*Article* **Characterisation of** *Listeria monocytogenes* **Isolates from Hunted Game and Game Meat from Finland**

**Maria Fredriksson-Ahomaa 1,\*, Mikaela Sauvala 1, Paula Kurittu 1, Viivi Heljanko 1, Annamari Heikinheimo 1,2 and Peter Paulsen <sup>3</sup>**


**Abstract:** *Listeria monocytogenes* is an important foodborne zoonotic bacterium. It is a heterogeneous species that can be classified into lineages, serogroups, clonal complexes, and sequence types. Only scarce information exists on the properties of *L. monocytogenes* from game and game meat. We characterised 75 *L. monocytogenes* isolates from various game sources found in Finland between 2012 and 2020. The genetic diversity, presence of virulence and antimicrobial genes were studied with whole genome sequencing. Most (89%) of the isolates belonged to phylogenetic lineage (Lin) II and serogroup (SG) IIa. SGs IVb (8%) and IIb (3%) of Lin I were sporadically identified. In total, 18 clonal complexes and 21 sequence types (STs) were obtained. The most frequent STs were ST451 (21%), ST585 (12%) and ST37 (11%) found in different sample types between 2012 and 2020. We observed 10 clusters, formed by closely related isolates with 0–10 allelic differences. Most (79%) of the virulence genes were found in all of the *L. monocytogenes* isolates. Only *fos*X and *lin* were found out of 46 antimicrobial resistance genes. Our results demonstrate that potentially virulent and antimicrobial-sensitive *L. monocytogenes* isolates associated with human listeriosis are commonly found in hunted game and game meat in Finland.

**Keywords:** *Listeria monocytogenes*; game; whole genome sequencing; sequence type; virulence; antimicrobial resistance

#### **1. Introduction**

*Listeria monocytogenes* has emerged over recent decades as an important foodborne pathogen responsible for numerous outbreaks [1]. *L. monocytogenes* is responsible for listeriosis, a disease affecting both humans and animals. Foodborne listeriosis typically causes a self-limited gastroenteritis among healthy people [2]. However, invasive infection leading to hospitalisation and even death may occur, especially among immunocompromised people [1]. Invasive listeriosis may also lead to abortion in pregnant women. The severity of listeriosis depends, inter alia, on the virulence of the bacterial strain [2]. Invasive listeriosis, in particular, requires antimicrobial treatment. Listeriosis had the highest proportion of hospitalised cases of all zoonoses in 2020 in the EU [3].

*L. monocytogenes* is a ubiquitous bacterium that can survive in a variety of environments and grow at low temperatures, e.g., in cold-stored foods [4]. Soil and decaying organic material are important sources of *L. monocytogenes*, and mammals and birds can spread this pathogen through faecal shedding [5]. *L. monocytogenes-*contaminated food is an important source attributed to human infections [6]. The consumption of contaminated food has been linked to both epidemic and sporadic listeriosis. Poor hygiene practices and inadequate sanitation procedures in the food processing industry can lead to listeriosis outbreaks [4,7].

**Citation:** Fredriksson-Ahomaa, M.; Sauvala, M.; Kurittu, P.; Heljanko, V.; Heikinheimo, A.; Paulsen, P. Characterisation of *Listeria monocytogenes* Isolates from Hunted Game and Game Meat from Finland. *Foods* **2022**, *11*, 3679. https:// doi.org/10.3390/foods11223679

Academic Editor: Arun K. Bhunia

Received: 11 October 2022 Accepted: 14 November 2022 Published: 17 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*L. monocytogenes* has sporadically been found in game animals and on game carcasses [8–11]. Detection rates of *L. monocytogenes* in deer and wild boar faeces have been low, varying between 0 and 6% [12]. However, this pathogen is more frequently present in the tonsils than in faeces [12–15]. In Spain, *L. monocytogenes* was detected in 44% and 41% of deer and wild boar tonsils, respectively [12]. Recently, *L. monocytogenes* was detected in 5% of deer carcasses in Austria using an antigen test [16] and in 12% of deer carcasses in Finland using a polymerase chain reaction (PCR) [17]. This relatively high prevalence of *L. monocytogenes* on deer carcasses shows the importance of observing hygiene practices during hunting and slaughtering.

*L. monocytogenes* is a very heterogeneous species, which can be divided into at least 14 serotypes based on variation in the somatic (O) and flagellar (H) antigens [18,19]. Over 95% of the human and food strains are linked to four (1/2a, 1/2b, 1/2c, and 4b) serotypes. Genetically, *L. monocytogenes* can be divided into four phylogenetic lineages (Lin), six serogroups (SGs), multiple clonal complexes (CCs), and sequence types (STs) [2,5]. Most clinical strains found in humans belong to Lin I (SGs IIb and IVb) and II (SG IIa) [20], whereas food strains more frequently belong to SG IIa [21,22].

Whole genome sequencing (WGS)—an accurate method with a high resolution—is currently becoming the method of choice for characterising *L. monocytogenes* isolates [20]. It has emerged as a powerful tool for outbreak investigations and is increasingly also used for the surveillance and monitoring of listeriosis [23]. Investigating the diversity of *L. monocytogenes* isolates from game and game meat will provide valuable information on the significance of game in the meat production chain and in human infections.

Studies on the genetic relationships between *L. monocytogenes* isolates from game sources remain scarce. Game and game meat may play an important role in the *L. monocytogenes* infection cycle. The aim of our study was to use WGS to investigate the diversity and genetic relationships between *L. monocytogenes* isolates from hunted game and game meat in Finland. Furthermore, we studied the presence of important virulence and resistance genes obtained from the WGS data.

#### **2. Materials and Methods**

#### *2.1. Listeria Monocytogenes Isolates*

*L. monocytogenes* has been detected in hunted game and game meat in Finland between 2012 and 2020, especially in deer and mallard meat (Table 1). We characterised a total of 75 *L. monocytogenes* isolates from various game sources in this study (Table 1). One isolate per positive sample from the earlier studies was characterised. The sampling plan and time frame differed between the earlier studies (Table 1). Moose, deer, and wild boar samples were collected from wild hunted animals and game bird samples from game birds that were farmed for hunting. Deer and mallard meat samples were collected from a small meat processing plant. *L. monocytogenes* isolates were found after PCR screening in our microbiological laboratory at the Department of Food Hygiene and Environmental Health of the Faculty of Veterinary Medicine, University of Helsinki, in Helsinki, Finland. PCR screening and isolation of the isolates have been described in earlier studies [17,24].

**Table 1.** Isolation rates of *Listeria monocytogenes* from hunted game and game meat in Finland between 2012 and 2020.


#### *2.2. Whole Genome Sequencing (WGS)*

DNA of *L. monocytogenes* isolates was purified from overnight enrichment at 37 ◦C in tryptic soya broth using PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbaden, CA, USA) according to the manufacturer's protocol. DNA quality was measured with a NanoDrop™ spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and DNA quantity with a Qubit fluorometer (ThermoFisher Scientific). WGS was performed on the Illumina platform by CeGaT (Center for Genomics and Transcriptomics, Tübingen, Germany). Illumina DNA Prep library preparation kit and NovaSeq6000 were used to generate 100 bp paired end reads. The short raw reads were assembled de novo using a Unicycler v0.4.8 assembler available on the PATRIC 3.6.12 platform (https://www.patricbrc. org/app/Assembly, accessed on 11 November 2022).

#### *2.3. Characterisation of Listeria Monocytogenes Isolates*

Species identification was confirmed in silico from the assemblies with KmerFinder v3.2 and SpeciesFinder v2.0 [26] available on the CGE (Center for Genomic Epidemiology) platform (http://www.genomicepidemiology.org/services/, accessed on 11 November 2022). In silico typing using 7-gene multi-locus sequence typing (MLST) [27] was performed on the CGE and BIGSdb-Lm (https://bigsdb.pasteur.fr/listeria/, accessed on 11 November 2022) platforms. STs obtained through the 7-gene MLST were grouped into CCs and phylogenetic Lin [27].

