Macroscopic sarcocystosis in a pig carcass from an Italian abattoir

Different food-safety institutions, including the European Food Safety Authority, encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs; among meat-producing animals, domestic pigs (Sus scrofa domesticus) can host two different Sarcocystis spp., that is Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis. Herein, we report for the first time the presence of macrocysts of Sarcocystis miescheriana in a domestic pig resulting in carcass condemnation. In North-West Italy, in June 2022 the carcass of a clinically healthy sow was condemned due to the detection of multifocal macroscopic whitish fusiform lesions. Affected muscle samples were submitted to histological and molecular analyses targeting the mtDNA cox1 and 18S rRNA genes. At gross examination and histology, well demarcated, oval or elongated macrocysts up to 8 mm in length characterized by a calcified central core surrounded by fibrosis were detected. The molecular amplification and sequencing of the cox1 mtDNA and 18S rRNA genes revealed the presence of Sarcocystis miescheriana DNA in all sampled macrocysts. Our study provides the first molecularly confirmed case of Sarcocystis miescheriana infection in a domestic pig in Italy. The present report highlights the need to increase data related to the occurrence and the prevalence of Sarcocystis spp. in meat-producing animals, and in wild and domestic pigs in particular, taking into account the zoonotic potential of Sarcocystis suihominis and the possible financial losses related to carcass discard due to macroscopic Sarcocystis spp. cysts.


Introduction
Sarcocystis is a neglected genus of intracellular protozoan parasites including more than 200 species infecting a wide range of vertebrates (Dubey et al. 2016).The two-host praypredator life cycle of Sarcocystis spp.includes carnivores and omnivores as definitive hosts, which become infected through the ingestion of muscle tissue containing mature sarcocysts, and herbivores, omnivores or carnivores as intermediate hosts, which become infected ingesting food, feed and water contaminated with faeces containing oocysts or sporocysts (Gazzonis et al. 2019).Three zoonotic species, that is S. suihominis, S. hominis and S. heydorni, can infect humans due to the consumption of undercooked or raw beef or pork (Dubey et al. 2015).
Among meat-producing animals, domestic pigs (Sus scrofa domesticus) can act as intermediate hosts of two Sarcocystis spp., that is S. miescheriana and S. suihominis, using canids and human or nonhuman primates, respectively, as definitive hosts.Humans, which become infected by eating raw or undercooked pork meat containing muscular S. suihominis cysts, can develop gastrointestinal symptoms including nausea, vomiting, stomachache and diarrhoea; nevertheless, most infections are paucisymptomatic or asymptomatic (Dubey et al. 2016).Both S. miescheriana and S. suihominis, usually cause subclinical infections in pigs.However, as proven by experimental infections, the ingestion of more than 1 million sporocysts can cause weight loss, thrombocytopenia, purpura of the skin, muscle tremors, dyspnoea, abortion and even the pig death (Daugschies et al. 1987).Actually, only one report of clinical sarcocystosis after natural infection has been described in pigs worldwide, determining the death of the affected domestic swine due to severe myocarditis; no gross lesions were observed at the necroscopy (Caspari et al. 2011).
Despite the lack of clinical cases in domestic swine, European surveys have shown a prevalence of Sarcocystis spp. in domestic pigs ranging from 3 to 43% (Dubey et al. 2016;Damriyasa et al. 2004;Pereira and Bermejo 1988); nevertheless, studies referring to the prevalence of sarcocystosis in domestic pigs reared in intensive indoor production system are rather dated, and most often did not distinguish between S. miescheriana and S. suihominis.Otherwise, the emphasis in recent years has been on prevalence studies in wild boars, showing higher infection rates than in domestic swine (Calero-Bernal et al. 2016;Coelho et al. 2015;Helman et al. 2022;Prakas et al. 2020).So far, no molecular investigations have been performed in Italy into the occurrence of S. suihominis and S. miescheriana in domestic swine.
Different food-safety institutions, including the European Food Safety Authority (EFSA), encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs (Taylor et al. 2010).However, while reports on bovine Sarcocystis spp.are growing, especially due to the fact that different Sarcocystis spp.have been associated with bovine eosinophilic myositis (Rubiola et al. 2020(Rubiola et al. , 2021)), there is no documentation of any species of Sarcocystis in pigs related to the presence of macroscopic sarcocysts causing carcass condemnation (Dubey et al. 2016).Furthermore, the quality of the meat of experimentally infected pigs, which has been investigated to estimate the economic impact of subclinical sarcocystosis, has not been reported to be reduced (Daugschies et al. 1987).Herein, the presence of macrocysts of S. miescheriana in a pig resulting in carcass condemnation is reported for the first time; furthermore, our study provides the first molecularly confirmed case of S. miescheriana infection in a domestic pig in Italy.

