Acyl modifications in bovine, porcine, and equine ghrelins.

Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology.

Characteristic Inflammatory Biomarkers in an Equine Model of Persistent Synovitis Induced By the Intra-Articular Administration of Monoiodoacetic Acid.

Persistent synovitis damages the articular cartilage in horses. To evaluate the effectiveness of treatment for synovitis using a model induced by intra-articular administration of monoiodoacetic acid (MIA), it is necessary to identify inflammatory biomarkers characteristic of the MIA model. Synovitis was induced by administering MIA into the unilateral antebrachiocarpal joints of five horses, and saline was injected into the contralateral joints as a control on day 0. Clinical and ultrasonographic examinations and synovial fluid collection were performed on days 0, 1, 2, 7, 14, 21, 28, and 35. Leukocyte, lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), and transforming growth factor-ß1 (TGF-ß1) concentrations in the synovial fluid were measured. Synovium was obtained after euthanasia on day 42 and histologically examined before quantification of the gene expression of inflammatory biomarkers by real-time PCR. Acute inflammatory symptoms persisted for approximately 2 weeks before returning to control levels. However, some indicators of chronic inflammation remained elevated until day 35. On day 42, synovitis continued histologically, with osteoclasts. The expressions of matrix metalloproteinase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), receptor activator of nuclear factor kappa-Β ligand (RANKL), and collagen type I α2 chain (Col1a2) were significantly higher in the MIA model than in the control. In the MIA model, representative inflammatory biomarkers in the chronic inflammatory stage were persistently expressed in both synovial fluid and tissue, suggesting that they may be useful for the assessment of the anti-inflammatory effect of drugs.

Rational determination of cefazolin dosage regimen in horses based on pharmacokinetics/pharmacodynamics principles and Monte Carlo simulations.

A pharmacokinetics/pharmacodynamics (PK/PD) approach was used to determine the best empirical dosage regimen of cefazolin (CEZ) after intramuscular (IM) administration of CEZ in horses. Seven horses received a single IM or intravenous (IV) administration of CEZ of 5 mg/kg bodyweight (BW) according to a crossover design. CEZ plasma concentrations were measured using LC-MS/MS. The plasma concentrations in these seven horses and those of six other horses obtained in a previous study with an IV CEZ dose of 10 mg/kg were modelled simultaneously using NonLinear Mixed-Effect modelling followed by Monte Carlo simulations to establish a rational dosage regimen. A 90% Probability of Target Attainment (PTA) for a PK/PD target of a free plasma concentration exceeding MIC90 (fT > MIC ) for 40% of the dosing interval was set for selecting an effective dosing regimen. The typical half-life of absorption and bioavailability after IM administration were 1.25 h and 96.8%, respectively. A CEZ dosage regimen of 5 mg/kg BW q12h IM administration achieved therapeutic concentrations to control both Streptococcus zooepidemicus and Staphylococcus aureus. For the same dose, the fT > MIC after IM administration was significantly longer than after IV administration, and the IM route should be favoured by clinicians for its efficiency and convenience.

Concentration of cephalothin in body fluids and tissue samples of Thoroughbred horses.

Cephalothin (CET) concentrations in body fluids (plasma, synovial fluid, pleural fluid, peritoneal fluid, and aqueous humor) and tissue samples (bone, lung, jejunum, hoof, and subcutaneous tissue) were investigated to consider the treatment of infectious diseases in horses. CET 22 mg/kg body weight was intravenously administered to 12 horses. Samples were collected from four different horses at 1, 3, and 5 hr after administration. The CET concentration in body fluids other than aqueous humor was maintained above the MIC90 values of Streptococcus zooepidemicus and Staphylococcus aureus until 5 hr, but it was not maintained above that of S. aureus in bone. CET (22 mg/kg twice a day) is effective for septic arthritis, pleuritis, and peritonitis caused by gram-positive bacteria but ineffective for osteomyelitis.

Incidence of carpal fractures and risk factors for recurrent fractures after arthroscopic removal of carpal chip fracture fragments in Thoroughbred racehorses.

