Ethyl pyruvate diminishes the inflammatory response to lipopolysaccharide infusion in horses

Reasons for performing the study: Endotoxaemia contributes to morbidity and mortality in horses with colic due to inflammatory cascade activation. Effective therapeutic interventions are limited for these horses. Ethyl pyruvate (EP), an anti‐inflammatory agent that alters the expression of proinflammatory cytokines, improved survival and organ function in sepsis and gastrointestinal injury in rodents and swine. Therapeutic efficacy of EP is unknown in endotoxaemic horses. Objectives: Determine the effects of EP on signs of endotoxaemia and expression of proinflammatory cytokines following administration of lipopolysaccharide (LPS) in horses. Methods: Horses received 30 ng/kg bwt LPS in saline to induce signs of endotoxaemia. Next, horses received lactated Ringer's solution (LRS), (n = 6), 150 mg/kg bwt EP in LRS, (n = 6), or 1.1 mg/kg bwt flunixin meglumine (FM), (n = 6). Controls received saline followed by LRS (n = 6). Physical examinations, behaviour pain scores and blood for clinical pathological testing and gene expression were obtained at predetermined intervals for 24 h. Results: Lipopolysaccharide infusion produced clinical and clinicopathological signs of endotoxaemia and increased expression of tumour necrosis factor alpha (TNFα), interleukin 6 (IL‐6) and IL‐8 (P<0.001) compared with controls. Leucopenia and neutropenia occurred in all horses that received LPS. Horses treated with EP and FM had significantly (P<0.0001) reduced pain scores compared with horses receiving LPS followed by LRS. Flunixin meglumine was significantly more effective at ameliorating fever compared with EP. Both EP and FM significantly diminished TNFα expression. Ethyl pyruvate significantly decreased, but FM significantly increased, IL‐6 expression. Neither EP nor FM altered IL‐8 expression. Conclusions and potential relevance: Ethyl pyruvate administered following LPS diminished the clinical effects of endotoxaemia and decreased proinflammatory gene expression in horses. Ethyl pyruvate suppressed expression of proinflammatory cytokines better than FM. However, FM was a superior anti‐pyretic compared with EP. Ethyl pyruvate may have therapeutic applications in endotoxaemic horses.     

Cardiovascular effects of intermittent or continuous treadmill conditioning in horses

Nine mature Quarter Horse mares were trained for ten weeks by continuous (n=4) or intermittent (n=5) treadmill work. Both groups of horses trotted at 3.3m/sec. The continuous work was performed at a 3% incline while the intermittent work was done at a 9% incline. Heart rate, cardiac output and blood lactate concentration were measured during a standard exercise test that included work on both treadmill grades before and after five and ten weeks of training. The two conditioning programs did not produce differences in the measured parameters of the two groups of horses. However, there was an overall conditioning effect observed in both groups of horses throughout the ten weeks of treadmill conditioning. Heart rate decreased (P<.05) during exercise on both treadmill grades after ten weeks of training. Lactate concentration decreased (P<.05) during the 9 percent grade trot after five weeks of training. Only slight further decreases occurred between week five and week ten of conditioning. Cardiac output increased (P<.05) between rest and exercise on both grades but there was no conditioning effect.     

Behavior of stabled horses provided continuous or intermittent access to drinking water

OBJECTIVE: To compare quantitative measures and clinical assessments of behavior as an indication of psychologic well-being of stabled horses provided drinking water continuously or via 1 of 3 intermittent delivery systems. ANIMALS: 22 Quarter Horse (QH) or QH-crossbred mares and 17 Belgian or Belgian-crossbred mares (study 1) and 24 QH or QH-crossbred mares and 18 Belgian or Belgian-crossbred mares (study 2). PROCEDURE: Stabled horses were provided water continuously or via 1 of 3 intermittent water delivery systems in 2 study periods during a 2-year period. Continuous 24-hour videotaped samples were used to compare quantitative measures and clinical assessments of behavior among groups provided water by the various water delivery systems. RESULTS: All horses had clinically normal behavior. Significant differences in well being were not detected among groups provided water by the various delivery systems. CONCLUSIONS AND CLINICAL RELEVANCE: Various continuous and intermittent water delivery systems can provide adequately for the psychologic well-being of stabled horses.     

