Prevalence and morphological and molecular characteristics of Sarcocystis bertrami in horses in China

Three cyst-forming Sarcocystis species have been identified in horsemeat; however, there exists considerable confusion concerning their relationships. Here, 74% (34/46) of the examined tissue samples from horses contained sarcocysts based on examination by light microscopy (LM), and the organism was identified as Sarcocystis bertrami based on cyst structure. The S. bertrami cysts were microscopic (up to 6750 μm in length) and exhibited a striated wall with 2.0–5.1 μm villar protrusions (vps) under LM. Transmission electron microscopy (TEM) observations showed that the vps were tightly packed, similar to “type 11c”. Four genetic markers (18S, 28S, ITS1 and the mitochondrial cox1 gene) of S. bertrami were sequenced and analyzed. The 28S and ITS1 sequences are the first records for Sarcocystis in horses. The newly obtained sequences of the 18S and cox1 genes both shared the highest similarities with those of S. bertrami and S. fayeri obtained from horses. Phylogenetic analysis based on the 18S, 28S and cox1 sequences revealed that S. bertrami and S. fayeri formed an independent clade within a group comprising Sarcocystis spp. from ruminants and pigs. Therefore, S. bertrami and S. fayeri are considered to represent the same species of Sarcocystis in horses, and S. fayeri is a junior synonym of Sarcocystis bertrami.

Traditionally, sarcocyst structure and life cycle are the two major criteria for naming a new species of Sarcocystis in a given intermediate host. However, the morphological characteristics of sarcocysts in horses have been observed to undergo some changes in various stages of development [6,17]. In the past decade, molecular analysis based on nucleotide sequences has been recommended as a useful and efficient tool for delineating or identifying species of Sarcocystis from the same or different hosts [10-12, 14, 15]. The 18S rDNA and mitochondrial cox1 sequences of S. bertrami and S. fayeri have recently been sequenced and deposited in GenBank. However, the relationship of the two species inferred from molecular data is still unclear owing to research work performed and published by different research teams almost simultaneously [19,23]. There are currently no records of the 28S rDNA and ITS-1 sequences of Sarcocystis spp. from horses available in GenBank.
In the present study, the prevalence of Sarcocystis species in horsemeat in China was investigated based on the morphological characteristics of the sarcocysts. Additionally, four genetic markers, 18S rDNA, 28S rDNA, ITS-1 and mitochondrial cox1 of the parasite were sequenced and analyzed to augment its descriptions and explore the relationship with Sarcocystis spp. in horses.

Morphological examination of sarcocysts from horses
The study protocol was approved by the Animal Ethics Committee of Yunnan University (permission number: AECYU2015021). Horsemeat serves as a food source for humans and is commonly marketed in China. In total, tissues from 46 horses were examined from two abattoirs, one in Kunming City and another in Shilin Prefecture, both of which are located in Yunnan Province, China, from October 2015 to June 2016. From each animal, fresh tissue samples (50 g each) from the esophagus, diaphragm, skeletal muscle, tongue, and heart were examined for sarcocysts. In the laboratory, 40 specimens of approximately 10 Â 3 mm in size from each collected sample were pressed and squeezed between two glass slides and then inspected using stereomicroscopy. Thereafter, individual sarcocysts were extracted and isolated from skeletal muscular fibers using needles and processed for light microscopy (LM), transmission electron microscopy (TEM) and DNA analysis.
For TEM, four sarcocysts (two from horse No. 3 and two from horse No. 8) were fixed in 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) at 4°C, postfixed in 1.0% osmium tetroxide in the same buffer, dehydrated in a graded alcohol series, and embedded in an Epon-Araldite mixture. Ultrathin sections were stained with uranyl acetate and lead citrate and then examined using a JEM100-CX transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 100 kV.
The ultrastructure of the sarcocysts was similar to "type 11c". The primary sarcocyst wall exhibited numerous vps, measuring 2.0-4.4 Â 0.3-0.8 lm (n = 15), which were often tightly packed and folded over the cyst surface (Fig. 1B). The vps contained bundled microtubules in the core, which penetrated diagonally into the ground substance and sometimes reached the interior border of the ground substance (Figs. 1B and 1C). The primary cyst wall presented minute undulations over the entire sarcocyst surface. There was a layer of ground substances of 0.7-1.2 lm (n = 12) in thickness immediately beneath the primary sarcocyst wall (Fig. 1C).

