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Rural Industries Research & Development Corporation
October 2003
RIRDC Publication No 03/089 RIRDC Project No US 82-A
Executive Summary
Inflammatory respiratory disease
continues to be a serious cause of wastage to the performance horse industries
in Australia and overseas. Substantial losses are incurred from inability
to train horses effectively, from costs of veterinary care and medication,
and decreased earning potential. In addition, up to 33% of young racehorses
in training can have lower airway inflammation without demonstrating any
clinical signs of respiratory disease, emphasising the serious welfare
implications of this important respiratory disorder.
Recently, studies have demonstrated the significance of specific bacteria and their association with neutrophilic inflammatory airway disease (IAD) in young racehorses, and have highlighted the lack of association with common equine respiratory viruses. However, these studies also underlined the importance of non-infectious environmental factors in producing IAD. Young racehorses housed in a range of stable conditions potentially are exposed to high burdens of aerosolised non-infectious dusts and gases in an almost continuous and cumulative manner. Spending most of their time (up to 22 hours) in looseboxes, racehorses are exposed to variably large burdens of dusts from bedding, feed, loosebox materials, and sources outside the loosebox such as from swept laneways, stable corridors and human and mechanical activity. Endotoxin, a major pro-inflammatory glycolipid component of Gram-negative bacterial cell walls, is one agent that is ubiquitously present in airborne dusts and, as in humans, may play an critical role in the genesis of IAD in young racehorses. In addition, along with the potential for exposure to high dust burdens, racehorses are placed under repeated stresses from transport, sales, variably intense training/racing regimens, intermingling, and housing in frequently poorly ventilated stables. A major outcome of these stressful events is likely to be an increased susceptibility to inhaled agents occurring as a result of corticosteroid-induced impairment in airway defences.
Under these conditions, many young racehorses may therefore be prone to development of lower airway inflammation, which may progress and be complicated by bacterial colonisation, given perpetuation of “stresses” and/or continued exposure to airborne agents. However, the relative contribution of environmental and stable factors, and specific non-infectious dust and gaseous agents over time (longitudinally), to development of IAD in young racehorses housed in stables in Sydney is unknown. In addition, the proportion of racehorses that develop IAD when housed in racetrack stables has not yet been determined under Australian conditions. Consequently, the major aims of the research presented in this report was to determine the incidence of IAD in young Thoroughbred racehorses, investigate as many non-infectious stable management and environmental risk factors for IAD, and to determine the concentrations of specific dust toxins (endotoxin and (1>3)-?-D-glucan) in stable environments and their association with IAD.
A cohort (or longitudinal) study, was designed and carried out between March 2000 and December 2001 using a cohort of 2 and 3-year-old thoroughbred racehorses drawn from a geographically well-defined metropolitan area in Sydney. All eligible, healthy 2 and 3-year-old racehorses that arrived at racetrack stables for training were followed for 4 weeks during which time historical, physical examination, stable management and meteorological information was gathered. In addition, upper and lower respiratory tract endoscopic examination was performed on all horses. From each horse, tracheal aspirate (TA) samples were collected, using a sterile guarded catheter, for cytological and bacteriological analysis, as were blood samples for serological analysis (to detect antibody responses to common respiratory viruses). The same data was gathered from every horse on 3 occasions (Day 0, 14 and 28).
It was found that more than a third of 2 and 3-year-old thoroughbred racehorses entering stables for training already had some form of lower airway inflammation (in which neutrophils or eosinophils or both predominated in TA samples). This most likely reflects exposure to airborne inflammogenic or allergenic agents, or (specifically in the case of eosinophilic IAD) perhaps irregular anthelmintic treatments at pre-training establishments prior to transport to training stables.
Of the racehorses that were IAD free upon arrival to racetrack stables, it was estimated that just over 40% developed neutrophilic lower airway inflammation within the first 2 weeks of stabling. Although in half of these horses IAD resolved during the next 2 weeks, this cumulative incidence of IAD nevertheless is very high and has important implications regarding possible progression of inflammation to clinical disease, decreased training potential and welfare concerns.
