Studies on the aetiopathogenesis of equine chronic obstructive pulmonary disease (COPD)

This thesis describes an investigation into the aetiopathogenesis of equine chronic obstructive pulmonary disease (COPD).To investigate the aetiology of equine COPD, control and asymptomatic COPD affected horses were given nebulised inhalation challenges with extracts of Micropolyspora faeni (MF), Aspergillus fumigatus (AF) and Thermoactinomyces vulgaris (TV). MF and AF challenges induced pulmonary disease, similar to naturally occurring COPD, only in the COPD affected horses, implicating MF and AF in the aetiology of equine COPD. The role of TV in the aetiology of equine COPD could, however, not be assessed as the TV challenges induced pulmonary inflammation in 2 control horses, which had been unaffected by hay and straw challenges i.e. 'natural challenges' (NC), indicating that the experimental TV challenge differed from the TV challenge which occurs during NC. The absence of pulmonary disease in control horses following MF, AF challenges and after NC suggests that equine COPD is a pulmonary hypersensitivity, rather than a non specific toxic response. In this study, bronchoalveolar lavage fluid (BALF) cytology examinations proved to be more useful for detecting pulmonary disease than clinical, pulmonary mechanics, arterial blood gas tensions and arterial pH examinations.The role of oil seed rape (OSR) (Brassica spp.) in the aetiology of equine pulmonary disease was investigated by exposing horses to a field of flowering B.campestrus and by experimental B.napus inhalation challenges. OSR had no detectable effect on control and asymptomatic COPD affected horses, suggesting that OSR is not a major cause of equine respiratory disease. However, the experimental B.napus inhalation challenges exacerbated pulmonary disease in some symptomatic COPD affected horses, presumably via non specific bronchial hyperresponsiveness/toxicity. Intradermal testing using a commercial B.napus pollen extract suggested that none of the horses investigated was hypersensitive to B.napus pollen antigens.As BALF is comprised of lavage fluid and pulmonary epithelial lining fluid (PELF) in variable proportions, quantitative comparisons of the cellular and molecular components in different BALF samples are valid only if the proportions of PELF in the BALF samples are standardised. Two BALF standardisation techniques, namely the urea and albumen dilution techniques, were evaluated in the horse. While both techniques were found to be satisfactory, the urea dilution technique was considered to be the more accurate.Comparison of the cellular and molecular components of BALF collected from 4 different lung segments of control and symptomatic COPD affected horses indicated that, in these horses, BALF components showed regional homogeneity. This suggests that the composition of PELF is uniform throughout the lungs of these horses and that a single BALF sample, collected from any lung lobe, is representative of the entire lung.The role of mast cells/basophils in the pathogenesis of equine COPD was investigated by quantifying histamine, an indicator of mast cell/basophil degranulation, in plasma, BALF and PELF of control and COPD affected horses, before and at 0.5 and 5h after NC. The PELF histamine concentrations of COPD affected horses were significantly increased only at 5h after NC. NC had no significant effect on the PELF histamine concentrations of control horses nor on the plasma and BALF histamine concentrations of either group. As the histamine concentrations of whole BALF lysates were significantly correlated with the numbers of metachromatically staining BALF cells, presumed to be mast cells and/or basophils, these findings support involvement of a late phase, mast cell/basophil mediated, hypersensitivity reaction in the pathogenesis of equine COPD.Quantification of tryptase, an inflammatory mediator which offers potential advantages over histamine as an indicator of mast cell degranulation, in equine serum and BALF, using a commercial radioimmunoassay kit for human tryptase, was unsuccessful.The role of lymphocytes in the pathogenesis of equine COPD was investigated by determining the lymphocyte phenotype distributions of peripheral blood (PB) and BALF from control and COPD affected horses, before and after NC. Prior to NC, asymptomatic COPD affected horses had a significantly higher proportion of BALF B lymphocytes than control horses, suggesting that these cells have a role in the pathogenesis of equine COPD. NC significantly increased the ratios of CD4+, T helper/inducer lymphocytes and significantly reduced the ratios of CD8+, T suppressor/cytotoxic lymphocytes in BALF from COPD affected horses, suggesting that T lymphocytes have an important role in the pathogenesis of equine COPD.Intradermal mould antigen testing was evaluated as a diagnostic technique for equine COPD. The intradermal endpoint litres of control horses for AF, MF and TV were not significantly different from those of COPD affected horses, suggesting that this technique is of limited value in the diagnosis of equine COPD. Furthermore, the lack of correlation between the intradermal endpoint titres for each antigen and the changes in pulmonary mechanics, arterial blood gas tensions and with BALF neutrophil ratios which had followed previous MF, AF and TV inhalation challenges and NC suggests divergence of equine dermal and pulmonary reactivities to these antigens.Local transendoscopic endobronchial antigen challenge, which has proved to be a valuable clinical and research technique in the study of human pulmonary hypersensitivity, was evaluated in the horse. As local endobronchial challenges with phosphate buffered saline, MF extract and mouldy hay extract induced a non specific pulmonary neutrophilia in both control and asymptomatic COPD affected horses and elicited endoscopically visible responses in a proportion of horses from both these groups, this technique was considered to be of limited value as a clinical and research technique in the study of equine COPD

