Sarcocystis spp. in domestic sheep in Kunming City, China: prevalence, morphology, and molecular characteristics

Sheep (Ovis aries) are intermediate hosts for at least six named species of Sarcocystis: S. tenella, S. arieticanis, S. gigantea, S. medusiformis, S. mihoensis, and S. microps. Here, only two species, S. tenella and S. arieticanis, were found in 79 of 86 sheep (91.9%) in Kunming, China, based on their morphological characteristics. Four genetic markers, i.e., 18S rRNA gene, 28S rRNA gene, mitochondrial cox1 gene, and ITS-1 region, were sequenced and characterized for the two species of Sarcocystis. Sequences of the three former markers for S. tenella shared high identities with those of S. capracanis in goats, i.e., 99.0%, 98.3%, and 93.6%, respectively; the same three marker sequences of S. arieticanis shared high identities with those of S. hircicanis in goats, i.e., 98.5%, 96.5%, and 92.5%, respectively. No sequences in GenBank were found to significantly resemble the ITS-1 regions of S. tenella and S. arieticanis. Identities of the four genetic markers for S. tenella and S. arieticanis were 96.3%, 95.4%, 82.5%, and 66.2%, respectively.


Introduction
Sarcocystis spp. are cyst-forming intracellular protozoan parasites with an obligate two-host life cycle between predators as definitive hosts and prey animals as intermediate hosts.
Sarcocystis tenella and S. arieticanis produce microscopic sarcocysts transmitted by canids, while S. gigantea and S. medusiformis produce macroscopic cysts transmitted by felids [2]. The remaining two species, S. mihoensis and S. microps transmitted by canids, are unusual or rare species of Sarcocystis; S. mihoensis, reported only from Japan, produces macroscopic sarcocysts [17]; however, S. microps, reported only once from China, produces microscopic sarcocysts [18]. Natural infections by Sarcocystis spp. in domestic sheep have been investigated in various countries throughout the world, with prevalence ranging from 9.0 to 100% depending on the detection methodology [2]. However, the prevalence of Sarcocystis spp. in domestic sheep in China is largely unknown.
The ultrastructure of sarcocysts is traditionally a reliable character for clarifying different Sarcocystis species in a given intermediate host. However, with more morphologically similar sarcocysts described from different, but closely related intermediate hosts, confusions or disputes have emerged concerning the relationships of these Sarcocystis species. For example, Sarcocystis spp. in cattle and water buffalo have been regarded as separate species based on host specificity; however, sarcocysts in bobcats (Felis rufus), domestic cats (F. catus), Florida panthers and cougars (F. concolor), and cheetahs (Acinonyx jubatus) were all identified as S. felis, on the basis of the morphological similarities [2]. It is therefore an urgent need, even a must, to delineate or reassess descriptions of extant or new species of Sarcocystis in different hosts, using different markers for clarifying their relationships. However, only limited molecular sequences for Sarcocystis spp. in sheep are presently provided in GenBank.
Therefore, the aims of the present study were (i) to investigate the prevalence of Sarcocystis spp. in domestic sheep in China based on the morphological characteristics of the sarcocysts, and (ii) to characterize these species using the 18S rRNA gene (18S rRNA), 28S rRNA gene (28S rRNA), mitochondrial cox1 gene (cox1), and ITS-1 (ITS-1) region for clarifying their descriptions.

Morphological observation of sarcocysts
In total, tissues from 86 sheep were examined from an abattoir in Kunming City in China from March to November 2015. From each animal, fresh tissue samples from the esophagus, diaphragm, skeletal muscles, tongue, and heart were examined for sarcocysts. In the laboratory, 0.5 mm pieces of muscle from each collected sample were pressed and squeezed between two glass slides to inspect sarcocysts using stereomicroscopy. Sarcocysts were isolated from muscular fibers using dissecting needles and processed for light microscopy (LM), transmission electron microscopy (TEM), and DNA analysis.
For TEM, sarcocysts were fixed in 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) at 4°C and post-fixed in 1% osmium tetroxide in the same buffer, then dehydrated in graded alcohols and embedded in epon-araldite mixture. Ultrathin sections were stained with uranyl acetate and lead citrate and then examined using a JEM100-CX transmission electron microscope at 80 kV. For DNA isolation, individual cysts were stored in sterile water at À20°C prior to processing.

