Co-Occurrence of Equine Asthma and Pharyngeal Lymphoid Hyperplasia in Pleasure Horses

Abstract

With the increasing awareness of the “united airway disease” theory, more horses, not only sport horses but also pleasure horses, undergo detailed examinations of the respiratory tract. Using endoscopy, equine asthma (EA) is most commonly diagnosed in the lower airway, while pharyngeal lymphoid hyperplasia (PLH) is common in the upper airway. Grading EA as mild–moderate (MEA) and severe (SEA), this study aims to compare the co-occurrence and investigate the possible relationship between the clinical symptoms and endoscopic signs of MEA/SEA and PLH in pleasure horses. In this retrospective study, 80 out of 93 pleasure horses suspected of EA were enrolled and underwent a standardized protocol for a complete airway examination, including resting endoscopy with mucus accumulation assessment and cytology. The obtained results were scored and analyzed. In the studied pleasure horses, PLH co-occurred more frequently in horses with EA than without (p < 0.0001) and more in horses with SEA than with MEA (p = 0.025). However, when EA and PLH co-occurred, the severity of the clinical symptoms of EA did not increase (p > 0.05). In both EA and PLH, the amount of tracheal and nasopharyngeal mucus increased with the severity of the disease; however, it was positively correlated (ρ = 0.33; p = 0.02) only in SEA horses. In conclusion, it is likely that EA is often accompanied by PLH; however, PLH did not play a role in increasing the severity of EA’s clinical symptoms. The role of the severity of accumulated mucus in the lower and upper airways when EA/PLH co-occur requires further research to confirm the morphological and functional unity of the respiratory tract, aligning with the concept of “united airways disease”.

1. Introduction

In human medicine, the concept of unified airways has been developed, recognizing that the nature of inflammation in the upper and lower airways is usually the same [1]. The upper and lower airways are contiguous, sharing functional linkages and similar histological pictures [2]. Upper airway diseases, such as rhinitis, chronic rhinosinusitis, and nasal polyps, have been linked to concurrent lower airway disorders like asthma, chronic obstructive disease, cystic fibrosis, and obstructive sleep apnea [3,4,5]. A similar link has been postulated in equine medicine [6,7,8,9,10,11,12,13], predominantly between equine asthma (EA) and various lower and upper airway diseases. EA is the most common equine lower airway disease associated with an increase in the negative pressure in the airways due to chronic airway inflammation, bronchospasm, excessive mucus secretion, and airway remodeling [14]. EA is graded based on clinical severity from mild–moderate equine asthma (MEA) to severe equine asthma (SEA) [14]. It is estimated that MEA affects approximately 68–77% of the pleasure horse population in the northern hemisphere, while SEA affects up to 14–17% [14]. In racehorses, bronchoalveolar lavage fluid (BALf) cytology revealed evidence of MEA in 80% of examined horses [15,16]. However, MEA-affected horses often do not show clinical symptoms of lower airway disease [17], leading to underdiagnosis until EA exacerbation or the accumulation of signs from another airway disorder.

Accumulating evidence has revealed links between MEA and exercise-induced pulmonary hemorrhage (EIPH) as well as between EIPH and upper airway obstruction (UAO). In both cases, negative pressure in the airways creates a risk of disruption of the alveolar–capillary membrane [12], similar to cases of UAO and negative pressure pulmonary edema (NPPE) [18] in humans. Moreover, in horses with concurrent MEA and EIPH, asthmatic inflammation and bronchoconstriction increase intrapleural pressure, leading to bleeding. However, EIPH can occur regardless of whether UAO or EA is present [13]. EA has also been linked with the occurrence of palatal disorders (PD), such as palatal instability (PI) and dorsal displacement of soft palate (DDSP) [6,7]. Both MEA and SEA have been reported as one of the factors exacerbating or causing DDSP [6]. In pleasure horses with PI and concurrent SEA, PI may resolve after the inflammation of lower airways has been treated [7]. Also, in sport horses with concurrent PI and MEA, as well as those with nasopharyngeal collapse and MEA, a similar outcome after anti-inflammatory treatment has been reported [11]. The exact basis of this connection is still not fully understood; however, the impact of increased negative pressure due to lower respiratory tract obstruction and the spreading of the inflammatory process is suspected [6]. Therefore, pharyngeal lymphoid hyperplasia (PLH), another inflammatory disease of the upper airways, and its co-occurrence with EA is of interest to equine practitioners [19,20]. PLH is characterized by the inflammation of local lymphoid tissue in the nasopharyngeal region [21], with an etiology involving the exposure to various irritants, allergens, and viral or bacterial agents [22]. PLH has been shown to predispose horses to UAO, including DDSP, nasopharyngeal collapse, aryepiglottic fold, and nasopharyngeal collapse [23]. The reason for this link is believed to be neural inflammation of the pharyngeal branch of the vagus nerve, leading to the neuromuscular dysfunction of the upper airway and increased negative pressure in the lower airway [24].