Assembled sequence data of 55 *L. monocytogenes* isolates were genotyped with core genome MLST (cgMLST) based on 1748 genes [28] using the open-source tool available on the BIGSdb-Lm platform. The nearest cgMLST profile (CT) from the database was recorded. Additionally, cgMLST targeting 1701 genes was performed using Ridom SeqSphere+ software v7.7.5 (Ridom GmbH, Muenster, Germany) [29] and the results were visualised with a minimum spanning tree (MST). Isolates forming a cluster (CL) displayed a maximum of 10 allelic differences from each other. The CLs were shaded in grey, and the number of allelic differences between the isolates was indicated on the connecting lines. Using the default parameters in the Ridom software, (1) STs, (2) PCR serogroups (SGs), (3) virulence genes and (4) antimicrobial resistance genes were also determined. Presence of the virulence genes was additionally studied with the VirulenceFinder 2.0 available on the CGE platform and on the Virulence Factor Database (VFDB) [30] (http://www.mgc.ac.cn/VFs/, accessed on 11 November 2022). In total, the presence of 33 virulence genes and 46 AMR genes among the 55 *L. monocytogenes* isolates was recorded.

#### **3. Results**

In total, 75 *L. monocytogenes* isolates from 75 hunted game and game meat samples isolated in Finland between 2012 and 2020—were serotyped and characterised by sevengene MLST (Table 2). Most (89%) of the isolates belonged to serotype 1/2a and were found in all sample types. Serotypes 4b and 2b were identified among 8% and 3% of the isolates, respectively. *L. monocytogenes* 4b was found on deer carcasses (n = 3), wild boar organs (n=2) and in pheasant faeces (n = 1) (Table 2). *L. monocytogenes* 2b was only found in mallard faeces.

Based on the seven-gene MLST, 75 *L. monocytogenes* isolates from hunted game and game meat samples (n = 75) were classified into Lin I and II, 18 CCs and 21 STs (Table 3). Most of the isolates (89%) belonged to Lin II, including all serotype 1/2a isolates. Lin I included isolates of serotypes 1/2b and 4b. ST451 (16/75) was the most frequent ST followed by ST585 (9/75) and ST37 (8/75). These STs were found from different sample types between 2012 and 2020 (Table 3). ST451 and ST37 have frequently been identified in human listeriosis during recent years in Finland (Table 3). In total, 8 out of 21 STs found in game have been identified in cases of human listeriosis in Finland between 2016 and 2021. Most (17/75) of the isolates from wild boars hunted in 2016 belonged to several (11/21) STs (Figure 1). *L. monocytogenes* isolates were also frequently found from mallard faeces (15/75)

and mallard meat (13/75) (Table 2). These samples were contaminated with less (5/75) STs compared with wild boar organs.

**Table 2.** Serotypes and sequence type (STs) using 7-gene multi-locus sequence typing (MLST) of 75 *Listeria monocytogenes* isolates obtained from hunted game and game meat in Finland between 2012 and 2022.


**Table 3.** Characteristics of 75 *Listeria monocytogenes* isolates from hunted game and game meat from Finland between 2012 and 2020.


<sup>a</sup> Sequence types in bold have recently been published in other European countries [31–33]. <sup>b</sup> Reporting year of the most common sequence types found in human listeriosis in Finland during 2016–2021.

A subset of 55 out of 75 *L. monocytogenes* isolates were characterised with cgMLST based on 1748 genes. In total, 35 CTs among 21 STs were obtained using the BIGSdb-Lm platform (Table 4). Overall, 10 CTs (CT5208, 11797, 20896, 20939, 25365, 26674, 26763, 28125, 28250, and 28251) included more than one *L. monocytogenes* isolate. In total, 10 CLs (CL1, 8, 18, 155, 412, 451a, 451b, 451c, 451d, and 585) formed by closely related genotypes were obtained with the cgMLST based on 1701 genes using the Ridom software (Table 4). Isolates belonging to the same CL showed an allelic difference between 0 and 10 (Figure 2). All CLs obtained by Ridom software had their own CT obtained from the BIGSSdb-Lm platform (Table 4). Most (9/10) of the CLs included isolates of SG IIa. CL1 included two undistinguishable isolates of SG IVb, both found from wild boar organs. Five CLs (CL1, 18, 155, 412 and 451c) included very closely related isolates, each with 0 to 5 allelic differences (Figure 2). Each of these CLs included *L. monocytogenes* isolates found from one source during the same year (Table 4). CL18 and CL412 were formed by isolates from mallard meat and CL155 and CL451c from deer meat. The other CLs (CL8, 451a, 451b, 451d and 585) were formed by closely related isolates (0 to 10 allele differences) found from different sources between 2012 and 2019 (Table 4).

**Table 4.** Distribution of different serogroups (SGs), MLST (STs) and cgMLST (CTs) profiles, clusters (CLs) and virulence profiles (VPs) among 55 *Listeria monocytogenes* isolates from hunted game and game meat between 2012 and 2020 in Finland.


<sup>a</sup> ST based on 7-gene MLST using the BIGSdb-Lm platform and Ridom software. <sup>b</sup> Nearest CT based on cgMLST (1748 target genes) using the BIGSdb-Lm platform. <sup>c</sup> CL based cgMLST (1708 target genes) using the Ridom software. <sup>d</sup> VPs using the Ridom software and CGE platform.

**Figure 1.** Sequence types (ST), which include at least three *Listeria monocytogenes* isolates, found in hunted game and game meat in Finland between 2012 and 2020.



**Figure 2.** Minimum spanning tree of 55 *Listeria monocytogenes* isolates from hunted game and game meat in Finland during 2012–2020. The tree was calculated in Ridom SeqSphere+ with 1708 core genome multi-locus sequence typing (MLST) targets and 7-gene MLST targets (pairwise ignoring missing values, logarithmic scale). Nodes are coloured according to sequence type. Number of allelic differences between the isolates are indicated on the connecting lines. Clusters are shaded in grey and a cluster distance threshold of maximum10 was used according to Ruppitsch et al. [29].

We studied the presence of 33 virulence genes available in the Ridom software [34] among the 55 *L. monocytogenes* isolates. Most (26/33) of the genes were detected in all isolates. Seven virulence genes (*act*, *ami*, *aut*, *inl*F, *inl*J, *lap*B and *vip*) were not present in all isolates. We designed 12 virulence profiles (VPs) based on these genes (Table 5). All virulence genes (VP0) were detected in 12 (22%) *L. monocytogenes* isolates, all belonging to SG IIa. The most frequently missing genes were *ami* and *vip*, which were missing in 42% and 38% of *L. monocytogenes* isolates, respectively. The VP did not correlate with ST, but isolates belonging to the same cluster mostly (66%) had the same VP (Table 4).


**Table 5.** Virulence profiles detected among 55 *Listeria monocytogenes* isolates.

<sup>a</sup> Isolates with sequence types in bold belong to serogroup IVb.

We studied the presence of 46 AMR genes available in the Ridom software. Only the *fos*X and *lin* genes were detected in all 55 *L. monocytogenes* isolates.

#### **4. Discussion**

*L. monocytogenes* is a common finding in hunted game and game meat in Finland. Most of the *L. monocytogenes* isolates originating from game belonged to serotype 1/2a (SG IIa, Lin II) but serotype 4b (SG IVb, Lin I) was also found. *L. monocytogenes* strains belonging to SG IIa/Lin II and SG IVb/Lin I are responsible for the largest share of listeriosis [20,35,36]. However, SG IVb is more frequently associated with human diseases and outbreaks than SG IIa, which is more often identified among isolates found in animal, environmental and food samples [6,18,21]. Recently, *L. monocytogenes* IIa and IVb were found in deer and wild boar tonsils in Spain [12]. Serotyping and serogrouping provide useful information about *L. monocytogenes* isolates found in epidemiological studies, surveys and during monitoring.

