Prevalence, Virulence and Antimicrobial Susceptibility of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes in European Wild Boar (Sus scrofa) Hunted in Tuscany (Central Italy)

Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated; of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeria monocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.


Introduction
The "One Health" idea started at the beginning of the 2000s. This approach is based on the concept that human and animal health are strictly linked and bound to the health of the ecosystems in which they coexist [1]. This concept is envisaged and implemented by the World Organization for Animal Health (OIE) as a collaborative global approach to understanding the risks for humans and animals, including domestic and wild animals, and ecosystem health as a unique entity [2].
Within the One Health approach, zoonosis plays an important role in the interaction between humans, domestic animals, and wildlife. Following European Food Safety Au-

Antimicrobial Resistance
Several patterns of antimicrobial resistance have been found in the 18 tested Salmonella strains (Table 1). In total, 10 out of 18 (55.6%) were resistant to streptomycin, 2 of 18 (11.1%) to cephalothin, and 1 of 18 (5.56%) to imipenem. Moreover, one isolate (6.67%) showed resistance simultaneously to tetracycline, enrofloxacin, nitrofurantoin and nalidixic acid, as well as to streptomycin. In total, 5 out of 18 (27.8%) isolates were susceptible to all tested antimicrobials.

Virulence Genes
Excluding the strains S345 and S346, all isolates presented at least one virulence gene ( Table 1). The most detected genes were sopE in 11 isolates out of 18 (61.1%), pipB in 10 of 18 (55.6%), and sodCI in 8 of 18 (44.4%). The genes spvR and spvC were found in association in 7 of the 18 isolates (38.9%). A low percentage of Salmonella cultures were positive for sopB (27.8%) and for mgtC (11.1%). None of the tested isolates carried the gene gipA.

Listeria monocytogenes 2.3.1. Isolation and Characterization
Only one Listeria monocytogenes isolate (0.35%) was obtained from the 287 collected wild boar. This strain was isolated from the rectal swab of the wild boar C147 during the 2018/2019 hunting season. This isolate belonged to serogroup IVb serovar 4b, as determined by its positive score for genes prfA, ORF2819 and ORF2110, used for molecular characterization.

Antimicrobial Resistance
The only Listeria monocytogenes strain showed resistance to cefoxitin, cefotaxime and nalidixic acid.

Statistical Analysis
Concerning the three different pathogens, no statistical differences (p > 0.05) were recorded regarding their prevalence in relation to sex, age, hunting seasons and province of sampling.