PLH is graded based on the clinical symptoms and endoscopic appearance of the nasopharyngeal mucosa from 0, indicating no signs of PLH, to 4, indicating severe PLH [21]. In young horses up to 5 years old, PLH is a common, self-limiting condition [22]. Saulez et al. reported grade 2–4 PLH in 63% of thoroughbred horses, noting that in young horses (3 vs. 4 years old), grade 3–4 PLH prevailed [25]. Hobo et al. revealed grade 3–4 PLH in 37% of 2-year-old thoroughbred racehorses; this decreased to nearly 0% in horses > 6 years [26]. Importantly, in young horses, PLH overlaps in time with the onset of training and performance career [22]. Many authors believe that the grade of PLH does not affect horse performance [19,25,26], although affected individuals may show clinical symptoms of airway disease. Clinical symptoms, also seen in older horses over 5 years of age, include abnormal respiratory sounds, coughing, and epistaxis [22], while endoscopically, the extent of pharyngeal follicular hyperplasia and the nasopharyngeal mucus quantity in the nasopharyngeal region are evaluated [20]. As clinical symptoms are non-specific and may accompany many airway diseases [25], a detailed examination of the entire respiratory tract, including resting endoscopy, is needed for an accurate differential diagnosis. As a resting endoscopy with tracheal mucus evaluation and BALf cytology is a ‘golden standard’ for the diagnosis of EA [17], the endoscopic signs of PLH and EA can be assessed during the same examination.

Therefore, this study aims to investigate the possible relationship between the endoscopic signs of two common diseases of the upper and lower airways in pleasure horses, considering PLH and EA. We hypothesize that (1) PLH co-occurs more frequently in horses with EA than without and with SEA than with MEA; (2) when EA and PLH co-occur, the severity of clinical symptoms of EA increases with the grade of PLH; (3) when EA and PLH co-occur, the amount of tracheal and nasopharyngeal mucus accumulation is related.

2. Materials and Methods

2.1. Horses

A prospective observational study enrolled ninety-three (n = 93) horses suspected of EA. All studied horses were privately owned clinical patients, used for pleasure (did not participate in sports competitions) at the preliminary level of training (working under saddle in walk, trot, and canter). Details regarding the environment were obtained in the form of a survey answered by the owners. Feeding regimen, housing type, stable ventilation, stable cleaning, hay and straw storage, and access to the paddock were considered.

Owners provided written consent for the horse’s inclusion in the study. Horses were examined from July 2020 to April 2024 by the field services of the Equine Clinic at Warsaw University of Life Sciences. Inclusion criteria included a history of suspected EA, while exclusion criteria included treatment with corticosteroids, bronchodilators, antibiotics, and/or allergen immunotherapy within four weeks before the examination. Twelve horses were excluded due to the administration of corticosteroids (7 horses), antibiotics (3 horses), and bronchodilators (2 horses), resulting in a final study group of eighty-one horses (n = 81). All horses underwent a standardized protocol for a complete airway examination, including a detailed respiratory tract examination, resting airway endoscopy, tracheal wash (TW) cytology, and BALf cytology. The entire examination protocol comprised standard diagnostic tests performed as a part of veterinary clinical examinations. Based on the results, the horses were classified as diseased (EA studied group; n = 71) and healthy (EA-free control group; n = 10), as described in Section 3.

2.2. Detailed Respiratory Tract Examination and Resting Endoscopy

A detailed respiratory tract examination was performed and quantified using the modified score system [7], which allows for the scoring of clinical symptoms such as respiratory rate, nasal discharge, tracheal and thoracic auscultation, nasal flare, cough, and abdominal lift. As a numerical result of a detailed respiratory tract examination, each horse was characterized by a sum of clinical scores, as shown in

Table 1. Clinical symptoms scored based on the detailed respiratory tract examination.

Clinical Signs	Descriptor	Score
Respiratory rate	<16	0
	17–20	1
	21–30	2
	>30	3
Nasal discharge	none	0
	serous	1
	mucopurulent/epistaxis	2
Tracheal auscultation	normal tracheal sounds	0
	slight increase	1
	clearly audible increased	2
	crackles and wheezing present	3
Thorax auscultation	normal pulmonary sounds	0
	slight increased pulmonary sounds	1
	clearly audible increased pulmonary sounds	2
	crackles and wheezing present	3
Nostril flare	none	0
	present	1
Cough score	none	0
	coughs at specific times of day (feeding/exercising/making beds)	1
	frequent cough	2
Abdominal lift	none	0
	slight flattening of ventral flank	1
	obvious abdominal lift and “heave line”	2
Maximum score		16

. The scores were assigned by two independent clinicians.

