First report of Sarcocystis spp. (Apicomplexa, Sarcocystidae) in Lagostomus maximus (Desmarest, 1917) (Rodentia, Chinchillidae) in Argentina

Sarcocystis is a genus of intracellular parasitic protozoa that infects various species of mammals, birds, and reptiles worldwide. At least 46 Sarcocystis species naturally infect rodents as intermediate hosts producing tissue cysts. This study aimed to provide the first report and molecular characterisation of Sarcocystis spp. in muscles from plains viscacha (Lagostomus maximus) in Argentina. Muscle samples of 53 plains viscachas from three provinces of Argentina were processed by homogenisation and optical microscopy to detect tissue cysts. Positive samples were analysed by PCR-sequencing, using the following markers: 18S rRNA, ITS1, and coxI. The 18S rRNA and coxI consensus sequences were aligned with other sequences from Sarcocystis spp., and phylogenetic trees were constructed. Of all animals processed, 13.2% (7/53) harboured Sarcocystis sp. cysts. 18S rRNA consensus sequences were obtained from four muscle samples and one individual cyst, and they showed 99.88–100% similarity, except for the cyst sequence, which showed 97.11% homology. Similarities of only 96–97% were recorded in the 18S rRNA fragment with other Sarcocystis spp. whose sequences are available in the GenBank. The five coxI fragment sequences obtained were 100% identical and showed an identity of 99.41–99.48% with S. canis. For ITS1 only short and low-quality sequences were obtained. In the phylogenetic trees, all the sequences from plains viscachas were positioned together in a branch separated from other Sarcocystis spp. These results could be related to new Sarcocystis spp. producing sarcocysts in plains viscachas. Besides, comprehensive cyst morphological analysis using TEM from the new Sarcocystis species will allow a description of the cyst wall ultrastructure. In this sense, further studies are needed to deepen these findings and elucidate other potential intermediate and possible definitive hosts.


Introduction
Sarcocystis spp. are intracellular protozoan parasites belonging to the cyst-forming coccidia, closely related to other apicomplexans such as Besnoitia spp., Neospora caninum and Toxoplasma gondii (Dubey et al., 2016). Most species have a predator-prey cycle producing sarcocysts in the muscles of intermediate hosts (IH) or prey and oocysts/sporocysts in the intestinal mucosa of definitive hosts (DH) carnivores and omnivores. About 200 species of this genus have been reported, infecting mammals, birds, and reptiles worldwide (Dubey et al., 2016). Humans can be hosts of some species, both as DH (S. hominis and S. heydorni in muscles from cattle and S. suihominis in muscles of swine) or IH (S. nesbitti with snakes as definitive host) (Fayer et al., 2015;Dubey et al., 2016;Rosenthal, 2021).
The plains viscacha, Lagostomus maximus (Desmarest, 1917) (Rodentia, Chinchillidae), is endemic in South America and inhabits southeastern Bolivia, western Paraguay, and northern, eastern, and central Argentina. This rodent species, herbivorous, fossorial, and with nocturnal habits, forms colonies of up to several dozen individuals (Jackson et al., 1996;Spotorno and Patton, 2015). The plains viscacha presents economic importance since it damages fields for the construction of its underground burrows ("vizcachera"), and it is also used as bushmeat (Jackson et al., 1996;Spotorno and Patton, 2015). Within Chinchillidae family, the presence of Sarcocystis microti in Chinchilla laniger and infections attributable to S. canis in Chinchilla sp. were reported (Rakich et al., 1992;Dubey et al., 2016). In addition, the presence of tissue cysts in various internal organs of L. maximus, presumably from a Besnoitia sp., has been reported in Argentina (Cwirenbaum et al., 2021). Different methods have been applied for the diagnosis of sarcocystosis, with marked differences in sensitivity and specificity (Moré et al., 2011;Dubey et al., 2016;Juozaitytė-Ngugu et al., 2021;Prakas et al., 2021;Rudaitytė-Lukošienė et al., 2022). The differentiation of species can be done through cysts' morphological studies but with certain limitations since the morphological features of some species are similar (Dubey et al., 2016). In recent years, studies based on molecular biology (PCR and sequencing) have allowed the identification and differentiation of several Sarcocystis spp., being more practical, precise, and reliable than traditional morphological methods (Gazzonis et al., 2019;Gjerde, 2013;Huang et al., 2019;Moré et al., 2013;Rubiola et al., 2020;Rosenthal, 2021). In this respect, genetic markers such as 18S rRNA (18S ribosomal RNA), coxI (mitochondrial cytochrome c oxidase subunit I), and ITS1 (internal transcribed spacer 1) have allowed reliable diagnosis and the identification of phylogenetic relationships among Sarcocystis species (Gazzonis et al., 2019;Huang et al., 2019;Prakas et al., 2020).
The present study provides the first report and molecular characterisation of a Sarcocystis spp. producing cysts in muscles of L. maximus in Argentina.

