The illegal rearing and slaughtering of pigs in the wild on the Mediterranean island of Sardinia favor an increase in the biomass of Trichinella britovi in wild boars (Sus scrofa) but do not affect the serological prevalence of infection

Background Worms of the nematode genus Trichinella are zoonotic pathogens with a worldwide distribution. The first report of Trichinella on the Mediterranean island of Sardinia was for Trichinella britovi, one of the four species of this genus circulating in Europe, which was identified in 2005 following an outbreak of trichinellosis in humans due to the consumption of pork from pigs reared in the wild. Since then, T. britovi larvae have been repeatedly isolated from free-ranging pigs, foxes (Vulpes vulpes) and wild boars (Sus scrofa) sampled in the central-eastern region of the island (Orgosolo municipality), but have never been isolated from samples from other areas of the island. The aim of this study was to investigate the parasitological and serological prevalence of T. britovi infection in wild boars in Sardinia over space [eight wild boar hunting management units (HMUs)] and time (seven wild boar hunting seasons). Methods Muscle and serum samples of boars killed in the 2014–2015 to 2020–2121 hunting seasons were collected from eight HMUs of central and south-western Sardinia. Trichinella sp. larvae were detected by artificial digestion of predilection muscles. A total of 4111 serum samples of wild boar were collected from the investigated HMUs and tested by enzyme-linked immunosorbent assay as a screening test and by western blot as a confirmatory test using excretory/secretory antigens. Results Trichinella britovi muscle larvae were detected in six (0.03%) of the 17,786 wild boars tested. All of the Trichinella sp.-positive wild boars had been hunted in Orgosolo municipality (central-eastern area of the island), except for one, hunted in a neighboring municipality. An overall serological prevalence of 3.8% (95% confidence interval, 3.3–4.5) was detected by western blot. No statistical differences were detected between the HMUs where T. britovi larvae were detected in wild boars, foxes, and free-ranging pigs and those where wild boars, foxes and free-ranging pigs tested negative. Conclusions The serological prevalence did not vary between the wild boar populations in which the larval load was detectable by artificial digestion (Orgosolo municipality) and those in which the larval load was below the detection limit. Furthermore, the serological prevalence of anti-Trichinella immunoglobulin G in the wild boar populations remained constant during the study period, which covered seven wild boar hunting seasons. As the transmission events (i.e., the serological prevalence) are stable, the high biomass of the parasite in Orgosolo municipality can only have arisen as a consequence of factors independent of its natural cycle, i.e., the presence of a high number of free-ranging pigs, and the concomitant presence of African swine fever, due to illegal pig slaughtering in the field. This epidemiological situation suggests that the natural cycle of T. britovi may be influenced by inappropriate pig husbandry and slaughtering practices. Graphical Abstract


Background
Foodborne zoonotic nematodes of the genus Trichinella are widespread and circulate on most continents.Wild animals play an important role as reservoir hosts of these parasites [1].Of the 13 described species of Trichinella, four (Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis and Trichinella papuae) are often found in naturally infected swine, and T. spiralis is the most frequently documented species due to its high reproductive rate and persistence of its larvae in the muscles of infected animals [2][3][4].In Europe, although T. britovi has mainly been detected in carnivorous mammals, it has also been found in 21.2% of free-ranging pigs and 50% of wild boars (Sus scrofa) (International Trichinella Reference Center, www.trich inell osis.org).In swine, T. britovi shows a low reproductive capacity index and infective larvae survive for no longer than 6 months in muscle [4].
In the Mediterranean islands, T. britovi was detected for the first time in a free-ranging pig in Corsica, France in 2004; in the following year, this parasite was detected in Sardinia, Italy during an outbreak of trichinellosis in humans caused by the consumption of sausages made with the pork of a free-ranging pig [5,6].Since the discovery of T. britovi in these two Mediterranean islands, numerous studies have been carried out to monitor the circulation of this nematode among domestic pigs reared in industrial farms, family farms, or free-ranging in the wild, and in wild boars and red foxes (Vulpes vulpes) [6][7][8][9][10].These studies showed that T. britovi was present in free-ranging and backyard pigs, domestic dogs, wild boars, and red foxes, in numerous foci in Corsica, whereas in Sardinia it was only detected in free-ranging pigs, wild boars, and red foxes sampled in the Orgosolo municipality (central-eastern area of the island).In the period 2005-2022, four outbreaks of trichinellosis in humans (a total of 30 cases) and a single case, all due to the consumption of meat from free-ranging pigs or wild boar were reported in Sardinia [7,9].Furthermore, an outbreak of trichinellosis in mainland France was also documented, which arose from the consumption of infected pork products imported from Corsica [10].A subsequent study based on microsatellite analysis indicated that T. britovi had probably been introduced into these two Mediterranean islands through two or more independent events that took place during the Middle Pleistocene and Early Holocene [11].Anti-Trichinella antibodies were recently detected in free-ranging black pigs in the Italian island of Sicily, the largest Mediterranean island [12].
The aim of this study was to investigate the parasitological and serological prevalence of T. britovi infection in wild boars in Sardinia over time (a total of seven wild boar hunting seasons, from 2014-2015 to 2020-2021) and space (a total of eight wild boar hunting areas in central and south-western Sardinia).Furthermore, the serological prevalence in hunting dogs and the parasitological prevalence of T. britovi infection in pigs illegally reared in the wild, and in red foxes, were also investigated to fully evaluate the epidemiology of T. britovi in Sardinia.

