subjective opinion was that sevoflurane afforded good control of anesthetic depth during induction, maintenance, and recovery. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of sevoflurane together with neuromuscular blocking drugs provides stable and easily controllable anesthetic management of horses for elective thoracotomy and cardiac manipulation.

Measurement for breath concentration of hydrogen and methane in horses.

This study concerns the establishment of a simple testing method for breath concentration of hydrogen and methane in horses. Twenty-eight healthy thoroughbreds and 24 Arabians were used. Breath samples were collected using one-minute closed circulatory respiration through an aluminum bag filled with 10 liters of pure oxygen, which was mounted on the subjects by means of a face mask. Breath samples obtained, were analyzed by gas chromatography. A significant correlation in both hydrogen and methane levels was observed for samples collected at separate times. These findings confirmed the usefulness of our approach for testing breath concentrations of hydrogen and methane in horses.

Transferability of cephalothin to the alveolar cavity in thoroughbreds.

Five Thoroughbreds were classified into 4 groups according to the administration method used for saline solution (saline), ambroxol, and cephalothin sodium (cephalothin). In group A, cephalothin was injected intravenously after oral administration of ambroxol. In group B, cephalothin was injected intravenously after oral administration of saline. Groups C and D were used as control groups. The dose of cephalothin or ambroxol was clinically administrated. Venous blood and bronchoalveolar lavage fluid (BALF) were sampled from each group. In groups A and B, cephalothin concentrations in plasma reached their maximum level 5 min after cephalothin administration and then declined over time. In plasma obtained from groups A and B, there were no significant differences in pharmacokinetic parameters (T1/2, Kel, Vd). By contrast, cephalothin concentrations in BALF reached their peak at 180 min after cephalothin administration in both groups A and B and maintained a relatively high level even after 300 min. These findings indicate that cephalothin requires a relatively long period of time to move from the blood stream to the alveolar cavity, but once transferred to the alveolar cavity, it is preserved for a long time. In groups A and B, cephalothin concentrations in BALF were approximately at the same level. However, in group A, total protein in BALF was lower at 60, 180, and 300 min than the other groups. Then, cephalothin concentration was adjusted to total protein in BALF. After adjustment to total protein in BALF, group A showed a concentration level of cephalothin approximately 1.5-fold higher than that of group B. This suggests that the transferability of cephalothin to the alveolar cavity improves as a result of the oral administration of ambroxol.

Purification and biochemical characterization of equine pulmonary surfactant protein D.

OBJECTIVE: To characterize surfactant protein D (SP-D) isolated from bronchoalveolar lavage fluid (BALF) of healthy horses. SAMPLE POPULATION: BALF from 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) without history or clinical signs of respiratory tract disease. PROCEDURE: BALF was obtained and centrifuged at 33,000 X g. The supernatant was applied to a mannose-Sepharose 6B affinity column in the presence of calcium, and the bound protein fraction was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis; amino acid composition was determined and partial sequencing was done. Phospholipid binding and liposome aggregation assay were performed, using purified proteins. RESULTS: The protein isolated by use of mannose affinity matrices was SP-D. It bound carbohydrates and phosphatidylinositol, which are the characteristic features of SP-D isolated from other animal species. Amino acid analysis and partial primary sequence of the isolated protein indicated high homology with rat and human SP-D. Furthermore, immunoblot analysis indicated that equine SP-D reacted with human and rat SP-D-specific antibodies. CONCLUSION AND CLINICAL RELEVANCE: SP-D exists in equine lungs; its measurement may be useful in evaluating equine lung disease.

Purification and biochemical characterization of pulmonary surfactant protein A of horses.

OBJECTIVE: To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses. ANIMALS: 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) that did not have a history or clinical signs of respiratory tract disease. PROCEDURE: Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 X g. Lipid was removed from precipitated fractions by means of extraction with 1-butanol, and organic solvent-insoluble protein precipitates were dialyzed against Tris buffer. The suspension was centrifuged, and supernatant was placed in a mannose-Sepharose affinity column, with calcium. The bound protein fraction was analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and amino acid sequencing. A liposome-aggregation assay was also performed, using purified proteins. RESULTS: Protein isolated by use of mannose-affinity matrices was identified as surfactant protein A (SP-A). It had carbohydrate-binding and phospholipid-aggregation properties characteristic of SP-A isolated from other animal species. The partial primary sequence of the isolated protein had high homology with rat and human SP-A. Furthermore, the equine SP-A reacted with anti-human and anti-rat SP-A specific antibodies. CONCLUSION: Analysis of these findings indicated the existence of SP-A in pulmonary tissues of horses. CLINICAL RELEVANCE: Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses.

