Evaluation of nebulised hay dust suspensions (HDS) for the diagnosis and investigation of heaves. 1: Preparation and composition of HDS

Currently, heaves is investigated by exposing susceptible horses to dusty hay. Consequently, the response will be dependent on the organic dust content and composition of the hay. It was hypothesised that the use of a hay dust suspension (HDS) would reduce the variability of the challenge and therefore standardise experimental protocols. Furthermore, analysis of HDS would also permit further investigation of the organic dust components responsible for the response. Three hay dust suspensions (HDS-1, 2 and 3) were prepared for use in the diagnosis and investigation of heaves. HDS were produced from fine dust particles, comprising mostly fungal spores, collected from 3 batches of dusty hay. HDS-1 and 3 were analysed for endotoxin, beta-D-glucan and protein concentrations, general protease activity and enumeration and size distribution of particulates. Protease activity was mainly attributable to a 28 kDa serine protease and to 85 kDa and 160 kDa metalloproteases. The particulate and soluble components of HDS could be aerosolised by jet nebulisation. We therefore conclude that detailed analysis of HDS is possible, that such a challenge system provides a method of standardising experimental protocols and that all components of HDS (both soluble and particulate) can be delivered to the lung using standard nebulisation techniques. For the above reasons, nebulised HDS offers considerable advantages over conventional hay/straw challenge for the diagnosis and investigation of heaves.     

Evaluation of cytokine production by equine alveolar macrophages exposed to lipopolysaccharide, Aspergillus fumigatus, and a suspension of hay dust

OBJECTIVE: To evaluate cytokine production by equine alveolar macrophages after exposure to lipopolysaccharide (LPS), Aspergillus fumigatus, and hay dust, and determine the effect of clenbuterol on the cytokine response. ANIMALS: 6 horses. PROCEDURE: Alveolar macrophages were exposed to PBS solution (negative control), LPS, hyphae and conidia of Aspergillus fumigatus (AF), or a suspension of hay dust (HDS) and incubated for 24 hours at 37 degrees C. Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta were measured in the supernatant. The procedure was repeated with cells that were concurrently incubated with 0.5 microM clenbuterol. RESULTS: Exposure to HDS and AF significantly increased production of TNF-alpha by equine alveolar macrophages. The increase in TNF-alpha produced in response to HDS and AF was 5 and 7 times as great, respectively, as the increase measured in response to LPS. The concentration of IL-1beta in the supernatant was significantly increased after exposure of cells to AF. Clenbuterol was effective at inhibiting TNF-alpha production by cells exposed to LPS, HDS, or AF. CONCLUSIONS AND CLINICAL RELEVANCE: Increased production of TNF-alpha and IL-1 indicated that the pro-inflammatory cytokines produced by alveolar macrophages in response to allergens may play a role in recurrent airway obstruction (RAO) in horses. Equine alveolar macrophages are not only a primary pulmonary defense mechanism but may also influence the pathogenesis of equine RAO. The beta2-adrenoceptor agonist clenbuterol, a drug that is commonly used for treatment of equine RAO, promotes immediate bronchodilation and may also contribute to downward modulation of the inflammatory response.     

Evaluation of nebulised hay dust suspensions (HDS) for the diagnosis and investigation of heaves. 3: Effect of fractionation of HDS

