John Mehaffy

Muramic Acid, Endotoxin, 3-Hydroxy Fatty Acids, and Ergosterol Content Explain Monocyte and Epithelial Cell Inflammatory Responses to Agricultural Dusts

In agricultural and other environments, inhalation of airborne microorganisms is linked to respiratory disease development. Bacterial endotoxins, peptidoglycans, and fungi are potential causative agents, but relative microbial characterization and inflammatory comparisons amongst agricultural dusts are not well described. The aim of this study was to determine the distribution of microbial endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, and ergosterol and evaluate inflammatory responses in human monocytes and bronchial epithelial cells with various dust samples. Settled surface dust was obtained from five environments: swine facility, dairy barn, grain elevator, domestic home (no pets), and domestic home with dog. Endotoxin concentration was determined by recombinant factor C (rFC). 3-OHFA, muramic acid, and ergosterol were measured using gas chromatography–mass spectrometry. Dust-induced inflammatory cytokine secretion in human monocytes and bronchial epithelial cells was evaluated. Endotoxin-independent dust-induced inflammatory responses were evaluated. Endotoxin and 3-OHFA levels were highest in agricultural dusts. Muramic acid, endotoxin, 3-OHFA, and ergosterol were detected in dusts samples. Muramic acid was highest in animal farming dusts. Ergosterol was most significant in grain elevator dust. Agricultural dusts induced monocyte tumor necrosis factor (TNF) α, interleukin (IL)-6, IL-8, and epithelial cell IL-6 and IL-8 secretion. Monocyte and epithelial IL-6 and IL-8 secretion was not dependent on endotoxin. House dust(s) induced monocyte TNFα, IL-6, and IL-8 secretion. Swine facility dust generally produced elevated responses compared to other dusts. Agricultural dusts are complex with significant microbial component contribution. Large animal farming dust(s)-induced inflammation is not entirely dependent on endotoxin. Addition of muramic acid to endotoxin in large animal farming environment monitoring is warranted.

Evaluation of the Limulus Amebocyte Lysate and Recombinant Factor C Assays for Assessment of Airborne Endotoxin

ABSTRACT As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) samplers, Button samplers, closed-face cassettes, and cyclone samplers. Field sampling was performed in 10 livestock production facilities, including those housing swine, chicken, turkey, dairy cows, cattle, and horses. Laboratory sampling was performed in exposure chambers using resuspended airborne dust collected in five livestock facilities. Paired samples were extracted in pyrogen-free water with 0.05% Tween 20 and analyzed using LAL and rFC assays. In 402 field sample pairs there was excellent agreement between endotoxin concentrations determined by LAL and rFC (r = 0.93; P < 0.0001). In 510 laboratory sample pairs there was also excellent agreement between the two assays (r = 0.86; P < 0.0001). Correlations for subgroups of facility or dust type ranged from 0.65 to 0.96. Mixed-model analysis of variance (ANOVA) for the field studies showed significant interactions of facility-sampler and facility-assay. rFC/LAL ratios of the geometric means were 0.9 to 1.14 for the samplers (not significantly different from 1.0). The data from this study demonstrate that the LAL assay and the rFC assay return similar estimates of exposure in livestock facilities. Both methods provided suitable lower limits of detection such that all but 19 of 1,824 samples were quantifiable.

Endotoxin Exposure and Inflammation Markers Among Agricultural Workers in Colorado and Nebraska

