Next Article in Journal
Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group Rickettsia Exposure
Previous Article in Journal
Challenges in Tick-Borne Pathogen Detection: The Case for Babesia spp. Identification in the Tick Vector
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 10
Issue 2
10.3390/pathogens10020093
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Giovanni Cilia
1,
Barbara Turchi
1,
Filippo Fratini
1,*,
Stefano Bilei
2,
Teresa Bossù
2,
Maria Laura De Marchis
2,
Domenico Cerri
1,
Maria Irene Pacini
1 and
Fabrizio Bertelloni
1
1
Department of Veterinary Sciences, University of Pisa, 56124 Pisa, Italy
2
Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, 00178 Rome, Italy
*
Author to whom correspondence should be addressed.
Pathogens 2021, 10(2), 93; https://doi.org/10.3390/pathogens10020093
Submission received: 30 December 2020 / Revised: 15 January 2021 / Accepted: 19 January 2021 / Published: 20 January 2021
Browse Figure
Versions Notes

Abstract

:
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.

1. 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 Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC) reports, salmonellosis, yersinosis and listeriosis are three of the most important zoonoses spreading in Europe, with around 91,622, 6823 and 2480 human cases per year, respectively [3]. Moreover, focusing on Italy, the last reported numbers of cases of salmonellosis, yersiniosis and listeriosis in humans were 3635, 14 and 180, respectively [3].
Wild animals could be responsible for this phenomenon due to contact with domestic animals, especially breeds in extensive farms, and to humans, due to their behaviour, which is more anthropized [4,5].
Among the impact of wildlife on the “One Health” approach, wild boar represents an important animal not only because its population constantly increased in number in Europe in the last few decades, but also because this animal is a reservoir for several zoonoses [6,7,8,9,10,11,12,13]. Some human categories, such as hunters, have a high risk of contracting infections from wildlife due to the contact with animal carcasses; however, considering the constant increasing of this animal’s population and its “colonization” of urban or suburban areas, common people could potentially come in contact with them, or with their secretions and excretions [14]. Although the direct transmission from wild boar to humans could be of great importance for public health, the contact and consequent sharing of pathogens between wild and domestic animals could amplify this problems [15]. Indeed, it is well documented that, especially in areas where extensive or semi-extensive farms are present, there is a high possibility of the transmission of bacteria and virus between wild boars and bred animals, in particular domestic pigs, but also ruminants [16,17,18,19,20].
Salmonella is a Gram-negative, rod-shaped, flagellated, and facultative type of anaerobes bacterium out of the family Enterobacteriaceae [21,22]. The Salmonella genus is divided into two broad species: Salmonella enterica and Salmonella bongori [21,22]. For Salmonella enterica, more than 2600 serovars have been isolated and described, and many of these are causes of illnesses in both humans and animals [21,23,24].
Yersinia enterocolitica belong to the Enterobacteriaceae family too, and are the causative agent of yersiniosis, an important zoonosis with symptoms ranging from mild, self-limiting diarrhoea to acute mesenteric lymphadenitis, and can sometimes develop into parenteral forms [25,26,27]. The bacterium has been divided into more than 70 serotypes based on differences in the structure of the somatic antigen, and into six biotypes based on its biochemical characteristics [25,27,28].
Listeria monocytogenes is a Gram-positive bacterium, which is facultative intracellular, and it causes listeriosis in humans and animals [29,30,31]. Listeria monocytogenes is diffused worldwide and spreads in every environment, such as soil, water and feces [29,30,31]. More than 90% of the Listeria infection epidemics and sporadic cases were carried out by strains that belong to 3 (1/2a, 1/2b and 4b) of the 13 serovars [29,32].
Wild boar, as well as domestic pigs, could be a possible reservoir of Salmonella, Yersinia and Listeria [33,34,35,36,37,38,39]. The constant screenings of various zoonotic pathogens are necessary due to the constant increases in wild boar population, and due to the consumption of meat.
The aim of this investigation was to investigate the occurrence of pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes in the wild boar population in the Tuscany region (Central Italy) during two consecutive hunting seasons.

2. Results

Rectal swabs, spleens and livers were collected from a total of 287 hunted wild boar. In total, 200 wild boar were sampled during the 2018/2019 hunting season (75 from Grosseto province, 58 from Pisa province, 55 from Siena province, and 12 from Livorno province). In addition, 87 animals were sampled during the 2019/2020 hunting seasons (38 from Pisa, 37 from Grosseto and 12 from Lucca) (Figure 1). Sampling was performed in collaboration with hunters, in relation to their availability. For this reason, the sample size could not be predicted beforehand, and for the Siena, Livorno and Lucca provinces, sampling was performed only during one hunting season.

2.1. Salmonella spp.

2.1.1. Isolation and Characterization

In total, 12 of the 287 (4.18%) animals scored positive, from which 18 Salmonella strains (6.27%) were isolated from collected wild boar samples, and 8 and 10 came from the 2018/2019 and from 2019/2020 hunting seasons, respectively. Detailed serotype characterization and the relationship between each isolate and wild boar organs are reported in Table 1. The isolates included the following: seven Salmonella enterica subspecies diarizonae serotype 50:r:1,5,7; four Salmonella enterica subspecies houtenae serotype 1,40:z4,z23; two Salmonella enterica subspecies enterica serotype Newport; two Salmonella enterica subspecies enterica serotype Kottbus; one Salmonella enterica subspecies enterica serotype London; one Salmonella enterica subspecies enterica serotype Infantis; and one Salmonella enterica subspecies enterica serotype Rubislaw.

2.1.2. 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.

2.1.3. 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.

2.2. Yersinia enterocolitica

2.2.1. Isolation and Characterization

In total, 71 Yersinia isolates (24.7%) were obtained from wild boar rectal swabs. Only 54 of these latter (18.8% of total) were biochemically confirmed as Yersinia enterocolitica (Table 2), while the other 17 were Yersinia frederiksenii or Yersinia intermedia. In total, 33 and 21 Yersinia enterocolotica cultures were collected during the 2018/2019 and 2019/2020 hunting seasons, respectively. Among the Yersinia enterocolitca isolates, 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 (Table 2).

