Introduction

Wild ungulate populations are nowadays in expansion, both numerically and in terms of habitat range. Particularly, the number of wild boars is increasing in Europe and Italy, although accurate data on species demography are not available due to the lack of harmonized census (Ramanzin et al. 2010). The overabundance due to the increase of wild boar populations has led to both ecological concerns and conflicts with human activities. In addition, risks posed by the transmission of zoonotic and animal-specific pathogens may increase. Indeed, wild boars are a reservoir of bacteria, viruses, and parasites, potential sources of infectious diseases in livestock and humans (Meng et al. 2009; Martin et al. 2011; Ranucci et al. 2013).

Particularly, the frequency of contact among wild boars, livestock, and humans has increased during the last years due to ecological changes in the environment, such as the increased cultivation of agricultural materials for energy generation and urbanization. Measures of control have been set up in many countries and regions in order to limit the overabundance of wild boar populations, by both hunting plans and scheduled management programs (Meng et al. 2009).

As a consequence of the increased number of hunted wild boars, the consumption of game meat incremented (Ramanzin et al. 2010). Indeed, the changes in food habits have led the consumers to choose alternative food including game meat, frequently considered more sustainable and healthy than meat from farmed animals (Ruiz-Fons 2017). The consumption of raw or undercooked meat (i.e., roast beef, tartare) besides cured meats (salami, saucisson, chorizo) may increase the risk of transmission of those pathogens that otherwise would be devitalized by cooking (Ruiz-Fons 2017). In Europe, the safety of game meat is regulated by the European food hygiene regulations (Reg. EC. 852, 853, 854/2004), providing rules for production, commercialization, and meat inspection of meat from wild animals. Nevertheless, for certain pathogens, the health surveillance programs and the report of epidemiological data are not harmonized and standardized among Member States (Kijlstra and Jongert 2008). Therefore, it is necessary to establish appropriate monitoring measures for wild boars destined for human consumption that include also those food-borne pathogens that are currently not subjected to the obligation of surveillance.

Among food-borne parasites affecting wild boars, Toxoplasma gondii, Trichinella spp., and Alaria alata are of superior importance. T. gondii is a ubiquitous protozoan parasite infecting a wide range of hosts and different host cells (Dubey 2010). Toxoplasmosis is one of the most common parasitic zoonoses worldwide, affecting one third of human population, and the most common in the European Union (EFSA 2007). The clinical outcome of the infection is influenced by several factors (host immune systems, parasite load) including the parasite genotype involved in the infection. The consumption of raw or undercooked meat of livestock containing tissue cysts is considered an important source of infection for humans; the increased consumption of game meat, including wild boars, is considered an emerging risk factor for T. gondii infection in humans (Tenter et al. 2000). Surveys on T. gondii infection in wild boars carried out in Europe show a high variability in seroprevalence values in the different countries, as well as in the same country, ranging from less than 10 to until 50% (Berger-Schoch et al. 2011a; Closa-Sebastia et al. 2011; Opsteegh et al. 2011; Beral et al. 2012; Deksne and Kirjusina 2013; Pastiu et al. 2013; Coelho et al. 2014; Jokelainen et al. 2015; Racka et al. 2015; Touloudi et al. 2015; Wallander et al. 2015; Witkowski et al. 2015; Roqueplo et al. 2017). In Italy, prevalence values between 14 and 23% were reported (Ranucci et al. 2013; Ferroglio et al. 2014; Sarno et al. 2014). Only few data on circulating genotypes in wild boars in Europe are available: genotype II was found in France (Richomme et al. 2009), while in Spain the circulation of all the three types was demonstrated in wild boars (Calero-Bernal et al. 2013; Calero-Bernal et al. 2015).