Grazing livestock are exposed to terrestrial cyanobacteria

While toxins from aquatic cyanobacteria are a well-recognised cause of disease in birds and animals, exposure of grazing livestock to terrestrial cyanobacteria has not been described. This study identified terrestrial cyanobacteria, predominantlyPhormidiumspp., in the biofilm of plants from most livestock fields investigated. Lower numbers of other cyanobacteria, microalgae and fungi were present on many plants. Cyanobacterial 16S rDNA, predominantly fromPhormidiumspp., was detected in all samples tested, including 6 plant washings, 1 soil sample and ileal contents from 2 grazing horses. Further work was performed to test the hypothesis that ingestion of cyanotoxins contributes to the pathogenesis of some currently unexplained diseases of grazing horses, including equine grass sickness (EGS), equine motor neuron disease (EMND) and hepatopathy.Phormidiumpopulation density was significantly higher on EGS fields than on control fields. The cyanobacterial neurotoxic amino acid 2,4-diaminobutyric acid (DAB) was detected in plant washings from EGS fields, but worst case scenario estimations suggested the dose would be insufficient to cause disease. Neither DAB nor the cyanobacterial neurotoxins β-N-methylamino-L-alanine and N-(2-aminoethyl) glycine were detected in neural tissue from 6 EGS horses, 2 EMND horses and 7 control horses.Phormidiumwas present in low numbers on plants where horses had unexplained hepatopathy. This study did not yield evidence linking known cyanotoxins with disease in grazing horses. However, further study is warranted to identify and quantify toxins produced by cyanobacteria on livestock fields, and determine whether, under appropriate conditions, known or unknown cyanotoxins contribute to currently unexplained diseases in grazing livestock

Nonprimate hepaciviruses in domestic horses, United kingdom

Although the origin of hepatitis C virus infections in humans remains undetermined, a close homolog of this virus, termed canine hepacivirus (CHV) and found in respiratory secretions of dogs, provides evidence for a wider distribution of hepaciviruses in mammals. We determined frequencies of active infection among dogs and other mammals in the United Kingdom. Samples from dogs (46 respiratory, 99 plasma, 45 autopsy samples) were CHV negative by PCR. Screening of 362 samples from cats, horses, donkeys, rodents, and pigs identified 3 (2%) positive samples from 142 horses. These samples were genetically divergent from CHV and nonprimate hepaciviruses that horses were infected with during 2012 in New York state, USA. Investigation of infected horses demonstrated nonprimate hepacivirus persistence, high viral loads in plasma (105–107 RNA copies/mL), and liver function test results usually within reference ranges, although several values ranged from high normal to mildly elevated. Disease associations and host range of nonprimate hepaciviruses warrant further investigation

Constitutive apoptosis in equine peripheral blood neutrophils in vitro

AbstractThe aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone

Comparative Susceptibility of Sheep of Different Origins, Breeds and PRNP Genotypes to Challenge with Bovine Spongiform Encephalopathy and Scrapie

Sheep are natural hosts of the prion disease, scrapie. They are also susceptible to experimental challenge with various scrapie strains and with bovine spongiform encephalopathy (BSE), which affects cattle and has been accidentally transmitted to a range of other species, including man. Incidence and incubation period of clinical disease in sheep following inoculation is controlled by the PRNP gene, which has different alleles defined on the basis of polymorphisms, particularly at codons 136, 154 and 171, although other codons are associated with survival time, and the exact responses of the sheep may be influenced by other breed-related differences. Here we report the results of a long term single study of experimental scrapie and BSE susceptibility of sheep of Cheviot, Poll Dorset and Suffolk breeds, originating from New Zealand and of a wide range of susceptible and resistant PRNP genotypes. Responses were compared with those of sheep from a closed Cheviot flock of UK origin (Roslin Cheviot flock). The unusually long observation period (6-8 years for most, but up to 12 years for others) allows us to draw robust conclusions about rates of survival of animals previously regarded as resistant to infection, particularly PRNP heterozygotes, and is the most comprehensive such study reported to date. BSE inoculation by an intracerebral route produced disease in all genotype groups with differing incubation periods, although M112T and L141F polymorphisms seemed to give some protection. Scrapie isolate SSBP/1, which has the shortest incubation period in sheep with at least one VRQ PRNP allele, also produced disease following sub-cutaneous inoculation in ARQ/ARQ animals of New Zealand origin, but ARQ/ARQ sheep from the Roslin flock survived the challenge. Our results demonstrate that the links between PRNP genotype and clinical prion disease in sheep are much less secure than previously thought, and may break down when, for example, a different breed of sheep is moved into a new flock