BACKGROUND: We aimed to investigate the recent incidence of carpal fractures and the risk factors for recurrent ipsilateral fractures after arthroscopic removal of clinically active unilateral carpal chip fracture fragments in Thoroughbred racehorses. METHODS: The findings for horses managed under the Japan Racing Association that developed carpal bone fractures between 2014 and 2018 were retrospectively reviewed. The proportion of cases that developed a recurrent carpal fracture in the originally affected joint was calculated, and the risk factors for recurrent fractures were analysed. RESULTS: In total, 2858 carpal fractures were recorded in the study period (incidence, 0.8%). Of the 554 horses that resumed racing after the treatment of the unilateral major carpal chip fracture, 144 had a recurrent fracture (26.0%). Chip fractures of the third carpal bone (odds ratio [OR]: 3.7) or a combination of the distal end of the radius and intermediate carpal bone (OR: 3.0) were associated with a significantly higher risk of recurrent fractures than the distal aspect of the radial carpal bone. CONCLUSIONS: The incidence of carpal fractures remained similar to that reported in Japan in the 1990s. The rate of recurrent ipsilateral fractures differed among lesion sites.

Medication control of flunixin in racing horses: Possible detection times using Monte Carlo simulations.

BACKGROUND: For medication control in several jurisdictions, withdrawal time is the period of refrain from racing after drug administration. It is set by adding a safety period to an experimental detection time. However, there are no reports of statistical analyses of detection time for the determination of withdrawal time in flunixin meglumine-treated horses. OBJECTIVE: To analyse the population pharmacokinetics of flunixin in horses through the generation of a dataset for detection time statistical analysis and predictions via Monte Carlo simulation. STUDY DESIGN: Experimental study. METHODS: Drug plasma and urine concentrations following single intravenous administration of flunixin 1.1 mg/kg bodyweight (BW) in 10 horses and multiple administration of q 24 hours for 5 days in 10 horses were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Data were modelled using a nonlinear mixed effect model followed by Monte Carlo simulation. Irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were calculated using the Toutain approach. Detection times were obtained considering the time after the last administration for selected quantiles of 5000 hypothetical horses under the international screening limit (ISL) proposed by the International Federation of Horseracing Authorities (plasma: 1 ng/mL, urine; 100 ng/mL). RESULTS: For a regimen of 1.1 mg/kg BW q 24 hours, the IPC and IUC values were 2.0 and 73.0 ng/mL respectively. Detection times in plasma above the ISL for 90% of simulated horses were estimated as 74 hours after a single 1.1 mg/kg dose administration, 149 and 199 hours after multiple doses over 5 days at either 24- or 12-hour intervals respectively. Corresponding detection times in urine were 46, 68 and 104 hours respectively. MAIN LIMITATION: Only female horses were investigated. CONCLUSIONS: Statistical detection times for different flunixin meglumine regimens indicated a delay of detection time in plasma after multiple administrations under ISL.

Toe white line separation associated with the notch (crena marginalis) on the distal phalanges in Thoroughbred yearling horses.

The shape of the white line of the hoof is closely related to the shape of the notch on the dorsal distal bearing border of the distal phalanx (P3). In this study, a radiographic survey of the P3 of both forelimbs of 163 Thoroughbred yearling horses was conducted. The correlation of the depth and width of the notch were analyzed with the toe white line separation grades (0 to 3). As a result, the toe white line separation grade increased, the depth and the width of the notch also increased significantly. Radiographic examination of the P3 of the forelimbs might be useful for deciding whether to implement hoof care to prevent onset of toe white line separation.

Optimal double-spin method for maximizing the concentration of platelets in equine platelet-rich plasma.

This study optimized the double-spin conditions for preparing equine platelet-rich plasma (PRP): leukocyte-rich PRP (L-PRP) and leukocyte-poor PRP (P-PRP). Whole blood samples were centrifuged at various double-spin conditions. Both L-PRP and P-PRP were prepared at each stage, and complete blood counts and growth factor concentrations were compared. Samples centrifuged at 160 × 900 g, 160 × 2,000 g, and 400 × 2,000 g exhibited the highest platelet counts. P-PRP had significantly lower leukocyte and erythrocyte contents than L-PRP, especially at 400 × 2,000 g. No significant differences were observed in growth factor concentrations. Our data suggest that optimum L-PRP preparation should include centrifugation under the aforementioned conditions, whereas centrifugation at 400 × 2,000 g is optimal for P-PRP.