Trichinellosis is another zoonotic disease caused by the consumption of raw or undercooked meat and meat-derived products containing infective larvae, having serious and sometimes fatal outcomes (Alban et al. 2011). After pig, wild boar meat is the second most important source of human trichinellosis, as demonstrated by human outbreaks reported in recent years in several countries, including Italy (Romano et al. 2011; Fichi et al. 2015; Turiac et al. 2017). In Europe, Regulation 1375/2015 specifies the official procedures for the inspection of Trichinella in meat from all susceptible (domestic and wild) animals destined for human consumption.

Moreover, wild boars are paratenic hosts of the mesocercarial stage (Distomum musculorum suis) of the trematode A. alata. Definitive hosts are carnivores (canides, felides, and mustelides), while snails and amphibians are intermediate hosts of the parasite. Initially, it was supposed that A. alata mesocercarial stage in wild boars would not represent a risk for consumers; however, the consumption of raw or undercooked meat from infected game in North America has been demonstrated to cause the disease in humans, known as alariosis, which manifests with a range of different clinical signs, even serious, and sometimes with fatal consequences (Moehl et al. 2009). A. alata has already been reported in different European wild boar populations (Jaksic et al. 2002; Paulsen et al. 2012; Riehn et al. 2012; Paulsen et al. 2013; Berger and Paulsen 2014; Portier et al. 2014). In Italy, A. alata has been detected in three foxes and in a dog from Northern Italy (Ferroglio et al. 2012; Fiocchi et al. 2016), while no epidemiological data for A. alata in wild boars in Italy are available so far.

Considering the lack of data regarding zoonotic parasites in wild boars in Italy, we carried out an epidemiological survey on selected pathogens, i.e., T. gondii, Trichinella spp., and A. alata, in wild boars hunted for human consumption in Italy, with the aim to evaluate the risk for humans posed by the consumption of meat derived from wild boars.

Materials and methods

Study area

The Val Grande National Park is located in the Piedmont region, in the Central Italians Alps northwest of Italy (45° 56′ N, 8° 32′ E). The area is mainly hilly and mountainous and the altitude ranges from 201 to 2193 m.a.s.l. The alpine climate is characterized by cold winter (average temperature 1.5 °C) and warm summers (average temperature 21.1 °C); the study area is very rainy in all season (average rainfall 1130 mm), with heavy snowfalls in winter. Due to the low altitude and to the proximity to the Lake Maggiore, the whole surface of the Park at altitudes comprised between 1000 and 1600 m.a.s.l. is covered by broadleaved and mixed woodlands, with a predominance of chestnut (Castanea sativa) and beech (Fagus sylvatica) trees and to a lesser extent conifers, mainly Norway spruce (Picea abies) and European silver fir (Abies alba). At higher altitude, woods are replaced by bushes of green alder (Alnus viridis) and rowan (Sorbus aucuparia). The rivers Rio Valgrande and Rio Pogallo run within the two principal valleys, Val Grande and Val Pogallo, respectively, feeding the rich waterways of the Park. The environment is thus characterized by wetlands and bogs. The Park was selected as the study area for its abundance in wild fauna; particularly, due to the high population density of wild boars, a depopulation management plan regulated by the Park was settled in order to reduce their impact on environment.

Study population

One hundred wild boars (Sus scrofa) regularly hunted for human consumption throughout two subsequent hunting seasons (2015–2016 throughout October and December) were sampled during the post-mortem inspection activities at the control center. Individual epidemiological data regarding age, gender, weight, and killing place were collected. Age was determined by the dental table according to Boitani and Mattei (1991): sampled animals were categorized into red (6–10 months), subadult (11–14 months), adult 1 (15–18 months), adult 2 (19–24 months), adult 3 (25–36 months), and adult 4 (> 36 months).

