Eric Richard

Sub-clinical diseases affecting performance in Standardbred trotters: diagnostic methods and predictive parameters.

The objectives of this study were to determine the prevalence of sub-clinical diseases in poorly-performing Standardbred horses, compare their physiological response to exercise with control horses, and identify predictive parameters of poor-performance. Fifty horses underwent thorough clinical and ancillary examinations, including haematological and biochemical evaluation, Doppler echocardiography, standardised exercise tests (SETs) on both treadmill and racetrack, treadmill video-endoscopy and collection of respiratory fluids. Most of the poorly-performing horses exhibited many concomitant diseases. The most frequently diagnosed problems involved the lower and upper respiratory tract and the musculoskeletal system. Poor-performers had lower speeds at a blood lactate (LA) concentration of 4mmol/L (V(LA4)) and a heart rate (HR) of 200bpm (V(200)) on treadmill and racetrack, as well as lower values for haematological parameters, plasma angiotensin-converting enzyme and antioxidants, compared to control horses. Problems of the respiratory system were the most frequently diagnosed sub-clinical diseases affecting performance. SETs, together with some blood markers, may be useful as a non-specific diagnostic tool for early detection of diseases that may affect performance.

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.

Laboratory findings in respiratory fluids of the poorly-performing horse

Any disorder impairing a performance horses ability to ventilate its lungs and exchange oxygen compromises exercise performance in any discipline. Since bronchoalveolar lavage was described in horses in the early 1980s, laboratory evaluation of respiratory fluids, along with clinical and functional assessment of the respiratory system, has become a relevant step in the diagnosis of respiratory disease affecting performance. The aim of this review is to provide objective information to assist clinicians in interpreting laboratory findings by (1) summarising published cytological references values in both clinically healthy horses and those with various airway diseases, (2) assessing the influence of physiological circumstances, such as exercise, on the cytological evaluation, (3) discussing the relationship between cytological and microbiological analyses, clinical signs and respiratory function, and (4) suggesting how this latter relationship may affect performance.

Herpesviruses in respiratory liquids of horses: Putative implication in airway inflammation and association with cytological features

The objectives of this study were to estimate the prevalence and the potential role of equine herpesviruses (EHVs) detection in both bronchoalveolar lavage (BAL) and tracheal wash (TW). The population included a control group (CTL; 37 TW and 25 BAL) and a pathological group (PAT; 259 TW and 387 BAL), including horses either suffering from respiratory diseases including syndrome of tracheal inflammation, inflammatory airway disease, recurrent airway obstruction, or submitted to respiratory investigation because of exercise intolerance or poor performance. Each respiratory liquid was submitted to a standardised cytological analysis, mentioning the morphological abnormalities of exfoliated epithelial cells (ECAb) and ciliocytophthoria (CCPh) as markers of potential viral infection, as well as PCR assays including a consensus PCR and virus-specific PCR for both equine alphaherpesviruses (EHV-1; EHV-4) and gammaherpesviruses (EHV-2; EHV-5). The EHV infections were more prevalent in the TW of PAT group (P=0.004), with the highest prevalence being for EHV-2 (P=0.006). The EHV detection in BALs was not significantly different between groups. The EHVs detection in TW was correlated to the polymorphonuclear neutrophil (PMN) counts in the respiratory liquid but not with CCPh or ECAb. CCPh or ECAb were associated with both consensus PCR and EHV-2 and EHV-5 virus-type PCR in the BAL only. The significant detection of EHVs in the TW of PAT group in association with the PMN increased counts could lead to further investigations about their putative role in equine syndrome of tracheal inflammation.

Influence of subclinical inflammatory airway disease on equine respiratory function evaluated by impulse oscillometry

REASONS FOR PERFORMING STUDY Inflammatory airway disease (IAD) is a nonseptic condition of the lower respiratory tract. Its negative impact on respiratory function has previously been described using either forced expiration or forced oscillations techniques. However, sedation or drug-induced bronchoconstriction were usually required. The impulse oscillometry system (IOS) is a noninvasive and sensitive respiratory function test validated in horses, which could be useful to evaluate IAD-affected horses without further procedures. OBJECTIVES To determine the sensitivity of IOS in detecting alterations of the respiratory function in subclinically IAD-affected horses without inducing bronchoprovocation and to characterise their respiratory impedance according to frequency for each respiratory phase. METHODS Pulmonary function was evaluated at rest by IOS in 34 Standardbred trotters. According to the cytology of bronchoalveolar lavage fluid (BALF), 19 horses were defined as IAD-affected and 15 horses were used as control (CTL). Total respiratory resistance (Rrs) and reactance (Xrs) from 1-20 Hz as well as their inspiratory and expiratory components were compared between groups. RESULTS A significant increase of Rrs at the lower frequencies (R1-10 Hz) as well as a significant decrease of Xrs beyond 5 Hz (X5-20 Hz) was observed in IAD compared to CTL horses. IOS-data was also significantly different between inspiration and expiration in IAD-affected horses. In the whole population, both BALF eosinophil and mast cell counts were significantly correlated with IOS measurements. CONCLUSIONS Functional respiratory impairment may be measured, even in the absence of clinical signs of disease. In IAD-affected horses, the different parameters of respiratory function (Rrs or Xrs) may vary depending on the inflammatory cell profiles represented in BALF. POTENTIAL RELEVANCE Impulse oscillometry could be used in a routine clinical setting as a noninvasive method for early detection of subclinical respiratory disease and of the results of treatment in horses.

