Analysis of the presence of Beta-hemolytic streptococci and cytological profiles in horse tracheal lavage samples with diverse clinical respiratory symptoms

Abstract

Background

Riding clubs face significant financial losses due to upper respiratory infections caused by Beta-hemolytic (β-hemolytic) streptococci, leading to strangles and related diseases that negatively impact the health and performance of horses. Our objective was to determine the relationship between these inflammatory cells, the presence of β-hemolytic streptococcal species, and the clinical symptoms exhibited by the horses. This study investigated the presence of β-hemolytic streptococcus in 133 tracheal lavage samples from horses exhibiting various clinical respiratory symptoms and quantified the populations of neutrophils, eosinophils, macrophages, lymphocytes, and mast cells. The horses included in this study exhibited a variety of clinical respiratory symptoms categorized into groups: (I) Horses with no apperent symptoms (Control Group), (II) Horses with only coughing, (III) Horses with only lymph node swelling, (IV) Horses with coughing and lymph node swelling, (V) Horses with all symptoms (coughing, lymph node swelling and nasal discharge). After obtaining the bacterial cultures from tracheal wash samples, additional diagnostic tests were performed to identify the suspected colonies. The cytological analysis determined the proportions of inflammatory cells, while the statistical analysis assessed the significance of differences in inflammatory cell populations and pathogen presence across the different symptom groups.

Results

Among 133 horses categorized into five clinical groups, S. zooepidemicus was isolated in 18% overall, with the highest isolation rate in horses exhibiting both coughing and lymph node swelling (36%). Statistical analysis showed a significant difference in S. zooepidemicus prevalence between groups (p = 0.019), with post hoc tests confirming significance between asymptomatic horses and those with combined symptoms. Neutrophil proportions differed significantly among groups (p = 0.010), with the highest counts in symptomatic horses with both coughing and lymph node swelling. Furthermore, bacteriologically positive samples exhibited significantly higher neutrophil counts (median 83.5, IQR 76) than negatives (median 22, IQR 46, p < 0.001), suggesting an acute inflammatory response linked to infection. In contrast, macrophage, lymphocyte, and eosinophil counts showed no significant variation among groups or infection status.

Conclusions

Horses with coughing and lymph node swelling showed the highest prevalence of β-hemolytic streptococci. S. zooepidemicus infection was associated with increased neutrophils and decreased macrophages and lymphocytes, highlighting the role of neutrophils in inflammation. Asymptomatic horses may also carry the bacteria, indicating their role as carriers. Distinct inflammatory profiles correspond to clinical signs, underscoring the complexity of equine respiratory disease and the value of cytology and tracheal wash in diagnosis.

Background

Streptococci can cause diseases in a variety of animals, including humans, and can be specific to a particular host or spread between species [1]. Beta-hemolytic (β-hemolytic) streptococci species are thought to be opportunistic commensals in horses, but can also cause diseases in other domestic animals such as cattle, sheep, goats, pigs, dogs, and cats [2]. Riding clubs suffer significant financial losses due to upper respiratory tract infections caused by β-hemolytic streptococci, which can cause strangles and strangles-like disease in horses [3]. Respiratory diseases in horses are a significant health concern, affecting performance and welfare, especially strangles, which is one of the most dangerous respiratory diseases that can affect horses and a major cause of both public and financial losses globally [4]. The inflammatory response in the respiratory tract involves various cell types, including neutrophils, eosinophils, macrophages, lymphocytes, and mast cells, each playing a critical role in pathogen defense and inflammation. The airway epithelium serves as the primary interface between inhaled substances, such as microorganisms, airborne allergens, and environmental pollutants, and the host respiratory system [5].

The three main β-hemolytic streptococci species that cause serious and economically significant diseases in horses are Streptococcus equi subsp. equi (S. equi), Streptococcus dysgalactia subsp. equisimilis (S. equisimilis), and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) [6]. Some horses that survive the disease may develop into “silent carriers” of the bacteria in their upper respiratory tracts. Among all infectious agents, S. zooepidemicus most likely causes the largest number of diseases in horses [7]. However, S. zooepidemicus is commonly regarded as an opportunistic pathogen; therefore, its role in disease outbreaks may be ignored [8].

