Multiple exposures to swine barn air induce lung inflammation and airway hyper-responsiveness.

Abstract

Swine farmers repeatedly exposed to the barn air suffer from respiratory diseases. However the mechanisms of lung dysfunction following repeated exposures to the barn air are still largely unknown. Therefore, we tested a hypothesis in a rat model that multiple interrupted exposures to the barn air will cause chronic lung inflammation and decline in lung function.
Rats were exposed either to swine barn (8 hours/day for either one or five or 20 days) or ambient air. After the exposure periods, airway hyper-responsiveness (AHR) to methacholine (Mch) was measured and rats were euthanized to collect bronchoalveolar lavage fluid (BALF), blood and lung tissues. Barn air was sampled to determine endotoxin levels and microbial load.
The air in the barn used in this study had a very high concentration of endotoxin (15361.75 +/- 7712.16 EU/m3). Rats exposed to barn air for one and five days showed increase in AHR compared to the 20-day exposed and controls. Lungs from the exposed groups were inflamed as indicated by recruitment of neutrophils in all three exposed groups and eosinophils and an increase in numbers of airway epithelial goblet cells in 5- and 20-day exposure groups. Rats exposed to the barn air for one day or 20 days had more total leukocytes in the BALF and 20-day exposed rats had more airway epithelial goblet cells compared to the controls and those subjected to 1 and 5 exposures (P < 0.05). Bronchus-associated lymphoid tissue (BALT) in the lungs of rats exposed for 20 days contained germinal centers and mitotic cells suggesting activation. There were no differences in the airway smooth muscle cell volume or septal macrophage recruitment among the groups.
We conclude that multiple exposures to endotoxin-containing swine barn air induce AHR, increase in mucus-containing airway epithelial cells and lung inflammation. The data also show that prolonged multiple exposures may also induce adaptation in AHR response in the exposed subjects.

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BioMed Central
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Respiratory Research
Open Access
Research
Multiple exposures to swine barn air induce lung inflammation and
airway hyper-responsiveness
Chandrashekhar Charavaryamath1, Kyathanahalli S Janardhan1,
Hugh G Townsend2, Philip Willson3 and Baljit Singh*1
Address: 1Immunology Research Group and Departments of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, S7N 5B4,
Canada, 2Large Animal Clinical Sciences, University of Saskatchewan, Saskatoon, S7N 5B4, Canada and 3Vaccine and Infectious Disease
Organization, University of Saskatchewan, Saskatoon, S7N 5B4, Canada
Email: Chandrashekhar Charavaryamath - c.chandru@usask.ca; Kyathanahalli S Janardhan - janardhan.ks@usask.ca;
Hugh G Townsend - hugh.townsend@usask.ca; Philip Willson - philip.willson@usask.ca; Baljit Singh* - baljit.singh@usask.ca
* Corresponding author
Abstract
Background: Swine farmers repeatedly exposed to the barn air suffer from respiratory diseases.
However the mechanisms of lung dysfunction following repeated exposures to the barn air are still
largely unknown. Therefore, we tested a hypothesis in a rat model that multiple interrupted
exposures to the barn air will cause chronic lung inflammation and decline in lung function.
Methods: Rats were exposed either to swine barn (8 hours/day for either one or five or 20 days)
or ambient air. After the exposure periods, airway hyper-responsiveness (AHR) to methacholine
(Mch) was measured and rats were euthanized to collect bronchoalveolar lavage fluid (BALF),
blood and lung tissues. Barn air was sampled to determine endotoxin levels and microbial load.
Results: The air in the barn used in this study had a very high concentration of endotoxin
(15361.75 ± 7712.16 EU/m3). Rats exposed to barn air for one and five days showed increase in
AHR compared to the 20-day exposed and controls. Lungs from the exposed groups were inflamed
as indicated by recruitment of neutrophils in all three exposed groups and eosinophils and an
increase in numbers of airway epithelial goblet cells in 5- and 20-day exposure groups. Rats exposed
to the barn air for one day or 20 days had more total leukocytes in the BALF and 20-day exposed
rats had more airway epithelial goblet cells compared to the controls and those subjected to 1 and
5 exposures (P < 0.05). Bronchus-associated lymphoid tissue (BALT) in the lungs of rats exposed
for 20 days contained germinal centers and mitotic cells suggesting activation. There were no
differences in the airway smooth muscle cell volume or septal macrophage recruitment among the
groups.