Very little is known about the genetic diversity of *L. monocytogenes* isolates from game origin [12]. We found several CCs and STs in hunted game and game meat from Finland showing a large genetic diversity among the *L. monocytogenes* isolates studied. This was expected because *L. monocytogenes* isolates were found from various sources and locations during a ten-year period [17,24,25]. All CCs identified among our hunted game and game meat isolates from Finland have recently been identified among various environmental and animal sources in Europe [5]. In our data, the most common CC was CC11 (25%), which included three STs: ST11, ST400 and ST451. CC11 is also a prevalent clonal type found in Europe [23,31]. Most (67%) of the STs found in our study have also been found in Europe from various sources [23,31,32]. Several (7/21) STs found in game in our study have been associated with human listeriosis in Finland (https://thl.fi/en/web/infectiousdiseases-and-vaccinations accessed on 11 November 2022). ST451 (21%) was the most frequently found ST in our study, as it was found in different sample types between 2012 and 2020. This type has also been reported in human listeriosis in Finland yearly between 2017 and 2021. ST451 is a common universal ST found in humans, animals, foods, and the environment in Europe [23,31,32,37]. To obtain more accurate information about the link between human and game isolates, STs based on the core or whole genome (cgMLST or wgMLST) should be used instead of seven-gene MLST.

Wild boar organs were contaminated with several *L. monocytogenes* isolates of different STs. This is very understandable because the isolates originated from wild boars hunted in various geographical locations in Finland [25]. Fewer STs were found in isolates from deer and mallard meat than from wild boar. Deer and mallard meat were processed in one meat processing plant each, which may explain the limited genetic diversity among the meat isolates. Interestingly, only 4 STs were identified among 13 *L. monocytogenes* SG IIa isolates from mallard faeces. The hunted mallards were reared and fed in a natural pond before being hunted, which could be a common contamination source for the mallards [24]. *L. monocytogenes* is relatively commonly found in various environments, and *L. monocytogenes*contaminated soil and water are therefore important *L. monocytogenes* sources [5,38]. ST18, ST20, ST37, ST91 and ST451, identified among our game isolates, are reportedly common STs among isolates from environmental samples in Finland [37] and Latvia [31].

We identified some CLs of *L. monocytogenes* isolates with 0 to 10 allelic differences among the hunted game and game meat isolates using cgMLST, which is the method capable of differentiating related strains from unrelated ones [39]. Very closely related isolates, with a maximum of five allelic differences, were found in five CLs, and they originated from the same source and year, which could explain the high genetic similarity and may indicate a common source of contamination. Three very closely related *L. monocytogenes* isolates forming CL18—were isolated from mallard meat originated from various mallards sampled on the same day in the same game meat processing plant, indicating a cross-contamination during processing. In CL412, four very closely related isolates from mallards were sampled on two different days in the same plant, indicating a plant contamination possibly due to inadequate cleaning. Deer meat isolates also formed two clusters—CL155 and CL451c both with three very closely related isolates. The isolates in CL155 and CL451c were from deer meat samples cut on different days in the same plant. Cross-contamination during meat cutting occurs easily if working hygiene is poor. *L. monocytogenes* can easily persist in the plant, and thorough cleaning of the meat processing plant after each working day is therefore very important.

*L. monocytogenes* has shown heterogeneity in its virulence [35,40]. Virulence factors are essential for adapting *L. monocytogenes* to spread optimally within the environment [35]. The virulence of *L. monocytogenes* is encoded by a wide range of virulence genes [2]. In our study, most (79%) of the 33 studied virulence genes were present in all 55 *L. monocytogenes* isolates of game origin in Finland. The *act*A gene located on the *Listeria* pathogenicity island (LIPI-1) was missing in only one isolate (a deer carcass isolate). LIP-I is composed of important virulence genes (including *act*A, *hly, mpl*, *plc*A, *plc*B, *prf*A and *orf*X) and is necessary for intracellular survival and spread from cell to cell [35]. LPI-1 is typically present in all *L. monocytogenes* strains [2,41]. This *actA*-negative deer carcass isolate (with VP3b, SG IIa and ST412) also missed the *lap*B (coding for an adhesion protein) and *vip* (coding for an invasion protein) genes, indicating a reduced virulence in this isolate. The most frequently missing virulence gene *ami*, which is coding an autolysin protein for adherence, was not found in 42% of the isolates. However, the meaning of *ami* in the virulence remains unclear. The invasion gene *aut* was missing only in the isolates belonging to SG IVb. All SG IVb isolates (with ST1, ST4 and ST249) were *aut*-negative. The three SG IVb isolates belonging to ST1 were also *inl*J-negative. The *aut* gene codes for an autolysin protein needed for invasion and the *inl*J for an internalin protein needed for adherence [35]. How the absence of these two genes affects the virulence of *L. monocytogenes* IVb isolates needs to be further studied. Typically, ST1 (CC1) and ST4 (CC4) have been associated with clinical cases more often than other STs [35,41].

AMR is a serious public health issue due to increasing resistance. There is also a trend of increasing AMR among *L. monocytogenes* strains of animal and food origin. Resistance, e.g., to penicillin, ampicillin, gentamycin, streptomycin, tetracycline, and trimethoprimsulfamethoxazole has been reported [42,43]. However, in our study, only the *fos*X and *lin* genes were detected. These two genes were present in all 55 *L. monocytogenes* isolates. Earlier studies have shown *fos*X and *lin* to be present in nearly all *L. monocytogenes* isolates [42]. This can be explained by native resistance to fosfomycin and lincosamides reported in *L. monocytogenes* strains [34,43]. Our results indicate that *L. monocytogenes* of game and

game meat origin found in Finland are so far sensitive to antimicrobials. One explanation may be that hunted game in Finland have no access to feed contaminated with resistant *L. monocytogenes* strains.

#### **5. Conclusions**

In this study, we analysed the sequence data of *L. monocytogenes* isolates of game origin using tools available on open-source platforms and Ridom software. Our study demonstrates that game meat is contaminated with various STs associated with human listeriosis. All *L. monocytogenes* isolates were potentially pathogenic, carrying most important virulence genes. No acquired AMR genes were found, indicating that all isolates were sensitive to most of the important antimicrobials used to treat listeriosis. Some of the isolates from mallard and deer meat belonged to CLs that were formed by very closely related isolates, indicating common contamination sources. Contaminated game meat may pose a public health problem, and game meat should therefore be handled and stored correctly.

**Author Contributions:** Conceptualisation: all authors; Investigation: M.S., P.K. and V.H.; Methodology: M.S. and A.H.; Software: P.K. and V.H.; Visualisation: P.K.; Validation: all authors; Analysis: M.F.-A.; Writing—original draft preparation: M.F.-A.; Writing—review and editing: P.P.; Resources: M.F.-A., A.H. and P.P.; Supervision, project administration and funding acquisition: M.F.-A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was partly funded by the "Kyllikki ja Uolevi Lehikoinen" (IQUJA:10019) and "Walter Ehrström" Foundations. Proofreading and APC were funded by the University of Helsinki, Finland.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** Open access funding provided by University of Helsinki. Maria Stark is gratefully acknowledged for her technical assistance.

