Morphological and molecular characteristics of Sarcocystis sp. in donkeys from China

Abstract

There is considerable confusion concerning the relationships among species of Sarcocystis found in donkeys and horses. Here, sarcocysts obtained from donkeys (Equus asinus) were morphologically and molecularly characterized. Sarcocysts were diagnosed in 12 of 32 (37.5%) Chinese donkeys and could be divided into two types, thin-walled and thick-walled, with the aid of light microscopy (LM). The thin-walled sarcocysts were macroscopic (up to 4856 × 320 µm in size) and had short club-like protrusions of up to 2.7 µm; the thick-walled sarcocysts were microscopic (up to 3750 × 135 µm in size) and had villar protrusions of up to 5.4 µm. The ultrastructures of the two types exhibited highly similar morphological characteristics, including bundled microtubules in the core of the villar protrusions penetrating diagonally into the ground substance, similar to wall type 11c. Three genetic markers, 18S rDNA, 28S rDNA, and mitochondrial cox1, were sequenced and analyzed in the two types. The three genetic markers presented high intraspecific similarity between the two types, ranging from 97.2–99.5%, 97.8–99.6% and 99.0 − 99.9%, respectively. The comparison of these sequences with those of Sarcocystis spp. previously deposited in GenBank showed that the newly obtained sequences of 18S rDNA, 28S rDNA, and mitochondrial cox1 presented identities of 90.0 − 97.5%, 94.7 − 95.1% and 82.6 − 84.5%, respectively, with those of S. bertrami and S. fayeri obtained from horses. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based on the mitochondrial cox1 sequences of donkey and horse sarcocysts revealed three fragments (196, 243 and 641 bp) and two fragments (416 and 644 bp), respectively, upon digestion with the restriction enzymes EcoRI and HinfI. Phylogenetic analysis based on 18S rDNA or mitochondrial cox1 sequences revealed that donkey sarcocysts formed an individual clade with Sarcocystis spp. obtained from horses. Based on the divergence of the three sarcocyst genetic markers, especially mitochondrial cox1, between donkeys and horses, the Sarcocystis sp. sarcocysts in donkeys were different with those obtained from horses. The cross-infection of Sarcocystis between donkeys and horses needs to be attempted for further confirmation in the future.