Single-cell transcriptomics delineates the immune cell landscape in equine lower airways and reveals upregulation of FKBP5 in horses with asthma.

Equine asthma (EA) is a heterogenous, complex disease, with a significant negative impact on horse welfare and performance. EA and human asthma share fundamental similarities, making EA a useful model for studying the disease. One relevant sample type for investigating chronic lung inflammation is bronchoalveolar lavage fluid (BALF), which provides a snapshot of the immune cells present in the alveolar space. To investigate the immune cell landscape of the respiratory tract in horses with mild-to-moderate equine asthma (mEA) and healthy controls, single-cell RNA sequencing was conducted on equine BALF cells. We characterized the major immune cell populations present in equine BALF, as well as subtypes thereof. Interestingly, the most significantly upregulated gene discovered in cases of mEA was FKBP5, a chaperone protein involved in regulating the activity of the glucocorticoid receptor.

The Effect of Insect Bite Hypersensitivity on Movement Activity and Behaviour of the Horse.

Insect bite hypersensitivity (IBH) associated with Culicoides biting midges is a common allergic skin disease in horses, reducing the welfare of affected horses. This study investigated the effect of IBH on animal welfare and behaviour and assessed a new prophylactic insect repellent. In total, 30 horses were recruited for a prospective cross-over and case-control study. Clinical signs of IBH, inflammatory markers in skin biopsies and behavioural data (direct observations, motion index) were scored longitudinally during two consecutive summers. No differences were observed in the total number of itching behaviours or motion index between IBH-affected horses and controls, but higher numbers of itching behaviours were observed in the evening. IBH-affected horses showed both clinical and histopathological signs of inflammatory skin lesions, with even short periods of scratching being associated with moderate/severe inflammatory skin lesions. In order to improve the welfare of the IBH-affected horses, they should be stabled/given extra protection in the evening and even short-term exposure to Culicoides should be avoided. Preliminary results showed that the repellent tested can be used as a safe and non-toxic prophylactic to potentially reduce allergen exposure in horses with IBH, but further studies are needed to determine its efficacy.

Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.

BACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi.

Effectiveness of Cleaning and Sanitation of Stable Environment and Riding Equipment Following Contamination With Streptococcus equi Subsp. equi.

Streptococcus equi subsp. equi (S. equi) is transmitted via contact with infected horses or fomites such as equipment or surfaces of the stable environment. Effective cleaning and sanitation is essential to minimize risk of fomite-associated infections. This study assessed the effectiveness of cleaning and sanitation of experimentally S. equi contaminated materials and equipment found in stables. Wood, concrete, plastic, leather halters, leather gloves and polyester webbing halters were inoculated with a 24-hour culture S. equi laboratory strain. In addition, selected materials were inoculated with a clinical strain of S. equi. Three days post inoculation all materials were sampled for retention of viable S. equi and a subset of each material was cleaned and sanitized. After an additional 2 days all treated and untreated materials were sampled for continued retention of viable S. equi. Separate subsets of contaminated polyester halter material were washed at 40°C with or without drying at 70°C, or washed at 60°C. After cleaning and sanitation, all samples except polyester halters were culture negative. Even before cleaning and sanitation leather appears to poorly support survival of S. equi. After washing at 40°C and tumble drying, 14 of 16 halters were culture positive, however culture negative when washed at 60°C. Routine cleaning and sanitation of fomites contaminated with S. equi was generally effective to eliminate viable bacteria. However, survival between materials and strains differed, with leather poorly permissive to S. equi survival even without cleaning, whereas polyester webbing halters retained viable S. equi even after washing at temperatures of 40°C.

Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses.

Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.

Clinical features and treatment response to differentiate idiopathic peritonitis from non-strangulating intestinal infarction of the pelvic flexure associated with Strongylus vulgaris infection in the horse.