To investigate the relative importance of inhaled particulates and soluble components in the response to inhaled hay dust suspension (HDS), heaves (previously termed chronic obstructive pulmonary disease; n = 7) and control (n = 6) horses were given inhalation challenges with whole and fractionated HDS. Fractionation was achieved by centrifugation to yield supernatant (SUP) and particulate debris. The particulates were then washed repeatedly in saline to produce a washed particulate (WP) fraction which comprised mainly fungal spores, and a wash fraction (WF) which comprised saline and soluble components washed from the surface of the particulates. Inhalation of HDS and SUP induced a significant airway neutrophilia in both groups, with the magnitude of the response being significantly greater in heaves horses. SUP induced significantly less airway neutrophilia than HDS in both groups, despite the endotoxin and protease content of HDS and SUP being comparable. WP and WF induced only a slight airway neutrophilia in heaves horses. However, a combined challenge with SUP and WP induced a neutrophilic response approaching the magnitude of that following HDS challenge, indicating that dust particulates contribute to the pulmonary recruitment of neutrophils in heaves. Consequently, inhalation challenge with HDS, which contains both particulates and soluble dust components, may be a more useful tool for the diagnosis and investigation of heaves than aqueous dust extracts, which contain only soluble components.     

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. OBJECTIVE: To identify components of organic dust responsible for elevated MMP levels in equine airways. METHODS: Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. RESULTS: HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. CONCLUSIONS: The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. POTENTIAL RELEVANCE: Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.     

Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.     

Characterization of house dust mite and scabies mite allergens by use of canine serum antibodies

OBJECTIVE: To identify the major allergenic proteins from the 3 main species of dust mites to which dogs react (Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei) and evaluate the potential cross-reactivity of dust mite allergens with antigens from the ectoparasitic mite Sarcoptes scabiei var canis. SAMPLE POPULATION: Sera from 83 dogs with atopic dermatitis. PROCEDURE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting using serum from atopic dogs was used to identify IgE-binding proteins in extracts of the 4 mite species. RESULTS: Sera of atopic dogs contained IgE against 23, 17, 25, and 17 allergens from D. farinae, D. pteronyssinus, E. maynei, and S. scabiei, respectively. Unlike the situation for humans, the major allergens for dogs are mostly proteins that are larger than 90 kd molecular weight. Dermatophagoides farinae and E. maynei appear to be more allergenic for dogs than is D. pteronyssinus. Some dogs with serum IgE against dust mites also had IgE against antigens of S. scabiei var canis. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple dust mite allergens induce an IgE response in dogs. These allergens are mostly greater than 90 kd molecular weight.     

Dermatophagoides mites in house dust as an allergen source in atopic dogs

Thirty dogs with a clinical diagnosis of atopy were skin tested with 58 allergens including an aqueous house dust mite extract and a crude house dust extract. Sixty percent of the dogs had positive skin reactions to both the house dust mite and house dust antigens. In atopic dogs, house dust mites appear to be an important allergen source in house dust extracts but are not the only major source.     

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.     

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.     

Prevalence of house dust mites and dermatophagoides group 1 antigens collected from bedding, skin and hair coat of dogs in south-west England

The house dust mites Dermatophagoides farinae (Df) and D. pteronyssinus (Dpt) are commonly implicated as allergens causing canine atopic dermatitis in the UK. However, there are few studies that characterize the exposure of UK pet dogs to these mites. The objectives of this study were to determine the prevalence of the mite species on the skin, hair coat and bedding of a population of pet dogs. Dust samples (n = 68) were collected from both dogs and their beds using a standardized vacuuming technique and stored at -20 degrees C. Mites were identified using accepted morphological criteria. House dust mite allergen concentrations were assayed using standardized ELISA for Dpt and Df group 1 allergens (Der p 1 and Der f 1). Mites were identified in 15/68 samples (22%) and Dpt was the most common. Df mites were not present. Der p 1 allergens were detected in 60% of samples, and Der f 1 in 6% of samples. There were no significant differences between the number of Der p 1 positive samples from dogs and the number of those from their bedding, or between the average Der p 1 concentrations from dogs and the number of those from their bedding. Contrary to studies elsewhere in Europe and the USA, these findings support studies of human asthma patients in the UK, where exposure to Df is rare, but to Dpt is common. As the prevalence of positive intradermal and serological reactions to Df in atopic dogs is high, further investigations are warranted to clarify true Df hypersensitivity or potential immunological cross-reactivity between mite allergens.