Sample collection
Muscle samples (tongue, diaphragm, masseter, heart, and hind limbs) were collected from a total of 53 plains viscacha specimens from three provinces of Argentina: Buenos Aires (n = 28), Entre Ríos (n = 12) and Santiago del Estero (n = 13), between 2017 and 2022 (Table 1). They were transported refrigerated (4 • C) and immediately processed or kept frozen (− 20 • C) until processing. Samples of each animal were analysed according to the methodology described by Moré et al. (2011). Briefly, 5-10 g of pooled muscle were grounded in a tissue homogeniser with the addition of 50 ml phosphate-buffered saline (PBS -pH 7.2) and then filtered, using a strainer with gauze, collected in a 50 ml tube, and centrifuged at 600×g for 5 min. Approximately 3 ml homogenate aliquots were placed in a Petri dish, diluted with PBS and observed in an inverted microscope at 40× magnification (Nikon, TMZ). An aliquot of each homogenate was collected in 1.5 ml DNase-free microtubes and preserved at − 20 • C for molecular studies. Samples containing at least one Sarcocystis sp. cyst (complete or a fraction) were considered positive.

Molecular analysis
The DNA was extracted from pooled muscle homogenates (only for positive samples at microscopy) and individual cysts using a commercial kit according to the manufacturer's instructions (PuriPrep T-kit, Inbio Highway, Argentina). The lysis step was carried out overnight at 55 • C. Each DNA extraction routine was conducted with a process control (a sample using only the kit solutions). The DNA samples were processed by PCR to three marker fragments: 18S rRNA, coxI (cysts and muscle homogenates), and ITS1 (only homogenates). All the amplifications were done in a thermocycler (T18, Ivema, Argentina), using a recombinant TaqDNA polymerase (Invitrogen™, Brazil), and each PCR routine included a negative control (extraction process control), a no template control (NTC, ultrapure water), and a positive control (S. falcatula-like DNA; Origlia et al., 2022). Fragments of 18S rRNA of 850 bp were amplified by PCR using SarcoFext and SarcoRext primers in a final volume of 25 μl using a previously described protocol (Moré et al., 2013).
Regarding the coxI gene marker, fragments of around 1100 bp were amplified by PCR using SF1 and SR5 primers (Gjerde, 2013) in a final volume of 25 μl under the following conditions: initial denaturation at 94 • C for 5 min, 45 cycles of 94 • C for 30 s, 53 • C for 30 s and 72 • C for 90 s, and final extension at 72 • C for 5 min.
Regarding the ITS1 marker, fragments from about 1200 bp were amplified using SU1F and 5.8SR2 primers (Gjerde, 2014)  Five μl of each PCR product were examined in 1.5% agarose gel stained with SYBRsafe and observed in a blue light transilluminator (Invitrogen, USA). In cases where the PCRs were negative, new PCRs with 1/10 dilutions of DNA samples were carried out (potential presence of inhibitors).
Amplicons of the 18S rRNA, coxI, and ITS1 PCR (with an estimated concentration of at least 40 ng/μl) were purified using a commercial kit according to the manufacturer's instructions (Wizard SV clean-up system, Promega). They were submitted for Sanger sequencing to Macrogen Inc., South Korea (http://www.macrogen.com), together with the two primers used for each amplification. Sequences obtained were aligned and analysed using the Geneious software (R9 version) (https ://www.geneious.com). The consensus sequences obtained were compared with others reported in GenBank by nucleotide BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The 18S rRNA and coxI consensus sequences obtained were aligned with other sequences of Sarcocystis spp. using small mammals, birds, and ruminants as IH and distance trees were constructed using the Neighbor-Joining method based on the Tamura-Nei genetic distance model, with 1000 bootstraps and using T. gondii 18S rRNA (M97703) and coxI (JX473257) sequence as the outgroup to root the tree, respectively (Geneious, R9).