Materials and methods
In June 2022, the carcass of a clinically healthy sow was condemned at slaughter due to the presence of multifocal macroscopic whitish lesions detected during the post-mortem meat inspection.The Large-White x Landrace sow was born in a commercial pig farm in Piedmont, North-West Italy.After weaning, the pig was purchased by an indoor farm located in the same area, where it was reared in confined housing for four years until it was bought by a nearby fattening pig farm and sent to slaughter after three months.No signs of disease nor abnormalities in body weight were recorded during the lifetime.The sow carcass was condemned due to the extensive involvement of different muscles, including masseter, tongue, neck, oesophagus, diaphragm, shoulders and back muscles and skeletal muscles of the fore and hind limbs.Affected muscle samples were submitted by the slaughterhouse to the Department of Veterinary Sciences (University of Turin, IT) for etiological diagnosis.
A gross examination of the affected muscle samples was performed; tissues affected by lesions were fixed in 10% buffered formalin and stored at room temperature for histopathological examination.Fixed samples were trimmed, embedded in paraffin wax, sectioned at 4 μm, stained with haematoxylin and eosin (H&E) and examined using a light microscope.
Ten of the detected lesions were isolated, excised and stored at − 20 °C for molecular examination.DNA extraction was performed using DNeasy Blood and Tissue Kit (Qiagen, Germany), according to the manufacturer's tissue protocol; the DNA was eluted in 100 µl of Buffer AE and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).Additionally, ten grams of healthy muscle were collected from different portion of the muscle samples and processed as described by Moré et al. (2011) in order to obtain a muscle homogenate; the resulting pellet was resuspended in 20 ml of phosphate buffered saline (PBS) and observed in an inverted microscope at 40 × magnification (Nikon TMS).
All DNA samples were initially tested for the presence of Taenia spp.DNA by PCR targeting a fragment of the cytochrome C oxidase subunit I mitochondrial gene (mtDNA cox1) using primer pair JB3 -JB4.5 as described by Bowles et al. (1992), and for the presence of Sarcocystis spp.DNA by PCR using the Sarcocystis genus-specific primer pair SARf -SARr amplifying a small portion of the 18S rRNA gene, as described by Vangeel et al. (2007).Positive samples were subsequently further molecularly characterised targeting the partial mtDNA cox1 gene with primers SF1 and SR11 (~ 1100 bp) as described by Gazzonis et al. (2019).The complete 18S rRNA gene of one out of ten samples was amplified using the primer sets ERIB1 -S2 (Barta et al. 1997;Fischer and Odening 1998) and S3 -Primer BSarc (Fischer and Odening 1998; Gjerde 2014).Each reaction mixture contained 2.5 µl of template DNA.The sequences of all primers used are reported in Table 1.
PCR products amplified using primers SARf -SARr, SF1 -SR11, ERIB1 -S2 and S3 -Primer BSarc were purified (ExoSAP-IT, Thermo Fisher Scientific, USA) and sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA); sequence analysis was performed on an Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA).Forward and reverse sequences, as well as the 18S rRNA overlapping sequences, were manually assembled into consensus sequences using the Alignment Explorer within MEGA X (Kumar et al. 2016).The nucleotide sequences were analysed using the BLASTN sequence similarity search at the NCBI database.Phylogenetic analyses of the mtDNA Cox1 gene and the 18S rRNA gene sequences were conducted separately.With reference to the mtDNA cox1 gene, a total of 70 partial sequences from 54 taxa were used in the analysis, including 9 out of 10 new sequences generated in the present study; as for the 18S rRNA gene, a total of 60 sequences from 54 taxa were used in the analysis, including the new sequence generated in the present study.Multiple alignments of the generated sequences and Sarcocystis spp.sequences retrieved from GenBank were obtained by using the ClustalW algorithm in MEGA X.The phylogenetic trees were reconstructed using the neighborjoining method within MEGA X.As for the mtDNA cox1, sequences longer than 1020 bp were truncated so that the final alignment comprised 1020 positions with no gaps.In both analyses of the mtDNA cox1 gene and the 18S rRNA gene, the coccidian parasite Neospora caninum was used as outgroup species to root the trees.The phylogeny was tested with the bootstrap method (1000 replicates).Sarcocystis spp.reference sequences included in the analyses are reported in the phylogenetic trees.The DNA Sequence Polymorphism (DnaSP) software (version 6.12.03) was used to calculate intraspecific genetic variation measurements.

