Variability in plasma concentrations of methylprednisolone 6 days after intrasynovial injection of methylprednisolone acetate in racing horses: A field study.

BACKGROUND: Methylprednisolone (MP) acetate is a commonly used corticosteroid for suppression of inflammation in synovial structures in horses. Its use is often regulated in equine sports by plasma MP concentrations. OBJECTIVES: To describe variability in MP plasma concentrations after MP acetate injection in different synovial structures and with co-administration with hyaluronic acid (HA). STUDY DESIGN: Field study in actively racing horses in three disciplines (Thoroughbred, Standardbred and Quarter Horse). METHODS: Seventy-six horses (15 Thoroughbreds, 20 Standardbreds and 41 Quarter Horses) were included in the study. Injection of any synovial structure with a total body dose of 100 mg MP acetate was permitted, data were grouped according to the synovial structure injected and co-administration with HA. Plasma was collected before injection and at 6 days post-injection. Per cent censored data (below the limit of quantification) for each synovial structure were determined, and summary statistics generated by Robust Regression on Order. Differences between synovial structures and co-administration with HA were identified by ANOVA with Tukey's post hoc testing. RESULTS: The MP plasma concentration at 6 days for injection for the entire group (mean ± standard deviation [s.d.], pg/mL) was 96 ± 104. Metacarpophalangeal (MCP) plasma concentrations contained 86% censored data and could not be included in the statistical analysis. The carpal joints (CJO) group had a lower plasma MP concentration (P<0.05) than the distal tarsal joints (DTJ) or medial femorotibial (MFT), the no HA (NHA) group had a lower plasma MP concentration (P<0.05) than HA. MAIN LIMITATIONS: The synovial structures injected varied by racing discipline, so this study was unable to identify any differences between disciplines. CONCLUSIONS: Practitioners should be aware that injection of DTJ, CS and MFT joints, and combining MP acetate with HA may prolong its clearance, and withdrawal times for competition in regulated equine sports.

Serological and molecular diagnosis of Ehrlichia canis and associated risk factors in dogs domiciled in western Cuba.

Ehrlichia canis is a rickettsia transmitted by the tick, Rhipicephalus sanguineus, and is the causative agent of canine monocytic ehrlichiosis (CME). In Cuba, the first diagnosis of CME was made in 2001, but few studies have since investigated this disease locally. The objective of this study was to estimate the prevalence of E. canis in dogs domiciled in four municipalities within the western region of Cuba and determine the associated risk factors. Blood was drawn from 378 selected dogs living in four municipalities in two provinces of western Cuba. From the total number of samples, 206 plasma samples were selected to perform an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against E. canis. Using the original 378 samples of extracted DNA, a nested polymerase chain reaction (nPCR) was performed to amplify a specific fragment of the 16S rRNA gene of E. canis. Analysis of the 206 plasma samples revealed a total of 162 animals that were seropositive for E. canis (78.64%) with a density index between 109.5 and 970.7. In contrast, 179 samples were positive based on the nPCR assay (47.35%). As well, there was a high concordance (kappa = 0.7), calculated through the Kappa index, between the animals found to be positive based on nPCR and those determined based on ELISA. The analysis of risk factors showed that residing in the municipality of Boyeros in addition to having a history of infestation by ticks increases the probability of having a positive result based on nPCR.

Plasma firocoxib concentrations after intra-articular injection of autologous conditioned serum prepared from firocoxib positive horses.

Orthobiologics such as autologous conditioned serum (ACS) are often used to treat joint disease in horses. Because ACS is generated from the horse's own blood, any medication administered at the time of preparation would likely be present in stored ACS, which could lead to an inadvertent positive drug test following intra-articular (IA) injection. The main objective of this study was to determine if ACS prepared from firocoxib positive horses could result in detectable plasma concentrations of the drug following IA injection. Firocoxib was administered to six horses at 0.1mg/kg PO twice at a 24h interval. Blood was obtained at 4h following the second dose and transferred to a separate syringe (Arthrex IRAP II) for ACS preparation. Plasma and ACS concentrations of firocoxib were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). When horses were confirmed firocoxib negative, 7.5mL of ACS was injected into both tarsocrural joints. Blood samples were collected at 0, 4, 8, 12, 24, and 48h, and firocoxib concentration was measured. Mean (±standard error of the mean, SEM) plasma concentration of firocoxib 4h following the second dose was 33.3±4.72ng/mL. Mean (±SEM) firocoxib concentration in ACS was 35.4±4.47ng/mL. Fourteen days following the second and last dose of firocoxib, mean plasma concentration was below the lower limit of detection (LOD=1ng/mL) in all horses. Following IA injection of ACS, plasma concentrations of firocoxib remained below LOD at all times in all horses. ACS generated from horses with therapeutic plasma concentrations of firocoxib did not contain sufficient firocoxib to lead to a positive plasma drug test following IA administration.

