Prevalence of Farm and Slaughterhouse Workers Carrying Shiga Toxin-Producing Escherichia coli in Korea.

OBJECTIVES: The aim of this study was to investigate the distribution of Shiga toxin (Stx) gene-positive stool samples from dairy farmer and slaughterhouse workers in Gyeonggi-Do province.
METHODS: A total of 621 samples from healthy farmers and 198 samples from slaughterhouse workers were screened by polymerase chain reaction (PCR) for Shiga toxigenic Escherichia coli (STEC) infection on stool samples.
RESULTS: The PCR product of Stx-encoding genes was detected in 21 (3.4%) of 621 farmers and 15 (7.6%) of 198 slaughterhouse workers' stool samples. Distribution of the Stx PCR positive workers by age increment revealed an increase in STEC infection with age increment in both workers. Distribution of the Stx PCR positive workers by working years revealed an increase in STEC infection with working years in farmers.
CONCLUSION: These results of the study show that slaughterhouse workers are at higher risk of STEC infection than farmers. In addition, slaughterhouse workers have a more potential source of food contamination of STEC and transmission.

1. Introduction

Enterohemorrhagic Escherichia coli (EHEC) comprises a subset of serotypes of Shiga toxin (Stx)-producing E coli (STEC) that has been associated with bloody diarrhoea and hemolytic uremic syndrome (HUS) in industrialized countries [1,2]. The majority of the EHEC infections worldwide are caused by strains of serotype O157:H7 [3,4].

Stxs and Vero toxins (VTs) are considered to be the major virulence factors of EHEC and comprise a family of structurally related cytotoxins with similar biological activity. The two main groups consist of Stx1 and Stx2. Stx1 is nearly identical to the toxin of Shigella dysenteriae type 1 and Stx2, which shares less than 60% amino acid sequence with Stx1 [5]. Whereas Stx1 exhibits only slight sequence variations, several variants of Stx2 with altered antigenic or biologic characteristics have been described [6,7].

Most of the reports have been concerned mainly with the associations between the virulence factors of STEC, disease, and transmission [4,8]. In Korea and elsewhere,cattle are considered to be a main reservoir of STEC. In national surveillance programs, the incidence of STEC-related diseases and the isolation rates of STEC have been reported, but there have been no reports concerned of STEC surveillance in dairy farmers and slaughterhouse workers in same region. Gyeonggi-Do province is nearest dairy farm region to Seoul, the capital city of Korea. Dairy farm and Slaughterhouse in Gyeonggi-Do province shared 41.0% dairy farm (3143/7657) and 12.8% Slaughterhouse (10/78) in Korea [9].

Dairy farmers are in direct contact with dairy cattle nearly every day and have an increased possibility of becoming infected with STEC. Also, slaughterhouse workers contact cattle every day and have high a possibility of STEC infection. Therefore, the purpose of this study was to assess the prevalence of STEC both dairy farmers and slaughterhouse workers to evaluate the relationship between STEC infection and work fields as well as to determine STEC infection as a potential source of food contamination and transmission.

2. Materials and Methods

2.1. Stool sample collection

Fecal samples of dairy farmers in November 2008 through April 2009 and 621 samples were collected. For the slaughterhouse workers, a total of 198 fecal samples from October 2007 to April 2008 were collected. We divided the slaughterhouse workers into four categories: inspector, slaughterer, residual products handler, and livestock hygiene controller.

2.2. DNA manipulations and genetic techniques

A loopful of human stool sample was directly inoculated into 3 ml of Tryptic Soy Broth (Oxoid, Basingstoke, Hampshire, United Kingdom) for enrichment and incubated overnight at 37℃ with shaking. After incubation, the enriched broth culture was used to isolate chromosomal DNA. Chromosomal DNA was purified using the GenomicPrep Cell and Tissue DNA isolation kit (Amersham Biosciences, Netherlands). All polymerace chain reactions (PCRs) were performed with the Expanded High Fidelity Polymerase System (Roche) or Taq polymerase (Takara, Japan) according to the manufacturer’s instructions. The primers used for amplification are listed in Supplemental Table 1.

Table 1.

Distribution of stool samples showing Stx positive by polymerase chain reaction compared with work fields and sex

Sex	Farmer		Slaughterer
	Positive(%)*	Negative(%)	Positive(%)**	Negative(%)
Male	3.8	61.3	7.7	83.6
Female	2.7	35.2	0.5	16.4
Total	3.4	96.6	7.6	92.4

*p = 0.641.**p < 0.05 by Chi-square trend test for analysis.

2.3. Statistical analysis

The data collected were evaluated using the SPSS 14.0 statistical package. The distribution of stx PCR positive samples from workers by the field of work, age and working year was analyzed using the Chi-square test.

3. Results

3.1. Screening for Stx-encoding genes

Of the 621 farmers who underwent stool examination results related to EHEC infection by PCR examination, 21 patients (3.4%) were positive. Of the 198 slaughterhouse workers, 15 workers (7.6%) were positive (Table 1). In an EHEC infection survey by sex-related test in farmers, the stx PCR test was positive in 15 out of 396 male farmers (3.8%), and 6 people out of the 225 female farmers (2.7%).

In slaughterhouse workers, 198 stool samples were tested; 14 male workers (7.7%) were positive and only 1 woman out of 31 (0.5%) was positive.

