Prevalence of Yersinia enterocolitica and Yersinia pseudotuberculosis in wild boars in the Basque Country, northern Spain

Background Yersiniosis is a zoonosis widely distributed in Europe and swine carry different serotypes of Yersinia enterocolitica and Y. pseudotuberculosis. The aim of this study was to determine the prevalence of Y. enterocolitica and Y. pseudotuberculosis in wild boars in northern Spain. The blood of wild boars (n = 505) was sampled between 2001 and 2012. Seroprevalence was determined in 490 serum samples with an indirect enzyme-linked immunosorbent assay. Seventy-two of the animals were also examined for the presence of Y. enterocolitica or Y. pseudotuberculosis in the tonsils with real-time polymerase chain reaction. All the tonsils were analysed twice, directly and after cold enrichment in phosphate-buffered saline supplemented with 1 % mannitol and 0.15 % bile salts. Results Antibodies directed against Y. enterocolitica and Y. pseudotuberculosis were detected in 52.5 % of the animals. Yersinia enterocolitica was detected with real-time polymerase chain reaction in 33.3 % of the wild boars and Y. pseudotuberculosis in 25 %. Significant differences were observed according to the sampling year, and the highest prevalence was during winter and spring. The highest antibody levels and Y. enterocolitica prevalence were observed in mountainous areas at altitudes higher than 600 m, with very cold winters, and with the highest annual rainfall for each dominant climate. Areas with low and medium livestock populations were associated with the highest seroprevalence of Yersinia spp. in wild boars, whereas areas with high ovine populations had the highest prevalence of Y. enterocolitica. Conclusions This study shows that Y. enterocolitica and Y. pseudotuberculosis are highly prevalent among wild boars in the Basque country, with Y. enterocolitica most prevalent. The risk of infection among wild boars is influenced by the season and the area in which they live. Electronic supplementary material The online version of this article (doi:10.1186/s13028-016-0184-9) contains supplementary material, which is available to authorized users.


Background
Yersiniosis is the fourth most frequently reported foodborne zoonosis in humans in Europe, although the number of reported cases of Yersinia infection has continued to decrease since 2007 [1]. The genus Yersinia is composed of several species, but only Y. pestis, Y. pseudotuberculosis and some Y. enterocolitica strains are human pathogens [1].
Pigs are assumed to be the main reservoir of human pathogenic Y. enterocolitica, and serotypes isolated from pig samples, such as 4/O:3, are the same that cause human disease in Europe [1]. Yersinia pseudotuberculosis has also been frequently isolated from pigs and these animals might be a source of human 2/O:3 infections [2].
Wild animals constitute a very important factor in the epidemiology of Yersinia infection [3,4], and wild boars (Sus scrofa) are considered an important reservoir of enteropathogenic Yersinia [5]. A great variety of serotypes, including those that cause human infections, have been isolated from wild boars in Europe [3,5,6], although some Y. enterocolitica strains differ from those in domestic pigs [2].

Open Access
Acta Veterinaria Scandinavica More studies are required to understand the real role of wild boars in the epidemiology of yersiniosis. During the last two decades, the wild boar population has increased significantly in Europe [7], favouring their contact with livestock and the transmission of diseases [8]. Interest in wild boars as a meat source has also increased, thus increasing the risk of the transmission of food-borne diseases [9].
The prevalence of pathogenic Yersinia spp. in Spanish wild boars is unknown. Therefore, the aim of this study was to determine the prevalence of Y. enterocolitica and Y. pseudotuberculosis in wild boars in northern Spain.

Study area
The Basque country is located in northern Spain, limited by the Cantabrian coastline and distributed in eight regions, defined according to rainfall, temperature, altitude and the dominant vegetation [10,11]. Climatologically, the Atlantic slope (northern part) is moderate in terms of temperature, but very rainy, whereas the Mediterranean slope (southern part) is less rainy, with warmer summers and colder winters.

