Morphological and molecular characterization of Sarcocystis wenzeli in chickens (Gallus gallus) in China

There has been considerable confusion concerning the number and classification of Sarcocystis spp. in chickens. Scarce nucleotide data of Sarcocystis spp. from chickens are provided in GenBank. The study aimed to investigate the morphological and molecular characteristics of Sarcocystis spp. found in chickens in China. Tissues from 33 chickens were collected in 2019. Sarcocysts were observed using light (LM) and transmission electron microscopy (TEM). Individual sarcocysts from different chickens were selected for DNA extraction, and five loci, 18S rDNA, 28S rDNA, ITS1 region, the mitochondrial cox1 gene and the apicoplastic rpoB gene, were amplified from each sarcocyst, sequenced and analyzed. Only S. wenzeli was found in 14 of 33 (42.4%) chickens. Under LM, the sarcocysts were microscopic and exhibited palisade-like villar protrusions measuring 1.5–2.8 μm. Ultrastructurally, the sarcocyst wall contained numerous stubby hill-like villar protrusions. The protrusions included scattered microtubules, which extended from the tips of the protrusions into the ground substance. The five loci were successfully sequenced and the sequences deposited in GenBank. At 18S rDNA, ITS1 and cox1, the most similar sequences in GenBank were those of Sarcocystis sp. obtained from the brains of chickens, i.e. 99.9–100%, 98.1–98.5% and 99.3% identity, respectively. The five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) showed different levels of interspecific sequence similarity with other closely related species of Sarcocystis (e.g. 99.8%, 99.0–99.2%, 89.3–89.7%, 98.5%, and 97.5%, respectively, with S. anasi). Phylogenetic analysis based on four of the loci (18S rDNA, cox1, rpoB and ITS1) revealed that S. wenzeli formed an independent clade with Sarcocystis spp. that utilize geese or ducks as intermediate hosts and canines as the known or presumed definitive host. To our knowledge, the sequences of 28S rDNA and rpoB reported here constitute the first records of genetic markers of Sarcocystis spp. in chickens. Based on molecular analysis, S. wenzeli might be responsible for the neurological disease in chickens, and ITS1 and rpoB are more suitable for discriminating it from closely related Sarcocystis spp. Phylogenetic analysis revealed that S. wenzeli presents a close relationship with Sarcocystis spp. in geese or ducks.

, S. gallinarum Krause and Goranoff [2] and S. wenzeli (Wenzel et al.) Odening 1997 [3,4]. However, there has been considerable confusion concerning the number and classification of species of Sarcocystis in chickens owing to the imperfection of the original description [5].
The correct identification of Sarcocystis species that might infect chickens is crucial for sarcocystosis control and prevention. The ultrastructure of sarcocysts is traditionally a reliable characteristic for identifying different Sarcocystis species in a given host. Currently, PCR assays and sequencing procedures are considered much more practical, accurate, and reliable methods for the delineation and identification of Sarcocystis species than traditional methods based on morphological characteristics [6,7]. However, there are only one 18S rDNA sequence (783 bp), one ITS1 region (ITS1) sequence (923 bp) and one mitochondrial cox1 gene sequence (547 bp) of Sarcocystis sp. in chickens currently deposited in GenBank. All these nucleotide sequences were obtained from Sarcocystis in chickens associated with neurological lesions in Brazil, and the parasite was closely related to S. anasi and S. albifronsi [8]. Nevertheless, Sarcocystis sp, found by these authors was not identified to the species level.
Therefore, the aims of the present study were: (i) to obtain data on the prevalence of Sarcocysits in chickens using morphological characteristics; to (ii) sequence and analyze the near-complete 18S rDNA, 28S rDNA, ITS1, cox1 and apicoplastic rpoB gene (rpoB) of Sarcocystis species found in chickens in order to augment the species descriptions; and (iii) to investigate phylogenetic relationships of Sarcocystis species in chickens with known species of fowl-infecting Sarcocystis spp. using 18S rDNA, cox1, rpoB and ITS1 sequences.

