Dairy workers are at an increased risk of exposure to airborne biological particulate matter (bioaerosols) commonly found in livestock operations ^1–6. Dairy-specific bioaerosols exist as complex mixtures of dust that contain biologically derived particles, including allergens (e.g., dander), microbial communities, and pro-inflammatory constituents (e.g., endotoxin, muramic acid, and β-glucans) ^3,7–9. Spanning an aerodynamic diameter of 0 μm to 100 μm, bioaerosols encompass the inhalable fraction that impacts in the nasopharyngeal region (e.g., nose, mouth, and pharynx) ⁴. Once inhaled, these microbial constituents have the capacity to elicit inflammatory immune responses in the upper airways and are likely driving the increased prevalence of rhinosinusitis and contributing to chronic obstructive pulmonary disease (COPD), asthma, allergic rhinitis, and respiratory pneumonitis well-documented in dairy workers ^10–14. Furthermore, bioaerosol exposure seems to significantly shift nasal microbiome composition in workers’ noses and may provide protection against pathogens ^6,15–18.

Previous studies have demonstrated that bioaerosol exposure concentrations in dairy operations can vary by several orders of magnitude, ranging from 0.22 to 9.8 mg/m^{3 3,8,9,19,20}. Currently, no enforceable standards exist for occupational bioaerosol exposure or its inflammatory constituents, making the clinical relevance of measured exposures difficult to determine without robust outcome data. However, a recommended exposure limit for bioaerosols of (2.4 mg/m³) was suggested in a publication by Reynolds et al. ²¹. Furthermore, the Dutch Expert Committee on Occupational Safety developed the former Dutch standard for endotoxin, an inflammatory component of gram-negative bacterial cell walls, of 90 endotoxin units per meter cubed (EU/m³) ²². Previous research comparing worker endotoxin exposures across 30 dairies in the High Plains region of the U.S. found that 89% of sampled workers exceeded the former Dutch standard ³. Based on reported concentrations, dairy workers are exposed to bioaerosols and inflammagens exceeding recommended occupational limits resulting in a disproportionate burden of respiratory diseases and chronic allergies. Consequently, bioaerosol exposure and efforts to mitigate it continue to be a major industry concern.

Dairy industry practices including 24-hour production schedules, extended work shifts, contact with livestock, and use of heavy equipment create a unique exposure environment compared to other agricultural industries ^23,24. The size of dairies has also increased dramatically as median herd size has grown from just 80 in 1987 to 1300 in 2007²⁵. A notable shift towards larger operations working with bigger herds (>500 head) has altered production scale and dramatically changed the workplace dynamics of dairy operations ^23,24,26,27. Worker demographics have also contributed to these challenges, as reliance on an under experienced, immunologically naïve immigrant workforce (>90% Hispanic/Latino) has led to a highly exposed and highly susceptible workforce ^23,27,28.

Interventions attempting to control bioaerosols exposure and resultant respiratory illnesses in dairy settings have been encouraging, but major challenges regarding long term efficacy and economic feasibility remain ^6,29,30. Through guidance from the High Plains Intermountain Center for Agricultural Health and Safety (HICAHS) Advisory Board, we identified a low-cost intervention with the potential to improve dairy worker respiratory outcomes. Previously, we pilot tested the effectiveness of a hypertonic saline (HTS) nasal lavage ¹⁹, based on previous in vitro and in vivo studies, to modulate the pro- and anti-inflammatory response ^31–37. Hypertonic saline has been shown to inhibit release of pro-inflammatory cytokines that signal for an inflammatory immune response and stimulate release of anti-inflammatory cytokines that limit inflammatory immune responses ^31–37. Dairy workers receiving HTS nasal rinses across five consecutive days experienced a significant increase in anti-inflammatory cytokine interleukin (IL)-10 compared to those receiving a normotonic saline (NTS), but also produced conflicting increases in IL-6 and IL-8 pro-inflammatory cytokines ¹⁹. We aimed to build off of this result with a larger cohort to better understand the immunomodulatory effects of HTS in dairy workers.

Potential side effects from regular nasal rinses on the resident microbial communities present in the nose were not evaluated in the pilot study. Diverse resident microbiomes help maintain the stability and health of their environment, in this case the upper airways ^38,39. Previous studies have demonstrated a role for microbes and pathogens in the development of mucosal inflammation ^40,41. Therefore, perturbations to microbial diversity from hypertonic rinses could compromise stability and have adverse inflammatory effects. Consequently, the viability of HTS rinses as an intervention for respiratory disease in dairy workers required further testing in a larger cohort. We hypothesized that administering pre-and post-shift HTS nasal rinse to dairy workers would reduce the pro-inflammatory cytokine response and increase anti-inflammatory production when compared to an NTS control rinse. We also hypothesized the effect would be cumulative and would not cause perturbations or washout resident protective microbiota.