Study area
The central-eastern area of Sardinia, including the Orgosolo municipality, where T. britovi has been documented in illegally reared free-ranging pigs as well as in wild animals (i.e., red foxes and wild boar), was chosen as the study area (Fig. 1) [9].This area is characterized by thick vegetation, mountain ranges and limited water sources, a mild climate during the spring-summer period, and winters characterized by low temperatures and frost.The area is agricultural with a low human population density (17.6 inhabitants/km 2 ), and several of the areas inhabited by humans are characterized by social deprivation [13,14].African swine fever (ASF) has been endemic in Sardinia since 1978 [15].Between 2011 and 2016, wild boar health was managed for the control of ASF based on areas in which wild boar are hunted.From 2016 onwards, these areas were revised and named hunting management units (HMUs); the HMUs are bordered Conclusions The serological prevalence did not vary between the wild boar populations in which the larval load was detectable by artificial digestion (Orgosolo municipality) and those in which the larval load was below the detection limit.Furthermore, the serological prevalence of anti-Trichinella immunoglobulin G in the wild boar populations remained constant during the study period, which covered seven wild boar hunting seasons.As the transmission events (i.e., the serological prevalence) are stable, the high biomass of the parasite in Orgosolo municipality can only have arisen as a consequence of factors independent of its natural cycle, i.e., the presence of a high number of free-ranging pigs, and the concomitant presence of African swine fever, due to illegal pig slaughtering in the field.This epidemiological situation suggests that the natural cycle of T. britovi may be influenced by inappropriate pig husbandry and slaughtering practices.
by natural barriers that limit the movements of animals [16].The wild boar hunting season (1 November-31 January) is currently managed within these HMUs to control the spread of ASF, with the obligation that the hunters provide blood and muscle samples from each wild boar hunted [17].Numerous wild boar HMUs located in the middle of Sardinia can be characterized by the historical presence of pigs illegally reared in the wild [18].Since 2016, action has been taken against the practice of raising pigs in the wild within the remit of the most recent eradication program for ASF, whereby pigs are culled and then sampled for the presence of ASF and Trichinella spp.[19].

Sampling areas
Seven HMUs located in the central-eastern area of the island, bordering the Orgosolo municipality, were included in this study.The HMUs are located in municipalities of Nuoro province, municipalities of the neighboring Ogliastra province, and some municipalities of Sassari province, which borders Nuoro (Fig. 1).Furthermore, serum and muscle samples collected between 2015 and 2016 from wild boars hunted in a south-western area of Sardinia (Carbonia province), about 130 km from the Orgosolo focus, were used as control samples from a potentially Trichinella-free area.

Detection of Trichinella spp. larvae
A total of 17,786 wild boar muscle samples from the seven wild boar hunting seasons from 2014-2015 to 2020-2021 were included in this study.Muscle samples (50 g) were collected from the diaphragm, tongue and/or masseter and tested by artificial digestion at the Istituto Zooprofilattico Sperimentale della Sardegna, in accordance with regulation 2015/1375 of the European Commission [20].Briefly, 100-g pools of muscle of 20 wild boars (5.0 g per boar) were tested.If the pool of muscles tested positive, 20-g samples from each of five animals were pooled and tested again.When Trichinella sp.larvae were detected in a pooled sample from five animals, more 20-g samples from each individual animal were tested again.From 2017 to 2021, a 5-to 10-g sample of tongue or masseter was collected under the ASF eradication program from 1189 pigs illegally reared in the wild in Nuoro province.These pigs were culled by the veterinary services under the protection of the police due to the dangerous social situation [19].When a pool of 10-20 pig muscle samples tested positive for Trichinella sp.larvae it was not possible to trace back the positive pig(s) in the pool due to a lack of additional muscle samples.Twenty grams of the tibialis anterior muscle of 141 red foxes, 5 g of head muscles of eight martens (Martes martes), four weasels (Mustela nivalis), and a wild cat (Felis silvestris) were also tested by digestion.Larvae were kept in 90% ethyl alcohol for further identification to species level by multiplex polymerase chain reaction at the Istituto Superiore di Sanità (ISS), Rome, in accordance with Pozio and La Rosa [21].
As a control, muscle samples from wild boars from south-western Sardinia (Carbonia province) collected between 2015 and 2016 were tested to detect Trichinella spp.larvae by digestion in accordance with European Commission regulation 2015/1375 [20].

