Airborne detection of Equid alphaherpesvirus 1 (EHV-1) at international equestrian events

Airborne detection of Equid alphaherpesvirus 1 (EHV-1) at international equestrian events | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Equine Veterinary Journal This is a preprint and has not been peer reviewed. Data may be preliminary. 2 June 2025 V1 Latest version Share on Airborne detection of Equid alphaherpesvirus 1 (EHV-1) at international equestrian events Authors : Amjad Khan 0000-0002-0879-5376 [email protected] , Eduard Jose-Cunilleras 0000-0002-4536-7717 , Emma Hyde , Edward Olajide , Maria Polo 0009-0005-3375-9755 , and Lutz Goehring 0000-0001-8493-0675 Authors Info & Affiliations https://doi.org/10.22541/au.174886928.83443800/v1 378 views 189 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Equid alphaherpesvirus 1 (EHV-1) remains a major concern for the equine industry, with recent outbreaks at international equestrian events highlighting the need for improved surveillance during competitions. Objectives: To investigate EHV-1 and -4 presence in shared airspaces and environmental surfaces in direct contact with horses at international equestrian events, and to evaluate air sampling as a surveillance alternative to individual horse testing. Study design: A cross-sectional field study. Methods: Air (37) and surface samples (205) were collected from temporary stabling facilities across six events in Spain and 2 in the U.S. at different seasons over 10 months. Viral DNA (genomic copies) was measured and quantitated by quantitative and digital PCR. During all Spain events, we sampled air twice, early evening vs nighttime. Results: EHV-1 was frequently detected in the air in 20/28 samples in Spain, in all 3 samples from Florida, and in 3/6 samples from Kentucky, U.S. Surface samples were positive 15.6% in Spain, 26.3% in Florida, and 0.0% in Kentucky. EHV-4 was found in air samples at rates of 12/28 in Spain, 5/6 in Kentucky, and 0/3 in Florida, with surface detection of 1.6% in Spain and 7% in Kentucky. No significant differences were observed between daytime (high activity) and nighttime (low activity) airborne viral loads, suggesting that daytime horse movement and management practices had minimal impact on detection levels in our study. A positive correlation (Cohen’s κ= 0.401) and moderate agreement between surface and air positivity for EHV-1 emphasized the potential of air sampling as a non-invasive, cohort-based surveillance tool. Main limitations: The study did not isolate infectious viruses, only molecular detection was employed, which limits conclusions about transmission risk. Conclusions: These findings underscore the utility of air sampling for early EHV-1 detection at mass gatherings, although further validation is needed to connect genomic detection to active shedding and viral infectivity. Airborne detection of Equid alphaherpesvirus 1 (EHV-1) at international equestrian events Abstract Background: Equid alphaherpesvirus 1 (EHV-1) remains a major concern for the equine industry, with recent outbreaks at international equestrian events highlighting the need for improved surveillance during competitions. Objectives: To investigate EHV-1 and -4 presence in shared airspaces and environmental surfaces in direct contact with horses at international equestrian events, and to evaluate air sampling as a surveillance alternative to individual horse testing. Study design: A cross-sectional field study. Methods: Air (37) and surface samples (205) were collected from temporary stabling facilities across six events in Spain and 2 in the U.S. at different seasons over 10 months. Viral DNA (genomic copies) was measured and quantitated by quantitative and digital PCR. During all Spain events, we sampled air twice, early evening vs nighttime. Results: EHV-1 was frequently detected in the air in 20/28 samples in Spain, in all 3 samples from Florida, and in 3/6 samples from Kentucky, U.S. Surface samples were positive 15.6% in Spain, 26.3% in Florida, and 0.0% in Kentucky. EHV-4 was found in air samples at rates of 12/28 in Spain, 5/6 in Kentucky, and 0/3 in Florida, with surface detection of 1.6% in Spain and 7% in Kentucky. No significant differences were observed between daytime (high activity) and nighttime (low activity) airborne viral loads, suggesting that daytime horse movement and management practices had minimal impact on detection levels in our study. A positive correlation (Cohen’s κ= 0.401) and moderate agreement between surface and air positivity for EHV-1 emphasized the potential of air sampling as a non-invasive, cohort-based surveillance tool. Main limitations: The study did not isolate infectious viruses, only molecular detection was employed, which limits conclusions about