2.2.2. Antimicrobial Resistance

High levels of antimicrobial resistance were detected in Yersinia enterocolitica (Table 2). All strains (100%) exhibited resistance to cephalothin. Moreover, resistance was reported in 38 out of 54 (70.4%) for amoxicillin-clavulanic acid, 30 of 54 (55.6%) for ampicillin, and 20 of 54 (37.0%) for cefoxitin. In total, 3 out of 54 (3.70%) isolates were resistant to aztreonam, nalidixic acid and nitrofurantoin. Finally, only 1 of the 54 (1.85%) strains was resistant to streptomycin, cefotaxime, tetracycline and enrofloxacin. None of the isolates showed resistance to imipenem, chloramphenicol, gentamycin, and sulfamethoxazole-trimethoprim.

2.2.3. Virulence Genes

A total of 63.0% of the isolates presented at least one virulence gene (Table 2). The most detected genes were ystA in 14 out of 54 isolates (25.9%), inv in 13 of 54 (24.1%), ail in 12 of 54 (22.2%), ystB in 10 of 54 (18.5%) and virF in 8 of 54 (14.8%).

2.3. 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.

2.3.2. Antimicrobial Resistance

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

2.4. 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.

3. 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 non-pathogenic 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].

4. Materials and Methods

4.1. 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 semi-extensive 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.

4.2. 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].
Listeria monocytogenes isolation was carried out according to Demaître et al. [84] from collected rectal swabs. Suspected L. monocytogenes isolates were confirmed by PCR based on the prfA gene [85]. Confirmed L. monocytogenes isolates were serotyped by multi-step PCR assays, to identify the following serotype groups: IIa (serovars 1/2a and 3a), IIc (serovars 1/2c and 3c), IIb (serovars 1/2b, 3b, and 7), and IVb (serovars 4b, 4d, and 4e) [86,87].

4.3. 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 employed: amoxicillin-clavulanic acid (AMC; 30 µg), ampicillin (AMP; 10 µg), aztreonam (ATM; 30 µg), cephalothin (KF; 30 µg), cefotaxime (CTX; 30 µg), cefoxitin (FOX; 30 µg), chloramphenicol (C; 30 µg), enrofloxacin (ENR; 5 µg), gentamycin (CN; 10 µg), imipenem (IPM; 10 µg), nalidixic acid (NA; 2 µg), nitrofurantoin (F; 300 µg), streptomycin (S; 10 µg), sulfamethoxazole-trimethoprim (STX; 25 µg), and tetracycline (TE; 30 µg). The zone diameter interpretive criteria suggested by Clinical & Laboratory Standards Institute (CLSI) were used [72].

4.4. 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.
Concerning the Salmonella spp. isolated, the presence of the following genes linked to virulence was evaluated using primers and protocols reported by other authors: mgtC, pipB, sopB, spvR, spvC, gipA, sodCI, sopE [89,90,91,92,93].
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].

4.5. Statistical Analysis

Data were analysed with a chi-square (X2) 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.

5. 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 subspecies 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.

Author Contributions

Conceptualization, G.C., F.F. and F.B.; investigation, G.C., B.T., S.B., T.B., M.L.D.M., M.I.P. and F.B.; data curation, G.C., B.T., M.I.P., F.F., D.C. and F.B.; writing—original draft preparation, G.C. and F.B.; writing—review and editing, G.C., B.T., F.F., S.B., T.B., M.L.D.M., M.I.P., D.C. and F.B.; supervision, D.C. and F.F.; funding acquisition, F.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the University of Pisa, grant number PRA_2018_56.

