Serological Prevalence of Enteropathogenic Yersinia spp. in Pigs and Wild Boars from Different Production Systems in the Moravian Region,Czech Republic

Abstract

Human yersiniosis caused by pathogenic Yersinia spp. is one of the most common reported zoonoses in the European Union and pigs are considered as the major reservoir of these bacteria. Serological testing represents a suitable method to obtain information about the prevalence of enteropathogenic Yersinia spp. in food animals. The prevalence of antibodies against enteropathogenic Yersinia spp. was studied in 319 slaughtered pigs and 135 wild boars from different production systems in the Moravian region (Czech Republic) using a commercially available ELISA test (an apparent prevalence). The seroprevalence was significantly associated with the type of breeding system, with the lowest seroprevalence being observed in household-raised pigs (13/29, 44.8%). No significant difference between the prevalence of anti-Yersinia antibodies in conventional (146/180, 81.1%) and organic pigs (92/110, 83.6%) was found. Antibodies were found in 65.9% (89/135) of wild boars without a significant difference between adult (23/41, 56.1%) and young (66/94, 70.2%) animals. Seropositivity was significantly higher in domestic (251/319, 78.7% in total) compared to feral pigs. A Bayesian approach taking into account the sensitivity and specificity of the ELISA test was used to estimate the true prevalence of anti-Yersinia antibodies in pigs and wild boars. According to our results, domestic pigs and wild boars proved to be an important reservoir of enteropathogenic Yersinia in the Czech Republic. Attention should be paid to good hygienic practice during slaughtering and handling of meat to prevent meat contamination and subsequently human infection.

Introduction

Yersiniosis is a gastrointestinal infection in humans most often caused by Yersinia enterocolitica, which is primarily transmitted by food such as pork products, and to a lesser extent by Y. pseudotuberculosis often in conjunction with contaminated water and vegetables (Galindo et al., 2011; EFSA and ECDC, 2015). In the European Union, yersiniosis was the third most frequently reported zoonosis in 2013 and Y. enterocolitica was as causative agent isolated from almost 99% of the confirmed cases (EFSA and ECDC, 2015). Infection in humans is usually characterized by gastroenteritis and mesenteric lymphadenitis with diarrhea, vomiting, mild fever, and abdominal pain. Postinfection complications such as reactive arthritis or erythema nodosum, and in rare cases septicemia, can also emerge (Galindo et al., 2011).

Pigs are considered as the most important reservoir of human pathogenic Y. enterocolitica, but both enteropathogenic yersiniae have been found in domestic and feral pigs (Fredriksson-Ahomaa et al., 2009; Novoslavskij et al., 2013; Sannö et al., 2014). Pigs are mostly asymptomatic carriers with the presence of bacteria in the tonsils and intestinal contents (Nesbakken et al., 2006; Laukkanen et al., 2008; Van Damme et al., 2014), and infected pigs cannot be distinguished using ante- and postmortem meat inspection practices. During slaughter procedures, bacteria can contaminate the carcass and, moreover, cross-contamination between carcasses can occur (Nesbakken et al., 2003; Laukkanen et al., 2009). The European Food Safety Authority (EFSA) considers Y. enterocolitica as one of the most relevant biological hazards in the context of meat inspection of swine (EFSA, 2011).

The wild boar population has increased in Europe, including in the Czech Republic (Anonymous, 2015) and the demand for game meat is also currently increasing. Wild boars represent a reservoir of many pathogenic agents, including Yersinia for domestic animals and for humans (Wacheck et al., 2010; Sannö et al., 2014). The enduring popularity of outdoor farming of domestic pigs may raise the risk of contact between wild and domestic animals and the risk of pathogen transmission.

Bacteriological examination of tonsils is the best indicator of the presence of pathogenic Yersinia in pigs (Nesbakken et al., 2006; Fredriksson-Ahomaa et al., 2009; Van Damme et al., 2014). However, serological testing, which holds the advantage of being more convenient and faster, is another possibility for diagnosis of subclinical zoonoses in food animals with regard to food safety and public health aspects (Meemken et al., 2014; Felin et al., 2015). Virulence of pathogenic Yersinia spp. (Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis-not found in Europe) is associated with the virulence plasmid pYV encoding yersinia outer proteins (Yops), which are immunogenic in animals and humans. Their presence indicates active infection involving bacterial replication (Robins-Browne et al., 1993; Galindo et al., 2011). The detection of antibodies against Yops was previously used to get information about the prevalence of pathogenic Yersinia in domestic and feral pigs (Fredriksson-Ahomaa et al., 2009; von Altrock et al., 2011; Vanantwerpen et al., 2014).

