Miia Riihimäki

Influence of horse stable environment on human airways

BackgroundMany people spend considerable amount of time each day in equine stable environments either as employees in the care and training of horses or in leisure activity. However, there are few studies available on how the stable environment affects human airways. This study examined in one horse stable qualitative differences in indoor air during winter and late summer conditions and assessed whether air quality was associated with clinically detectable respiratory signs or alterations to selected biomarkers of inflammation and lung function in stable personnel.MethodsThe horse stable environment and stable-workers (n = 13) in one stable were investigated three times; first in the winter, second in the interjacent late summer and the third time in the following winter stabling period. The stable measurements included levels of ammonia, hydrogen sulphide, total and respirable dust, airborne horse allergen, microorganisms, endotoxin and glucan. The stable-workers completed a questionnaire on respiratory symptoms, underwent nasal lavage with subsequent analysis of inflammation markers, and performed repeated measurements of pulmonary function.ResultsMeasurements in the horse stable showed low organic dust levels and high horse allergen levels. Increased viable level of fungi in the air indicated a growing source in the stable. Air particle load as well as 1,3-β-glucan was higher at the two winter time-points, whereas endotoxin levels were higher at the summer time-point. Two stable-workers showed signs of bronchial obstruction with increased PEF-variability, increased inflammation biomarkers relating to reported allergy, cold or smoking and reported partly work-related symptoms. Furthermore, two other stable-workers reported work-related airway symptoms, of which one had doctors diagnosed asthma which was well treated.ConclusionBiomarkers involved in the development of airway diseases have been studied in relation to environmental exposure levels in equine stables. Respirable dust and 1,3-β-glucan levels were increased at winter stabling conditions. Some employees (3/13) had signs of bronchial obstruction, which may be aggravated by working in the stable environment. This study contributes to the identification of suitable biomarkers to monitor the indoor horse stable environment and the personnel. An improved management of the stable climate will be beneficial for the health of both stable workers and horses.

Partial divergence of cytokine mRNA expression in bronchial tissues compared to bronchoalveolar lavage cells in horses with recurrent airway obstruction.

The aim of this study was to investigate mRNA levels of cytokines in bronchial epithelium in horses with recurrent airway obstruction (RAO) during acute crisis and remission. Additionally, cytokine mRNA levels in endobronchial biopsies and bronchoalveolar lavage (BAL) cells were compared. Seven RAO horses were examined while in respiratory crisis following provocation and again while in remission after 2 months on pasture, during which time six healthy horses on pasture were also examined. Quantitative real-time PCR (RT-PCR) was used to assess mRNA expression for cytokines IL-5, IL-6, IL-8, IL-10, IL-17 and transforming growth factor beta1 (TGF-beta1) in endobronchial biopsies and bronchoalveolar lavage. Expression of IL-8 mRNA was significantly upregulated during crisis in both endobronchial biopsies and BAL cells (p=0.036), while there was a similar trend for upregulation of IL-10 mRNA only in BAL cells that approached significance (p=0.059). Moreover, during crisis the expression of IL-8 mRNA in BAL cells was positively correlated to relative IL-6 mRNA expression (r(s)=0.971, p=0.001) and bronchial epithelial expression of IL-10 and TGF-beta1 mRNA were positively correlated (r(s)=0.943, p=0.005). In comparing the relationship of mRNA expression in BAL to biopsy in individual RAO horses, there was a positive correlation with IL-6 to IL-8 mRNA expression in BAL during respiratory crisis (r(s)=0.971, p=0.001) that also correlated positively with IL-8 expression in biopsies on pasture (r(s)=0.986, p<0.0001 for both). Regarding RAO horses at pasture versus controls neither the cytokine mRNA levels in endobronchial biopsy nor in BAL cells differed significantly. These results further support previous findings that IL-8 mRNA in both BAL cells and bronchial epithelium is upregulated in RAO horses during crisis. However, apart from IL-8, it appears that expression of other cytokines, including IL-5, IL-6, IL-10, IL-17 and TGF-beta1 in bronchial epithelium does not necessarily mirror cytokine expression in BAL cells in individual horses with RAO. Accordingly, examination of markers of inflammation in endobronchial tissue provides complementary but not necessarily identical information to that obtained in BAL cells. Given the potential for repeated sampling over time bronchial biopsy can serve as an invaluable additional tool for investigation of time-dependent changes in inflammatory process in this animal model of asthma.

Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-α in endobronchial biopsies in horses with recurrent airway obstruction

BackgroundThe aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-α, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission.ResultsDespite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls.In comparing real-time PCR analysis to results of immunohistochemistry only IL-10 mRNA and protein levels in RAO horses on pasture were significantly correlated (rs = 0.893, p = 0.007). Curiously, in controls examined on pasture the TNF-α protein level was positively correlated to IL-10 mRNA expression (rs = 0.967, p = 0.007) and negatively correlated to IL-6 mRNA expression (rs = -0.971, p = 0.001).ConclusionGiven the complementary relationship of assessing cytokines directly by immunohistochemistry, or indirectly by PCR to mRNA, the lack of significant changes in either mRNA or protein levels of IL-6, IL-10 or TNF-α mRNA in RAO horses in exacerbation suggests that these particular cytokines in bronchial tissue may not play a substantive role in the active inflammation of this disease. To support this contention further studies examining time dependency of expression of IL-6, IL-10 or TNF-α are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.

Viral load of equine herpesviruses 2 and 5 in nasal swabs of actively racing Standardbred trotters: Temporal relationship of shedding to clinical findings and poor performance

The equine gamma herpesviruses 2 and 5 (EHV-2 and -5) have frequently been observed in the equine population and until recently presumed low to nonpathogenic. However, recent reports linking presence of equine gamma herpesviruses with clinical signs of mild to severe lung disease, suggest that the role of these viruses in respiratory disease and poor performance syndrome is still unclear. Moreover, baseline data regarding the temporal pattern of shedding of EHV-2 and EHV-5 within stables and within individual actively racing horses have been lacking. In a prospective longitudinal study, we followed elite racing Standardbred trotters at monthly intervals for 13 months, to investigate whether the amount of EHV-2 and EHV-5 shedded in nasal secretions varied over time within and between individual horses. Sixty-six elite horses were investigated by analyzing nasal swabs and serum samples, a health check and evaluation of athletic performance monthly during the study period. Nasal swabs were analyzed with two newly developed qPCR assays for EHV-2 and EHV-5, respectively. Of 663 samples, 197 (30%) were positive for EHV-2 and 492 (74%) positive for EHV-5. Furthermore, 176 (27%) of the samples were positive for both EHV-2 and EHV-5 simultaneously. There was considerable variation in the amount and frequency of shedding of EHV-2 and EHV-5 within and between individual horses. Viral load varied seasonally, but neither EHV-2 nor EHV-5 viral peaks were associated with clinical respiratory disease and/or poor performance in racing Standardbred trotters.

Air Quality in Horse Stables

A large number of people are highly engaged in the equine industry around the world, especially in industrialised countries. For example, in the USA about 4.6 million Americans are directly involved in the equine industry, and in Sweden the industry provides full-time employment to over 30 000 people. Horse riding is the second biggest sport in Sweden after soccer, engaging approximately 6% of the population, and is by far the biggest sport for girls. Consequently, many people spend a considerable portion of their day in stable environments, either as workers in the care and training of horses, or in their leisure time. The type of buildings used for stabling horses can vary widely. Some are newly built and thus designed solely for housing horses, whereas others have been converted or “retrofitted” from barns used for other farming activities, such as housing cattle or for feed storage. However, regardless of design these buildings have many common requirements for indoor environment, including selection of type of feed and bedding, and the need for continual removal of faeces and urine. Moreover, the building ventilation must provide adequate exchange of fresh air to the horses’ living space to regulate the stable temperature and remove moisture, stable gases and organic debris produced by the horses and their activities. There is no need to remove excess heat in stables in the winter in northern climates. However, this puts more focus on ventilation, which must rid the stables of moisture, odour, and ammonia that have built up in the more closed environment of the stable. Moisture originates from horse’s respiration, faeces and urine, and other stable activities such as showering the animals, and general stable cleaning. Accompanying this moisture build-up is increased risk of condensation, intense odour, and increased ammonia release from bedding that ventilation must remove. How much ventilation should be provided? Ventilation of one air change per hour (ACH) means that the total volume of air in the stable is replaced in one hour. Guidelines for horse stables suggest providing between 4 to 8 air changes per hour to reduce mould spore contamination, minimize condensation, and reduce moisture, odour, and ammonia accumulation (Horse Stable Ventilation, 2003). Thus, for the mean of 6 ACH a stable’s ventilation system must be capable of a complete air change every 10 minutes. With the inevitable seasonal appearance of subzero winter weather, horse owners occasionally work against these principles of ventilation in an unheated building by trying

