GENERAL INTRODUCTION
In animal houses, especially those for pigs and poultry, air quality can be seriously impaired by high dust concentrations (Wathes et al 1997; Takai et al
Animal housing environments pose health risks not only to humans working in these settings but potentially also to the animals residing there Research indicates that these facilities significantly contribute to increased particle concentrations in the surrounding air due to the emission of particles in exhausted air.
Dust in animal houses is characterized by its biological activity, containing organic compounds from animals (such as skin, hair, and feathers), feed, feces, bedding materials, and various microbes like viruses, bacteria, fungi, and dust mites This dust is significantly more concentrated in the air of animal housing, often ten to one hundred times higher than in typical office environments
The size of airborne dust particles is a crucial characteristic that affects their behavior, transport in the air, and the selection of control technologies Additionally, particle size plays a significant role in determining the impact of dust on both human and animal health Dust particles are typically categorized into three size classes, with those smaller than a certain threshold posing different health risks.
Particles measuring 10 micrometers (PM10), 2.5 micrometers (PM2.5), and 1.0 micrometers (PM1) are significant contributors to health issues due to their ability to infiltrate the respiratory system Research indicates that smaller particles penetrate deeper into the respiratory tract, posing greater risks to both human and animal health While various studies have explored particle size and distribution within animal housing, most have focused specifically on certain types of facilities, such as pig buildings.
(Maghirang et al 1997; Lee et al 2008); and cattle feedlots (Sweeten et al
In a study by Lee et al (2006), the impact of various farming activities on personal dust exposure across different sizes was examined in pig, poultry, and dairy farms However, a comprehensive analysis of particle size distribution (PSD) using the same instrument across diverse species and housing configurations remains unexplored Due to the variability in dust concentrations over time and space (Maghirang et al 1997), sampling was conducted in spring and summer across two animal houses for each species and housing type This research aimed to assess the particle size distribution, both in terms of counts and mass, within different commercial animal houses in the Netherlands.
In a study conducted in the Netherlands, PM10 mass and particle size distribution (PSD) were analyzed across 13 different animal species and housing types, with measurements taken at two farms during spring and summer 2009 The species/housing combinations included broilers, layers in both floor and aviary systems, broiler breeders, turkeys, piglets, fattening pigs in traditional and modern low-emission housing (with both dry and wet feed), sows in individual and group housing, dairy cattle, and mink Detailed housing systems and conditions for each species are provided in Tables 1a, 1b, and 1c.
PM10 mass concentrations and particle size distribution (PSD) were measured using aerosol spectrometers that operate on the light-scattering principle These instruments detect individual particles through scattered light photometry within an optical measuring cell, with the intensity of the scattered light indicating particle size Calibration with standardized dust enables consistent comparisons of measurements when the dust source or type is primarily the same.
PM10 mass concentrations were measured using a DustTrak aerosol monitor model 8520, a portable and battery-operated laser-photometer manufactured by TSI Inc This device provides real-time measurements through 90° light scattering and can detect aerosol mass concentrations ranging from 0.0001 to 100 mg m^-3, with a sampling air flow rate of 1.7 L min^-1 Factory calibrated to the respirable fraction of standard ISO 12103-1, A1 test dust, the DustTrak enables reliable comparisons between measurements.
Particle size distribution was analyzed using a Grimm model 1.109 aerosol spectrometer, which measured particle counts across 31 size ranges, with lower limits ranging from 0.25 μm to 32 μm The largest size range, exceeding 32 μm, was excluded from the analysis due to an undefined upper limit The instrument operated at a sampling airflow rate of 1.2 L/min, with data collected at one-minute intervals Mean values for each location and measurement day were utilized in the analysis to provide accurate insights into particle size distribution.
