Microscopic detection and molecular characterization of Sarcocystis miescheriana in wild boars (Sus scrofa): �rst report from Greece

The genus Sarcocystis includes protozoan parasites with an indirect life cycle. Sarcocystis spp. can infect various animal species and humans, causing sarcocystosis, a parasitosis of economic importance and zoonotic concern. Wild boars can act as intermediate hosts for Sarcocystis mieshieriana and the zoonotic Sarcocystis suihominis that infects humans by consumption of raw or undercooked infected swine meat. In the present study, diaphragmatic muscle tissue of 123 wild boars hunted in northern Greece were examined to determine the frequency of Sarcocystis spp. The samples were examined by tissue compression and molecular techniques. Under light microscopy, 34 out of 123 (27.6%) wild boars tested positive for Sarcocystis spp., while a higher infection prevalence (75%) was revealed by multiplex PCR performed in 100 of the samples. The partial mtDNA cox1 gene (~1100 bp) of 20 samples tested positive for S. miescheriana by multiplex PCR was ampli�ed and sequenced. Sarcocystis miescheriana was identi�ed as the only species involved in these infections. This is the �rst study on the prevalence of Sarcocystis spp. in wild animals in Greece. Further large-scale surveys are needed to assess the prevalence and species of this parasite in Greece and to design e�cient control and preventive measures in a One Health perspective.


Introduction
The genus Sarcocystis comprises more than 200 protozoan parasite species that exhibit a worldwide distribution and can infect a wide range of animals and humans (Fayer et al. 2015;Taylor et al. 2016).Sarcocystis spp.have an indirect life cycle, involving a predator-de nitive host, where sexual reproduction (gametogony) and oocyst formation take place within the small intestine, and a prayintermediate host, where asexual reproduction (merogony) occurs in the vascular endothelium and striated muscles (Dubey et al. 2016).The de nitive hosts excrete sporocysts in their faeces, which, when ingested by an appropriate intermediate host, reach the muscles where tissue cysts, known as sarcocysts, develop.The life cycle is completed when a de nitive host consumes sarcocysts in the tissues of an intermediate host through predation or scavenging (Dubey et al. 2016).
At least two Sarcocystis species can be found in the tissues of domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa), i.e., Sarcocystis miescheriana and Sarcocystis suihominis, with canids, and humans/nonhuman primates as de nitive hosts, respectively.A third species, Sarcocystis porcifelis, has been reported in the former Soviet Union in 1976; however the validity of S. porcifelis is uncertain (Dubey et al. 2016).Sarcocystosis in domestic pigs may result in weight loss, abortion, dyspnea, muscle tremors, and even death, depending on the initial infectious load of sporocysts (Dubey et al. 2016).
Nonetheless, most infections are paucisymptomatic or asymptomatic.Moreover, macroscopic sarcocystosis leading to meat condemnation has been recently documented in pigs (Rubiola et al. 2023).Human intestinal sarcocystosis is acquired by consuming undercooked pork infected with S. suihominis.The infection can remain asymptomatic but may also manifest with gastrointestinal symptoms (Dubey et al. 2016).In heavy infections symptoms start 6 to 8 hours after raw meat consumption and include inappetence, nausea, stomach ache, vomiting, diarrhoea and rapid pulse, a clinical picture associated with the pathogenicity or even toxicity of S. suihominis to humans (Fayer et al. 2015).
To date, there is no information regarding the presence and prevalence of Sarcocystis infection in wild boars in Greece.Over the past few years, there has been a rise in the number of wild boars hunted for human consumption in the country, especially after the rst African swine fever cases, when wild boar hunting was legally expanded for population control.Against this background, and taking into consideration the public health concerns, it is important to investigate the presence of food-borne zoonotic parasites in this animal species.In a previous study, the prevalence of Trichinella spp.and Alaria alata was examined in muscle samples of wild boars hunted in northern Greece (Dimzas et al. 2021).The aims of the present study were to investigate the occurrence of Sarcocystis spp. in wild boars and to characterize the Sarcocystis species involved, in order to identify potential risks for public health linked to the presence of S. suihominis.