Molecular characterization of 18S rDNA
Genomic DNA was extracted from five individual sarcocysts, and the 18S rDNA, 28S rDNA, mitochondrial cox1 and ITS-1 sequences were amplified successfully using their DNA as templates. Four 18S rDNA nucleotide sequences for cysts 3, 8, 10 and 15, two 28S rDNA sequences for cysts 3 and 10, three mitochondrial cox1 sequences for cysts 3, 8 and 26, and five ITS-1 sequences for cysts 3, 8, 10, 15 and 26 were successfully assembled, and all of the sequences including the primers were deposited in GenBank.
The four 18S rDNA nucleotide sequences (MH025625-MH025628) were 1592-1594 bp in length and shared an identity of 99.7-99.9% (average 99.8% identity). The most similar sequences in GenBank were those of S. bertrami from
The phylogenetic tree inferred from the 18S rDNA sequences revealed that the newly obtained sequences of S. bertrami formed an individual clade with those of S. fayeri from horsemeat from Japan (AB661437), Canada (AB972443) and Italy (LC171838) within a group comprising S. miescheriana (JN256123) and S. suihominis (KP732435) from pigs using canids and primates as definitive hosts, respectively, and Sarcocystis spp. from ruminants with primates or felids as definitive hosts (Fig. 2).

Molecular characterization of 28S rDNA
The two 28S rDNA nucleotide sequences (MH025629 and MH025630) were 3456 bp and 3449 bp in length, respectively, and shared 99.2% identity. The most similar sequence in Gen-Bank was that of S. miescheriana (AF076902) from a pig, but the identity was only 90.4-90.5%.
The phylogenetic tree based on the 28S rDNA sequences revealed that S. bertrami formed a clade with S. miescheriana (AF076902), S. gigantea (U85706) from sheep and S. moulei (AF012884) from goats, the last two of which use felids as definitive hosts (Fig. 3).
The phylogenetic tree based on the mitochondrial cox1 sequences revealed that the newly obtained sequences of S. bertrami formed an individual clade with those of S. bertrami from horsemeat from China (KY399753) and S. fayeri from horsemeat from Japan (LC171856), Canada (LC171850) and Italy (LC171857) within a group comprising S. miescheriana (LC349978), S. suihominis (MH404228), and Sarcocystis spp. from ruminants with felids as known or suspected definitive hosts (Fig. 4).

Molecular characterization of ITS-1
The five ITS-1 nucleotide sequences (MH025634-MH25638) were 934-936 bp in length and shared an identity of 96.7-98.3% (average 97.4% identity). BLAST searches using only the ITS-1 region sequences of approximately  660 bp from S. bertrami revealed that no sequences in GenBank shared significant similarities with them.
To date, three Sarcocystis species, S. bertrami, S. equicanis and S. fayeri, have been identified from horsemeat, all of which have the dog as the definitive host. However, there is considerable confusion concerning the validity of the above species due to their similar life cycles and morphology. On the basis of  TEM morphology, Dubey et al. (2016) suggested that there are two valid species of Sarcocystis in horses: a thick-walled species (S. fayeri), with a "type 11a" sarcocyst wall, and a thinwalled species (S. bertrami synonym S. equicanis), with a "type 11c" sarcocyst wall. The critical distinction between the two sarcocyst wall types is that "type 11a" exhibits nearly upright vps, but "type 11c" exhibits packed and folded vps [4]. The sarcocysts reported here were diagnosed as S. bertrami based on their similarity with "type 11c", which has been demonstrated previously for S. equicanis from European horses [13], for Sarcocystis sp. from Mongolian horses [20] and for S. bertrami from Chinese horses [23].
Nucleotide sequence analysis has proven to be a useful tool for delineating or identifying species of Sarcocystis from the same or different hosts, and different genetic markers have shown different levels of intra-or inter-specific sequence diversity [14,15]. There are only sequences of 18S rDNA and mitochondrial cox1 from S. bertrami and S. fayeri currently deposited in GenBank. In our analysis, the newly obtained 18S rDNA sequences exhibited up to 100% identity (average 98.0% and 96.5% identity, respectively) with those of S. bertrami and S. fayeri from horses provided in GenBank; the newly obtained mitochondrial cox1 sequences shared the highest identity of 98.0-99.8% (average 99.0%) with those of S. fayeri, followed by S. bertrami (98.1-99.2% identity, average 98.7% identity). A possible explanation for the high similarities of the two parasites is that both represent the same species of Sarcocystis in horses. The protrusions observed via TEM may appear to be upright or folded depending on the plane of the cut section. Phylogenetic analysis based on the 18S rDNA, 28S rDNA and mitochondrial cox1 sequences also confirmed the close relationship between S. bertrami and S. fayeri.
In conclusion, we found a high prevalence rate of Sarcocystis in horses in China, and only S. bertrami was identified based on the cyst ultrastructure. Based on 18S rDNA and mitochondrial cox1, S. bertrami and S. fayeri are inferred to represent the same species from horses. According to the general rules of the International Code of Zoological Nomenclature, S. fayeri should be considered a junior synonym of S. bertrami.