Air samples using Grimm were collected for short durations to avoid contamination in high dust environments Both Grimm and DustTrak samples were gathered inside and outside each animal house, with indoor samplers positioned approximately 1.5 meters above the floor and near the air outlet, while maintaining a distance of at least 1.5 meters from ventilators to ensure accurate exhaust air representation and minimize the impact of high air velocities (Hinds, 1999) In naturally ventilated buildings, samplers were placed 5 to 8 meters from the ridge air outlet Indoor sampling commenced immediately after installation for 60 minutes, but only the final 30 minutes were analyzed to eliminate potential human disturbance effects All measurements were conducted between 10:00 and 15:00 hours, with sampling periods illustrated in relation to the diurnal PM10 concentration patterns for various animal categories (Winkel et al., 2011), although these patterns were not recorded on the same days as the measurements Outdoor sampling also began immediately after.
Table 1a Characteristic of the animal houses (n = 26) in this study
Production cycle (weeks of age)
Sampling moments (weeks of age)
Inside/outside conditions Housing Feed
Automatically dispensed crumbs and pellets
Side inlet, ventilators in end wall
Layer_floor 1 3,850 8.8 - 9 18 - 75 52 14.8C, 75.0%/11.4C, 88.9% Floor with bedding, slattedhopper, laying nests
Automatically dispensed crumbs and pellets
Side inlet, ventilators in end wall
Layer_aviary 1 25,650 17 - 18 18 - 75 52 13.6C, 72.7%/11.4C, 88.9% Floor with bedding, slattedhopper, laying nests
Automatically dispensed crumbs and pellets
Side inlet, ventilators in end wall
Broiler_breeder 1 3,698 7.7 - 8.5 20 - 60 29 20.4C, 47.8%/18.6C, 28.2% Floor with bedding, slattedhopper, laying nests
Automatically dispensed crumbs and pellets
Side inlet, ventilators in end wall
Automatically dispensed crumbs and pellets
Natural ventilation with open ridge and side inlets
Table 1b Characteristic of the naimal houses ( n &) in this study (Continued)
Production cycle (weeks of age)
Sampling moments (weeks of age)
Automatically dispensed crumbs and pellets
Ceiling inlet ventilator in ceiling
Door inlet ventilator in ceiling
Fat_pig_trad 1 60 10/11 - 25/27 17 20.8C, 54.4%/14.8C, 33.5% Partially slatted
Automatically dispensed crumbs and pellets
Ceiling inlet ventilator in ceiling Door inlet ventilator in ceiling
Fat_pig_mod_dry 1 132 1.0 - 1.3 10/11 - 25/27 22 22.7C, 53.2%/19.9C, 48.1% Partially slatted
Automatically dispensed crumbs and pellets
Floor inlet, ventilator in ceiling
Fat_pig_mod_wet 1 144 22 25.4C, 64.2%/18.4C, 64.9% Partially slatted
Liquid feeding Floor inlet, ventilator in ceiling
Sow_individual 1 32 Diverse Diverse 21.2C, 60.3%/19.8C, 57.3% Partially slatted
Door inlet ventilator in ceiling
Table 1c Characteristic of the animal houses (n = 26) in this study (Continued)
Production cycle (weeks of age)
Sampling moments (weeks of age)
Sow_group 1 46 0,4 Diverse Diverse 19.7C, 56.6%/22.2C, 31.2% Partially slatted with feeding crates
Automatically dispensed crumbs and pellets
Ceiling inlet ventilator in ceiling 25.7C, 61.9%/21.6C, 64.9%
2 34 0.3 - 0.4 Diverse Diverse 19.4C, 82.1%/14.2C, 33.8% Valves inlet, ventilator in ceiling 23.6C, 54.4%/19.9C55.2%
Cattle 1 51 Diverse Diverse 19.8C, 51.2%/19.9C, 56.6% Cubicle house
Roughage (maize and grass silage) two times/day
Naturally ventilate with side and cutains and ridge
Mink 1 9,015 4 - 5 48 - 52 7 18.9C, 71.7%/17.9C, 72.9% Cages Feeding wet day feed 3 times/day
Naturally ventilate with side and cutains and ridge
Note: (1) : per m 2 basic floor space, so excluding floor space at tiers
Figure 1 Time periods in which the samplings were done, given in relation to the diurnal pattern of PM10 concentrations for the various animal categories
Temperature and relative humidity levels inside and outside the animal house were monitored using Escort ilog data loggers The recorded data were averaged for each sampling day and are presented in Table 1.