Field sampling
The study was conducted on wild boars from Thrace and Macedonia, two regions of northern Greece.
The studied areas were selected due to the high wild boar population density, and the proximity to the European inland and other Balkan countries with high wild boar populations (Pittiglio et

Tissue Compression
From each animal, 1g from the diaphragm was examined by the tissue compression method (Forbes et al. 2003).Brie y, the samples were cut into smaller pieces (rice grain size), compressed between two glass plates (compressorium) and examined under the stereomicroscope (20-40× magni cation) and the optical microscope (100× magni cation).Key morphological features (size, shape, and structure of the sarcocyst wall) were used for identi cation of the parasite species (Dubey et al. 2016).

Molecular detection and characterization
A 25 g aliquot sample of homogenised muscle from the diaphragm of each animal was processed for DNA extraction, performed based on the protocol described by (Chassalevris et al. 2020) with slight modi cations.Brie y, 450 μl of the tissue were transferred to a Lysis buffer [4.2 M guanidinium isothiocyanate, 1.2 Μ guanidinium hydrochloride, 50 mM Tris-HCl (pH 7.5), 25 mM EDTA, 1% N-Lauroylsarcosine, 2% Triton X-100, pH 7.0] to a nal volume of 2400 μl and vortexed rigorously.An aliquot (250 μl) of this material was transferred to a microcentrifuge tube and mixed with Lysis buffer (850 μl).Finally, 600 μl of phenol (equilibrated, stabilized, pH 7.8 -8.2) and 200 μl of chloroform-isoamyl alcohol (24:1) were added.The next steps of the procedure were performed according to a previously published protocol (Chassalevris et al. 2020).A quantity of 60 μl of preheated (75 °C) elution buffer (5 mM Tris-HCl, pH 8.5) was used for the retrieval of DNA.included as positive control.The presence of species-speci c PCR products of the expected size (~400 bp and ~140 bp amplicons for S. suihominis and S. miescheriana, respectively) was determined through electrophoresis on 2% agarose gels stained with SYBR safe stain (Invitrogen, Carlsbad, CA) followed by visualization in a Bio-Rad Molecular Imager ChemiDoc (Bio-Rad Laboratories, Hercules, California, USA).
The partial mtDNA cox1 gene of a subset of samples (n=20), which resulted positive by multiplex PCR, was ampli ed using primers SF1 and SR11 (~1100 bp), following the protocol described by Gazzonis et al. (2019) (Gazzonis et al. 2019), in order to further molecularly characterize the detected species.PCR products were puri ed using ExoSAP-IT TM PCR Product Cleanup Reagent (Thermo Fisher Scienti c, USA); sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem, ThermoFisher Scienti c, Inc., USA); sequence analysis was performed on a SeqStudio Genetic Analyzer (Applied Biosystems, ThermoFisher Scienti c, Waltham, MA).Forward and reverse sequences were merged into consensus sequences using MEGA11 (Kumar et al. 2016).The nucleotide sequences were analysed with the BLASTN sequence similarity search within the NCBI database.The phylogenetic analysis was conducted using 78 partial sequences from 56 taxa, including 20 new sequences generated in the present study.Sequences were trimmed at both ends, ensuring that all aligned sequences started and ended at the same nucleotide positions.The ClustalW algorithm in MEGA11 was used to obtain multiple alignments of the generated sequences and Sarcocystis spp.sequences retrieved from GenBank.The phylogenetic trees were reconstructed using the Neighbour-Joining (NJ) method within MEGA11; evolutionary distances were computed using the p-distance algorithm.The phylogenetic tree was rooted using Neospora caninum as outgroup species.The phylogeny was tested with the bootstrap method (1000 replicates).Accession numbers of the sequences included in the phylogenetic analyses are shown within the tree.

Results
A total of 34 out of 123 (27.6%) wild boars from Greece tested Sarcocystis spp.positive by microscopy after tissue compression.Under light microscope, all detected sarcocysts were morphologically identical, belonging to one type.Ovoid elongated cysts (up to 1500 μm long and 200 μm wide) with thick wall (2.8-6.0 μm), serrated surface, with numerous internal septa dividing the cyst into compartments were observed.The villar protrusions on the sarcocyst wall were up to 5.0 μm long and 1.3 μm wide (Fig. 1).Moreover, banana-shaped (17.5-20.0×4.8-5.0 μm) bradyzoites were lling the cysts.According to these morphological characteristics, the sarcocysts were identi ed as S. miescheriana.
