R. S. Pirie

Pulmonary and systemic effects of inhaled endotoxin in control and heaves horses

To investigate whether inhaled endotoxin contributes to airway inflammation and dysfunction in stabled horses, control (n = 6) and asymptomatic heaves (previously termed chronic obstructive pulmonary disease)-susceptible (n = 7) horses were given inhalation challenges with 20, 200 and 2,000 microg of soluble Salmonella typhimurium Ra60 lipopolysaccharide (LPS). LPS inhalation induced a dose-dependent neutrophilic airway inflammatory response in both groups. Inhalation with 2,000 microg of LPS also induced detectable lung dysfunction in the heaves group. LPS inhalation did not alter clinical score, tracheal secretion volume or airway reactivity in either group. The no-response thresholds were lower for the heaves group (<20 microg for airway inflammation; 200 to 2,000 microg for lung dysfunction) than for the control group (20 to 200 microg for airway inflammation; >2,000 microg for lung dysfunction). To enable comparison of these threshold levels with airborne endotoxin concentrations in stables, horses also received a 5 h duration hay/straw challenge, during which the total and respirable airborne endotoxin concentrations were determined. Comparison of the effects of acute LPS inhalation and hay/straw challenges suggest that inhaled endotoxin is not the sole cause of heaves. However, it is likely that it contributes to airway inflammation, both in heaves horses in concert with other inhalants, and in normal horses when they are exposed to high levels in poor stable environments.

Recurrent airway obstruction: A review

Recurrent airway obstruction is a widely recognised airway disorder, characterised by hypersensitivity-mediated neutrophilic airway inflammation and lower airway obstruction in a subpopulation of horses when exposed to suboptimal environments high in airborne organic dust. Over the past decade, numerous studies have further advanced our understanding of different aspects of the disease. These include clarification of the important inhaled airborne agents responsible for disease induction, improving our understanding of the underlying genetic basis of disease susceptibility and unveiling the fundamental immunological mechanisms leading to establishment of the classic disease phenotype. This review, as well as giving a clinical overview of recurrent airway obstruction, summarises much of the work in these areas that have culminated in a more thorough understanding of this debilitating disease.

Inhalation of organic dusts and lipopolysaccharide increases gelatinolytic matrix metalloproteinases (MMPs) in the lungs of heaves horses

We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the majority of gelatinolytic activity comprising pro- and active MMP-9. The 5 h duration hay/straw challenge increased BALF gelatinolytic MMP activity in heaves horses at 5 and 24 h after the start of this challenge, with activity returning to baseline by Day 4. In contrast to hay/straw and HDS challenges, LPS inhalation increased BALF gelatinolytic MMP activity in both groups. For all challenges, absolute BALF neutrophil counts were highly significantly correlated (P<0.0001) with levels of proMMP-9 and active MMP-9, but not with levels of MMP-2 (P>0.05). As gelatinolytic MMPs are pro-inflammatory agents, they may contribute to lung dysfunction and tissue destruction in heaves horses exposed to airborne organic stable dusts.

Equine grass sickness

Equine grass sickness (EGS; equine dysautonomia) is a polyneuronopathy affecting both the central and the peripheral nervous systems of horses. As the name implies, EGS almost exclusively affects grazing horses, resulting in the development of a characteristic array of clinical signs, most of which can be attributed to neuronal degeneration in the autonomic and enteric nervous systems. Varying disease severities occur, largely determined by the extent of neuronal degeneration in the myenteric and submucous plexuses of the enteric nervous system. Extensive neuronal degeneration, as seen in acute and subacute forms of EGS, results in intestinal dysmotility, the severity of which is incompatible with survival. In comparison, a proportion of chronic forms of EGS, characterised by less severe neuronal degeneration, will survive. Despite extensive research efforts since EGS was first reported over 100 years ago, the precise aetiology remains elusive. This article reviews much of the scientific literature on EGS, covering epidemiology, pathology, diagnosis, treatment and aetiological hypotheses.