Sample collection
Wild boar samples were collected within the context of a wildlife health surveillance program in the Basque Country. In total, 505 wild boars were sampled between 2001 and 2012, during which time 490 serum samples were obtained, and in the last 3 years, 72 tonsils were also collected. Both serum and tonsil samples were obtained from only 57 animals. Most of the animals studied (90 %) had been shot by accredited hunters, and samples were taken in the field in collaboration with competent local authorities, and 8 % were obtained from wildlife rehabilitation centres. The cause of death and the health status of these animals were not recorded. The remaining samples (2 %) were obtained from animals found dead or run over, and necropsies were performed in the laboratory. No significant lesions, except physical trauma, were observed in these animals. The samples were collected in individual containers, properly identified and stored at −20 °C until analysis. The details of each animal, including its sex, age, and the date and geographic location of collection were recorded. The animals were classified into two groups according to age: young, including piglets (<1 year) and yearlings (1-2 years); and adults (>2 years). Details of the animals are given in Tables 1 and 2.

Real-time polymerase chain reaction
The tonsil samples (1-5 g) were weighed and aseptically cut into small pieces. Approximately 150 mg of each tonsil was disrupted and homogenised with 30 chrome-steel beads (1.3 mm) (Biospec Products, Bartlesville, OK, USA) and 750 µL of TE buffer using the TissueLyser system (Qiagen, Hilden, Germany). DNA was extracted from 200 µL of the supernatant for direct real-time polymerase chain reaction (rt-PCR) analysis. The rest of each tonsil sample was mixed with phosphate-buffered saline (PBS) supplemented with 1 % mannitol (Fluka, Seelze, Germany) and 0.15 % bile salts (Fluka, Seelze, Germany) (PBS-MSB), diluted 1:10 and homogenised in a stomacher (Lab-Blender 80, Cole-Parmer, Vernon Hills, IL, USA) until homogeneity. The mixture was incubated for 14 days at 4 °C. DNA was extracted from 200 µL of the supernatant and used as the template for rt-PCR.
DNA extraction was performed with the QIAamp ® DNA Blood Mini Kit (Qiagen), according to the manufacturer's instructions, with minor modifications [12], and the DNA was measured with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Inc.). DNA (150-200 ng) was used to detect Yersinia with the TaqMan rt-PCR assay in three independent reactions, using the Applied Biosystems 7500 Real-Time PCR System and The Express qPCR Supermix, universal kit (Invitrogen ™ ), according to the supplier's recommendations. Yersinia enterocolitica was detected with the amplification of the ail gene [13], using a previously described procedure [12]. To detect all the Y. pseudotuberculosis serotypes, the wzz and ail genes were amplified in two independent reactions [12,14,15]. Amplification of the ail gene detects all serotypes but O:11 and O:12, and amplification of the wzz gene detects all serotypes but O:6 and O:7 [14,15]. A sample was considered positive for Y. enterocolitica or Y. pseudotuberculosis when at least one positive result was obtained in the direct reaction or after enrichment in any of the three rt-PCRs used.

Enzyme-linked immunosorbent assay
The presence of antibodies directed against pathogenic Yersinia was determined with a commercial indirect enzyme-linked immunosorbent assay (ELISA) specific for swine (PIGTYPE ® YOPSCREEN, Labor Diagnostic, Leipzig, Germany), according to the manufacturer's instructions. The optical density (OD) was measured in an ELISA Multiskan (Thermo Labsystem) spectrophotometer at 450 nm. The ratio between the sample OD and the positive control OD (S/P ratio) was calculated. Samples with an S/P ratio ≥0.3 were considered positive.

Bacteriology
Selective cefsulodin-irgasan-novobiocin (CIN) agar (bioMérieux, Marcy l'Etoile, France) and CHROMagar ™ Y. enterocolitica (CHROMagar, Paris, France) agar were inoculated with 20 µL of the rt-PCR-positive tonsil mixtures and incubated at 30 °C for 24-48 h to isolate the  Yersinia strains. Red CIN agar "bull's-eye" colonies surrounded with a transparent area of 1 mm and mauve CHROMagar ™ colonies were selected. The selected colonies were homogenised in 500 µL of PBS, and 50 µL of this mixture was incubated for 10 min at 100 °C in a water bath and then for 10 min on ice. The mixture was then centrifuged for 10 min at 15,600×g and 5 µL of the supernatant was used for Y. enterocolitica and Y. pseudotuberculosis identification with rt-PCR, with the procedures described above. The colonies were also streaked directly onto triple sugar iron agar (Oxoid Ltd, Basingstoke, England) and onto blood agar (bioMérieux) and identified with the VITEK system (bioMérieux), using a previously reported protocol [12]. The Yersinia strains were serotyped with slide agglutination using commercial Y. . Yersinia pseudotuberculosis was also serotyped with O-genotyping, using a conventional multiplex PCR, according to Bogdanovich et al. [16].