β-hemolytic streptococci are traditionally categorized into Lancefield serogroups [9]. Using at least one of the tests such as APIStrep, STR Rapid ID32, or VITEK2 GP-ID in conjunction with the AccuProbe Group B Streptococcus Culture ID Test (Gen-Probe, San Diego, CA, USA), the laboratories identified S. zooepidemicus to the species level with varying results [2].

Horses affected by respiratory infections often exhibit a range of clinical symptoms that can significantly impact their overall health and performance of the animal. According to Chanter [10], these horses typically experience a loss of appetite, fever, and depression, which leads to weight loss, lethargy, and reduced physical activity, particularly detrimental for those raised for competition or sport. Respiratory symptoms such as nasal discharge, coughing, sneezing, and increased mucus production compromise the horse’s natural defense mechanisms [11]. Coughing, which can result from both infectious and noninfectious causes, is a prominent symptom that further complicates the horse’s health and well-being [12]. Additionally, lymph node swelling is commonly observed in cases of upper respiratory infections. These combined effects can hinder the horse’s ability to recover and continue performing at optimal levels. Based on more proximal respiratory tract sampling using tracheal wash (TW) cytology, cough has been linked to neutrophilic inflammation and excessive mucus release [13,14,15]. To our knowledge, there are no recent studies published that have classified horses based on the presence of upper respiratory tract clinical signs, either individually or in combination, and examined both the distribution of cytological profiles among these groups and their relationship with the presence of β-hemolytic streptococci. Therefore, this study aims to provide a more detailed analysis of the following aspects for the first time: (i) the presence of β-hemolytic streptococci in relation to different upper respiratory tract symptoms (ii) the distribution of inflammatory cells according to upper respiratory tract symptoms, (iii) and the relationship between the presence of β-hemolytic streptococci and inflammatory cells.

Methods

Sample collection

In this study, the samples were collected from horses that were brought to the Jockey Club of Türkiye Equine Hospital for routine examinations. Permission to publish the data obtained from these samples was granted by the General Directorate of Jockey Club of Türkiye. A total of 149 horses were sampled via tracheal lavage. Initially, the horses were classified into eight clinical groups based on their presenting symptoms. However, due to the relatively small sample sizes of some groups, these groups were excluded from the statistical analysis to maintain the robustness and validity of the results. Consequently, the statistical evaluation was conducted on the remaining five groups, comprising a total of 133 horses. (Group I Horses with no apperent symptoms, Group II Horses with only coughing, Group III Horses with only lymph node swelling, Group IV Horses with coughing and lymph node swelling, Group V Horses with all symptoms) (Table 1). In this study, each horse was assigned to only one group based on its specific combination of clinical symptoms. The groups were designed to be mutually exclusive, meaning that no individual horse was included in more than one group. For example, a horse showing both coughing and lymph node swelling was categorized under the group defined for that specific combination (Group IV), and was not included in any other group such as the “only coughing” or " lymph node swelling " groups.

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Sedation was administered in horses that exhibited aggressive behavior during the tracheal lavage procedure to ensure a safe and effective intervention. For this purpose, a combination of 0.5 mL of Detomidine hydrochloride (Domosedan®, 10 mg/mL) and 0.3 mL of Butorphanol tartrate (Butomidor®, 10 mg/mL) was administered intravenously. The dosages were determined based on the horse’s clinical condition and body weight.

The tracheal lavage procedure was performed after confirming that an adequate level of sedation had been achieved.

The sampled horses ranged in age from 2 to 5 years old, with a mean age of 2.8 years. Each of the horses that were tested was kept in closed barns at the Veliefendi Racetrack in Istanbul. All the tracheal lavage samples were collected according to Hoffman [16], and Rossi et al. [17].

Through the endoscopy biopsy channel, a sterile polyethylene tube (Jorgen Kruuse A.S. Langeskov, Denmark) was inserted to collect secretions from the distal end of the trachea that was next to the carina. A 40-mL sterile solution containing 0.9% sodium chloride was injected into each horse, and roughly 60% of the instilled solution was aspirated back into a 60-mL sterile syringe. The samples were collected and then immediately transferred to a 4.5 mL ethylenediaminetetraacetic acid (EDTA) tube (Diasera A.S. Izmir, Türkiye) for cytologic examination and a 10 mL plane tube for bacteriology (Diasera A.S. Izmir, Türkiye).