Conclusion: We conclude that multiple exposures to endotoxin-containing swine barn air induce
AHR, increase in mucus-containing airway epithelial cells and lung inflammation. The data also show
that prolonged multiple exposures may also induce adaptation in AHR response in the exposed
subjects.
Published: 02 June 2005
Respiratory Research 2005, 6:50doi:10.1186/1465-9921-6-50
Received: 02 October 2004
Accepted: 02 June 2005
This article is available from: http://respiratory-research.com/content/6/1/50
© 2005 Charavaryamath et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Respiratory diseases in agricultural workers are one of the
earliest recognized occupational hazards [1]. Swine farm-
ers work in confined buildings in close proximity to a
large number of pigs and are exposed to toxic gasses such
as ammonia and hydrogen sulfide, and to high levels of
dust and endotoxins [2]. Exposure to such toxic bio aero-
sols including endotoxins in the barn air is a risk factor for
the development of chronic respiratory symptoms and
lung dysfunction [3-5]. Workers exposed to barn air report
significantly higher frequencies of respiratory symptoms,
cold, chest illness and pneumonia [2,3]. The severity of
lung irritation and respiratory symptoms increases during
winter and is also related to the number of working hours
[6]. Single, 3–5 hour exposure of naïve, healthy, non-
smoking subjects to swine barn air increases IL-6 in serum
and IL-6 and IL-8 in nasal lavage and inflammatory cells
in bronchoalveolar lavage fluid (BALF) [7,8]. Further-
more, pig barn dust stimulates IL-8 and IL-6 release from
human bronchial epithelial cells in vitro [9]. Collectively,
these data show that a single exposure to the barn air ini-
tiates acute lung inflammation.
Although swine barn workers are repeatedly exposed to
barn air, majority of studies have focused on the acute
pulmonary effects of single exposure [7,10]. Multiple
exposures to barn air are linked to chronic lung inflamma-
tion including chronic bronchitis, decline in lung func-
tion and higher incidence of asthma [3,11,12]. Pig
farmers with an average exposure history of 10.5 years and
a daily exposure of 6.6 hours show significantly lower
forced expiratory volume in one second (FEV1) and forced
vital capacity (FVC) compared to unexposed control sub-
jects [3]. Interestingly, acutely exposed naïve volunteers,
show significantly more lung dysfunction, AHR, increase
in cytokine levels and inflammatory cell numbers in
blood and nasal lavage compared to the pig barn workers
repeatedly exposed to the barn air [7,13,11]. These data
suggest induction of an adaptive response in subjects
repeatedly exposed to the barn air.
There is paucity of data on in situ cellular and molecular
changes following multiple exposures to pig barn air. This
is largely because of lack of an animal model to investigate
the physiological impact of exposure to barn air. There-
fore, we decided to undertake an in vivo single and multi-
ple exposure study using rats to characterize cellular and
molecular responses. We hypothesized that single and
multiple exposures to swine barn air will induce lung
inflammation and a decline in lung function. The data
show that single and multiple exposures cause increase in
AHR, inflammatory cells in BALF, mucus cells in the air-
ways and lung inflammation.
Methods
Rats and treatment groups
The experimental protocols were approved by the Univer-
sity of Saskatchewan Campus Committee on Animal Care
and experiments were conducted according to the Cana-
dian Council on Animal Care Guidelines. Specific patho-
gen-free, six-week-old, male, Sprague-Dawley rats
(Charles River Laboratories, Canada) were maintained in
the animal care unit of Western College of Veterinary
Medicine. Rats were randomly divided into four groups (n
= 6 each). All personnel involved in collection and analy-
ses of samples were blinded to the treatment groups.
Exposure to swine barn air
We selected a regular commercial swine barn in the village
of Aberdeen in Saskatchewan. The barn chosen for study
had 60 dry sows and three boars. These pigs were fed with
ground barley. Rat cages were hung from the barn ceiling
at an approximate height of two meters above the floor.
Groups of rats were exposed to barn air either for eight-
hours for one-day, 5 days or for four cycles of 5 days (8
hours/day) each followed by 2 days in normal ambient air
after every cycle. When rats were not exposed to the barn
air, they were kept with the control animals in normal
ambient air. Control rats were treated similarly except that
they were not exposed to the barn air.