Mass of particles in the different size ranges were calculated as follows:
The mass of particles within a specific size range, denoted as Mi (mg m -3), is calculated using the midpoint diameter (di) which represents the average diameter between the upper and lower limits of that size range, measured in micrometers (µm) Additionally, the density of these particles, represented as ρi (mg mm -3), is crucial for understanding their physical properties and behavior in various applications.
Fi = number of particles in size range i per unit of volume, m -3
In the analysis of particulate matter concentration, particles of all sizes were assumed to have a spherical shape and unit density The measured particle counts and mass were categorized into four classes: PM1 (0.25 - 1.0 µm), PM1-2.5 (1.0 - 2.5 µm), PM2.5-10 (2.5 - 10 µm), and PM10-32 (10 - 32 µm) Following a loge-transformation, ANOVA was employed to assess the impact of animal category on particle counts and mass, with significant differences identified at P-values below 0.05 using Bonferroni’s two-tailed t-test Additionally, correlation coefficients between particle counts across different size ranges were calculated, and the influence of external climate factors (temperature and relative humidity) on particle counts, as well as on count median diameter (CMD) and mass median diameter (MMD), was evaluated through multiple linear regression analysis The analysis revealed that the model did not significantly benefit from interaction terms, as indicated by P-values greater than 0.05 All statistical analyses were conducted using Genstat software (Genstat, 2008).
Particle sizes and their distribution can be represented through various methods and characterized by distinct equations for both particle numbers and mass In our study, we standardized the measured values using the equations provided by Zhang (2004a) The specific formulas employed to describe particle size distribution are outlined in our findings.
- Count median diameter (CMD, àm)
Most particle size distributions exhibit a skewed pattern with a long right tail, yet the median is frequently utilized for analysis The characteristic mean diameter (CMD) is defined as the diameter at which half of the particles in a sample are smaller and the other half are larger The calculation of CMD is performed using Equation 2.
Fi = number of particles in size range i, m -3 di = midpoint diameter of particles in size range i, àm
N = total number of particles (sum of all size ranges) , m -3
- Standardized number fraction distribution (Δfi, àm -1 )
SIZE DISTRIBUTION OF AIRBORNE PARTICLES IN ANIMAL
Airborne fine dust serves as a carrier for various substances, including gases, microorganisms, and endotoxins, as noted by several researchers (Aarnink and Wagemans, 1997; Aarnink et al., 2003; Gustafsson, 1999; Takai et al., 1998; Tielen et al., 1978; Collins and Algers, 1986) This organic dust can originate from animals, comprising elements such as skin, hair, and feathers (collagen), as well as from arthropods (chitin), feed, feces, and bedding materials (Aarnink et al., 2004, 2005; Aarnink and Ellen, 2007).
Poultry houses exhibited the highest dust concentrations and emission rates for airborne microbes, with bacteria levels reaching 3.5 x 10^9 cfu/h and fungi at 3.6 x 10^7 cfu/h In comparison, pig and cattle stables showed lower dust levels, highlighting the significant respiratory risks associated with poultry environments.
Inhaled endotoxin levels in animal housing can reach 1 μg/h, influenced by factors such as stock density, animal age, ventilation, and dust (Seedorf et al., 1998; Collins and Algers, 1986; Murch, 2001) Dust from animal houses is notably more biologically active, containing organic compounds, viruses, bacteria, fungi, endotoxins, parasites, and dust mites, with concentrations up to 100 times higher than that found in offices (Muller and Wieser, 1987) This dust varies in size and shape, composed of dander, hair, feed dust, and fecal materials, with particles ranging from less than 1 to 100 μm in diameter Smaller respiratory particles, particularly those under 10 μm, pose significant health risks as they can penetrate deep into the lungs, similar to tobacco smoke (Collins and Algers, 1986; Wathes, 1995).