Resting endoscopy was performed following the international guidelines [27,28] using a flexible video bronchoscope (8 × 2000 mm, Karl Storz, Berlin, Germany). Horses were sedated with detomidine hydrochloride (Domosedan; Orion Corporation, Espoo, Finland; 0.01 mg/kg b.w i.v.) and butorphanol (Torbugesic; Zoetis Polska Sp. z o.o., Warsaw, Poland; 0.01 mg/kg b.w i.v.), with doses determined based on the horse’s body weight. The nasopharynx, larynx, trachea, and tracheal septum were visually examined. The nasopharynx mucus was assessed quantitatively by visual evaluation, whereas the tracheal mucus was assessed quantitatively and qualitatively by visual evaluation and cytology. The scoring of both quantitative assessments is described in Section 2.4 and Section 2.5.

2.3. Tracheal Wash and Bronchoalveolar Lavage Fluid

TW and BALf were collected following a standardized protocol [29]. TW was collected using a sterile polytetrafluroethylene catheter (KRUUSE, Langeskov, Denmark) passed through the working channel of the endoscope into the trachea. Sterile normal saline (30 mL; 0.9% NaCl; Baxter, Warsaw, Poland) was infused into the trachea, and a sample was collected from the tracheal puddle. The TW sample was placed in EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for cytology (Becton Dickinson, Franklin Lakes, NJ, USA).

The BALf protocol was then performed after infusing 20 mL of lidocaine solution (Lidor 20 mg/mL, Richter Pharma, Wels, Austria) into the tracheal septum area. BALf was collected using a determined catheter (KRUUSE, Langeskov, Denmark) wedged into a bronchus. Sterile normal saline (250 mL; 0.9% NaCl; Baxter, Warsaw, Poland) was infused into the bronchus and aspirated immediately. The BALf sample was placed in EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for cytology.

The TW and BALF samples were centrifuged (300× g for 10 min). The supernatant was decanted, and the pellet was smeared on the slide. The smears were dried, fixated using Cytofix (Samko, Warsaw, Poland), and sent to the commercial veterinary laboratory (Laboklin Laboratory, Warsaw, Poland) for cytology.

2.4. Classification of Equine Asthma

The presence and severity of EA were assessed using the EA scores described in

Table 2. Scoring of EA based on the clinical signs, endoscopic signs, and cytology of TW and BALf.

Endoscopic Signs	Descriptor	Score
Neutrophil percentage in BALf	<5%	0
	5–20	1
	20–60%	2
	>60%	3
Neutrophil percentage in TW	<20%	0
	20–60%	1
	>60%	2
Tracheal mucus accumulation	None	0
	little, multiple small blobs	1
	moderate, larger blobs	2
	confluent or stream–forming thick white to yellow	3
	pool forming mucus	4
	profuse amounts of mucus	5

in connection with the clinical scores. The TW and BALf samples were qualitatively assessed using neutrophil percentages following Couëtil et al. [17]. Based on the TW and BALf neutrophil percentage, horses were assigned into one of three categories: (1) normal TW: <20% neutrophils and normal BALf: <5% neutrophils; (2) moderate inflammation in TW: >20%; ≤60% and in BALf: >5%; ≤20% neutrophils; and (3) severe inflammation in TW: >60% and in BALf: >20% neutrophils [17].

EA was confirmed when the neutrophil percentage exceeded >5%, and the horse was assigned to category 2 or 3. Horses assigned to category 1 were classified as EA-free. Next, for the classification of EA severity, the clinical staging sheet developed by Tilley et al. [30] was used and scored, as shown in Table 1. Based on clinical symptom scoring, horses with confirmed EA (TW/BALf categories 2 and 3) were classified as MEA or SEA.

MEA was recognized when the clinical symptom score was ≤4 points and BALf neutrophil percentage was <60%. SEA was recognized when the clinical symptom score was >4 points, and BALf neutrophil percentage was >60%.

Additionally, tracheal mucus accumulation was quantitatively assessed using on a 0–5 point scale following Gerber et al. [27,31] (Table 2).

2.5. Classification of Pharyngeal Lymphoid Hyperplasia

The presence and grade of PLH were assessed using a visual assessment of the nasopharyngeal mucosa using a 0–4 points PLH scale following Holcombe et al. [22] (

Table 3. Scoring of PLH based on the endoscopic signs.