In the Czech Republic, the number of human yersiniosis infections has shown a slightly increasing trend in recent years (dr. Cestmir Benes, The National Institute of Public Health, Czech Republic, personal communication). To the best of our knowledge, there is a lack of data regarding the prevalence of enteropathogenic Yersinia in pigs and wild boars in the Czech Republic. The aim of this study was to provide data about the serological prevalence of enteropathogenic Yersinia in pigs and wild boars at the time of slaughter intended for human consumption and coming from different production systems in the Moravian region of the Czech Republic.

Materials and Methods

Sera or meat juices (obtained by subsequent freezing–thawing of ∼50 g of diaphragmatic tissue) of pigs (n = 319) were collected in four slaughterhouses between 2012 and 2014. The conventional (n = 180, 5–6 months of age), organic (n = 110, 8–10 months of age), and household-raised pigs (n = 29, 8–10 months of age) originated from seven conventional farms (10–40 pigs per farm), one organic farm, and 16 breeders (one to three pigs per breeder) from the Moravian region in the Czech Republic. The proportion of pigs reared in an organic farming system (1,860 pigs in 2013) represents only ∼0.1% of all pigs reared in the Czech Republic (Anonymous, 2013). A large proportion of these organic pigs (nearly 800 animals) are raised in the only organic farm in this region with its own slaughterhouse. It is currently the largest organic farm in the Czech Republic. To obtain a sufficient number of samples from organic pigs (raised with outdoor access, straw bedding, organic feed, and without the use of antibiotics or drug treatments), pigs from this organic farm were sampled repeatedly (seven times in 2012–2013, 9–19 pigs per sampling day).

In addition, meat juice samples from the diaphragms of a total of 135 hunted wild boars were obtained. The wild boars came from four hunting areas in the Moravian region (free-ranging animals, 27–40 wild boars per hunting area). The animals were shot during the hunting seasons in autumn 2013 and 2014. Approximate ages (determined on the evaluation of the dental table) of the pigs were recorded and animals were divided into two groups: under 1 year (n = 94) and adult animals (n = 41).

Sera and meat juices samples were stored at −20°C until examination. The prevalence of anti-Yersinia antibodies (an apparent prevalence) was examined using a commercial Pigtype Yopscreen ELISA kit (Labor Diagnostik, Leipzig, Germany) based on recombinant Yops expressed by pathogenic Yersinia strains according to the manufacturer's instructions. The optical density (OD) was determined with a spectrophotometer (Sunrise™ Microplate Reader, Tecan Austria GmbH), and an OD value of 0.3 was used as cutoff value. The ELISA test is licensed both for blood serum and meat juice samples.

The relationship between the seroprevalence and the type of breeding system was assessed using a chi-squared test for independence followed by Fisher's exact post hoc tests with Bonferroni's correction of p-values. The association between the seroprevalence and the age of wild boars and between the seroprevalences in domestic and feral pigs (Table 1) was assessed using Fisher's exact test. Tests were performed using the GraphPad Prism 5.04 program (GraphPad, Inc., San Diego, CA). Differences in the prevalence were considered statistically significant if p < 0.05.

Table 1.

Prevalence of Anti-Yersinia Antibodies in Domestic Pigs and Wild Boars from the Moravian Region, Czech Republic

	Domestic pigs				Wild boars
Type of production system	Conventional	Organic	Household-raised	Total	≤1 year	>1 year	Total
No. of animals	180	110	29	319	94	41	135
No. of positive animals	146	92	13	251	66	23	89
Apparent prevalence (%)	81.1^A	83.6^A	44.8^B	78.7^a	70.2^A	56.1^A	65.9^b
True prevalence (%)	97.0, 89.5–99.9	97.3,89.3–99.9	54.2, 30.5–78.4	96.2, 88.9–99.8	86.9, 73.1–98.0	68.5, 47.1–88.8	81.9, 69.5–94.1
Sensitivity¹(%)				80.8, 76.6–85.1			79.4, 73.7–84.6
Specificity¹(%)				96.7, 84.5–99.8			96.7, 85.1–99.8

A,B

Capital letters were used for expression of statistically significant differences within the group of domestic pigs (chi-squared test for independence followed by Fisher's exact post hoc tests with Bonferroni's correction of p-values) and within the group of wild boars (Fisher's exact test). Values of prevalence with different superscripts were significantly different (p < 0.01).

a,b

Small letters were used for expression of statistically significant differences between the group of domestic pigs and the group of wild boars. Values of total prevalence with different superscripts were significantly different (p < 0.01, Fisher's exact test).