Out of the 123 samples, DNA quantity was su cient for further analysis in 100 cases, which were subsequently examined using the above-mentioned multiplex PCR protocol.Out of these 100 individual samples, S. miescheriana DNA was detected in 75% of the wild boar carcasses (75/100).No muscle sample resulted positive for S. suihominis by multiplex PCR.
Of the 34 microscopically positive samples, 4 were not examined by multiplex PCR (insu cient DNA) and 4 resulted negative.Thus, the multiplex PCR protocol con rmed the infection in 26 of the microscopically positive samples and revealed infection in 49 additional samples (negative by microscopy).In the 100 samples examined by both microscopy and multiplex PCR, 79 samples (79%) were Sarcocystis spp.positive at least in one of the methods (Table 1).

Conclusion
In the present study a high prevalence of S. miescheriana was recorded in wild boars in northern Greece.The zoonotic food-borne S. suihominis was not found but should still be considered as a possible pathogen in the area.Conventional, non-molecular diagnostic methods are simple and time/cost e cient and can be applied in studies, as a rst step.The molecular methods are more sensitive and can be used for epizootiological studies, and for the identi cation of the parasite at the species level.Wild boar populations are increasing and come in close proximity to human environments.As a result, they may play an important role in perpetuating the life cycle of a variety of parasites including zoonotic species, increasing the risk for human and domestic animal health. Sarcocystis al. 2018; ENETwild Consortium et al. 2018).During the 2019-2020 hunting season (i.e., October 2019 to January 2020), in the frame of a previously published study (Dimzas et al. 2021), diaphragm samples were collected from 128 wild boars.Volunteering hunters were trained on sampling procedures from culled animals.All samples were placed individually in labelled plastic bags, stored at 4-7 C and sent to the Laboratory of Parasitology and Parasitic Diseases, School of Veterinary Medicine, Aristotle University of Thessaloniki, the latest 48 hours after collection.Out of the initial 128 wild boars, samples from 123 individuals were deemed su cient for inclusion in the present study.
The extracted DNA of each sample was sent to the Laboratory of Parasitology at the Department of Veterinary Medicine and Animal Productions of University of Naples Federico II for molecular examinations.A species-speci c multiplex PCR assay targeting the cytochrome C oxidase subunit I mitochondrial (mtDNA cox1) gene was used to detect and differentiate S. miescheriana and S. suihominis following the protocol described by Paci co et al. (2023) (Paci co et al. 2023).The multiplex PCR mixture contained 2.5 μl of template DNA (5-20 ng/μl), 0.5 μM of each primer (Cox1 SM, Cox1 S and Cox1 M), 2 mM MgCl2, 0.2 mM of each dNTP, 1 U Platinum Taq DNA polymerase, 10 x PCR Buffer and distilled water to a total volume of 25 μl.The cycling pro le included a denaturation step at 95 °C for 3 min, followed by 35 cycles at 95 °C for 60 s, 56 °C for 60 s and 72 °C for 30 s and nal extension 72 °C for 3 min.DNA from a collection of Sarcocystis spp.positive samples isolated from wild boar carcasses in the Department of Veterinary Medicine and Animal Productions of University of Naples Federico II were miescheriana cysts from wild boar meat in Greece.Left: banana-shaped bradyzoites exiting a ruptured cyst.Right: villar protrusions (2.8-5.0 μm long and 1.3-1.8μm wide) covering the cyst wall.

Table 1 .