Institutional Review Board Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Rock, M.; Buntain, B.J.; Hatfield, J.M.; Hallgrímsson, B. Animal-human connections, “one health,” and the syndemic approach to prevention. Soc. Sci. Med. 2009, 68, 991–995. [Google Scholar] [CrossRef] [PubMed]
  2. Ryu, S.; Kim, B.I.; Lim, J.S.; Tan, C.S.; Chun, B.C. One health perspectives on emerging public health threats. J. Prev. Med. Public Health 2017, 50, 411–414. [Google Scholar] [CrossRef] [PubMed]
  3. EFSA, (European Food Safety Authority); ECDC, (European Centre for Disease Prevention and Control). The European Union one health 2018 zoonoses report. EFSA J. 2019, 17, 276. [Google Scholar]
  4. Bertelloni, F.; Turchi, B.; Vattiata, E.; Viola, P.; Pardini, S.; Cerri, D.; Fratini, F. Serological survey on Leptospira infection in slaughtered swine in North-Central Italy. Epidemiol. Infect. 2018, 146, 1275–1280. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Cahill, S.; Llimona, F.; Cabañeros, L.; Calomardo, F. Characteristics of wild boar (Sus scrofa) habituation to urban areas in the Collserola Natural Park (Barcelona) and comparison with other locations. Anim. Biodivers. Conserv. 2012, 35, 221–233. [Google Scholar] [CrossRef]
  6. Massei, G.; Kindberg, J.; Licoppe, A.; Gačić, D.; Šprem, N.; Kamler, J.; Baubet, E.; Hohmann, U.; Monaco, A.; Ozoliņš, J.; et al. Wild boar populations up, numbers of hunters down? A review of trends and implications for Europe. Pest Manag. Sci. 2015, 71, 492–500. [Google Scholar] [CrossRef]
  7. Bertelloni, F.; Mazzei, M.; Cilia, G.; Forzan, M.; Felicioli, A.; Sagona, S.; Bandecchi, P.; Turchi, B.; Cerri, D.; Fratini, F. Serological survey on bacterial and viral pathogens in wild boars hunted in tuscany. Ecohealth 2020, 17, 85–93. [Google Scholar] [CrossRef]
  8. Pacini, M.I.; Forzan, M.; Cilia, G.; Bernardini, L.; Marzoli, F.; Pedonese, F.; Bandecchi, P.; Fratini, F.; Mazzei, M. Detection of pseudorabies virus in wild boar foetus. Animals 2020, 10, 366. [Google Scholar] [CrossRef] [Green Version]
  9. Cilia, G.; Bertelloni, F.; Mignone, W.; Spina, S.; Berio, E.; Razzuoli, E.; Vencia, W.; Franco, V.; Cecchi, F.; Bogi, S.; et al. Molecular detection of Leptospira spp. in wild boar (Sus scrofa) hunted in Liguria region (Italy). Comp. Immunol. Microbiol. Infect. Dis. 2020, 68, 101410. [Google Scholar] [CrossRef]
  10. Montagnaro, S.; De Martinis, C.; Sasso, S.; Ciarcia, R.; Damiano, S.; Auletta, L.; Iovane, V.; Zottola, T.; Pagnini, U. Viral and antibody prevalence of hepatitis e in european wild boars (Sus scrofa) and hunters at zoonotic risk in the Latium Region. J. Comp. Pathol. 2015, 153, 1–8. [Google Scholar] [CrossRef]
  11. Ruiz-Fons, F.; Segalés, J.; Gortázar, C. A review of viral diseases of the European wild boar: Effects of population dynamics and reservoir rôle. Vet. J. 2008, 176, 158–169. [Google Scholar] [CrossRef] [PubMed]
  12. Cilia, G.; Bertelloni, F.; Piredda, I.; Ponti, M.N.; Turchi, B.; Cantinle, C.; Parisi, F.; Pinzauti, P.; Armani, A.; Palmas, B.; et al. Presence of pathogenic Leptospira spp. in the reproductive system and fetuses of wild boars (Sus scrofa) in Italy. PLoS Negl. Trop. Dis. 2020, 14, e0008982. [Google Scholar] [CrossRef] [PubMed]
  13. Bertelloni, F.; Cilia, G.; Bogi, S.; Ebani, V.V.; Turini, L.; Nuvoloni, R.; Cerri, D.; Fratini, F.; Turchi, B. Pathotypes and antimicrobial susceptibility of Escherichia coli isolated from wild boar (Sus scrofa) in Tuscany. Animals 2020, 10, 744. [Google Scholar] [CrossRef] [PubMed]
  14. Ruiz-Fons, F. A review of the current status of relevant zoonotic pathogens in wild swine (Sus scrofa ) populations: Changes modulating the risk of transmission to humans. Transbound. Emerg. Dis. 2017, 64, 68–88. [Google Scholar] [CrossRef]
  15. Kock, R. Drivers of disease emergence and spread: Is wildlife to blame? Onderstepoort J. Vet. Res. 2014, 81, 1–4. [Google Scholar] [CrossRef]
  16. Meng, X.J.; Lindsay, D.S.; Sriranganathan, N. Wild boars as sources for infectious diseases in livestock and humans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2009, 364, 2697–2707. [Google Scholar] [CrossRef] [Green Version]
  17. Barth, S.; Geue, L.; Hinsching, A.; Jenckel, M.; Schlosser, J.; Eiden, M.; Pietschmann, J.; Menge, C.; Beer, M.; Groschup, M.; et al. Experimental evaluation of faecal escherichia coli and hepatitis E virus as biological indicators of contacts between domestic pigs and eurasian wild boar. Transbound. Emerg. Dis. 2017, 64, 487–494. [Google Scholar] [CrossRef] [PubMed]
  18. Fretin, D.; Mori, M.; Czaplicki, G.; Quinet, C.; Maquet, B.; Godfroid, J.; Saegerman, C. Unexpected Brucella suis biovar 2 Infection in a dairy cow, Belgium. Emerg. Infect. Dis. 2013, 19, 2053–2054. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Barlozzari, G.; Franco, A.; Macrì, G.; Lorenzetti, S.; Maggiori, F.; Dottarelli, S.; Maurelli, M.; Di Giannatale, E.; Tittarelli, M.; Battisti, A.; et al. First report of Brucella suis biovar 2 in a semi free-range pig farm, Italy. Vet. Ital. 2015, 51, 151–154. [Google Scholar] [PubMed]