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

#### **References**


**Caterina Altissimi 1, Clara Noé-Nordberg 2, David Ranucci <sup>1</sup> and Peter Paulsen 3,\***


**Abstract:** The wild boar is an abundant game species with high reproduction rates. The management of the wild boar population by hunting contributes to the meat supply and can help to avoid a spillover of transmissible animal diseases to domestic pigs, thus compromising food security. By the same token, wild boar can carry foodborne zoonotic pathogens, impacting food safety. We reviewed literature from 2012–2022 on biological hazards, which are considered in European Union legislation and in international standards on animal health. We identified 15 viral, 10 bacterial, and 5 parasitic agents and selected those nine bacteria that are zoonotic and can be transmitted to humans via food. The prevalence of *Campylobacter*, *Listeria monocytogenes*, *Salmonella*, Shiga toxin-producing *E. coli*, and *Yersinia enterocolitica* on muscle surfaces or in muscle tissues of wild boar varied from 0 to ca. 70%. One experimental study reported the transmission and survival of *Mycobacterium* on wild boar meat. *Brucella*, *Coxiella burnetii*, *Listeria monocytogenes*, and Mycobacteria have been isolated from the liver and spleen. For *Brucella*, studies stressed the occupational exposure risk, but no indication of meat-borne transmission was evident. Furthermore, the transmission of *C. burnetii* is most likely via vectors (i.e., ticks). In the absence of more detailed data for the European Union, it is advisable to focus on the efficacy of current game meat inspection and food safety management systems.

**Keywords:** wildlife; game meat; *Salmonella*; *Listeria*; *Campylobacter*; *Yersinia*; mycobacteria; verotoxinogenic *E. coli*; *Brucella*; *Staphylococcus aureus*

#### **1. Introduction**

During the last decade, numbers of wild ungulates, in particular wild boars, have been rising significantly worldwide, generating environmental, economic, public health, and social concerns. Wild boar is the most widespread species due to its high adaptability and fertility rate, and its spread has been facilitated by climate change, the abandonment of rural areas, reforestation, a lack of predators, animal introductions, and supplementary feeding for hunting purposes [1–4]. The high density of this expanding species is causing, in particular, in Europe, not only relevant damages to agriculture and ecosystems and an increase in road accidents but also increases the risk of transmission of pathogens from wild boar to humans, livestock, and domestic animals [5,6]. The synanthropic behavior of wild boars is an important co-factor in creating disease-transmission scenarios [7]. Furthermore, the attention being paid to wild boar population control is leading to an increase in the availability of game meat. Additionally, the market has to face different harvesting practices, the wider distribution of this product, and, simultaneously, guarantee its safety aspects. In this context, it is of the utmost importance to understand the epidemiological situation and the major hazards due to the consumption of such meat.

Indeed, it has been highlighted by several authors how wild boar could act as a reservoir, playing an important role in the maintenance, circulation, and diffusion of certain

**Citation:** Altissimi, C.;

Noé-Nordberg, C.; Ranucci, D.; Paulsen, P. Presence of Foodborne Bacteria in Wild Boar and Wild Boar Meat—A Literature Survey for the Period 2012–2022. *Foods* **2023**, *12*, 1689. https://doi.org/10.3390/ foods12081689

Academic Editor: Evandro Leite de Souza

Received: 22 March 2023 Revised: 12 April 2023 Accepted: 15 April 2023 Published: 18 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pathogens for humans and animals [8–12]. In particular, the same authors focused their attention on the most relevant bacterial food hazards that: cause disease to wild boar and can be present in the meat (e.g., *Brucella* spp., *Mycobacterium tuberculosis* complex); are harbored in the gut or other tissues and then transferred to the meat during processing (e.g., *Salmonella* spp., *Campylobacter* spp., *Escherichia coli*, *Yersinia enterocolitica*); contaminate the carcass due to their presence on animal skin and in the environment (e.g., Listeria spp., *Staphylococcus aureus*).

In a framework of global health, it is essential to consider not only zoonotic diseases but also animal diseases with an impact on food security. The aim of this review is to give an overview of publications from the period 2012–2022 on the presence of biological hazards in the wild boar population. In particular, foodborne zoonotic bacteria commonly reported in meat from domestic animals will be the focus, and their presence in wild boars will be reviewed.

#### **2. Materials and Methods**

A list of infectious agents was compiled, combining zoonotic agents included in compulsory monitoring in the European Union (Directive 2003/99/EC List A) [13], zoonotic agents monitored according to the epidemiological situation (Directive 2003/99/EC List B) [13], swine and multiple species diseases, infections, and infestations listed by the World Organisation for Animal Health (OIE), and the most common agents responsible for foodborne outbreaks reported from the European Food Safety Authority (EFSA) during the period 2015–2020 and in the EU Rapid Alert System for Food and Feed (RASFF).

For each agent on the list, a literature search was conducted on SCOPUS using the name of the selected pathogen or the related disease combined with the search string: "wild" AND "boar" OR "feral AND pig" OR "warthog". During the literature search, biological hazards that do not concern wild boars were excluded. The search was then adjusted for (i) the time period 2012–2022, (ii) document type as article or review, and (iii) English as the selected language. Papers about the prevalence and control strategy of selected diseases were considered, whereas articles reporting solely detection methods were included only if relevant for the interpretation of results. Although our work focuses on the relevance of wild boar (meat) in the European Union, we included references from other countries in view of imports of wild boar meat from third countries in the EU; similarly, studies on feral pigs and warthogs were included.

We also report the number of publications per agent and year as a proxy for the relevance of the agent and the interest and effort of the scientific community in this topic [14]. From this long list of biological hazards specifically addressed in national legislation or by international organizations, we selected those with evidence that they are actually transmitted via the handling, processing, and consumption of porcine meat and meat products.

#### **3. Results**

#### *3.1. Overview of Biological Hazards in Wild Boar and Their Impact on Food Safety and Security*

The array of biological agents addressed in EU legislation and international organizations such as the OIE is displayed in Table 1. Information on zoonotic potential and mode of transmission was taken from OIE, EFSA, and ECDC documentation. Notably, not all agents are zoonotic, and not all zoonotic agents are transmitted by meat. Among the pre-selected (i.e., taken from EU and OIE documents) infectious agents, no scientific literature was retrieved for two viruses and one bacterial genus. A clear increase (i.e., more than one doubling) in the average number of publications per year in the period 2017–2022 compared with that from 2012–2017 was noted for the viral diseases African swine fever, West Nile fever, and Japan encephalitis; the bacterium *Listeria*; and the parasite genera *Cryptosporidium*, *Cysticercus*, and *Echinococcus*.


**Table 1.** Agents or diseases of wild boar covered in the literature survey (2012–2022), their coverage in legislation, and the number of pertinent publications.

V = virus; B = bacterium; P = parasite; f = facultative, according to the epidemiological situation; m = mandatory; \* = multi-resistant *St. aureus*.

For a detailed review of the occurrence and significance of biological hazards, we focused on bacteria since these are the main causative agents for foodborne diseases reported in the EU [15].

#### *3.2. Occurrence and Prevalence of Selected Zoonotic Bacteria in Wild Boar* 3.2.1. *Brucella*

Brucella (B.) are gram-negative, nonsporeforming, aerobic, short-rod bacteria that include several pathogenic species. In the EU, monitoring of brucellosis is compulsory (Directive 2003/99/EC List A) [13]. In ruminants, swine, and dogs, infection with the agents causes diseases of the reproductive system, e.g., abortion or epididymitis. Symptomless carriers can excrete the pathogen, e.g., via milk. Small ruminants with mastitis caused by Brucella-melitensis can excrete the pathogen via milk. Ingestion of raw milk, inhalation, or close contact with infected animals or parts thereof (e.g., when dressing hunted wild game) can lead to human infections. These may resemble a feverish flu, whereas more severe courses involve splenomegaly and splenic or hepatic abscesses. In 2021, cattle livestock in 21 EU member states was officially free from brucellosis (*B. abortus*, *B. melitensis*, and *B. suis*), and as regards small ruminant livestock, 20 member states were officially free from the pathogen. In 2021, 162 human cases were reported, two of them foodborne. In 2020, there were also 2 cases linked to the consumption of sheep meat products, with *B. melitensis* being the causative species [15]. In the EU rapid alert system for food and feed (RASFF), no notification of the presence of Brucella in food was found.