Barn air sampling for endotoxin analysis
We sampled the barn air twice weekly to determine endo-
toxin levels as described previously [14]. Briefly, we col-
lected airborne barn dust onto a pre-weighted, binder-free
glass fibre inline filter (SKC Edmonton, Canada) hung at
the level of rat cages. Barn air was drawn through the sam-
pler (DuPont Air Sampler) for eight hours on each sam-
pling day. The average flow-rate of the sampler was noted
before and after each sampling period. Filters were desic-
cated before and after sampling. After weighing, the filters
were placed in 50-mL polypropylene centrifuge tubes and
were stored at 4°C until endotoxin analysis.
Endotoxin analysis was performed as described elsewhere
[14]. Briefly, the filters with collected dust were washed
individually in centrifuge tubes with 10 mL of sterile pyro-
gen-free water (DIN 00624721; Astra Pharma Inc; Missis-
sauga, ON, Canada) followed by incubation for one-hour
at room temperature in a sonicating water bath. Serial
two-fold dilutions of the supernatant fluids were analyzed
for Gram-negative bacterial endotoxin using an end-point
assay kit as recommended by the manufacturer (model
QCL-1000; Cambrex Bioscience Inc.; Walkersville, MD).
The endotoxin standard (Escherichia coli O111:B4) was
used in duplicate at four concentrations (0.1 to 1.0 endo-
toxin units (EU)/mL) in each assay to generate the stand-
ard curve. The lower detection limit was 0.1 EU/mL,
which is equivalent to 1.0 EU per filter. The sampling time

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and flow rate were used to calculate the concentration of
endotoxin in air (EU/m3).
Viable microbial count
Viable microbial count was achieved using a six-stage via-
ble cascade impactor (Graseby, Smyrna, GA). Air samples
were collected from the vicinity of the rat cages hung from
the ceiling of the barn by using a vacuum pump that was
attached to the impactor capable of drawing air through
the impactor at a rate of 1 ft3/ min (28.3 L/min). Six media
plates of Tryptic Soy Agar with 5% sheep's blood were
placed in the sampler and airborne microbes were directly
collected onto 20 mL of media in 100 mm petri dishes.
The air was drawn through the impactor for a duration of
15 seconds. The procedure was performed twice every
week. The cascade impactor was cleaned thoroughly with
70% ethanol between each collection event. The plates
were incubated at 37°C for 18–24 hours, and the colonies
were counted using the positive-hole method correcting
for microbial coincidence [15].
Measurement of airway hyper-responsiveness
AHR was measured in awake control and exposed rats in
response to increasing concentrations of methacholine
(Mch) using head-out whole body plethysmography [16].
Air was supplied to the head and body compartments of
the plethysmograph through a small animal ventilator
(Kent Scientific, Litchfield, CT) and changes in respiratory
airflow were monitored using a flow sensor (TRS3300;
Kent Scientific, Litchfield, CT) linked via a preamplifier
and A/D board (Kent Scientific) to a computer-driven
real-time data acquisition/analysis system (DasyLab 5.5;
DasyTec USA, Amherst, NH). The compartment of the
plethysmograph, which accommodates the animal's
head, was connected to an ultrasonic nebuliser (UltraNeb
99; Devilbiss Co., Somerset, PA) to expose the rats to Mch
(Sigma Chemical Co. St. Louis, MO) [17,18]. Each rat was
sequentially exposed to aerosols of saline alone (Mch 0
mg/ml) and then increasing doses of Mch diluted in saline
(0.75, 1.5 and 3.0 mg/mL) and Flow@50%Tve1 (lung air-
flow at 50% of the expiratory tidal volume) was noted for
saline and each of the Mch concentrations.
Blood, bronchoalveolar lavage, tissue collection and
processing
At the end of the exposure period, rats were euthanized (1
mg xylazine and 10 mg ketamine / 100 g) and blood,
BALF and lung samples were collected. Blood was col-
lected by cardiac puncture for differential and total leuko-
cyte counts. BALF was collected by washing the whole
lung with 3 ml of ice cold Hanks Balanced Salt Solution
(Sigma Chemicals Co., St. Louis, MO). Three pieces from
each lung lobe (left and right) were fixed in 4% parafor-
maldehyde for 16 hours and embedded in paraffin for
light microscopy. Haematoxylin and eosin stained sec-
tions were used for histopathological evaluation of pul-
monary inflammation.
Quantification of mucus-producing cells
Mucus-producing goblet cells were quantified in lung sec-
tions stained with Periodic-acid Schiff (PAS) reagent [19].
