C. E. Medina-Torres

Prevalence of Clostridium difficile in horses

Fecal samples were collected to establish the apparent prevalence of Clostridium difficile shedding in Standardbred and Thoroughbred racehorses housed at 4 racetracks and 2 breeding facilities, and in horses admitted to a referral large animal clinic. Forty-one (7.59%) of 540 racetrack horses, seven (5.83%) of 120 breeding farm horses, and four (4.88%) out of 82 horses admitted to the referral clinic were culture-positive for C. difficile. An overall fecal culture prevalence of 7.01% for C. difficile was identified in 742 fecal samples. PCR-ribotyping and toxin gene identification was performed and seventeen 17 PCR-ribotypes were identified among the 52 C. difficile isolates.

Continuous digital hypothermia initiated after the onset of lameness prevents lamellar failure in the oligofructose laminitis model.

REASONS FOR PERFORMING STUDY Prophylactic digital hypothermia reduces the severity of acute laminitis experimentally but there is no evidence for its efficacy as a treatment once lameness has already developed. OBJECTIVES To investigate the therapeutic effects of digital hypothermia, applied after the onset of lameness, in an experimental acute laminitis model. STUDY DESIGN Randomised, controlled (within subject), blinded, experimental trial. METHODS Eight Standardbred horses underwent laminitis induction using the oligofructose model. Once lameness was detected at the walk, one forelimb was continuously cooled (CRYO), with the other forelimb maintained at ambient temperature (NON-RX). Dorsal lamellar sections (proximal, middle and distal) harvested 36 h after the onset of lameness/initiation of cryotherapy were analysed by 2 blinded observers: laminitis pathology was scored (0 [normal] to 4 [severe]) and morphometric analyses performed. RESULTS Median (interquartile range) histological scores were greater (P<0.05) in NON-RX (proximal 2.8 [2.5-4]; middle 3.5 [2-4]; distal 2.5 [2-3.8]) compared with CRYO limbs (proximal 0.5 [0.5-1.4]; middle 1 [0.6-1]; distal 0.75 [0.5-1]). There was complete physical separation of lamellar dermis from epidermis (score of 4) in 4 of the NON-RX feet at one or more section level(s), which was not observed in any CRYO sections. Histomorphometry was thus limited to sections that remained intact; there was a trend of increased total (TELL) and secondary (SELL) epidermal lamellar length and decreased secondary epidermal lamellar width (SELW) in NON-RX limbs compared with CRYO at all 3 levels; differences were significant (P<0.05) for SELL and SELW in the distal sections. CONCLUSIONS Digital hypothermia reduced the severity of lamellar injury and prevented lamellar structural failure (complete dermoepidermal separation) when initiated at the detection of lameness in an acute laminitis model. This study provides the first evidence to support the use of therapeutic digital hypothermia as a treatment for acute laminitis.

Validation of a commercial enzyme immunoassay for detection of Clostridium difficile toxins in feces of horses with acute diarrhea.

BACKGROUND Clostridium difficile infection (CDI) is a recognized cause of colitis in the horse. Identification of its toxins is important for management of individual cases and for prevention of transmission and zoonosis. In humans, CDI diagnosis is performed with enzyme immunoassays, none of which have been validated for horses. HYPOTHESIS/OBJECTIVES (1) Establish which test for CDI diagnosis was more frequently used by diagnostic laboratories, (2) determine the identified tests performance, sensitivity, and specificity, and (3) validate its use in diarrheic horses. ANIMALS Samples were obtained from 72 horses presented with acute diarrhea and hospitalized at the Ontario Veterinary College, University of Guelph. METHODS A survey was conducted to establish which of the tests for CDI diagnosis in horses is most commonly used throughout North America. A questionnaire was sent to all laboratories registered in the Veterinary Infection Control Society and the American Association of Veterinary Laboratory Diagnosticians. The performance of the test was evaluated by comparison to a cell cytotoxicity assay (CTA), the accepted Gold Standard for C. difficile toxin detection. RESULTS The Techlab C. difficile Tox A/B II ELISA was the most frequently used test. Compared with the CTA, no significant difference was observed, and a good level of agreement (93%) was obtained. The diagnostic performance of the ELISA test was adequate (84% sensitivity and 96% specificity). CONCLUSIONS AND CLINICAL IMPORTANCE Results demonstrate that the Techlab C. difficile Tox A/B II ELISA is a reliable, adequate, and practical tool for identification of C. difficile toxins in horse feces.