  20. Bertelloni, F.; Turchi, B.; Cerri, D.; Pinzauti, P.; Fratini, F. Leptospira spp. and Brucella ovis seroprevalence in sheep: Preliminary results of one year surveillance program. J. Hell. Vet. Med. Soc. 2017, 68, 567–572. [Google Scholar] [CrossRef] [Green Version]
  21. Jajere, S.M. A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors, host specificity and adaptation and antimicrobial resistance including multidrug resistance. Vet. World 2019, 12, 504–521. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Eng, S.-K.; Pusparajah, P.; Ab Mutalib, N.-S.; Ser, H.-L.; Chan, K.-G.; Lee, L.-H. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Front. Life Sci. 2015, 8, 284–293. [Google Scholar] [CrossRef] [Green Version]
  23. Bertelloni, F.; Tosi, G.; Massi, P.; Fiorentini, L.; Parigi, M.; Cerri, D.; Ebani, V.V. Some pathogenic characters of paratyphoid Salmonella enterica strains isolated from poultry. Asian Pac. J. Trop. Med. 2017, 10, 1161–1166. [Google Scholar] [CrossRef] [PubMed]
  24. Mezal, E.H.; Sabol, A.; Khan, M.A.; Ali, N.; Stefanova, R.; Khan, A.A. Isolation and molecular characterization of Salmonella enterica serovar Enteritidis from poultry house and clinical samples during 2010. Food Microbiol. 2014, 38, 67–74. [Google Scholar] [CrossRef] [PubMed]
  25. Bottone, E.J. Yersinia enterocolitica: The charisma continues. Clin. Microbiol. Rev. 1997, 10, 257–276. [Google Scholar] [CrossRef]
  26. Wren, B.W. The Yersiniae—A model genus to study the rapid evolution of bacterial pathogens. Nat. Rev. Microbiol. 2003, 1, 55–64. [Google Scholar] [CrossRef]
  27. Tirziu, E.; Cumpanasoiu, C.; Gros, R.V.; Seres, M. Yersinia enterocolitica Monographic Study. J. Anim. Sci. Biotechnol. 2011, 44, 144–149. [Google Scholar]
  28. Wauters, G.; Kandolo, K.; Janssens, M. Revised biogrouping scheme of Yersinia enterocolitica. Contrib. Microbiol. Immunol. 1987, 9, 14–21. [Google Scholar]
  29. Farber, J.M.; Peterkin, P.I. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 1991, 55, 476–511. [Google Scholar] [CrossRef]
  30. McLauchlin, J. Animal and human listeriosis: A shared problem? Vet. J. 1997, 153, 3–5. [Google Scholar] [CrossRef]
  31. Vázquez-Boland, J.A.; Kuhn, M.; Berche, P.; Chakraborty, T.; Domínguez-Bernal, G.; Goebel, W.; González-Zorn, B.; Wehland, J.; Kreft, J. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 2001, 14, 584–640. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Kathariou, S. Listeria monocytogenes virulence and pathogenicity, a food safety perspective. J. Food Prot. 2002, 65, 1811–1829. [Google Scholar] [CrossRef] [PubMed]
  33. Torres, R.T.; Fernandes, J.; Carvalho, J.; Cunha, M.V.; Caetano, T.; Mendo, S.; Serrano, E.; Fonseca, C. Wild boar as a reservoir of antimicrobial resistance. Sci. Total Environ. 2019, 717, 135001. [Google Scholar] [CrossRef] [PubMed]
  34. Zottola, T.; Montagnaro, S.; Magnapera, C.; Sasso, S.; De Martino, L.; Bragagnolo, A.; D’Amici, L.; Condoleo, R.; Pisanelli, G.; Iovane, G.; et al. Prevalence and antimicrobial susceptibility of Salmonella in European wild boar (Sus scrofa); Latium Region—Italy. Comp. Immunol. Microbiol. Infect. Dis. 2013, 36, 161–168. [Google Scholar] [CrossRef]
  35. Sannö, A.; Jacobson, M.; Sterner, S.; Thisted-Lambertz, S.; Aspán, A. The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA–data for Y. enterocolitica and Y. pseudotuberculosis. J. Microbiol. Methods 2018, 150, 32–38. [Google Scholar] [CrossRef] [PubMed]
  36. Takahashi, T.; Kabeya, H.; Sato, S.; Yamazaki, A.; Kamata, Y.; Taira, K.; Asakura, H.; Sugiyama, H.; Takai, S.; Maruyama, S. Prevalence of yersinia among wild sika deer (Cervus nippon) and boars (sus scrofa) in Japan. J. Wildl. Dis. 2020, 56, 270–277. [Google Scholar] [CrossRef]
  37. Wacheck, S.; Giezendanner, N.; König, M.; Fredriksson-Ahomaa, M.; Stephan, R. Phenotypical and genotypical traits of Listeria monocytogens strains isolated from tonsils of wild boars hunted in Switzerland. Int. J. Infect. Dis. 2010, 14, e162. [Google Scholar] [CrossRef] [Green Version]
  38. Sarangi, L.N.; Panda, H.K. Occurrence of Listeria species in different captive wild animals of Nandankanan Zoo, Baranga, Odisha, India. J. Threat. Taxa 2013, 5, 3542–3547. [Google Scholar] [CrossRef] [Green Version]
  39. Sannö, A.; Rosendal, T.; Aspán, A.; Backhans, A.; Jacobson, M. Distribution of enteropathogenic Yersinia spp. and Salmonella spp. in the Swedish wild boar population, and assessment of risk factors that may affect their prevalence. Acta Vet. Scand. 2018, 60, 1–9. [Google Scholar] [CrossRef] [Green Version]
  40. Cilia, G.; Bertelloni, F.; Angelini, M.; Cerri, D.; Fratini, F. Leptospira Survey in Wild Boar (Sus scrofa) Hunted in Tuscany, Central Italy. Pathogens 2020, 9, 377. [Google Scholar] [CrossRef]
  41. Botti, V.; Valérie Navillod, F.; Domenis, L.; Orusa, R.; Pepe, E.; Robetto, S.; Guidetti, C. Salmonella spp. and antibiotic-resistant strains in wild mammals and birds in north-western Italy from 2002 to 2010. Vet. Ital. 2013, 49, 195–202. [Google Scholar] [PubMed]