Serosurvey of anti-Trichinella immunoglobulin G in wild boars and hunting dogs
The sample size needed to detect a minimum serum prevalence of anti-Trichinella immunoglobulin G (IgG) of 1% (with a 95% confidence interval; CI) was calculated for each HMU by using EpiTools (copyright 2014 AusVet Animal Health Services) [17,22,23]; there was one exception to this, HMU 28, located in south-western Carbonia province, for which the sample size needed to detect a minimum serum prevalence of anti-Trichinella IgG of 4% (with a 95% CI) was calculated instead (Table 1).A further 10-20% of samples for every HMU were collected to account for sample dropout.Furthermore, 203 serum samples of hunting dogs from the Orgosolo municipality were included in this study because these act as sentinel animals for the monitoring of infections with Trichinella spp. in wildlife [24].An aliquot of each serum sample was preserved at -20 °C until use.

Serological tests
Wild boar serum samples were first screened by using a commercial enzyme-linked immunosorbent assay (ELISA) kit (PrioCHECK Porcine Trichinella; Applied Biosystem) at the Istituto Zooprofilattico Sperimentale della Sardegna, Nuoro.Then, ELISA-positive sera were forwarded to the ISS in Rome under dry ice for confirmatory testing by western blot (WB) using excretory/ secretory antigens, in accordance with Gómez-Morales et al. [25,26].Dog serum samples were first tested by an in-house ELISA at ISS, then ELISA-positive sera were tested by using WB with excretory/secretory antigens in accordance with Gómez-Morales et al. [24].Serum samples were also tested to detect ASF antigen and antibodies [16,23].

Discussion
The wild boars of the existing population in Sardinia are descended from animals introduced into the island no earlier than the 7th millennium B.C., and probably originate from domesticated pigs that returned to their wild state [27].Since T. britovi had already been present on the island for at least 3000 years [11] when these swine were introduced, it is not surprising that anti-Trichinella IgG are uniformly distributed in the present-day wild boar population of Sardinia.
From 2005 to 2021, Trichinella britovi larvae were only detected in Sardinia in wild animals (wild boars and red foxes) and in free-ranging pigs in the Orgosolo municipality [6,7,9; and data presented here].The present study shows that the serological prevalence does not vary between the wild boar populations of the HMU in which the larval load is detectable by artificial digestion,  i.e., HMU 9, and those of the HMUs in which the larval load is below the limit of detection, 1.0-1.9larvae/g [28].These epidemiological data agree with laboratory data that showed that T. britovi larvae survive in swine muscles for up to 6 months after infection, whereas the antibody response persists for at least 2 years after infection [4].
The fact that the biomass of T. britovi in the Orgosolo municipality (HMU 9) was higher than that of the other hunting areas with similar antibody prevalence, and where the parasite has never been detected in the muscles of pigs reared illegally in the wild or in wild animals, indicates that the pathogen's transmission events among susceptible animals were constant over time (four hunting seasons) and space (eight HMUs of central and south-western Sardinia).As the transmission events were stable, the presence of a high biomass of the parasite in the Orgosolo municipality can only be due to factors independent of those of the natural cycle, i.e., the presence of a high number of free-ranging pigs and the concomitant presence of ASF due to their illegal slaughtering in the field.ASF has been endemic in Sardinia since 1978, and there were active foci of this virus in the investigated areas until 2018 [13,15,29].In HMU 9, cyclical outbreaks of ASF reported in domestic pigs illegally reared in the wild were associated with high mortality and the presence of infected carcasses in the wild and thus the possibility of more, new T. britovi infections in scavenging animals.
From 2005 to 2021, as many as 25 domestic pigs (mainly sows) illegally reared in the forest of the Orgosolo municipality for family self-consumption were found to be positive for T. britovi larvae [7, 9; the present study].The higher larval biomass and prevalence of infection in pigs (0.1-43 larvae/g; 0.5%) than wild boars (0.6-7.5 larvae/g; 0.03%) indicate the impact of free-ranging pigs on the epidemiology of T. britovi in the Orgosolo municipality.
The parasitological prevalence detected by artificial digestion (0.03%) and the serological prevalence of anti-Trichinella IgG detected by WB (3.8%) in wild boars in Sardinia are similar to the those (0.07% and 2.2%, respectively) reported for wild boars in five continental Italian regions where T. britovi is circulating among susceptible wild animals [26].Serological tests do not allow recent infections to be distinguished from past ones, but we assumed that most of the wild boars found to be seropositive only harbored residual antibodies and had no infective larvae in their muscles.
The fox is one of the main natural reservoirs of T. britovi in Europe.T. britovi prevalence in this carnivore in the present study (4.2%) is similar to that detected in other Italian regions [30], but much higher than the average prevalence in red foxes of the European Union in recent years [31].
The anti-Trichinella IgG prevalence of 1.5% of hunting dogs in Orgosolo municipality confirms the results of previous investigations, which showed that they can act as sentinel animals for the monitoring of Trichinella sp.infections in wildlife [24].A bias of this study was the lack of testing of sera of hunting dogs from the HMUs where T. britovi larvae have never been detected in wild boars or foxes.
From 2002 to 2023, a total of 185 human cases of trichinellosis reported in Italy were due to the consumption of wild boar meat from hunting, of which 155 cases were caused by T. britovi and 30 by T. pseudospiralis [32-34; unpublished data,MA GM].The presence of T. britovi in wild boars represents a threat to hunters and their families because the carcasses of some of the hunted animals escape official controls and thus are considered a health risk.