Results
At gross examination, skeletal muscles showed multifocal, whitish, well demarcated, fusiform lesions up to 8 mm in length and 3 mm in diameter described as macrocysts; most of the cysts appeared solid and released whitish necrotic material when squeezed.Macroscopic appearance of the affected muscle samples is shown in Fig. 1.Histologically, the three measured club-shaped cysts were 950-2900 × 600-900 μm in size.Most of them were characterized by a calcified central core surrounded by fibrosis and a slight non suppurative inflammatory reaction composed by lymphocytes, macrophages and rare plasma cells (Fig. 2).A thin rim of atrophic, degenerated muscle fibres was present at the periphery.Multifocal areas of fibrosis were also detected in the affected muscles.
All collected samples tested negative for the presence of Taenia spp.DNA.The healthy muscle homogenate tested negative for the presence of sarcocysts by direct microscopic examination.On the other hand, the initial screening of excised cysts for Sarcocystidae DNA by PCR assay targeting Sarcocystis genus revealed the presence of Sarcocystis spp.DNA in all sampled cysts.Sanger sequencing of the amplified products resulted in ~ 180 bp fragments showing OP681505-OP681509, ON211345-ON211346) and less than 80.4% identity with other Sarcocystis spp.sequences available in GenBank.Based on these results, it was confirmed that all excised macrocysts belonged to S. miescheriana (Fig. 3).One out of 10 cox1 sequences was removed from further analysis due to the shorter length (716 bp).The phylogenetic analysis on the basis of cox1 sequences showed close relationships of S. miescheriana sequences from the present study with S. miescheriana from Italian wild boar and from Latvian and Lithuanian wild boar (Fig. 3a).The 9 sequences represented 5 haplotypes (haplotype diversity: 0.861 ± 0,00760); 13 polymorphic (segregating) sites were detected among the 1020 nucleotide positions covered by all haplotypes.The sequence divergence among the generated S. miescheriana sequences ranged from 0.001 to 0.01%.While the average number of nucleotide differences within the newly obtained S. miescheriana sequences was 5.2, the average number of nucleotide differences between the 100% identity with each other and with S. miescheriana GenBank entries (accession no.MH404230, KX008292, JN256123, MT066104-MT066113).All the ten DNA samples corresponding to the cysts were therefore submitted to the partial amplification and sequencing of the mtDNA cox1 gene using primers SF1 -SR11 and one DNA sample was characterised at complete 18S rRNA gene.