transmission risk. Conclusions: These findings underscore the utility of air sampling for early EHV-1 detection at mass gatherings, although further validation is needed to connect genomic detection to active shedding and viral infectivity. KEYWORDS Air-sampling, EHV-2, EHV-4, digital PCR 1 INTRODUCTION Equid alphaherpesvirus 1 (EHV-1) infections continue to pose a significant threat to equine health, welfare, and the equine industry worldwide. EHV-1 infection initially appears as a respiratory tract infection, but it can also cause abortion/neonatal death, and equine herpesvirus-associated myeloencephalopathy (EHM). The latter condition is an ischemic (mostly) spinal cord condition that affects a horse’s gait and posture and can lead to recumbency. 1 EHV-1 initially replicates in the upper respiratory tract from where it also can spread horizontally through direct and indirect contacts (including spread via fomites and airborne transmission) to other horses. 2 Due to EHM and the horizontal transmission of EHV-1, outbreaks can lead to euthanasia or temporary to permanent loss of performance in affected horses. Furthermore, its contagiousness requires quarantine and travel restrictions, and there are significant emotional and economic tolls inflicted on the equine industry, ranging from disrupted training schedules to event cancellations, and extensive management challenges. 3 During first-time infection, herpesviruses establish lifelong latency within the host, remaining dormant and capable of reactivation. Reactivation from latency, resulting in a return to active replication at the site of the original infection, is called recrudescence. For EHV-1, recrudescence signifies a return to the respiratory tract with replication and the potential for horizontal spread to other horses. Reactivation of latency is linked to stressors such as transportation, mingling, immunosuppression, and likely other factors that are currently unknown. 4 Many EHV-1 outbreaks with EHM occur at boarding or training facilities after horses return from events with commingling or after a recent history of horses passing through a facility. Recent outbreaks of EHV-1 with EHM have occurred at major equestrian events, some with subsequent spread into home facilities causing satellite outbreaks. 5,6 A recent outbreak of EHV-1 infection with EHM fatalities at an international equestrian event in Valencia, Spain, in 2021, triggered the International Equestrian Federation (FEI, Switzerland) to investigate early/earliest detection and heightened surveillance of EHV-1 during events. However, as reactivation from latency and recrudescence can occur at any time point between event entry and departure, surveillance cannot be restricted to intake exams only and must be continuous throughout the event, including as many as possible event participants. Our group and others have already shown the validity of alternative sampling techniques such as nasal wipes of the outer nares, and environmental swabs from in-contact areas over the current gold standard, nasal swabs inserted into one or both nasal passages of a horse. 7 However, individual sampling is labor-intensive, cumbersome, and can be accompanied by low tolerance from horses and owners alike. Furthermore, individual horse sampling or sampling in its direct environment inadvertently comes with an increased risk for fomite transmission of pathogens. Shared airspace and wastewater sampling became effective methods of assessing groups in shared environments (Maryam et al., 2023). 8 In a previous study, we compared 2 sampling protocols for stable or barn air using Equid gammaherpesvirus -2 (EHV-2), a common respiratory tract pathogen of young horses, as a model. 7 Here, we wanted to investigate whether shared air space sampling during equestrian events can detect any of the respiratory tract-associated Equid herpesviruses -1, -2, and -4. On select locations, we also evaluated whether results differ between afternoon/evening and nighttime sampling periods. 2 MATERIALS AND METHODS 2.1 Study locations This observational study was conducted at FEI-sanctioned equestrian (showjumping, dressage, eventing) events in Spain (March/April 2024; four events) and in the United States (Florida, December 2023; Kentucky, August 2024). Upon arrival, we selected temporary stabling facilities, large tent-like structures holding 40-200 horses at each venue, based on accessibility and occupancy. Within these, we identified subunits within a structure, each with 10 stalls (boxes) arranged adjacent to a central aisle. The aisles were either configured parallel to the tent’s long axis (Kentucky and Spain), or perpendicular to it (Florida). The Kentucky set-up was one long central aisle, while events in Spain had a parallel