Clinical assesment

Before sample collection, all horses were clinically examined and categorized based on the presence of respiratory symptoms into spesificgroups as previously defined in Table 1. All symptoms in our horses were systematically assessed based on the presence of nasal discharge, coughing at rest, and pharyngeal lymphoid hyperplasia. These parameters were selected because they are key clinical indicators of respiratory health and inflammation.

Cytological techniques

The TW samples were centrifuged at 400 g for 5 min to prepare the slides. As previously mentioned, the slides were prepared from the sediment of the centrifuged TW samples [18]. May Grünwald quick stain (GBL-Gul Biyoloji A.S. Istanbul, Türkiye) was applied to the air-dried slides in accordance with the manufacturer’s instructions. The slides were examined under a microscope with a 100X magnification, and 200 cells were counted to determine the relative proportions (%) of mast cells, neutrophils, macrophages, lymphocytes, and eosinophils [19]. Consequently, stained smears from each sample were analyzed for a differential count of inflammatory cells, with the proportions of each cell type reported as a percentage of the total inflammatory cell count [20].

Bacteriology

The TW samples were subjected to bacterial culture in accordance with the methods previously outlined in some research [21, 22]. The suspected colonies were identified through a series of tests to determine their biochemical profile, including Gram staining (GBL, Istanbul), the catalase test, the latex agglutination test (Oxoid Ltd., Basingstoke, UK), to determine serological group, and the API 20 Strep ID kit, following the manufacturer’s instructions (bioMerieux S.A., Marcy l’Etoile, France). The cultures were checked for the development of bacterial colonies after the incubation period, as these colonies were thought to be pathogenic to horses, according to previous research [23, 24].

Isolation of β-hemolytic streptococci from tracheal lavage samples were carried out as described before [25, 26]. Briefly, the samples were inoculated in 5% sheep blood agar in both aerobic and microaerobic (5% CO2) conditions at 37 °C. The culture plates were incubated 24–48 h for β-hemolytic streptococci growth. Cultures were reported positive for β-hemolytic streptococci when pure or predominant growth of the microorganism was observed, as described earlier [27].

Statistical analysis

Horses exhibiting similar symptoms were assigned to the same group to aid in the statistical analysis. The classification was carefully constructed to avoid overlapping, ensuring that each group represents a distinct clinical presentation. Although some symptoms may be shared between groups, the inclusion criteria for each group were based on unique combinations of symptoms, and each horse fits into only one category accordingly. All statistical analyses were performed using SPSS Statistics software version 29.0 (IBM, New York, USA). To assess the normality of the data distribution, the Shapiro-Wilk test was performed for each inflammatory cell type. Consequently, the non-parametric Kruskal-Wallis test was applied to determine whether there were significant differences in cell distributions among symptom groups (95% CI). Following the Kruskal-Wallis test, pairwise comparisons were conducted using the Mann-Whitney U test to identify specific group pairs with statistically significant differences. We conducted a Chi-square test to investigate the association between the presence of β-hemolytic streptococcci and different symptom groups in horses. Furthermore, Mann-Whitney U Test was performed to analyze the significant difference between the percentages of each inflammatory cell type and the presence of β-hemolytic streptococci. Finally, to control for the risk of Type I error due to all multiple comparisons, the Bonferroni correction was applied to adjust the significance level.

Results

A total of 133 horses were categorized into five mutually exclusive groups according to their clinical presentations. Group I (n = 38, 28.6%) consisted of clinically healthy horses with no observable symptoms and was designated as the control group. Group II (n = 24, 18.0%) included horses presenting only with coughing, while Group III (n = 27, 20.3%) comprised those exhibiting only lymph node swelling. Group IV (n = 25, 18.8%) represented horses showing both coughing and lymph node swelling. Group V (n = 19, 14.3%) included horses displaying all three clinical signs: coughing, lymph node swelling, and nasal discharge. Importantly, each horse was allocated to a single group based on the unique combination of its clinical signs, ensuring that no overlapping occurred between groups.

The distribution of S. zooepidemicus presence varied significantly across the symptom groups (Table 2). Out of a total of 133 horses, S. zooepidemicus was isolated in 24 cases (18.04%). When evaluated by clinical symptom groups, the presence of S. zooepidemicus was detected in 3 of 38 horses with no apparent symptoms (7.89%), in 3 of 24 horses with only coughing (12.5%), in 3 of 27 horses with only lymph node swelling (11.11%), in 9 of 25 horses with both coughing and lymph node swelling (36%), and in 6 of 19 horses exhibiting all symptoms (31.6%). The highest isolation rate was observed in horses showing both coughing and lymph node swelling, followed by those with all clinical signs.

