Antibodies against Yersinia among farmers and slaughterhouse workers.

Antibodies to immunoglobulins (Ig) M, G, and A against Yersinia enterocolitica serotypes O:3, O:5, O:8, and O:9 and Yersinia pseudotuberculosis serotypes I and III were analyzed by enzyme immunoassay of the serum samples of 161 slaughterhouse workers, 147 pig farmers, and 114 grain or berry farmers. The crude risk ratios for elevated serum antibody concentrations were calculated with the use of the grain and berry farmers as the reference population. The risk for an elevated Y enterocolitica O:3 Ig G concentration was 3.0 (95% confidence interval (95% CI) 1.3-7.1) for the pig farmers and 1.8 (95% CI 0.7-4.4) for the slaughterhouse workers and the respective risks for elevated Y enterocolitica O:9 Ig G were 2.4 (95% CI 1.1-5.5) and 1.7 (95% CI 0.7-4.0). Since these two serotypes are commonly associated with swine, the increased number of subjects with elevated antibody levels could be causally related to occupational contact with this animal.

A comprehensive review published in 1988 lists 20 occupational zoonoses found among agricultural workers who breed animals. In addition, nine zoonoses were associated with workers in facilities which proces s animal products (I). Yersiniaspecies were not included in either group. In a recent Finnish study, slaughterhouse workers were observed to have elevated antibody levels against Y enterocolitica 0 :3 twice as frequently as blood donors, and it was concluded that yersinia infections are an occupational health risk to workers slaughtering swine in slaughterhouses (2). There is only one report in Russian concerning Yersinia spp and agricultural work, but no detailed figures are given in it (3). Y enterocolitica has been isolated from several different animal species, animal products, and a variety of inanimate reservoirs, including lakes, streams, vegetables, and soil (4). Swine is a recognized reservoir for human pathogenic Yenterocolitica in Europe and Japan, where serotypes 0 :3 and 0:9 have been isolated from the tongue, throat, tons ils, cecal contents, and feces of swine (5)(6)(7)(8).
The most common clinical manifestation of yersinosis is enterocolitis, which may mimic appendicitis. Focal extraintestinal infections and bacteremia are less common occurrences. Even if enterocolitis is generally seen in young children, the postinfection complications, including reactive arthritis and erythema nodosum, occur mainly in adults (4).
The documented transmission of Yenterocolitica to humans has occurred primarily through the ingestion of contaminated foods, water, and milk (4). The consumption of contaminated raw pork has been suspected to account for many sporadic cases of infection (4), but the documentation supporting this suspicion is scarce . In Belgium, the consumption of raw pork was found to be associated with Yenterocolitica infections (9) . Outbreaks of such infections have been caused by well or spring water (10, 11) and contaminated milk (12)(13)(14). There is no documentation of outbreaks associated with contaminated pork. Household preparation of chitterlings made of pig intestines caused an outbreak of 15 Yenterocolitica 0:3 infections in the United States (15). In Finland, outbreaks have been documented among nurses caring for patients with yersinia diarrhea and among patients in the same hospital wards (16,17), as well as among families of patients (18).
The documented prevalence of Yersinia isolation from swine is high in Denmark, where 80-1000/0 of swine is reported to harbor Y enterocolitica in their tonsils (19). The percentage of swine herds from which the isolation of Yenterocolitica succeeded from the ton sils in the southwest of Finland is 35 (20). The reported prevalences depend on the isolation methods employed (21,22).
The purpose of this study was to determine whether occupational groups in close contact with swine production or swine slaughtering could have an increased risk of elevated antibody content to the common pathogenic Y enterocolitica and Y pseudotuberculosis serotypes. In occupations which have close contact with swine (ie, among pig farmers and slaughterhouse workers) an elevated risk was found for a high anti-body content of Y enterocolitica 0:3 and 0 :9 in the immunoglobulin (lg) G class.

Subjects and methods
One hundred and seventy-two persons working on the kill floor of six swine-processing slaughterhouses situated in central and northern Finland were asked to participate. Blood samples were obtained from 161 (940/0) of these persons.