Data analysis
All statistical analyses were performed in the SAS 9.3 software. The official 2009 livestock census data were obtained from the Basque Statistics Institute (http://www.eustat.es) for each region and the PROC RANK Statement was used to classify each region as containing high, medium, or low numbers of each species. The relationships between Yersinia prevalence and the different independent variables studied (sex, age, sampling year, season, natural region, slope and livestock numbers) were examined statistically using the χ 2 or Fisher's test. The simple kappa coefficient of agreement was used to determine the degree of agreement between the ELISA and PCR results when applied to the same animal. A t test was used to compare the ELISA S/P ratios between the PCR-positive and -negative animals. Differences were considered significant at P < 0.05.
Yersinia infection was detected with rt-PCR in 51. Seroprevalence was higher in the adult animals than in the young animals (P < 0.0001; Table 1), but no significant differences were observed according to age with PCR (P = 0.2157; Table 2).
Significant differences were observed according to the sampling year. The highest seroprevalence was detected in 2002 and in 2005-2006, although in 2002, only 10 samples were analysed (P < 0.0001; Table 1). The prevalence of Y. enterocolitica was highest in 2010 (P = 0.0213) and that of Y. pseudotuberculosis was highest in 2012 (P < 0.0001; Table 2).
The overall seroprevalence was highest in winter and spring (P < 0.0001; Table 1). The prevalence of Y. pseudotuberculosis was highest in spring (P = 0.0305), but no significant difference was observed in the prevalence of Y. enterocolitica between seasons (P = 0.3180; Table 2). Statistically significant differences were observed in the seroprevalence of Yersinia spp. according to the slope and region of habitation (P < 0.0001; Table 1). These differences were also significant for Y. enterocolitica and regions (P = 0.0096; Table 2). The geographic distribution of the positive samples is illustrated in Fig. 2.   Fig. 1 Distribution of ELISA S/P ratios for Yersinia spp. detected in 490 wild boars. Each of the two box plots represents the summary statistics for the S/P ratios of the ELISA-positive and -negative samples. Boxes represent the 25 and 75 % percentiles; the horizontal lines indicate the median values for the S/P ratios; the diamond shapes represent the mean S/P ratios; and the vertical lines extend from the minimum S/P ratios to the maximum ratios Higher seroprevalence was observed in areas with small livestock populations (caprine, P < 0.0001) or medium livestock populations (bovine, P = 0.0011; ovine, P < 0.0001) ( Table 1), whereas Y. enterocolitica prevalence was highest in areas with large ovine populations (P = 0.0012; Table 2).
Two isolates of Y. pseudotuberculosis and two of Y. enterocolitica were collected from four different wild boars. The Y. pseudotuberculosis isolates were obtained on CIN agar, one with direct plating and the other after enrichment. Both Y. enterocolitica isolates were obtained after enrichment, one on CIN agar and the other on CHROMagar ™ . The identities of Y. enterocolitica and Y. pseudotuberculosis were confirmed for each isolate with rt-PCR amplification of the ail gene. No agglutination was detected when the Y. pseudotuberculosis isolates were serotyped with the antisera used, but both isolates were identified as serotype O:1c with multiplex O-gene amplification. It was not possible to serotype the Y. enterocolitica isolates because of contamination.
Of the 57 wild boars analysed with rt-PCR and ELISA, 13 were positive and 19 were negative with both techniques, seven animals were positive only according to ELISA, and 18 animals were positive only according to rt-PCR (κ index = 0.1452). No differences were observed in the ELISA S/P ratios when the Y. enterocolitica-rt-PCRpositive and -negative animals were compared. However, the Y. pseudotuberculosis-positive animals had higher S/P ratios (mean 0.53; 95 % CI 0.21-0.86) than the Y. pseudotuberculosis-negative animals (mean 0.23; 95 % CI 0.12-0.35; P = 0.0249).