3.2. Distribution of age of Stx positive workers

We divided the age of workers into five categories: below 30, 30 to 39, 40 to 49, 50 to 59, and over 60 years, and surveyed the age distribution of Stx positive workers. Results of farmers revealed that within the groups 40 years or older–‘40 to 49’ and ‘50 to 59’–they shared the rate of 1.0% and 4.9% positive and the ‘over 60’ group showed the highest rate of 5.8%, and the difference among age classes was significant based on the Chi-square test (p = 0.01; Table 2). In slaughterhouse workers in the ‘40 to 49’ and ‘50 to 59’ groups, they shared the rate of 2.5% and 3.0% and the ‘over 60’ group showed a rate of 2.0% with a Chi-square test of 0.118. Thus, the results showed that as the worker age increased, the percentage of Stx positivity increased in the farmers.

Table 2.

Distribution of stool samples showing Stx positive by polymerase chain reaction compared with age

Age (yrs)	Farmer		Slaughterer
	Positive*(%)	Negative(%)	Positive**(%)	Negative(%)
≤29	0.0	2.3	0.0	1.5
30-39	0.0	4.9	0.0	14.1
40-49	1.0	32.8	2.5	27.8
50-59	4.9	43.4	3.0	32.3
≥60	5.8	13.0	2.0	15.2
Total#	3.4	96.6	7.6	92.4

. *p = 0.01.**p = 0.118 by Chi-square trend test for analysis.

3.3. Distribution of working year of Stx positive workers

We also surveyed the distribution pattern of the working year of Stx positive workers on the basis of

a questionnaire. The results in farmers indicated that the rate of Stx positivity at ‘over 30’ years of work was 5.6% and was the highest value. The percentage values of Stx positivity increase with years of work (p = 0.052; Table 3). In slaughterhouse workers, the rate of Stx positivity at ‘below 10’ years of work was 3.0% and was the highest value (p = 0.235).

Table 3.

Distribution of stool samples showing Stx positive by polymerase chain reaction compared with working years

Working year	Farmer		Slaughterer
	Positive*(%)	Negative(%)	Positive**(%)	Negative(%)
≤10	1.4	11.7	3.0	39.4
10-19	1.2	27.5	1.0	15.7
20-29	4.8	44.3	1.5	12.1
≥30	5.6	10.8	0.5	26.8
Total#	3.5	96.5	6.1	93.9

*p = 0.052.**p = 0.235 by Chi-square trend test for analysis.

3.4. Distribution of workfields of Stx positive workers in slaughterhouse workers

We divided the slaughterhouse workers into four categories: inspector, slaughterer, residual products handler, and livestock hygiene controller (Table 4).

Table 4.

Distribution of stool samples showing Stx positive by polymerase chain reaction compared with slaughterhouse work fields

Work fields	Total	Positive	%*
Slaughterer	113	10	8.9
Livestock hygiene controller	22	1	4.5
Residual products handlers	63	4	6.3
Total	198	15	7.6

*p = 0.046 by Chi-square trend test for analysis.

The prevalence of Stx was found to be 6.3% (4/63) in residual products handlers, 8.9% (10/113) in slaughterers, and 4.5% (1/22) in livestock hygiene controllers. In this study, the prevalence of Stx in dairy farmers was 3.4% (21/621), which is lower than in the slaughterers but similar to that in the livestock hygiene controllers.

4. Discussion

Cattle are considered to be the main reservoir of STEC [10-12]. Recently, in a study of the prevalence

study of STEC O157 in Swedish dairy herds, a rate of 8.9% STEC O157 was found [13,14]. In France, the prevalence of STEC was found to be 34.9% [15]. In India, the prevalence of STEC in cows and calves was found to be 18.9% and in dairy cattle was 32.4% [16]. Furthermore, in a study of Canadian dairy farm families, approximately 6% of the individuals were STEC carriers [17-19]. And in a follow-up study, the prevalence of Stx in dairy farmers was 5.3% (3/57) [20].

Among meat-processing company employees, the prevalence of STEC was as high as 9% [21,22]. Also, our previous survey results showed that 5.6% were STEC carriers in slaughterhouse workers [23]. Based on these reports, there is a distinct difference in the prevalence of STEC for dairy cattle by country, thus the altered trend in the prevalence of STEC for dairy farmers and slaughterhouse workers may have originated from different countries and different seasons. The majority of the reported human cases of STEC infection in the United Kingdom have been shown to occur in the warmer season [24]. This study was conducted in the colder season (Dairy farmer: November to April; slaughterhouse workers: October to April), thus the prevalence of STEC could have been influenced.

In this study, Stx PCR screening results of the dairy farmer was 3.4% and slaughterhouse workers was 7.6%. Stx PCR positive of slaughterhouse workers are more than two times higher than dairy farmers represented that slaughterhouse workers in EHEC are exposed to more than double that indicated. Among the three work fields in the slaughterhouse workers, the slaughterer showed the highest value and livestock hygiene controllers (3.4%) showed values near that of the dairy farmer. And, in dairy farmers, with STEC infection, age, working year are showed trend, as age and working years increased, the proportion of stx-positive farmers. Therefore, as increase as the contact with cattle, increase the risk of STEC infection in farmer and slaughterhouse workers.

All stx PCR positive workers had no symptoms of EHEC infection. The lack of disease associated with STEC infection in work field workers is interesting and may reflect protection associated with immunity induced by previous exposure. The role of asymptomatic human carriers as a source of contamination and, thus, the importance of personal hygiene measures in dairy farm and in the meat-processing slaughterhouse should not be underestimated. We hope that there will be increased attention to the STEC infection from cattle to diminish the risks of infection and transmission. These results of the study show that slaughterhouse workers are at higher risk of STEC infection than farmers. In addition, slaughterhouse workers are a more potential source of food contamination and transmission.

Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phrp.2011.11.045