A total of 45 dairy workers participated in the study for up to five consecutive days at five dairies in the High Plains region. With participation ranging from one to five days, 344 nasal lavages and 178 PBZ samples were collected and analyzed. Participation duration and animal contact time by group, shown in Table 1, were evenly distributed between groups. Geometric mean dust and endotoxin concentrations for the treatment group were 0.36 mg/m³ and 52.99 EU/m³, respectively. Geometric mean dust and endotoxin concentrations for the control group were 0.36 mg/m³ and 56.81 EU/m³, respectively. PBZ dust and endotoxin concentrations stratified by day and treatment group are presented in Table 2.

From the ten pro- and anti-inflammatory biomarkers measured from each nasal lavage, IFN-γ, IL-12p70, and IL-4 were removed for not meeting our criteria requiring at least 70% of values to be above the limit of detection. Group level comparisons among the remaining seven biomarkers indicated concentrations of TNF-α were significantly higher in the treatment group compared to the control group (p=0.018). Log adjusted biomarker concentrations averaged by treatment group, day, and time are presented in picograms per milliliter (pg/ml) in Figures 1–4 below. A summary of results from the linear mixed effects model fits are presented in Table 3.

The dust model did not reveal sufficient evidence of an effect on average log mg/m³ between treatment groups (HTS and control) (p=0.95), day (p=0.826), or an interaction effect between treatment and day (p=0.703). Similarly, the endotoxin model did not indicate evidence of an effect on average log EU/m³ between treatment groups (p=0.781), among days (p=0.651), or an interaction effect between treatment and day (p=0.458). These results indicate similar dust and endotoxin exposure between treatment groups and sampling days. Therefore, exposure was not included as a variable in the biomarker model.

The biomarker model provided evidence of an interaction effect on average log concentrations between day and time of day for anti-inflammatory cytokine IL-10 (p=0.004). However, neither day nor time of day were significant main effects. After adjusting for multiple comparisons using Holm’s procedure, there was evidence that average log IL-10 concentrations were higher on day 5 than day 1 in AM samples (p=0.026). To visualize the interaction in absence of main effects, we created a plot of the model estimated marginal means shown in Figure 5. The plot illustrates a crossover interaction for IL-10 production between pre-shift (AM) and post-shift (PM) samples with AM samples exhibiting a positive trend while PM samples exhibit a negative trend across the 5-day sampling. There was no evidence of an interaction effect on average log concentrations between treatment, day, and time of day among all biomarkers. Further, there was not sufficient evidence of an interaction effect on average log concentrations between treatment and day or treatment and time among all biomarkers. Analysis of main effects provided evidence of differences in average log concentrations between time of day (AM and PM) for IL-13 (p=0.047), IL-1β (p<0.001), IL-6 (p<0.001), IL-8 (p<0.001), and TNF-α (p=0.024). Post-shift cytokine concentrations were higher than pre-shift cytokine concentrations for each significant result above. There was no evidence of differences in average log concentrations between treatment and control groups for any of the seven-modeled inflammatory biomarkers. There was no evidence of a difference in average log concentration between days among all biomarkers.

Microbiome diversity analyses revealed no significant differences in participant nasal microbiome alpha diversity, a measure of within sample diversity, between treatment and control groups. Beta diversity, a measure of between sample diversity, was not significantly different between group. There was evidence of differences in beta diversity between pre-shift and post-shift lavages (p=0.002) indicating a work shift related change in nasal microbiome. There was not sufficient evidence of differences in alpha diversity between pre and post shift. Additionally, there were not significant differences in alpha and beta diversity by day.

Exposure to bioaerosols and associated inflammagens (e.g., endotoxin) present in the dairy environment is associated with an increased risk of upper and lower respiratory disease. Past efforts to control dairy bioaerosol exposure have been mostly ineffective and cost prohibitive. In this study, pre- and post-shift hypertonic saline nasal lavages produced potentially advantageous albeit conflicting effects. Pre-shift pro-inflammatory cytokine concentrations were significantly lower than post-shift concentrations for IL-13, IL-1B, IL-6, IL-8, and TNF-⍺, confirming a work-related pro-inflammatory nasal response. Additionally, visual analysis of IL-10 anti-inflammatory concentrations shown in Figure 1 indicate an increasing anti-inflammatory response in both groups across the five-day sample period. However, no significant differences were observed in the model between treatment groups or between day among all biomarkers. Evidence of a significant interaction effect was found for IL-10 between day and time of day (p=0.004). Visual inspection of the model estimated marginal means (Figure 5) suggests a successful increase in log IL-10 across sample days among pre-shift samples while post-shift samples exhibit a downward trend. This crossover could be explained by IL-10s ability to auto regulate itself ⁵¹. Essentially, upregulated pre-shift IL-10 may be auto regulating itself and inhibiting a large post-shift IL-10 response.