configuration. Usually, there were 1-3 traveling parties per subunit that were chosen, and occupancy was between 60-100%. Unoccupied stalls were used for storage or staging. All units relied primarily on natural ventilation, with completely open sidings (rolled up tarp or completely open structures) in Florida and Kentucky, or adjustable openings in Spain. Portable fans were common and positioned by individual horse owners in proximity to their horses throughout the stable in Kentucky. Despite organizational subdivisions in the stable units, airspace was always considered ‘shared’ with other units. We switched units for sampling daily, except during the Florida event, where the same unit was sampled on 3 consecutive days. We collected two sample types daily. We collected surface (environmental) samples from individual stalls of a unit, and we collected air over 6 hours between 22:00 to 04:00. During the 4 Spain events additional air samples from the same units were also collected between 16:00 and 22:00. 2.2 Sample collection Each sampling day was considered an independent event. When possible, we collected surfaces and air samples before the animals were moved into the stalls (Spain & Ky) as a control sample. We collected environmental swabs in the mornings (Florida, Kentucky) or mid-day (Spain) after an air sampling period. In detail, we collected from a 3x5 cm surface area between two metal bars of a stall’s access gate. A saline-wetted polyurethane sponge (80 mm2) with a 10cm long plastic shaft (VWR® Whirl-Pak Sterile Sponge Probe) was wiped over the surface. The sponge was then inserted into a 2mL container and closed after the shaft was cut off with tweezers. For an air sample, we used a Coriolis Compact portable air sampler (Bertin Technologies SAS, France) at its maximum capacity of 50 L/min. In each subunit, we chose an empty stall if available, or a central point on either the left or right side of the aisle. Some variation in the placement of the sampler was dictated by logistics. The opening of the one-time use sampling cone was fixed at 150cm measured from the floor and pointed towards most of the occupied stalls. Cones were exchanged in the mornings (Florida and Kentucky), or twice per day, at 16:00 and 22:00 during the Spain events, and before collecting any of the surface samples. 2.3 Sample Transport and Processing All samples were transported to a laboratory under stringent protocols to prevent cross-contamination during collection and before extraction with pre-labeling and decontaminated containers. Upon arrival, all samples went through an (outside) decontamination step with 10% bleach in a separate dedicated sample-receiving area before entering the main laboratory. All samples were processed in a biosafety cabinet. First, we added 5 mL of PBS to rinse the collection cones and this volume was transferred into 15 mL tubes and stored. Then, 1 mL of PBS was added to each surface sample tube, closed, and pulse-vortexed for 90 seconds. All samples were stored at −20°C until further analysis. 7 2.4 DNA Extraction and Molecular Analysis Random batches of 11 samples were thawed and accompanied by a negative control PBS (extraction control) for DNA extraction. Each sample was vortexed for 10 seconds, followed by brief centrifugation. DNA was extracted from 200 uL of the original sample using DNeasy Blood & Tissue Kit (Qiagen, Radnor, PA, USA) following the manufacturer’s protocol. Extracted DNA was analyzed for the presence of EHV-2 (control), and EHV-1 and EHV-4 using quantitative PCR (qPCR) for all samples (Quant Studio 7 system: Applied Biosystems, Foster City, CA, USA) initially. Air samples were all re-analyzed by digital PCR (dPCR) using Quant Studio Absolute Q Digital PCR system (ThermoFisher Scientific Inc., Florence, KY, USA) for absolute quantification. Then, all positive qPCR positive surface samples were also analyzed by dPCR. Both systems use the same primers and probe for EHV-2, -1, and -4 (sequences in supplementary data S1) in a single plex assay. The total volume per reaction (10uL) was composed of either a reaction of 5.5 µL of a master mix (TaqPathTM qPCR Master Mix, ThermoFisher Scientific, Florence, KY, USA and primer in a ratio of (1:10 primer-probe to master mix)) or Absolute Q DNA Digital PCR Master Mix™ (ThermoFisher Scientific) for qPCR or dPCR, respectively and 4.5 uL of extracted DNA as template. Reagents for PCR reactions were mixed through an automated setup (epMotion® 5075, Hamburg, Germany). Plates were sealed, centrifuged at 500×g for 2 minutes, and subjected to standard thermal cycling protocols. EHV-2, EHV-1, and EHV-4 positive samples were reported qualitatively as presence or absence and semi-quantitatively as cycle threshold (CT) values. 