True prevalence of anti-Yersinia antibodies, sensitivity and specificity of the ELISA test were estimated by Bayesian approach (posterior estimates) with iterative simulation based on Gibb's sampler. The first row shows median and the second row shows 95% probability interval. The sensitivity and specificity of the ELISA test. The first value = median, the second value = 95% probability interval.

The true prevalence of antibodies against enteropathogenic Yersinia spp. in pigs and wild boars was estimated from an apparent prevalence using a Bayesian approach (Joseph et al., 1995). This method provides a reliable estimate of the true disease prevalence and the parameters of diagnostic tests in the absence of a gold standard. Noninformative (uniform) prior distribution Beta(1, 1) was assigned as the prior distribution of the true prevalence. To estimate the prior distributions for sensitivity and specificity of the used commercial ELISA kit, information about medians and 95% probability intervals published in the article of Vilar et al. (2015) were used: sensitivity = 79.5% (73.6–84.8%) and specificity = 96.9% (85.3–99.9%). These values are more conservative than those reported by the manufacturer of the ELISA test (sensitivity = >99%, specificity = 100%). The corresponding distribution Beta(161.7, 41.94) for sensitivity and Beta(24.05, 1.067) for specificity were used as prior distributions. Posterior estimates of the true prevalence, sensitivity, and specificity were obtained by iterative simulation based on Gibb's sampler (http://epitools.ausvet.com.au/content.php?page=OneTest) with a total number of iterations = 25,000 and number of iterations to be discarded = 5,000.

Results

The results acquired directly from the commercially available ELISA test represent an apparent prevalence of antibodies against enteropathogenic Yersinia spp. in pigs and wild boars from different production systems in the Moravian region, Czech Republic (Table 1). The seroprevalence was found to be significantly associated with the type of breeding system (p < 0.01, chi-squared test for independence). The lowest seroprevalence was observed in the household-raised pigs (44.8%), the difference between animals from conventional (81.1%) and organic breeding systems (83.6%) was not statistically significant (p > 0.05, Table 1). Antibodies were found in 65.9% of wild boars without statistically significant difference (p > 0.05) between adult and young (under 1 year old) animals (Table 1). In total, anti-Yersinia antibodies were detected more frequently in domestic than in feral pigs (p < 0.01, Table 1). The true prevalence of anti-Yersinia antibodies in domestic pigs (total prevalence 96.2%) and in wild boars (81.9%,Table 1) was estimated from the aforementioned apparent prevalence adjusting for the sensitivity and specificity of the ELISA test using a Bayesian approach (see Materials and Methods section).

Discussion

Human yersiniosis, mostly caused by Y. enterocolitica, is an important foodborne zoonosis, even if it has exhibited a decreasing trend in the European Union in the last years (EFSA and ECDC, 2015). Pigs, mostly without showing any visible symptoms, are considered to be an important source for enteropathogenic Yersinia spp. (Novoslavskij et al., 2013; Vanantwerpen et al., 2014), however, pathogenic isolates have seldom been recovered from pork or other foods (Laukkanen-Ninios et al., 2014). Although seropositivity does not directly demonstrate the presence of bacteria, there is a high correlation between the presence of anti-Yersinia antibodies and positive tonsil culture even at the time of slaughter (Nesbakken et al., 2003, 2006; Van Damme et al., 2014). Especially tonsils of infected pigs are important sources of contamination of carcasses and offal during slaughter.

According to previous studies, the housing system appears to be an important factor in the prevalence of both pathogenic Yersinia spp. A higher prevalence of Y. enterocolitica was described in pigs from conventional and high-capacity farms than from organic farms. In contrast, Y. pseudotuberculosis is more frequently detected in organically farmed and feral pigs due to their more frequent contact with pests and other animals and the outside environment (Nowak et al., 2006; Fredriksson-Ahomaa et al., 2009; Laukkanen et al., 2009; Ortiz Martínez et al., 2010). In our study, the applied ELISA kit cannot distinguish between infections with individual pathogenic Yersinia species. This could be the reason that we did not find a significant difference in the seroprevalence between conventional and organic pigs.

