Detection of Sarcocystis aucheniae in blood of llama using a duplex semi-nested PCR assay and its association with cyst infestation

Abstract

The protozoon Sarcocystis aucheniae is the causative agent of South American camelid (SAC) sarcocystosis. Infections are characterized by the presence of cysts in muscles which are in size and appearance similar to rice grains. As consumption of insufficiently cooked infected meat produces gastroenteritis, cyst-containing SAC meat is confiscated by sanitary authorities or depreciated with serious economic consequences for SAC breeders. In this work, a duplex semi-nested PCR was designed to simultaneously detect parasite and llama DNA in host blood samples. Species-specific regions of S. aucheniae 18S rRNA gene and Lama glama 16S mitochondrial gene were amplified, yielding bands of 583 and 257 bp, respectively, and separated by gel electrophoresis. The method proved to be highly sensitive, with a detection limit lower than one parasite per milliliter blood, and the inclusion of primers to detect llama-specific DNA resulted useful as a methodological control. Blood samples collected from llamas of Argentina and Bolivia (n = 225) were analyzed using this method, and 18.7 % resulted positive for S. aucheniae. No correlation was found between PCR results and llama age, sex or the finding of macroscopic cysts in meat after slaughter. Lack of molecular detection in the blood of some llamas harboring macrocysts suggests that parasite circulation in the bloodstream after encystment is under the detection threshold of the test or even absent, while PCR positive results in cyst-infested animals suggests that prior exposure to the parasite does not impede subsequent infections. The described method can be useful to detect active foci of infection, to assess the effectiveness of parasiticide treatments, and for the surveillance and tracing of definitive hosts.

Keywords: Bioinformatics; Microbiology; Molecular biology.

Fig. 1

General scheme of the semi-nested PCR for the amplification of the hypervariable region of S. aucheniae 18S rRNA gene.

Fig. 2

Molecular detection of S. aucheniae DNA by semi-nested PCR. Fragments of S. aucheniae 18S rRNA gene were amplified by semi-nested PCR from DNA samples extracted from a S. aucheniae macrocyst using the protocol described in Martín et al. (2016) (lane 1, amplicon size ∼400 bp) or in the present work (lane 2, amplicon size ∼550 bp). A duplex format of this assay was set up, including primers to amplify a ∼257 bp fragment of Lama glama DNA isolated from blood of llama. Lanes 3 and 4 show a positive and a negative result, respectively. M: 1 kb Plus DNA marker.

Fig. 3

Alignment of S. aucheniae 18S rRNA sequences obtained from llama blood and cysts from llama, guanaco and alpaca. Sequences were truncated to correspond in size to the amplicon as obtained after semi-nested PCR amplification of DNA extracted from llama blood (MG832003). The determined sequence MG832003 was aligned with KT382799 (nt 1233 to 1775) from a cyst found in a guanaco of Santa Cruz, Argentina, AF017123K (nt 1233 to 1768), from a cyst of an alpaca from Australia, and KF383266 (nt 1237 to 1774), KF383267 (nt 1238 to 1773), KF383268 (nt 1235 to 1771), and KU527117 (nt 1235 to 1779) obtained from cysts found in llamas of Jujuy, Argentina. Identity percentages with respect to MG832003 are shown at the end of the alignment.