2.5 Statistical Analysis Descriptive statistics of the data were performed using IBM SPSS Statistics version (IBM® 29.0.2). Data analysis was conducted using GraphPad Prism (10.5.0 Windows) to construct plots. Data management and summary chart construction were done through Microsoft Excel (Microsoft Excel 2024). Descriptive results are presented as frequency distribution, and targeted gB gene mean values comparison across studies for each virus type was done through SPSS. Correlation analysis was conducted through R software(version 4.4 1) using Cohen’s Kappa analysis. 9 3 RESULTS Environmental and air samples were collected on consecutive event days during 6 international equestrian events. A total of 205 surface and 37 air samples were collected with an additional 16 surface controls and 3 control air samples collected prior to population. All results are presented in Fig.1. There was no detection of EHV genome copies before the population. Also, there were no reports of clinical cases during any event, defined as ‘febrile’; ‘cough’, or ‘with sudden neurologic gait anomalies. EHV-2 genome copies were detected during most events in either air or surface samples. The only exception was Spain 1, where both sample types were negative for EHV-2. EHV-1 genome copies on surfaces were detected during the Florida competition and during all Spain events at least once. At any of the competitions throughout the year, at least one air sample was positive for the EHV-1 genome on at least one day. When the number of positive samples increased in an aisle, it did not automatically increase air sample copy numbers (Spain 3, Figure 1). Typically, EHV-1 copy numbers in air samples were detected between 0.5-8x102 copies/m3. The exception was the Florida event where copy numbers in the air were detected between 3-8x103 copies/m3. Here, the increased copy number in air corroborated with high EHV-1 copy number detection (1.2x106 copies/cm2 surface area) in surface samples of 2 stall gates suggesting a productive shedder in this environment. EHV-4 results were like EHV-1 results; however, detection of EHV-4 genome copies on surfaces was mostly limited to the Kentucky event (with 2 surface samples also positive during Spain 3). EHV-4 in air was either undetectable (Florida), or of low abundance (between 50-500 copies/ m3) during all Spain events. However, we detected EHV-4 in moderate abundance in the air (2-3x 103 EHV-4 copies/m3) with concomitant moderate abundance in individual surface samples during the Kentucky event (Figure 1). During all Spain events, we sampled air of an aisle during 2-time slots: slot 1 (16:00-22:00) and slot 2 (22:00-04:00) with a sampling cone exchange at 22:00 and at 16:00 the next day. There was no significant difference in EHV-1 or -4 copy number detection between the 2 time slots (Figure 2). In Florida, EHV-1 was detected on 5 of 19 tested surface samples and in 3 of 3 air samples. EHV-4 was also detected at higher concentrations at varying levels. qPCR testing showed EHV-1 positivity, while none of the surface or air samples were found positive for EHV-4 (Table 1). Samples from Spain were collected at different locations during similar times of the year. The study showed positivity rates of 15.6% (20/128) and 71.4% (20/28) for EHV-1 in the surface and air samples, respectively, while EHV-4 positivity was 1.6% (2/128) in surface samples and 42.9% (12/128) in air samples. Whereas, in samples from the Kentucky event, EHV-1 was undetectable in 58 surface samples while detected in 3 out of 6 air samples (low copy numbers). EHV-4 was detected in 7% of all surface samples and present in 83.3% (5/6) of air samples. To compare the viral particle load detected on surface samples across all studies and the reliability of target detection by the air sampler, we calculated digital PCR results (copies/uL template) to quantify the viral load as viral genomic copies/cm2 in surface samples and copies/m3 in air samples(Figure 3). A similar quantitative detection pattern was found in air samples in our results for EHV-2, and EHV-4. But unexpectedly the mean copies detected were higher for EHV-1 in air samples across all studies in comparison to surface contamination, as compared to EHV-2 and -4 (Figure 3). Figure 3 shows a significant correlation between surface and viral particle detection in air samples considering when all studies were analyzed as a single group. Outliers, presented in the graphs separately, could be attributed to a large contributor resulting in high target genome detection in air and on surfaces (Figure 3). EHV-1 and -4 detection load via air sampling was significantly higher as compared to EHV-2 used as a calibrator. It is evident from the results that with higher surface contamination, we had a higher load of viral particle detection in air samples for respective days of sampling. Our data demonstrates a strong predictive relationship between surface positivity and airborne detection of EHV-1, as evidenced by a Positive Predictive Value (PPV) of 90.9% (10/11 days) (Table 2). This indicates that when surfaces tested positive, the air was concurrently positive in over 90% of cases, at a 95% confidence interval (58.7%-99.8%). While Cohen’s Kappa (K=0.401, ”moderate” per Landis & Koch) reflected overall agreement between surface and air sampling, the PPV directly tested our hypothesis by quantifying the reliability of surface positivity as an indicator for airborne target genome detection during on-air sampling. 