1. ENDOTOXIN-MEDIATED IMMUNE ALTERATIONS IN PIG HUSBANDRY WORKERS IN KOREA
Katharine Roque1, Kyung Min Shin1, Ji Hoon Jo1, Jae Hee Lee1,
Hyoung Ah Kim2, Yong Heo1,3
1 School of Graduate Studies, Dept. of Occupational Health, Catholic University of Daegu, Republic of Korea
2 Dept. of Preventive Medicine, The Catholic University of Korea
3Dept. of Occupational Health, Catholic University of Daegu, Republic of Korea

2. Introduction Occupational hazards to animal husbandry workers
Chemical hazards :
- Acute and chronic dermal, ocular and respiratory diseases from exposure to
ammonia, H2S, CO, CO2, SO2, Nox
- Dermatitis and respiratory tract irritation by detergents, disinfecting agents
(formaldehyde)
Biological hazards :
- Primarily organic dusts from feathers, dander, feed and bedding,
hay and grain, microorganisms (endotoxin)
- Immunologically mediated diseases :organic dust toxicity syndrome,
occupational asthma, chronic obstructive pulmonary disease, allergic alveolitis,
hypersensitivity pneumonitis, farmer’s lung disease, dermatitis.
- Recently, endotoxin-mediated occurrence of respiratory allergic diseases is actively investigated in European countries and the Unites States.
But not much studies were performed in Korea.

3. - heat resistant suggesting persistence in the environment
Potency of endotoxin :
- Very small amount enough to non specifically stimulate immune systems
- heat resistant suggesting persistence in the environment
Endotoxin structure
(adopted from Liu AH, J Allergy Clin Immunol 109:379, 2002)

4. (Liu AH, J Allergy Clin Immunol 109:379, 2002)

5. Objective
To evaluate the possible relationship of immune health status of the pig husbandry workers in Korea with endotoxin concentration extracted from dusts collected inside swine building.
To attract attention to importance of endotoxin exposure in indoor occupational or environmental settings for health outcomes including respiratory allergic disease.

6. Materials and Methods I. A. Study subjects B. Serum analysis
Table 1. Mean age and work duration (husbandry workers) of study subjects at same residential area in Gyeonggi Province (I-cheon)
Group
No. of subjects
Mean±SD
Age
Work duration (years)
Swine husbandry worker (2001) 25
47.5 ± 9.2
13.7 ± 7.9
Swine husbandry worker (2012) 12
40.7 ± 7.7
15.4 ± 8.5
Control subjects (2001) 7
39.4 ± 13.2
B. Serum analysis
Serum IgE, a typical marker of allergic response was measured using ELISA kit (IBL, Germany).
Allergen-specific IgE – RIDA Allergy Screen Panel. (positive reaction: ≥0.7 EU/ml)

7. Materials and Methods II. A. Study subjects
Table 2. Demographic characteristics of the selected 10 swine husbandry workers from 2012 that were further evaluated for cytokine production and differential cell counts
Group
No. of subjects
Mean±SD
Age (year)
Swine husbandry work duration (year)
Daily work hour in swine husbandry indoors
High endotoxin exposure (> 20 EU/m3) 5
44.2 ± 12.4
16.8 ± 8.3
5.2 ± 3.3
Low endotoxin exposure (≤ 20 EU/m3)
40.8 ± 8.2
13.5 ± 8.2
7.8 ± 4.5

8. Sykes et al., 2011. Waste Management 31:423
Rylander Int. J. Occup. Environ. Health 3 (suppl.):s32-s36

9. Materials and Methods
B. Particulate matter collection inside pig confinement buildings was done using a PVC membrane filter and mini volume air sampler (NIOSH standard).
C. Endotoxin (EU/m3) evaluation from the dust was done by Limulus Amebocyte Lysate Kinetic QCL method
D. Blood samples analysis : 10 ml/subject (2012)
Hematology – 1 ml/subject, (automated hematology analyzer)
Peripheral blood mononuclear cells (PBMC) – 5 ml whole blood, Ficoll density gradient centrifugation resuspended in complete RPMI.
T cell activation assay – 1x106 PBMC in 5 ng phorbol 12-myristate 13-acetate (PMA) ng ionomycin for 48 hrs in 5% CO2 incubator
Enzyme-linked immunosorbent assay (ELISA) – interleukin-4,13 and 12; Interferon-ɣ and Tumor necrosis factor-α e

10. Results *

11. Results
Table 3. Number and percentage of study subjects positive* to each respiratory allergen
Allergen
Swine husbandry worker (2001)
Swine husbandry worker (2012)
Control subjects (2001)
No. (%)
Golden rod
14 (56)
0 (0)
1 (14)
Pigweed
3 (43)
Russian thistle
13 (52)
3 (25)
4 (57)
Dandelion
10 (40)
2 (29)
Mugwort
8 (32)
1 (8)
Ragweed
Alternaria alternata
6 (24)
2 (16)
Aspergillus fumigatus
2 (8)
Cladosporium herbarum
4 (16)
Penicillium notatum
5 (20)
Cat
1 (4)
Dog
3 (12)
Cockroach
11 (44)
4 (33)
Housedust
Dermatophagoides farinae
9 (36)
5 (42)
6 (86)
Dermatophagoides pteronyssinus
12 (48)
Sweet vernal grass
Reed
Pine
Ox-eye-daisy
Total
25 (100)
12 (100)
7 (100)
Distribution of allergen positive subjects among the 2001 and 2012 swine husbandry workers, and the control subjects was statistically significant (p=0.025, x²=56.973, d.f.=38) by x² test. *Positive reaction to each allergen: ≥0.7 EU/ml (class 2).

12. Results
Table 4. Level of endotoxin in total or respirable dust collected from swine farms at 2012
GM (GSD)*
Statistical significance (p value)
High endotoxin exposure group
Low endotoxin exposure group
Total dust (mg/m3)
0.53 (2.88)
0.21 (13.50)
Respirable dust (mg/m3)
0.73 (1.23)
0.24 (9.04)
Endotoxin in total dust (EU/m3)
(4.75)
0.95 (8.04)
0.008
Endotoxin in respirable dust (EU/m3)
1.20 (4.19)
0.24 (2.01)
0.036
*GM: geometric, GSD: geometric standard deviation.

13. Results

14. Results

15. Summary
Type I or Immediate hypersensitivity allergic reaction could occur among swine workers exposed to indoor allergens based on the significantly high levels of serum IgE.
House dust mites (Dermatophagoides pteronysinnus, D. farinae) and cockroach are the major indoor allergens inside swine barns especially for 2012 subjects.
Exposure to high levels of endotoxin could be modulated towards type-2 immune responses among swine husbandry workers leading to allergic reactions as indicated by lower IFNƴ:IL4 ratio plus high level of interleukins 4 and 13.

16. Inhalation in indoor environment settings
Working hypothesis
Th1-type immune development
Cellular immunity
Autoimmunity
IL2
IFNɣ
TNF
APC
Dust (ENDOTOXIN)
Inhalation in indoor environment settings
Naïve T cell
IL5
IL4
IL13 * **
Eosinophils
Th2-type immune development
IgG1, IgE
Activated T cell
Humoral immunity
Asthma, allergy

17. Thank you. Occupational and Environmental Toxicology Laboratory,
Dept. of Occupational Health, CUD