Images were captured with the 20× objective lens of an
Olympus microscope (Olympus BH2) connected to a dig-
ital camera (DVC Digital Camera, Diagnostic Video Cam-
era Company, Austin, TX 78736-7735). The images were
analysed using image analyses software (Northern
Eclipse, version 6; Empix Imaging Inc., Mississauga, ON,
Canada). Only those bronchi with a length to width ratio
of less than 2.5 were selected for counting PAS-positive
cells so as to minimize the error that might arise from tan-
gential sectioning [20]. The PAS-positive goblet cells were
counted manually and normalized to the length of the
bronchial epithelial perimeter on the basal side, and
expressed as the number of PAS-positive cells per mm of
basement membrane.
Immunohistochemistry
Lung sections were processed for immunohistochemistry
as described previously [21]. Briefly, the sections were
deparaffinized, hydrated and incubated with 5% hydro-
gen peroxide for 30 minutes to quench endogenous per-
oxidase, treated with pepsin (2 mg/ml in 0.01 N HCl) for
45 minutes to unmask the antigens and blocked with 1%
bovine serum albumin for 30 minutes. Sections were
incubated with primary antibodies against rat macro-
phage (1:400; ED-1, Serotec Inc. NC, USA) or monoclonal
mouse anti-human smooth muscle actin (1:50; clone
1A4; DAKO A/S, Denmark), followed by appropriate
biotinylated or horseradish peroxidase (HRP)-conjugated
secondary antibodies (1:150; DAKO A/S, Denmark). Sec-
tions incubated with biotinylated antibodies were incu-
bated with HRP conjugated streptavidin (1:300, DAKO A/
S, Denmark) before color development. The reaction was
visualized using a color development kit (VECTOR-VIP,
Vector laboratories, USA). Controls consisted of staining
without primary antibody or with isotype matched immu-
noglobulin instead of primary antibody.
Quantification of macrophages and airway smooth muscle
ED-1 positive macrophages in the septa were counted in
20-high power fields (using 40× objective covering an
area of 9.6 mm2). For smooth muscle quantification, a
method described by Leigh et al. [22] was followed with a
slight modification. A line was drawn along the outer bor-
der of the positively stained smooth muscle area and total
stained area within that circle was measured using North-
ern Eclipse image analyses software. Next, a similar line
was drawn along the inner border of the airway smooth
muscle area to demarcate and measure the stained area.
Stained area within the line drawn along smooth muscle

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inner border was deducted from the stained area within
line drawn along smooth muscle outer border, to obtain
the total stained area of airway smooth muscle. This total
stained area of airway smooth muscle was normalized to
the length of the outer perimeter of the airway smooth
muscle, and results were expressed as, smooth muscle
stained area in mm2 per mm of airway smooth muscle
perimeter.
Statistical analyses
All data were expressed as mean ± SD. Group differences
were examined for significance using one-way analysis of
variance or two-way repeated measures analysis of vari-
ance with Fishers LSD as post hoc test (Sigma Stat Version
2.0, SPSS Inc., Chicago, IL 60611). Significance was estab-
lished at P < 0.05.
Results
Barn air characterization
The mean endotoxin concentration in the swine barn air
for the period of exposure was 15361.75 ± 7712.16 EU/
m3 of air. The amount of endotoxin in air samples from
the room where control animals were kept (normal ambi-
ent air) was below the level of detection. The levels of
endotoxin in the barn air in our study are much higher
than those reported by other researchers [23,3]. The total
viable aerobic bacterial counts in the barn air during the
exposure period are shown in Table 1. Air samples col-
lected from the room where control rats were kept did not
yield any bacterial colonies.
Airway hyper-responsiveness (AHR)
Inhalation of increasing concentrations of Mch caused
decrease in airflow (Flow@50%Tve1) indicating airway
reactivity and broncho-constriction. The data showed
group differences in percent decrease in Flow@50%Tve1
(Figure 1; P < 0.001). Both 1- and 5-day exposed rats
showed increased AHR compared to controls (P < 0.001)
and 20-day exposed (P < 0.05). However, there were no
differences in AHR between the control and 20-day
exposed (P = 0.207) and 5-day and 1-day (P = 0.249)
exposed rats.
BALF cell counts
There were differences in total leukocyte counts in BALF
among the four groups (Figure 2A; P < 0.001). The one
day exposure group had higher BALF total leukocytes
compared to the control, 5-day or 20-day exposed rats (P
< 0.001). The 20-day exposed animals contained higher
numbers of total leukocytes than control (P = 0.01) and
those exposed for 5 days (P = 0.008). BALF total leuko-
cytes were not different between control and 5-day
exposed rats (P = 0.932).