Single-dose pharmacokinetics of orally administered metronidazole and intravenously administered imipenem in healthy horses and computer-based simulation of pleural fluid concentrations with multiple dosing.

OBJECTIVE: To determine plasma pharmacokinetics of metronidazole and imipenem following administration of a single dose PO (metronidazole, 15 mg/kg) or IV (imipenem, 10 mg/kg) in healthy Thoroughbreds and simulate pleural fluid concentrations following multiple dose administration every 8 hours. ANIMALS: 4 healthy Thoroughbreds. PROCEDURES: Metronidazole and imipenem were administered, and samples of plasma and pleural fluid were collected at predetermined time points. Minimum concentrations of metronidazole and imipenem that inhibited growth of 90% of isolates (MIC90), including 22 clinical Bacteroides isolates from horses with pleuropneumonia, were calculated. For the computer simulation, the target ratio for area under the pleural fluid concentration-versus-time curve during 24 hours to the MIC90 for metronidazole was > 70, and the target percentage of time per day that the pleural fluid concentration of imipenem exceeded the MIC90 was > 50%. RESULTS: Mean ± SD pleural fluid concentrations of metronidazole and imipenem were 12.7 ± 3.3 µg/mL and 12.1 ± 0.9 µg/mL, respectively, 1 hour after administration and 4.9 ± 0.85 µg/mL and 0.3 ± 0.08 µg/mL, respectively, 8 hours after administration. For both antimicrobials, concentrations in the pleural fluid and plasma were similar. The ratio for area under the pleural fluid concentration-versus-time curve during 24 hours to the MIC90 for metronidazole was 84.9, and the percentage of time per day the pleural fluid concentration of imipenem exceeded the MIC90 was 70.9%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that administration of metronidazole (15 mg/kg, PO, q 8 h) or imipenem (10 mg/kg, IV, q 8 h) resulted in their accumulation in the pleural fluid in healthy horses and concentrations were likely to be effective for the treatment of pneumonia and pleuropneumonia caused by Bacteroides spp.

Isolation of equine papillomavirus type 1 from racing horse in Japan.

Although many studies have been conducted worldwide for Equus caballus papillomavirus (EcPV), limited information is available on the virus in Japan. We recently collected one classical viral papillomatosis sample (E150904) from a racing horse in Japan. Papillomavirus infection was confirmed by histopathology, immunohistochemistry and PCR assays, and the sample was diagnosed as epithelial papilloma. Full-length genome of the virus was cloned and sequenced. It was 7,613 bp in length and had the same genome organization with reported EcPV-1. Moreover, phylogenetic analysis based on L1 gene revealed that the infection was caused by a variant of EcPV-1. This is the first report of EcPV infection in Japan, and would further contribute to the molecular epidemiological and pathological studies for EcPV.

Does the injection of platelet-rich plasma induce changes in the gene expression and morphology of intact Thoroughbred skeletal muscle?

Platelet-rich plasma (PRP) therapy is promising for treating skeletal muscle injuries in human athletes by promoting muscle regeneration. It might also be useful for treating muscle injuries in equine athletes. In the present study, muscle regeneration induced by injection of PRP into intact muscle of Thoroughbred was investigated. Autologous PRP and saline were injected twice into intact left and right gluteus medius muscles of seven clinically healthy Thoroughbreds. Muscle samples were collected from the injection sites by needle biopsy at 2 and 7 days after PRP injection. Immunohistochemical staining to identify the types of myosin heavy chains (MHCs) and satellite cells was performed to compare morphological changes among intact (pre-injection), saline-, and PRP-injected muscles. The expression of marker genes related to muscle regeneration (MHC-I, MHC-II, and embryonic MHC [MHC-e]), satellite cell activity (CK, Pax7, MyoD, and myogenin), and proinflammatory and promyogenic cytokines (IL-6, IGF-1, and HGF) was analyzed and compared between saline- and PRP-injected muscles. There were no obvious morphological differences among the three treatments. There were no significant differences in gene expression associated with satellite cell activity between saline and PRP injection at 7 days after injection. MHC genes showed significantly higher expression levels with PRP than with saline, including MHC-e at 2 days and MHC-I at 7 days after injection. It is suggested that injection of PRP into intact skeletal muscle does not induce specific morphological changes, but upregulate the expression of genes related to muscle regeneration.

