Tatiana Art

Subclinical diseases underlying poor performance in endurance horses: diagnostic methods and predictive tests

Thirty-eight endurance horses underwent clinical and ancillary examinations, including haematological and biochemical evaluation, standardised exercise tests both on a treadmill and in the field, Doppler echocardiography, impulse oscillometry, video endoscopy and collection of respiratory fluids. All of the examined poorly performing horses were affected by subclinical diseases, and most of them had multiple concomitant disorders. On the contrary, the well-performing horses were free of any subclinical disease. The most frequently diagnosed diseases were respiratory disorders, followed by musculoskeletal and cardiac problems. Poor performers exhibited lower speeds at blood lactate concentration of 4 mmol/l (VLA4) and at heart rates of 160 (V160) and 200 bpm (V200) on the treadmill and in the field, as well as slower recovery of heart rate.

Inhalation with NDS27 attenuates pulmonary neutrophilic inflammation in recurrent airway obstruction

RECURRENT airway obstruction (RAO) or heaves is a chronic respiratory disease that affects mature horses. It is characterised by neutrophilic inflammation and delayed neutrophilic apoptosis (Turlej and others 2001) leading to lower airway inflammation, bronchoconstriction and mucus accumulation. Curcumin has been demonstrated to have a potent inhibitory effect on neutrophilic migration and myeloperoxidase (MPO) release and to induce neutrophilic apoptosis (Hu and others 2005). However, due to its low bioavailability and solubility, its clinical use is limited (Anand and others 2007). The aim of the study was to test the effects of inhalation with the modified highly soluble curcumin derivate NDS27 (World Intellectual Property Organization 2009), on clinical signs, broncho-alveolar lavage fluids (BALF) cytology, and lung function in RAO-affected horses.nnFive RAO-susceptible horses from the University of Lieges research horses herd were used for this blinded randomised cross-over study. The study was approved by the Committee for the Use of Animals of the University of Liege (approval number 611). Horses were kept on low dust wood shavings and were fed with haylage before the experiments and during the two-month wash-out period. Clinical exacerbation of RAO was induced by exposure to dusty hay in all five horses on two different occasions, two months apart. Environmental conditions were maintained during the whole treatment period. All measurements were performed before (T1) and after a treatment …

Effect of strenuous exercise and ex vivo TLR3 and TLR4 stimulation on inflammatory gene expression in equine pulmonary leukocytes.

The effects of strenuous exercise and ex vivo stimulation of TLR3 and TLR4 pathways on the expression of six inflammatory genes in equine pulmonary leukocytes were investigated. The genes tested were interferon-beta (IFN-β), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interferon gamma-induced protein 10 (IP-10), chemokine (c-c motif) ligand 5 (RANTES) and tumor necrosis factor-alpha (TNF-α). We hypothesized that strenuous exercise would modulate basal gene expression on one hand and modulate the response to bacterial lipopolysaccharide (LPS) and to polyinosinic:polycytidylic acid (Poly IC) on the other hand. Eight young Thoroughbred mares were selected for the experiment. Bronchoalveolar lavages were performed on horses 48 h before and 24h after the completion of treadmill exercise until fatigue. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was used to quantify cytokine expression in pulmonary leukocytes. Target gene expression was normalized to the expression of three housekeeping genes (HKG). There were no significant differences in the mRNA expression of the six cytokines between pre-exercise and post-exercise cells. LPS and Poly IC induced respectively significant increases of TNF-α, IFN-β, IL-6, IL-1β, and TNF-α, IFN-β, IP-10 and RANTES, both before and after exercise. However, exercise induced a significant decrease of the genes response to LPS and Poly IC. These findings may suggest that strenuous treadmill exercise exerts a deleterious effect on part of the pulmonary immune response in horses 24h following an intense physical activity.

Expression microarrays in equine sciences

Microarrays have become an important research tool for life science researchers. Expression microarrays are capable of profiling the gene expression pattern of tens of thousands of genes in a single experiment. It appears to be the platform of choice for parallel gene expression profiling. Various equine-specific gene expression microarrays have been generated and used. However, homologous microarrays are not yet commercially available for the horse. An alternative is the use of heterologous microarrays, mainly microarrays specific for mice or humans. Although the use of microarrays in equine research is still in its infancy, gene expression microarrays have shown their potential in equine research. This review presents the previous, current and potential use of expression microarrays in equine research.

