Abstract
This study aimed to investigate the prevalence of antibodies against pathogenic Yersinia such as Y. enterocolitica and Y. pseudotuberculosis in domestic pigs. A total of 650 serum samples from pigs in nine regions of the Chiba Prefecture in Japan, were tested using plasmid-encoded Yersinia outer membrane protein (Yops) antigen ELISA. The cutoff value was calculated using 20 pathogenic Yersinia-free pig serum samples. According to the cutoff value, 246 (37.8%) pigs from seven regions were considered seropositive for pathogenic Yersinia during the study period. These results indicate that pathogenic Yersinia is widespread in pigs in Chiba, which may become the source of human yersiniosis in this region.
Keywords: domestic pig, ELISA, Yersinia, Yersinia outer membrane protein
Pathogenic Yersinia, Yersinia enterocolitica and Y. pseudotuberculosis are important foodborne pathogens [2, 4]. Pathogenic Yersinia causes gastroenteritis, with clinical symptoms including abdominal pain, diarrhea, and fever. Recently, Y. enterocolitica serotype O8, which is highly pathogenic to humans, has spread not only in Japan [3, 10] but also in some European countries [13, 15,16,17]. Pigs are considered the most important natural reservoir of pathogenic Yersinia [1, 8]. Some researchers have reported that the presence of antibodies indicates that the animal was exposed to the pathogen at some stage in its life, even though seropositive animals no longer carry the pathogen [19]. Notably, Yersinia outermembrane proteins (Yops) antigen ELISA has been reported to be a more specific and novel method for the diagnosis of yersiniosis [7, 21]. Therefore, the detection of antibodies against pathogenic Yersinia is useful for determining the prevalence of these pathogens in pigs. However, there is no report on serological research on pathogenic Yersinia in pigs in Japan, although a few reports on Yops ELISA in pigs have been published overseas [20]. Therefore, we investigated the prevalence of pathogenic Yersinia antibodies in pigs kept in the Chiba Prefecture, Japan, using Yops antigen, which is commonly expressed by pathogenic Yersinia.
A total of 650 pig serum samples were collected at slaughterhouse from pig farms located in nine regions of the Chiba Prefecture from 2017−2021. The serum samples were collected from 6-months-old domestic pigs, stored at −20°C until use, and inactivated at 56°C for 30 min before use. Yops were prepared using a previously described method [12]. The ELISA was performed according to the method described by Iwata et al. [12]. Briefly, ELISA was performed in flat-bottomed 96-well microtiter plates (MaxiSorp; Nunc, Roskilde, Denmark). The plates were coated with 250 μg of Yops antigen/mL (50 μL/well) in phosphate buffer saline (pH 7.2) and incubated overnight at 4°C. The wells were then blocked with Diluent/Blocking Concentrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD, USA) at 25°C for 15 min. In each of the three wells assigned for individual serum samples, the wells were loaded with the sample (1:40 dilution in Wash solution; Kirkegaard and Perry Laboratories) and incubated at 37°C for 1 hr. The plates were then washed three times with Wash solution and incubated with peroxidase-conjugated Protein G (1:1,000 dilution in Diluent/Blocking Concentrate; Invitrogen Co., Carlsbad, CA, USA) at 25°C for 1 hr. After five washes, the plates were incubated with the ABTS (KPL) substrate for 10 min at 25°C, and the optical density (OD) was measured at 405 nm using a Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Additionally, 20 pig serum samples that showed negative agglutination to pathogenic Y. enterocolitica and Y. pseudotuberculosis serotypes were used as negative controls. Serum obtained from pigs experimentally infected with Y. enterocolitica O8 was used as the positive control. The cutoff value was calculated as the average OD of the negative serum sample plus three standard deviations (SD), as described by Iwata et al. [12]. The Yops antibodies were considered positive when the OD value was higher than the cutoff value. The cutoff value calculated in this study was 0.144. Statistical analyses were performed using the χ2 test and Fisher’s exact test.
Of the 650 pig serum samples tested, 246 (37.8%) samples were seropositive for pathogenic Yersinia (Fig. 1). The Yersinia seropositive pigs were detected in seven regions of the Chiba Prefecture ranging from 24.0–70.0% in each region (Table 1), with significant differences observed in the rate of seropositive pigs among the regions. These results indicate that pathogenic Yersinia are widely prevalent in the Chiba Prefecture. Additionally, Yersinia seropositive pigs were detected during all of the survey years, with positivity rates over the years ranging from 25.4–55.0% (Table 2). Notably, Yersinia seropositivity rates in 2020 and 2021 were relatively lower than those in other years; however, the reason for this is unclear. Vanantwerpen et al. [20] reported that 66% of 7,047 pigs in Belgium tested using the Yops ELISA were positive. Several countries, including Belgium, have implemented this strategy. However, to date, no seroepidemiological survey of pathogenic Yersinia in pigs has been performed in Japan. This is the first seroepidemiological study on pathogenic Yersinia in pigs in Japan, and our results indicate that domestic pigs may be a source of human yersiniosis in the Chiba Prefecture.