Discussion
This investigation confirmed the role of wild boar as a carrier of Salmonella spp. and Yersinia enterocolitica in the Tuscany region. While, regarding Listeria monocytogenes, only one animal was positive, the role of the wild boar in the epidemiology of this bacterium in this area remains unknown, and it probably cannot be considered as a reservoir. Interestingly, only one case of co-infection was recorded (wild boar C263), whereby, from a rectal swab, Salmonella enterica subspecies houtenae serotype 1,40:z4,z23 and Yersinia enterocolitica biotype 1 were isolated.
A total of 4.18% of the sampled wild boar scored positive for Salmonella spp. infection. Very similar prevalence was reported in two studies carried out in the Lazio region (7.2%) and in North-West Italy (10.8%) [34,41], as well as in two studies performed in Spain (7.70%) and Sweden (10.0%) [42,43]. On the other hand, in research performed in Switzerland (12.0%) and Portugal (22.0%), the prevalence was higher than that reported in this investigation [44][45][46][47]. Concerning serotype, the most detected strains were Salmonella enterica subspecies diarizonae serotype 50:r:1,5,7 (7 isolates), followed by subspecies houtenae serotype 1,40:z4,z23 (4 isolates), Newport and Kottbus (2 isolates for each serotype) and London, Infantis and Rubislaw (1 isolates for each). Salmonella ser. 50:r:1,5,7 and Salmonella enterica subspecies houtenae serotype 1,40:z4,z23 were also isolated from the wild boar sampled in other northern (Lombardy, Piedmont, Liguria and Valle d'Aosta) and central (Latium) Italian regions, as well as in Spain [34,41,42,48], highlighting the circulation of these Salmonella serovars in North-Central Italy and in other Mediterranean countries. On the other hand, the serotype Newport was reported only in the Lombardy region and Spain [42,48], while the serotype Kottbus only in the Lombardy and Latium regions [34,48]. Moreover, the serotype Infantis was previously identified in the wild boar hunted in the North of Italy [41,48]. At the best of the authors' knowledge, this investigation reported the first isolation in Italy from wild boar samples of serotypes Rubislaw and London.
Half of the positive specimens (six animals) presented multi-organ infection. Indeed C12, C141, C196, C218 and C217 scored positive for Salmonella infection in two, or all, of the investigated organs, probably with the same strains. Interestingly, in the spleen and liver of wild boar 209, a co-infection by two different serotypes was reported. None of the isolated Salmonella enterica strains proved to be resistant to more than one antibiotic, except for serotype Infantis, isolated from wild boar C103 spleen. The highest percentage of resistance was recorded for streptomycin (55.6%), while low resistances were reported to cephalothin (11.1%) and imipenem (5.56%). These data agree with reports focusing on the antimicrobial resistance of wild boar [34,41,49,50] and swine isolates [23,51,52], especially for the Salmonella enterica serovars that are less diffused. Concerning pathogenic characteristics, the presence of some virulence genes, located on Salmonella pathogenicity island 3 (SPI-3) and SPI-5, prophages and plasmids, was investigated. The most-detected gene was sopE-it was found in three S. sub. diarizonae ser. 50:r:1,5,7, three S. sub. houtenae ser. 1,40:z4,z23, two S. ser. Kottbus and one S. ser. Rubislaw examined strains. Another gene that was highly detected was sodCI, found in four S. sub. diarizonae ser. 50:r:1,5,7, two S. sub. houtenae ser. 1,40:z4,z23, one S. ser. Infantis and one S. ser. Rubislaw. The genes sopE and sodCI, both carried by phages, are more often associated with serotypes Enteritidis and Typhimurium, respectively, and they are rarely detected in other serotypes [23,53,54]. The obtained results expand the bacterial hosts spectrum of these phages, and suggest a possible reservoir for bacteriophages harboring virulence genes among salmonellae circulating in wild animals. The genes spvRC were detected in seven isolates belonging to S. sub. houtenae ser. 1,40:z4,z23, and three S. sub. diarizonae ser. 50:r:1,5,7. Both genes are part of the Salmonella Plasmid Virulence (spv) that is associated with more virulent serotypes, such as Typhimurium or Enteritidis. The detection of spv in unusual serotypes is not uncommon, but suggests a more wide diffusion of this plasmid and the potential high virulence of these serotypes too [55]. In total, 10 and 5 strains harboured the genes pipB and sopB, respectively, both located on SPI-5. Only two isolates had these two genes in association, suggesting a possible fragmenting acquisition of SPI-5, which it is not as highly conserved as other SPIs, or the partial genetic leak of this SPI. Finally, only two isolates, one S. ser. Rubislaw and one S. sub. diarizonae ser. 50:r:1,5,7, harboured the gene mgtC located on SPI-3. Overall, genes located on prophages and plasmids were detected more frequently than genes located on genomic SPI. This could suggest, as might be expected, a large diffusion of these highly mobile elements.
In this investigation, Yersinia enterocolitica was isolated from 18.8% of the samples. In other studies carried out in Sweden, Germany and Poland, the culture-positive prevalence was very similar, ranging from 13.2% to 20% [35,56,57]. On the other hand, in research performed in Japan and Poland the prevalence was higher, from 50% to 74% [36,58,59], while in other studies done in Sweden and in northern Italy, it was lower, at around 7% [43,60]. These data suggest a high variability in isolation rates, probably linked to the different geographic areas and time of sampling. This observation stresses the importance of a constant monitoring of the prevalence of infectious agents among wildlife, which could be influenced by many different factors. Furthermore, the diffusion of Yersinia enterocolitica biotypes in wild boar found in this investigation highlighted that biotype 1 was the most prevalent, followed by biotype 3, biotype 2, biotype 4 and biotype 5. These data are in accordance with other previously published work, wherein biotype 1 was the most frequently detected in wild boar [36,39,56,58,59]. The detected Yersinia enterocolitica was poorly virulent or non-pathogenic; indeed, most isolates harboured only one or none of the investigated virulence genes. The presence of few virulence genes in Yersinia enterocolitica isolates was also reported by other authors, highlighting that wild boar is a reservoir for nonpathogenic or less virulent strains [36,58,59,61]. Furthermore, this finding could be linked to the distribution of the detected biotypes. Indeed, in biotypes considered more virulent, as well as biotypes 3 and 5, more than one gene associated with virulence was found. Pathogenic bacteria belonging to this species were found in wild boar specimens, as well as non-pathogenic ones, strengthening the idea, suggested by some studies, that this animal could act as an accidental Yersinia carrier [43,60]. In this investigation, the ystA virulence gene was the most prevalent, as demonstrated for other wild boar isolates [62]. Moreover, several wild boar isolates scored positive for the inv [60], ail [59,62,63] and ystB [58,61] virulence genes, as obtained from samples here investigated. virF is a plasmid regulatory gene located on the virulence plasmid designated pYV; the plasmid genes encoded by this plasmid guide the invasion of Yersinia enterocolitica and enable bacteria to survive inside the human host, and for this reasons they are considered essential for pathogenesis [64]. All these observations confirm the low virulence of Y. enterocolitica strains circulating among wild boar, despite the high isolation rate. However, most investigated strains had the genes for enterotoxins production, which seem to play an important role in Yersinia-induced diarrhoea [65]. Antimicrobial resistance tests revealed a high resistance to penicillins and cephalosporins. In detail, all isolates showed resistance to cephalothin (100%), while high resistances were reported to amoxicillin-clavulanic acid (70.4%), ampicillin (55.6%) and cefoxitin (37.0%). The high resistance to cephalothin was well reported in isolates from domestic swine [66], livestock [67,68] and food [69][70][71]. Moreover, the resistance to penicillins and cephalosporins, including amoxicillin-clavulanic acid, ampicillin and cefoxitin, were also documented in wild boar Yersinia enterocolitica isolates [36,56]. For this bacterial species, an intrinsic resistance to β-lactams was suggested, and the obtained results seem to confirm these data [72]. Excluding this class of antimicrobials, a very low level of resistance was recorded among isolates, suggesting the scant involvement of Yersinia enterocolitica in this threatening phenomenon.
Only one Listeria monocytogenes isolate was obtained from the wild boar rectal swab. The same low infection ratio was previously reported in studies carried out in Japan and the Russian Federation [73,74]. On the other hand, some studies reported a high infection rate, although in these cases isolations from tonsils in particular were recorded [75][76][77][78][79]. These differences could be related to the geographic area, time and sampled organs. Moreover, most of the Listeria monocytogenes strains isolated from wild boar belong to serogroup IVb serovar 4b [74,79], as well as the one isolated in this investigation. Usually, the presence of Listeria monocytogenes serogroup IVb could be linked to the invasive strains that colonize wild animals that inhabit pristine environments, and so is related to wildlife with little contact with domestic animals and/or humans [79].