Antibiotic prophylaxis of lower respiratory tract contamination in horses confined with head elevation for 24 or 48 hours

Objective To evaluate the administration of procaine penicillin prior to or during confinement with head elevation as a means of reducing the associated accumulation of inflammatory lower respiratory tract secretions and increased numbers of bacteria within the lower respiratory tract of confined horses. Design and Procedure Two experiments were conducted to evaluate the efficacy of different dose rates and dosing frequencies. In experiment A a single low dose (15,000 IU/kg) of procaine penicillin was administered to four horses immediately prior to confinement with head elevation for 48 hours. The systemic leucocyte response, gross and cytologic characteristics of transtracheal aspirate and bacterial numbers in lower respiratory tract samples were compared with corresponding samples from two horses confined with heads elevated but not given penicillin. The efficacy of higher dose rates (20,000 IU/kg and 40,000 IU/kg) given before and during confinement with heads elevated for 24 hours was evaluated in experiment B. Results Treatment with procaine penicillin had no effect on the systemic leucocyte response or on the accumulation of inflammatory lower respiratory tract secretions at any of the dosing schedules evaluated. The number of bacteria isolated from trans-tracheal samples was reduced at 12 hours for treated horses in experiment A and at 24 hours for experiment B. β-haemolytic Streptococcus spp were not isolated from treated horses in either experiment. Bacterial species isolated from treated horses were predominantly Pasteurella and/or Actinobacillus spp, however, members of the family Enterobacteriaceaé and a Staphylococcus sp were isolated from treated horses. One treated horse in experiment A developed clinically apparent pulmonary disease. Conclusions The prophylactic administration of penicillin before or during confinement did not reliably reduce bacterial numbers or prevent the accumulation of purulent lower respiratory tract secretions in horses confined with their heads elevated. Numbers of β-haemolytic Streptococcus spp were reduced following treatment, suggesting that the repeated administration of procaine penicillin may have some merit as part of a strategy to prevent transport-associated respiratory disease. However, methods directed at minimising the duration of confinement with head elevation, augmentation of the clearance of accumulated secretions and prompt identification of animals in which airway inflammation has extended to the pulmonary parenchyma remain the best ways of minimising transport-associated respiratory disease.     

Effects of intermittent and continuous administration of decoquinate on bovine coccidiosis in male calves

Male Holstein calves were each inoculated with 350,000 sporulated oocysts of Eimeria bovis. Two calves were given decoquinate (0.5 mg/kg of body weight) continuously in dry feed for 29 days, and 2 calves each were given 0.5, 1, or 1.5 mg of decoquinate/kg on an every 2nd-or 3rd-day schedule for 29 days. Calves given decoquinate continuously did not discharge oocysts but had slightly loose feces. In general, the number of oocysts discharged increased and fecal consistency decreased as the time between feeding of medicated feed increased. Calves given 0.5 or 1.5 mg of decoquinate/kg every 3rd day discharged more oocysts and had more diarrhea than did calves given 1 mg of decoquinate/kg every 3rd day. At postinoculation day 29, calves were euthanatized. At necropsy, intestinal tissues of calves given decoquinate were mostly normal. Apparently, reduced infections along with the elapsed time were sufficient to resolve most intestinal lesions caused by the coccidia. Decoquinate was most effective when fed continuously at 0.5 mg/kg. However, when fed at 1 or 1.5 mg of decoquinate/kg every 2nd day or 1.5 mg of decoquinate/kg every 3rd day, oocyst production was reduced and clinical coccidiosis was prevented.     

Effects of continuous oral administration of phenylbutazone on biomarkers of cartilage and bone metabolism in horses

OBJECTIVE: To evaluate the effects of continuous oral administration of phenylbutazone on serum and synovial fluid biomarkers of skeletal matrix metabolism in horses. ANIMALS: 11 adult female horses without clinical or radiographic evidence of joint disease. PROCEDURES: Horses were randomly assigned to control or treatment groups. Phenylbutazone was administered orally twice daily at a dose of 4.4 mg/kg for 3 days to the treatment group and subsequently at a dose of 2.2 mg/kg for 7 days. Serum and radiocarpal synovial fluid samples were obtained at baseline and thereafter at regular intervals for 4 weeks. Biomarkers of cartilage aggrecan synthesis (chondroitin sulfate 846) and type II collagen synthesis (procollagen type II C-propeptide) and degradation (collagen type II cleavage) were assayed. Biomarkers of bone synthesis (osteocalcin) and resorption (C-terminal telopeptide of type I collagen) were also measured. RESULTS: No significant differences were found between control and treatment groups or temporally for the biomarkers chondroitin sulfate 846, procollagen type II C-propeptide, collagen type II cleavage, and C-terminal telopeptide of type I collagen in serum or synovial fluid. A significant increase in osteocalcin concentration occurred in synovial fluid during treatment in the treated group. No treatment effect was detected for serum osteocalcin concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that continuous phenylbutazone administration at recommended doses altered some biomarkers in healthy equine joints after short periods of administration. Increased osteocalcin concentration may indicate an undetermined anabolic effect of phenylbutazone administration on periarticular bone or transient induction of osteogenesis in articular chondrocytes or a mesenchymal subpopulation of synoviocytes.     

