Yersinia pseudotuberculosis Prevalence and Diversity in Wild Boars in Northeast Germany

Abstract

In this study, the prevalence of Yersinia pseudotuberculosis in wild boars in northeast Germany was determined. For that purpose, the tonsils of 503 wild boars were sampled. The presence of Y. pseudotuberculosis was studied by diagnostic PCR. Positive samples were analyzed by cultural detection using a modified cold enrichment protocol. Ten Y. pseudotuberculosis isolates were obtained, which were characterized by biotyping, molecular serotyping, and multilocus sequence typing (MLST). In addition, whole-genome sequences and the antimicrobial susceptibility of the isolates were analyzed. Yersinia pseudotuberculosis was isolated from male and female animals, most of which were younger than 1 year. A prevalence of 2% (10/503) was determined by cultural detection, while 6.4% (32/503) of the animals were positive by PCR. The isolates belonged to the biotypes 1 and 2 and serotypes O:1a (n = 7), O:1b (n = 2), and O:4a (n = 1). MLST analysis revealed three sequence types, ST9, ST23, and ST42. Except one isolate, all isolates revealed a strong resistance to colistin. The relationship of the isolates was studied by whole-genome sequencing demonstrating that they belonged to four clades, exhibiting five different pulsed-field gel electrophoresis (PFGE) restriction patterns and a diverse composition of virulence genes. Six isolates harbored the virulence plasmid pYV. Besides two isolates, all isolates contained ail and inv genes and a complete or incomplete high-pathogenicity island (HPI). None of them possessed a gene for the superantigen YPM. The study shows that various Y. pseudotuberculosis strains exist in wild boars in northeast Germany, which may pose a risk to humans.IMPORTANCEYersinia pseudotuberculosis is a foodborne pathogen whose occurrence is poorly understood. One reason for this situation is the difficulty in isolating the species. The methods developed for the isolation of Yersinia enterocolitica are not well suited for Y. pseudotuberculosis We therefore designed a protocol which enabled the isolation of Y. pseudotuberculosis from a relatively high proportion of PCR-positive wild boar tonsils. The study indicates that wild boars in northeast Germany may carry a variety of Y. pseudotuberculosis strains, which differ in terms of their pathogenic potential and other properties. Since wild boars are widely distributed in German forests and even populate cities such as Berlin, they may transmit yersiniae to other animals and crop plants and may thus cause human infections through the consumption of contaminated food. Therefore, the prevalence of Y. pseudotuberculosis should be determined also in other animals and regions to learn more about the natural reservoir of this species.

Keywords: Yersinia pseudotuberculosis; antimicrobial resistance; cultural detection; virulence; whole-genome sequencing; wild boars.

FIG 1

Effect of 0.25% KOH on the recovery of seven Y. pseudotuberculosis strains. Average values of all strains (serotypes O:1a, O:1b, O:3, O:4, and O:5) are shown. The experiments were performed in triplicates; bars indicate standard deviations.

FIG 2

Growth of Y. pseudotuberculosis at 4°C with and without preincubation at room temperature. Mean values (CFU/ml) from 16 Y. pseudotuberculosis strains (serotypes O:1 to O:5) are shown; bars indicate standard deviations. d, days.

FIG 3

Relationship of the isolates. (A) Clustering determined by whole-genome analysis. Also shown are typing results and the virulence gene content. (B) Numbers of SNPs identified in each isolate.