Various methods can be used for determination of the seroprevalence, and differences in sensitivity and specificity between diagnostic tests can influence the obtained results and their direct comparison. Using the same ELISA kit for prevalence surveys, we detected a relatively much higher seroprevalence in conventional pigs reared in the Czech Republic than was found in pigs from conventional fattening herds in Germany (64.1%, von Altrock et al., 2011) and in pigs at slaughter in Belgium (65.1% and 66%; Van Damme et al., 2014 and Vanantwerpen et al., 2014, respectively). Differences in management practices on farms producing fattening pigs can explain these differences between studies because many farm factors seem to influence the serological prevalence of Yersinia: hygienic measures in herds, access of other farm animals or pets, type of housing, the use of bedding, the way of feeding and watering, herd size, and so on (Skjerve et al., 1998; Laukkanen et al., 2009; Virtanen et al., 2011; von Altrock et al., 2011). Because of the low number of investigated farms from a limited geographic region, our results require further investigations to confirm and to explain the high prevalence of enteropathogenic Yersinia spp. in pigs in the Czech Republic.

Nevertheless, the lowest seroprevalence among domestic pigs was observed in the household-raised pigs. These animals are raised either individually or in small groups, and the contact between animals and between animals and contaminated environment (pen floor, feed, or water) is not as close as on farms. High population density was previously described as a risk factor associated with high prevalence of Y. enterocolitica and Y. pseudotuberculosis in pigs and animal-to-animal transmission as an important factor in the spread of Yersinia infection (Skjerve et al., 1998; Laukkanen et al., 2008, 2009; Virtanen et al., 2011).

Wild boars appear to be frequent carriers of foodborne pathogens, including both pathogenic Yersinia spp. (Al Dahouk et al., 2005; Sannö et al., 2014). Y. enterocolitica is more frequently detected but the prevalence of Y. pseudotuberculosis is also high because of frequent contact with wild animals, pests, and the outside environment (Laukkanen et al., 2008; Fredriksson-Ahomaa et al., 2009; Sannö et al., 2014). The overall prevalence of anti-Yersinia antibodies in wild boars in our study was found to be very similar to the results of studies carried out in Switzerland (65%, Fredriksson-Ahomaa et al., 2009) and in Germany (62.6%, Al Dahouk et al., 2005).

The increase of antibodies with age in domestic and feral pigs was reported in previous studies (Al Dahouk et al., 2005; Nesbakken et al., 2006; Fredriksson-Ahomaa et al., 2009). This could be due to prolonged exposure to Yersinia spp. during the lifetime of the animals. Unlike these studies, we found a slightly higher seroprevalence (not statistically significant) in wild boars under 1 year old compared with adult ones. Our result could be influenced by the age limit chosen for the grouping of animals (under and over 1 year of age). Fredriksson-Ahomaa et al. (2009) found the lowest prevalence of anti-Yersinia antibodies in wild boars less than 6 months of age. In contrast to seroprevalence, it was shown that especially young animals were important carriers of pathogenic Yersinia (Nesbakken et al., 2006; Fredriksson-Ahomaa et al., 2009). This poses a risk to human health because hunters tend to prefer young individuals because of the higher meat quality.

The seroprevalence in feral pigs was significantly lower compared with the overall seroprevalence in domestic pigs in our study. This could be due to the fact that in comparison to wild boars, domestic pigs are bred under more intensive conditions with higher animal density. Under such conditions, the transmission of pathogenic Yersinia from positive to negative animals is unavoidable (Skjerve et al., 1998; Fredriksson-Ahomaa et al., 2009; Virtanen et al., 2011).

Using Bayesian analysis taking into account the sensitivity and specificity of the used ELISA test, an extremely high true prevalence of antibodies against enteropathogenic Yersinia spp. in domestic pigs and wild boars in the Czech Republic was estimated. A similar approach was applied in the study of Vilar et al. (2015) to calculate the true prevalence of Yersinia in serum samples from pigs in Finland, however, more farms were involved (n = 16). They estimated the lower true prevalence (67.5%) of anti-Yersinia antibodies in slaughter-age pigs in Finland in comparison with our study (96.2%).

Conclusion

Domestic pigs and wild boars proved to be an important reservoir of enteropathogenic Yersinia in the Czech Republic. To prevent their transmission to humans, attention should be paid to good hygienic practice during slaughtering and handling of meat, including sufficient heat treatment before consumption. Further studies are needed to confirm conclusions about the prevalence in domestic pigs from different production systems.

Footnotes

Acknowledgments

This work was supported by grant QJ1210113 (Ministry of Agriculture of the Czech Republic) and project LO1218 under the NPU I program (Ministry of Education, Youth and Sports of the Czech Republic). Neysan Donnelly (Max-Planck-Institute of Biochemistry, Germany) and Ludmila Faldikova (Veterinary Research Institute, Czech Republic) are acknowledged for grammatical corrections.

Disclosure Statement

No competing financial interests exist.

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