  42. Molino, G.M.; Sánchez, G.A.; Pérez, R.D.; Blanco, G.P.; Molina, Q.A.; Pérez, R.J.; Cano, M.F.E.; Horrillo, C.R.; Salcedo, H.-d.-M.J.; Llario, F.P. Prevalence of Salmonella spp. in tonsils, mandibular lymph nodes and faeces of wild boar from Spain and genetic relationship between isolates. Transbound. Emerg. Dis. 2019, 66, 1218–1226. [Google Scholar] [CrossRef] [PubMed]
  43. Sannö, A.; Aspán, A.; Hestvik, G.; Jacobson, M. Presence of Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis and Escherichia coli O157:H7 in wild boars. Epidemiol. Infect. 2014, 142, 2542–2547. [Google Scholar] [CrossRef] [PubMed]
  44. Wacheck, S.; Fredriksson-Ahomaa, M.; König, M.; Stolle, A.; Stephan, R. Wild Boars as an important reservoir for foodborne pathogens. Foodborne Pathog. Dis. 2010, 7, 307–312. [Google Scholar] [CrossRef] [PubMed]
  45. Vieira-Pinto, M.; Morais, L.; Caleja, C.; Themudo, P.; Torres, C.; Igrejas, G.; Poeta, P.; Martins, C. Salmonella sp. in Game (Sus scrofa and Oryctolagus cuniculus ). Foodborne Pathog. Dis. 2011, 8, 739–740. [Google Scholar] [CrossRef]
  46. Wahlström, H.; Tysén, E.; Engvall, E.O.; Brändström, B.; Eriksson, E.; Mörner, T.; Vågsholm, I. Survey of Campylobacter species, VTEC 0157 and Salmonella species in Swedish wildlife. Vet. Rec. 2003, 153, 74–80. [Google Scholar] [CrossRef]
  47. Vieira-Pinto, M.; Morais, L.; Caleja, C.; Themudo, P.; Aranha, J.; Torres, C.; Igrejas, G.; Poeta, P.; Martins, C. Salmonella spp. in wild boar (Sus scrofa): A public and animal health concern. In Game Meat Hygiene in Focus; Wageningen Academic Publishers: Wageningen, The Netherlands, 2011; pp. 131–136. [Google Scholar]
  48. Chiari, M.; Zanoni, M.; Tagliabue, S.; Lavazza, A.; Alborali, L.G. Salmonella serotypes in wild boars (Sus scrofa) hunted in northern Italy. Acta Vet. Scand. 2013, 55, 42. [Google Scholar] [CrossRef] [Green Version]
  49. Methner, U.; Heller, M.; Bocklisch, H. Salmonella enterica subspecies enterica serovar Choleraesuis in a wild boar population in Germany. Eur. J. Wildl. Res. 2010, 56, 493–502. [Google Scholar] [CrossRef]
  50. Caleja, C.; de Toro, M.; Gonçalves, A.; Themudo, P.; Vieira-Pinto, M.; Monteiro, D.; Rodrigues, J.; Sáenz, Y.; Carvalho, C.; Igrejas, G.; et al. Antimicrobial resistance and class I integrons in Salmonella enterica isolates from wild boars and Bísaro pigs. Int. Microbiol. 2011, 14, 19–24. [Google Scholar]
  51. Bonardi, S.; Alpigiani, I.; Bruini, I.; Barilli, E.; Brindani, F.; Morganti, M.; Cavallini, P.; Bolzoni, L.; Pongolini, S. Detection of Salmonella enterica in pigs at slaughter and comparison with human isolates in Italy. Int. J. Food Microbiol. 2016, 218, 44–50. [Google Scholar] [CrossRef]
  52. Fois, F.; Piras, F.; Torpdahl, M.; Mazza, R.; Consolati, S.G.; Spanu, C.; Scarano, C.; De Santis, E.P.L. Occurrence, characterization, and antimicrobial susceptibility of salmonella enterica in slaughtered pigs in Sardinia. J. Food Sci. 2017, 82, 969–976. [Google Scholar] [CrossRef] [PubMed]
  53. Proroga, Y.T.R.; Mancusi, A.; Peruzy, M.F.; Carullo, M.R.; Montone, A.M.I.; Fulgione, A.; Capuano, F. Characterization of salmonella typhimurium and its monophasic variant 1,4, [5],12:i:- isolated from different sources. Folia Microbiol. 2019, 64, 711–718. [Google Scholar] [CrossRef] [PubMed]
  54. Nguyen Thi, H.; Pham, T.-T.-T.; Turchi, B.; Fratini, F.; Virginia Ebani, V.; Cerri, D.; Bertelloni, F. Characterization of salmonella spp. isolates from swine: Virulence and antimicrobial resistance. Animals 2020, 10, 2418. [Google Scholar] [CrossRef] [PubMed]
  55. Bertelloni, F.; Chemaly, M.; Cerri, D.; Le Gall, F.; Ebani, V.V. Salmonella infection in healthy pet reptiles: Bacteriological isolation and study of some pathogenic characters. Acta Microbiol. Immunol. Hung. 2016, 63, 203–216. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Von Altrock, A.; Seinige, D.; Kehrenberg, C. Yersinia enterocolitica isolates from wild boars hunted in Lower Saxony, Germany. Appl. Environ. Microbiol. 2015, 81, 4835–4840. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Bancerz-Kisiel, A.; Platt-Samoraj, A.; Szczerba-Turek, A.; SyczyŁo, K.; Szweda, W. The first pathogenic Yersinia enterocolitica bioserotype 4/O: 3 strain isolated from a hunted wild boar (Sus scrofa) in Poland. Epidemiol. Infect. 2015, 143, 2758–2765. [Google Scholar] [CrossRef]
  58. Bancerz-Kisiel, A.; Socha, P.; Szweda, W. Detection and characterisation of Yersinia enterocolitica strains in cold-stored carcasses of large game animals in Poland. Vet. J. 2016, 208, 102–103. [Google Scholar] [CrossRef]
  59. Syczyło, K.; Platt-Samoraj, A.; Bancerz-Kisiel, A.; Szczerba-Turek, A.; Pajdak-Czaus, J.; Łabuć, S.; Procajło, Z.; Socha, P.; Chuzhebayeva, G.; Szweda, W. The prevalence of Yersinia enterocolitica in game animals in Poland. PLoS ONE 2018, 13, e0195136. [Google Scholar] [CrossRef]
