Sarcocystis spp. in chickens may cause severe myositis  and occasionally neurological disease [8, 15]. Sarcocystis infection in chickens has been reported in Hungary , Bulgaria , Russia , Papua New Guinea , Australia , Germany , the Czech Republic , Azerbaijan , China , Iran  and Brazil . 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. Banana-shaped 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 . 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. . 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 . 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 , but was higher than the 8.9% (17/191) prevalence based on microscopic detection reported in China in 2012 . 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  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 , 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 . The sequences of the five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) of S. wenzeli exhibited different levels of similarity compared with closely related Sarcocystis species, sharing 99.8%, 99.0–99.2%, 89.3–89.7%, 98.5% and 97.5% identity with the corresponding sequences of S. anasi, and 99.5%, 98.3–98.5%, 78.8–79.0%, 96.4%, and 95.9% identity with those of S. rileyi. Therefore, ITS1 and rpoB appeared to be more suitable to for distinguishing S. wenzeli from other Sarcocystis spp., especially closely related species of Sarcocystis, than the 18S rDNA, 28S rDNA and cox1 loci.
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 .