Conclusions
The presence of ASF in the population of wild boars in Sardinia and the coexistence of these animals with herds of pigs reared in the wild that have escaped any veterinary control have certainly favored an increase of the T. britovi biomass in the last two decades, which has led to outbreaks of trichinellosis among humans.Artificial digestion can be used to determine the larval load of infected meat that is sufficient to cause clinical infection in individuals who eat it [28].However, this diagnostic method cannot detect low parasitic loads that are of sufficient magnitude to perpetuate the cycle of T. britovi in the wild.The results of this investigation suggest that, in an insular context under a Mediterranean climate, T. britovi can circulate in susceptible wildlife at a very low prevalence and, notably, the larval load is lower than that which can be detected by artificial digestion, as has also been observed in Sicily.However, inadequate pig rearing and slaughtering practices can modify the natural cycle, as shown in the municipality of Orgosolo.The results of this study indicate the following: that programs need to be implemented throughout the region for the surveillance of T. britovi in animals that are hunted and in farmed pigs; that the breeding of pigs in the wild needs to be prohibited; and above all, educational programs need to be implemented for breeders, hunters, and consumers of boar meat and pork.Fox carcasses left in the field after the animals have been skinned, and the discarding of entrails/offal and other parts of hunted wild boars in hunting areas, and those of pigs following their illegal slaughter, favor the transmission of the parasite among scavenging omnivorous and carnivorous mammals.It was shown that the education of hunters and the organization of a collection and storage service for slaughter leftovers led to a drastic reduction in the prevalence of T. spiralis infection in an animal population [35].Thus, health authorities should provide a service for the disposal of animal carcasses and offal, and for the training of hunters and pig owners for the management of waste and byproducts, in accordance with the relevant European Union regulations.
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Fig. 1
Fig. 1 Map of hunting management units in Sardinia where samples were collected from hunted wild animals and from pigs illegally reared in the wild

Table 2 )
. No Trichinella sp.larvae were detected in marten, weasel, or wild cat muscles.Most of the Trichinella sp.-positive animals originated from the Orgosolo municipality.Only one

Table 1
Sample size of wild boar (Sus scrofa) serum samples needed to detect a minimum prevalence of 1% with a 95% confidence level of anti-Trichinella immunoglobulin G, based on wild boar hunting management unit (HMU) surface area and boar density Trichinella sp.-positive wild boar had been hunted in the neighboring municipality of Oliena.All of the larvae were identified as T. britovi.As part of the ASF eradication plan, 1189 domestic pigs reared in the wild were killed and tested for Trichinella sp.(487 pigs from HMU 9; 483 from HMU 11; 124 from HMU 23; 83 from HMU 6; and 12 from HMU 5).Positive pooled samples were only found for HMU 9.

Table 2
Trichinella britovi larvae detected in wild animals and in pigs illegally reared in the wild in the seven HMUs from 2014 to 2021 a Free-ranging pigs slaughtered in the field by veterinary services for the eradication of African swine fever b Pig slaughtered at home for self-consumption c Positive pools: the number of Trichinella sp.-infected pigs in each positive pool was unknown (for more details, see "Methods")