Endoscopic Signs	Descriptor	Scores
Nasopharyngeal mucosa	no visible lymphoid tissue	0
	few small white follicles over the dorsal wall	1
	numerous small follicles on the dorsal and lateral nasopharyngeal wall	2
	hyperemic follicles over the entire dorsal and lateral wall	3
	large edematous hyperemic follicles coalesce broad-based polypoid aggregate	4
Nasopharyngeal mucus accumulation	no visible mucus	0
	little blobs of mucus presented ventrally and abaxially to the epiglottis	1
	confluent mucus presented ventrally and dorsally to the epiglottis	2
	large amount of dense mucus	3

). Based on the PLH scale, horses were classified as PLH-free (grade 0), grade 1 PLH, grade 2 PLH, grade 3 PLH, or grade 4 PLH, as shown in Figure 1. Additionally, the nasopharyngeal mucus was quantitatively assessed using a modified 0–3 points scale following Koblinger et al. [20] (Table 3).

2.6. Statistical Analysis

Statistical analyses were performed using the GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA), with the level of significance set at p < 0.05.

2.6.1. Descriptive Statistics

Descriptive statistics (number of horses classified as EA-free, MEA, and SEA; number of horses as classified as grades 0–4 PLH; percentage of horses showing evidence of the studied environmental descriptors) were used for the characterization of the collected data. The age of horses in each group was presented using ranges (minimum and maximum age) and the mean ± standard deviation (SD). Then, data series were tested for normality using the Shapiro–Wilk test. Since the data were not normally distributed, non-parametric tests were selected for further comparisons and the plot results were presented using the medians, interquartile ranges, and minimum and maximum values.

2.6.2. Co-Occurrence of EA with PLH

The co-occurrence of EA with PLH in pleasure horses was compared using a Chi-square test. Data were presented in consecutive rows as the number of horses classified in grade 0–4 PLH for EA-free, MEA, and SEA groups separately, and then compared between EA-free, MEA, and SEA groups and next between MEA and SEA groups. The number of horses was supported by the percentage in relation to group size and total sample size, respectively.

2.6.3. Severity of Clinical Symptoms

Clinical symptom scores were compared using the Mann–Whitney test between MEA and SEA groups. Then, each group was subdivided into grade 0–3 PLH subgroups and compared between PLH grades using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test.

2.6.4. Tracheal and Nasopharyngeal Mucus Accumulation

As different scales were used to assess tracheal and nasopharyngeal mucus accumulation (0–5 points and 0–3 points, respectively), data were normalized using a standardization protocol before comparison. A standardized amount of mucus was compared between locations (trachea, nasopharyngeal area) using the Wilcoxon–Signed–Rank test. Next, for each location, a standardized amount of mucus was compared between EA-related groups and PLH-related groups, separately. The Kruskal–Wallis test, followed by Dunn’s multiple comparisons test, was used. Then, Spearman’s rank correlation coefficient (ρ) was calculated between a standardized amount of tracheal mucus and nasopharyngeal mucus for each EA-related and PLH-related pair of data series. Correlations were considered significant for p < 0.05.

2.6.5. Correlations with Age and Environmental Descriptors

The Spearman’s rank correlation coefficient (ρ) was calculated between disease classifiers (clinical symptoms scores; EA presence and severity; PLH grades; tracheal mucus scores; nasopharyngeal mucus scores) and age and environmental descriptors (feeding regimen, housing type, stable ventilation, stable cleaning, hay and straw storage, and access to the paddock). Correlations were considered significant for p < 0.05.

3. Results

3.1. Descriptive Statistics Results

This study enrolled thirty-six mares and forty-five geldings of different breeds (fifty Polish Half-Bred horses, ten Thoroughbreds, four Silesian horses, three Arabian horses, three Belgian horses, two Hanoverian horses, two Hucul horses, two Konik Polski horses, two Tinkers, two Welsh ponies, and one Fiord), aged between 4 and 26 years (mean ± SD: 12.9 ± 4.6).

Considering EA occurrence and severity classification, ten horses (seven mares and three geldings, aged between 7 and 17 years (mean ± SD: 10.9 ± 3.6)) were classified as EA-free; twenty-three horses (seven mares and sixteen geldings, aged between 4 and 19 years (mean ± SD: 11.2 ± 3.8)) were classified as MEA; whereas, forty-eight horses (twenty-two mares and twenty-six geldings, aged between 4 and 26 years (mean ± SD: 14.1 ± 4.7)) were classified as SEA.