As regards wild boar and Brucella, 96 documents were retrieved. Those reporting prevalence data were included in Table 2 (seropositivity) and Table 3 (DNA or viable bacteria). With respect to serological testing, the cross-reactivity with the Yersinia enterocolitica O9 antigen is a well-known issue. More recent methods may overcome this problem [16]. Some authors present seroprevalences corrected for cross-reactivity [17]. When tissues/organs of the animal were tested by bacteriological culturing, or PCR, blood, lymphatic organs, genital organs, and fetuses were examined. There was no study on Brucella in muscle tissue or commonly consumed organs, e.g., liver, from wild boar. When Brucella species and biovars are explicitly reported, it is mainly B. suis biovar 2.

While no documented cases of meat-borne brucellosis could be retrieved, several cases of brucellosis in humans hunting wild boar and dressing wild boar carcasses have been published; most reports are from the USA [18–21], but also from France [22] and Australia [23]. In two cases, neurological disorders [18,23] were reported, and in one case, arterial and venous thromboses were reported [20], which are otherwise rarely observed [24]. Similarly, dogs frequently in contact with wild boar are at risk of seropositivity to Brucella [25–27].


**Table 2.** Prevalence of *Brucella* spp. antibodies in wild boars (2012–2022), by country and continent.


**Table 2.** *Cont.*

BAPA = Buffered Acidified Plate Antigen, CFT = Complement Fixation Test, RBT = Rose-Bengal-Test, RIV = Rivanol Agglutination, 2MET = 2-Mercapto-Ethanol.

**Table 3.** Prevalence of *Brucella* spp. (viable bacteria or DNA) in wild boar (2012–2022), by country and continent.


#### 3.2.2. *Campylobacter*

*Campylobacter* is a genus of gram-negative, nonsporeforming, microaerophilic, motile spiral-shaped bacteria, with *C. jejuni* and *C. coli* as the main species involved in Campylobacteriosis. The principal symptoms of *Campylobacter* infections are diarrhea, abdominal pain, fever, headache, nausea, and vomiting. The disease is usually self-limiting, and death is rare except in severe cases in elderly people, very young children, or immunocompromised patients [55]. In 2021, campylobacteriosis was the zoonosis with the highest number of human cases reported in the EU, with 127,840 cases of illness and 10,469 hospitalizations. With respect to foodborne outbreaks, it was the fourth most frequently reported agent with

249 outbreaks, 1051 cases, and 134 hospitalizations [15]. *Campylobacter* is common in food animals such as poultry, pigs, and cattle, and the main transmission route is via meat and meat products, as well as raw milk and milk products.

Twenty-two articles have been published from 2012 to 2022 regarding the prevalence of *Campylobacter* in wild boars, five of which were excluded as not relevant. The main matrix considered for the isolation of *Campylobacter* is feces, as reported in Table 4. The references highlighted the role of wild boars as a possible source of *Campylobacter* infection due to the prevalence of *Campylobacter* spp. in feces samples, albeit in a variable range from 12.5% [56] to 66% [57]. Several species have been isolated from fecal samples in varying prevalence ranges, e.g., *C. lanienae* from 1.2% [56] to 69% [58], *C. hyointestinalis* from 0.8% [59] to 22.1% [60], *C. coli* from 0.8% [56] to 16.3% [58], and *C. jejuni* from 0% [61] to 4.1% [58] of samples. As suggested by [59], the degree of urbanization of some areas populated by wild boars could have a relationship with the detection frequency of some *Campylobacter* species; in particular, *C. lanienae* was more frequently isolated in low urbanizations areas, suggesting that this pathogen could be interconnected with the kind of diet available.

During the period considered, only two studies were conducted on carcasses, and they presented similar results, with a prevalence of *Campylobacter* spp. of 11.1% [62] and 16.7% [63]. Peruzy et al. [64] investigated the presence of *Campylobacter* in wild boar meat samples, but the pathogen was not detected.

To date, the EU has set food processing hygiene criteria for *Campylobacter* only for poultry [65].


**Table 4.** Prevalence of *Campylobacter* spp. in wild boar (2012–2022) feces or on carcasses or meat.


**Table 4.** *Cont.*

WB = wild boars.

#### 3.2.3. *Coxiella burnetii*—Q-Fever

*Coxiella burnetii* is a gram-positive short-rod bacterium that grows aerobically within but also outside of host cells. It can form spores and persist under dry and acidic conditions. The bacterium is not only excreted via effluents, but several tick species can act as vectors for the pathogen. Infection of humans can occur via contact with effluents, ingestion of contaminated food, and inhalation of aerosolized pathogens, but also by tick bites. Infection causes a feverish disease (Q-fever) with pneumonia, followed by affections of the heart, liver, and spleen. In the EU, human cases are notifiable. Data indicate that the number of human cases as well as prevalence in animals is declining. However, monitoring of farm and wild animals is not harmonized in the EU [15]. At least 347 of the 460 confirmed human cases of Q-fever in 2021 were acquired within the EU, and the pathogen was prevalent in 5.2%, 5.9%, and 16.5% of samples from cattle, goats, and sheep, respectively. Since not all member states submitted data, the reported percentages are not necessarily representative of the EU [15]. Studies conducted on *C. burnetii* and wild boar can be grouped into three categories: (i) those on ticks collected from wild boars or from hunters or dogs in frequent contact with wild boars; (ii) those on serum or spleen samples from wild boar; and (iii) studies on the genetic diversity of *C. burnetii*.

Within Europe, studies originated in Spain and Italy (Table 5). DNA from *C. burnetii* was detected in 1.9% of spleen samples [69], and antibodies were found in 5.5% of serum samples [70] from wild boar in Spain. In studies from Italy, the pathogen was not recovered from wild boar samples but from ticks feeding on wild boars (0.5%; [71]) and from dogs in contact with wild boars (5.1%; [72]). Wild boar is not a specific or primary host for the pathogen [73], but since the agent is occasionally detected in tissues from wild boar, hunters and consumers handling and processing wild boar (meat) are both occupationally and dietary exposed. Similarly, hunters and dogs often in contact with wild boars are at risk of exposure to tick-borne pathogens, among them *C. burnetii* [71].

**Table 5.** Presence of *Coxiella burnetii* or antibodies in wild boar or in vectors associated with wild boar, according to country and continent, 2012–2022.




No notifications regarding the presence of *C. burnetii* in foods were listed in the EU rapid alarm system (RASFF).

#### 3.2.4. *Listeria monocytogenes*

Listeriosis is a zoonotic disease caused by *Listeria monocytogenes*, a gram-positive, nonsporeforming, facultatively anaerobic bacterium. Foodborne listeriosis is one of the most severe diseases, causing septicemia, neurologic disorders, and reproductive disorders. Pregnant women, elderly people, and individuals with weakened immune systems are at risk for severe courses of the disease. *Listeria* is a ubiquitous microorganism that thrives in soil, water, vegetables, and the digestive tracts of animals. It can survive and proliferate in different environmental conditions since it is tolerating a wide range of pH and temperatures [80]. The main transmission route of *Listeria* is through the ingestion of contaminated food [15].