New methods for isolation of keratolytic bacteria inducing intractable hoof wall cavity (Gidoh) in a horse; double screening procedures of the horn powder agar-translucency test and horn zymography.

To establish a new system to isolate keratolytic bacteria from the hoof wall cavity (Gidoh) of a racehorse, we invented the horn powder agar-translucency (HoPAT) test and horn zymography (HZ). Using routine bacteriological techniques and these methods, we isolated five strains of keratolytic soil bacteria, which were then identified by means of 16S ribosomal RNA (rRNA) gene sequencing analysis. The findings from the study on the horse suggested that Brevibacterium luteolum played the main role in the local fragility of the hoof, eventually forming a Gidoh in coordination with four other strains of keratolytic bacteria. The double screening procedures of the HoPAT test and HZ were useful and easy techniques for isolating the keratolytic bacteria from the horn lesions.

Population doubling level-dependent change of secreted glycosaminoglycan in equine bone marrow-derived mesenchymal stem cells.

In regenerative medicine using transplantation of mesenchymal stem cells (MSCs), the importance of regulating the quality of MSCs has been well recognized; however, there is little information concerning the relationship between the population doubling level (PDL) and the stemness of MSCs in equine medicine. In this study, we showed that the amount of glycosaminoglycan (GAG) secreted by bone marrow-derived MSCs (BMSCs) decreases with increase of PDL. Enzymatic digestion and two-dimensional electrophoresis revealed that a main component of GAG produced by BMSCs was hyaluronan with a small amount of chondroitin sulfate. Increase of PDL downregulated the expression of MSC CD markers, including CD44, CD73, CD90, CD105, and CD146, along with loss of differentiation capacity. Thus, the effect of hyaluronan supplement to the growth medium on both expression of CD markers and the tri-lineage potential of BMSCs was evaluated. Expression of CD73 and CD90 was preserved by continuous addition of hyaluronan to the growth medium, whereas mRNA levels corresponding to CD44, CD105 and CD146 were not preserved by supplementation of hyaluronan. BMSCs subcultured with hyaluronan-supplemented growth medium to PDL-12 showed osteogenic capacity, however adipogenic and chondrogenic activities at PDL-12 were not preserved by exogenous hyaluronan. These results suggest that downregulation of CD44, CD105 and CD146 might not affect the osteogenic capacity. Taken together, the results suggested that supplementation of hyaluronan to the growth medium might be effective at maintaining the osteogenic capacity of equine BMSCs.

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

We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a racehorse. A 5-year-old mare developed punctate keratitis after racing. The corneal ulcer continued to expand despite ophthalmic antimicrobial therapy. On day 6, a conjunctival graft surgery was performed. The mare was euthanized, following colitis and laminitis development on day 10. MRSA was isolated from the corneal swab taken at the time of euthanasia. Immunohistochemical analysis demonstrated gram-positive and anti-S. aureus monoclonal antibody-positive cocci infiltration of the corneal stroma; and a diagnosis of MRSA ulcerative keratitis was made. An ophthalmic antimicrobial against the isolated MRSA did not improve the ocular lesion. The MRSA strain was found to be staphylococcal cassette chromosome mec type II, a strain frequently isolated from humans in Japan.

Effects of aluminum hinged shoes on the structure of contracted feet in Thoroughbred yearlings.

We applied aluminum hinged shoes (AHSs) to the club foot-associated contracted feet of 11 Thoroughbred yearlings to examine the effects of the shoes on the shape of the hoof and third phalanx (P III). After 3 months of AHS use, the size of the affected hooves increased significantly, reaching the approximate size of the healthy contralateral hooves with respect to the maximum lateral width of the foot, the mean ratio of the bearing border width to the coronary band width, and the mean ratio of the solar surface width to the articular surface width. These results suggest that the AHSs corrected the contracted feet in these yearling horses.

Regulation of Tenomodulin Expression Via Wnt/ß-catenin Signaling in Equine Bone Marrow-derived Mesenchymal Stem Cells.

Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/ß-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of ß-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/ß-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/ß-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel.

Anatomical location and culture of equine corneal epithelial stem cells.