The adverse respiratory effects of agricultural dust inhalation are mediated in part by endotoxin, a constituent of gram-negative bacterial cell walls. This study quantified personal work-shift exposures to inhalable dust, endotoxin, and its reactive 3-hydroxy fatty acid (3-OHFA) constituents among workers in grain elevators, cattle feedlots, dairies, and on corn farms. Exposures were compared with post-work-shift nasal lavage fluid inflammation markers and respiratory symptoms. Breathing-zone personal air monitoring was performed over one work shift to quantify inhalable dust (Institute of Medicine samplers), endotoxin (recombinant factor C [rFC] assay), and 3-OHFA (gas chromatography/mass spectrometry). Post-shift nasal lavage fluids were assayed for polymorphonuclear neutrophils (PMN), myeloperoxidase (MPO), interleukin 8 (IL-8), albumin, and eosinophilic cation protein (ECP) concentrations. The geometric mean (GSD) of endotoxin exposure (rFC assay) among the 125 male participants was 888 ± (6.5) EU/m3, and 93% exceeded the proposed exposure limit (50 EU/m3). Mean PMN, MPO, albumin, and ECP levels were two- to threefold higher among workers in the upper quartile of 3‐OHFA exposure compared to the lowest exposure quartile. Even numbered 3-OHFA were most strongly associated with nasal inflammation. Symptom prevalence was not elevated among exposed workers, possibly due to endotoxin tolerance or a healthy worker effect in this population. This is the first study to evaluate the relationship between endotoxins 3-OHFA constituents in agricultural dust and nasal airway inflammation. More research is needed to characterize the extent to which these agents contribute to respiratory disease among agricultural workers.

Recombinant Factor C (rFC) Assay and Gas Chromatography/Mass Spectrometry (GC/MS) Analysis of Endotoxin Variability in Four Agricultural Dusts

Endotoxin exposure is a significant concern in agricultural environments due to relatively high exposure levels. The goals of this study were to determine patterns of 3-hydroxy fatty acid (3-OHFA) distribution in dusts from four types of agricultural environments (dairy, cattle feedlot, grain elevator, and corn farm) and to evaluate correlations between the results of gas chromatography/mass spectrometry (GC/MS) analysis (total endotoxin) and biological recombinant factor C (rFC) assay (free bioactive endotoxin). An existing GC/MS-MS method (for house dust) was modified to reduce sample handling and optimized for small amount (<1 mg) of agricultural dusts using GC/EI-MS. A total of 134 breathing zone samples using Institute of Occupational Medicine (IOM) inhalable samplers were collected from agricultural workers in Colorado and Nebraska. Livestock dusts contained approximately two times higher concentrations of 3-OHFAs than grain dusts. Patterns of 3-OHFA distribution and proportion of each individual 3-OHFA varied by dust type. The rank order of Pearson correlations between the biological rFC assay and the modified GC/EI-MS results was feedlot (0.72) > dairy (0.53) > corn farm (0.33) > grain elevator (0.11). In livestock environments, both odd- and even-numbered carbon chain length 3-OHFAs correlated with rFC assay response. The GC/EI-MS method should be especially useful for identification of specific 3-OHFAs for endotoxins from various agricultural environments and may provide useful information for evaluating the relationship between bacterial exposure and respiratory disease among agricultural workers.

Field and Wind Tunnel Comparison of Four Aerosol Samplers Using Agricultural Dusts

Occupational lung disease is a significant problem among agricultural workers exposed to organic dusts. Measurements of exposure in agricultural environments in the USA have traditionally been conducted using 37-mm closed-face cassettes (CFCs) and respirable Cyclones. Inhalable aerosol samplers offer significant improvement for dose estimation studies to reduce respiratory disease. The goals of this study were to determine correction factors between the inhalable samplers (IOM and Button) and the CFC and Cyclone for dusts sampled in livestock buildings and to determine whether these factors vary among livestock types. Determination of these correction factors will allow comparison between inhalable measurements and historical measurements. Ten sets of samples were collected in swine, chicken, turkey, and dairy facilities in both Colorado and Iowa. Pairs of each sampling device were attached to the front and back of a rotating mannequin. Laboratory studies using a still-air chamber and a wind tunnel provided information regarding the effect of wind speed on sampler performance. Overall, the IOM had the lowest coefficient of variation (best precision) and was least affected by changes in wind speed. The performance of the Button was negatively impacted in poultry environments where larger (feather) particulates clogged the holes in the initial screen. The CFC/IOM ratios are important for comparisons between newer and older studies. Wind speed and dust type were both important factors affecting ratios. Based on the field studies (Table 6), a ratio of 0.56 is suggested as a conversion factor for the CFC/IOM (average for all environments because of no statistical difference). Suggested conversion factors for the Button/IOM are swine (0.57), chicken (0.80), turkey (0.53), and dairy (0.67). Any attempt to apply a conversion factor between the Cyclone and inhalable samplers is not recommended.