Considering PLH occurrence and severity classification, eight horses (four mares and four geldings, aged between 8 and 17 years (mean ± SD: 11.6 ± 3.2)) were classified as grade 0 (PLH-free); twenty-three horses (twelve mares and eleven geldings, aged between 7 and 17 years (mean ± SD: 14.0 ± 4.8)) were classified as grade 1; thirty horses (eleven mares and nineteen geldings, aged between 6 and 26 years (mean ± SD: 12.8 ± 5.0)) were classified as grade 2; nineteen horses (eight mares and eleven geldings; aged between 4 and 20 years (mean ± SD: 12.5 ± 3.6)) were classified as grade 3; and one horse (mare; age 4 years) was classified as grade 4. Because only one horse was classified as grade 4 PLH, further statistical analysis was performed on 80 horses ranging from grade 0 to 3 PLH.

The percentage of horses showing evidence of the studied environmental descriptors was as follows. Considering the feeding regimen, 65.4% of the studied horses were fed hay and concentrates, 12.3% of horses were fed soaked hay and concentrates, 7.4% of horses were fed only hay, and 14.8% of horses were fed only soaked hay. Among the types of housing, 18.5% of studied horses were housed in stables with boxes with access to the outside or with a window, 16.0% of horses were housed in the free-range stable, and 65.4% of horses were housed in stables with boxes with access to the corridor. Stable ventilation in 39.5% of the stables was very good; in 34.6% of the stables, it was good; in 22.2% of the stables, it was poor; and in 3.7% of the stables, it was bad. In the case of free-range housing, the ventilation was always assessed as very good. Given the stable cleaning, 4.9% of stables were cleaned more often than twice a day, 60.5% of stables were cleaned twice a day, 19.8% of stables were cleaned once a day, and 14.8% of stables were cleaned less than once a day. In 66.7% of the stables, hay and straw were stored in a building outside the stable, 18.5% in the stable in a separate box/storage space, and 14.8% in the stable in the attic. All studied horses had daily access to the paddock, including 51.9% of horses to grassy pasture and 48.1% of horses to the sandy paddock.

3.2. Co-Occurrence of EA with PLH

In EA-free horses, no PLH (grade 0) was found in six horses (60.0%) and grade 1 PLH in four horses (40.0%), whereas no horses with grade 2 and 3 PLH were found. In MEA horses, no PLH (grade 0) was found in two horses (8.7%), grade 1 PLH in eight horses (34.8%), grade 2 PLH in eleven horses (47.8%), and grade 3 also in two horses (8.7%). In SEA horses, grade 1 PLH was found in 11 horses (23.4%), grade 2 PLH in 19 horses (40.4%), and grade 3 in 17 horses (36.2%); so that all horses with SEA showed endoscopic signs of PLH. Comparing the pattern of PLH occurrence between EA-related groups, PLH appeared more frequently in EA horses than EA-free horses (p < 0.0001), as well as with SEA than with MEA (p = 0.025) (

Table 4. The co-occurrence of equine asthma (EA; including EA-free, mild–moderate equine asthma (MEA), severe equine asthma (SEA) horses) with pharyngeal lymphoid hyperplasia (PLH; including grades 0–3) in horses before and after severe equine asthma.

EA		EA-Free	MEA	SEA	Total
PLH	Grade 0	6 (60.0%; 7.5%)	2 (8.7%; 2.5%)	0	8 (10.0%)
	Grade 1	4 (40.0%; 5.0%)	8 (34.8%; 10.0%)	11 (23.4%; 13.8%)	23 (28.8%)
	Grade 2	0	11 (47.8%; 13.8%)	19 (40.4%; 23.8%)	30 (37.5%)
	Grade 3	0	2 (8.7%; 2.5%)	17 (36.2%; 21.3%)	19 (23.8%)
	Total	10 (100%; 12.5%)	23 (100%; 28.8%)	47 (100%; 58.8%)	80 (100%; 100%)
EA-free/MEA/SEA: Chi-square test; p < 0.0001			MEA/SEA: Chi-square test; p = 0.025

).

3.3. Severity of Clinical Symptoms

In horses with confirmed EA, the severity of clinical symptoms reflected by the clinical symptoms scores was significantly higher in SEA than in MEA horses (Figure 2A; p < 0.0001). Therefore, the PLH grade-related differences were compared in SEA and MEA groups, separately. However, in both groups MEA (Figure 2B; p = 0.16) and SEA (Figure 2C; p = 0.09) groups, there were no differences in the severity of clinical symptoms between grades of PLH.