Effect of lipopolysaccharide infusion on gene expression of inflammatory cytokines in normal horses in vivo

Horses are exquisitely sensitive to bacterial endotoxin and endotoxaemia is common in colic cases. In this study, gene expression of inflammatory cytokines was characterised in the blood of healthy horses following i.v. administration of lipopolysaccharide (LPS). Six horses received an LPS infusion and 6 controls received an equivalent volume of saline. Gene expression of genes encoding interleukin (IL)‐1α, IL‐1β, IL‐6, IL‐8, and tumour necrosis factor‐α (TNF‐α) was quantified by real‐time PCR. Gene expression of all inflammatory cytokines was upregulated following administration of LPS. Interleukin‐1α, IL‐1β, IL‐8 and TNF‐α gene expression peaked at 60 min, while IL‐6 expression peaked at 90 min post LPS infusion. Interleukin‐1β and IL‐6 messenger RNA expression levels were above the baseline values 3 h post LPS infusion, whereas IL‐1α, IL‐8 and TNF‐α expression levels returned to baseline values by 3 h after LPS infusion. It was concluded that LPS infusion upregulated gene expression of inflammatory cytokines in the blood of healthy horses.     

Effects of acute hyperinsulinemia on inflammatory proteins in horses

Laminitis is a painful, inflammatory disease of the equine hoof that often results in euthanasia. Elevated plasma insulin concentrations are a predictive factor for laminitis, and in previously healthy horses and ponies, laminitis was induced by infusion of insulin. Thus, we chose to determine if an infusion of insulin would increase plasma concentrations of inflammatory cytokines and cytokine mRNA abundance in subcutaneous adipose tissue, skeletal muscle, and white blood cells. Ten mature Thoroughbred mares received an insulin infusion that elevated plasma insulin concentrations for 6h or an equivalent volume of isotonic saline in a switchback design. Insulin infusion altered plasma concentrations of both TNF (P=0.037) and IL-6 (P=0.044), but did not result in consistent changes to either skeletal muscle or adipose tissue cytokine mRNA. Insulin may be involved in the production of inflammatory cytokines, and this could be a mechanism for insulin increasing the risk of laminitis.     

Effects of continuous versus intermittent transport on plasma constituents and antibody response of lambs

Recommendations for transportation of lambs, horses, calves, and pigs from a committee of the European Commission, which required rest stops of 6 or 24 h, every 8 h, were evaluated using Rambouillet x Suffolk lambs. The lambs of 17.6 +/- 0.5 kg of BW were randomly assigned to 1 of 3 treatments: transported for 22 h (continuous; n = 15); transported for 8 h, unloaded and rested for 6 h, transported for 8 h, unloaded and rested for 24 h, transported for 6 h (rested, n = 15); or remained in the home pasture throughout the study (control, n = 16). Off-trailer rest with food and water occurred in novel pens. Food deprivation in the continuous lambs was reflected by a decrease (P < 0.001) in plasma concentrations of glucose and an increase (P < 0.02) in plasma concentrations of blood urea nitrogen, creatinine, and total bilirubin relative to rested or control lambs. Electrolytes varied within and among all 3 treatments (P < 0.05), but no distinct pattern indicating dehydration was evident. Serum concentrations of cortisol were elevated in continuous and rested lambs compared with control lambs at 22 h (P < 0.05). Plasma immunoglobulin G antibody response to ovalbumin was suppressed (P < 0.05) in the continuous and rested lambs relative to the control lambs. Differences (P < 0.05) between continuous and rested lambs indicated the rest stops were sufficient to maintain BW during transport; however, these results were confounded by the control lambs losing a similar (P = 0.50) percentage of their initial BW as the continuous lambs at 22 h. The rest stops eliminated the physiological indicators of food deprivation and maintained BW but did not alleviate evidence of immunosuppression, and 52 h was required to complete the otherwise 22-h-long trip.     

