Ultrastructural Description of Sarcocystis Sp. in Cardiac Muscle of Naturally Infected Alpacas (Vicugna pacos).

Background: Recently, it was proposed the name of Sarcocystis masoni n. sp. for the Sarcocystis that causes microcyst in skeletal muscle of South American camelids. However, there are no ultrastructural reports of microcysts of Sarcocystis in cardiac muscle of alpacas. This study reports ultrastructural features of microcysts of Sarcocystis sp. from cardiac muscle of naturally infected alpacas. Methods: Thirty alpacas (age range: three to five years) from the province of Junin, Peruvian Central Andes, were included in this study in January 2015. Cardiac muscle samples were evaluated by histology and transmission electron microscopy. Results: Bradyzoites in cysts had typical characteristics of Apicomplexa including organelles, a large nucleus, micronemes, dense bodies, and polysaccharide granules. Moreover, cysts had a thin wall with numerous, short, finger-like shapes with rounded tip protrusions (0.51 × 0.17 μm). Conclusion: Sarcocystis sp. from the heart and S. masoni n. sp. from the skeletal muscle have similar ultrastructural characteristics.


Introduction
arcocystis spp. cause sarcocystosis in humans and animals worldwide. Two zoonotic species, S. suihominis and S. hominis, cause the parasitic food-borne disease sarcocystosis and are both acquired by the consumption of undercooked meat. Furthermore, sarcocystosis negatively affects food safety that results in economic losses by producing condemnation in slaughterhouses (1,2).
In the high Andean region of Peru, South American camelids (SAC) are an important economic resource. The alpaca is a SAC that acts as an intermediate host of the two recognized Sarcocystis spp. affecting South American camelids. S. aucheniae develops macrocysts in striated skeletal muscles while S. sp. (wellknown as a S. "lamacanis") produces microcysts mainly in cardiac muscle and there are biological and genetic differences between them (3)(4)(5)(6). Recently, Moré et al (7) considered Sarcocystis sp. "lamacanis" as nomen nudum, proposing the name S. masoni for Sarcocystis sp. forming microcysts in skeletal muscle of SAC.
Ultrastructural characterization of the primary cyst wall (CW) is considered the most important criteria in differentiating Sarcocystis spp. (8,9). Despite this, there are scarce ultrastructural descriptions of Sarcocystis from alpacas, and there is no information confirming that Sarcocystis sp. "lamacanis" infecting cardiac muscle and S. masoni n. sp. in skeletal muscle are the same species.
Therefore, the aim of this study was to report the ultrastructural features of Sarcocytis sp. from cardiac muscle of naturally infected alpaca of the Peruvian Andes.

Materials and Methods
Samples of cardiac muscle were obtained from thirty alpacas (age range: three to five years) from the province of Junin in the Central Andean region of Peru 3,300 meters above sea level in January 2015. Samples of cardiac muscle (2 cm 2 ) per animal were obtained from the Ninancaca slaughterhouse (Junin). Half of each sample was fixed in 10% buffered formalin for histological analysis, and the other half was placed in 2% glutaraldehyde in Sorensen's phosphate buffer 0.1 M (pH 7.2) for 24 h as the first step for transmission electron microscopy (TEM) analysis.
For histology, samples were processed following the standard hematoxylin and eosin staining procedures in the Laboratory of Animal Pathology of the National Service of Animal Health, Peru. Samples were screened for Sarcocystis spp. microcysts under low magnification with a light microscope. Heart tissues containing microcysts were submitted for TEM analysis to the Electron Microscopy Department at the Adolfo Lutz Institute, Brazil.
Fragments of fixed cardiac muscle were fixed in 1% Osmium Tetroxide, dehydrated in acetone series, and embedded in Epoxy resin. Semithin sections were stained with 1% metilene blue and examined by light microscopy at high power magnification to select areas of muscle containing cysts.
Ultrathin sections were obtained in a Sorvall Ultramicrotome, stained with uranyl acetate and lead citrate and observed under a JEOL JEM1011 Transmission Electron Microscopy operating at 80kV. Images were recorded with a Gatan 785 ES 1000W Erlangshen camera.

