Bilateral bronchoalveolar lavage cytology profiles in a warmblood horse population during a 1-year period.

BACKGROUND: Bronchoalveolar lavage (BAL) cytology results from 1 lung might not be representative of both lungs. OBJECTIVES: To determine whether the lung site sampled would influence the horse's BAL cytology profile, and if a pooled BAL sample would be superior with regard to BAL cytology diagnosis in a cohort of healthy and subclinical asthmatic warmblood horses. ANIMALS: Fifty-nine horses in 2021 and 70 horses in 2022, the follow-up included 53 of the same in each year. METHODS: A cross-sectional study with follow-up included BAL cytology samples from individual lungs and from pooled BAL samples. The BAL samples were enumerated and differential cell count were applied to categorize the horses as control or with airway inflammation (AI). RESULTS: Bronchoalveolar lavage mast cell count was higher in left lung compared to right lung (2021; median 1.6 [range, 0.6-3.3] vs 1.2 [0.7-1.5] P = .009, 2022; median 3.1 [2.1-4.2] vs 2.4 [1.7-3.4], P < .001) and compared to pooled samples (2022; median 2.6 [1.7-3.7], P < .001). Between year 2021 and 2022, 17 of the horses had changes in BAL cytology from control to AI or vice versa. CONCLUSIONS AND CLINICAL IMPORTANCE: Pooled BAL sample was the least reliable for detecting AI, and was not representative of the overall lung condition.

Atrial fibrillation as a risk factor for exercise-induced pulmonary haemorrhage following a standardised exercise test.

BACKGROUND: Atrial fibrillation (AF) has been proposed as a risk factor for exercise-induced pulmonary haemorrhage (EIPH) due to increased pressure in the left atrium. OBJECTIVE: To evaluate if AF was associated with EIPH following a standardised exercise test (SET) to fatigue. STUDY DESIGN: Two-arm controlled experiment. METHODS: Ten untrained Standardbred mares mean (standard deviation [SD]) age 6 (2) years performed a SET on the treadmill in sinus rhythm (SR) (SET1) and 25-44 days after induction of self-sustained AF (SET2). AF was induced by tachypacing using a pacing device. Endoscopy, including tracheal wash and bronchoalveolar lavage (BAL), was performed 48-72 h before and 24 h after the two SETs. In addition, endoscopic grading of tracheal blood was performed 2 h after each SET. RESULTS: After SET1, none of the horses showed blood in the trachea, and two horses showed erythrophagocytosis. Following SET2, two horses had grade 1 blood in the trachea and free erythrocytes and erythrophagocytosis in the BAL, while another two horses had erythrophagocytosis in the BAL. In SET2, the overall performance on the treadmill was decreased with a lower maximum velocity (SET1 10.3 ± 0.8 m/s vs. SET2 8.9 ± 0.9 m/s, p = 0.004), a higher heart rate (284 ± 21 vs. 221 ± 18 bpm, p = 0.003) and more abnormal QRS complexes (p < 0.001) compared with SET1. CONCLUSIONS: Two horses showed signs of EIPH, resulting in visible blood in the trachea, when exercising in AF compared with SR. However, a possible link between EIPH, pulmonary pressure and AF needs to be further elucidated.

Characterization of Influenza D Virus in Danish Calves.

Influenza D virus (IDV) was first described in 2011 and has been found to mainly circulate among cattle and swine populations worldwide. Nasal swab samples were collected from 100 Danish calf herds (83 dairy and 17 veal herds) from 2018-2020. Influenza D virus was detected in 12 of the herds. Samples with the lowest cycle quantification value were selected for full genome sequencing. A hemagglutinin-esterase fusion (HEF) gene sequence from a Danish IDV collected in 2015 was also included in this study. Phylogenetic analysis showed that viruses from seven of the IDV-positive herds belonged to the D/OK lineage and clustered together in the HEF tree with the IDV collected in 2015. Viruses from the four other herds belonged to the D/660 lineage, where three of the viruses clustered closely together, while the fourth virus was more phylogenetically distant in all gene segments. The high level of genetic similarity between viruses from two different herds involved in calf trading suggests that transmission occurred through the movement of calves. This study is, to our knowledge, the first to describe the characterization of IDV in calves in Denmark.

Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle.

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system "Fluidigm BioMark HD" based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

Evaluation of Treatment With Respiratory Gene Technology and Serum in a Group of Standard Bred Racehorses With Cytological Evidence of Mild Equine Asthma.