The use of phenylbutazone in the horse.

This review presents a brief historical prospective of the genesis of regulated medication in the US racing industry of which the nonsteroidal anti-inflammatory drug (NSAID) phenylbutazone (PBZ) is the focus. It presents some historical guideposts in the development of the current rules on the use of PBZ by racing jurisdictions in the US. Based on its prevalent use, PBZ remains a focus of attention. The review examines the information presented in a number of different models used to determine the effects and duration of PBZ in the horse. They include naturally occurring lameness and reversible-induced lameness models that directly examine the effects and duration of the administration of various doses of PBZ. The review also examines indirect plasma and tissue models studying the suppression of the release of arachidonic acid-derived mediators of inflammation. The majority of studies suggest an effect of PBZ at 24 h at 4.4 mg/kg. This reflects and substantiates the opinion of many clinical veterinarians, many of whom will not perform a prepurchase lameness examination unless the horse is free of NSAID. This remains the opinion of many regulatory veterinarians responsible for the prerace examination of race horses that they wish to examine a horse without the possibility of an NSAID interfering with the examination and masking possible musculoskeletal conditions. Based on scientific studies, residual effects of PBZ remain at 24 h. The impact of sustained effect on the health and welfare of the horse and its contribution to injuries during competition remains problematic.

Screen and confirmation of PEG-epoetin ß in equine plasma.

Methods have been developed to screen for and confirm darbepoetin alfa, recombinant human EPO, and methoxy polyethylene glycol-epoetin ß (PEG-epoetin ß) in horse plasma. All three methods screen samples with an enzyme-linked immunosorbent assay (ELISA) and confirm by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This report focuses on PEG-epoetin ß. The ELISA assay was able to detect PEG-epoetin ß at 0.02 ng/mL in 50 µL of horse plasma. Many samples had high background levels of immunoreactivity; however, introducing polyethylene glycol 6000 (PEG 6000) into the samples before the ELISA assay removed the high background and increased the apparent concentrations of PEG-epoetin ß. In samples collected following the administration of 100 µg of PEG-epoetin ß by the intravenous (IV), intramuscular (IM) and subcutaneous (SC) routes, PEG-epoetin ß was detectable up to 72, 144, and 120 h, respectively. The samples were prepared for LC-MS/MS analysis by extraction with anti-rHuEPO-antibodies-coated Dynabeads followed by digestion with trypsin. The LC-MS/MS confirmation method used the multiple reaction monitoring (MRM) scan mode to monitor four precursor-product ion transitions of the EPO-derived peptide T6. All four transitions of T6 were detectable with S/N > 3. The limit of confirmation for PEG-epoetin ß was 1.0 ng/mL in 2 mL of horse plasma. The method successfully confirmed the presence of PEG-epoetin ß in a sample collected from a Mircera®-treated horse. Compared to PEG-epoetin ß, better sensitivity was achieved for darbepoetin alfa and recombinant human EPO. Darbepoetin alfa was detected in horse plasma four days after IM administration of 100 µg.

Low dose exogenous erythropoietin elicits an ergogenic effect in standardbred horses.

REASONS FOR PERFORMING STUDY: Recombinant human erythropoietin (rhuEPO) causes an increase in red blood cell production and aerobic capacity in other species; however, data are lacking on effects in the horse. HYPOTHESIS: This study tested the hypothesis that rhuEPO administration would alter red cell volume (RCV), aerobic capacity (VO2max) and indices of anaerobic power. METHODS: Eight healthy, unfit mares accustomed to the laboratory and experimental protocols were randomly assigned to either a control (CON, n = 4; 3 ml saline 3 times/week for 3 weeks) or EPO group (EPO, n = 4, 50 iu/kg bwt rhuEPO/3 ml saline 3 times/week for 3 weeks). Exercise tests (GXT) were performed on a treadmill (6% incline), 1 week before and 1 week after treatment. The GXT started at 4 m/sec, with a 1 m/sec increase every 60 sec until the horse reached fatigue. Oxygen uptake was measured via an open flow indirect calorimeter. Blood samples were collected before, during (each step) and 2 and 15 min post GXT to measure packed cell volume (PCV), haemoglobin concentration (Hb), blood lactate concentration (LA) and plasma protein concentration (TP). Plasma volume (PV) was measured using Evans Blue dye. Blood volume (BV) and RCV were calculated using PCV from the 8 m/sec step of the GXT. RESULTS: There were no alterations (P>0.05) in any parameters in CON horses. By week 3, EPO produced increases (P<0.05) in resting PCV (37 +/- 2 vs. 51 +/- 2) and Hb (37%). RCV (26%) and VO2max (19%) increased, but BV did not change (P>0.05) due to decreased PV (-11%, P<0.05). There was a significant increase in velocity at VO2max and LApeak for horses treated with rhuEPO and substantial decrease (P<0.05) in VO2 recovery time when the pretreatment GXT was compared to the post treatment GXT. No differences (P<0.05) were detected for TP, VLA4, run time or Vmax. CONCLUSIONS: Low dose rhuEPO administration increases RCV and aerobic capacity without altering anaerobic power. POTENTIAL RELEVANCE: This study demonstrates that rhuEPO enhances aerobic capacity and exercise performance, a question relevant to racing authorities.

