Molecular Detection of Cattle Sarcocystis Spp. In North-West Italy Targeting Cox1 And 18S Genes Highlights Its Association With Bovine Eosinophilic Myositis.


 Background: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa due to the evidences supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based either on morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis.Methods: To reach our aim, individual cattle samples from BEM condemned carcasses (N=54) and randomly sampled carcasses (N=59) were obtained from Piedmont slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S and cox1 genes. PCR products amplified using the genus specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis, were sequenced to achieve species identification.Results: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in 2 carcasses.Conclusions: Our study contributes to update the data on the prevalence of the different Sarcocystis spp. in cattle in Italy and emphasize the role of S. hominis and S. bovifelis as the major sarcosporidian species involved.


Background
Sarcocystis species are protozoan parasites belonging to the phylum Apicomplexa. The genus Sarcocystis consists of more than 200 species characterized by a worldwide distribution, three of which -S. hominis, S.heydorni and S. suihominis -are known to use humans as de nitive hosts [1].
Among meat producing animals, cattle (Bos Taurus) are common intermediate hosts of Sarcocystis, whose prevalence in muscle can reach up to 95-100% [2]. Although there has recently been confusion about the validity and classi cation of several Sarcocystis spp. from cattle, it is now generally agreed that bovine muscle tissue can harbor at least six Sarcocystis spp., the well-known S. cruzi, S. hirsuta and S. hominis, with felids, canids and humans, respectively, as de nitive hosts, and the recently added S. bovifelis, S. bovini and S. heydorni, with felids and primates as de nitive hosts [3].
The consumption of raw or undercooked beef meat constitutes an important risk factor for humans, who become infected by ingesting muscular sarcocysts [4]. Symptoms of intestinal sarcocystosis, such as nausea, abdominal pain and diarrhea, can have a wide range of intensity, depending on the number of ingested cysts and on the immune response of the host, though most infections go unnoticed [4]. In addition to the zoonotic potential, there is increasing interest around these protozoa in the food industry due to the evidence of their association with bovine eosinophilic myositis (BEM), a speci c in ammatory myopathy with multifocal grey-green lesions which leads to carcass condemnation and considerable economic losses [5]. Worldwide, BEM reported prevalence in slaughtered cattle ranges from 0.002% to 5% [6]. This data might appear inconsistent with the high prevalence of Sarcocystis in cattle; in this regard, few evidences have pointed out as possible explanation that BEM might be associated with one or more Sarcocystis species [6].
Prevalence data reported from cattle in Italy are consistent with European reports, revealing a Sarcocystis spp. prevalence of 96% [7], 80% [8], 91% [9] and 88% [10]. All these studies have been based on morphological techniques or on molecular techniques targeting the nuclear small subunit (18S) rDNA gene; however, the suitability of this locus for distinguishing between closely related Sarcocystis spp. has recently been challenged [11]. Indeed, though public databases contain mostly 18S rDNA sequences due to its high use for Sarcocystis identi cation, Cytochrome C Oxidase subunit I mitochondrial (mtDNA cox1) gene is actually seen as the most promising tool to differentiate closely related Sarcocystis spp. [3,12]. In particular, as highlighted by Moré et al. [13], sequence differences between S. hominis, S. bovifelis and S. bovini are approximately 3% of the 18S rDNA gene; therefore, using only size differences of the ampli ed 18S rDNA fragments may result in the misidenti cation of these species [2,13].
In the light of this, data resulting from the prevalence studies carried out in Italy in the last years might have led to an overestimation of S. hominis prevalence [14], apparently ranging from 42,7% [8] to 68% [10].
Thus, the aim of the present study was to evaluate the prevalence of the different Sarcocystis spp. in Italian slaughter cattle and in BEM condemned carcasses, focusing on the hypothesis that BEM might be associated with speci c Sarcocystis spp. [6,9].