4 DISCUSSIONS We ventured into this study with two questions. First, we wanted to learn how common it is to detect potentially debilitating EHV-1 (and EHV-4) at large horse gatherings, and secondly, whether we can use shared air space sampling as a surveillance tool for viral (genome) detection instead of collecting individual samples from (fractions of) participants. First, we were surprised by the frequency of EHV-1 detection, both on surfaces and in air specimens collected in stable air during equestrian events, with the current belief that it should be a rare event for a single horse to reactivate EHV-1 from latency followed by replication of the virus in the respiratory tract with subsequent horizontal spread. 10,11 However, our data collected at independent events on different continents suggests that EHV-1 presence is likely more common than previously expected. Our data also suggests that EHV-1 genome detection could be more frequent during winter (Florida) and spring events (Spain 1-4), rather than during (late) summer events (Kentucky). While our findings could be due to chance, seasonality of EHV-1 circulation among horses has been suggested before, 12 and it is a yearly recurring phenomenon that an increase in EHV-1 outbreaks with EHM in the northern hemisphere occurs during winter and spring months. 13 It has been speculated whether poor ventilation of barns during outbreaks in the winter months increases the risk for horizontal spread and disease propagation and is therefore the reason for an increase in EHM outbreaks each year. However, our data suggests differently, with a steady year-round presence of EHV-2 antigen and summer-polarized EHV-4 antigen presence during these events, which contradicts ‘ventilation’ as an explanation of this pattern. Alternatively, different factors drive EHV-1 reactivation which makes EHV-1 a rare find during summer events. We explored air sampling over individual horse environment sampling mainly for reasons of economics and efficiency. Furthermore, approaching an animal for individual sampling comes with mild to moderate discomfort and irritation, as well as biosecurity breeches and an increased risk for fomite transfer. Here, we used a second-best option for ‘individual nasal swab’ sampling via ‘environmental’ sampling and compared individual results to ‘cohort sampling’ using a stationary air sampling protocol. Both sampling techniques have been validated and compared to gold standard nasal swab sampling using EHV-2 as a surrogate for EHV-1 (or -4) respiratory tract shedding. 7,14 Both samples will provide a different yet indirect assessment of the horse’s respiratory tract. Environmental samples may contain smears, large and small(er) droplets, and aerosols as well as various (in)organic particles containing EHV-1 target genome copy deposits. Depending on the distance to the shedding source, the sampled areas may contain variable target genome numbers, and numbers likely increase over time due to accumulation. Surface samples are not exclusively linked to deposits of the horse in the sampled stall. Airborne (in)organic matter deposits containing target genome copies may also originate from neighboring horses, even from a neighboring stable unit. In addition, surface particle load is likely affected by manual cleaning or wiping by humans. Aerosolization of an EHV-1 suspension in the form of a dose of modified-live virus vaccine formula and nebulized, still allowed detection of EHV-1 target genome copies at a distance of 10m. Bovid alphaherpesvirus 1 transmission caused seroconversion in sentinel calves at a 4m distance from the shedding source. 15 Air samples are different, as they are more likely to contain a mixture of airborne (in)organic particles and target copies including variable-size aerosols originating directly from a horse’s respiratory tract. Large(r) droplets loaded with high target copy numbers are less likely to be captured in an air sample. Their weight determines the radius of deposition away from the shedding source, which favors the surface samples for effective detection. Air specimens will also contain air from neighboring units, further supporting the variability of results and a moderate correlation coefficient (k=0.4) between the two sampling techniques. 