Sample collection

Approximately 150 g of diaphragm muscle tissue samples was collected from wild boars during the mandatory inspection of carcasses at a game-handling establishment. Samples were placed in plastic bags, then stored and refrigerated until arrival at the laboratory. For T. gondii, an aliquot of 25 g was submitted to a frost/defrost thermal treatment according to Nöckler et al. (2005) to extract meat juice, that was stored at − 20 °C until analysis; another aliquot (25 g) was mechanically homogenized and then stored at − 20 °C until molecular analysis. Other two aliquots were used for the detection of A. alata mesocercariae (30 g) and Trichinella spp. (20 g); both performed within 24 h after arrival at the laboratory. An additional aliquot (50 g) was stored at − 20 °C for further analysis, in case of a positive finding in the pools of samples examined for Trichinella spp.

T. gondii antibody detection and molecular analysis

Meat juice samples were analyzed using an indirect multispecies ELISA validated for the detection of anti-T. gondii antibodies in meat juice from multiple species including swine (ID Screen® Toxoplasmosis Indirect Multi-species, IDvet, Montpellier, France), according to the manufacturer’s instructions. Positive and negative control sera provided with the kit were used as controls. For each sample, the resulting values were calculated, applying the formula supplied in the kit: S/P %  = OD sample − OD − negative control/OD positive control − OD negative control) × 100. Samples with SP% ≥ 50% were considered positive.

On animals showing antibodies anti-T. gondii, diaphragm muscle homogenate was processed to extract genomic DNA using a commercial kit (NucleoSpin®Tissue, Macherey-Nagel, Germany), following the manufacturer’s instructions. As screening, DNA samples were analyzed using a single-tube nested PCR targeting a region of 227 bp of the ITS-1 region as described by Hurtado et al. (2001). Then, on nested-PCR positive samples, multilocus PCR for BTUB and GRA6 loci according to Su et al. (2010) was performed for genotyping purpose; moreover, a conventional PCR was carried out to amplify B1 gene, using the primers described by Papini et al. (2017). Positive (Zanzani et al. 2016) and negative (non-template) controls were inserted in each run. PCR products were run on 1.5% agarose gel containing 0.05% ethidium bromide in TBE buffer electrophoresis and visualized under UV light on a transilluminator. Bands of the expected size were excised from agarose gel, purified with a commercial kit (NucleoSpin® Gel and PCR Clean-up, Macherey-Nagel, Germany), and finally sent for sequencing in both directions to a commercial service (Metabion, Martinsried, Germany). Obtained sequences were manually assembled and compared to available T. gondii sequences using BLASTn software (https://www.ncbi.nlm.nih.gov/blast/).

Trichinella spp. detection

The magnetic stirrer method for pooled sample digestion, described as reference method in Regulation (EC) No. 1375/2015, was employed for Trichinella detection. Pools of five individuals each were inspected; since the technique is standardized for the analysis of a total amount of 100 g, for each individual, 20 g of diaphragm were examined. In case of positive pool, individual samples were independently examined.

A. alata mesocercariae detection and molecular analysis

On a sample of 30 g of diaphragm, the larva migration technique (AMT) was performed as described by Riehn et al. (2010). This technique is based on the tendency of the mesocercariae to move actively out of the infected tissue and on their high affinity to liquids, comparatively to the Baermann technique for nematodes. The recovered larvae were identified by morphological features using light microscopy according to Odening (1961) and Johnson (1968). Subsequently, molecular techniques were performed to confirm the morphological identification of A. alata mesocercariae. Collected mesocercariae were washed with saline solution and stored in 70% ethanol until further examination. Genomic DNA extraction was performed using a commercial kit (NucleoSpin®Tissue, Macherey-Nagel, Germany), following the manufacturer’s instructions. A region of around 300 base pairs was amplified by PCR according to the protocol described by Riehn et al. (2011). A positive control, an A. alata specimen previously identified by the German Federal Institute of Risk Assessment (BfR), and a negative (non-template) control were used in each amplification. Amplicons were separated electrophoretically in 1% agarose gel and visualized on UV transilluminator (Alpha Innotech, distribution by Biozym Scientific, Hessisch Oldendorf, Germany).