Direct microscopy examination
Sarcocystis sp. cysts were observed in 7 of 53 (13.2%) plains viscachas. One specimen was from Bahía Samborombón, Buenos Aires province, and the other six were from Estancia Los Quebrachitos, Santiago del Estero province. Samples showed a low number of sarcocysts (1-4 per analysed aliquot), except for two samples from Santiago del Estero that showed a moderate number of sarcocysts (≥4 per analysed aliquot) ( Table 1). All sarcocysts were microscopic, septate, and thin-walled, appearing to belong to one morphological type. Most positive samples showed cyst portions of 150-200 μm (Fig. 1), and the few complete cysts measured up to 500 μm. Bradyzoites presented a banana-shaped structure.

Molecular analysis
Out of seven positive samples by optical microscopy analysis, five were positive by PCR for 18S rRNA and ITS1, and six for cox1, all of them by using muscles homogenate DNA. Four samples gave PCR products in the three markers suitable for sequencing, while another was sequenced only in ITS1 and cox1 markers. All the PCR samples were from Santiago del Estero province. The sequences obtained were deposited in the GenBank under accession numbers: OP937321, OP937322, OP937323, OP937324, OP937325 for the 18S rRNA, and OP936996, OP936997, OP936998, OP936999 and OP937000 for coxI.
Seven samples were processed for 18S rRNA PCR from individualised cysts; only one was positive and with appropriate PCR products for sequencing. All the negative samples were repeated at 1/10 dilution and resulted negative again. Five consensus sequences (four from homogenates and one from a single cyst), ranging from 847 to 877 bp were obtained for 18S rRNA. The chromatograms from the homogenates showed some double picks and a background signal. The sequences were 99.88-100% similar (only one single base difference), except for the sequence from the individual cyst, which shows a 97.11% homology with the other four. All the sequences showed only 96.89-97.37% identity with reported sequences of S. singaporensis (KY513624), S. zuoi (KU341120 and JQ029113), S. nesbitti (HF544323), S. attenuati (MZ826982), S. masoni (KU527108) and S. tarandi (GQ250970).
For coxI, five consensus sequences (each from a muscle homogenate sample), ranging from 989 to 1014 bp, were obtained and showed a 100% identity. The single cyst DNA resulted weak positive and was not sequenced. The sequences showed an identity of 99.41-99.48% with S. canis (KX721497) from the brain of a dolphin (Tursiops aduncus), Sarcocystis sp. from micromammals in Spain (MT418689), and Sarcocystis sp. ex Pantherophis alleghaniensis (KU891603).
Sequences of ITS1 were shorter than expected, of low quality (with several double peaks), and consensus sequences could not be constructed. The distance tree constructed with 39 18S rRNA sequences showed that all Sarcocystis spp. sequences from this study were grouped in a branch with a consensus support of 91%. The closest species was S. nesbitti, forming a sister group (Fig. 2). The distance tree constructed with 39 coxI sequences positioned the sequences of this study in a wellsupported group separated from the rest of the Sarcocystis species. In a sister clade were positioned sequences of Sarcocystis sp. from Mus spretus in Spain and Eastern rat snake in the USA, as well as S. canis sequences, with a support of 91% (Fig. 3).