The clinical significance of Nicoletella semolina in horses with respiratory disorders and a screening of the bacterial flora in the airways of horses.

Nicoletella semolina, a member of the family Pasteurellaceae, can be isolated from the airways of horses with respiratory disorders. However, its role as a potential or opportunistic pathogen is not clear nor is its presence as part of the normal flora. We therefore investigated the presence and bacterial load of N. semolina in healthy and diseased horses. Samples from a healthy control group were compared with samples from the routine analysis of horses with a clinical history of respiratory disorders. A total of 1770 nose swabs and 1132 tracheal aspirate samples were analysed and subjected to conventional bacteriological examination. N. semolina was isolated from 12 (6%) of 207 nose samples from the healthy control group and from 42 (3%) of 1563 samples from horses with respiratory disorders. In tracheal aspirate, N. semolina was isolated from 7 (3%) of 211 samples from the control group and 49 (5%) of 921 samples from horses with respiratory disorders. Other bacteria were also isolated in laboratory analyses, the most commonly isolated bacterium in both the control group and the respiratory disorders group being Streptococcus equi subsp. zooepidemicus. It was isolated in 21% of tracheal aspirate from the control group and 33% of those from horses with respiratory disorders. In conclusion, N. semolina is not a primary pathogenic bacterium, as it was isolated at similar frequencies in horses with respiratory disorders and those in the healthy control group.

Decreased Clinical Severity of Strangles in Weanlings Associated with Restricted Seroconversion to Optimized Streptococcus equi ssp equi Assays

Background Streptococcus equi ssp. equi causes characteristic clinical signs that are most severe in young horses, including fever, purulent nasal discharge, and lymph node abscessation in the head region. Hypothesis/Objectives Clinical, serologic, and microbiologic factors related to unexpectedly mild disease severity in a natural outbreak of strangles in immunologically naïve weanlings were investigated. Animals One‐hundred and twelve warmblood weanlings. Methods Prospective longitudinal observational study of a natural outbreak of strangles. The entire cohort was examined at the peak of the outbreak by deep nasal swabs for culture and quantitative PCR (qPCR) for the presence of S. equi and clinically and serologically in a sequential manner by an optimized ELISA from the index case throughout the outbreak until resolution. Descriptive statistics were calculated and comparisons made using a nondirectional Wilcoxon signed‐rank test. Results Outbreak morbidity was 53%, with 9 of 14 horses culture positive and 26 of 53 horses qPCR positive for S. equi lacking clinical signs characteristic of strangles. By resolution, 91 of 112 had seroconverted to Antigen A by ELISA but seroconversion to antigen C (part of the SeM protein) was minimal. Sequencing of the isolates detected no alterations in the SeM protein, but identified a 61 bp deletion in the gene SEQ_0402. Conclusions and Clinical Importance Absence of clinical signs alone in naïve horses may be an insufficient criterion to release horses from strangles quarantine measures. Restricted seroconversion to antigen C may have been associated with decreased clinical severity. The role of a minor gene deletion in SEQ_0402 in the virulence of S. equi warrants further investigation.