The effect of weightbearing and limb load cycling on equine lamellar perfusion and energy metabolism measured using tissue microdialysis

REASONS FOR PERFORMING STUDY Lamellar perfusion is thought to be affected by weightbearing and limb load cycling; this may be critical in the development of supporting limb laminitis. OBJECTIVES To document the effects of unilateral weightbearing and altered limb load cycling on lamellar energy metabolism and perfusion. STUDY DESIGN Randomised, controlled (within subject), experimental trial. METHODS Nine Standardbred horses were instrumented with microdialysis probes in the foot lamellar tissue and skin (over the tail base). Urea (20 mmol/l) was added to the perfusate. Samples were collected every 15 min for a 1 h control period, then during periods of unilateral weightbearing (opposite limb held off the ground for 1 h); enhanced static limb load cycling (instrumented limb lifted every 10 s for 30 min); reduced limb load cycling activity (i.v. detomidine sedation) and continuous walking (30 min). Dialysate concentrations of glucose, lactate, pyruvate and urea were measured and lactate:glucose (L:G) and lactate:pyruvate (L:P) ratios were calculated. For each intervention, values were compared with baseline using nonparametric statistical testing. RESULTS Lamellar dialysate glucose increased and L:G decreased significantly during enhanced static limb load cycling. Glucose and pyruvate increased, and L:G, L:P and urea decreased significantly during walking. Simultaneous skin dialysate values did not change significantly. There were no significant dialysate changes during unilateral weightbearing or after detomidine administration, but only the latter resulted in a significant decrease in limb load cycling frequency. CONCLUSIONS Increases in limb load cycling frequency (particularly walking) caused dialysate changes consistent with increased lamellar perfusion. Unilateral weightbearing (1 h) and a sedation-induced reduction in limb load cycling frequency did not have a detectable effect on lamellar perfusion. More research is needed to confirm the role of hypoperfusion in supporting limb laminitis, but strategies to increase limb load cycling may be important for prevention.

Equine lamellar energy metabolism studied using tissue microdialysis

Failure of lamellar energy metabolism may contribute to the pathophysiology of equine laminitis. Tissue microdialysis has the potential to dynamically monitor lamellar energy balance over time. The objectives of this study were to develop a minimally invasive lamellar microdialysis technique and use it to measure normal lamellar energy metabolite concentrations over 24 h. Microdialysis probes were placed (through the white line) into either the lamellar dermis (LAM) (n = 6) or the sublamellar dermis (SUBLAM) (n = 6) and perfused continuously over a 24 h study period. Probes were placed in the skin dermis (SKIN) for simultaneous comparison to LAM (n = 6). Samples were collected every 2 h and analysed for glucose, lactate, pyruvate, urea and glycerol concentrations. LAM was further compared with SUBLAM by simultaneous placement and sampling in four feet from two horses over 4 h. Horses were monitored for lameness, and either clinically evaluated for 1 month after probe removal (n = 4) or subjected to histological evaluation of the probe site (n = 10). There were no deleterious clinical effects of probe placement and the histological response was mild. Sample fluid recovery and metabolite concentrations were stable for 24 h. Glucose was lower (and lactate:glucose ratio higher) in LAM compared with SUBLAM and SKIN (P < 0.05). Pyruvate was lower in SUBLAM than SKIN and urea was lower in LAM than SKIN (P < 0.05). These differences suggest lower perfusion and increased glucose consumption in LAM compared with SUBLAM and SKIN. In conclusion, lamellar tissue microdialysis was well tolerated and may be useful for determining the contribution of energy failure in laminitis pathogenesis.

Hypoxia and a hypoxia mimetic up-regulate matrix metalloproteinase 2 and 9 in equine laminar keratinocytes

The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.

Microdialysis measurements of lamellar perfusion and energy metabolism during the development of laminitis in the oligofructose model

REASONS FOR PERFORMING STUDY Failure of lamellar energy metabolism, with or without ischaemia, may be important in the pathophysiology of sepsis-associated laminitis. OBJECTIVES To examine lamellar perfusion and energy balance during laminitis development in the oligofructose model using tissue microdialysis. STUDY DESIGN In vivo experiment. METHODS Six Standardbred horses underwent laminitis induction using the oligofructose model (OFT group) and 6 horses were untreated controls (CON group). Microdialysis probes were placed in the lamellar tissue of one forelimb (all horses) as well as the skin dermis of the tail in OFT horses. Dialysate and plasma samples were collected every 2 h for 24 h and concentrations of energy metabolites (glucose, lactate, pyruvate) and standard indices of energy metabolism (lactate to glucose ratio [L:G] and lactate to pyruvate ratio [L:P]) determined. Microdialysis urea clearance was used to estimate changes in tissue perfusion. Data were analysed nonparametrically. RESULTS Median glucose concentration decreased to <30% of baseline by 8 h in OFT lamellar (P = <0.01) and skin (P<0.01) dialysate. Lactate increased mildly in skin dialysate (P = 0.04) and plasma (P = 0.05) but not lamellar dialysate in OFT horses. Median pyruvate concentration decreased to <50% of baseline in OFT lamellar dialysate (P = 0.03). A >5-fold increase in median L:G compared with baseline occurred in OFT lamellar and skin dialysate (P<0.03). From a baseline of <20, median L:P increased to a peak of 80 in OFT skin and 38.7 in OFT lamellar dialysates (P<0.02); however, OFT lamellar dialysate L:P was not significantly different from CON. Urea concentration decreased significantly in OFT lamellar dialysate (increased urea clearance) but not in OFT skin or CON lamellar dialysate. CONCLUSIONS Increased lamellar perfusion occurred during the development of sepsis-associated laminitis in the oligofructose model. Glucose concentrations in the lamellar interstitium decreased, suggesting increased glucose consumption but there was no definitive evidence of lamellar energy failure.