Thermospray liquid chromatography-mass spectrometry of corticosteroids.

A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of steroidal hormones. Using a Nova-Pak C18 reversed-phase column and isocratic elution with a solvent comprised of 25 mM ammonium formate in 30% acetonitrile, corticosteroids were separated within 10 min. This solvent also permitted ultraviolet absorbance detection down to 220 nm with low-nanogram sensitivity. The use of acetonitrile was favourable for thermospray mass spectrometric analysis because mass spectra were obtained with a pseudomolecular ion as the base peak. A combination of liquid chromatography, ultraviolet absorbance detection and thermospray mass spectrometry provided a sensitive and reliable method for unequivocal confirmation of the presence of steroidal drugs in equine urine.

Identification of detomidine carboxylic acid as the major urinary metabolite of detomidine in the horse.

Horse urine was investigated for metabolites by chromatography and mass spectrometry following the oral administration of the large animal analgesic sedative detomidine to two stallions and intravenous administration of [3H]-detomidine to a mare. Detomidine carboxylic acid and hydroxydetomidine glucuronic acid conjugate were identified in the urine after the oral doses. In addition, traces of free hydroxydetomidine were observed. About half of the radioactivity of [3H]-detomidine was excreted in the urine in 12 h after the i.v. dose (80 micrograms/kg). Most of the excretion occurred between 5 and 12 h in contrast to urine output which was highest 2-5 h after the dosing. The major radioactive metabolite in the urine was detomidine carboxylic acid. It comprised more than two thirds of the total metabolites in all the urine fractions collected. Its excretion profile was similar to that of total radioactivity. Hydroxydetomidine glucuronide was also excreted. It contributed 10-20% of the total metabolites in the urine. The free aglycone was only seen in the samples collected during the peak urine flow. A minor metabolite was tentatively characterized as the glucuronide of N-hydroxydetomidine.

Absence of cadmium in the blood of horses fed oats grown on municipal sludge-amended soil.

Effluents from a number of industries which are typically treated in municipal sewage plants result in cadmium-containing sludge. Disposal of such sewage sludge by application to agricultural land can result in uptake of cadmium by crops. In this study, oats were grown on soils which had been amended with sewage sludge from Syracuse, New York. The cadmium concentration in the sludge-grown oats was 1.79 ppm dry weight. Horses were fed the sludge-grown oats for 6 weeks during which time blood samples were taken for the determination of cadmium. No significant differences (p greater than 0.05) were found in the concentration of cadmium in the blood of the horses consuming the oats compared with that in the blood before feeding began (control). The well-known efficient accumulation of renal cadmium by horses may account for a lack of significantly increased cadmium in blood during the feeding period.

The effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study.

Phenylbutazone, a non-steroidal anti-inflammatory drug known to produce intestinal erosions, was administered intravenously (13.46 mg/kg bodyweight) to 12 horses which were killed after 24, 48, 72 and 96 h. Eight untreated horses served as controls. Annular erosions in the duodenum and mucosal necrosis in the colon were seen after 48 h which progressed in severity. The erosions were characterised by sloughing of the surface epithelium, subepithelial cleft and bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries and microthrombosis. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Ultrastructurally, there was swelling of the endothelium of capillaries and small vessels, and of pericyte and smooth muscle cytoplasm in arterioles. In capillaries and post capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and oedema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury associated with the formation of duodenal and colonic erosions in horses. The duodenal and colonic mucosa were assayed at 48 and 96 h for prostacyclin and PGE2. There was no statistically significant difference between prostaglandin levels in the mucosa of control and treated horses. It was concluded that there was no correlation between mucosal prostaglandin levels and intestinal erosions after 48 h.

The effects of phenylbutazone on the morphology and prostaglandin concentrations of the pyloric mucosa of the equine stomach.