Prevalence of natural infections
Sarcocysts were found in 79 of 86 sheep (91.9%). Two morphologically distinct sarcocysts (S. tenella with a thick cyst wall and S. arieticanis with a thin cyst wall) were observed by LM observation (Figs. 1A, 2A). Sarcocysts of S. tenella were found in 73 sheep (84.9%), and were more common than those of S. arieticanis, found in 46 sheep (53.5%). The distribution of the two parasites in different organs is shown in Table 1.

Molecular characterization of the 18S rRNA
The two 18S rRNA nucleotide sequences, each from an individual sarcocyst of S. tenella, were 1,832 bp in length, and completely identical; as a result, only one sequence (MF039329) was submitted to GenBank. The most similar sequences in GenBank were those of S. tenella (KC209734 and KC209737) from sheep (99. 8

Molecular characterization of the ITS-1
The two ITS-1 nucleotide sequences (MF039318 and MF039319), each from a sarcocyst of S. tenella, were 784 bp and 787 bp in length, respectively; the identity between them was 96.7%, and the differences included 23 nucleotide substitutions and three nucleotide deletions. The two ITS-1 nucleotide sequences (MF039320 and MF039321), each from a sarcocyst of S. arieticanis, were 784 bp and 786 bp in length, respectively; the identity between them was 97.0%, and the differences included 20 nucleotide substitutions and four nucleotide deletions. Blast search only using the ITS-1 region, of approximately 525 bp for S. tenella and 520 bp for S. arieticanis, revealed that no sequences shared significant similarities with them. The identity of the new ITS-1 nucleotide sequences for the two species of Sarcocystis in sheep was 65.7-69.7%, and a mean of 66.2%.
The phylogenetic analyses based on either the 18S rRNA, 28S rRNA, or cox1 sequences all clustered the new sequences

Discussion
Sarcocystis spp. are among the most common parasites in domestic ruminants, and some of them can generate significant economic losses when causing clinical and subclinical disease. Up to now, at least six species of Sarcocystis have been named in sheep; however, only four species, i.e., S. tenella, S. arieticanis, S. gigantea, and S. medusiformis, have been frequently found in different areas, especially in Asia and Latin America [2]. In the present study, sarcocysts were common in sheep (91.9%), but only microscopic sarcocysts for S. tenella and S. arieticanis were found, and both parasites were transmitted by canids, which reflects the significant role played by dogs, rather than cats, in the transmission of these parasites in the area.
Thus, the two sarcocyst TEM types (14 and 7) in domestic sheep are common in the tissues of different, but closely related, ruminant animals; however, the relationships between these morphologically similar Sarcocystis spp. are not very clear. It is not easy to complete cross-transmission for Sarcocystis spp. using large experimental animals in the laboratory. Accordingly, characterizing sequences of different genetic markers should be a useful tool to differentiate these species of Sarcocystis or delineate their phylogenetic relationships. For example, Sarcocystis sp. in chamois has been designated as S. tenella based on the similarities of their 18S rRNA and cox1 sequences [10].
In the present study, four genetic markers (18S rRNA, 28S rRNA, cox1, and ITS-1) for Sarcocystis spp. in sheep were sequenced and characterized. Among them, sequences of cox1 for S. arieticanis and ITS-1 for Sarcocystis spp. in sheep were the first records in GenBank. When blasting these sequences in GenBank, sequences of 18S rRNA, 28S rRNA, and cox1 for S. tenella shared high identities with those of S. capracanis, i.e., 99.0%, 98.3%, and 93.6%, respectively; S. hircicanis shared high identities with S. arieticanis, i.e., 98.5%, 96.5%, and 92.5%, respectively. Therefore, mitochondrial cox1 seemed to perform better than 18S rRNA and 28S rRNA for distinguishing S. tenella from S. capracanis, and S. arieticanis from S. hircicanis. The high identity (99.0%) between 18S rRNA sequences for S. tenella and S. capracanis has even led to a debate whether sheep and goat can harbor the same Sarcocystis species [3,5]. Blast search suggested that no sequences in GenBank had significant similarity with the ITS-1 regions of S. tenella and S. arieticanis. However, when comparing the new sequences of the four genetic markers (18S rRNA, 28S rRNA, cox1, and ITS-1) for S. tenella and S. arieticanis, the sequence identities were 96.3%, 95.4%, 82.5%, and 66.2%, respectively. Thus, the ITS-1 region could be more useful for discriminating closely related Sarcocystis spp. because of its high divergence.