Statistical analysis

For each pathogen, prevalence of infection was calculated considering different categories (age, sex, year of sampling), according to Bush et al. (1997). For T. gondii, analysis of risk factors associated with the infections was performed. A generalized linear model (GLM) with binary logistic distribution was performed using as dependent variable a binary outcome, on the basis of presence/absence of antibodies anti-T. gondii. The following response variables were used in the model: age (ordinal variable), sex (category variable), and weight (continuous variable). The model was developed through a backward selection procedure (significance level to remove variables from the model = 0.05), based on AIC values. Statistical analysis was performed using SPSS software (Statistical Package for Social Science, IBM SPSS Statistics for Windows, Version 19.0., Chicago, IL, USA).

Results

A hundred wild boars, 33 females and 67 males, were included in the survey. The body weight of the wild boars ranged between 12 and 86 kg (arithmetic mean 38.5; median 35; standard deviation 18.8). The male wild boars weighed on average 39.4 kg (min–max 12–86; median 35; standard deviation 19.2), while the females were a little bit lighter on average (min–max 13–68; arithmetic mean 36.8; median 3835; standard deviation 18.2). Both young (30 red and 7 subadult) and adult (63) animals were sampled (Table 1).

Table 1 Baseline characteristics of wild boars sampled in Italy and Toxoplasma gondii seroprevalence values for each considered category
Full size table

Ninety-seven meat juice samples from wild boars were analyzed for the presence of antibodies anti-T. gondii in meat juice samples; three animals could not be included because obtained meat juice was not sufficient for the analysis. Of these, 42 samples were found positive for anti-T. gondii antibodies, representing a prevalence of 43.3% (95% CI 33.9–53.2). Prevalence was similar in wild boars hunted in 2015 (23/53; P 43.4%; 95% CI 30.9–56.7) and in 2016 (19/44; P 43.2%; 95% CI 29.7–57.8). Seropositive samples showed an average S/P% of 100.1 (standard deviation 33.7). Considering individual data, young animals showed higher prevalence (17/34; P = 50%; 95% CI 34.1–65.9) than adults (25/63; P 39.7%; 95% CI 28.5–52), while in males and females seroprevalence values were recorded at 43.1 and 43.8%, respectively. Data related to the prevalence values in the considered categories are summarized in Table 1.

Of the 42 seropositive wild boars, only three samples resulted positive for T. gondii to the screening nested PCR. Subsequent sequencing of ITS-1 fragments confirmed the identity of T. gondii (homology of 100%). Considering the following genotyping analysis, amplicons were successfully sequenced only for sample ID WB-04 (GRA6, BTUB, and B1) and for WB-54 (B1) (Table 2). Sequencing of the amplified GRA6, BTUB, and B1 fragments showed that all of them belonged to type II, with a 99% homology (Table 2). Sequences obtained were submitted to GenBank under the accession number MH094810 to MH094814.

Table 2 Toxoplasma gondii genotype patterns in wild boars sampled in Italy
Full size table

Trichinella spp. larvae were not found in any of the sampled wild boars (0/100; P = 0%; 95% IC 0–0.4).

Only one animal (1/100; P = 1%; 95% IC 0.1–5.45) was found positive to A. alata mesocercariae: this was a female adult 2 wild boar, with a bodyweight of 43 kg. The isolated larva was identified by both morphological examination and PCR.

Since no larvae of Trichinella and only one mesocercariae of Alaria was found in the surveyed wild boars, statistical analysis on risk factors was performed only on results obtained on T. gondii: no associations between T. gondii infection and the considered risk factors were highlighted, resulting in all the variables introduced in the model (age, sex, and weight) are statistically not associated to the infection (P value > 0.05).

Discussion

The present study contributed to update data on selected food-borne pathogens (i.e., T. gondii, Trichinella, and A. alata), showing a zoonotic potential in wild boars destined for human consumption. The European Food Safety Authority highlighted the necessity of a continuous monitoring of T. gondii infection in animal species destined for human consumption (EFSA 2007); indeed, epidemiological studies have been carried out also in the study area, particularly in domestic species (Gazzonis et al. 2015; Papini et al. 2017; Gazzonis et al. 2018). Moreover, the monitoring of T. gondii infection has been indicated as high priority, not only in domestic animals but also in game meat, including wild boars (EFSA 2013). In the monitoring and surveillance of T. gondii infection, serological surveys represent an important diagnostic tool for their applicability on large scale. In this study, antibody detection was carried out on meat juice using a commercial ELISA validated for analysis on this biological substrate. Meat juice has been proven as suitable for the screening of T. gondii infection in different meat-supplying animals (Berger-Schoch et al. 2011a; Meemken et al. 2014). Meat juice is easier to collect than blood, especially when it is difficult or even impossible to collect blood ante mortem and there are obvious logistic difficulties in obtaining post-mortem serum samples, as in the case of hunted, wild animals (Coelho et al. 2015).

A high spread of the infection was confirmed by the presence of antibodies anti-T. gondii in a huge portion of investigated wild boars. The recorded seroprevalence value (43.3%) is similar to those reported in Europe, as recently reviewed (Rostami et al. 2017b), although other epidemiological studies reported lower prevalence values (< 10 up to 16%) in wild boars in Italy, Greece, and Switzerland (Berger-Schoch et al. 2011a; Ranucci et al. 2013; Ferroglio et al. 2014; Touloudi et al. 2015). In order to verify any possible association with T. gondii infection, individual data were considered in a statistical analysis of risk factors. The prevalence of T. gondii antibodies was higher in young (50%) than in adult animals (39.7%) although either the age or the weight of the animals did not result in enhancing the risk of T. gondii infection. Data available in literature shows contradictory results: some authors reported higher seroprevalence values in adult animals (Richomme et al. 2009; Ranucci et al. 2013; Coelho et al. 2014; Racka et al. 2015; Wallander et al. 2015; Calero-Bernal et al. 2016; Reiterová et al. 2016), while in other studies no significant differences among age classes were registered (Gauss et al. 2005; Antolová et al. 2007; Richomme et al. 2010; Opsteegh et al. 2011; Jokelainen et al. 2012; Jokelainen et al. 2015). Indeed, contrary to what was already demonstrated in pigs, a significant effect of age on seroprevalence, like lifelong persistence of T. gondii antibodies, has not been demonstrated to date in wild boars (Opsteegh et al. 2011). Similarly, any association between T. gondii prevalence and gender was not observed, in agreement with other studies (Gauss et al. 2005; Closa-Sebastia et al. 2011; Jokelainen et al. 2015).

Furthermore, the presence of T. gondii was verified through the molecular detection of the parasite DNA in the diaphragm muscle samples. Out of 42 seropositive wild boars, only three animals (7.1%) were positive in the PCR. Although the serological status reflects the infectivity of meat from these animals (Dubey 2009), the lack of detection of T. gondii cysts in the skeletal muscle of seropositive animals has been also reported in previous studies. In a study conducted in Slovakia, only one (5.5%) out of 18 seropositive animals (n = 91) was positive in the PCR (Luptakova et al. 2010); in Switzerland, 10 (6.7%) out of 150 sampled wild boars showed antibodies anti-T. gondii but in only one (10%) of these animals T. gondii DNA was detected (Berger-Schoch et al. 2011a; Berger-Schoch et al. 2011b). A lack in sensitivity of molecular analysis could be explained by the limited sample size, random distribution of tissue cysts, and low numbers of T. gondii tissue cysts in the tissues tested (Hill et al. 2006).

Nevertheless, molecular techniques allow the identification of T. gondii strains involved in the infection. Indeed, to correctly evaluate the risk for humans posed by the consumption of meat of infected animals, the determination of circulating T. gondii genotypes is crucial, since illness severity and clinical aspects of toxoplasmosis may depend on the virulence of T. gondii strains involved (Dubey 2009). Our results confirmed the circulation of genotype II in the surveyed wild boars, the lineage more frequently involved in clinical cases of human toxoplasmosis (Su et al. 2010).

The results update information on the spread of T. gondii genotypes circulating in European wild boar populations (Richomme et al. 2009; Calero-Bernal et al. 2013; Calero-Bernal et al. 2015); the report of all three clonal types (I, II, and III) in European wild boar populations suggests the possibility that various parasite strains are involved in the infection. Indeed, a higher possibility of sexual recombination potentially able to generate T. gondii strains with new biological properties, such as an increased virulence for humans, has been suggested for the sylvatic cycle (Grigg and Sundar 2009). From a public health perspective, the genetic variability of T. gondii underlines the importance of the continuous monitoring of the circulation of T. gondii genotypes among wild game populations.

Human trichinellosis is a rare but serious human disease and outbreaks are still found within the EU (Murrell and Pozio 2011). Recent data from EFSA reported 101 confirmed cases of trichinellosis in 2016 from 27 member states of EU. With a notification rate of 0.02 cases per 100,000 population, the number of cases decreases if compared to previous years (EFSA and ECDC, 2017). Transmission to humans is usually domestically acquired by the consumption of raw or undercooked meat from infected pigs or horses (domestic cycle) or from infected wildlife (sylvatic cycle), including wild boars (Ranque et al. 2000; Gari-Toussaint et al. 2005; Rostami et al. 2017a).

In the present study, none of the 100 analyzed wild boars presented larvae of Trichinella spp. Indeed, in 2016, only 256 infected animals were identified out of the 1,152,650 wild boars tested in Europe (P = 0.02%), with only 9 positive wild boars reported in Italy out of 147,930 tested (P = 0.006%). The number of infected wild boars in Europe is decreasing from 0.14% positive animals in 2012 to 0.02% in 2016 probably due to a corresponding decrease of the infection in both sylvatic and domestic cycle (EFSA and ECDC, 2017). However, the increasing number of wild boars in Europe, as well as other wildlife species (foxes and raccoon dogs), may increase Trichinella biomass circulating among wild animals (Kirjusina et al. 2015; Pannwitz et al. 2010). For this reason, a systematic Trichinella inspection in wild boars remains an essential risk-reducing measure.

Finally, the present survey reports the finding of A. alata mesocercariae in a wild boar in Italy. The low prevalence value registered in our survey (P = 1%) is similar to those previously reported in Croatia (1.8%) (Jaksic et al. 2002), France (0.6%) (Portier et al. 2014), and Hungary (1.6%) (Berger and Paulsen 2014), although other surveys in Europe reported higher prevalence values, ranging from 6 to 11.5% (Paulsen et al. 2012; Riehn et al. 2012; Paulsen et al. 2013). This finding demonstrates for the first time the circulation of the trematode in Italian wild boar populations, with the morphological identification of the trematode larva confirmed by molecular techniques. Indeed, in Italy, A. alata was previously reported in red foxes (Fiocchi et al. 2016) and in a domestic dog (Ferroglio et al. 2012). Considering the potential risk for humans to acquire alariosis by the consumption of game meat and the lack of information on the prevalence of A. alata in wild boars in Italy, it is necessary to implement a regular monitoring of the parasite in wild animals, particularly those destined for human consumption (Moehl et al. 2009).

Conclusions

Data obtained from this study emphasize the importance of infected wild boar meat as a source of zoonotic infections and support the need of monitoring and surveillance of food-borne pathogens in wild animals, including wild boars. Noteworthy, this is the first finding of A. alata mesocercariae in wild boars in Italy. The circulation of the trematode among wild animals in the country has been confirmed, although further studies are necessary to evaluate the prevalence of this potential zoonotic parasite in Italian wild boar populations and thus correctly assess the consequent risk for humans.