Phenylbutazone, a nonsteroidal anti-inflammatory drug known to produce gastric ulcers, was administered intravenously (13.46 mg/kg body weight) daily to 12 horses. Horses were euthanatized daily after 24, 48, 72, and 96 hours following the initial injection. Eight untreated horses served as controls. Small multifocal pyloric erosions were seen after 24 hours and then progressed in severity over time. The erosions were characterized by sloughing of the surface epithelium, subepithelial bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries, and microthrombosis of the capillaries of the pyloric mucosa. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Eroded pyloric mucosa and adjacent macroscopically intact mucosa were examined ultrastructurally. In both the macroscopically eroded mucosa and multifocally in the adjacent macroscopically uneroded mucosa, there was cellular swelling of the endothelium, pericytes, and smooth muscle cells of arterioles. In capillaries and post-capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and edema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury that is associated with the formation of pyloric erosions in horses. The pyloric mucosa of six horses was assayed for prostacyclin and prostaglandin E2 at 48 and 96 hours following the initial injection. There was no statistically significant difference between prostaglandin concentrations in the mucosa of control and treated horses. It was concluded that there was little correlation between pyloric mucosal prostaglandin concentrations and pyloric erosions after 48 hours.

Changes in articular cartilage after intra-articular injections of methylprednisolone acetate in horses.

Eight mature horses with no prior signs of joint disease or history of intra-articular therapy were treated with 8 weekly intra-articular injections of methylprednisolone acetate. Treatments were given at a dose of 120 mg/joint into the right radiocarpal and intercarpal joints, with the left joints as untreated controls. Articular cartilage samples were obtained at necropsy 1, 4, and 8 weeks after the last injection. Compared with controls, cartilage from injected joints had a loss of hematoxylin basophilia and decreased intensity of staining in safranin O fast green dye. Chondrocyte necrosis and hypocellularity were observed in all samples of cartilage from treated joints. Proteoglycan content and its rate of synthesis were reduced. There was a progressive loss of proteoglycan content, whereas proteoglycan synthesis increased somewhat 4 and 8 weeks after treatment. Collagen content was unchanged, but its rate of synthesis was markedly inhibited. Collagen synthesis did not recover, but remained decreased at 5 to 15% of the values from untreated cartilage. Water percentage was increased, but fibronectin content was not significantly different. A single injection of methylprednisolone acetate was also given into the right metacarpophalangeal joints of 3 of the 8 horses in this group, with the left joints serving as untreated controls. Sixteen weeks after the treatment, cartilage of the treated joints had a loss of histochemical staining and proteoglycan content was reduced to 50% of control values. The mean rate of proteoglycan synthesis and mean fibronectin content were increased, but the differences were not statistically significant (P greater than 0.05). Other variables were essentially unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Ameliorative effects of reduced food-borne fluoride on reproduction in silver foxes.

Reduction of ingested fluoride in a skulk of silver foxes resulted in the reduction of fluoride burden, decreased neonatal mortality and increased kit production during a two breeding and whelping season period.

Background lead levels in dairy cattle: updated values for 1986.

Lead poisoning continues to be a problem in cattle. The toxicologic significance of blood lead levels requires the differentiation of background blood lead from toxic levels. A ten-fold variation of background blood lead levels reported in previous studies has prompted us to conduct our own survey. Our results indicate significantly lower background blood lead levels in New York State cattle analyzed in 1986 than in those levels previously reported elsewhere.

Fluoride intoxication in dairy calves.

Chronic fluoride intoxication in dairy cattle, caused by feeding fluoride contaminated commercial feed, was previously described in a dairy herd. Dental fluorosis and a catastrophic decrease in milk yield were the foremost findings. In calves born to the fluoride intoxicated cows, congenital fluorosis was manifested by brown discoloration of enamel, enamel hypoplasia, brown mottling of bone, severe retardation of cartilage cell differentiation, atrophy of osteoblasts, osteopenia, atrophy of bone marrow cells, serous atrophy of bone marrow fat and severely stunted growth.

Track condition and racing injuries in thoroughbred horses.

The incidences of fractures and soft tissue injuries during 68397 starts of thoroughbred horses at New York Racing Association tracks were analyzed concerning track condition, dirt and turf tracks, environmental conditions, length of races, location of fractures on the track, and age of horses. It was concluded that the conditions evaluated are of no importance in the occurrence of racing injuries to thoroughbred horses.

Toxic effects of food-borne fluoride in silver foxes.

Chronic ingestion of excessive amounts of fluoride from commercial fox food is associated with agalactia in vixens resulting in the starvation deaths of large numbers of kits in three fox herds. Evidence of infectious disease or poor management could not be found and a causal relationship between fluoride and high kit mortality is suggested.

Milk production of cows fed fluoride contaminated commercial feed.

A commercial feed concentrate and a mineral mix with excessive amounts of fluoride were introduced into a Holstein dairy herd with an average milk production well above national standards. Milk production decreased drastically, and during the following 6 years the deficit in milk production in the herd ranging from 52 to 120 milking cows was 1.5 million Kg (3 1/4 million lbs.). The tolerance levels set by the National Academy of Sciences for fluoride ingestion by lactating cow were found to be inadequate.