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This table outlines the group names, symptoms, and the number of horses exhibiting Streptococcus zooepidemicus presence in each group. The total number of horses in the study was 133, with 24 horses testing positive for S. zooepidemicus.

S. zooepidemicus typically showed β-hemolysis on blood agar as shown in Fig. 1.

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The comparison of S. zooepidemicus prevalence among the groups showed a significant difference (χ², p = 0.019). After applying the Bonferroni correction (adjusted α = 0.005), only the comparison between Group I (no apparent symptoms) and Group IV (coughing and lymph node swelling) remained significant (p = 0.005).

The distribution of inflammatory cells varied notably among the clinical groups (Fig. 2).

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Figure 2 Inflammatory cell profiles in horses with different clinical signs. Macrophages predominated in Groups 1, 2, and 3, while neutrophils were higher in Groups 4 and 5; lymphocyte and eosinophil levels were low in all groups.

No mast cells were detected. The distribution of cytological cells across symptom groups was examined, and the results are presented in Fig. 3. According to this figure neutrophils differed significantly among groups (p = 0.010), with horses showing both coughing and lymph node swelling (Group IV) exhibiting the highest levels, suggesting an inflammatory response linked to combined symptoms. Post hoc analysis with Bonferroni correction revealed significant differences between Group I and Group IV, as well as between Group III and Group IV. In contrast, macrophage, lymphocyte, and eosinophil proportions showed no significant variation across groups, indicating that these cell types were not markedly affected by the presence or combination of clinical signs.

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Figure 3 Different letters above the boxes indicate significant differences between groups based on post hoc Bonferroni test (p < 0.05). Data are presented as median (line) and interquartile range (box), with whiskers representing minimum and maximum values. Group I Horses with no apperent symptoms, Group II Horses with only coughing, Group III Horses with only lymph node swelling, Group IV Horses with coughing and lymph node swelling, Group V Horses with all symptoms.

The distribution of inflammatory cell types differed significantly between S. zooepidemicus-positive and -negative samples, with infection being associated with increased neutrophil proportions and reduced macrophage and lymphocyte proportions (p < 0.001 for all). Neutrophil counts were significantly higher in bacteriologically positive samples (median 83.5, IQR 76) compared with negative samples (median 22, IQR 46). Eosinophil proportions were low in both groups and did not differ significantly (p = 0.802).

Discussions

Respiratory diseases in horses are common across all ages and disciplines. Especially in performance horses, they can lead to economic losses and put animal welfare at risk [28]. Common diagnostic procedures in equine medicine include respiratory endoscopy, cytological analysis, obtaining a TW sample for bacterial culture, BAL, and various thoracic imaging techniques [29, 30]. While PCR is a sensitive method for detecting β-hemolytic streptococci, studies such as those by Timur and Ekinci [31], have shown that culture and commercial biochemical tests can also effectively identify the pathogen. Similarly, in our study, we utilized this approach to detect β-hemolytic streptococci, offering a cost-effective and accessible alternative for diagnosis. These methods, though less sensitive, remain valuable in settings where PCR might not be feasible. Since non-specific clinical indicators might make diagnosis difficult, TW and respiratory tract endoscopy are frequently utilized during initial testing because of their convenience and speed. However, the lack of a widely recognized cytological reference range makes it difficult to analyze tracheal wash fluid [29]. Accurate history taking and a comprehensive clinical examination are essential for the clinical diagnosis of respiratory diseases in horses. Bronchoscopy and cytological investigation of the fluid extracted from the respiratory system by bronchoalveolar lavage and TW are specific methods for diagnosing lower airway disease in horses [32].

In a study by Erol et al. [6], S. zooepidemicus was the most frequently isolated bacterium (72%) from the lower respiratory tract of adult horses. However, among the potentially pathogenic bacteria, S. zooepidemicus was the most commonly cultured, as it is also a frequent commensal of the upper respiratory tract [24]. In our study, we identified S. zooepidemicus as the only β-hemolytic streptococcal species, detected in 18.04% of 133 horses. Our results suggest that the combination of coughing and lymph node swelling, and possibly the presence of all symptoms, may serve as more reliable indicators for the presence of β-hemolytic streptococci in horses with respiratory issues.