Study Area and Sampling
The investigated area (Tuscany region) is a very extensive area which comprises different ecosystems, from mountains to hilly areas, and also reaches the sea. The area, very rich in vegetation, is characterized by sandy coasts, swamps, wetlands, forests, and agriculture and farm areas. Many types of different wild animals are present, in particular wild boars (Sus scrofa), foxes (Vulpes vulpes), roe deer (Capreolus capreolus), fallow deer (Dama dama), hares (Lepus europaeus), hedgehogs (Erinaceus europaeus), badgers (Meles meles), porcupines (Hystrix cristata), wolf (Canis lupus), marmot (Marmota marmota), red squirrel (Sciurus vulgaris), common rabbit (Oryctolagus cuniculus) and different small rodent species, as well as a wide range of birds. Some zones of these areas host farm animals, in particular pig, cattle, sheep and horses, which are mainly bred in extensive or semiextensive conditions. This area is also characterized by the significant presence of hunting activity, in particular for wild boar. During hunting season, the numbers of this animal specimen that are hunted per year range from 100,000 to 150,000 [80].
During two hunting seasons (from November 2018 to January 2019 and from November 2019 to January 2020), rectal swab, spleen, and liver were collected from wild boar. All specimens were sampled from the Tuscany region (Central Italy), in detail from the provinces of Pisa, Livorno, Siena, and Grosseto. All animals included in the study were hunted during the authorized hunting season, following regional hunting law (Regolamento di Attuazione della Legge Regionale 12 Gennaio 1994, N. 3 D.P.G.R. 48/R/2017). No animals were specifically sacrificed for this study's purpose. Sampling was performed just before slaughtering procedures, and within 4 h after collection, swabs and organs were transported to the infectious disease laboratory of the Department of Veterinary Sciences, University of Pisa. During sampling, the hunting area, sex, and age of each animal were recorded. In particular, age was evaluated after assessing the degree of tooth eruption and wear of teeth of the lower jaw [81]. Due to the sampling being performed in collaboration with hunter companies, the sample size could not be predicted beforehand, and the authors collected samples from all possible hunted specimens.