Long term dynamics of a Streptococcus equi ssp equi outbreak, assessed by qPCR and culture and seM sequencing in silent carriers of strangles

The aim of the study was to use culture, qPCR and seM sequencing to map Streptococcus equi subspec. equi (S.equi) isolates in long term carrier animals. A strangles outbreak affecting 41 Icelandic horses was followed to determine strangles free status using nasal and/or guttural pouch lavages collected serially on eleven separate occasions over 13 months. Ten persistent carriers, of which eight had repeated culture positive samples for S. equi, were selected for the study. Of 115 samples collected, 61 were S. equi positive on qPCR; from which 32 were also culture positive. Amplification of parts of the gene encoding the M-protein seM was performed on isolated colony material (n = 32) or, where only PCR product was obtained, directly on the DNA sample (n = 29) with a nested amplification approach. The seM sequence could be determined for six of the 29 samples that were solely qPCR positive. The outbreak was due to a S. equi strain of seM type 72. Three months after initial sampling isolates from two horses had seM gene sequences with one amino acid change. After six months S. equi with truncated seM genes were found in two horses; one variant in a single horse once, and in the other horse a variant that persisted and that was later identified in two additional horses. Non- mucoid S. equi colonies were found in two horses. Importantly, after acute strangles outbreaks many horses not only remain persistently qPCR positive for S. equi but are also intermittently culture positive.

Seasonal Variation in Tracheal Mucous and Bronchoalveolar Lavage Cytology for Adult Clinically Healthy Stabled Horses

ABSTRACT Seasonal changes affect the concentration of dust particles and aeroallergens in the equine stable environment and are hypothesized to change airway cytology in healthy stabled horses. The objective of the study was to evaluate if seasonal changes from May to November influenced the lower airway health cytological findings in clinically healthy stabled horses. Endoscopic examination, including tracheal aspiration (TA) and bronchoalveolar lavage (BAL) cytology was performed in November and May on 63 clinically healthy stabled Danish Warmblood geldings, aged 12.9 (±4.6) years. Tracheal aspiration and BAL cytology were evaluated via May‐Grünwald‐Giemsa stain after cytocentrifugation of samples. Five‐hundred cells were identified microscopically as macrophages, lymphocytes, neutrophils, eosinophils, or mast cells. A significant difference in climatic conditions with lower temperatures (P < .001), fewer sunlight hours (P = .004), less precipitation (P = .006), and higher humidity (P < .001) was found in November compared to May. A significantly higher percentage of neutrophils (Median 6.10%, Quartiles 4.00–8.80, P < .001) and a higher tracheal mucus score (Median 3.50%, Quartiles 1.95–5.20, P = .010) were found during November compared to May period. Furthermore, a positive correlation between TA and BAL neutrophils was found for both sampling times. Mild winter weather conditions with lower temperatures, fewer minutes of sunlight, and a higher percent humidity were associated with significantly higher BAL neutrophil percentage and significantly higher mucus score in stabled clinical healthy horses. The clear seasonal influence on BAL neutrophilia and tracheal mucous findings in clinically normal horse needs to be included in the working diagnosis of asthma in horses. HIGHLIGHTSSignificant increase in tracheal mucus score in November compared to MaySignificant increase in BAL neutrophils in November compared to MayPositive correlation between TA and BAL neutrophilsNovember sampling period had significant fewer hours of sun than May sampling periodNovember sampling period had significant higher humidity than May sampling period.

Equine Airway Mast Cells are Sensitive to Cell Death Induced by Lysosomotropic Agents.

Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine‐ and human‐cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of in vivo‐derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of equine in vivo‐derived mast cells. Bronchoalveolar lavage (BAL) fluids from horses were incubated with LLME at 15–100 μm for up to 48 h. The overall cell viability was unaffected by 15 μm LLME up to 48 h, whereas a relatively modest drop in total cell counts (~30%) was seen at the highest LLME dose used. In contrast to the relatively low effect on total cell counts, LLME efficiently and dose dependently reduced the number of mast cells in BAL fluids, with an almost complete depletion (96%) of mast cells after 24 h of incubation with 100 μm LLME. A significant but less dramatic reduction (up to ~45%) of lymphocytes was also seen, whereas macrophages and neutrophils were essentially resistant. The appearance of apoptotic bodies suggested a mechanism involving apoptosis rather than necrosis. These findings suggest that equine airway mast cells are highly sensitive to lysosomotropic agents. Possibly, lysosomotropic agents could be of therapeutic value to treat disorders involving harmful accumulation of mast cells in the airways.