An important part of dust is formed by microbes (Collins and Algers,
Pathogen-associated molecular patterns (PAMPs) from microbes, such as lipopolysaccharides (LPS) from gram-negative bacteria and β-glucans (BGL) from yeast, play a crucial role in immune responses by binding to toll-like receptors on antigen presenting cells (APCs) in mammals and poultry These interactions trigger specific immune responses, including T-helper (TH)2-mediated antibody production, TH1-mediated cellular inflammation, and TH17 responses In chicken houses, high concentrations of airborne PAMPs, ranging from 240 to 13,400 endotoxin units/m³, are present, with some reports indicating levels as high as 63 μg/m³ Chickens are exposed to these PAMPs through inhalation, cloacal contact, ocular exposure, and oral intake, highlighting the importance of understanding their impact on poultry health.
In a dose-dependent fashion, PAMP modulated primary and secondary immune responses of layers and BWG (Ploegaert et al 2007; Parmentier et al.,
2008), whereas signs of cannibalistic behavior were found in PAMP-sensitized layers (Parmentier et al., 2009) In broilers, PAMP such as LPS increased pulmonary arterial pressure after i.v exposure (Chapman et al., 2005;
Wideman et al., 2004), decreased respiratory capacity and caused death
This study investigates the impact of concurrent intratracheal challenges with various components, including LPS, LTA, BGL, chitin, heat-inactivated dust, and human serum albumin (HuSA), to model the interactions of airborne PAMPs and protein antigens on systemic antibody responses and body weight gain (BWG) in broilers Previous research indicated interactions between HuSA and LPS in layer birds, prompting this study to explore whether broilers exhibit similar susceptibility to intratracheal immunization with HuSA The findings aim to enhance our understanding of immune responses in broiler chickens exposed to diverse dust components in their environment.
Our research investigated the impact of dust and its components, particularly PAMP, on specific antibody responses in broilers following repeated intratracheal challenges We also examined the immune response to a significant dust component, LPS, in broilers compared to layers and how various treatments influenced this response Additionally, we explored the potential effects of concurrent challenges with dust, dust components, and HuSA on body weight gain (BWG) and heart parameters The implications of our findings are thoroughly discussed.
The experiment involved 101 slow-growing Hubbard JA 957 female broilers, aged three weeks, housed in sawdust-covered floor pens throughout the study Two barns were partitioned into nine smaller pens, accommodating 12 birds in Groups 1 to 8 and 5 birds in Group 9 The lighting schedule consisted of 14 hours of light and 10 hours of darkness, with temperatures maintained between 18°C and 24°C during the observation period The birds were provided with ad libitum access to a standard broiler diet containing 204 g/kg of crude protein and 2,859 kcal of metabolizable energy per kg, along with unrestricted access to water via drinking nipples Vaccinations against Newcastle disease, Infectious Bursal disease (Gumboro), and Infectious Bronchitis were administered at hatch using live vaccines.
The experiment was approved by the Animal Welfare Committee of Wageningen University according to Dutch law
Human serum albumin (lot H3383), Escherichia coli derived LPS (lot
L2880-017K4097), LTA from Staphylococcus aureus (lot L2515-105K4061) Chitin from crab shells (lot C7170-065K7026), and BGL derived from
Saccharomyces cerevisiae, (Zymosan A, Z-4250, lot Z4250-084K1220) were from Sigma Chemical Co.(St Louis, MO) NH3 was from Merck KGaA (Darmstadt, Germany)
At three weeks of age, all birds in Groups 1 to 8 were intratracheally challenged with 0.5 mg HuSA in 0.5 ml PBS over two consecutive days, totaling 1 mg HuSA The challenges involved using a blunted anal cannula for administration Group 1 received only HuSA in PBS, while Group 2 was administered 0.5 mg HuSA and 0.5 mg chitin, and Group 3 received 0.5 mg HuSA and 0.5 mg LTA Group 4 was challenged with 0.5 mg HuSA and 0.5 mg heat-inactivated dust, and Group 5 received 0.5 mg HuSA and 0.5 mg LPS Group 6 was given 0.5 mg HuSA and 0.5 mg BGL, whereas Group 7 received a combination of 0.5 mg HuSA with 0.25 mg LPS, 0.25 mg LTA, 0.25 mg chitin, and 0.25 mg BGL Group 8 was administered 0.5 mg HuSA and 100 ppm NH3, and Group 9 received only PBS Blood samples were collected on days 0, 3, 7, 10, 14, 21, and 28 post-primary challenge, with a secondary challenge administered on day 28 Body weight was measured before and after challenges, and at ten weeks of age, the birds were euthanized for heart measurements.