Twelve studies have been found from 2012 to 2022 regarding the presence of *Listeria* spp. in wild boar carcasses, meat, and related products, two of which were excluded as not relevant (Table 6). *Listeria monocytogenes* was detected by many authors in tonsil samples, highlighting this organ as the preferred matrix for the presence and detection of *Listeria* [63,81,82]. Fredriksson-Ahomaa et al. [39] found *L. monocytogenes* in 48% of spleen and kidney samples from wild boars. Almost all isolates belonged to serotype 2a, except for two isolates identified as serotype 4b. The presence of *Listeria* in tonsils and in visceral organs underlines the necessity of particular attention during handling and evisceration of wild boar carcasses.

Regarding the presence of *Listeria* in wild boar meat products, Roila et al. [83] did not detect the pathogen in wild boar salami, whereas Lucchini et al. [84] isolated *Listeria* spp. in 65% of cured game meat sausages. Three species were identified: *L. monocytogenes,* 24%*; L. innocua*, 32% and *L. welshimeri,* 8%. Counts of *L. monocytogenes* were, however, always below the legal limit of 100 cfu/g set by Regulation (EC) 2073/2005 [65].

In the years 2020–2022, 340 notifications regarding the presence of *L. monocytogenes* in foods were listed in the EU rapid alarm system RASFF, of which 82 implicated meat and meat products; there was no explicit mention of game meat or wild boar meat in particular.


#### **Table 6.** Presence of *Listeria* sp. in wild boar, 2012–2022.

#### 3.2.5. *Mycobacterium tuberculosis* Complex

*Mycobacterium tuberculosis* complex is a group of *mycobacteria* that include *M. tuberculosis,* the major cause of human tuberculosis (TB), and other genetically related species that affect livestock and wild animals but are also implicated in human disease [87,88]. Among these species, in the last decade, *M. bovis* [89–115], *M. caprae* [89,104,111,116,117], and *M. microti* [118–124] have been frequently reported from wild boar, feral pigs, and warthogs in different countries.

The MTC bacteria can cause localized granulomas (primary complex) after entering the host through the respiratory or digestive tract, and when the organism´s immune system cannot contain it (which can be the case in the elderly, children, and in people with compromised immune systems), it may be followed by primary or secondaryreactivated TB. Meningitis, extrapulmonary granulomas, miliary tuberculosis, and other disseminated/generalized forms are only a few examples of the various manifestations, along with a variety of clinical symptoms [125]. *M. bovis* is usually transmitted through oral ingestion, and therefore the extrapulmonary lesions in humans are more frequent than for *M. tuberculosis* [126]. In wild boar, the main primary complex is usually located in the submandibular and retropharyngeal lymph nodes, where the MTC is most frequently isolated [89,98,105,117,122,127,128]. Lesions were also reported in the tonsils, lung, mediastinal lymph nodes, spleen, liver, and kidney [106,117,127,128]. The lesion in the

lymph nodes is characterized by caseous or necrotic-calcified tubercles that are defined as tuberculosis-like lesions (TBLL), as other *mycobacteria* different from MTC (e.g., *M. avium subsp. hominissuis*) could cause the same lesion [119,129–131]. *M. bovis* and *M. caprae* could also be detected (isolated/PCR) in lymph nodes without visible lesions [94,105,128,131]. Wild boar is reported for MTC shedding through the oral, nasal, and fecal routes [132], and therefore animal aggregation areas could result in contaminated water and soil and the maintenance of the infection in wildlife and livestock [118,133,134].

In addition, 214 studies regarding MTC and non-MTC in wild *Suidae* species have been found in the literature over the considered period, but only 35 were related to prevalence studies of MTC and were therefore considered. These studies were performed both by serology (Table 7) and by isolation or direct identification of *mycobacteria* in organs and tissues (Table 8). The prevalence of MTC varies between countries and between regions/counties inside each nation (e.g., Spain), but also due to the investigated matrix and the diagnostic methods adopted [94,98,135]. In this context, some studies were performed to define the sensitivity of different diagnostic tools on sera and on organs and tissues [94,96,119,136]. The serological prevalence of MTC in wild boar is generally conducted over multi-year studies and ranged from 87.7% in Montes de Toledo and Doñana National Park (Spain) [132] to near 0% in the USA [137]. The prevalence of MTC isolation in tissue and organs, considering studies conducted on more than 100 subjects, ranges from 64.2% for *M. microti* in the Lombardia region (Italy) [123] to 1.1% for *M. bovis* in the Basque Country (Spain) [89].

The presence of MTC in wild boar is still recognized as one of the main barriers to the eradication of the disease in livestock and, subsequently, in humans, particularly when extensive pastoral systems are implemented and there is an interface between farmed and wild animals [93,100,101,104,111,133,138,139]. Although the disease is notifiable in many countries (such as Europe and the United States), its control in wild boar is primarily restricted to standard visual game meat inspection, which is thought to be insufficient to find primary complex and small lesions [117], especially as post-mortem inspection could be carried out also by trained hunters [EC Regulation 853/2004 [140]]. Even the cultural method for bacterial isolation is less effective than other diagnostic tools (e.g., screening PCR directly performed on target tissues, such as head lymph nodes, even when no TBLL are detected) [94,136]. Another topic to be considered is the free movement of wildlife that could spread the disease in different geographic areas. The identification and longterm monitoring of the genotype/spoligotype existing in a territory may aid in specific surveillance plans and control actions [100,141].

Despite the role of wild boar as a reservoir for MTC and the possible transmission through food [11], wild boar meat and meat products as a source for human infection are reported only by Clausi et al. [142]. In this study, PCR tests revealed the presence of MTC DNA on the carcass surface of wild boar without TBLL, but no *Mycobacterium* spp. could be isolated. Clausi et al. [142] added lymph nodes with active TBLL (*M. bovis*) to meat batter during sausage processing. Although live bacteria could be isolated only at day 23 after the contamination of the sausages (neither before nor after), bacterial DNA was detected (PCR) throughout the entire study period (end of sampling at day 41). When *M. bovis* (105 CFU/g) was directly added during sausage manufacturing, it was isolated for up to 22 days of ripening. When meat surfaces were experimentally contaminated with *M. bovis,* the bacterium could be recovered after frozen storage for over 5 months [142]. The role of wild boar meat and derived raw meat products could therefore be further investigated, even if other authors consider meat a negligible source of human infection [117].


**Table 7.** Seroprevalence of MTC in wild boar, feral pigs, and warthogs, 2012–2022.


**Table 8.** Prevalence of *Mycobacterium* spp. in wild boar, feral pigs and warthog organs and tissues, 2012–2022.

*Foods* **2023**, *12*, 1689


**Table 8.** *Cont.*

*Foods* **2023**, *12*, 1689


**Table 8.** *Cont.*

#### 3.2.6. *Salmonella*

Salmonellosis is an enteric infection caused by species of the *Salmonella* genus other than *Salmonella* Typhi and *Salmonella* Paratyphi. Salmonellae are gram-negative bacteria belonging to the Enterobacteriaceae family. They are motile, nonsporeforming, aerobic, or facultatively anaerobic. The transmission of this infection occurs principally by the fecal-oral route: the ingestion of contaminated food or water, contact with infected animals, feces or contaminated environments. The main symptoms of salmonellosis are diarrhea, abdominal cramps, vomiting, and fever. The severity and course of the disease are related to the serotype, the number of microorganisms ingested, and the individual's immune system [147]. *Salmonella* spp. is widely spread for its ability to infect several animal species and survive in different environmental conditions with a wide range of temperatures (2–54 ◦C) and pH values (3.7–9.4) [148].

Salmonellosis is a public health issue, and it was the second zoonosis reported in the EU in 2021, with 60,050 confirmed human cases, 11,785 hospitalisations, and 71 fatalities [15]. The *Salmonella* genus consists of two species: *Salmonella bongori* and *Salmonella enterica*, the latter divided into six subspecies and several serotypes [149]. The main *Salmonella* serovars implicated in human infections in 2020 and 2021 were *S.* Enteritidis, *S.* Typhimurium, monophasic *S.* Typhimurium (1,4, [5],12:i:-), *S.* Infantis, and *S.* Derby [15,150].