BACKGROUND: Peritonitis in horses secondary to non-strangulating infarction (NSII) has a guarded prognosis, even after intestinal resection. In contrast, horses with idiopathic peritonitis respond well to medical treatment. Affected horses in both cases often show signs of both colic and systemic inflammation, but early diagnosis is crucial for optimal treatment and an accurate prognosis. One cause of NSII is thrombus formation secondary to Strongylus vulgaris larval migration. There has been a documented increase in S. vulgaris prevalence in Sweden since the implementation of selective anthelmintic treatment in 2007, which subsequently could result in a rise in NSII cases. In a retrospective clinical study, medical records from cases diagnosed with NSII of the pelvic flexure or idiopathic peritonitis from three equine referral hospitals in Sweden during 2017-2020 were reviewed. Information including demographic data, relevant medical history, and clinical- and laboratory parameters were obtained from patient records. To facilitate the differentiation between cases of idiopathic peritonitis and cases with confirmed NSII of the pelvic flexure, the aim of the study was to compare clinical and laboratory parameters, clinical progression and initial response to antimicrobial treatment. A secondary aim was to compare survival-rates. RESULTS: Horses with NSII (n = 20) were significantly more likely to present during the winter months with a poorer response to medical treatment within 48 h. Cases of idiopathic peritonitis (n = 107) had a 100% survival rate with medical treatment, although one case required surgical correction of a colon displacement. In comparison, all confirmed NSII cases were non-responsive to antimicrobial treatment, with a survival rate to discharge of 50% after colon resection. Specific rectal findings and peripheral blood neutropenia were strongly associated with NSII. CONCLUSIONS: In Sweden, idiopathic peritonitis cases still predominate over S. vulgaris associated NSII cases and have an excellent survival rate with antimicrobial treatment. However, horses presenting with septic peritonitis during the winter months with a palpable rectal mass and displaying fever and colic signs beyond 48 h of medical treatment are likely to suffer from NSII of the pelvic flexure and should be considered for abdominal surgery.

Repeated nasopharyngeal lavage predicts freedom from silent carriage of Streptococcus equi after a strangles outbreak.

BACKGROUND: The value of repeated nasopharyngeal lavage (NPL) to detect silent carriers of Streptococcus equi has not been investigated. HYPOTHESIS/OBJECTIVES: Determine if results of serial testing for S. equi by NPL predicts subsequent true carrier status as determined by both NPL and guttural pouch lavage. ANIMALS: An outbreak of strangles with 100% morbidity in 41 mature Icelandic horses was followed prospectively to investigate development of silent carriers. All were initially positive to S. equi on NPL. The farm was closed to horse movement during the entire study. METHODS: Prospective observational study. Testing for S. equi was performed by NPL at weeks 18, 28, 29, and 30 postindex case and subsequently at week 45 by both NPL and guttural pouch lavage. Carrier status at week 45 was compared to results obtained at weeks 18, 28, 29, and 30. Descriptive statistics were calculated. Comparisons were made using Fisher's exact test or the Freeman-Halton extension with a P < .05 level of significance. RESULTS: Of 24 noncarriers at week 45, only 4 horses were negative on all 3 consecutive weekly NPL samples at weeks 28 to 30. However, 10 of the 11 horses with at least 3 negative NPL obtained from weeks 18, 28, 29, and 30 were S. equi-free at week 45 (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Repeated NPL on at least 3 separate occasions can assist in predicting S. equi carrier-free status in horses after recovery from a strangles outbreak.

Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers.

Streptococcus equi subsp. equi (SEE) is a host-restricted bacterium that causes the common infectious upper respiratory disease known as strangles in horses. Perpetuation of SEE infection appears attributable to inapparent carrier horses because it neither persists long-term in the environment nor infects other host mammals or vectors, and infection results in short-lived immunity. Whether pathogen factors enable SEE to remain in horses without causing clinical signs remains poorly understood. Thus, our objective was to use next-generation sequencing technologies to characterize the genome, methylome, and transcriptome of isolates of SEE from horses with acute clinical strangles and inapparent carrier horses-including isolates recovered from individual horses sampled repeatedly-to assess pathogen-associated changes that might reflect specific adaptions of SEE to the host that contribute to inapparent carriage. The accessory genome elements and methylome of SEE isolates from Sweden and Pennsylvania revealed no significant or consistent differences between acute clinical and inapparent carrier isolates of SEE. RNA sequencing of SEE isolates from Pennsylvania demonstrated no genes that were differentially expressed between acute clinical and inapparent carrier isolates of SEE. The absence of specific, consistent changes in the accessory genomes, methylomes, and transcriptomes of acute clinical and inapparent carrier isolates of SEE indicates that adaptations of SEE to the host are unlikely to explain the carrier state of SEE. Efforts to understand the carrier state of SEE should instead focus on host factors.

Markers of long term silent carriers of Streptococcus equi ssp. equi in horses.

BACKGROUND: Difficulty in detection of silent carriers of Streptococcus equi is a key reason for its continued spread to immunologically naïve groups of horses. OBJECTIVE: To determine whether clinical examination, markers of inflammation, or serology differentiate silent carriers of S. equi in recovered comingled horses. ANIMALS: Ninety-eight warmblood yearlings and 72 unaffected mares on a large breeding farm (outbreak A), 38 mature Icelandic horses at a riding stable (outbreak B), and 27 mixed breed horses at a boarding stable (outbreak C). METHODS: Prospective observational study 6 months to 2 years after strangles outbreaks. Carriers were defined as any animal positive on culture or qPCR to S. equi from nasopharyngeal lavage or guttural pouch endoscopy and lavage. Most horses had complete physical exams and 1 group included evaluation of white blood cell counts and serum amyloid A. Sera from all horses was tested for antibodies to antigens A and C of S. equi using an enhanced indirect ELISA. Descriptive statistics were calculated. Data were compared using paired t tests, Wilcoxon ranked test, chi square, or the Fishers exact test. Significance was set at P < .05. RESULTS: Apart from weanlings at 6 months in outbreak A, there was no significant association between any clinical markers or serology with carrier state (P = .06-1). Moreover, 3/12 culture positive carriers were seronegative to S. equi. CONCLUSIONS AND CLINICAL IMPORTANCE: Silent carriers of S. equi do not differ clinically or on markers of inflammation to their noncarrier herd-mates. Moreover, serology alone will not distinguish carriers in comingled horses.

