Morphological and Molecular Characterization of Sarcocystis Wenzeli in Chickens (Gallus gallus) in China

Abstract

Background: There has been considerable confusion concerning the number and classification of Sarcocystis spp. found in chickens. To date, no nucleotide sequences of Sarcocystis spp. from chickens have been provided in GenBank. The present study aimed to investigate the morphological and molecular characteristics of Sarcocystis spp. in chickens in China.Methods: 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, the ITS-1 region (ITS-1), the mitochondrial cox1 gene (cox1), and the apicoplastic rpoB gene (rpoB), were amplified from each sarcocyst, cloned, sequenced and analyzed.Results: 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. The bradyzoites were lancet-shaped, measuring 9.2–12.6 × 1.5–3.5 μm. Ultrastructurally, the sarcocyst wall contained numerous stubby hill-like villar protrusions that were up to 1.2 μm long and 1.0 μm wide. The protrusions included scattered microtubules, which extended from the tips of the protrusions into the ground substance. The intraspecific similarity of the five loci (18S rDNA, 28S rDNA, ITS-1, cox1, and rpoB) among the newly obtained sequences ranged from 99.8–100%, 99.7–100%, 99.0–99.9%, 100% and 98.9–100%, respectively. The five loci showed different levels of interspecific sequence similarity with 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 three of the loci (18S rDNA, cox1, and rpoB) revealed that S. wenzeli formed an independent clade with Sarcocystis spp. that employ geese or ducks as intermediate hosts and canines as the known or presumed definitive host.Conclusions: The sequences of 18S rDNA, 28S rDNA, ITS-1, cox1 and rpoB from S. wenzeli reported here constitute the first records of genetic markers of Sarcocystis from chickens made available in GenBank. Based on sequence analysis, ITS-1 and rpoB are more suitable for discriminating S. wenzeli from closely related species of Sarcocystis. Phylogenetic analysis revealed that S. wenzeli presents a close relationship with Sarcocystis spp. from geese or ducks.

Sarcocystis cristata sp. nov. (Apicomplexa, Sarcocystidae) in the imported great blue turaco Corythaeola cristata (Aves, Musophagidae)

“…Sarcocystis cristata is a separate species from Sarcocystis sp. isolated from chicken and S. wenzeli, which are the same species, as stated by Pan et al [19]. The latter two species and five other congeners formed a recurrent group of species reported in several previous works [e.g.…”

“…For Sarcocystis species using birds as intermediate hosts, the ITS1 region was the most conclusive locus to clearly separate the new species from its congeners, while other loci were scarcely variable to distinguish species, although all genetic markers supported the designation of the new species. Apparently, the ITS1 region is more sensitive to the genetic differences among species, as mentioned by Prakas et al [24] and Pan et al [19], who also found a similar utility of molecular analysis while studying S. fulicae in Fulica atra from Lithuania and S. wenzeli in G. gallus from China, respectively.…”

“…hawks, eagles) as definitive host [see e.g. 9, 21-25], while the second grouped the new species and others allegedly using mammals as definitive hosts, such as S. albifronsi in Arctic fox (Alopex lagopus) from Lithuania [26], S. anasi and S. rileyi in V. vulpes and racoon dog (Nyctereutes procyonoides) from Lithuania [27] and Germany [28], S. atraii probably in American mink (Neovison vison) and red fox (Vulpes vulpes) from Egypt [29], S. chloropusae maybe in V. vulpes [30] and S. wenzeli experimentally in dogs and cats [19]. With the most conclusive locus (ITS1 region), the clade formed by S. cristata sp.…”

“…nov. and those of S. anasi, S. albifronsi and S. wenzeli (99.8% each) in the mallard duck (Anas platyrhynchos) (EU553477), the white-fronted goose (Anser albifrons), both from Lithuania (EU502868) and chicken (Gallus gallus) from China (MT756990), respectively; Sarcocystis sp. isolate from chicken (most likely S. wenzeli, see [19]) (99.7%) from Brazil (MN845627); S. rileyi (99.5%) in the common eider (Somateria mollissima) in Norway (KJ396583); S. atraii and S. chloropusae (99.2% each) in the common coot (Fulica atra) (KJ810606) and the common moorhen (Gallinula chloropus) (KJ810604), respectively, both from Egypt. The 28S rRNA locus (1350 bp) was most similar to those of S. wenzeli (99−99.2%) (MT756986-8), S. albifronsi (98.8%)…”