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

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.

- 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)

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

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.

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)

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

Sykes et al., 2011. Waste Management 31:423 Rylander. 1997. Int. J. Occup. Environ. Health 3 (suppl.):s32-s36

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) + 500 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

Results *

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).

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) 109.35 (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.

Results

Results

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.

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

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