Molecular diversity of cox1 and LSU rDNA sequences of Sarcocystis bertrami (syn. S. fayeri) (Apicomplexa: Eucoccidiorida: Sarcocystidae) in horses.

Overview

• This research investigates the genetic diversity of Sarcocystis bertrami, a parasite found in horsemeat, by analyzing specific DNA sequences to clarify the relationship between two morphotypes previously thought to be separate species.
• The study confirms that the two morphotypes, S. bertrami and S. fayeri, are actually the same species and explores the genetic variation within and between individual parasites from different geographic locations.

Background

• Sarcocystis parasites can contaminate raw horsemeat and cause food poisoning, thus representing a public health concern.
• Two distinct morphotypes of sarcocysts found in horsemeat, named based on their villar protrusions as Sarcocystis fayeri (upright protrusions) and Sarcocystis bertrami (folded protrusions), have historically been considered separate species.
• Recent molecular studies focusing on ribosomal RNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (cox1) genes suggested these two forms might be the same species (conspecific), but these studies used only a small number of samples.

Study Objectives

• To further investigate the genetic diversity of S. bertrami and S. fayeri by analyzing cox1 and LSU rDNA sequences from sarcocysts collected from horsemeat.
• To confirm whether the two morphotypes really represent a single species across horses from different geographic origins.
• To examine intra-individual and inter-individual genetic variation within the parasite populations.

Materials and Methods

• Samples collected from 150 horses: 76 from Canada, 41 from France, and 33 reared in Japan.
• Muscle tissues were inspected for presence of sarcocysts; positive detections were 71, 2, and 3 horses respectively from the three regions.
• A total of 58 sarcocysts were analyzed for their mitochondrial cox1 gene and large subunit (LSU) rDNA sequences.
• Sequences from this study were compared with existing sequences in GenBank for S. bertrami, S. fayeri, and related species.
• Cloning and sequencing involved 211 LSU rDNA sequences obtained from 54 sarcocysts to assess sequence variation.

Results

• Newly acquired cox1 sequences and those retrieved from the database showed that all morphotypes belonged to the same species, confirming conspecificity.
• Significant inter-individual variation was detected in cox1 sequences even among sarcocysts from the same horse, demonstrating genetic diversity at the population level.
• No intra-individual variation in cox1 was observed, indicating mitochondrial DNA sequences were consistent within each parasite.
• Some sequences identified as pseudogenes (NUMTs—mitochondrial DNA sequences inserted in the nuclear genome) were obtained due to the use of certain primers, highlighting potential methodological artifacts.
• LSU rDNA sequences showed robust intra-individual and inter-individual variation, revealing a heterogeneous mix of ribosomal RNA gene copies within individual parasites (intragenomic mosaicism).
• No geographic subpopulations were identified, suggesting the species is genetically mixed across regions.

Conclusions

• The two sarcocyst morphotypes infecting horses, S. bertrami and S. fayeri, are the same species genetically.
• This study expands understanding of genetic diversity within this species by analyzing multilocus DNA sequences from many samples geographically spread, confirming no division into subspecies by region.
• Intragenomic variation in ribosomal DNA highlights complexity in genetic structure that must be considered in molecular diagnostics.
• Care must be taken to avoid amplifying pseudogene sequences that could confound genetic analyses.
• These findings improve accuracy in parasite identification important for public health monitoring and horsemeat safety assessments.