Sample collection and processing
From January 2012 to July 2020, striated muscle samples from 54 BEM condemned carcasses were submitted by different slaughterhouses located in North-West Italy to the Laboratory of Food Inspection at the Department of Veterinary Sciences (University of Turin, IT) for etiological con rmation. Muscle samples were macroscopically examined for the presence of typical focal or diffuse grey-green lesions; detected lesions were excised and stored at -20° C for further analysis. Simultaneously, in 2019-2020 the diaphragm muscles of 59 slaughter cattle were collected from Piedmont slaughterhouses, for a total of 113 individual cattle samples. Tissue samples were collected by veterinarians during post-mortem inspections of slaughtered animals and then transported to the laboratory at refrigeration temperature; 25 mg of tissue for each individual muscle sample were collected and stored at -20°C until further analysis.
2.2 DNA extraction and molecular detection of Sarcocystis spp.
DNA extraction was performed using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to manufacturer's tissue protocol; the lysis step was carried out at 56°C overnight with Proteinase K. DNA samples were eluted in 50 μl of Elution Buffer and kept frozen at -20°C. The identi cation of different Sarcocystis spp. was performed through the application of the multiplex-PCR assay described by Rubiola et al. [14] targeting the 18S rDNA gene and the mtDNA cox1 gene. The multiplex-PCR contained 2.5 μl of template DNA (5-20 ng/μl), 0,5 mM of each primer, Sarco Rev, Sar F, Hirsuta, Cruzi, COI HB, COI H and COI B, 2 mM MgCl2, 0.2 mM of each dNTP, 1 U Platinum Taq DNA polymerase, 10 x PCR Buffer and RNasefree water to a total volume of 25 μl. The ampli cation was performed in an Applied Biosystems 2720 Thermal Cycler (AppliedBiosystems, CA, USA) with the following cycling pro le: a denaturation step at 95°C for 3 min, followed by 35 cycles at 95 °C for 60 s, 58 °C for 60 s and 72 °C for 30 s and nal extension 72 °C for 3 min. In each PCR run, 2.5 μl of DNA from a collection of Sarcocystis positive samples isolated from cattle striated muscle in the Department of Veterinary Science of Turin University [9,14,15] were used as positive controls while extracted DNA from negative cattle muscles as well as reagent blanks were included as negative controls. PCR products were observed in 2% agarose gel stained with SYBR safe stain (Invitrogen, Carlsbad, CA) and observed in a blue light transilluminator (Invitrogen, Groningen, The Netherlands).

Sanger sequencing and phylogenetic analysis
PCR products ampli ed through the use of the genus speci c primer set in absence of the speci c fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis, were sequenced to achieve species identi cation. PCR amplicons were puri ed with Exo-Sap treatment ( Fig 3a).