Descriptive information on relevant risk factors suggested that hay-type fed (straw-like versus lucerne or clover), and ventilation quality (as determined by window(s) open in the loosebox and carbon dioxide concentrations in boxes), was the most important determinants of neutrophilic IAD in racehorses. In addition, it was found that single endoscopic examinations of proximal lower airways and identification of minor tracheal discharge is unreliable as a means to determine presence of neutrophilic IAD. Finally, no horses in this study were infected with equine influenza virus although most had previously been exposed to Equine Herpes Virus-1 and -4 or recently been infected with Equine Rhinitis Virus A and B.
When all relevant variables were examined together, a number of significant risk factors were identified for neutrophilic IAD in young thoroughbred racehorses. Young racehorses were significantly less likely to develop neutrophilic IAD if housed in looseboxes with at least one open window or if all four walls were open to constant ventilation, after controlling for other confounding risk factors. This result emphasises the importance of stable and individual box ventilation in preventing lower airway inflammation. In addition, young racehorses were almost 3-4 times more likely to develop IAD if exposed on average to greater than 5.7 mm/2weeks of evaporation. Although reasons are unclear, this result may reflect levels of humidity, ambient temperature and wind, each of which may contribute to detrimental effects on respiratory defences and airborne dust levels in stables and looseboxes from bedding and feed.
Finally, sex, trainer, stable area, factors related to current training, bedding type, quality and age, other stable management factors and infection with common respiratory viruses were not significantly associated with development of neutrophilic IAD in this population of young racehorses.
In order to determine the association between lower airway inflammation and inhaled endotoxin and (1>3)-?-D-glucan found in airborne dust in stables, a case-control study was nested within the cohort study. Dust samples, for determination of endotoxin and (1>3)-?-D-glucan concentrations, and ammonia and carbon dioxide samples were collected only from selected cases and controls. A horse was classified as a case if cytological examination of smears from a TA sample collected either on Day 14 or Day 28 demonstrated > 20% neutrophils (and . 5% eosinophils) on differential count of at least 300 inflammatory cells. In contrast, controls were horses with . 20% neutrophils and . 5% eosinophils on cytological examination of smears from a TA sample.
It was found that a significant linear (or exposure-response) relationship existed between the average percentage of neutrophils in lower airways and exposure to high (>4-5ng/m3) respirable endotoxin concentrations in breathing zone dust. In addition, young racehorses that became cases (as defined) and developed neutrophilic IAD were approximately 4 times more likely to have been exposed to either low (<1.2ng/m3) or high (>4.2ng/m3) concentrations of endotoxin. These associations controlled for the possible confounding effects of many other variables, highlighting the importance of endotoxin alone in the genesis of neutrophilic lower airway inflammation. The effect of endotoxin was likely the result of activation of macrophages and epithelial cells in airways and subsequent release of pro-inflammatory mediators. Racehorses with naïve airways probably responded to lower endotoxin concentrations whereas airways in horses that had adapted to cumulative prior exposures required much higher concentrations to induce a significant neutrophilic airway inflammation. Finally, although mould exposure is a major environmental factor implicated in a variety of respiratory disorders, (1>3)-?-D-glucan, a fungal cell wall component, was not found to be associated with neutrophilic airway inflammation in this population of young racehorses.
In conclusion, results of this study indicate that endotoxin from dust sources (such as feed, bedding and sources outside boxes) in stables is associated with neutrophilic IAD in 2 and 3 year old thoroughbred racehorses in Australia. As in humans, the severity of this airway inflammation is related to the endotoxin concentration of the inhaled dust. Ventilation quality and meteorological conditions, particularly evaporation level, in looseboxes and stables also were significant risk factors for IAD. This has important practical implications, specifically for stable and ventilation design, and feed and bedding management.
Horses housed in environments that encourage high dust exposure may be more prone to the early developmental stages of IAD and therefore as training progresses may be more likely to develop bacterial complications and clinical respiratory disease.