Parasite Occurrence and Parasite Management in Swedish Horses Presenting with Gastrointestinal Disease-A Case-Control Study.

All grazing horses are exposed to intestinal parasites, which have the potential to cause gastrointestinal disease. In Sweden, there is a concern about an increase in parasite-related equine gastrointestinal disease, in particular Strongylus vulgaris, since the implementation of prescription-only anthelmintics approximately 10 years ago. In a prospective case-control study, parasitological status, using fecal analyses for strongyle egg counts, the presence of Anoplocephala perfoliata eggs and S. vulgaris Polymerase chain reaction (PCR) as well as serology for S. vulgaris, were compared between horses presenting with or without gastrointestinal disease at a University hospital during a one-year period. Information regarding anthelmintic routines and pasture management was gathered with an owner-filled questionnaire. Although the prevalence of S. vulgaris PCR was 5.5%, 62% of horses were positive in the enzyme-linked immunosorbent assay (ELISA) test and horses with peritonitis showed higher antibody levels for S. vulgaris, as compared to other diagnoses or controls. Overall, 36% of the horse owners used only fecal egg counts (FEC), 32% used FEC combined with specific diagnostics for S. vulgaris or A. perfoliata, and 29% dewormed routinely without prior parasite diagnostics. Effective management methods to reduce the parasitic burden on pastures were rare and considering exposure to S. vulgaris appears high; the study indicates a need for education in specific fecal diagnostics and pasture management.

Potential for residual contamination by Streptococcus equi subspp equi of endoscopes and twitches used in diagnosis of carriers of strangles.

BACKGROUND: Endoscopic examinations are essential for diagnosis and treatment of strangles (S equi infection) in horses. However, even after disinfection, endoscopes may retain viable bacteria or bacterial DNA. Twitches are commonly used during endoscopic examinations and can thus also potentially transmit the organism to other horses. OBJECTIVES: To evaluate the efficacy of different disinfectant methods to eliminate S equi from experimentally contaminated endoscopes and twitches and the effectiveness of field disinfection of endoscopes used in sampling carriers of S equi. STUDY DESIGN: Experimental contamination and observational field study. METHODS: One endoscope and 30 twitches were contaminated with standardised S equi broth solutions. The endoscope was disinfected following three protocols using various disinfectants for manual disinfection. A fourth protocol used an automated endoscope reprocessor (AER). The twitches (n = 30) were disinfected following eight different disinfecting protocols. Three endoscopes used in sampling for silent carriers were disinfected following a field-based protocol. After each protocol the endoscopes and twitches were sampled for S equi by culture and qPCR. RESULTS: Following experimental contamination all endoscope disinfection protocols, apart from 1/6 of the ethanol protocol were S equi culture negative. However, no endoscope disinfection protocol completely eliminated retention of S equi DNA. Field disinfection of endoscopes after sampling carriers yielded no culture positives and all but one (13/14) were qPCR negative. All twitches disinfected following experimental contamination were culture negative but sodium hypochlorite was the only disinfectant that completely eliminated detection of S equi DNA. MAIN LIMITATIONS: Experimental contamination may not reflect the numbers of S equi transferred to endoscopes or twitches during use on silent carriers and purulent secretions from infected horses may influence survival of S equi. CONCLUSIONS: While most disinfection methods appear to ensure removal of cultivable S equi, residual DNA can remain on both endoscopes and twitches.

Influence of penicillin treatment of horses with strangles on seropositivity to Streptococcus equi ssp. equi-specific antibodies.