Identification of equid herpesvirus-5 in respiratory liquids: A retrospective study of 785 samples taken in 2006–2007

During a case control study undertaken in 2006-2007, a screening and consensus polymerase chain reaction (PCR) was performed to evaluate the potential role of equid herpesviruses (EHV) in several occurrences of respiratory disorders in 661 horses. Of 785 bronchoalveolar or tracheal lavage fluid samples submitted for analysis, 20 were positive for EHV-5 DNA by sequential analysis of the consensus PCR product. Nineteen of those samples were confirmed using a specific EHV-5 PCR. No particular changes in cytological profile could be associated with the detection of EHV-5 in contrast to suggestions in previous reports of natural or experimental respiratory viral infections in horses or ponies. This is the first description of EHV-5 isolation in equine respiratory fluids in Europe, but further investigations are needed to determine the potential pathogenic role of this gammaherpesvirus in the horse.

Detection and quantitation of equid gammaherpesviruses (EHV-2, EHV-5) in nasal swabs using an accredited standardised quantitative PCR method.

Equid gammaherpesviruses-2 and -5 are involved in respiratory problems, with potential clinical manifestations such as nasal discharge, pharyngitis and swollen lymph nodes. These viruses are sometimes associated with a poor-performance syndrome, which may result in a significant and negative economic impact for the horse industry. The aim of the present study was to develop and validate quantitative PCR methods for the detection and quantitation of EHV-2 and EHV-5 in equine respiratory fluids. Two distinct tests were characterised: (a) for the qPCR alone and (b) for the whole method (extraction and qPCR) according to the standard model AFNOR XP U47-600-2 (viz., specificity, quantifiable sensibility, linearity, accuracy, range of application, trueness, precision, repeatability and precision of reproducibility). EHV-2 and EHV-5 detection were performed on nasal swabs collected from 172 horses, all of which exhibited clinical signs of respiratory disease. The data revealed a high rate of EHV-2/EHV-5 co-detection that was correlated significantly with age. Viral load of EHV-2 was significantly higher in young horses whereas viral load of EHV-5 was not significantly different with age.

Serum concentration of surfactant protein D in horses with lower airway inflammation

REASONS FOR PERFORMING STUDY Surfactant protein D (SP-D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP-D are routinely used as biomarkers for pulmonary injury. To date, serum SP-D levels have only been investigated in horses in an experimental model of bacterial airway infection. OBJECTIVES To compare serum SP-D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). METHODS Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD-affected and 20 classified as controls. Serum SP-D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. RESULTS Serum concentrations of SP-D in IAD-affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD-affected group, no significant correlation was found between serum SP-D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP-D levels. CONCLUSIONS This is the first study determining serum SP-D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP-D levels. POTENTIAL RELEVANCE Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.

Molecular detection of Theileria equi and Babesia caballi in the bone marrow of asymptomatic horses.

Equine piroplasmosis is a tick-borne disease, the aetiological agents of which are either Theileria equi or Babesia caballi parasites. Piroplasmosis is commonly encountered in acute or sub-acute clinical forms although clinically recovered horses may remain asymptomatic but infected for several years. The clinical detection of such apparently healthy carrier horses (that serve as a host for subsequent infecting ticks), remains a worldwide challenge for controlling the spread of the disease. The aim of the present paper is to report on the detection of both T. equi and B. caballi by PCR in the bone marrow of naturally infected asymptomatic horses. Among 35 bone marrow samples evaluated for orthopaedic clinical research purposes, three samples from clinically healthy horses were found to be positive for T. equi, one of which was also positive for B. caballi. Even if the precise localisation of these parasites as well as the underlying mechanisms for persistence still remains unknown, one should not exclude bone marrow as a potential reservoir site for T. equi and B. caballi in infected asymptomatic horses. We suggest that, this possible localisation site (the bone marrow) should be considered as a therapeutic target when treating parasitic infection in apparently healthy horses.

Experimental model of equine alveolar macrophage stimulation with TLR ligands.

Pulmonary diseases are common in horses and have a major economic impact on the equine industry. Some of them could be associated with an inadequate immune response in the lung, but methods to evaluate this response in horses are lacking. The aim of this study was to develop and validate an experimental model that could be applied in several physiological and pathological conditions to assess the innate immune response of equine pulmonary cells. Equine alveolar macrophages (AMs) obtained from bronchoalveolar lavages were isolated from other cells by adhesion. TLR2, 3, and 4 expression in AMs was studied and their responses to commercial ligands (respectively FSL-1, Poly(I:C), and LPS) were evaluated after determination of the appropriate dose and time of incubation. TLR responses were assessed by measuring cytokine production using (1) gene expression of TNFα, IFNβ, Il-1β, and IFNα by qPCR (indirect method); and (2) cytokine production for TNFα and IFNβ by ELISA (direct method). TLR 2, 3, and 4 were expressed by AMs. TLR 2 stimulation with 10 ng/mL of FSL-1 during 3h significantly increased IL-1β and TNFα gene expression. TLR 3 stimulation with 1000 ng/mL of Poly(I:C) during 1h increased IFNβ, IFNα, Il-1β and TNFα expression. TLR 4 stimulation with 100 ng/mL of LPS during 3h increased TNFα, IFNβ, and Il-1β expression. Results obtained by ELISA quantification of TNFα and IFNβ produced by AMs following stimulation during 6h were similar: FSL-1 increased TNFα production but not IFNβ, Poly(I:C) and LPS increased production of IFNβ and TNFα. In conclusion, pulmonary innate immunity of horses can be assessed ex vivo by measuring cytokine production following stimulation of AMs with TLR agonists. This experimental model could be applied under several conditions especially to improve the understanding of equine respiratory disease pathogenesis, and to suggest novel therapeutic opportunities.