The pig farmers studied came from a smaller geographic area, the Kuopio district, in the eastern part of Finland . The pig farmers were chosen from the register of the local cooperative meat-proces sing company, which covers virtually all the swine producers in the area . Those farmers involved in milk or beef production were excluded, in addition to tho se who were over 65 years of age. One hundred and fortyseven (71%) pig farmers, out of the 206 invited, participated in the blood sampling.
The farmers comprising the reference population lived in the same area, but their main line of production was either grain or berries with no pork, beef, or milk production. They were identified from the register of the local agricultural advisory association, which covers about 60-80% of the farmers in the district. One hundred and fourteen (70%) blood samples were obtained from the 162 farmers of the reference population invited to take part.
Only one serum sample per farm was taken. All of the farms were run by owner families, and, in each case, the family member who spent the most time tending the swine, grain , or berries was asked to take part in the sampling.
Finally, 200 blood donor sample s from the same geographic area as the farmers (the Kuopio district) were obtained with which the cutoff poin t for the antibody positivity was determined. No other information regarding the donors (ie, age, gender) was known.
In general, the slaughterhouse workers were somewhat younger than the farmers. However, within the 35-to 55-year-old age bracket, which covered the majority of the subject s, there were no marked differences between the groups (table 1). There were 33 (22%) women among the pig farmers, 17 (11%) among the slaughterhouse workers, and 14 (12%) among the grain and berr y farmer s. Almost half (48%) of the slaughterhouse workers were smokers at the time of the blood sampling in comparison with 240/0 of the pig farmers and 17% of the grain and berry farmers.
The Ig M, Ig G, and Ig A antibodies to Y'enterocolitica 0 :3, 0:5, 0 :8, and 0 :9 and Y pseudotuberculosis I and III were measured by enzyme immunoassay (23). The preparation of sodium dodecyl sulfate extracts of whole Yersinia bacteria to be used as antigen has been described elsewhere (24). Pol ystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with bacterial antigens in pho sphate buffered saline (PBS) (0.1 mol/I, pH 7.5, 100 Ill/well) . The plates were saturated with 1% normal sheep serum in PBS (100 Ill/well) .
Patient serum samples at a dilution of 1:250 were incubated on the plates for 2 h at 37°C. Thereafter, For each well 75 III of swine antihuman Ig M , Ig G, or Ig A conjugated with alkaline phosphatase (Orion Diagnosti ca, Espoo, Finland), diluted 1:250, 1:300, and 1:250, respectively, was incubated on the plates overnight at room temperature. Freshly prepared p-nitrophenol phosphate in a buffer solution of diethanolamine and magnesium chloride (1 mg/ml; Orion Diagnostica) was added and incubated for 30 min at 37°C. The reaction was stopped with 1 M sodium hydro xide. The opt ical densit y was measured with a Titertek Multiscan Photometer (Labsystems, Helsinki, Finland) at a wavelength of 405 nm. The results were given in enzyme immunoassay units (EIU) compared with a strongly positive reference serum: 1 U was 1/100 of the corresponding antibody concentration in the reference serum. The samples were tested as duplicates, and the results were expressed as mean values. The serum samples were analyzed in three series, each containing blood donor samples, but otherwise the analyzing laborator y did not know from which study group the samples originated.
The cutoff point for the antibody positivity of different serotypes in the three Ig classes was determined from the results of the 200 blood donor samples. The cutoff point was chosen to be the mean of all the blood donor samples plus two standard deviations of the mean .
Analyzing four serotypes of Yenterocolitica and two serot ypes of Y pseudotuberculosis in three Ig classes yielded 18 different sets of results. Possible crossreactions were investigated from a correlation matrix of results expressed in EIU. In the same way, possible cross-positi vities were looked for in the cross-tabulation of the positive values of different analyses.
No attempt was made to standardize the crude risk ratio s. The relat ive risk (or risk ratio) of Yersinia antibody positivity was calculated with its 95% confidence interval (95% CI) (25).   The risk ra tios comparing pig farmers or slaughterhouse workers to the farm ers of the reference population rema ined below two for the other anti body groups, as the dat a in tabl e 2 suggest.