16 When air sampling detected high target gene abundance during the Florida and Kentucky events for EHV-1 and -4, respectively, high abundance was also present in surface samples indicating high output within the sampled unit (Figure 1) and should prompt individual horse sampling for the detection of high-volume shedding horses. However, a threshold determination to distinguish between low-, moderate, or high-shedding animals still needs to be determined. To assess possible variation in airborne target genome load, air sample collection was performed at 2 different time intervals: once during (early) evening with increased activity and at nighttime (low activity). We aimed to assess whether differences in horse activity levels-such as aisles walk-through, post-exercise cool-down, vocalization, eating but also human activities (stall cleaning) can influence target genome detection in our air samples. However, no significant differences were recorded between the two sampling times. These findings suggest that sampling timing may not critically bias results in similar equine barn settings, but due to the small sample size further studies are needed to optimize group sampling strategies. EHV-1 genome copy detection in air and surface samples, even at low concentrations and intermittently, signifies that at some time point, at least a proportion of infection-capable virus was released into the environment. Interestingly, all samples (air and surfaces), that we collected before populating the stable, were negative for EHV (any of 3) genome copies. Stalls in stables had only been cleaned (stripped) from manure and bedding from a previous occupant without any (power)washing or disinfection. We can assume that most detected target genome copies were recently released by a horse’s respiratory tract and not so much by persistent DNA from previous rounds of stabling. We know that infectious virus in the environment is deactivated quickly due to environmental circumstances. 17 However, our findings indicate that Equid herpesviruses are circulating at equestrian events, and there is reason to believe that under the right circumstances, e.g., exposure time to infectious virus, contact frequencies between horses, variable immune status, viral spread, and infection pressure, could become volatile. Air sampling detected viral genome sequences repeatedly during these investigations, also occasionally in moderate abundance (Florida 23; Kentucky 24). Abundance increased with proximity to high positivity in surface samples, which were probably chance circumstances considering the size of these events. Because of the costs of equipment and logistics, routine air surveillance, at least for herpesviruses at equestrian events, is unlikely to become a viable tool soon. It may be different for other respiratory tract pathogens. Our results: however, indicate that we need to better understand the dynamics of a herpetic viral cloud at horse gatherings, and likely at home facilities as well. In the meantime, an important message should go out to event management and participants to limit direct and indirect contact (including possible fomite transmission) between horses. This message should also extend to home facilities, where one should be thoughtful and vigilant upon the return of horses from any type of commingling event. With the advent of several point-of-care solutions and a steady increase in specific testing capacities and locations, we suggest increased testing. 4.1 LIMITATIONS OF THE STUDY Our study’s key limitations include the inability to confirm the infectivity of detected viral DNA, as virus isolation was not attempted, but it was not our goal; here, we only aimed to evaluate air sampling as a potential non-invasive surveillance tool. Surface contamination could reflect cumulative deposition over days of competition, complicating temporal associations. 5 CONCLUSIONS Our findings demonstrate that air sampling can detect airborne EHV-1 and EHV-4 target genome copies in shared equine environments. Our findings on surface and air sample analysis also indicate the relative abundance and appearance frequency at events of commingling, which requires further investigation. 6 FUNDING: This study was funded by a grant from the International Equestrian Federation (FEI), Lausanne, Switzerland. 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Koch. “An Application of Hierarchical Kappa-Type Statistics in the Assessment of Majority Agreement among Multiple Observers.” Biometrics. 