Overall, 80 articles regarding *Salmonella* in wild boars have been found in the literature from 2012 to 2022, seven of which are reviews [10,11,150–155], and 28 articles were not considered relevant for this study. The prevalence of *Salmonella* in the wild boar population has been studied through the analysis of different matrices. Some authors investigated the seroprevalence from blood serum, diaphragm, or muscle samples, achieving different percentages: 1.27% (141/1103) [156], 3.6% (14/393) [157], 4.3% (4/94) [158], 5% (1/20) [159], 17% (21/126) [160], 19.3% (52/269) [161], 38% (69/181) [39], and 66.5% (255/383) [162]. Testing of serum samples can reveal the presence of antibodies against *Salmonella* spp. in wild boars but not the presence of the microorganism on carcass surfaces or meat. The prevalence of *Salmonella* spp. in other matrices such as feces, spleen, kidney, submandibular lymph nodes, ileocecal lymph nodes, mesenteric lymph nodes, and tonsils is reported in Table 9, which shows that feces are the main investigated samples with a prevalence range of 0% to 43%. As shown in Table 10, the prevalence of *Salmonella* spp. in wild boar carcasses is between 0% and 2.5%, while in meat samples it ranges from 0% to 35.7%. This wide variability could be due to different geographic sampling areas, sampling methods, and the hygienic level of process procedures and the environment. The presence of *Salmonella* in wild boar cured meat products was investigated only by Roila et al. [83] in wild boar salami. *Salmonella enterica* serovar typhimurium and *Salmonella enterica* serovar Rissen were found in different batches of meat batter and salami after 7 days of curing, but in the final product after 60 days of aging, *Salmonella* spp. were not detected. However, it was not possible to specify if wild boar had been the source of *Salmonella* since the salami were made with 50% wild boar meat and 50% pork meat.


**Table 9.** Prevalence of *Salmonella* spp. in wild boar, feces, lymphatic tissues, and inner organs, 2012–2022.


**Table 9.** *Cont.*

**Table 10.** Prevalence of *Salmonella* spp. in wild boar meat and carcasses, 2012–2022.



**Table 10.** *Cont.*

In order to reduce the risk of infection, it is recommended to pay particular attention to the skinning and evisceration processes, maintain the cool chain, have a good hygienic level during meat cutting, and to cook the final product.

#### 3.2.7. *Staphylococcus aureus*

*Staphylococcus aureus* is a gram-positive, spherical, nonsporeforming, coagulase-positive, aerobic or anaerobic, facultative, halophilic bacterium with the tendency to aggregate in "grape-like" clusters. The usual habitat of this commensal microorganism is the skin and nose of healthy humans and animals, but in some cases, it could lead to a wide range of clinical infections such as bacteremia, endocarditis, pneumonia, infections of the skin and soft tissues, mastitis, and bone and joint infections [182,183]. Some *S. aureus* strains may develop resistance to beta-lactam antibiotics, which are widely used to treat infections, and these strains are termed methicillin-resistant *Staphylococcus aureus* (MRSA). MRSA used to be associated mainly with hospital-related infections, but recently this strain has been found also in people without any contact with hospitals and, in companion animals, livestock, and wild animals [184]. There is an increasing interest in understanding the role of wild boars as possible reservoirs of *S. aureus* and MRSA in particular. About this topic, it has been found in 27 articles from 2012 to 2022, 14 of which were relevant for this study. The majority of studies performed nasal swabs for the detection of *S. aureus*, with a variable prevalence as shown in Table 11. Sousa et al. [185] considered both oral and nasal swabs, with a prevalence of *S. aureus* of 33%. Both studies from Porrero et al. [186,187] considered skin and nasal swabs; in the first study, they found 0.86% of animals positive for MRSA, of which 62.5% were detected from skin swabs and 37.5% from nasal swabs, and only one wild boar was positive in both the skin and nasal samples. Instead, Porrero et al. [187] noticed a higher percentage of positives for *S. aureus* in the nasal sample rather than in skin swabs, but without skin swabs, 18.25% of positives for wild boars would not have been detected. Only Traversa et al. [188] considered lymph nodes for the detection of *S. aureus* in wild boar and revealed a prevalence of 3.2%. No studies on the presence of *S. aureus* in carcasses, raw meat, or processed meat were retrieved in our literature survey.


**Table 11.** Prevalence of MRSA on wild boar mucosal membranes and in lymphatic organs, 2012–2022.

MRSA: methicillin-resistant *Staphylococcus aureus*; MSSA: methicillin-susceptible *Staphylococcus aureus* (MSSA); CoPS: coagulase positive *Staphylococcus.*

#### 3.2.8. Verotoxinogenic/Shigatoxinogenic *E. coli*

Verotoxinogenic/Shigatoxinogenic *E. coli* (VTEC/STEC) form a group of pathogenic *E. coli* (gram-positive short-rods) that elaborate Shiga-like toxins together with other virulence factors. Infections in humans can range from bloody diarrhea to life threatening coagulopathy and renal failure/hemolytic-uremic syndrome. Originally associated with the presence of the O157 antigen, a number of strains with other O-serotypes have been identified as STEC. It has been proposed to use *stx*-gene typing to assess the pathogenicity of STEC (EFSA 2020). In particular, *E. coli* with genes encoding for the stx-2 gene and the virulence factor intimin (*eae*) are associated with severe courses of the disease [15]. In 2021, 6084 confirmed cases were reported in the EU, with 901 hospitalizations and 18 fatalities. From the 5 strong evidence outbreaks, 3 were attributable to meat or meat products [15]. In many animal species, asymptomatic STEC carriers are the rule. In particular, ruminants do not show symptoms since they lack vascular receptors for the Shiga-toxins [195]. A survey of notifications in the RASFF revealed no cases of wild boar meat contamination with STEC.

As regards wild boar, the literature search retrieved 27 documents. The definitions for pathogenic *E. coli* were not consistent between the studies. In 12 studies, the prevalence of STEC was reported, ranging from 0 to 28.3% (Table 12). Data on meat were reported in merely four studies, with a prevalence ranging from 0 to nearly 43% (Table 13). A more detailed view of other isolates with pathogenic potential and antimicrobial resistance described in the studies is outside the scope of our review. E.g., one study reported the isolation of STEC from wild boars with the additional feature of producing enterotoxins (*sta1* and *stb* genes), causing oedema disease [196].

Three studies reported the transmission of STEC from the feces of wild boar to fresh produce [197,198] or to recreational waters [199]. Although not the primary focus of this review, the studies highlight indirect transmission routes of pathogenic bacteria to humans.

**Table 12.** Prevalence of Shiga toxin-forming *E. coli* in wild boar, fecal samples, lymphatic organs, 2012–2022.


**Table 13.** Prevalence of Shiga toxin-forming *E. coli* in wild boar meat and carcasses.


#### 3.2.9. Yersinia

The Enterobacteriaceae family includes the food-borne pathogen *Yersinia enterocolitica,* responsible for yersiniosis in humans, a gastrointestinal disease that could simulate appendicitis and can cause mesenteric lymphadenitis, reactive arthritis, erythema nodosum, and conjunctivitis [208,209]. The disease appears to be widespread, with ca. 6800 cases in Europe in 2020 and 100,000 illnesses every year in the USA [EFSA, 2022; CDC, 2016] [15,210]. The epidemiological situation could be even more severe, as the role of biotype 1A in human infection and disease symptoms (considered non-pathogenic compared to biotypes 1B, 2, 3, 4 and 5) is still debated and therefore underestimated [211].