The correlations of antibod y values expressed in BID for the three antibodies, when the risk ratio s exceeded two, were rath er low, as seen in table 3. Table 4 shows the numbers of observed and expected jo intly positives of the same serotypes.

Results
The most remarkable differences between the groups were found for the Ig G class antibodies against Yenterocolitica serotypes 0 :3 and 0 :9 and for Ig M class antibod ies against Yenterocolitica 0 :5 (table 2). The risk ratio of positive Y enterocolitica 0 :3 Ig G antibodie s was 3.0 (95 % CI 1.3-7.1) for the pig farmer s Discussion Y enterocolitica 0 :3 and 0 :9 are the two sero types associated with swine (4). The workers in occupations which involved close contact with swine had more frequently elevated Ig G class ant ibodies against these serotypes. The Ig G class is the type which stays elevated for the greatest length of time after yersinosis with no postinfectious arthritis (26). In the case of Yersinia-triggered reactive arthritis both Ig A and, to a less extent, Ig G stay elevated (26). In a previous Finnish study, II (50%) of 22 enteritis patients who did not suffer from arthritis and 28 (720/0) of 39 subjects with reactive arthritis were found to have Ig G class antibodies against Yenterocolitica 0:3 at 12-16 months after the onset of the infection. For the Ig A class antibodies the respective results were 7 (32%) for the nonarthritis cases and 33 (85%) for the arthritis subjects (27). However, it is not known how long Ig G antibodies persist after entirely asymptomatic enteric infection.
Although the number of subjects in this study was relatively limited, the 95 % confidence interval of the risk ratios of Yenterocolitica 0:3 Ig G and 0:9 Ig G positivity were above unity for the pig farmers . For the slaughterhouse workers, the risk ratios might have been biased because the slaughterhouse workers had a more urban way of living than the rural reference population. Thus the risk of positivity could have been underestimated for the slaughterhouse workers.
The increased risk of positivity of Yenterocolitica 0:5 Ig M class observed among the pig farmers and the slaughterhouse workers might be a result of the marked cross-positivity between Y enterocolitica 0:5 Ig M with both Y enterocolitica 0:3 Ig G and Y enterocolitica 0 :9 Ig G, as seen in table 4. On the other hand, the correlations of the antibody concentration expressed in EIU were rather low between these groups, and therefore suggest that the cross-positivity was not primarily due to cross-reactivity in the analysis, but rather caused by some external factor which is a common determinant of the three Y enterocolitica serotypes. However, when the wide confidence intervals are taken into account, the elevated risk ratio of Ig M class antibodies against Y enterocolitica 0:5 might be a product of chance.
The results of this study are somewhat similar to the findings of Merilahti-Palo et al (2) in a previous study of butchers living in the southwest of Finland. In that study, slaughterhouse workers were found to have an increased number of antibodies against Yenterocolitica 0 :3, but not against the other serotypes tested, namely, Y enterocolitica 0:9 and Y pseudotuberculosis IA and III (2). Even if the method of selecting the cutoff value between elevated and "normal" antibody concentration was similar to ours, there were some differences in the study design and analysis. In the design, we used blood donors only for the evaluation of the cutoff value. Merilahti-Palo et al used blood donors as the reference group. In our study the comparability of effects and populations (28) was more valid for the comparison of pig farmers with grain or berry farmers, but, as has already been discussed, the comparison with slaughterhouse workers with farmers may be biased.
In the analysis, Merilahti-Palo et al considered a subject to have antibodies against Yersinia if he or she was found to be positive in anyone of the three Ig classes. For Yenteroco/itica 0:3 the greatest difference between the slaughterhouse workers and the blood donors was in the Ig A class (9% positives among the slaughterhouse workers versus 3% among the blood donors). For the Ig G class the respective percentages were 9 and 6 (2). Expressed in terms of the risk ratio, this finding means 1.5, which is about the same as we observed, namely, 1.8.
Our results show that occupational contact with swine is associated with an increased number of workers having elevated antibody concentrations against those Y enterocolitica serotypes found in swine. This association was found to be more obvious among pig farmers than among slaughterhouse workers. Whether diseases caused by these serotypes of Yenterocolitica are more prevalent in occupations involving contact with swine needs to be studied.