Discussion
Cattle sarcocystosis is gaining importance as one of the causes of bovine eosinophilic myositis, a speci c in ammatory myopathy which leads to serious economic outcomes in the beef sector [2,9,19]. Thus, species identi cation of intra-lesional Sarcocystis is crucial to better understand the contribution of speci c species to BEM pathogenesis and to explain the low prevalence of BEM lesions [14], despite the high prevalence of sarcocysts in cattle population. Therefore, the aim of this study was to evaluate the presence of Sarcocystis spp. in Italian slaughter cattle and in BEM condemned carcasses, in order to update the prevalence data reported from cattle in Italy in light of the recent taxonomic revision of cattle Sarcocystis and in order to evaluate the hypothesis that BEM might be associated with speci c Sarcocystis spp. [6].
In our study, the 67.8% prevalence of Sarcocystis spp. in diaphragm samples randomly taken from Piedmont slaughterhouses (n=59) is compatible with the high prevalence previously reported [7][8][9][10]. S. cruzi has been con rmed as the most common species, followed by S. bovifelis and S. hominis, while S. hirsuta DNA was only detected in 1.7% of samples. Notably, the 8.5% prevalence of S. hominis here detected is much lower than previously reported (43-68%), while S. bovifelis has never been considered in prevalence studies carried out in Italy so far [7][8][9][10], since at that time it still had to be described [20]. These ndings highlight the previous overestimation of S. hominis prevalence due to the detection techniques based on the lower discriminative 18S rDNA gene; besides, this evidence suggests that S. bovifelis might have been misidenti ed with S. hominis, thus explaining its absence in all previous prevalence studies carried out in Italy [14]. Considering the high detection of S. cruzi, followed by S. bovifelis and S. hominis, the prevalence data reported in our study shows most resemblance to that of Hungary and Netherlands [21,22], while in Germany and Lithuania a higher prevalence of S. hirsuta is reported [23,24].
The presence of Sarcocystis spp. in BEM condemned carcasses differed signi cantly with respect to the previously described group of randomly sampled slaughter cattle. In particular, the detection of Sarcocystis spp. DNA in 90.7% of the BEM condemned carcasses was signi cantly higher than in unaffected cattle. This nding con rms the association of Sarcocystis spp. with BEM lesions, though the presence of Sarcocystis spp. is not exclusively associated with lesions typical for bovine eosinophilic myositis [22]. Besides, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was signi cantly higher in samples isolated from BEM condemned carcasses than in samples isolated from randomly sampled slaughter cattle. This nding supports the hypothesis that BEM might be associated with speci c Sarcocystis spp. Literature on this topic is confusing since several species have been reported in association with eosinophilic lesions [5,6,22,[25][26][27][28]; among these studies, both thin-and thick-walled Sarcocystis spp. are reported, including S. hominis [5,6,22,25] and S. cruzi [28]. However, most of these reports have been based on morphological identi cation, which cannot discriminate among closely related Sarcocystis spp.; besides, this method is affected by the damage of the cyst walls which is often present in BEM lesions [6]. Molecular detection techniques were applied by Vangeel et al. [6] and the majority of intralesional Sarcocystis were found to be S. hominis, though also S. cruzi and S. hirsuta were found in BEM lesions; however, S. bovifelis wasn't recognized as a different Sarcocystis spp. until its resurrection in 2016 [20]. Our ndings lead us to hypothesize a major role for S. hominis and S. bovifelis in bovine eosinophilic myositis. To corroborate our current ndings, as well as to update knowledge on this disease, which leads to serious economic outcomes in the beef sector, further research is required. In particular, multiple sampling in BEM affected carcasses, involving both intralesional and extralesional tissue might be performed to evaluate the presence of different Sarcocystis spp. outside and inside lesions.
In the present study, an unidenti ed species was detected in two carcasses; although the small size of the sequenced fragment cannot give consistent molecular results, as highlighted by the low bootstrap values reported in the phylogenetic tree (Fig 2), this species seems to be most closely related to Sarcocystis spp. with thick walled, villar-like protrusions from bovids and cervids. Interestingly, as suggested by the high percentage of identity reported in the phylogenetic analysis (Fig. 2), a 177 bp 18S rDNA fragment of this unidenti ed species has already been sequenced in association with BEM lesions in Belgium [6], though no further research was performed at that time. Further investigations are needed to characterize this putative new species and investigate its cycle and possible role in BEM pathogenesis.
The detection of the zoonotic S. hominis con rms the established transmission cycle between cattle and humans in Italy, pointing out the risk for the consumer of raw or undercooked beef. Human intestinal sarcocystosis is well documented in the literature, both in asymptomatic patients and in patients with gastrointestinal symptoms [4]. Recently, the presence of S. hominis in 6 patients hospitalized with gastrointestinal symptoms has been reported in Piedmont region, North-West Italy, which is well known for raw beef consumption [14]. Therefore, epidemiological data on this and other actually undetected zoonotic species must be considered of importance from a public health perspective.

Conclusions
In conclusion, the results of our study contribute to update the data on the prevalence of the different Sarcocystis spp. from cattle in Italy, highlighting the previous overestimation of S. hominis due to the use of morphological methods or ineffective 18S rDNA-based molecular techniques. Besides, our ndings contribute to the understanding of the importance of different Sarcocystis spp. in BEM pathogenesis, emphasizing the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved.

Declarations
Ethics approval and consent to participate Not applicable: for this type of formal study consent is not required.

Consent for publication
Not applicable.

Availability of data and materials
All datasets generated for this study are included in article/additional les. The sequences generated in the present study are available in GenBank database with accession numbers MW582306, MW582307.

Competing interests
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.

Funding
Not applicable: this research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.
Authors' contributions SR performed the experiments and the data analysis and wrote the manuscript. TC an FC conceived and designed the experiments. FP assisted the experiments. DV carried out the sampling. All authors reviewed and approved the nal manuscript.  Neighbor-joining phylogenetic tree for members of the Sarcocystidae based on 18S rDNA sequences of 37 Sarcocystis spp. and including the unidenti ed Sarcocystis spp. sequences isolated in this study (in bold) and three GenBank entries (accession no. FN394500.1, FN394498.1, FN394499.1) corresponding to unidenti ed Sarcocystis spp. (6). Toxoplasma gondii and Neospora caninum were used as outgroups.