  60. Bonardi, S.; Brémont, S.; Vismarra, A.; Poli, I.; Diegoli, G.; Bolzoni, L.; Corradi, M.; Gilioli, S.; Le Guern, A.S. Is Yersinia bercovieri Surpassing Yersinia enterocolitica in Wild Boars (Sus scrofa)? Ecohealth 2020, 17, 388–392. [Google Scholar] [CrossRef]
  61. Bancerz-Kisiel, A.; Szczerba-Turek, A.; Platt-Samoraj, A.; Michalczyk, M.; Szweda, W. A study of single nucleotide polymorphism in the ystB gene of Yersinia enterocolitica strains isolated from various wild animal species. Ann. Agric. Environ. Med. 2017, 24, 56–61. [Google Scholar] [CrossRef]
  62. Weiner, M.; Kubajka, M.; Szulowski, K.; Iwaniak, W.; Krajewska, M.; Lipiec, M. Genotypic virulence markers of Yersinia enterocolitica O:9 isolated from pigs and wild boars serologically positive and negative for brucellosis. Bull. Vet. Inst. Pulawy 2014, 58, 541–545. [Google Scholar] [CrossRef] [Green Version]
  63. Fredriksson-Ahomaa, M.; Wacheck, S.; Bonke, R.; Stephan, R. Different enteropathogenic Yersinia strains found in wild boars and domestic pigs. Foodborne Pathog. Dis. 2011, 8, 733–737. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Fàbrega, A.; Vila, J. Yersinia enterocolitica: Pathogenesis, virulence and antimicrobial resistance. Enferm. Infecc. Microbiol. Clin. 2012, 30, 24–32. [Google Scholar] [CrossRef] [PubMed]
  65. Bancerz-Kisiel, A.; Pieczywek, M.; Łada, P.; Szweda, W. The most important virulence markers of yersinia enterocolitica and their role during infection. Genes 2018, 9, 235. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Terentjeva, M.; Bẽrziņš, A. Prevalence and antimicrobial resistance of Yersinia enterocolitica and Yersinia pseudotuberculosis in Slaughter Pigs in Latvia. J. Food Prot. 2010, 73, 1335–1338. [Google Scholar] [CrossRef]
  67. Baumgartner, A.; Küffer, M.; Suter, D.; Jemmi, T.; Rohner, P. Antimicrobial resistance of Yersinia enterocolitica strains from human patients, pigs and retail pork in Switzerland. Int. J. Food Microbiol. 2007, 115, 110–114. [Google Scholar] [CrossRef]
  68. Von Altrock, A.; Roesler, U.; Merle, R.; Waldmann, K.H. Prevalence of pathogenic Yersinia enterocolitica strains on liver surfaces of pigs and their antimicrobial susceptibility. J. Food Prot. 2010, 73, 1680–1683. [Google Scholar] [CrossRef]
  69. Younis, G.; Mady, M.; Awad, A. Yersinia enterocolitica: Prevalence, virulence, and antimicrobial resistance from retail and processed meat in Egypt. Vet. World 2019, 12, 1078–1084. [Google Scholar] [CrossRef]
  70. Andualem, B.; Geyid, A. Antimicrobial responses of Yersinia enterocolitica isolates in comparison to other commonly encountered bacteria that causes diarrhoea. East Afr. Med. J. 2005, 82, 241–246. [Google Scholar] [CrossRef]
  71. Bhaduri, S.; Wesley, I.; Richards, H.; Draughon, A.; Wallace, M. Clonality and antibiotic susceptibility of Yersinia enterocolitica isolated from U.S. market weight hogs. Foodborne Pathog. Dis. 2009, 6, 351–356. [Google Scholar] [CrossRef] [Green Version]
  72. CLSI (Clinical and Laboratory Standards Institute). M100 Performance Standards for Antimicrobial Susceptibility Testing A CLSI Supplement for Global Application, 28th ed.; CLSI supplement M100; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
  73. Olga, V.L.; Natalia, R.N.; Marina, K.S.; Margarita, K.A.; Andrey, S.N.; Ekaterina, A.I.; Irina Yu, E.; Denis, K.V.; Svetlana, E.A.; Alexander, G.L. Diversity and pathogenic potential of listeria monocytogenes isolated from environmental sources in the Russian Federation. Int. J. Mod. Eng. Res. 2015, 5, 5–15. [Google Scholar]
  74. Hayashidani, H.; Kanzaki, N.; Kaneko, Y.; Okatani, A.T.; Taniguchi, T.; Kaneko, K.I.; Ogawa, M. Occurrence of Yersiniosis and listeriosis in wild boars in Japan. J. Wildl. Dis. 2002, 38, 202–205. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Bakulov, I.A.; Egorova, I.Y.; Fertikov, V.I.; Voronin, M.S. Spread of Listeria in fauna of the central region of Russia. Russ. Agric. Sci. 2010, 36, 58–60. [Google Scholar] [CrossRef]
  76. Stella, S.; Tirloni, E.; Castelli, E.; Colombo, F.; Bernardi, C. Microbiological evaluation of carcasses of wild boar hunted in a hill area of northern Italy. J. Food Prot. 2018, 81, 1519–1525. [Google Scholar] [CrossRef] [PubMed]
  77. Weindl, L.; Frank, E.; Ullrich, U.; Heurich, M.; Kleta, S.; Ellerbroek, L.; Gareis, M. Listeria monocytogenes in different specimens from healthy red deer and wild boars. Foodborne Pathog. Dis. 2016, 13, 391–397. [Google Scholar] [CrossRef] [PubMed]
  78. Kanai, Y.; Hayashidani, H.; Kaneko, K.I.; Ogawa, M.; Takahashi, T.; Nakamura, M. Occurrence of zoonotic bacteria in retail game meat in Japan with special reference to Erysipelothrix. J. Food Prot. 1997, 60, 328–331. [Google Scholar] [CrossRef] [PubMed]
  79. Zaytseva, E.; Ermolaeva, S.; Somov, G.P. Low genetic diversity and epidemiological significance of Listeria monocytogenes isolated from wild animals in the far east of Russia. Infect. Genet. Evol. 2007, 7, 736–742. [Google Scholar] [CrossRef]