Discussion
This study demonstrates that Y. enterocolitica and Y. pseudotuberculosis infections are widespread among the wild boars in northern Spain. The seroprevalence was high (52.5 %), although slightly lower than those detected in wild boars in Germany and Switzerland (62.6 and 65.0 %, respectively) [5,17]. The prevalence of Y. enterocolitica and Y. pseudotuberculosis can also be considered high (33.5 and 25 %, respectively) because their observed prevalence in wild boars in Europe ranges from 4.35 to 35 % for Y. enterocolitica and is around 20 % for Y. pseudotuberculosis [5,18,19]. Yersinia enterocolitica was more prevalent than Y. pseudotuberculosis, as is usually found in wild boars and pigs [5,20]. Mixed infections were detected in a proportion of the animals, as previously described [5], but the prevalence of Y. pseudotuberculosis (25 %) was higher than expected in wild boars or organically produced pigs, probably because they are in frequent contact with other infected wild species and livestock in extensive grazing systems [4,21]. The use of two different rt-PCR methods and the higher detection rates recorded when an enrichment step was included before rt-PCR, could also have improved the detection rate for Y. pseudotuberculosis [12].
The highest seroprevalence was detected in spring and winter, which is attributable to the highest Y. pseudotuberculosis prevalence recorded in spring and the (not significantly) highest Y. enterocolitica prevalence recorded in winter. To the best of our knowledge, the seasonality of Y. enterocolitica and Y. pseudotuberculosis infections has not been reported previously in wild boars. However, in other wildlife species, the disease is usually detected in the coldest months of the year [22] or from November to May, which is related to the birth of newborns [23].
The highest seroprevalence and presence of Y. enterocolitica were associated with mountainous areas at altitudes higher than 600 m, very cold winters, and the highest annual rainfall for each dominant climate. A similar trend was observed in pigs slaughtered in China, in which the incidence of Y. enterocolitica was higher in cold areas than in warm areas [24].
The highest prevalence of Y. enterocolitica was detected in areas with a high ovine presence. Sheep have been described as a reservoir of pathogenic Y. enterocolitica and Y. pseudotuberculosis [25,26], but little is known about the infection of sheep in Spain with pathogenic Yersinia or their relationship with the Yersinia species found in wild boars, although Yersinia is reported to cause sporadic abortion in sheep in the area studied [27]. In contrast, the highest Yersinia seroprevalence was associated with medium or low numbers of other livestock, suggesting that other wildlife species also contribute to the epidemiology of Yersinia infection among wild boars.
However, more studies are required to determine the real impact of pathogenic Yersinia on livestock in this area.
The rates of isolation were low, despite the use of two different culture media, including CHROMagar ™ , which is recommended for the isolation of Y. enterocolitica [28]. Y. enterocolitica pathogenicity therefore remains unknown, because the ail gene is an insufficient marker of virulence, and is also present in some Y. enterocolitica biotype 1A strains [29]. The only two Y. pseudotuberculosis strains isolated were identified as serotype O:1c. Little is known about the infection of animals or humans by serotype O:1c because the majority of studies have not included this subserotype. However, Y. pseudotuberculosis serotype O:1 has been described as one of the most commonly found serotypes infecting wild boars, pigs and humans in Europe [2,30,31]. This fact highlights the need for the better characterisation of its pathogenicity.
More efforts are required to isolate and characterise the Yersinia strains from infected wild boars in the Basque country to determine their pathogenicity and any potential risk they pose to humans and domestic species.

Conclusions
This study demonstrates that Y. enterocolitica and Y. pseudotuberculosis are highly prevalent among wild boars in the Basque Country, with Y. enterocolitica the most frequently found species. The risk of infection among wild boars is influenced by the season and the area in which the animals live.