3.4. Tracheal and Nasopharyngeal Mucus Accumulation

In EA-free horses, the amount of mucus was higher in the nasopharyngeal area than in the trachea (p = 0.002); however, no differences between locations were noted in MEA (p = 0.77) and SEA (p = 0.34) horses. In only SEA horses, the weak positive significant correlation (ρ = 0.33; p = 0.02) was found between tracheal and nasopharyngeal mucus amounts. In the trachea, the amount of mucus was low in EA-free horses, higher in MEA horses, and highest in SEA horses (p < 0.0001). Whereas in the nasopharyngeal area, the amount of mucus was low in EA-free and MEA horses and higher in SEA horses (p < 0.0001) (Figure 3A).

In PLH horses, the amount of mucus did not differ between locations regardless of PLH grades (p > 0.05), and no significant correlations were found (p > 0.05), respectively. Both in the trachea (p < 0.0001) and nasopharyngeal area (p < 0.0001), the amount of mucus was low in PLH-free horses (grade 0) and higher in grade 2–3 PLH horses. Moreover, the amount of tracheal mucus did not differ between grades 0 and 1 as well as grades 1 and 2, whereas the amount of nasopharyngeal mucus did not differ between grades 0 and 1 as well as grades 2 and 3 (Figure 3B).

3.5. Correlations with Age and Environmental Descriptors

Weak positive correlations were found between the horse’s age and clinical symptom scores (ρ = 0.32; p = 0.004); EA presence and severity (ρ = 0.32; p = 0.004), and amount of tracheal mucus (ρ = 0.26; p = 0.01). Considering environmental descriptors, clinical symptom scores were weakly negatively correlated with a feeding regimen with only soaked hay (ρ = −0.32; p = 0.003) as well as weakly positively correlated with bad ventilation in the stable (ρ = 0.27; p = 0.01) and cleaning the stable less than one a day (ρ = 0.31; p = 0.005). The presence and severity of EA were weakly and negatively correlated with only a feeding regimen with only soaked hay (ρ = −0.40; p = 0.002). PLH grades weakly negatively correlated with a feeding regimen with only soaked hay (ρ = −0.33; p = 0.003), housing in boxes with access to the outside or with a window (ρ = −0.24; p = 0.03), cleaning the stable twice a day (ρ = −0.26; p = 0.02), and storage of hay and straw in building outside the stable (ρ = −0.24; p = 0.03). The amount of tracheal mucus was weakly negatively correlated with the feeding regimen with only soaked hay (ρ = −0.29; p = 0.007) and weakly positively correlated with bad ventilation in the stable (ρ = 0.28; p = 0.01). Weak negative correlations were found between the amount of nasopharyngeal mucus and feeding regimen with only soaked hay (ρ = −0.26; p = 0.02), in boxes with access to the outside or with a window (ρ = −0.23; p = 0.04), very good (ρ = −0.19; p = 0.04) and good (ρ = −0.24; p = 0.03) stable ventilation, cleaning the stable twice a day (ρ = −0.25; p = 0.01), as well as storage of hay and straw in building outside the stable (ρ = −0.23; p = 0.04). Moreover, weak positive correlations were noted between the amount of nasopharyngeal mucus and cleaning the stable less than once a day (ρ = 0.30; p = 0.007) as well as storage of hay and straw in the stable in the attic (ρ = 0.23; p = 0.04) (

Table 5. Spearman correlation coefficients (ρ) between disease classifiers (clinical symptoms scores; equine asthma (EA) presence and severity; pharyngeal lymphoid hyperplasia (PLH) grades; tracheal mucus (TM) scores; nasopharyngeal mucus (NM) scores) and age and environmental descriptors (feeding regimen, housing type, stable ventilation, stable cleaning, hay and straw storage, and access to the paddock); ρ was provided when p < 0.05.

Age and Environment	Detail Descriptors	Clinical Symptoms	EA	PLH	TM	NM
Age		ρ = 0.32; p = 0.004	ρ = 0.32; p = 0.004	-	ρ = 0.26; p = 0.01	-
Feeding regimen	hay and concentrates	-	-	-	-	-
	soaked hay and concentrates	-	-	-	-	-
	only hay	-	-	-	-	-
	only soaked hay	ρ = −0.32; p = 0.003	ρ = −0.40; p = 0.002	ρ = −0.33; p = 0.003	ρ = −0.29; p = 0.007	ρ = −0.26; p = 0.02
Housing type	boxes & outside or window	-	-	ρ = −0.24; p = 0.03	-	ρ = −0.23; p = 0.04
	boxes & corridor	-	-	-	-	-
	free-range stable	-	-	-	-	-
Stable ventilation	very good	-	-	-	-	ρ = −0.19; p = 0.04
	good	-	-	-	-	ρ = −0.24; p = 0.03
	poor	-	-	-	-	-
	bad	ρ = 0.27; p = 0.01	-	-	ρ = 0.28; p = 0.01	-
Stable cleaning	>twice a day	-	-	-	-	-
	twice a day	-	-	ρ = −0.26; p = 0.02	-	ρ = −0.25; p = 0.01
	once a day	-	-	-	-	-
	<once a day	ρ = 0.31; p = 0.005	-	-	-	ρ = 0.30; p = 0.007
Hay and straw storage	building outside the stable	-	-	ρ = −0.24; p = 0.03	-	ρ = −0.23; p = 0.04
	in stable in separate box/storage space	-	-	-	-	-
	in stable in the attic	-	-	-	-	ρ = 0.23; p = 0.04
Daily access to the paddock	grassy pasture	-	-	-	-	-
	sandy paddock	-	-	-	-	-

).