Morphological examination of sarcocysts from chickens
In total, tissues from 33 chickens were collected from Jiaojiaqing village, Shizong County, Yunnan Province, located in southwestern China, in July and December 2019. These chickens were free ranging and were raised by the local peasants. From each chicken, fresh tissue samples of the skeletal muscles and heart were examined for sarcocysts. In the laboratory, 20 pieces of 3 mm muscle from each collected sample were pressed and squeezed between two glass slides and inspected using a stereomicroscope. Individual sarcocysts were extracted and isolated from muscle fibers using dissection needles and processed for light (LM) and transmission electron microscopy (TEM) and DNA analysis. For TEM, four sarcocysts (two from chicken no. 4 and two from chicken no. 10) 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 a graded alcohol series and embedded in an epon-alaldite 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 80 kV. For DNA isolation, individual cysts were stored in sterile water at −20 °C prior to processing.

DNA isolation, PCR amplification, cloning and sequence analysis
For DNA analysis, 5 individual sarcocysts isolated from different chickens were subjected to genomic DNA extraction using the TIANamp Genomic DNA Kit (Tiangen Biotech Ltd., Beijing, China) according to the manufacturer's instructions. The Sarcocystis species were characterized at 5 loci within the 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB genes. The near-complete 18S rDNA gene was amplified with the primer pair S1/SarDR [9,10]; the near-full-length 28S rDNA gene was amplified with the primer sets KL1/KL3, KL4/KL5b, and KL6/KL2 [11]; the complete ITS1 region was amplified with the primer pair P-ITSF/P-ITSR [10]; the partial cox1 gene was amplified with the primer pair SF1/COIRm [6]; and the partial rpoB fragment was amplified with the primer pair rpoBF2 (5'-ATT TTT GTG GAT ATG ATT TTG AAG ATG C-3') and rpoBR2 (5'-AGT TTA GAT CCA GTT CTA CCG-3'), designed using OLIGO 7.60 (Molecular Biology Insights, Inc., West Cascade, USA) based on highly conserved regions of the rpoB sequences of Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. deposited on GenBank. The PCR products were purified, cloned, sequenced, and assembled using the methods described in a previous report [12].
Phylogenetic analyses were conducted separately for the 18S rDNA, cox1, rpoB and ITS1 sequences using MEGA X software [13]. The selected sequences of the four loci of Sarcocystis spp. from various hosts were downloaded from GenBank, respectively, and aligned with the ClustalW program integrated in MEGA X applying a gap opening penalty of 10/10 and a gap extension penalty of 0.1/0.2 as pairwise and multiple alignment parameters, respectively. The alignment was subsequently checked visually; some sequences were truncated at both ends, so all sequences started and ended at the same nucleotide positions. The maximum parsimony (MP) trees were generated with a tree-bisection-regrafting (TBR) algorithm. The reliability of the MP phylograms was tested with the bootstrap method using 1000 replications.
In the case of 18S rDNA, the final alignment comprised a total of 29 nucleotide sequences from 26 taxa and 2100 aligned positions, and Besnoitia besnoiti (GenBank: DQ227418), N. caninum (GenBank: U16159) and T. gondii (GenBank: U03070) were chosen as outgroups to root the tree. At cox1, the final alignment comprised 28 cox1 nucleotide sequences from 28 species and 1020 aligned positions with no gaps, and T. gondii (GenBank: JX473253), Hammondia triffittae (GenBank: JX473247) and B. besnoiti (GenBank: XM029362743) were used as outgroup species. At rpoB, the final alignment comprised 20 rpoB nucleotide sequences from 19 species and 694 aligned positions with no gaps, and T. gondii (GenBank: AF095904) and N. aninum (GenBank: AF138960) were used as outgroup species to root the tree. At ITS1, the final alignment comprised 25 ITS1 nucleotide sequences from 21 species and 1206 aligned positions, and B. tarandi (GenBank: MH217579) and N. caninum (GenBank: U16159) were chosen as outgroups.
Four sarcocysts from both chickens were examined using TEM, all of which appeared to have walls that were ultrastructurally similar and closely resembled the "type 9k" cyst wall. The sarcocyst wall contained numerous stubby hill-like villar protrusions that were up to 1.2 μm long and 1.0 μm wide and were lined with an electrondense layer that appeared thicker at the tips of the protrusions (Fig. 1c). Within the protrusions, there were numerous scattered fine, electron-dense granules and scattered microtubules. The microtubules extended from the tips of the protrusions into the ground substance, where they crossed microtubules originating from neighboring protrusions (Fig. 1d). The protrusions were spaced at intervals of 0.3-1.1 μm from each other. Small invaginations of the primary wall were present on the lateral aspect of the protrusions and in the spaces between protrusions. The layer of ground substance beneath the protrusions was 0.3-0.4 μm in thickness; septa were evident within the cysts (Fig. 1c).
The five 28S rDNA sequences of S. wenzeli were 3279 bp in length and shared 99.7-100% identity (average 99.9% identity). Therefore, only 4 sequences (GenBank: MT756986-MT756989) were deposited in GenBank. The most similar sequence in GenBank was that of S. The five rpoB sequences of S. wenzeli were 844 bp in length and shared 98.9-100% identity, with an average identity of 99.3%, so only 2 sequences (MT761694 and MT761695) were submitted to GenBank. The most similar sequence in GenBank was that of S. anasi (MH138320) (97.5% identity), followed by those of S. albifronsi (MH138319) (97.4% identity) and S. rileyi (MF596308) (95.9% identity).
Although the newly obtained 18S rDNA, cox1 and ITS1 sequences had the highest similarity with homologous sequences of Sarcocystis sp. isolate Chicken-2016-DF-BR, only the ITS1 sequence of this isolate was included in the phylogenetic analysis because sequences of the other two loci of the isolate were shorter than the rest and it would lower the phylogenetic signal of the analysis. In the phylogenetic tree constructed based on 18S rDNA (Fig. 2), cox1 (Fig. 3), or rpoB (Fig. 4) sequences, S. wenzeli formed an individual clade with S. anasi, S. albifronsi and S. rileyi basal to a group comprising Sarcocysits spp. obtained from birds or terrestrial carnivores. Phylogenetic analysis based on ITS1 sequences (Fig. 5) revealed that S. wenzeli formed an individual clade with Sarcocysits sp. isolate Chicken-2016-DF-BR and S. rileyi, and this clade was within a group comprising Sarcocysits spp. obtained from birds or terrestrial carnivores.