Clinical, biochemical, and hygiene assessment of stabled horses provided continuous or intermittent access to drinking water

OBJECTIVE: To compare health, hydration status, and management of stabled pregnant mares provided drinking water continuously or via 1 of 3 intermittent delivery systems. ANIMALS: 22 Quarter Horse (QH) or QH-crossbred mares and 18 Belgian or Belgian-crossbred mares (study 1); 24 QH or QH-crossbred mares and 18 Belgian or Belgian-crossbred mares (study 2). PROCEDURE: Stabled horses were provided water continuously or via 1 of 3 intermittent water delivery systems in 2 study periods during a 2-year period. Body temperature, attitude, appetite, water intake, and urine output were recorded daily. Hygiene of each horse and the stable were assessed weekly. Clinical and biochemical measures of hydration were determined 3 times during each study. Clinical measures of hydration included skin turgor, gum moisture, capillary refill time, and fecal consistency. Biochemical measures of hydration included PCV, plasma total protein concentration, serum osmolality, plasma vasopressin concentration, urine specific gravity, and urine osmolality. RESULTS: All horses remained healthy. Stable hygiene was worse when horses had continuous access to water. Clinical and biochemical measures of hydration did not differ among water delivery systems. CONCLUSIONS AND CLINICAL RELEVANCE: Various continuous and intermittent water delivery systems provided adequate amounts of water to stabled horses to maintain health and hydration status. Providing intermittent access to water may be preferable on the basis of stable hygiene.     

Effect of intermittent oral administration of ponazuril on experimental Sarcocystis neurona infection of horses

OBJECTIVE: To evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against Sarcocystis neurona in horses inoculated intragastrically with S neurona sporocysts. ANIMALS: 20 healthy horses that were seronegative for S neurona-specific IgG. PROCEDURES: 5 control horses were neither inoculated with sporocysts nor treated. Other horses (5 horses/group) each received 612,500 S neurona sporocysts via nasogastric tube (day 0) and were not treated or were administered ponazuril (20 mg/kg, PO) every 7 days (beginning on day 5) or every 14 days (beginning on day 12) for 12 weeks. Blood and CSF samples were collected on day - 1 and then every 14 days after challenge for western blot assessment of immunoconversion. Clinical signs of equine protozoal myeloencephalitis (EPM) were monitored, and tissues were examined histologically after euthanasia. Results: Sera from all challenged horses yielded positive western blot results within 56 days. Immunoconversion in CSF was detected in only 2 of 5 horses that were treated weekly; all other challenged horses immunoconverted within 84 days. Weekly administration of ponazuril significantly reduced the antibody response against the S neurona 17-kd antigen in CSF. Neurologic signs consistent with EPM did not develop in any group; likewise, histologic examination of CNS tissue did not reveal protozoa or consistent degenerative or inflammatory changes. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of ponazuril every 7 days, but not every 14 days, significantly decreased intrathecal anti-S neurona antibody responses in horses inoculated with S neurona sporocysts. Protocols involving intermittent administration of ponazuril may have application in prevention of EPM.     

Effects of intravenous administration of peripheral blood‐derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses

BackgroundA systemic and dysregulated immune response to infection contributes to morbidity and mortality associated with sepsis. Peripheral blood‐derived mesenchymal stromal cells (PB‐MSC) mitigate inflammation in animal models of sepsis. Allogeneic PB‐MSC administered IV to horses is well‐tolerated but therapeutic benefits are unknown.HypothesisAfter IV lipopolysaccharide (LPS) infusion, horses treated with PB‐MSC would have less severe clinical signs, clinicopathological abnormalities, inflammatory cytokine gene expression, and oxidative stress compared to controls administered a placebo.AnimalsSixteen horses were included in this study.MethodsA randomized placebo‐controlled experimental trial was performed. Sixteen healthy horses were assigned to 1 of 2 treatment groups (1 × 10⁹ PB‐MSC or saline placebo). Treatments were administered 30 minutes after completion of LPS infusion of approximately 30 ng/kg. Clinical signs, clinicopathological variables, inflammatory cytokine gene expression, and oxidative stress markers were assessed at various time points over a 24‐hour period.ResultsA predictable response to IV LPS infusion was observed in all horses. At the dose administered, there was no significant effect of PB‐MSC on clinical signs, clinicopathological variables, or inflammatory cytokine gene expression at any time point. Antioxidant potential was not different between treatment groups, but intracellular ROS increased over time in the placebo group. Other variables that changed over time were likely due to effects of IV LPS infusion.Conclusions and Clinical ImportanceAdministration of allogeneic PB‐MSC did not cause clinically detectable adverse effects in healthy horses. The dose of PB‐MSC used here is unlikely to exert a beneficial effect in endotoxemic horses.