  80. Consiglio Regionale della Toscana. Nota Informativa Sull’attuazione delle Politiche Regionali n° 40; Consiglio Regionale della Toscana: Tuscany, Italy, 2018. [Google Scholar]
  81. Sáez-Royuela, C.; Gomariz, R.P.; Luis Tellería, J. Age determination of European wild boar. Wildl. Soc. Bull. 1989, 17, 326–329. [Google Scholar]
  82. Fredriksson-Ahomaa, M.; Korkeala, H. Low occurrence of pathogenic Yersinia enterocolitica in clinical, food, and environmental samples: A methodological problem. Clin. Microbiol. Rev. 2003, 16, 220–229. [Google Scholar] [CrossRef] [Green Version]
  83. Wang, X.; Cui, Z.; Jin, D.; Tang, L.; Xia, S.; Wang, H.; Xiao, Y.; Qiu, H.; Hao, Q.; Kan, B.; et al. Distribution of pathogenic Yersinia enterocolitica in China. Eur. J. Clin. Microbiol. Infect. Dis. 2009, 28, 1237–1244. [Google Scholar] [CrossRef]
  84. Demaître, N.; Van Damme, I.; De Zutter, L.; Geeraerd, A.H.; Rasschaert, G.; De Reu, K. Occurrence, distribution and diversity of Listeria monocytogenes contamination on beef and pig carcasses after slaughter. Meat Sci. 2020, 169, 108177. [Google Scholar] [CrossRef]
  85. D’Agostino, M.; Wagner, M.; Vazquez-Boland, J.A.; Kuchta, T.; Karpiskova, R.; Hoorfar, J.; Novella, S.; Scortti, M.; Ellison, J.; Murray, A.; et al. A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model—Towards an international standard. J. Food Prot. 2004, 67, 1646–1655. [Google Scholar] [CrossRef] [PubMed]
  86. Doumith, M.; Buchrieser, C.; Glaser, P.; Jacquet, C.; Martin, P. Differentiation of the major listeria monocytogenes serovars by multiplex PCR. J. Clin. Microbiol. 2004, 42, 3819–3822. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. Borucki, M.K.; Call, D.R. Listeria monocytogenes serotype identification by PCR. J. Clin. Microbiol. 2003, 41, 5537–5540. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  88. Clinical and Laboratory Standards Institute. M02-A12 Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard, 12th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2015. [Google Scholar]
  89. Skyberg, J.A.; Logue, C.M.; Nolan, L.K. Virulence genotyping of Salmonella spp. with multiplex PCR. Avian Dis. 2006, 50, 77–81. [Google Scholar] [CrossRef] [Green Version]
  90. Huehn, S.; La Ragione, R.M.; Anjum, M.; Saunders, M.; Woodward, M.J.; Bunge, C.; Helmuth, R.; Hauser, E.; Guerra, B.; Beutlich, J.; et al. Virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in Europe. Foodborne Pathog. Dis. 2010, 7, 523–535. [Google Scholar] [CrossRef]
  91. Paban Bhowmick, P.; Devegowda, D.; Karunasagar, I. Virulotyping of seafood associated Salmonella enterica subsp. enterica isolated from Southwest coast of India. Res. Artic. Biotechnol. Bioinf. Bioeng. 2011, 1, 63–69. [Google Scholar]
  92. Parvathi, A.; Vijayan, J.; Murali, G.; Chandran, P. Comparative virulence genotyping and antimicrobial susceptibility profiling of environmental and clinical salmonella enterica from Cochin, India. Curr. Microbiol. 2011, 62, 21–26. [Google Scholar] [CrossRef]
  93. Karasova, D.; Havlickova, H.; Sisak, F.; Rychlik, I. Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection. Vet. Microbiol. 2009, 139, 304–309. [Google Scholar] [CrossRef]
  94. Thoerner, P.; Kingombe, C.I.B.; Bögli-Stuber, K.; Bissig-Choisat, B.; Wassenaar, T.M.; Frey, J.; Jemmi, T. PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution. Appl. Environ. Microbiol. 2003, 69, 1810–1816. [Google Scholar] [CrossRef] [Green Version]
  95. Thisted Lambertz, S.; Danielsson-Tham, M.L. Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis. Appl. Environ. Microbiol. 2005, 71, 3674–3681. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  96. Falcão, J.P.; Falcão, D.P.; Pitondo-Silva, A.; Malaspina, A.C.; Brocchi, M. Molecular typing and virulence markers of Yersinia enterocolitica strains from human, animal and food origins isolated between 1968 and 2000 in Brazil. J. Med. Microbiol. 2006, 55, 1539–1548. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Pathogens 10 00093 g001 550
Figure 1. Geographical distribution of the sampling area included in the study (Tuscany region, Central Italy). The number of sampled hunted wild boar per province is indicated in relation to hunting seasons. (A) Hunting season 2018/2019. (B) Hunting season 2019/2020. (This figure was adapted from Cilia et al., 2020 [40]).
Figure 1. Geographical distribution of the sampling area included in the study (Tuscany region, Central Italy). The number of sampled hunted wild boar per province is indicated in relation to hunting seasons. (A) Hunting season 2018/2019. (B) Hunting season 2019/2020. (This figure was adapted from Cilia et al., 2020 [40]).
Pathogens 10 00093 g001
Table
Table 1. Virulence genes and antimicrobial resistance profiles of analysed and characterized Salmonella spp. strains in relation to samples of isolation.
Table 1. Virulence genes and antimicrobial resistance profiles of analysed and characterized Salmonella spp. strains in relation to samples of isolation.
IsolateSerotypeSourceWild BoarProvinceHunting SeasonVirulence Genes ProfileAntimicrobial Resistance Profile