Discussion
Sarcocystis spp. in chickens may cause severe myositis [14] and occasionally neurological disease [8,15]. Sarcocystis infection in chickens has been reported in Hungary [1], Bulgaria [2], Russia [16], Papua New Guinea [14], Australia [14], Germany [3], the Czech Republic [17], Azerbaijan [18], China [19], Iran [20] and Brazil [8]. Three species of Sarcocystis, S. horvathi, S. wenzeli and S. gallinarum, have been proposed to be responsible for the sarcocysts observed in muscle tissues of chickens. The sarcocysts found in chickens have been divided into two types based on the shape of the bradyzoites. Bananashaped sarcocysts are considered to be produced by S. horvathi, described in 1908, which is synonymous with S. gallinarum whereas lancet-shaped sarcocysts are attributed to S. wenzeli, described in 1982 [5]. The ultrastructure of the sarcocysts of S. wenzeli has been described in detail previously [19,21] and is similar to the type 9k sarcocyst wall classified by Dubey et al. [5]. It is worth noting that morphologically similar sarcocysts have been observed in the lesser snow geese (Anser caerulescens) in Saskatchewan, although the species has not been named [22]. The fine structure of the sarcocysts of S. horvathi and S. gallinarum is still unclear. In our materials, only the sarcocysts of S. wenzeli were found and identified, based on the observation of lancet-shaped bradyzoites and the TEM analysis of sarcocysts. The 42.4% (14/33) prevalence rate of Sarcocystis identified in chickens was lower than the 94.78% (37/39) prevalence recently surveyed in Iran using the digestive method [20], but was higher than the 8.9% (17/191) prevalence based on microscopic detection reported in China in 2012 [19]. It needs to be stressed that only squash preparation was used to search for mature sarcocysts in tissues of chickens in the present study. Therefore, the prevalence rate of Sarcocystis surveyed in the village should be underestimated because of the low sensitivity of the method.
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 revealed different levels of intra-or interspecific sequence diversity [6,7,12]. There are only one 18S rDNA sequence, one ITS1 sequence and one cox1 sequence of Sarcocystis sp. obtained from brains of two chickens in Brazil currently available in GenBank. In the present study, five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) from S. wenzeli were sequenced and analyzed, to the best of our knowledge, for the first time. Among them, 28S rDNA and rpoB constitutes the first records of Sarcocystis species in chickens. In our analysis, the sequences of the five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) of this parasite presented high intraspecific similarities of 99.8-100%, 99.7-100%, 99.0-99.9%, 100%, and 98.9-100%, respectively. When comparing these sequences with those deposited on GenBank, sequences of 18S rDNA, ITS1 and cox1 of S. wenzeli shared high similarities with those of Sarcocystis sp. isolate Chicken-2016-DF-BR obtained from brains of two chickens, i.e. 99.9-100%, 98.1-98.5%, and 99.3% identity, respectively. Therefore, the unrecognized species of Sarcocystis associated with meningoencephalitis in chickens from Brazil in 2020 [8] could be inferred as S. wenzeli owing to the high similarities of the three loci. The first case of Sarcocysits-associated encephalitis in chickens was diagnosed in the USA in 1995 [15], and the species of Sarcocysits was not identified because of no sarcocysts observed in brain samples of chickens, similar to the case occurred in Brazil in 2020 [8]. The sequences of the five