A significant association was observed between bacterial culture positivity and the distribution of inflammatory cells. It appeared that the amount of inflammatory cells varied when comparing samples with and without bacteria. In response to a bacterial infection, TW cytology results showed a decrease in macrophages and an increase in neutrophils, as reported by Fernandes et al. [33]. Similarly, our findings suggest that the proliferation of S. zooepidemicus coincides with a shift toward neutrophilic inflammation, while the relative decrease in macrophages and lymphocytes may reflect the host’s acute inflammatory response dynamics.

The distributions of inflammatory cells in TW samples were recorded as neutrophils (44.78%), lymphocytes (6.49%), eosinophils (1.48%), and macrophages (47.22%) in 133 horses. As it was depicted in Fig. 3, asymptomatic horses (Group 1) exhibited a macrophage-dominant profile with moderate neutrophils, low lymphocytes, and low eosinophils, consistent with the absence of active inflammation. Horses showing only coughing (Group 2) displayed a similar neutrophil level but slightly higher macrophages and reduced lymphocytes and eosinophils, indicating a mild, macrophage-predominant response. In horses with only lymph node swelling (Group 3), macrophages reached their highest level, accompanied by stable neutrophils and low lymphocytes, suggesting a chronic or resolving inflammatory phase. In contrast, horses with both coughing and lymph node swelling (Group 4) showed a marked shift toward neutrophilia with decreased macrophages and lymphocytes, consistent with acute inflammatory activity. Finally, horses exhibiting all symptoms (Group 5) had high neutrophils and moderate macrophages, indicating a predominantly neutrophilic response with some lymphocytic involvement. This pattern suggests that the severity and type of clinical signs are associated with distinct inflammatory cell profiles, potentially reflecting different stages or types of immune activation.

In our study, eosinophil proportions were consistently low across all clinical groups, with no statistically significant differences observed (p = 0.802), and mast cells were entirely absent. These findings align with previous reports by Depecker et al. [34] and Morini et al. [35], which also documented minimal or undetectable levels of eosinophils and mast cells in bronchoalveolar lavage fluid of horses. In contrast, higher eosinophil counts in respiratory secretions are typically associated with allergic bronchitis and parasitic infections, where they serve as indicators of hypersensitivity reactions [19]. In the same study by Fernandes et al. [33], cytological results showed a predominance of macrophages in the control group, which exhibited no clinical signs. Similarly, our study found that macrophages were dominant in horses with no symptoms (Group I) (Fig. 2). There have been reports of widespread neutrophils in tracheal wash samples in lower respiratory tract inflammations due to septic and nonseptic causes [19, 24]. Considering that β-hemolytic streptococci are thought to cause airway inflammation, Holcombe et al. [36] reported that it was characterized by higher neutrophilia in stabled horses. Since an increase in neutrophil count in tracheal lavage has been associated with an increase in tracheal mucus score [37], the elevated neutrophil percentages in symptomatic groups (horses with coughing and lymph node swelling and horses with all symptoms - coughing, lymph node swelling, nasal discharge) with excessive secretions, such as cough and nasal discharge, suggest this correlation. A previous study reported that neutrophilic tracheitis is commonly associated with increased mucus production. However, elevated neutrophil counts in tracheal lavage fluid alone, without accompanying mucus, have not been considered a significant risk factor [36]. While the results of Rossi et al. [17] similarly suggested that visible mucus in the trachea is likely to indicate neutrophilic airway inflammation, our study extends this understanding by identifying a potential association between neutrophilic inflammation and horses exhibiting both coughing and lymph node swelling (Fig. 3, p = 0.01). In our study, similar to the findings of Laus et al. [38], we observed that even asymptomatic horses (Group I), regardless of whether β-hemolytic streptococci was detected, showed a neutrophilic inflammatory response. Despite the thorough routine health screenings showing no other clinical diseases in the studied racehorses, our study did not include comprehensive screening for all possible pathogens beyond β-hemolytic streptococci. Consequently, subclinical infections or other inflammatory conditions not identified in this study may have contributed to the neutrophilic inflammatory responses observed, especially in asymptomatic horses. Future studies involving broader pathogen detection and detailed clinical evaluations are necessary to better understand these findings. This suggests that the presence of neutrophils, indicating inflammation, can occur independently of the pathogen’s presence. Such results highlight that inflammation may be present even in the absence of detectable β-hemolytic streptococci and point to the complexity of interpreting inflammatory responses in horses with and without clinical signs. Additionally, it is known that exercise increases the neutrophil percentage in tracheal lavage [39, 40].