2.4 Humoral immune response to HuSA and LPS
Plasma samples from all birds were analyzed for total antibody titers to HuSA and LPS using ELISA at multiple time points: days 0, 3, 7, 10, 14, 21, and 28 following the initial exposure to airborne dust components and HuSA.
In this study, 96-well plates were coated with 100 µL of either 4 µg/mL of HuSA or 4 µg/mL of LPS Following a wash with PBS containing 0.05% Tween, the plates were incubated at room temperature for 60 minutes with serial four-step double dilutions of plasma in PBS that included 1% horse serum and 0.05% Tween The binding of total antibodies to either HuSA or LPS antigen was subsequently detected.
The study involved a one-hour incubation at room temperature using a 1:20,000 dilution of rabbit anti-chicken IgGH+L peroxidase conjugate in PBS with 1% horse serum and 0.05% Tween The binding of immunoglobulin M (IgM) and IgG antibodies to human serum albumin (HuSA) or lipopolysaccharides (LPS) was assessed daily Following incubation with serial dilutions of plasma and thorough washing, the bound isotype-specific antibodies were detected using a 1:20,000 dilution of goat anti-chicken IgM and goat anti-chicken IgGFc, both coupled to peroxidase Finally, tetramethylbenzidine and 0.05% hydrogen peroxide were added for further analysis.
The reaction was conducted at room temperature for 10 minutes and subsequently halted by adding 50 µL of H2SO4 Extinction levels were measured at a wavelength of 450 nm using a Multiscan device from Labsystems in Helsinki, Finland A pooled plasma sample from all birds on day 7 served as the positive standard The titers were calculated as log2 values of the dilutions that resulted in an extinction closest to 50% of Emax, with Emax defined as the highest mean extinction of the standard positive plasma present on each microtiter plate.
The study analyzed primary and secondary antibody titers (total, IgM, and IgG) to HuSA and LPS using a 3-way ANOVA, assessing the effects of dust components (PAMP), time, and their interactions with repeated measures, accounting for birds nested within PAMP treatment A 1-way ANOVA was conducted to evaluate differences in body weight gain (BWG) following primary and secondary challenges with dust components, as well as heart characteristics (weight, length, width, and relative heart weight) at slaughter Body weight at 3 weeks was considered a covariate for intratracheal treatment effects on BW gain throughout the experimental period, while body weight at 10 weeks served as a covariate for heart characteristics at the study's conclusion All statistical analyses adhered to SAS Institute GLM procedures (SAS, 1990).
The kinetics of total antibody (Ab) responses, including IgM and IgG titers to HuSA, were analyzed in plasma from birds immunized intratracheally with various dust components and HuSA, as illustrated in Figures 1, 2, and 3 Table 1 presents the least square means of the total (IgT) and specific isotype (IgM, IgG) antibody titers to HuSA, measured over four weeks following primary immunization and three weeks after secondary immunization with different airborne dust components and HuSA.
3.1 Primary Total Antibody Responses to HuSA
Total primary antibody titers to HuSA were significantly influenced by the interaction between treatment and time (P < 0.0001) The highest titers were observed 10 days post-immunization in BGL-treated (Group 6) and NH3-treated birds (Group 8), while other treatment groups peaked at 7 days Nontreated birds (Group 9) exhibited low titers and were excluded from analysis Birds challenged with BGL (Group 6) or a cocktail of LPS, LTA, BGL, and chitin (Group 7) showed significantly higher total antibody titers to HuSA At day 7 post-primary challenge, all dust component treatments resulted in notably higher titers compared to birds challenged only with HuSA (Group 1) Additionally, at days 10, 14, 21, and 28, BGL-challenged birds (Group 6) continued to demonstrate significantly elevated total antibody titers to HuSA.