Morphological and Molecular Characterization, and Demonstration of a Definitive Host, for Sarcocystis masoni from an Alpaca (Vicugna pacos) in China

Abstract

Only 18S rDNA sequences of Sarcocystis spp. in South American camelids (SACs) are deposited in GenBank as references, and the definitive host of S. masoni in SACs is still unclear. Here, S. masoni sarcocysts detected in an alpaca (Vicugna pacos) in China were investigated with the aid of light (LM) and transmission electron (TEM) microscopy, and characterized using four genetic markers, i.e., 18S rDNA, 28S rDNA and ITS, and the mitochondrial cox1. Additionally, the life cycle of the parasite was completed via experimental animal infection. Under LM, S. masoni sarcocysts exhibited numerous 1.3-2.1 μm conical protrusions. Under TEM, the sarcocyst wall contained conical, cylindrical, or irregular-shaped villar protrusions, similar to type 9j. Two dogs (Canis familiaris) fed S. masoni sarcocysts shed sporocysts with a prepatent period of 8-9 days. The newly obtained 18S rDNA sequences showed 98.4-100% identity with those of S. masoni in SACs previously deposited in GenBank. Interestingly, the newly obtained sequences of 18S rDNA and mitochondrial cox1 shared 99.6-100% and 98.2-98.5% identity, respectively, with those of S. cameli in dromedary camels (Camelus dromedaries). Phylogenetic analysis based on sequences of 18S rDNA, 28S rDNA, or mitochondrial cox1 revealed that S. masoni has a close relationship with Sarcocystis spp. in ruminants. The relationship between S. masoni and S. cameli deserves to be further clarified in the future.

Keywords: Sarcocystis masoni; alpaca; life cycle; morphological and molecular characterization.

Figure 1

Morphological characteristics of Sarcocystis masoni sarcocysts isolated from the myocardium of an alpaca. (a) Overview of a sarcocyst (unstained, light microscopy, LM). (b) Sarcocyst bounded by short conical villar protrusions (vp) (unstained, LM). (c,d) Diagonal section of a sarcocyst (under transmission electron microscopy, TEM). Sarcocyst surrounded by host cell (hc). The sarcocyst wall exhibits numerous villar protrusions (vp) lined by an electron-dense parasitophorous vacuolar membrane (pvm). Knob-like structures (ks) present on the surface of the pvm. Each vp contains scattered microtubules (mt) in its core, which extend from the tip of the vp into the ground substance (gs).

Figure 2

Light micrograph of Sarcocystis masoni sporulated oocyst and sporocysts in the feces of experimentally infected dogs (unstained). (a) Sporulated oocyst. (b) Sporocyst.

Figure 3

Results of PCR with primers SF1/SR9 and restriction enzyme digestion with ClaI and AvaII of sarcocyst and oocyst DNA from Sarcocystis masoni. M, molecular mass marker; Sms, S. masoni sarcocyst; Smo, S. masoni oocyst; P, PCR product; D, digestion of PCR product with ClaI and AvaII.

Figure 4

Phylogenetic trees of selected Sarcocystis species. The trees constructed based on (a) 18S rDNA sequences, (b) 28S rDNA sequences, and (c) mitochondrial cox1 sequences using maximum likelihood (ML) with the Tamura 3-parameter, Hasegawa–Kishino–Yano, and Kimura 2-parameter models, respectively. The values between the branches represent bootstrap values per 1000 replicates, and values below 50% are not shown. (a) The six newly obtained 18S rDNA sequences of S. masoni (ON533528–ON533533, shown in boldface) obtained from the alpaca formed an individual clade with those of S. cameli in the one-humped camel and S. masoni in the llama, alpaca, and guanaco. (b) The six new sequences of S. masoni (ON533536–ON533541, shown in boldface) obtained from an alpaca formed an individual clade within a group comprising Sarcocystis spp. in domestic ruminants. (c) The eight sequences of S. masoni (ON564410–ON564417, shown in boldface) obtained from the alpaca and two infected dogs formed an individual clade with S. cameli in the one-humped camel.