Training Modifies Innate Immune Responses in Blood Monocytes and in Pulmonary Alveolar Macrophages

In humans, strenuous exercise causes increased susceptibility to respiratory infections associated with down-regulated expression of Toll-like receptors (TLRs) and costimulatory and antigen-presenting molecules. Lower airway diseases are also a common problem in sport and racing horses. Because innate immunity plays an essential role in lung defense mechanisms, we assessed the effect of acute exercise and training on innate immune responses in two different compartments. Blood monocytes and pulmonary alveolar macrophages (PAMs) were collected from horses in untrained, moderately trained, intensively trained, and deconditioned states before and after a strenuous exercise test. The cells were analyzed for TLR messenger ribonucleic acid (mRNA) expression by real-time PCR in vitro, and cytokine production after in vitro stimulation with TLR ligands was measured by ELISA. Our results showed that training, but not acute exercise, modified the innate immune responses in both compartments. The mRNA expression of TLR3 was down-regulated by training in both cell types, whereas the expression of TLR4 was up-regulated in monocytes. Monocytes treated with LPS and a synthetic diacylated lipoprotein showed increased cytokine secretion in trained and deconditioned subjects, indicating the activation of cells at the systemic level. The production of TNF-α and IFN-β in nonstimulated and stimulated PAMs was decreased in trained and deconditioned horses and might therefore explain the increased susceptibility to respiratory infections. Our study reports a dissociation between the systemic and the lung response to training that is probably implicated in the systemic inflammation and in the pulmonary susceptibility to infection.

The innate immune response of equine bronchial epithelial cells is altered by training

Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway endoscopy. EBEC were grown in vitro by an explant method. The innate immune response of EBEC was evaluated in vitro by treatment with TLR ligands. TLR3 is the most strongly expressed TLR at the mRNA level in EBEC and stimulation of EBEC with Poly(I:C), an analog of viral dsRNA, triggers a strong secretion of IFN-β, TNF-α, IL-6 and CXCL8. We further evaluated the EBEC innate immune response in horses that underwent a 4-month-training program. While training had no effect on TLR mRNA expression in EBEC as well as in bronchial biopsies, it increased the production of IFN-β after stimulation with a TLR3 ligand and decreased the secretion of TNF-α and IL-6 after stimulation with a TLR2 and TLR3 ligand. These findings may be implicated in the increased risk for viral and bacterial infections observed in sport horses. Altogether, we report a successful model for the culture of EBEC that can be applied to the investigation of pathophysiologic conditions in longitudinal studies.

Effects of a P-class CpG-ODN administered by intramuscular injection on plasma cytokines and on white blood cells of healthy horses

Cytosine-phosphate-guanosine (CpG-ODN) has been described as a potent immunostimulatory agent in different species. No study reported the effect of a P-class CpG when administered systemically in healthy horses. The aim of this study was to evaluate the tolerance and the effect of an intramuscularly administered P-class CpG-ODN on hematology and on plasma cytokines (IFN-α, IL-10, TNF-α, IFN-γ) in 8 healthy horses. Intra-muscular CpG-ODN or placebo (PBS) was administered twice at a 7u202fdays-interval. Groups were inversed after 2 months of washout period. A physical examination, complete blood count (CBC) and plasma cytokine measurements were performed from 2u202fdays before injection up to 21u202fdays after injection. P-class CpG-ODN injection was well tolerated with minor side effects. After the first injection a significant transient drop in circulating total leukocytes, lymphocytes and an increase in monocytes were observed. A transient drop in eosinophils was also noted after each CpG injection. P-class CpG-ODN at a dose of 5u202fmg did not create major side effects in 7 horses, one horse showed transient pyrexia. A redistribution of white blood cells was observed in horses receiving CpG, but no change in plasma cytokines was observed at the indicated dose, route of administration and sampling times.

Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.