(Dubey et al. 2016;Rosenthal 2021)019)p. in wild boars in Greece examined by classical (tissue compression microscopy) and molecular (multiplex PCR) methods.The ampli cation of a subset of 20 samples that tested positive by multiplex PCR for the presence of S. miescheriana DNA, resulted in fragments of the expected size (~1100 bp) in all tested samples.Sanger sequencing of the ampli ed products resulted in 943-1047 bp fragments showing 94.7-99.The overall prevalence of Sarcocystis spp. in wild boars in Spain was 72.7% and the molecular identi cation of species in eight of the positive samples showed presence of S. miescheriana in seven and of S. suihominis in one(Calero-Bernal et al. 2016).The examination methods and the muscle sampled can in uence the detection of the parasite.According to previous studies(Coelho et al. 2015;Gazzonis et al. 2019), histological sections of the diaphragm, tongue, sublingual, and abdominal muscles had a greater load of sarcocysts than sections from the oesophagus and heart.Nevertheless, in other studies it was the cardiac muscle that was selected for analysis, considered the most suitable tissue for determining prevalence and intensity of Sarcocystis spp. in muscles is achieved by microscopic examination of compressed samples, histology, arti cial digestion, and molecular methods(Dubey et al. 2016).Microscopical examination of compressed samples and histology are useful techniques to study sarcocysts structures and can be used for screening and estimating the presence of infection.The identi cation of Sarcocystis spp.involves analysing the unique morphological characteristics of the cyst, i.e. dimensions, wall thickness, and internal structures(Fayer 2004;Dubey et al. 2016).Nevertheless, variances may be seen due to factors including the cyst's growth stage, its location, and the technique employed to x the histological sections(Coelho et al. 2015).Therefore, relying solely on morphological characteristics is insu cient for the identi cation of Sarcocystis spp.(Coelho et al. 2015).When conducting epizootiological surveys, compressorium is a more effective approach than histology, as it allows examination of a larger amount of tissue(Dubey et al. 2016).The arti cial digestion is highly sensitive and can detect even mild infections.Digestion of 50 g of tissue containing a small number of sarcocysts can release a substantial number of bradyzoites which can be detected by immunological and molecular methods(Dubey et al. 2016).Disadvantages of digestion include false negative results in cases of immature sarcocysts and low DNA quantities, while the destruction of the cyst wall may complicate the morphological diagnosis(Dubey et al. 2016).In the present study, the prevalence of Sarcocystis spp.found by microscopical examination of compressed tissue was 27.6%, but increased to 79% by associating molecular examination.Similarly, the prevalence of Sarcocystis spp. in wild boars in Portugal was 25.5% based on histology, and 73.8% according to molecular examination(Coelho et al. 2015).In another study in domestic pigs, the application of both microscopic and molecular detection proved that microscopic examination was less sensitive than classic and real time quantitative PCR, with a prevalence of Sarcocystis spp.found at 58.4%, 72.8% and 76.4%, respectively(Kaur et al. 2016).Molecular techniques can be employed to either detect or con rm the identi cation of Sarcocystispositive samples and are more sensitive than microscopy when applied after digestion (by pepsin or trypsin) of the muscle samples (Calero-Bernal et al. 2014; Kaur et al. 2016; Imre et al. 2017; Gazzonis et al. 2019).During the past few years, several studies used DNA based techniques for the identi cation of Sarcocystis spp. at species level (Coelho et al. 2015; Dubey et al. 2016; Imre et al. 2017; Gazzonis et al. 2019; Helman et al. 2023).The classi cation of Sarcocystis species was performed by molecular markers, including the cox1 mtDNA gene and the internal transcribed spacer 1 (ITS1) region (Gazzonis et al. 2019; Prakas et al. 2020), or included also the 18S rDNA and the 28S rDNA genes (Huang et al. 2019).Paci co et al., (2023) (Paci co et al. 2023) on the other hand developed a multiplex PCR protocol using the cox1 gene as a promising marker that can simultaneously detect both S. miescheriana and S. suihominis.This protocol was also applied in the present study, identifying S. miescheriana as the only species present in wild boars in northern Greece.The prevalence of S. suihominis in wild boars and/or feral pigs in the present and earlier studies in Europe, was low or zero (Gazzonis et al. 2019; Prakas et al. 2020; Paci co et al. 2023).Scarcity of S.suihominis is documented in areas where the parasite in humans is absent or rare.As a result, the dispersal and environmental contamination of sporocysts by human faeces is negligible.Data on human sarcocystosis are absent in Greece, with the exception of a single nding of intestinal sarcocystosis, where molecular identi cation of the parasite was not performed(Diakou 1997).Cultural cooking habits along with implementation of personal hygiene can in uence the spread of infection.A study conducted in India reported extensive environmental contamination and transmission of S. suihominis to pigs due to the consumption of raw or undercooked pork meat by humans in combination with poor sanitation(Kaur et al. 2016).Gazzonis et.al.