1. Association of chicken's immunological status with endotoxin level in husbandry indoor environment.
1Katharine Roque*, 1Ji Hoon Jo, 1Kyeong Min Shin,
1Gyeong Dong Lim, 3Hyoung Ah Kim, 2Yong Heo⌘
1Catholic University of Daegu, Graduate School, Department of Occupational Health
2Catholic University of Daegu, College of Bio-Medical Sciences, Dept. of Occupational Health
3Catholic University of Korea, College of Medicine Dept. of Preventive Medicine

2. Current situation of livestock production
Pig industry
Poultry industry
 2014 – China, 723 million heads
produced (Modern farmer)
Factory farms
2013 – BRF (Brazil), 1.88 billion heads produced (WATT Global Media)
Increased stocking density.
Animals are constantly exposed to bio-chemical hazards.
Animals have lowered resilience to diseases.

3. And it is heat resistant!!
Biological and chemical hazards in indoor husbandry environment
Endotoxin is the most common contaminant in dust particle
LPS
SO2
CO2
NO2
LPS CO
LPS
NH3
LPS
LPS
NH3
Bio-aerosol
NO2
LPS
It is so potent that even a very small amount is enough to non-specifically stimulate the immune system
And it is heat resistant!!

4. A major component of the outer membrane
What is endotoxin?
Virulence of O polysaccharide
1. Adhesion to tissues
2. Resistance to phagocytes
3. Carrier for toxic Lipid A
4. Protection from damaging reac tions with antibody and comple ment.
5. Antigenic variation
Virulence of Lipid A
1. Toxic
2. Mediates mainly the physiological activities of the LPS
Endotoxin structure
A major component of the outer membrane
of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria.

5. Impacts of endotoxin In human
Endotoxin exposure has been recognized as a causative agent or contributing factor for various pulmonary illnesses including asthma, organic dust toxic syndrome, or chronic obstructive pulmonary disease
(Liu AH, J Allergy Clin Immunol 109:379, 2002)
2. In animal in vivo and in vitro experiments a
Non-specific pathophysiological reactions, such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock and  death.
Up regulation of proinflammatory cytokine gene expression such as IL-1s, IL-6, and IL-8 was also observed from chicken heterophils in the presence of LPS

6. Objective
Only a few studies have been done for effects of endotoxin exposure on immunity of livestock wherein most studies were done in controlled experiments.
This study examines the association of the immunity of poultry, to dusts or endotoxin on the basis of its immunological responses to the mentioned environmental contaminants in their natural environment.

7. Materials and methods I.
Table 1. General husbandry characteristics of poultry farms investigated
Unit BS BE LK LG
Location
Yeoju,
Eumseong,
Chungbuk
province
Gangwha,
Incheon
metropolitan city
Gimpo,
Gyeonggi province
Temperature
33.06 ˚C
30.42˚C
26.81˚C
31.83˚C
Relative Humidity (%)
58.23
59.9
90.40
59.46
Chicken
house style
Open house
with winch curtains
with upper windows
Environmental
controlled
house without window
Broiler/laying
hen (breed)
Broiler
Laying hen
(Ross)
(Arbor Acres)
(Hy-Line Brown)
House space
(m2)
790
840
820
1,090
No.chicken
(head)
12,000
13,300
11,000
20,000
Stocking density
(m2/head)
0.066
0.063
0.075
0.055
Ventilation
Mechanical
fans
Tunnel
ventilation system

8. Materials and Methods
II. Particulate matter (PM10) collection inside poultry confinement buildings was done using a PVC membrane filter with 10 mm Dorr-Oliver nylon cyclone at flow rate of liters/min for 8 hours (NIOSH standard). Dust samplings were done from two different locations (1/3 and 2/3 distance from the exit) in each farm.
III. Endotoxin (EU/m3) evaluation from the dust was done by Limulus Amebocyte Lysate Kinetic QCL method and microplate spectrophotometer.
IV. Blood samples analysis : 3 ml/head (via wing vein)
Hematology – 0.5 ml/subject, (automated hematology analyzer)
Peripheral blood mononuclear cells (PBMC) – Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation (Ficoll-Paque Plus, GE Healthcare Life Sciences, Uppsala, Sweden).
Three-color flow cytometry (FACScan, BD, Franklin Lakes, NJ, USA) was used for analysis of peripheral lymphocyte subpopulation.
T cell activation assay – PBMC (106/ml) in complete RPMI medium were activated with 5 g concanavalin A for 72 hours in 41C, 5% CO2 incubator.
Enzyme-linked immunosorbent assay (ELISA) – IFN level in each culture supernatants, plasma cortisol and plasma IgY was determined by a sandwich ELISA method.

9. Results and Discussion
Table 2. Level of endotoxin in respirable dust collected from chicken farms* BS BE LK LG
Respirable dust
(mg/m3)
0.08±0.05
0.06±0.04
0.58±0.28
0.18±0.04
Endotoxin in
respirable dust
(EU/m3)
1.75±1.75
6.83±1.22
12.3±12.0
123.2±102.0
*Data are expressed as mean±SD.

10. Results and Discussion
Table 3. Red blood cell count among subjects BS BE LK LG
RBC (106/ul)*
2.05±0.05
2.1±0.05
2.07±0.03
2.2±0.054
Hemoglobin (g/dl)*
10±0.27
9.92±0.38
9.06±0.14
10.52±0.34
Hematocrit (%)*
23.76±0.52
23.56±0.58
23.28±0.23
25.75±0.63
Results are presented as mean±SD
*indicates P<0.05 significant difference between LK and LG

11. Results and Discussion
Table 4. Proportion (%) of lymphocyte subsets among peripheral blood
mononuclear cells of laying hens*
Farm
CD3+CD4+
CD3+CD8+
CD3+ TCRgd+
CD3-Ia+
T lymphocyte
B lymphocyte† LK
34.8±11.7
9.6±3.0
9.4±1.1
10.6±2.3 LG
34.6±4.4
8.2±2.4
11.7±3.3
5.7±0.9
*Data are expressed as mean±SEM. The number of chicken was 10 for each farm.
†Significant difference was assessed by Mann-Whitney Rank Sum Test (p<0.05).

12. Results and Discussion
Table 5. Levels of humoral or cellular immunity parameters for broiler chickens or laying hens*
Broilers
Layers BS BE LK LG
Plasma IgY
(mg/ml)
1.47±0.22
1.52±0.50
5.63±1.60
5.98±0.93
Interferon-g
(pg/ml)†
17.15±14.38‡
0.0±0.0
251.35±143.57
47.06±32.59
Plasma cortisol (ng/ml)
1.77±0.51
2.17±0.65
2.47±0.79
5.51±2.69
*Data are expressed as mean±SEM.
† Peripheral mononuclear cells (106) were stimulated with 5 mg concanavalin A at 41°C, 5% CO2 incubator for 72 hours and the culture supernatants were collected for interferon-g measurement.
‡Significant difference (p value) : BS vs. BE (0.024).

13. Summary ↑ Stocking density ↑ Endotoxin Negative relationship
↑ Plasma cortisol
↓ B cell
↓ Interferon-ƴ
Negative relationship

14. Conclusions
Chickens reared under higher stocking density and environmental endotoxin level could be susceptible to stress resulting in lowered resistance against various pathogenic insults including infection.

15. Thank you. Occupational and Environmental Toxicology Laboratory,
Dept. of Occupational Health, CUD
Park, Ji Hoon
Dr. Hyoung-Ah Kim