Alternative treatment options to glucocorticoids for equine asthma is desirable due to withdrawal time. The objective was to evaluate if serum and Respiratory Gene Technology (RGT), a commercial kit to produce autologous conditioned serum, was effective in reducing bronchoalveolar lavage (BAL) neutrophils and mast cells in racehorses with cytological evidence of mild equine asthma . Thirty-six Standardbred trotters in active training were enrolled in this randomized clinical trial; a healthy control group (n=11), a RGT group (n=12) and a serum group (n=13). Endoscopy including tracheal wash (TW) and BAL was performed before (T0), after a 6-week treatment period including 12 intramuscular injections of RGT or serum (T6) and as a follow-up 10 weeks after treatment (T16). A significant decrease in BAL neutrophils for the RGT group was found between T0 and T6 (P = .002, d=-1.51, CI: -2.43;-0.59) and for the serum group between T0-T6 (P = .002, d=-1.36, CI: -2.26;-0.46). Further, a significant decrease in BAL mast cells between T0-T6 for the both the RGT group (P = .019, d=-1.23, CI: -1.22;-0.34) and the serum group (P= .004, d=-0.81, CI: -1.65;0.04), and further between T0-T16 (RGT P= .011, d=-1.55, CI: -2.62;-0.48; serum P= .044, d=-0.65, CI: -1.68;-0.37). No significant difference in TW cytology was found for any of the time-points. Pro- and anti-inflammatory cytokines were regulated according to treatment. The control group showed no cytological differences between any time-point. Study results showed that intramuscular treatment with both RGT and serum was effective associated with reduction of BAL neutrophils and mast cells in horses with cytological evidence of mild equine asthma. Further large-scale studies are necessary.

Bronchoalveolar lavage fluid cytokine, cytology and IgE allergen in horses with equine asthma.

The pathophysiology of equine asthma (EA) is still not fully described, but the involvement of an allergic reaction is strongly suspected. This theory has led to the use of allergen-specific immunoglobulin (Ig)E tests to support a diagnosis of asthma. The objective of this descriptive study was to evaluate the correlation between four subgroups of EA (mastocytic mild equine asthma [MEA], neutrophilic MEA, mixed MEA, and severe equine asthma [SEA]), allergen specific IgE (measured in both serum and BALF) and mRNA expression of selected genes in bronchoalveolar lavage fluid (BALF). Serum and BALF were collected from 64 horses with a history of lower airway problems with or without poor performance. Differential cell counts from BALF were used to assign horses to one of four groups (mastocytic MEA; neutrophilic MEA, mixed MEA, and SEA). The expression of messenger RNA (mRNA) coding for IL4, IL5, IL8, IL10, TGFB, TNFA, toll-like receptor (TLR)4, IL1RA, IL1B, matrix metalloproteinase 8 (MMP8), TLR9, chemokine ligand 5 (CCL5) and cluster of differentiation (CD)14 in BALF were measured using reverse transcriptase (RT) quantitative PCR (qPCR). Allergen-specific IgE was measured in serum and BALF using an allergen-specific IgE ELISA test with the screening panel: house mites, storage mites, mould and pollen. As expected, the BALF neutrophil differential count correlated with mRNA expression of MMP-8 (r = 0.611, p < 0.001), TLR-4 (r = 0.540, p < 0.001), IL-1RA (r = 0.490, p < 0.001), IL-1ß (r = 0.463, p < 0.001) and IL-8 (r = 0.302, p = 0.015). Cytokine expression of IL-1ß (p = 0.014), MMP8 (p = 0.028) and IL-1RA (p = 0.037) was significantly higher in the SEA group compared to the MEA subgroups. The BALF mast cell count was correlated with allergen-specific IgE for insects (r = 0.370, p = 0.002) and pollen (r = 0.313, p = 0.011). Eosinophils in BALF were correlated with BALF mRNA expression of IL-4 (r = 0.340, p = 0.006) together with a significant correlation between BALF eosinophils and allergen-specific IgE for mites (r = 0.930, p < 0.001) and pollen in BALF (r = 0.837, p < 0.001). No correlation was found between allergen-specific IgE in serum and BALF for any of the allergen in the screening panel. Based on these results from allergen-specific IgE in horses with EA is not found in systemic circulation, and only the mastocytic and mixed subgroups of horses with EA had allergen-specific IgE in BALF. Further studies are needed to clarify the relationships identified here.

Age-related dynamics of pro-inflammatory cytokines in equine bronchoalveolar lavage (BAL) fluid and peripheral blood from horses managed on pasture.

The objectives of this study were to evaluate the natural age-related variation and compare the level of pro-inflammatory cytokines in the peripheral blood and lower airways of horses. The mRNA expression of IL-1ß, IL-6, IL-8 TNF-α TLR-4 in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMC) were studied by quantitative real time polymerase chain reaction (PCR) and differential cell count cytology from 44 horses of different ages. A significant age-related increase was found for the mRNA expression of IL-6, IL-8, TLR-4 and TNF-α in stimulated BAL cells and for TNF-α in stimulated PBMC. Furthermore, a significant decrease was found in the mRNA expression of IL-1ß and TNF-α in stimulated BAL cells compared to stimulated PBMC. In conclusion, continued low antigen exposure of horses in the pasture environment could lead to a tight regulation of mRNA gene expression in the airway space compared to the peripheral blood and favour an age-related accumulation of mRNA genes.