Is ventilation during maximal exercise limited by dynamic airway closure?

A study was undertaken to find if the reason why horses hypoventilate when running is that they experience expiratory flow limitation due to dynamic airway closure. To test this hypothesis, we measured peak expiratory flows on a Thoroughbred galloping on a treadmill and hypoventilating and compared those flows with the peak dynamically-limited flow that the same horse could achieve during a forced expiratory flow-volume manoeuvre. At the approximate lung volumes at which the horse was ventilating while running, it did not appear to be mechanically limited and appeared to have reserve capacity available potentially to increase its expiratory flow.

Changes in left ventricular dynamics during graded exercise.

Three mature Thoroughbred horses were prepared surgically with ultrasonic sonomicrometer crystals affixed to their ventricular pericardia. Signals from crystals recorded dimensions of axes across the left ventricle. Cubic algorithms were fitted to dimensional data to generate volume estimates that matched stroke volumes simultaneously measured using the Fick principle. As horses stood at rest or exercised at various intensities (approx 7, 12, 24, 47 and 100% maximal rate of O2 consumption VO2max[), left ventricular dimensions were recorded and 20 consecutive diastolic and systolic volumes calculated. Although Fick estimates detected no difference in stroke volume at different exercise intensities, sonomicrometer measurements of stroke volume were significantly lower at rest and higher at VO2max. These differences mirrored changes in end-diastolic volume, although end-systolic volume did not change. At all exercise intensities, stroke volume was most variable and end-diastolic volume the least. The pattern conforms to the Frank-Starling mechanism, and indicates that at high exercise intensities ventricular myocytes generate high pressures with higher myocardial wall stress due to the increased size of the chamber.

Cardiac output measurements using sonomicrometer crystals on the left ventricle at rest and exercise.

Eight horses were fitted surgically with 8 ultrasonic sonomicrometer crystals each attached to their left ventricular pericardia and a left atrial catheter. Three horses returned to treadmill performance with a maximum rate of oxygen consumption similar to their presurgical values. These horses were evaluated to determine how well sonomicrometer estimates of cardiac output agreed with those obtained by a steady-state method, the Fick principle. Variance between the 2 was similar to the coefficient of variation (approximately 12.5%) of the Fick estimates. We conclude that left ventricular sonomicrometer measurements in horses appear to give a reasonable estimate of ventricular dimensions and can, therefore, be used potentially to evaluate such variables as diastolic flow.

Chronological changes in superoxide-scavenging ability and lipid peroxide concentration of equine serum due to stress from exercise and transport.

It has been suggested that a variety of stresses on animals may accelerate their production of superoxide. Racehorses are considered to be exposed to substantial oxide stress due to transport and exercise for training and racing. To determine the effect of exercise and transport on racehorses in terms of superoxide and antioxidative ability, changes in the superoxide-scavenging ability of equine serum were observed using electron spin resonance (ESR). Changes in the concentration of lipid peroxide, which is produced in equine serum by superoxide, were also examined. The analysis revealed that lipid peroxide concentrations increased as a result of stress from exercise and transport. On the other hand, the superoxide-scavenging ability of equine serum showed a decline during transport, which is in sharp contrast to the increase seen immediately after a race due to the severe load exerted in exercise.

Characteristics of respiratory function during swimming exercise in thoroughbreds.

Equine respiratory patterns during swimming were examined in five normal horses. The experiment included a preliminary warming-up stage and 6 circuits of swimming around an annular pool of a 50-meter-circumference. The horses were examined for respiratory rates, intratracheal pressures, inspiratory time (TI), expiratory time (TE), respiratory cycle (T; TI + TE), heart rates, blood lactate concentrations, hematocrit and blood gases. The respiratory rates were maintained around 25/min. Blood gas values changed significantly during swimming. The intratracheal pressures during expiration and inspiration increased significantly with exercise duration compared to the immediately after the warming-up stage. The duty ratio (TI/T) averaged 0.33, which implied that the expiratory time was roughly doubled the inspiratory time. We considered that a longer expiratory time may limit sudden collapse of airways by water pressure during swimming and prevent a radical decrease of air space volume, thus maintains buoyancy.