4. Discussion

In the concept of a unified airway, each respiratory tract disease should be considered in a broader spectrum due to the possible presence of concurrent linkage disease. Widely, EA has been investigated to interfere with upper airway diseases [6,23]. In this study, we revealed a higher prevalence of PLH in horses with EA than in EA-free horses. Interestingly, the grade of PLH was not associated with the severity of EA symptoms; however, PLH appeared in all horses with SEA. This co-occurrence remains not fully determined; however, one may suggest the spreading of the inflammatory process occurred via the continuous airway epithelium and an increased reaction of pharyngeal lymphoid tissue as a result of asthma triggers, especially during the onset of symptoms exacerbation.

EA is a progressive condition with respiratory distress becoming more severe over the years [14], which is in agreement with our study, where clinical signs were positively correlated with a horse’s age. However, we cannot support the higher incidence of PLH in young horses < 5 years old [21,25,26] considering an older range group in our study. It is generally accepted that PLH results from exposure to environmental and infectious airborne antigens, while EA exacerbation occurs after exposure to the specific asthma triggers in predisposed individuals, such as the organic components of dust [22]. The role of infectious agents in EA pathogenesis needs further investigation. However, our study supports the environmental impact [28,32]. We reported more severe clinical signs of EA associated with poor ventilation and infrequent stable cleaning. Horses fed with soaked hay expressed fewer clinical signs; however, in those fed with soaked hay and concentrates, no direct correlation was found. It could be due to the low number of horses in that feed regimen, or it may suggest the impact of concentrate quality. It was already reported that the hygienic status of concentrates plays a role in maintaining proper equine respiratory tract health, as ventilation does not remove the dust challenge from horse nostrils when the animal is eating or sniffing [33]. Intemann et al. showed that concentrates play an important role in the emission of airborne allergens and have mold counts enough to induce coughing [34]. On the other hand Vandenput et al. [35] indicated that concentrates play a minor role as a source of aeroallergens in dust since the emission of dust is low. However, the association is still suggested.

Similarly, we found a positive correlation between PLH grade and (1) feeding with soaked hay and concentrates, (2) keeping in outside boxes, (3) frequent cleaning of the stable, and (4) outside storage of the bedding. We suggest that dust components are capable of initiating PLH, although opinions among researchers are still divided. Bagshaw et al. showed that racetrack location and race day quality index were associated with PLH severity in thoroughbred racehorses of all ages [36]. In contrast, Clarke et al. found no association between poor stable environments and PLH in 2-year-old horses [37].

Bacterial or viral infections of the respiratory tract may be linked to both PLH and EA; however, recent opinions are still divided [14]. Van Cleemput et al. showed that dust-derived proteases might disrupt airway epithelium integrity, leading to an increased infection rate of epithelial cells by equine herpesvirus type 1 (EHV-1) [38]. This is in agreement with Cook [39] and Blakeslee et al. [40], who confirmed the correlation between equine herpesvirus type 2 (EHV-2) detection and neutrophilia in tracheal wash, and Clarke et al. [37] who suggested PLH as a sequel to viral infection.

Similarly, the composition of the lower respiratory tract microbiota was associated with EA pathogenesis in young racehorses [37,38], while in older horses, this association has not been supported [8]. Lo Feudo et al. [8,23] reported that MEA horses were more positive for bacterial culture compared to the SEA horses, suggesting the same age-related association linked to the development of immunity. Interestingly, no association between PLH and bacterial culture was reported by present reports.

Both EA and PLH are associated with UAO diseases. In EA, bronchoconstriction, mucus accumulation, and airway remodeling lead to an increase in negative pressure [14]. The laryngeal mucosa is abundant in negative pressure receptors that are stimulated during obstruction and provide afferent information to the central nervous system, signaling the contraction of upper airway muscles that ultimately leads to the dilation and stabilization of the upper airway [41]. In the pharyngeal mucosa, mechanoreceptors of the pharyngeal branch of the vagus nerve are present and play a role in maintaining normal nasopharyngeal function [24].