Dose responses to inhalation of endotoxin, hay dust suspension and Aspergillus fumigatus extract in horses as measured by levels and activation of matrix metalloproteinase‐9

REASONS FOR PERFORMING STUDY Airway matrix metalloproteinases (MMPs) increase after endotoxin (LPS) exposure, but there are no reports describing dose-dependent increases or activation following exposure. OBJECTIVES To study matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) responses in bronchoalveolar lavage fluid (BALF) from heaves-susceptible and control horses following inhalation of hay dust suspension (HDS), LPS and Aspergillus fumigatus extract (AFE). METHODS Heaves-susceptible (n = 7) and control (n = 6) horses received inhalation challenges with 3 different doses of HDS and LPS. Heaves-susceptible horses (n = 6) also received 3 different doses of AFE and one dose of AFE depleted of endotoxin (AFE-LPS). BALF collected following inhalation challenges was analysed using gelatin zymography. Gelatinolytic bands were identified as complex, pro-MMP-9, active MMP-9, pro-MMP-2 and active MMP-2 based on molecular weights. RESULTS Each challenge substance induced a dose-dependent elevation in gelatinolytic activity. The dose-dependency was most evident for pro-MMP-9 and total MMP-9 levels in heaves-susceptible horses following LPS challenges. CONCLUSIONS There is a dose-dependent elevation in MMP-9 in BALF of heaves-susceptible and control horses following inhalation challenge with organic dust and some of its components, elevation being more marked in heaves-susceptible horses. Organic dust components vary in their pro-inflammatory potential. POTENTIAL RELEVANCE This study supports the role of MMPs in the pathogenesis of heaves and highlights the potential value of protease inhibitors in attenuating the airway inflammatory response to inhaled organic dust.

Prevalence of Clostridium perfringens in faeces and ileal contents from grass sickness affected horses: Comparisons with 3 control populations

REASONS FOR PERFORMING STUDY While previous studies have demonstrated an association between equine grass sickness (EGS) and the presence of Clostridium botulinum within ileal contents and faeces, no such associations with other intestinal-derived anaerobic bacteria have been extensively investigated. HYPOTHESIS The prevalence of C. perfringens in the ileal contents and faeces of EGS horses is greater than control horses; the detection of C. perfringens in faeces by ELISA could be diagnostically beneficial in a clinical setting. METHODS The prevalence of C. perfringens in faeces from EGS horses and healthy grazing control horses was determined by both selective culture and ELISA to permit both validation of the ELISA and inter-group comparisons. Additionally, the prevalence of C. perfringens (ELISA) in ileal contents from EGS horses was compared with that for control horses with nongastrointestinal disease. Finally, the prevalence of C. perfringens (ELISA) in faeces from EGS cases was compared with that from both horses with which they shared pasture at the time of disease onset and non-EGS colic horses. RESULTS When compared with culture, the ELISA had a sensitivity and specificity of 86 and 98%, respectively. The prevalence of C. perfringens in faeces as determined by both culture and ELISA was significantly higher (P<0.001) for EGS horses (7/9 and 15/37, respectively) than for healthy grazing controls (0/60 and 1/74, respectively). The prevalence of C. perfringens in ileal contents from EGS horses (5/10) was greater than that for horses with nongastrointestinal disease (1/12) at a level that approached significance (P = 0.056). EGS cases had a significantly greater prevalence of C. perfringens in faeces (15/37) than co-grazing horses (1/18) and colic (1/16) horses. The specificity (93%) and PPV (94%) of the detection of C. perfringens by ELISA on faecal samples in relation to disease status (EGS compared with colic horses) was good. Sensitivity (41%) and NPV (39%) were poor. CONCLUSIONS AND POTENTIAL RELEVANCE The use of a commercial ELISA to detect faecal C. perfringens may be diagnostically beneficial when differentiating EGS cases from colic cases, although further work is required to fully evaluate its potential.

Endoscopic and bacteriological findings in a chronic outbreak of strangles

Recently there has been increased awareness of the role of the carrier state in propagating Streptococcus equi var equi (S equi) infections (strangles), although the anatomical location of the organisms in chronic carriers has not been consistently established. This case report describes a chronic strangles outbreak in a riding school, that was monitored over six months by repeated clinical and endoscopic guttural pouch examinations. All asymptomatic horses that had positive S equi cultures on nasal swabs or guttural pouch lavages were found to have lesions in their guttural pouches. These lesions included empyema, chondroids and previously undescribed chronic discharging lesions on the floor of the medical compartment of the guttural pouches. These observations further support previous studies indicating the importance of investigating the guttural pouches in horses suspected to be asymptomatic carriers of this organism.