S345NewportLC12Siena2018/2019 KF
S346NewportSpC12Siena2018/2019 KF
S347LondonRC51Grosseto2018/2019pipB
S349InfantisSpC103Pisa2018/2019sopB, sodCITE, ENR, S, F, NA
S35250:r:1,5,7LC141Grosseto2018/2019sopB, sopES
S35350:r:1,5,7SpC141Grosseto2018/2019sopB, sopES
S35450:r:1,5,7SpC196Grosseto2018/2019pipB, spvC, spvR, sodCI
S35550:r:1,5,7RC196Grosseto2018/2019pipB, spvC, spvR, sodCI
S38250:r:1,5,7SpC203Grosseto2019/2020spvC, spvRS
S38350:r:1,5,7RC209Lucca2019/2020sopB, pipB, mgtC, sopE, sodCIIPM
S387RubislawLC209Lucca2019/2020sopB, pipB, mgtC, sopE, sodCIS
S38650:r:1,5,7RC216Grosseto2019/2020mgtC, sodCIS
S389KottbusRC218Grosseto2019/2020pipB, sopE
S390KottbusSpC218Grosseto2019/2020pipB, sopE
S3911,40:z4,z23RC263Lucca2019/2020spvC, spvRS
S3941,40:z4,z23LC270Lucca2019/2020spvC, spvR, sopES
S3921,40:z4,z23RC271Lucca2019/2020spvC, spvR, sopE, sodCIS
S3931,40:z4,z23LC271Lucca2019/2020spvC, spvR, sopE, sodCIS
L: liver; Sp: spleen; R: rectal swab; KF: cephalothin; TE: tetracycline; ENR: enrofloxacin; S: streptomycin; F: nitrofurantoin; NA: nalidixic acid; IPM: imipenem.
Table
Table 2. Virulence genes and antimicrobial resistance profiles of analysed Yersinia enterocolitica isolated.
Table 2. Virulence genes and antimicrobial resistance profiles of analysed Yersinia enterocolitica isolated.
IsolateBiotypeWild BoarProvinceHunting SeasonVirulence Genes ProfileAntimicrobial Resistance Profile
YC11C10Siena2018/2019 AMP, AMC, KF
YC21C11Siena2018/2019invKF, FOX
YC43C24Livorno2018/2019 AMP, AMC, KF, FOX
YC63C36Grosseto2018/2019ystAAMP, AMC, KF, FOX
YC73C37Grosseto2018/2019ailAMP, KF, FOX
YC113C23Livorno2018/2019virFAMP, KF, FOX
YC121C30Pisa2018/2019ystAAMP, KF
YC133C54Grosseto2018/2019ystA, ystB, invAMP, AMC, KF, FOX
YC143C53Grosseto2018/2019ailAMP, KF, FOX
YC151C49Livorno2018/2019virFAMP, KF
YC161C74Pisa2018/2019ystAAMP, KF
YC171C92Pisa2018/2019 AMP, KF, FOX
YC181C94Pisa2018/2019ail, ystBKF
YC203C56Grosseto2018/2019virF, invKF
YC212C48Siena2018/2019ystAKF
YC272C113Grosseto2018/2019 AMP, AMC, KF, FOX
YC291C140Grosseto2018/2019 AMP, AMC, KF, FOX
YC302C139Grosseto2018/2019ystAAMP, AMC, KF
YC313C124Siena2018/2019invAMP, KF
YC323C132Grosseto2018/2019ail, ystAKF
YC335C134Grosseto2018/2019ail, ystB, invKF
YC341C145Pisa2018/2019 AMP, AMC, KF, FOX
YC353C146Pisa2018/2019ail, ystA, ystBAMP, AMC, KF
YC372C149Pisa2018/2019 AMP, KF
YC382C150Pisa2018/2019 KF, F
YC392C151Pisa2018/2019 AMP, AMC, KF, FOX
YC443C163Grosseto2018/2019ail, ystBKF
YC452C172Pisa2018/2019 KF
YC463C173Pisa2018/2019ystAAMP, KF, S, ATM, NA
YC472C176Pisa2018/2019 KF, CTX, TE, ENR, FOX, ATM, NA
YC483C174Pisa2018/2019invAMP, ATM, KF, FOX
YC492C193Grosseto2018/2019virFAMP, KF
YC503C197Grosseto2018/2019 AMP, KF
YC513C202Grosseto2019/2020ail, invAMP, AMC, KF, FOX
YC521C229Grosseto2019/2020ystAAMC, KF
YC531C230Grosseto2019/2020 KF, FOX
YC541C232Pisa2019/2020 AMP, AMC, KF
YC551C241Pisa2019/2020virFAMP, AMC, KF, FOX
YC563C240Pisa2019/2020ailKF
YC574C244Pisa2019/2020ystB, invAMP, AMC, KF
YC581C249Grosseto2019/2020 AMP, AMC, KF, FOX
YC591C252Pisa2019/2020invAMP, KF
YC601C261Pisa2019/2020ystAAMP, AMC, KF
YC611C258Pisa2019/2020ystB, invAMP, AMC, KF, FOX
YC621C263Lucca2019/2020 AMP, AMC, KF, FOX
YC633C262Lucca2019/2020ail, virFAMC, KF
YC641C264Lucca2019/2020 AMC, KF, FOX
YC651C265Pisa2019/2020ystA, invAMP, AMC, KF, FOX
YC661C269Pisa2019/2020 AMP, AMC, KF, FOX
YC671C266Pisa2019/2020invAMP, AMC, KF
YC681C272Lucca2019/2020ystAAMP, AMC, KF
YC691C275Lucca2019/2020 AMP, AMC, KF
YC701C281Pisa2019/2020 AMC, KF
YC711C282Pisa2019/2020ystBAMC, KF
AMP: amoxicillin-clavulanic acid; AMC: ampicillin; KF: cephalothin; FOX; cefoxitin; S: streptomycin; ATM: aztreonam; NA: nalidixic acid; CTX: cefotaxime; TE: tetracycline; ENR: enrofloxacin; F: nitrofurantoin.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Cilia, G.; Turchi, B.; Fratini, F.; Bilei, S.; Bossù, T.; De Marchis, M.L.; Cerri, D.; Pacini, M.I.; Bertelloni, F. Prevalence, Virulence and Antimicrobial Susceptibility of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes in European Wild Boar (Sus scrofa) Hunted in Tuscany (Central Italy). Pathogens 2021, 10, 93. https://doi.org/10.3390/pathogens10020093

AMA Style

Cilia G, Turchi B, Fratini F, Bilei S, Bossù T, De Marchis ML, Cerri D, Pacini MI, Bertelloni F. Prevalence, Virulence and Antimicrobial Susceptibility of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes in European Wild Boar (Sus scrofa) Hunted in Tuscany (Central Italy). Pathogens. 2021; 10(2):93. https://doi.org/10.3390/pathogens10020093

Chicago/Turabian Style

Cilia, Giovanni, Barbara Turchi, Filippo Fratini, Stefano Bilei, Teresa Bossù, Maria Laura De Marchis, Domenico Cerri, Maria Irene Pacini, and Fabrizio Bertelloni. 2021. "Prevalence, Virulence and Antimicrobial Susceptibility of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes in European Wild Boar (Sus scrofa) Hunted in Tuscany (Central Italy)" Pathogens 10, no. 2: 93. https://doi.org/10.3390/pathogens10020093

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop