Wild boar are listed amongst the hundred most invasive species by the International Union for Conservation of Nature (Sales et al. 2017), and pose significant health concerns to humans and livestock (Meng et al. 2009; Rostami et al. 2017; Almeria and Dubey 2021; Abrantes and Vieira-Pinto 2023). Additionally, wild boar are considered the most important and widely distributed game animal species globally (Massei et al. 2015). For these reasons, this species acts as indicator for studies of the geographical variations of T. gondii prevalence and incidence (Ranucci et al. 2013), and offer a suitable biological model for investigating T. gondii infection dynamics (Beral et al. 2012). Alongside the increase in wild boar populations, several European countries, like France, Spain, Austria, and Germany are witnessing a growing wild game meat market, which pose health challenges linked to the risk of human infections (Fantechi et al. 2022). Notably, a recent study in Poland showed that higher consumption of wild game meat is correlated with lower safety concerns (Niewiadomska et al. 2020; Fantechi et al. 2022).
To the best of our knowledge, the present survey represents the first comprehensive epidemiological study of T. gondii prevalence in the wild boar population of a large Mediterranean island. The sample size in this study (n = 562) exceeded the minimum number of 377 samples initially required, thus strengthening the robustness of the experimental design and the conclusions. The 37.1% prevalence of T. gondii (via PCR testing) in the wild boar population indicates a widespread presence of the parasite throughout the island, in contrast with data from other studies previously conducted in intensively and extensively farmed animals from the same area (Scala et al. 2008; Pipia et al. 2018b; Dessì et al. 2022). Nevertheless, the T. gondii prevalence recorded by PCR is lower than that detected in other studies conducted in Southern Italy by Santoro et al.[12] (44%) and by Sgroi et al.(Sgroi et al. 2020) (39.6%), but higher than that in Northern Italy (16%) (Ferroglio et al. 2014). A study conducted in the Czech Republic recorded a low prevalence (8.8%) by PCR testing, and a higher seroprevalence rate (15.4%) (Slany et al. 2016), likely due to the use of diaphragmatic rather than cardiac tissue. The use of cardiac tissue provides a more accurate of epidemiological dynamics as it yields a higher number of infections among sampled animals, thus indicating that determining the prevalence of animals harbouring tissue cysts would be the most efficient use of resources (Opsteegh et al. 2016). Recently, several surveys have reported varying seroprevalence rates of T. gondii in wild boar worldwide, including Japan (22.7%) (Saito et al. 2021), Korea (36%) (Jeong et al. 2014), the United States (27.7%) (Sandfoss et al. 2011), Argentina (12.5%) (Winter et al. 2019) and Brazil (15.6%) (Brandão et al. 2019). Likewise, in Europe, studies have reported varying T. gondii seroprevalence, with the highest percentage reported in Poland (48%) (Puchalska et al. 2021), Sweden (50%) (Wallander et al. 2015), the Czech Republic (40%) (Račka et al. 2015) Romania (56.6%) (Grema et al. 2015), Corsica (55%) (Richomme et al. 2010) and Italy (53.1%) (Villa et al. 2023). Conversely, lower seroprevalence rates were recorded in the Slovak Republic (8.1%) (Antolová et al. 2007) and Switzerland (6.7%) (Berger-Schoch et al. 2011). The overall seroprevalence (24.5%) in our study is comparable to that recorded in Germany (24.40%) (Bier et al. 2020), and in the Netherlands (24.4%) (Opsteegh et al. 2011) but higher than the infection rates recorded in Spain (14%) (Lizana et al. 2021) and Central Italy (14%) (Ranucci et al. 2013). The variations in the data reported emphasize the challenges with comparing seroprevalences among different surveys, which can be attributed to geographical differences among areas, the absence of standardized diagnostic techniques, and the application of different epidemiological approaches (Veronesi et al. 2011). Moreover, it is essential to note that meat juice, examined in this study as a serological matrix, differs from blood serum and that ELISAs applied to this material are characterised by lower sensitivity than blood serum. However, specific antibody levels in meat juice depend on the muscle(s) chosen for sampling, with heart meat juice characterised by significantly higher levels of antibodies compared with the other muscles (Wallander et al. 2015). It is worth mentioning that PCR analysis detects nucleic acids from both live and dead T. gondii, while ELISA can only identify chronic infections via the detection of anti-Toxoplasma IgG (Dessì et al. 2022). Importantly, previous studies pointed out that the detection of T. gondii antibodies in wild boar is positively correlated with the presence of bradyzoites in the animal muscles (Bártová et al. 2006; Richomme et al. 2010). In our survey, animal gender and age were identified as risk factors for T. gondii infection, with significantly higher seroprevalences recorded in males (29.0%) compared to females (19.1%) and in adults (27.9%) compared with juveniles (14.7%). The higher seroprevalence in male wild boar is inconsistent with data from most studies that reported no gender-related difference in seroprevalence (Richomme et al. 2010; Opsteegh et al. 2011; Ranucci et al. 2013), may be explained with the larger home ranges of males compared to females (Laguna et al. 2022; Cavazza et al. 2023).The higher prevalence observed in adult wild boar is also inconsistent with data from other studies (Antolová et al. 2007; Opsteegh et al. 2011; Ranucci et al. 2013) and it may be related to longer exposure to the parasite compared with juveniles. However, whether anti-T. gondii immunity in wild boar is a lifelong condition remains to be determined. Opsteegh et al. (2011) showed that the animals up to 10 months of age show a step-increase in seroprevalence, with titres of anti-Toxoplasma antibodies stabilising thereafter, suggesting susceptibility to infection may remain throughout an animal’s life. Bernal and colleagues observed three cases of vertical transmission of toxoplasmosis. They found one with type III infection with no malformations, while the other two, one with type I infection and the other with types I and III infection, exhibited severe malformations. This indicates that the pathogenicity of different T. gondii types may vary in wild boars. (Calero-Bernal et al. 2013) Further research is needed to determine the role of vertical transmission of T. gondii and the congenital toxoplasmosis as well in perpetuating its life cycle. The prevalence for T. gondii identified in this study indicates that the parasite is widespread in Sardinia. Moreover, our data supports the hypothesis that infected wild boar may represent a key-sentinel of environmental contamination with T. gondii, due to their capacity for adaptation to different habitats, wide geographical distribution and high reproductive rates (Sgroi et al. 2020). Recreational hunting can impact wild boar social and spatial behaviour (Keuling and Massei 2021). For instance, a study in Catalonia (Spain) found six of 40 wild boar that had been ear-tagged and later culled by hunters at a mean linear distance of 45.8 km (min. 30, max. 89.8) from their origin (Casas-Díaz et al. 2013). Wild boar plays an important role in maintaining the T. gondii sylvatic cycle due to their scavenging and predatory behaviour and frequent interactions with a wide range of different hosts (including sheep, cattle, birds, rodents and foxes). The risk of human transmission is exacerbated by hunters’ malpractices, such as abandoning animal offal and muscle pieces in the environment following evisceration (Sgroi et al. 2019). The offal and viscera can be scavenged by other wildlife, including wild boar, predators (including cats) or pigs, thus amplifying the risks for transmission (Ranucci et al. 2013). The detection of T. gondii in wild boar meat highlights a potential risk to humans, particularly given local eating habits that differ from the traditional Italian cuisine, that envisages wild boar meat being cooked for several hours before consumption. The growing market demand for wild boar sausages and cured meat may lead to a significant increase in risk of human infections as already been highlighted (Richomme et al. 2010). These products have previously been shown to present a risk for infection with pathogenic microorganisms, including T. gondii (Hill and Dubey 2018; Fredericks et al. 2019). Indeed, sausages and fresh meat subjected to smoking, salting, drying, and injection of solutions containing sodium chloride, potassium lactate, and sodium lactate are intended for direct consumption, although some of these processes may inactivate bradyzoites (Hill et al. 2006; Kijlstra and Jongert 2008; Hill and Dubey 2018), however, the pathogen transmission is still possible. In particular, the "Salsiccia Sarda", a short-aged, cured salami, made with meat and pork fat, is a traditional Sardinian product included in the national list of traditional agri-food products (XXIII Revision of the list of traditional agri-food products produced, Italian Republic, 22.05.2023). The Salsiccia Sarda consists of a semi-dry, fermented sausage made from minced, fermented, dried and seasoned pork fat and meat, with added spices and food additives (nitrates, nitrites, glutamate). However, production is strongly influenced by family customs and recipes, resulting in great variability in the production process (Siddi et al. 2022). Curing time of > 12 months (typical of many Italian salami) is recommended to ensure the inactivation of the parasite (Fredericks et al. 2019). Nevertheless, for the Sardinian sausage, this time is reduced to a maximum of 20–25 days, thus potentially leading to any contaminating Toxoplasma cysts retaining their infectious properties throughout the curing process. In addition, Italian law does not enforce health checks on meat obtained from hunted wild boar. Biological samples from hunted wild boar are typically tested for the presence of African Swine Fever (ASF) and Trichinella spp. (according to the European Regulation 2015/1375), which outlines rules for Trichinella control in meat. However, no specific checks are required for other pathogens. This is justified by the fact that the hunted wild boar is considered for self-consumption and not intended for sale. However, this game meat is frequently offered in typical country restaurants (“agriturismo”) to locals and tourists alike, who often consume raw homemade products (e.g., sausages or “guanciale”). Due to this potential risk to public health, a citizen science approach is essential to obtain large-scale epidemiological data and to educate and raise public health awareness.
In our study, meat inspection specialists supported the hunting teams to improve slaughter hygiene and assist health monitoring of carcasses. Similar collaborations between veterinarians and hunters in southern Italy revealed a stable circulation of Trichinella britovi in wild boar meat (Sgroi et al. 2023), as well as a high molecular prevalence of T. gondii (39.6% out of 338 animals), indicating a tangible risk of infection for consumers (Sgroi et al. 2020). Notably, in 2005 has been documented an outbreak of trichinellosis in two Sardinia villages, which involved 11 people who had eaten raw sausages made from the same free-ranging sow (Pozio et al. 2006). Given the high prevalence of important foodborne zoonosis like toxoplasmosis and trichinellosis, we argue that the existing gap in legislation related to inspection of game meat should be addressed by both local and European authorities. Consumption of raw or undercooked wild boar meat may lead to infection, as evidenced by several reported cases of acute toxoplasmosis among hunters and their families who consumed meat from infected wild boar (Puchalska et al. 2021). In addition, hunters and their families can become infected during evisceration practices and at various stages of venison processing (Tenter et al. 2001). For instance, in three patients from Korea, T. gondii chorioretinitis was described following ingestion of raw liver and spleen from a wild pig (Choi et al. 1997; Meng et al. 2009). Most human infections reported in the US and in France are likely the result of ingestion of cysts contaminating undercooked meat, with ~ 84% of pregnant women presenting anti-T. gondii antibodies (De Barros et al. 2022). These data confirm that the consumption of raw or rare wild boar meats is likely to lead to T. gondii infection. To address this concern, the Biological Hazard Panel of the EFSA has recently proposed the implementation of Toxoplasma monitoring programmes (EFSA 2007). These programs aim to produce Toxoplasma-free meat via selection of seronegative animals and the introduction of validated tests and certification, leading to the issuance of ‘Toxoplasma-free meat’ label for Toxoplasma-free farms (Kijlstra and Jongert 2008). Alternatively, the development and standardization of methods for the testing of pathogen survival in dry-cured meat products may assist with mitigating the risk of Toxoplasma transmission to consumers (Herrero et al. 2016). These measures may significantly contribute to the reduction of zoonotic transmission of toxoplasmosis from wild board to humans and ensure safer consumption practices.