Sarcocystis wenzeli MT761695
Neospora caninum AF138960 Toxoplasma gondii AF095904  This study also established the phylogenetic relationships between S. wenzeli and Sarcocystis spp. in different hosts based on 18S rDNA, cox1, rpoB and ITS1 sequences. The topologies of the trees inferred from these sequences were highly similar and revealed that S. wenzeli presents a close relationship with Sarcocystis sp. isolate Chicken-2016-DF-BR, S. rileyi, S. albifronsi and S. anasi. The later three species utilize geese or ducks as intermediate hosts, and the definitive hosts of S. rileyi and S. albifronsi are canines, but that of S. anasi is still unknown [23,24]. Based on experimental infection, the definitive hosts of S. wenzeli were confirmed to be both cats and dogs [3,25], which is peculiar and differs from the situation for all known Sarcocystis spp. found in domestic animals, which use only either cats or dogs as their definitive host. However, Sarcocystis sporocysts were not found in the feces of cats fed breast muscle sample from over 2000 chickens from grocery stores in the USA, although the muscle was not examined microscopically for sarcocysts or bradyzoites [26].

Conclusions
In summary, we found a high prevalence rate of Sarcocystis in free-range chickens in China, and only S. wenzeli was identified based on the cyst ultrastructure. Five loci (18S rDNA, ITS1, 28S rDNA, cox1 and rpoB) of the parasite were sequenced, analyzed and deposited in Gen-Bank. Based on molecular analysis, S. wenzeli might be an agent caused neurological disease in chickens. Among these genetic markers, ITS1 and rpoB are more suitable for discrimination among closely related Sarcocystis species. Phylogenetic analysis revealed that S. wenzeli shows a close relationship with Sarcocystis spp. that use geese and/or ducks as intermediate hosts and canines as definitive hosts.