Pulmonary function reductions among potentially susceptible subgroups of agricultural workers in Colorado and Nebraska.

Objective: Organic dust inhalation has been associated with adverse respiratory responses among agricultural workers. We evaluated factors that may confer increased susceptibility to these health effects. Methods: We quantified personal work shift exposures to inhalable dust, endotoxin, and its 3-hydroxy fatty acid constituents, and evaluated changes in pulmonary function among 137 grain elevator, cattle feedlot, dairy, and corn farm workers. Results: Increased dust exposure was associated with work shift reductions in lung function. Although interpretation is limited because of small samples, a suggestion of stronger exposure–response relationships was observed among smokers, as well as workers reporting pesticide/herbicide application, asthma, or allergies, and those with genetic polymorphisms (TLR4) (Pinteraction ⩽ 0.05). Conclusions: A better understanding of factors leading to increased susceptibility of adverse respiratory outcomes is needed to optimize exposure reduction strategies and develop more comprehensive wellness programs.

Evaluation of Parlor Cleaning as an Intervention for Decreased Occupational Exposure to Dust and Endotoxin Among Dairy Parlor Workers—A Pilot Study

Agreater prevalence of respiratory symptoms has been observed among workers involved in animal production compared to other farmers and rural residents.(1) In addition, respiratory diseases such as byssinosis,(2) asthma,(3) allergic alveolitis,(4) chronic bronchitis,(5) and the organic dust toxic syndrome (ODTS)(6) have been reported among workers in the animal feed industry, slaughterhouses, compost facilities, and other agriculture-related industries.(7) Respiratory hazards such as organic dust, microorganisms, fungi, molds, and endotoxin are common in the dairy industry.(8) Exposure to organic dust and endotoxin may lead to pulmonary inflammation among dairy parlor workers.(9–11) Organic dusts are associated with intensive livestock operations, such as dairy, swine, and poultry production; however differences in the components of these organic dusts have been reported.(12,13) A substantial amount of work has evaluated the effects of organic dust exposure among swine workers, whereas less is known about these exposures among dairy parlor workers.(13–16) Sources of organic dust on dairy farms include feed/hay grinding and animal sources, such as hair and feces.(17) Little information is available on the impact of these exposures among workers in the dairy industry. However, a few studies have demonstrated an association of dust and endotoxin exposure with markers of lung inflammation among dairy parlor workers.(9,12,18) The primary task of dairy parlor workers is to milk the cows. Cows are moved through the parlor building using workers to guide the animals as well as mechanical gating systems. Cows enter the parlor on an elevated platform for milking. Cows are typically milked three times a day over a series of three 8-hr shifts.(19) The walkways and other surfaces that come into contact with the animals become soiled with animal waste. Workers use an automated cleaning system to remove animal waste from the parlor surfaces. Therefore, increasing the frequency in which these walkways and other surfaces are cleaned may reduce the concentration of aerosolized dust and endotoxin in the milking parlor. Parlor workers spend their work shift in proximity to cows and animal waste, which may be sources of inhalation exposure to organic dust and endotoxin. Therefore, these workers may be more exposed to inflammatory agents, as organic dust and endotoxin, compared to other workers on the dairy farm. The objective of our study was to assess occupational exposure to dust and endotoxin among dairy parlor workers. This study also evaluated the effectiveness of increasing the frequency of cleaning the dairy parlor surfaces on dust and endotoxin inhalation exposure among parlor workers.