Richard et al. [37] reported that the clinical significance of lymphocytes in tracheal lavage samples collected from horses is not yet clear. In our study, lymphocyte percentages remained consistently low across all clinical groups, with median values ranging between 2% and 5% (Fig. 3).This finding is consistent with the age range of the sampled horses (2 to 5 years, mean 2.8 years), as previous research by Pacheco et al. [41] has shown that older adult horses tend to exhibit higher lymphocyte proportions in bronchoalveolar lavage fluid compared to younger individuals. These results suggest that age-related differences in lymphocyte distribution should be considered when interpreting BAL cytology.

In the study by Jaramillo-Morales et al. [42], S. equi subspecies equi was detected in 13.8% of 19 horses, with 68% of these horses being asymptomatic carriers. In contrast, Laus et al. [38] reported that all horses exhibiting clinical signs tested positive for at least one β-hemolytic streptococci species, while many healthy horses also tested positive. Similarly, our control group (Group I), consisting of horses with no symptoms, showed that 3 out of 38 horses (7.9%) were carriers of β-hemolytic streptococci. These findings align with previous research, suggesting that while β-hemolytic streptococci are more commonly associated with clinical symptoms, asymptomatic carriers are also prevalent, underscoring the importance of considering both symptomatic and asymptomatic horses in diagnostic practices. However, a limitation of the present study is the lack of longitudinal follow-up and detailed clinical history, which prevents us from determining whether asymptomatic β-hemolytic streptococcal carriers later developed clinical disease or had previous respiratory episodes.

In comparison to the study by Pusterla et al. [43], which found a strong association between nasal discharge and anorexia with higher S. equi detection, our study observed the highest prevalence of S. zooepidemicus in horses with both coughing and lymph node swelling (Group IV, 36%), with a statistically significant difference compared to asymptomatic individuals (p = 0.005). These findings suggest a strong association between these two symptoms and the presence of S. zooepidemicus. Groups of horses with only coughing and horses with only lymph node swellingalso showed a lower prevalence of β-hemolytic streptococci (12.5% and 11%, respectively), which further supports the idea that specific combinations of symptoms are more likely to correlate with bacterial presence. The data suggests that horses with coughing and lymph node swelling, or those with all symptoms, are more likely to harbor β-hemolytic streptococci, while horses with isolated symptoms such as only coughing appear to have a lower association with the bacteria.

Hughes et al. [44] reported that, in their cytological evaluation of tracheal aspirates TA and BAL samples from racehorses, mast cells were predominantly found in samples taken from the horses’ distal and smaller airways, while eosinophils were mainly found in samples taken from the proximal and larger-caliber airways. However, they also emphasized that there were limited cell percentages of both eosinophils and mast cells in the lower respiratory tract. Similar, in our study, mast cells were not detected in the TA. These findings suggest that the cellular composition varies depending on the sampling site within the respiratory tract, indicating that the localization of the samples significantly influences the cytological results.

Conclusions

We found that horses exhibiting a combination of coughing and lymph node swelling, as well as those with all symptoms, had the highest prevalence of β-hemolytic streptococci, suggesting that these clinical signs are strongly associated with infection. Infection with S. zooepidemicus was linked to higher neutrophil counts and lower macrophage and lymphocyte counts. This highlights the key role of neutrophils in the inflammatory response to the Streptococcal infections. Moreover, this study reinforces that asymptomatic horses may still carry S. zooepidemicus, underscoring the potential for these horses to act as carriers, a factor that should be considered in diagnostic practices. Future studies including broader pathogen screening and clinical assessments would be necessary to clarify the exact causes of neutrophilic inflammation in these cases. Different clinical signs in horses correspond to distinct inflammatory cell profiles; neutrophil predominance in multiple symptomatic cases is associated with acute inflammation, whereas macrophage predominance in asymptomatic or mildly affected horses suggests chronic or resolving phases.These findings highlight the need for a nuanced approach when diagnosing respiratory diseases in horses. Overall, the results show that tracheal wash and cytological examination are good ways to diagnose infections in the upper respiratory tract, especially when more sensitive tests like PCR are not available.

Data availability

No datasets were generated or analysed during the current study.

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