Fig. 1.
Analysis of serum obtained from 650 domestic pigs in Chiba Prefecture, Japan. The vertical dashed line represents the cutoff value, which was calculated as 0.144.
Table 1. Positive rates of serum antibody to Yersinia outer membrane protein in domestic pigs from 9 regions in Chiba prefecture, Japan by region.
| Regions (city or town) | No. of Yersinia positive samples/ No. of samples tested (%) |
No. of Yersinia positive pig farms/ No. of pig farms tested (%) |
|||
|---|---|---|---|---|---|
| North East | A | 81 / 265 | (30.6)a | 31 / 36 | (86.1) |
| B | 83 / 175 | (47.4)b | 18 / 24 | (75.0) | |
| C | 18 / 75 | (24.0) | 7 / 10 | (70.0) | |
| D | 38 / 70 | (54.3)c | 9 / 12 | (75.0) | |
| E | 0 / 10 | (0.0) | 0 / 1 | (0.0) | |
| F | 15 / 30 | (50.0)d | 3 / 3 | (100.0) | |
| G | 0 / 5 | (0.0) | 0 / 1 | (0.0) | |
| H | 7 / 10 | (70.0)e | 1 / 1 | (100.0) | |
| Subtotal | 242 / 640 | (37.8) | 69 / 88 | (78.4) | |
| Central | I | 4 / 10 | (40.0)f | 1 / 1 | (100.0) |
| 246 / 650 | (37.8) | 70 / 89 | (78.7) | ||
Samples from 1 location were used only for the calculation of cutoff values and is not included in this table. The χ2 test and Fisher’s exact test were used to calculate significant differences. a A greater than E (P<0.05). b B greater than A, C, E and G (P<0.05). c D greater than A, C, E and G (P<0.05). d F greater than A, C, E and G (P<0.05). e H greater than A, C and E (P<0.05). f I greater than E (P<0.05).
Table 2. Positive rates of serum antibodies to Yersinia outer membrane protein in domestic pigs in Chiba prefecture, Japan by year.
| Sampling Year | No. of Yersinia positive samples/ No. of samples tested (%) |
No. of Yersinia positive pig farms/ No. of pig farms tested (%) |
||
|---|---|---|---|---|
| 2017 | 44 / 80 | (55.0)a | 13 / 16 | (81.3) |
| 2018 | 32 / 80 | (40.0)b | 11 / 16 | (68.8) |
| 2019 | 52 / 80 | (65.0)c | 13 / 16 | (81.3) |
| 2020 | 61 / 240 | (25.4) | 16 / 23* | (69.6) |
| 2021 | 57 / 170 | (33.5) | 15 / 16* | (93.8) |
| Total | 246 / 650 | (37.8) | 70 / 89 | (78.7) |
The χ2 test and Fisher’s exact test were used to calculate significant differences. a 2017 greater than 2020 and 2021 (P<0.05). b 2018 greater than 2020 (P<0.05). c 2019 greater than 2018, 2020 and 2021 (P<0.05). *Some farms were sampled in duplicate.
Yops, which are used as antigens for ELISA, are encoded in a 70 kb plasmid of pathogenic Yersinia [6, 18]. Pathogenic Yersinia infections elicit specific antibody responses against Yops in humans and animals, regardless of the pathogenic Yersinia species or serovars [5, 11, 14]. Therefore, the domestic pigs that were Yops-positive in the present study were previously infected with pathogenic Yersinia.
In the present study, it was difficult to identify the species and serotype of pathogenic Yersinia even when the serum samples were Yersinia positive. Therefore, in future, we will also aim to isolate the pathogenic Yersinia from the fecal specimens of pigs brought to slaughterhouses in the Chiba Prefecture, Japan. Fukushima et al. [9] indicated that high percent of fattening pigs were infected with pathogenic Yersinia just after weaning, and were no longer detected before slaughterhouse shipments. In the present study, we only determined the presence of IgG against Yops produced by pathogenic Yersinia. To obtain information on the history of infection with pathogenic Yersinia, IgM antibodies against Yops in the same pig serum should be investigated.
CONFLICT OF INTEREST
The authors have no conflicts of interest directly relevant to the content of this article.
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