Our results illustrate a less robust immunomodulatory effect of hypertonic saline on anti-inflammatory cytokine production in comparison to existing literature ^31–34. We demonstrated increases in IL-10 over time at a descriptive level in both groups, indicating HTS and NTS may be capable of upregulating the anti-inflammatory response. Still, model results revealed no significant differences by group or day. In contrast, in vivo testing of HTS was found to significantly increase IL-10 production in adult hemorrhagic trauma patients and produced a two-fold increase in peritoneal exudative macrophage IL-10 production compared to normotonic saline respectively ³³. The comparatively small effect in our study could be due to the much higher inflammatory state experienced by hemorrhagic patients or that cytokines were quantified from blood. Additionally, the muted anti-inflammatory effect we observed could be due to study design limitations surrounding the intervention. Previous studies investigating HTS’s anti-inflammatory properties were able to test biomarkers at various intervals after administration allowing for quantitation of effects over time ^33,35. Our study was only capable of estimating transient changes in biomarkers due to samples being post-administration lavages.

For pro-inflammatory cytokines, there was no observed reduction in concentration attributable to the HTS treatment, contrary to our hypothesis. This result is a departure from our pilot study that measured significantly higher IL-6 concentrations in the HTS treatment group ¹⁹. Earlier research found HTS treatment to be associated with significant decreases in TNF-a and IL-6 when compared to a normotonic saline treatment ³³. These studies were able to test lavage effects over time with multiple samples taken post-administration. Therefore, analysis of worker lavages as samples may be overlooking the true effect of the intervention on pro-inflammatory production. HTS can also illicit nasal mucosal irritation associated with transient increases in pro-inflammatory mediators. Analysis of post-administration lavages may be capturing this initial increase in inflammation while potentially missing subsequent decreases ^36,52.

Dust and endotoxin exposure concentrations provided by full-shift personal breathing zone samples were well below suggested agricultural exposure guidelines and were lower than most measures found in previous dairy environments ⁶. Unavoidable wet and cool weather conditions observed during sampling campaigns (snowstorm and ice storm) likely altered the dry dusty conditions regularly present in high plains dairy environments. Independent of cause, reduced bioaerosol exposure results in a less inflammagenic, cleaner environment and may be concealing the immunomodulatory effects of the HTS rinse.

When evaluating a workplace intervention, it is essential to consider potential side effects, barriers of adoption, and worker reception. The primary side effect of concern regarding frequent nasal irrigation is potential washout of healthy diverse microbial communities present in the nose. Microbiome diversity analyses revealed that microbiome alpha and beta diversity did not significantly change after five consecutive days of the intervention. Moreover, the higher salinity of HTS lavages did not significantly shift nasal microbiome diversity compared to NTS lavages. Considering the low economic cost of hypertonic saline and the short amount of time to self-administer a nasal rinse (~10 sec), this intervention exhibits few adoption barriers in contrast to previous efforts ^29,30. Positive anecdotal comments made by participants also illustrate worker acceptability for this intervention.

It is important to interpret the results from this study in the context of its limitations and strengths. Foremost, our sample size (n=45) may not have provided the adequate power needed to estimate the effects of HTS treatment over the full five days. Although we more than quadrupled the sample size from the pilot study, only 18 participants were able to complete all five days due to variable work schedules. Additionally, workers taking medication for existing respiratory conditions and workers unable to meet respiratory testing requirements were excluded due to participation requirements possibly introducing healthy worker bias. This study was also limited by only measuring inflammatory markers in post-administration lavages to estimate intervention efficacy. Consequently, we were only able to measure transient changes in inflammatory markers. Additional measurement of inflammation at different intervals post-administration was not feasible in this study due to the nature of dairy operation schedules. The exclusion of other dairy bioaerosol inflammagens (i.e., peptidoglycans, bacterial pathogens, and viruses) and relevant pro-inflammatory mediators (i.e., histamine and leukotrienes) is another study limitation. Future investigations should expand exposure characterization and measure additional mediators to better estimate intervention efficacy. Finally, this study was limited by the absence of a true control group receiving no lavage. Future studies should include participants receiving no rinse to elucidate whether the rinse itself provides protective effects regardless of tonicity.

Regular bioaerosol exposure in the dairy environment and the resulting burden of respiratory disease in workers demonstrates the need for a low-cost, low-burden intervention. Pre- and post-shift administration of a hypertonic saline nasal lavage over five days was found to have limited but encouraging effect on the inflammatory response. When compared to controls receiving normotonic saline lavages, we observed no significant differences in inflammatory response in the HTS group. However, both groups experienced an increase in anti-inflammatory IL-10 concentration indicating the rinse itself may provide immunomodulatory benefits, independent of tonicity. Based on these results, it remains unclear if saline nasal rinses provide significant health benefits for bioaerosol exposed dairy workers. Therefore, we recommend future studies that measure inflammation incrementally post lavage and introduce a no-lavage control group to better estimate efficacy.