Ready-to-eat foods are the major sources of human infection, especially as *Y. enterocolitica* can resist cold environments and even replicate at refrigeration temperatures [211]. Animals, especially pigs, are considered the main reservoir of the bacteria, which could be found mainly in the intestine and tonsils [212]. Nevertheless, the outbreaks reported in 2021 are related to prepared dishes and ready-to-eat vegetables [15], and no reports are available on wild boar meat as an outbreak source.

The database research retrieved 39 studies regarding *Y. enterocolitica* in wild boars and feral pigs between 2012–2022. The articles that reported studies on the prevalence of the microorganism in animal tissue, feces, or carcasses/muscles of wild boars were 21. Only two articles describe the prevalence of antibodies against *Y. enterocolitica* in animal blood samples. Papers on *Yersinia pseudotuberculosis* were not considered. Most of the studies were conducted in Europe (19 out of 21), especially in Italy (10 articles). Samples of different matrices were considered: eight studies on fecal samples, nine on organs different from muscles, four on carcass surfaces (external or internal), and four in muscles (Table 14).

The seroprevalence in wild boar was above 50% (in Finland and the Czech Republic), proving that the microorganism is widespread in this species. Fecal material is considered the main source of contamination of the carcass and, ultimately, of the meat. This contamination could happen during hunting (the precision of the shot), evisceration, or carcass processing and cutting [176,180]. Fecal sample positivity for *Y. enterocolitica* ranges from 0% (different Italian regions) to 74% (Japan). Thus, as for other genus belonging to the Enterobacteriaceae family, the fecal shedding could be intermittent [213]. Regarding organs and tissues that could harbour the microorganism in *Suidae*, the prevalence of the microorganism in the tonsils of wild boar ranges from 14% (Sweden) to 64% (Campania Region, Italy), with a higher percentage than in lymph nodes (ranging from 0% to 4.4%). The presence of the pathogen in such tissues could be considered during carcass processing to avoid the spread of the microorganism to the meat. Nonetheless, in wild boar, in contrast to the domestic pig, the head is removed during carcass dressing at cervical vertebrae level, thus the laryngeal and pharyngeal area is removed from the carcass at an early stage of the processing chain.

The presence of *Y. enterocolitica* on carcass surfaces ranges from 0% to 85.7%. Such a wide range could be due to different sampling methods and areas, but also to differences in the hygienic level of the process. The same might hold true for muscles, where the prevalence ranges from 0% to 71%. The wide range of prevalence denotes that, although wild boar can harbour microorganisms in the intestines and tonsils, the procedures to obtain the meat are relevant to prevent contamination of muscles. In this perspective, the training of the personnel, the presence of suitable structure and equipment, the correct hygienic procedure implementation, and standard sanitation operating procedures are of paramount importance.

Another important aspect that emerged from the literature survey is that the biotype most frequently observed in wild boar is 1A, the least pathogenic but also the most underrated of the *Y. enterocolitica* biotypes.


**Table 14.** Prevalence of *Yersinia enterocolitica* in wild boar, feral pigs and warthog.

*Foods* **2023**, *12*, 1689


#### **Table 14.** *Cont.*


**Table 14.** *Cont.*

#### **4. Conclusions**

The increasing popularity of meat from wild game is observed in many countries. Diseases in wildlife have often been seen as an issue or spill-over or spill-back of infection agents from farm animals, and exposure of humans and animals in frequent and close contact with wild animals has been studied to some extent. Additionally, while the presence of antibodies against a specific pathogen may be useful for epidemiological purposes, its value for the assessment of meat safety is primarily that the given pathogen must be considered a potential hazard. Similarly, the presence of pathogens in the feces and even in the lymph nodes of the digestive tract mainly indicates that the host organism can keep the pathogen under control. Similar to farm animals, it can be expected that stress, but also the dressing procedures after killing, can cause the spread of the pathogen on/in edible organs. Since these scenarios do not result in any typical lesion, the routine ante- and post-mortem examinations [226] will not give an indication of the presence of a certain pathogen, and minimizing the spread of the agent is a matter of good hygienic practice. However, if serological or other testing has demonstrated the presence of a certain pathogen in wildlife in a certain region, it would be wise to adopt hygienic precautions (i.e., no admittance of carcasses with "gut shots" in the food chain; or disinfecting knives after cutting in the tonsillar area).

For five (*Campylobacter*, *Listeria monocytogenes*, *Salmonella*, Shiga toxin-forming *E. coli,* and *Yersinia enterocolitica*) of the nine agents we reviewed, one or more studies dealt with the presence of the pathogen on muscle surfaces or muscle tissues of wild boar, with prevalences ranging from 0 to ca. 70%. One experimental study was retrieved on the transmission and survival of *Mycobacterium* on wild boar meat. As regards edible inner organs, the liver and spleen have been examined for the presence of *Brucella*, *Coxiella burnetii*, *Listeria monocytogenes,* and *Mycobacteria*, and the latter four agents have actually been recovered, albeit with varying percentages. For *Brucella,* human case reports and epidemiological studies in (hunting) dogs stressed the occupational exposure risk, but no indication of meat-borne transmission to humans was evidenced. Similarly, the mode of transmission of *C. burnetii* is more likely via vectors (i.e., ticks). In most studies, animals without specific histories or pathologies had been examined.

In essence, the literature we reviewed confirmed that food-borne pathogenic bacteria present in meat from domestic animals [15] and implicated in food-borne disease can also be found in wild boars, with varying prevalence and regional differences. It is unclear to what extent such differences are biased by sampling and analytical procedures. In the absence of more detailed data for the European Union, it might be advisable to focus on the efficacy of current game meat inspection [226] and handling practices [140] to minimize introduction in the game meat chain. Similarly, the implementation of HACCP-based food safety management systems [227] needs to be stressed.

With respect to the placing on the market of meat from wild hunted game, European Union legislation distinguishes an "approved" chain (i.e., the hunted game specimens are collected, post-mortem inspected, and processed in approved establishments) from an unapproved chain, which is largely subject to national regulation (for primary products, i.e., the eviscerated carcass, see Recital 10 and Article 1 of EC Regulation 852/2004 [228]; for processed or unprocessed products, see Recital 11 and Article 1 of EC Regulation

853/2004 [140]). This unapproved chain represents the supply of small quantities of wild game or wild game meat directly from the hunter to the final consumer or to local retail establishments directly supplying the final consumer [140].

Currently, there is no uniform way in which this unapproved sector is regulated in the member states; there is even no consistent definition of "small quantities of wild game or wild game meat" [140]. Admittedly, all national legislation has a common baseline represented by EC Regulation 178/2002 (in particular, Articles 14, 16–19; "safe food", traceability, identification of hazards, and management of risks) [229,230]. An in-depth and comprehensive consideration of said regulation should, in fact, be sufficient to warrant food safety. European Union member states have chosen different approaches [231,232], but there are no real metrics to assess how the systems actually perform in managing the consumers´ risk posed by the presence of foodborne pathogens in game meat.

**Author Contributions:** Conceptualization, C.A., P.P. and D.R.; methodology, C.A. and P.P.; validation, C.A., C.N.-N. and P.P.; formal analysis, C.A. and D.R.; investigation, C.A., P.P. and D.R.; data curation, C.A.; writing—original draft preparation, C.A., P.P., C.N.-N. and D.R.; writing—review and editing, C.A., P.P. and D.R.; supervision, P.P. and D.R.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** Open access funding by the University of Veterinary Medicine Vienna.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Italian Ministry of University and Research—P.O.N. Research and Innovation 2014–2020 (CCI 2014IT16M2OP005), Action IV.5. Project title: Game meat green safety.

**Conflicts of Interest:** The author, Clara Noé-Nordberg, was employed by the company Esterhazy Betriebe GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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