Light Microscopy description
All the 30 samples of alpaca cardiac muscle contained microcysts of Sarcocystis sp. "lamacanis". Microcysts were high in number (Fig. 1), but no inflammatory reaction was observed against the microcysts. All microcysts were similar in size, with a mean of 43.43 µm (range: 22.67-74.18 µm) in width, and 78.24 µm (range: 28.05-280.82 µm) in length.

Ultrastructural description
All of the microcysts were bounded by thin primary cyst walls (CW) having a mean of 33.9 nm (range: 3.1-74.2 nm) in thickness (Fig. 2). At the surface (Fig. 2), the CW formed tubu-S lar, short, villi projections or protrusions (VP) that were not seen in sections stained with hematoxylin and eosin (Fig. 1). The VP had a finger-like shape with rounded tips and were a mean of 0.51 μm (range: 0.16-1.06 μm) long and 0.17 µm (range: 0.04-0.56 µm) wide. The VP occasionally showed a mean of 0.29 µm base (range: 0.14-0.56µm) that was slightly wider than the mean 0.12 µm tip (range: 0.07-0.21µm) (Fig. 3). The VP were spaced a mean of 0.46 μm (range: 0.23-1.44 μm) apart, had longitudinally arranged microtubules and hairlike projections on the VP tip (Fig. 4) and an external electron dense layer, which seems to be vesicles in transverse section (Fig. 5).    (Fig.  2). The GL extended to the interior of the cysts and formed septa (SP) that subdivided the cysts into many compartments, enclosing numerous BZ (Fig. 6). All of the examined sarcocysts were mature as they contained many BZ (1.95-3.53 x 2.69-10.68 µm), which showed typical characteristics of other Sarcocystis spp., i.e. single membrane (24.47-31.72 nm thick), completely developed organelles (i.e., Rugose endoplasmic reticule, microtubules, mitochondria), a large nucleus, micronemes, and numerous polysaccharide and electron dense granules in the central and caudal region (Fig. 6).

Discussion
All the samples were positive for Sarcocystis sp. "lamacanis", which was expected because the sarcocystosis is a hyperendemic disease for SAC of the Andean region. Moreover, our results agree with other studies that reported frequencies from 87% to 100% (2). The ingrained practices related to the lack of knowledge of parasites transmission could favor sarcocystosis dissemination in Peruvian Highlands. For instance, Andean farmers feed their shepherd dogs with infected tissues during home slaughtered of SAC (3).
All sarcocystis species possess a wall that often has unique ultrastructural characteristics that are used to distinguish Sarcocystis spp. within the same intermediate host (10)(11)(12)(13)(14)(15). This is particularly useful to identify zoonotic and non-zoonotic Sarcocystis spp. species in cattle (15).
The present study reports a TEM description of S. sp. from alpaca cardiac muscle and our results show that the CW of Sarcocystis sp. has finger-like rounded tip protrusions. We depicted that Sarcocystis sp. from heart of alpacas has similar ultrastructural characteristics of CW to those of S. cameli, parasite of camels classified earlier (16) as "type 9j." Moreover, these characteristics are similar to those previously reported for S. masoni in the skeletal muscle of camelids (7). However, the microcysts and VP of S. masoni (7) were longer than in our description. Apparently, the microcysts of Sarcocystis sp "lamacanis" are shorter than those from skeletal muscle because cardiac muscle is a denser and less elastic tissue which could restrict microcysts growth. Similarly, microcysts of S. gigantea could had a different shape and size depending of the tissue they are found (17). Other parasitic cysts showed similar development restriction within dense and inelastic tissues, i.e. hydatid cyst in hepatic tissue (18).
The cyst wall of S. aucheniae has numerous cauliflower-like protrusions that are densely distributed (3,7). Therefore, it is confirmed that the Sarcocystis sp. producing microcysts in cardiac muscle of camels is a different species from the S. aucheniae. TEM also permits us to establish that the organelles of Sarcocystis sp. from heart show similar characteristics with other Sarcocystis spp.

Conclusion
This ultrastructural description of CW confirms that S. sp. affecting cardiac muscle of alpacas and S. masoni n. sp. reported from skeletal muscle of SAC are similar. These results increase the biological knowledge of Sarcocystis spp. in SAC.