It has been suggested that inflammation in this region may predispose horses to nasopharyngeal collapse, dorsal displacement of the soft palate, and aryepiglottic fold collapse [24,28]. Inflammatory follicles on the pharyngeal wall decrease airway size, potentially increasing negative pressure during inspiration, which results in the dorsal wall of the pharynx being pulled down and the soft palate being elevated [42]. Thus, if EA and PLH commonly co-occur, they may both play a role in the pathophysiology of UAO. Additionally, the incidence of PLH and some dynamic upper nasopharyngeal diseases appears to decrease as the horse ages [22].

The appearance of pharyngeal wall follicles varies between horses and may suggest the character of the inflammatory process, which may be acute or chronic. Red and edematous follicles indicate acute inflammation, while firm, white, and fibrotic follicles suggest a chronic process [42]. In our study, chronic pharyngeal follicles dominated, suggesting that the process might not be incidental.

Tracheal mucus accumulation correlates with clinical signs [43], cough, cytological indicators of airway inflammation [27], and poor performance [19]. Production of tracheal mucus can be initiated by inflammation or directly by irritants, including bacteria, viruses, and environmental contaminants. Variations in tracheal grades are observed in individuals depending on age, airway status, or even weather conditions, such as temperature and humidity [32,44,45]. In our study, there was no correlation between housing conditions and mucus accumulation. Only weak positive and weak negative correlations were found between the mucus score and bad ventilation and soaked hay feed regimen, respectively. This is in agreement with Koblinger et al., who found no association between housing conditions and mucus score [20].

Interestingly, in this study, we found a wide association between environmental conditions and nasopharyngeal mucus accumulation. To date, this hypothetical relationship has not been studied in a horse. We reported weak negative correlations between the amount of nasopharyngeal mucus and feeding regimen with only soaked hay, keeping horses in boxes with access to the paddock or a window, with very good or good stable ventilation, with stable cleaning twice a day, as well as storage of hay and straw in the building outside the stable. Interestingly, weak positive correlations were noted between the amount of nasopharyngeal mucus and cleaning the stable less than once a day, as well as with storage of hay and straw in the stable in the attic. This finding may reflect the independence in the upper and lower respiratory airway in terms of mucus secretion due to environmental irritants. In this study, tracheal and nasopharyngeal mucus accumulation increased with EA severity, similar to the research of Couëtil et al. [17], and PLH grades, similar to the research of Holcombe et al. [19]; however, most do not correlate between these two locations. In this study, tracheal and nasopharyngeal mucus accumulation increased with EA severity, similar to the research of Couëtil et al. [17], and PLH grades as observed in the Holcombe et al. [19] study. However, there was mostly no correlation observed between these two locations. Only a positive but weak correlation was observed in SEA, which may suggest that SEA horses produce such an excess of mucus in the trachea that, during expectoration [17], it also accumulates in the nasopharyngeal area, affecting the local mucus amount. This smaller number and the strength of correlations between the amount of mucus in the studied locations, compared to what was assumed in the third hypothesis, may reflect a certain independence between upper and lower airway inflammation [11,28], especially since Koblinger et al. noted a similar lack of correlation [20]. However, even if the PLH grade does not correlate with tracheal mucus grade, some associations may still be investigated. It has been shown that irritation of the nasopharynx and larynx receptors may increase mucus secretion from the trachea by triggering reflexes that run through the glossopharyngeal nerves, the superior laryngeal nerves, and the pulmonary vagus nerves, respectively [46,47]. While numerous studies on asthma are generally consistent, PLH has not received much attention. Therefore, further research is needed in a larger number of horses, in which sufficiently large groups with subsequent PLH grades will be EA-free and co-affected by MEA and SEA.

When considering the clinical usefulness of the presented results, the following limitations should be mentioned. The main limitation is the small sample size. Thus, a larger sample size should be enrolled to ensure that it is representative of the population, including healthy horses and both EA groups. Moreover, the study should be more adjusted considering the age of the horses, as the PLH has been shown to be age-related [48]. Finally, for a better understanding of the ongoing inflammatory process in equine airways, future research should pay more attention to the morphological and histological aspects. This approach would provide a more accurate and comprehensive understanding of the implications of the UAD concept in equine medicine.

5. Conclusions

EA is often accompanied by PLH, while PLH appeared more frequently in EA horses than EA-free horses as well as in SEA horses than in MEA horses. However, PLH did not play a role in increasing the severity of EA clinical symptoms. The role of the severity of accumulated mucus in the lower and upper airways when EA/PLH co-occur requires further research to confirm the morphological and functional unity of the respiratory tract, aligning with the concept of “united airways diseases”.