Bacterial Isolation and Characterization
Salmonella spp. isolation was performed as previously described [23] from collected rectal swabs, spleens and livers. All isolates were serotyped by the "Istituto Zooprofilattico Sperimentale Lazio e Toscana, Rome section".
Yersinia enterocolitica isolation was performed as was earlier reported [82,83] from collected rectal swabs. Biochemical tests were done to distinguish the biotypes of the isolates [25].

Antimicrobial Resistance
For all Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolates, the antimicrobial susceptibility was evaluated using the disc diffusion test on Mueller Hinton Agar (Oxoid, Ltd., Basingstoke, UK) [88]. The following antibiotics (Oxoid) were em-

Virulence Genes
From each isolate, DNA was extracted with Quick-DNA Plus Kits (Zymo Research, Irvine, CA, USA), following the manufacturer's instructions, from overnight bacterial cultures.
On the other hand, as regards Yersinia enterocolitica isolates, the presence of the following virulence genes was evaluated using primers and protocols previously published: ail, virF, ystA, ystB and inv [94][95][96].

Statistical Analysis
Data were analysed with a chi-square (X 2 ) test. The statistical test was used to evaluate the infection ratio of each pathogen in relationship to sex (male or female), age class (young, sub-adult, or adult), province (Pisa, Lucca, Livorno, Grosseto, or Siena) and hunting season (2018/2019 or 2019/2020). The statistical significance threshold was set at a p value ≤ 0.05.

Conclusions
In conclusion, the results of this investigation highlight that a great variability is present among Salmonella and Yersinia enterocolitica serotypes circulating in free-ranging wild boar. Some strains were more virulent than others, especially the isolates belonging to Salmonella enterica subspecies diarizonae serotype 50:r:1,5,7, serotype Rubislaw and sub-species houtenae serotype 1,40:z4,z23, and Yersinia enterocolitica serotypes 3 and 5. Moreover, the single Listeria monocytogenes serogroup IVb serovar 4b isolation seems to be strictly related to wild animal infections. Finally, this investigation confirms that many virulent Salmonella, Yersinia enterocolitica and Listeria monocytogenes strains circulate among wild boar, which represent a source of pathogenic bacteria for humans, especially for hunters and wildlife stakeholders.

Conflicts of Interest:
The authors declare no conflict of interest.