Discussion
In the present study, the presence of Sarcocystis cysts in L. maximus is reported for the first time. Furthermore, the identified species were characterised molecularly by sequencing two genetic fragments. So far, there are only three reports of detecting Sarcocystidae protozoan cysts in rodents of the Chinchillidae family (Rakich et al., 1992;Dubey et al., 2016) and only one in L. maximus (Cwirenbaum et al., 2021). The cysts described in L. maximus from a previous study in Argentina probably belong to Besnoitia sp. considering the type (without clear septa) and localisation (Cwirenbaum et al., 2021). The present study detected septated and thin-walled muscular cysts in 7/53 animals. The cysts observed resembled a Sarcocystis sp. However, further studies will allow accurate morphological description, particularly the TEM cyst-wall type as proposed for new species (Dubey et al., 2016). Regarding distribution, only one sample from 28 obtained in Buenos Aires province resulted positive, while 6/13 from Santiago del Estero were positive, and two showed a moderate number of cysts. Despite the low number of samples, it is possible to suggest that the potential DH of the observed Sarcocystis spp. should be a frequent predator of plains viscachas with a higher prevalence in Santiago del Estero.
Almost all the consensus sequences obtained in the present study from muscles homogenate DNA showed a high identity, suggesting the presence of a single species. However, the observation of some double picks and the background signal in these sequences could suggest that DNA of more than one species was amplified. Despite of this, we obtained good consensus sequences, probably because one of them is overrepresented in the mix. In addition, the 18S rRNA sequence obtained from a single cyst showed a low identity with the other four sequences. Altogether, indicates that at least two Sarcocystis spp. species were present in plains viscacha muscles. Nevertheless, all the obtained 18S rRNA sequences showed only around 96.9-97.4% identity with other Sarcocystis spp. sequences. These homology levels are lower than the recognized for a Sarcocystis sp. specific identification (Gjerde, 2013;