OBJECTIVE: To identify morphologically the locations of equine corneal epithelial stem cells (CESCs) and to culture these cells. ANIMALS STUDIED: We studied the eyes of 12 adult thoroughbred horses. PROCEDURES: Eye tissues were immunostained for two positive stem cell markers (p63, CK14) and one negative marker (CK3) to identify the locations of CESCs, so we could compare their immunostaining patterns with those of human stem cells previously reported. We compared the proliferation rates and morphological features of epithelial cells isolated from the corneal limbus and central cornea. RESULTS: Undifferentiated cells expressing the same immunostaining pattern as human CESCs were present in the equine corneal limbus. Cultured epithelial cells isolated from the limbus expressed the same immunostaining pattern that CESCs show histologically, but cells isolated from the central cornea did not proliferate and could not be evaluated. CONCLUSIONS: Equine CESCs were localized in the epithelial basal layer of the corneal limbus, where melanocytes reside. They could be cultured without loss of their undifferentiated nature. When collecting such stem cells, it may be useful to harvest and culture corneal epithelial tissues in the limbus where melanocytes serve as an indicator of the collecting area.

Active hyaluronidase 2 expression in the granulation tissue formed in the healing process of equine superficial digital flexor tendonitis.

Upregulation of hyaluronidase 2 (HYAL2), one of somatic hyaluronidase (HAase), was demonstrated in granulation tissue during the healing of equine superficial digital flexor tendon injuries. The activity of HAase was assessed by hyaluronan (HA)-containing gel zymography and in situ zymography using frozen sections obtained from normal and injured tendon tissues. Elevated HAase activity was identified in the extract from the tendinopathic tissues, with lower levels of the activity in normal tendons. In situ zymography using fluorescently-labeled HA demonstrated HAase activity in the granulation tissue formed in the injured region. In addition, in situ hybridization analysis indicated that fibroblastic cells in the granulation tissue of the injured tendon actively expressed HYAL2 but not HYAL1. Quantitative RT-PCR further confirmed a higher level of amplicons corresponding to HYAL2 in tendonitis-derived samples. These results suggest that elevated HYAL2 activity in the granulation tissue could participate in controlling the healing process in equine tendonitis.

Investigation of hydration processes of the equine hoof via nuclear magnetic resonance microscopy.

OBJECTIVE: To examine the distribution of water in hoof wall specimens of horses via nuclear magnetic resonance (NMR) microscopy and determine changes in water distribution during hydration. SAMPLE: 4 hoof wall specimens (2 obtained from the dorsum and 1 each obtained from the lateral quarter and lateral heel regions) of the stratum medium of healthy hooves of 1 horse. PROCEDURES: Equine hoof wall specimens were examined via NMR microscopy. Proton density-weighted 3-D images were acquired. Changes during water absorption were assessed on sequential images. RESULTS: The inner zone of the stratum medium had higher signals than did the outer zone. Areas of high signal intensity were evident in transverse images; these corresponded to the distribution of horn tubules. During water absorption, the increase in signal intensity started at the bottom of a specimen and extended to the upper region; it maintained the localization pattern observed before hydration. The relationship between the local maximal signals in areas corresponding to the horn tubules and minimal signal intensities in areas corresponding to the intertubular horn was similar and maintained approximately a linear distribution. CONCLUSIONS AND CLINICAL RELEVANCE: Based on the premise that signal intensity reflects water content, hydration in the equine hoof wall during water absorption occurred concurrently in the tubules and intertubular horn, and there was maintenance of the original water gradients. This technique can be applied for the assessment of pathophysiologic changes in the hoof wall on the basis of its hydration properties.

Treponemes-infected canker in a Japanese racehorse: efficacy of maggot debridement therapy.

A 3-year-old thoroughbred colt presented with canker on its left hind foot. Subsequent development of cottage cheese-like horns and dermatitis disturbed healing, despite the use of miscellaneous orthodox treatment approaches to the lesions. Histological examination revealed exudative and suppurative dermatitis, and proliferatively suppurative epidermitis infected with helically coiled treponemes. Total debridement under general anesthesia led to a temporary improvement, but the ground surface regenerated abnormal epidermis similar to that observed initially after surgery. Maggot debridement therapy (MDT) was attempted, which removed all the abnormal tissue. After MDT, general farriery trimming helped to correct the distorted ground surface, and the horse returned to constant training and eventually raced. This case shows that MDT was successfully used for treatment of an intractable and treponemes-infected canker.