A liquid chromatography-tandem mass spectrometry-based investigation of the lamellar interstitial metabolome in healthy horses and during experimental laminitis induction.

Lamellar bioenergetic failure is thought to contribute to laminitis pathogenesis but current knowledge of lamellar bioenergetic physiology is limited. Metabolomic analysis (MA) can systematically profile multiple metabolites. Applied to lamellar microdialysis samples (dialysate), lamellar bioenergetic changes during laminitis (the laminitis metabolome) can be characterised. The objectives of this study were to develop a technique for targeted MA of lamellar and skin dialysates in normal horses, and to compare the lamellar and plasma metabolomic profiles of normal horses with those from horses developing experimentally induced laminitis. Archived lamellar and skin dialysates (n = 7) and tissues (n = 6) from normal horses, and lamellar dialysate and plasma from horses given either 10 g/kg oligofructose (treatment group, OFT; n = 4) or sham (control group, CON; n = 4) were analysed. The concentrations of 44 intermediates of central carbon metabolism (CCM) were determined using liquid chromatography-tandem mass spectrometry. Data were analysed using multivariate (MVA) and univariate (UVA) analysis methods. The plasma metabolome appeared to be more variable than the lamellar metabolome by MVA, driven by malate, pyruvate, aconitate and glycolate. In lamellar dialysate, these metabolites decreased in OFT horses at the later time points. Plasma malate was markedly increased after 6 h in OFT horses. Plasma malate concentrations between OFT and CON at this time point were significantly different by UVA. MA of lamellar CCM was capable of differentiating horses developing experimental laminitis from controls. Lamellar malate, pyruvate, aconitate and glycolate, and plasma malate alone were identified as the source of differentiation between OFT and CON groups. These results highlighted clear discriminators between OFT and CON horses, suggesting that changes in energy metabolism occur locally in the lamellar tissue during laminitis development. The biological significance of these alterations requires further investigation.

Ultrafiltration of equine digital lamellar tissue.

There are no experimentally validated pharmacological means of preventing laminitis; however, locally acting pharmaceutical agents with the potential to prevent laminitis have been identified. Demonstrating therapeutic drug concentrations in lamellar tissue is essential for evaluating the efficacy of these agents. The aim of this study was to develop an experimental technique for repeatedly sampling lamellar interstitial fluid. A technique for placing ultrafiltration probes was developed in vitro using 15 cadaver limbs. Subsequently, lamellar ultrafiltration probes were placed in one forelimb in six living horses. Interstitial fluid was collected continuously from the probes as ultrafiltrate for 4 (n = 4) or 14 days (n = 2). The rate of ultrafiltrate collection was calculated every 12 h. Biochemical analyses were performed on ultrafiltrate collected on night 1 (12-24 h post-implantation) and night 4 (84-96 h post-implantation). Sections surrounding the probe and control tissue from the contralateral limb were harvested, stained with H&E and Massons trichrome and scored based on the tissue response to the probe. Ultrafiltration probes were placed in the lamellar tissue in all six horses. Ultrafiltrate was collected from these probes at 55 (30-63) μL/h (median [interquartile range]). Fluid production decreased significantly with time from night 3 onwards (P < 0.05). There was no significant change in the constituents of the ultrafiltrate between nights 1 and 4 (P > 0.05). The technique was well tolerated. This study demonstrates that ultrafiltration can be used to sample equine digital lamellar interstitial fluid, and has potential for measuring lamellar drug levels.

Immune response to intranasal modified-live EHV-1 vaccination in immunised equids

Friday 13.30–14.30 13.30–13.45 Cathcart Exhaled carbon monoxide as a marker for lower airway inflammation in thoroughbred racehorses 13.45–14.00 Hermange Cytology of bilateral bronchoalveolar lavage fluids: comparison of pooled and individual samples 14.00–14.15 Losada-Floriano Evaluation of the predictive value of the external laryngeal ultrasound in detecting equine upper airway disease 14.15–14.30 Barba In vitro cytokine production in response to equine influenza virus and Streptococcus equi subspecies zooepidemicus Friday 15.00–16.30 15.00–15.15 Medina-Torres Immune response to intranasal modified-life EHV-1 vaccination in immunised equids 15.15–15.30 Banse Impact of phenylbutazone on gastric glandular prostaglandin concentration and ulcer score 15.30–15.45 Westermann Evaluation of a blood sucrose test for the assessment of gastric ulcers in warmblood horses aimed at field conditions 15.45–16.00 Khan Evaluation of the rectal route of fluid administration in horses 16.00–16.15 Bakos Effects of fasting on serum concentrations of lipid mobilisation and hepatic parameters in horses 16.15–16.30 Kolk Acylcarnitine profile in endurance horses with and without metabolic dysfunction