(2019)(Gazzonisetal. 2019) commented that the main source of S. suihominis infections in wild boars is likely to be humans who have contracted the infection by consuming pork from domestic pigs, rather than from wild boars.Thus estimating the prevalence of the zoonotic S. suihominis in wild boars in a geographical region could give a rough estimation of the prevalence in domestic pigs in the same region(Gazzonis etal.2019).Recent research on the prevalence of Sarcocystis spp.infections in domestic pigs reared in intensive management methods in Europe is scant.The most recent data come from studies conducted in Germany the years 1978 and 2004, Austria in 1979, and Spain in 2006 (González et al. 2006; Gazzonis et al. 2019), while a recent report of sarcocystosis caused by S. miescheriana in a domestic pig in Italy highlights the need to increase data related to the occurrence of Sarcocystis spp. in domestic swine (Rubiola et al. 2023).Hunters can play a crucial role in perpetuating the cycle of S. miescheriana.Potentially infected carcasses or muscle tissues that are not discarded properly, left to the environment, increase the risk of infection to scavengers-wild carnivore nal hosts, thus contributing to the perpetuation of the parasite's sylvatic life cycle.Hunting dogs may also consume infected carcasses and disperse the parasite, not only in wildlife but also in areas with domestic pig farms, contributing to the domestic cycle of the parasite.Foodborne parasites belong to a group of pathogens that are often neglected.The long incubation period of many foodborne parasites, the fact that infected animals typically do not display any clinical signs and the tendency for subclinical disease to occur in humans are some of the reasons for this classi cation (van der Giessen et al. 2021).Cultural habits of consuming traditional pork products from backyard pigs or wild boars in a raw or undercooked state are the cause of sarcocystosis in humans.Thermally processed meat can be considered free of parasites.The freezing process can signi cantly decrease or even eradicate the infectious sarcocysts in meat.Depending on the size of the meat piece, sarcocysts can be effectively eliminated through overnight freezing in a household freezer(Dubey et al. 2016;Rosenthal 2021).Accordingly, sarcocysts can be killed by exposure to temperatures of 55°C or higher for 20 minutes or more.Sporocysts and oocysts can be killed by exposure to a temperature of 60°C for 1 minute, 55°C for 15 minutes, or 50°C for one hour(Dubey et al. 2016;Rosenthal 2021).In conclusion, adherence to proper cooking methods and adequate freezing techniques can effectively prevent the transmission of sarcocystosis to humans, ensuring the safety of swine consumption.
DiscussionTo the best of the authors' knowledge, this is the rst study on the prevalence of Sarcocystis spp. in wild boar in Greece, evidencing a monospeci c infection by S. miescheriana while also providing the rst S. miescheriana sequences from Greece.The high prevalence of infection found in the present study indicates that wild boars in northern Greece are highly exposed to this parasite.In wild boars, Sarcocystis infection has been extensively investigated in recent years in several European countries, showing high prevalences, not dissimilar to those found in Greece.Sarcocystis miescheriana was the only species found in the examined wild boars from Portugal (73.8%),Romania (60.4%),Lithuania (88.24%), and Latvia (87.1%)(Prakas 2011; Coelho et al. 2015; Calero-Bernal et al. 2016; Imre et al. 2017; Prakas et al. 2020).In wild boars from northern Italy, S. miescheriana was the predominant species (97%), followed by S. suihominis (1%) (Gazzonis et al. 2019).Accordingly, the prevalence of S. miescheriana was 80.7% in southern Italy, while no S. suihominis positive wild boars were found (Paci co et al. 2023).infection(Calero-Bernal et al. 2014; Kaur et al. 2016; Imre et al. 2017).It is possible that the low prevalence found in certain studies does not re ect the actual incidence of Sarcocystis spp.but is rather the result of less sensitive methods.In general, the detection of