Fig. 2.
Phylogenetic tree of 18S rRNA using the Neighbor-Joining method. Sequences obtained from muscles samples and individual cysts of Lagostomus maximus (VC46_1_cyst_B, VC46, VC44, VC38, and VC43) form a separate clade of other Sarcocystis species and S. nesbitti sequences formed a sister branch. Moré et al., 2013). Therefore, it is possible to suggest that the obtained 18S rRNA sequences from plains viscachas correspond to a two closely related and unreported species. On the other hand, the BLAST comparison of coxI sequences obtained in this study shows a high similarity with S. canis (99.48%) and a Sarcocystis sp. identified in Mus spretus muscles in Spain and faecal samples from Eastern rat snake (Pantherophis alleghaniensis) in the USA. Infections attributable to S. canis were reported in a wide range of hosts, including Canis lupus familiaris (initial report), Ursus americanus, Ursus maritimus, Zalophus californianus, Equus caballus, Eumetopias jubatus, Monachus schauinslandi, Stenella coeruleoalba and Chinchilla sp. (Rakich et al., 1992;Dubey et al., 2016). This species has a unique multiple host pattern, similar to S. neurona (Dubey et al., 2016). It is important to remark, that the primers used for cox1 are one universal (SF1) for Sarcocystis spp. and the reverse (SR5) is restricted to a group of species (Gjerde 2013). Therefore, we obtain good quality chromatograms (and without doble picks or background) for cox1 sequences probably by "selecting" one of the species present in muscles with the reverse primer.
Despite obtaining proper amplicons with validated primers for Sarcocystis spp. ITS1, the sequences were shorter than expected, and no consensus sequence was reached. This could be due to the DNA amplification of two or more different species (as previously mentioned for the 18S rRNA) as well as the high repeatability and variability reported for this marker, which could lead to reading problems in the Sanger sequencing (Gjerde, 2014). Future studies should be conducted using single cyst PCR to reach unique amplicons, as previously performed in other Sarcocystis spp. from rodent muscles (Prakas et al., 2019;Rudaitytė-Lukošienė et al., 2022).
The analysis of genetic distance trees with 18s rRNA and coxI sequences shows that all Sarcocystis sequences obtained in this study were grouped with high support and having as sister clade sequences from S. nesbitti, and S. canis and Sarcocystis sp. from micromammals and snake, respectively. In both trees, our sequences were separated from the other Sarcocystis spp. used in the alignments. However, considering that there is no higher homology at the 18S rRNA marker, the positioning in the trees could vary if sequences with higher degrees of similarity were available. Since S. nesbitti and Sarcocystis sp. from snake faeces show a closer genetic distance with Sarcocystis species studied here, snakes could be suggested to be the DH of some of them (Fayer et al., 2015;Dubey et al., 2016). Nevertheless, the close relation of cox1 sequences with those of S. canis, suggests that plain vizcachas could also being infected with this specie, as previously reported for Chinchilla sp. (Rakich et al., 1992). Sarcocystis canis is a species with multiple IH and uncertain DH, could indicate that hosts other than L. maximus may be infected, and even canids could be DH. Canids and snakes are found in sympatry with L. maximus and are considered possible natural predators of this rodent. It has also been reported that Boa constrictor could be a natural predator of the plains viscacha, and its distribution area includes Santiago del Estero province, where the most significant number of plains viscacha with Sarcocystis spp. cysts was found and also all the sequenced samples came from (Jackson et al., 1996;Arzamendia et al., 2021). Likewise, in Argentina, there are four species of canids whose distributions coincide to a greater or lesser extent with that of the plains viscachas in which Sarcocystis cysts were found: Canis lupus familiaris, Cerdocyon thous, Chrysocyon brachyurus, and Lycalopex gymnocercus (Cirignoli et al., 2019a(Cirignoli et al., , 2019bLartigau et al., 2019;Luengos Vidal et al., 2019). Nevertheless, further studies are needed to identify the DH of the Sarcocystis spp. producing sarcocysts in plains viscacha and clarify its IH range and zoonotic potential. Notably, plains viscacha is frequently consumed by humans, and the presence of Sarcocystis spp. could be underestimated.

Conclusions
In this paper, the molecular characterisation of a Sarcocystis species was carried out from L. maximus muscles, a host that had not been previously recorded for this genus. Given the host specificity that Sarcocystis spp. can present and the lower identity with other reported sequences for 18S rRNA marker, the species in plains viscacha from Argentina are potentially new species. In addition, regarding the coxI sequences obtained here, viscachas could be HI for a Sarcocystis canislike species. Regarding nomenclature, we considered the species to be named as new once further morphological descriptions become available. In addition, further studies are necessary to elucidate definitive hosts and the zoonotic potential of the species producing sarcocysts in plains viscacha.

Ethical statement
The research was conducted according to Argentine laws. Sample collection was carried out during fieldwork under official permission granted by the Dirección de Minería, Medio Ambiente y Recursos Naturales, province of Entre Ríos, the Dirección de Flora y Fauna, province of Buenos Aires, and the Dirección General de Bosques y Fauna, province of Santiago del Estero, and by the recommendations of the Guidelines for the capture, handling and care of mammals as approved by the American Society of Mammalogists (Animal Care and Use Committee 1998). No endangered species were involved in this study.

Author's contributions
VC and AMA collected the host specimens. VC, EH, and GM examined the muscle samples. GM did the molecular analysis. All authors discussed the result and contributed to the final manuscript.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.