Development and evaluation of an ultrasonic personal aerosol sampler

Abstract Assessing personal exposure to air pollution has long proven challenging due to technological limitations posed by the samplers themselves. Historically, wearable aerosol monitors have proven to be expensive, noisy, and burdensome. The objective of this work was to develop a new type of wearable monitor, an ultrasonic personal aerosol sampler (UPAS), to overcome many of the technological limitations in personal exposure assessment. The UPAS is a time‐integrated monitor that features a novel micropump that is virtually silent during operation. A suite of onboard environmental sensors integrated with this pump measure and record mass airflow (0.5–3.0 L/min, accurate within 5%), temperature, pressure, relative humidity, light intensity, and acceleration. Rapid development of the UPAS was made possible through recent advances in low‐cost electronics, open‐source programming platforms, and additive manufacturing for rapid prototyping. Interchangeable cyclone inlets provided a close match to the EPA PM2.5 mass criterion (within 5%) for device flows at either 1.0 or 2.0 L/min. Battery life varied from 23 to 45 hours depending on sample flow rate and selected filter media. Laboratory tests of the UPAS prototype demonstrate excellent agreement with equivalent federal reference method samplers for gravimetric analysis of PM2.5 across a broad range of concentrations.

Particulate matter, endotoxin, and worker respiratory health on large Californian dairies

Objective: To assess respiratory exposures and lung function in a cross-sectional study of California dairy workers. Methods: Exposure of 205 dairy and 45 control (vegetable processing) workers to particulate matter and endotoxin was monitored. Pre- and postshift spirometry and interviews were conducted. Results: Geometric mean inhalable and PM2.5 concentrations were 812 and 35.3 &mgr;g/m3 versus 481.9 and 19.6 &mgr;g/m3, respectively, for dairy and control workers. Endotoxin concentrations were 329 EU/m3 or 1122 pmol/m3 and 13.5 EU/m3 or 110 pmol/m3, respectively, for dairy and control workers. In a mixed-effects model, forced vital capacity decreased across a work shift by 24.5 mL (95% confidence interval, −44.7 to −4.3; P = 0.018) with log10 (total endotoxin) and by 22.0 mL (95% confidence interval, −43.2 to −0.08; P = 0.042) per hour worked. Conclusions: Modern California dairy endotoxin exposures and shift length were associated with a mild acute decrease in forced vital capacity.

Personal exposure of dairy workers to dust, endotoxin, muramic acid, ergosterol, and ammonia on large-scale dairies in the high plains Western United States

ABSTRACT Dairy workers experience a high degree of bioaerosol exposure, composed of an array of biological and chemical constituents, which have been tied to adverse health effects. A better understanding of the variation in the magnitude and composition of exposures by task is needed to inform worker protection strategies. To characterize the levels and types of exposures, 115 dairy workers grouped into three task categories on nine farms in the high plains Western United States underwent personal monitoring for inhalable dust, endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, ergosterol, and ammonia through one work shift. Eighty-nine percent of dairy workers were exposed to endotoxin at concentrations exceeding the recommended exposure guidelines (adjusted for a long work shift). The proportion of workers with exposures exceeding recommended guidelines was lower for inhalable dust (12%), and ammonia (1%). Ergosterol exposures were only measurable on 28% of samples, primarily among medical workers and feed handlers. Milking parlor workers were exposed to significantly higher inhalable dust, endotoxin, 3-OHFA, ammonia, and muramic acid concentrations compared to workers performing other tasks. Development of large modern dairies has successfully made progress in reducing worker exposures and lung disease prevalence. However, exposure to endotoxin, dust, and ammonia continues to present a significant risk to worker health on North American dairies, especially for workers in milking parlors. This study was among the first to concurrently evaluate occupational exposure to assayable endotoxin (lipid A), 3-hydroxy fatty acids or 3-OHFA (a chemical measure of cell bound and noncell-bound endotoxins), muramic acid, ergosterol, and ammonia among workers on Western U.S. dairies. There remains a need for cost-effective, culturally acceptable intervention strategies integrated in OHS Risk Management and production systems to further optimize worker health and farm productivity.