1977; 33(2): 363-74. https://doi.org/10.2307/2529786 10. Pusterla N, Hussey SB, Mapes S, Johnson C, Collier JR, Hill J, Lunn DP, Wilson WD. Molecular investigation of the viral kinetics of equine herpesvirus-1 in blood and nasal secretions of horses after corticosteroid-induced recrudescence of latent infection. J Vet Intern Med. 2010;24(5):1153-7. https://doi.org/10.1111/j.1939-1676.2010.0554.x 11. Smith FL, Watson JL, Spier SJ, Kilcoyne I, Mapes S, Sonder C, Pusterla N. Frequency of shedding of respiratory pathogens in horses recently imported to the United States. J Vet Intern Med. 2018;32(4):1436-1441. https://doi.org/10.1111/jvim.15145 12. Lawton K, Runk D, Hankin S, Mendonsa E, Hull D, Barnum S, Pusterla N. Detection of Selected Equine Respiratory Pathogens in Stall Samples Collected at a Multi-Week Equestrian Show during the Winter Months. 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Sci Rep. 2025; 15: 7192. https://doi.org/10.1038/s41598-025-91566-z 17. Saklou NT, Burgess BA, Ashton LV, Morley PS, Goehring LS. Environmental persistence of equid herpesvirus type-1. Equine Vet J. 2021;53(2):349-355. https://doi.org/10.1111/evj.13313 TABLES TABLE 1 qPCR positivity rates for EHV-2, EHV-1, and EHV-4 in surface and air samples across study locations. Surface Air Surface Air Surface Air Florida-USA 5/19 0/3 5/19 3/3 0/19 0/3 4 Spain 29/128 16/28 20/128 20/28 2/128 12/28 15 Kentucky-USA 6/58 6/6 0/58 3/6 10/57 5/6 7 Note: *qPCR positivity was reported as positive out of a total number of samples tested. TABLE 2 Agreement and predictive performance between surface and air sampling for detecting EHV-1 and EHV-4. Value Interpretation Value Interpretation Cohen’s Kappa (κ) 0.4 Moderate overall agreement (Landis & Koch scale) 0.1 Slight agreement beyond chance Positive Predictive Value (PPV) 90.9% When the surface was positive, the air was positive 90.9% of the time 75% When the surface was positive, the air was positive 75% of the time Overall Agreement 69.6% Total percentage of matching results 54.2% Total percentage of matching results Note: Comparison of surface and air sampling methods, and metrics are reported. Cohen’s Kappa (κ) quantifies overall agreement between surface and air sampling beyond chance, with values ranging from -1 (complete disagreement) to +1 (perfect agreement). PPV reflects the probability of detecting airborne viral genome copies when surfaces are positive. Data was derived from 23 sampling days from three different events. 95% confidence intervals have been used for key metrics (e.g., PPV: 58.7-99.8% for EHV-1). FIGURES LEGENDS 1. FIGURE 1 Summary chart of qualitative/quantitative EHV-2, EHV-1, and EHV-4 detection results in surface and air samples across study locations. Six study locations Florida (December 2023), Spain 1-4 (March/April 2024), Kentucky (early September 2024) (Row numbers 1-6: a sampled subunit of (max.) 10 stalls. Stall(s) (boxes) marked with ‘X’ - empty stall/alternative use. Color coding for individual stall: green-EHV-2; red-EHV-1; yellow-EHV-4. Columns ‘air samples’: target gene copy numbers/ m 3 of air sampled in the corresponding aisle). 2. FIGURE 2 Day versus night air sampling comparison for EHV-1 and -4 genomic particle detection. 3. FIGURE 3 Absolute quantification and comparison of EHV-1, EHV-2, and EHV-4 across all studies expressed as gB gene copies per cm 2 area on the surface and per m 3 of air in air samples (6 hours of continuous sampling= 18m 3 ), [only positive surface and air samples are included in the analysis; blue dashed lines represent the median value for surface samples, brown dashed line is the median for air samples at 95% C.I). Information & Authors Information Version history V1 Version 1 02 June 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Equine Veterinary Journal Authors Affiliations Amjad Khan 0000-0002-0879-5376 [email protected] University of Kentucky Maxwell H Gluck Equine Research Center View all articles by this author Eduard Jose-Cunilleras 0000-0002-4536-7717 Universitat Autonoma de Barcelona Unitat de Fisiologia Animal View all articles by this author Emma Hyde University of Kentucky Maxwell H Gluck Equine Research Center View all articles by this author Edward Olajide University of Kentucky Maxwell H Gluck Equine Research Center View all articles by this author Maria Polo 0009-0005-3375-9755 University of Kentucky Maxwell H Gluck Equine Research Center View all articles by this author Lutz Goehring 0000-0001-8493-0675 University of Kentucky Maxwell H Gluck Equine Research Center View all articles by this author Metrics & Citations Metrics Article Usage 378 views 189 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Amjad Khan, Eduard Jose-Cunilleras, Emma Hyde, et al. 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