Systemic concentrations of antioxidants and biomarkers of macromolecular oxidative damage in horses with grass sickness

REASONS FOR PERFORMING STUDY The aetiopathogenesis of equine grass sickness (EGS) is unknown. The role of free radical-mediated neuronal damage has not previously been investigated in this condition. OBJECTIVES To investigate the potential contribution of oxidative damage and antioxidant status to neurodegeneration in EGS. METHODS Systemic levels of surrogate biomarkers were determined in 10 horses with acute EGS and in 2 control populations; 10 healthy horses co-grazing with the 10 EGS horses at the onset of clinical disease, and 10 healthy mares grazing where EGS has not been reported. RESULTS EGS horses had alterations in levels of several antioxidants, consistent with oxidative stress, the acute phase response and/or the secondary metabolic complications of EGS. EGS horses had elevated plasma dihydroxyphenylalanine (DOPA) levels. CONCLUSIONS The elevated DOPA levels probably reflected a generalised disturbance of catecholamine metabolism rather than increased DOPA production via free radical-mediated oxidation of tyrosine. However, there was no evidence of systemic macromolecular oxidative damage. POTENTIAL CLINICAL RELEVANCE Further work is required to determine whether macromolecular oxidative damage occurring at the neuronal level contributes to EGS.

Clinical features and management of equine post operative ileus: Survey of diplomates of the European Colleges of Equine Internal Medicine (ECEIM) and Veterinary Surgeons (ECVS)

REASONS FOR PERFORMING STUDY There is a need for an improved understanding of equine post operative ileus (POI), in terms of both clinical definition and optimal management. Although the pharmacological strategies that are used to treat POI continue to evolve, little is known about the supplementary strategies used to prevent and manage this condition. OBJECTIVES To report the current strategies used to diagnose, prevent and manage POI following emergency abdominal surgeries. STUDY DESIGN Cross-sectional survey. METHODS An electronic survey invitation was sent by email to 306 European college diplomates (European Colleges of Equine Internal Medicine, ECEIM n = 120, and Veterinary Surgeons, ECVS n = 186). RESULTS The response rate was 33% (100 of 306). The median reported estimated incidence of POI was 10-20%. The presence of reflux on nasogastric intubation was the main criterion used to define POI. Lesions involving the small intestine were thought to be the leading risk factors for developing POI. Anti-inflammatory drugs, antimicrobial drugs and i.v. fluids were the primary preventative strategies when managing cases at high risk for POI. Flunixin meglumine and lidocaine were the drugs most commonly used to treat horses with POI. Supplementary preventative and treatment strategies for POI included control of endotoxaemia, fluid therapy, early ambulation and judicious timing of post operative feeding. CONCLUSIONS Appreciation of the potential risk factors associated with the development of POI appeared to have an impact on the choice of management strategies that are implemented. The majority of ECEIM and ECVS Diplomates in the survey used flunixin meglumine and lidocaine, often in combination, to treat horses with POI, which is likely to reflect the contributory role of inflammation in its pathophysiology. Various supplementary strategies were used to prevent and manage POI risk factors intraoperatively and post operatively.

In equine grass sickness, serum amyloid A and fibrinogen are elevated, and can aid differential diagnosis from non-inflammatory causes of colic.

Equine grass sickness (EGS) is a debilitating and often fatal neurodegenerative disease. A presumptive diagnosis of EGS may be made on the basis of clinical signs and subjective ancillary tests, but a definitive antemortem diagnosis can only be made following histopathological examination of intestinal biopsies. It has previously been reported that horses with EGS may show clinical and clinicopathological signs of systemic inflammation. The objective of this study was to (a) quantify acute inflammatory markers in blood samples collected from acute, subacute and chronic EGS cases, and (b) compare them with (i) clinically normal horses co-grazing with acute EGS cases (co-grazers), (ii) horses with other causes of colic and (iii) healthy horses. Serum amyloid A (SAA), serum activin A and plasma fibrinogen were quantified. There were marked increases in SAA and fibrinogen in EGS cases compared with healthy horses, co-grazers and non-inflammatory colic cases. The concentrations of SAA and fibrinogen in EGS cases were not significantly different from inflammatory colic cases. When concentrations of SAA, fibrinogen and activin A in each EGS subgroup were compared, no significant differences were detected. Activin A concentrations were significantly elevated in EGS cases and co-grazing horses; this could reflect the presence of subclinical disease in some horses that do not develop clinical signs of EGS, and suggests widespread exposure to the aetiological agent. When faced with sparse antemortem diagnostic techniques, identification of marked increases in acute phase protein concentrations may help to differentiate EGS from other causes of abdominal pain, such as intestinal obstructions; however, there could be diagnostic difficulty in differentiating other inflammatory abdominal conditions, such as peritonitis or enteritis.