Discussion
Reports concerning the occurrence of Sarcocystis spp. in livestock in EU countries are scarce and difficult to interpret (Taylor et al. 2010).Regulation (EU) 2019/627, repealing regulation (EU) 854/2004, covers Sarcocystis spp.under the blanket term "zoonotic agents", recommending their detection in beef and pork and stating that carcasses infected with parasites have to be declared unfit for human consumption (Commission Regulation (EU), 2019).In this context, Sarcocystis spp.infections in domestic swine were not paid much attention in the veterinary field, as natural infections   Helman et al. 2022;Kaur et al. 2016;Prakas et al. 2020).Our report stresses the need to increase data related to the occurrence of Sarcocystis spp. in wild boars and domestic pigs at national and international level, pointing out the possible presence of Sarcocystis spp.cycling in pigs raised in intensive conditions.
Although S. miescheriana cannot infect humans, and therefore is not a direct risk for the consumer of raw or undercooked meat, its detection highlights, on one hand, the possible economic damage related to carcass discard due to macroscopic Sarcocystis spp.sarcocysts, and on the other hand, the presence of a biosecurity breach.The infection source of the present case was not investigated, and therefore cannot be unequivocally stated; nevertheless, two owned dogs were recorded in the indoor farm where the sow was reared for four years.Thus, the domestic dogs are most likely the source of S. miescheriana sporocysts, which might have been introduced in pigpens despite the absence of shared areas.
In conclusion, to the best of our knowledge, our study reports the first molecularly confirmed case of S. miescheriana infection in a domestic pig in Italy; furthermore, it reports the first condemnation of a pig carcass due to the presence of disseminated Sarcocystis macrocysts.The present study highlights the need to increase data related to the occurrence and prevalence of Sarcocystis spp. in wild and domestic pigs, taking into account the zoonotic potential of S. suihominis and the possible carcass discard due to macroscopic Sarcocystis spp.cysts detection at slaughter.do not usually cause clinical symptoms nor gross lesions visible at slaughter (Dubey et al. 2016).
The current study reports for the first time the presence of disseminated macroscopic S. miescheriana cysts in a domestic pig, resulting in carcass condemnation.As stated by Dubey et al. (2016), there is no documentation about the economic impact of swine sarcocystosis; indeed, although swine carcass discard due to suspected sarcocystosis has been reported in the past, there was no associated evidence supporting the presence of any Sarcocystis spp. or macroscopic sarcocyst.It is therefore unclear whether sarcocysts were actually present or the lesions were misidentified (Dubey et al. 2016).Herein the presence of whitish, sometimes calcified cysts up to 8 mm in length is described.As shown in the present report, the presence of macroscopic cysts can possibly involve the entire carcass, thereby leading to carcass condemnation and economic losses, in the absence of clinical signs.As previously hypothesized by Caspari et al. (2011), due to the high hygiene standards and close housing systems adopted by pig farms in the last decades, pigs natural immunisation with S. miescheriana might be decreased, thereby allowing the emergence of clinical cases or abnormal response of the host tissue to the infection.Nevertheless, further studies are needed to monitor and investigate that which actually remain occasional findings.
The molecular identification and characterization of the etiological agent involved in the present report has allowed to rule out the presence of other parasites which might be associated to the detection of degenerated cysts or other macroscopic lesions, e.g.calcified cysticerci (González et al. 2006).The phylogenetic analysis on the basis of mtDNA cox1 and 18S rRNA sequences and the intraspecific genetic variation analysis showed close relationships of S. miescheriana isolated in the present study and S. miescheriana isolated from Latvian, Lithuanian and Italian wild boars.On the other hand, our isolates were genetically distinct from S. miescheriana isolates originating from Chinese domestic pig and Japanese wild boars (Gazzonis et al. 2019;Huang et al. 2019), thereby showing the genetic similarity of the samples to be dependent upon their geographical distances as previously observed by Prakas et al. (2020).
Moreover, herein the first molecularly confirmed case of S. miescheriana infection in a domestic pig in Italy is reported.So far, no investigations into the occurrence and prevalence of S. suihominis and S. miescheriana have been conducted in domestic swine in Italy.Likewise, at EU and global level, emphasis in recent years has been placed upon prevalence studies in wild boars, feral pigs and free-ranging domestic pigs, while there are no recent investigations on the occurrence of sarcocystosis in domestic pigs kept under indoor intensive systems (Calero-Bernal et al. 2015, 2016;

Fig. 2 Fig. 1
Fig. 2 Microscopic appearance of the detected cysts.(A-B) Crosssection of the cyst with mineralized central area A: 25X; B: 100x.(C) Cross section of a cyst surrounded by a slight inflammation, fibrosis and atrophy of the muscle fibres; 25X.(D) Higher magnification of figure C showing the non-suppurative inflammatory infiltrate and fibrosis surrounding the cyst and the multifocal calcification in the core; 200x.Haematoxylin and eosin stain generated S. miescheriana sequences (n = 9) and the homologous fragments of S. miescheriana isolated from Italian wild boars (n = 43; MH404185-MH404227), Lithuanian and Latvian wild boars (n = 22; MT070614-MT070635), Chinese pigs (n = 3; MK867462-MK867464) and Japanese wild boars (n = 4; LC349977-LC349980) corresponded to 13.2, 9.5, 33.4 and 51.4 respectively.Sequences generated in the present study were submitted in GenBank under accession numbers OQ472889 and OQ472068-OQ472077.

Fig. 3
Fig. 3 Phylogenetic trees for members of the Sarcocystidae family based on 70 partial sequences from 54 taxa of the cox1 gene (a) and 60 sequences of the near-full-length 18S rRNA gene of 54 taxa (b), inferred using the neighbour-joining method.Six out of nine cox1 generated sequences have been collapsed.The percentages of replicate