The increased BALF total leukocytes in single exposure
group, compared to control, 5- and 20-day exposed rats,
were characterised by increased absolute neutrophil, mac-
rophage and lymphocyte numbers (Figure 2B-D, P <
0.001). Increased BALF total leukocytes in 20-day exposed
rats were characterized by increased absolute neutrophil
(from controls, P = 0.022) and macrophage (control and
5-day exposed rats, P < 0.001) numbers. BALF absolute
eosinophil numbers did not differ among the four groups
(P = 0.178).
Blood cell counts
There was no difference among the groups for total leuko-
cyte counts (Figure 3A; P = 0.090). However, the absolute
neutrophil numbers were different among the four groups
(Figure 3B; P < 0.001). Rats exposed for 20 days showed
higher absolute neutrophil numbers compared to the
control and those exposed for 1 or 5 days (P < 0.001). Fur-
thermore, rats exposed for 1 day showed higher blood
absolute neutrophils when compared to 5-day exposed
rats (P = 0.038). Blood absolute monocyte numbers did
not differ among the four groups (Figure 3C; P = 0.122).
Blood absolute lymphocyte numbers were different
among the four groups (Figure 4D; P < 0.001). Compared
to 20-day exposed, control (P = 0.003), 1-day (P < 0.001)
and 5-day (P = 0.011) exposed rats showed increased
numbers of blood absolute lymphocytes.
Histopathology
Lung sections from control rats showed normal histology
(Figure 4A) while those exposed for 1 day, 5 (Figure 4B-C)
or 20 days (not shown) showed neutrophil infiltration
into the lung tissue. Lung sections from 5-day (Figure 4D)
and 20-day (not shown) exposed rats manifested perivas-
cular and peribronchial eosinophil infiltration. Bronchus-
associated lymphoid tissue (BALT) showed germinal cen-
tres and mitotic cells indicating BALT activation in rats
exposed for 20 days (Figure 4F) compared to the controls
(Figure 4E) or those subjected to 1 and 5 exposures (data
not shown).
Mucus cell quantification
Because PAS method stains mucus as pink, it is commonly
used as a method to identify mucus-containing cells
Table 1: The total, respirable and non-respirable aerobic viable
bacterial count (CFU/m3 of air sampled) from the barn air
ClassificationViable aerobic bacterial
count × 104 (CFU/m3 of
sampled air)*
Total
Respirable
Non-respirable
12.10 ± 8.47
4.85 ± 4.97
7.26 ± 7.50
* Viable bacterial counts are expressed as Mean ± SD.

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(Figure 5). Morphometric data revealed more PAS-posi-
tive mucus-containing goblet cells in the airways of rats
exposed for 5 or 20 days compared to the controls (5-day:
P = 0.040; 20-day: P < 0.001) and 1-day (5-day: P = 0.007;
20-day: P < 0.001) exposed rats (Figure 5A-D). Further-
more, rats exposed 20 times contained more airway
mucus cells compared to the 5-day exposure group (P <
0.001). There was no difference between control and 1-
day exposed rats (P = 0.435).
Quantification of ED-1 positive macrophages
The numbers of macrophages in the alveolar septa,
stained with ED-1 antibody were not different among the
four groups (Figure 6, P = 0.350).
Immunohistochemical quantification for smooth muscle
actin (SMA)
We used anti-human SMA antibody, which cross reacts
with rat tissue to stain smooth muscles around the
bronchi, bronchioles and blood vessels. Morphometric
analyses showed no differences in smooth muscle area
among the groups (Figure 7, P = 0.681).
Discussion
We report in vivo and in situ data using an animal model
on the effects of single and multiple exposures to the
swine barn air. The data show that exposures to swine
barn air induce an initial increase in AHR in one and five
day exposed rats followed by an adaptive response in 20-
day exposed rats; the 20-day group resembled the con-
trols. Swine barn exposure induced lung inflammation in
Airway hyper-responsiveness
Figure 1
Airway hyper-responsiveness. Airway hyperresponsiveness to methacholine challenge in rats was measured using a whole-
body head-out plathysmograph. Compared to controls, both 1-day and 5-day (P < 0.001) exposed rats showed increased air-
way hyperresponsiveness. Compared to 20-day exposed rats, 5-day (P = 0.001) and 1-day (P = 0.014) exposed rats showed
increased airway hyper-responsiveness There was no difference between control and 20-day exposed (P = 0.207) and 1-day
and 5-day exposed (P = 0.249) rats. *: Significantly different from other groups as indicated by line/s.