BACKGROUND: Antibiotic treatment of horses with strangles is reported to impair the development of immunity to subsequent exposure to Streptococcus equi ssp equi (S. equi). However, apart from a single clinical report, evidence-based studies for this hypothesis are lacking. HYPOTHESIS/OBJECTIVE: To determine whether penicillin treatment during clinical strangles influences the development or persistence of seropositivity to S. equi-specific antibodies. ANIMALS: A natural outbreak of strangles with 100% morbidity in 41 unvaccinated mature Icelandic horses. METHODS: A prospective longitudinal study of acute clinical strangles from onset through full recovery approximately 10 months after the index case. Horses were monitored clinically 6 times for S. equi, as well as serologically for antibodies to antigens A and C of S. equi using an enhanced indirect ELISA. Seven horses received penicillin within 11 days of onset of fever (Group 1), 5 between 16 and 22 days after onset of fever (Group 2), and the remainder (Group 3, n = 29) received no antibiotics during clinical disease. The proportions of seropositive horses in each group were compared using an extension of Fisher's exact test with P < .05 as the level of significance. RESULTS: Although all horses were seropositive to S. equi within 2 months of the index case, significantly fewer horses treated early (Group 1) remained seropositive by 4 to 6 months (P = .04 and .02, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Findings support earlier suggestions that penicillin administered during acute strangles can interfere with persistence of humoral immunity to S. equi.

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.

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.

Installation of mechanical ventilation in a horse stable: effects on air quality and human and equine airways.

OBJECTIVES: To examine the effects of installing a mechanical ventilation system at a riding-school stable on indoor air quality and human and horse airways. METHODS: The intervention was the installation of mechanical ventilation in a riding-school stable. Carbon dioxide (CO2), ammonia, particles, horse allergen, microorganisms and endotoxins were measured in the stable. The stable-workers and riding-students completed a questionnaire and underwent the following tests: analysis of nasal lavage for inflammation biomarkers; levels of exhaled nitrogen oxide (NO); measurements of daily peak-expiratory flow (PEF). The horses were examined clinically by airway endoscopy and bronchoalveolar lavage (BAL) and were analysed for cytology and biomarkers. RESULTS: Levels of CO2 were nearly halved and airborne horse allergen levels were markedly reduced (5-0.8 kU/m3) after the intervention. A decreased level of ultrafine particles was observed (8000-5400 particles/cm3) after the intervention, while total and respirable dust levels were mainly unchanged (200 and 130 µg/m3). Levels of microorganisms in surface samples decreased following the intervention, whereas airborne microorganisms and endotoxin increased. There was no significant change in human symptoms, PEF-variability, exhaled NO or inflammatory biomarkers in the nasal lavage. In horses, the mean score of lower airway mucus was significantly reduced together with the mean level of expression of interleukin-6 mRNA in BAL cells after the intervention. CONCLUSIONS: The installation of a mechanical ventilation system resulted in an increased air exchange rate, as demonstrated by reduced levels of CO2, ammonia, ultrafine particles and horse allergen. There was no significant clinical effect on human airways, but there was a tendency for reduced inflammation markers. The results on the horses may indicate less impact on their airways after the intervention.

Influence of horse stable environment on human airways.

BACKGROUND: Many 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. METHODS: The 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. RESULTS: Measurements 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-beta-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 doctor's diagnosed asthma which was well treated. CONCLUSION: Biomarkers involved in the development of airway diseases have been studied in relation to environmental exposure levels in equine stables. Respirable dust and 1,3-beta-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.

Markers of respiratory inflammation in horses in relation to seasonal changes in air quality in a conventional racing stable.

Airborne factors in a conventionally managed racing stable and markers of pulmonary inflammation in the stabled horses were investigated on 3 occasions at 6-month intervals, including 2 winter periods and the intervening summer period. The stable measurements included inside and outside ambient temperature and relative humidity, levels of total and respirable dust, endotoxin, and 1,3-beta-glucan. Horses (n = 12) were examined in detail clinically as well as by endoscopy, bronchoalveolar lavage (BAL) cytology, and real-time polymerase chain reaction (RT-PCR) analysis of BAL-cells for IL-6 and IL-10 mRNA. Indoor measurements showed low dust levels irrespective of season. Inhalable dust, as well as 1,3-beta-glucan, were higher during the winter stabling period, whereas endotoxin levels were higher during summer. Complete data from all sampling occasions to be used for further evaluation was obtained for only 8 of the horses. There was a trend for elevation of BAL neutrophils in the horses during winter stabling that coincided with a 3.7-fold increased expression of IL-6 mRNA in BAL cells (P = 0.014). Compared to summer sampling, IL-10 mRNA expression was significantly upregulated in only 1 of the winter sampling occasions, implicating influence on immune regulation by factor/s apart from seasonal differences. Our findings suggest up-regulation of innate immunity in the airways of stabled horses; in particular involving IL-6 in association with mild elevations in respirable dust, 1,3-beta-glucan, and/or cold ambient air. However, given that this study was observational, other unmeasured environmental factors associated with winter stabling need to be considered.

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

BACKGROUND: The 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-alpha, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission. RESULTS: Despite 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-alpha 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). CONCLUSION: Given 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-alpha 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-alpha are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.