Purpose: In different apicomplexan parasites, multicopy organellar DNA, such as mitochondrion genomes, have been used for detection and molecular epidemiology analysis. We developed a workflow, to obtain complete mitochondrial genome sequences from contaminated foods and clinical samples, for differentiation of C. cayetanensis isolates.
Methods: The 6,274 bp long C. cayetanensis mitochondrial genome, in four overlapping amplicons from genomic DNA, extracted from cilantro and spiked with oocysts and extracted from clinical stool samples, was amplified by PCR. DNA sequence libraries of the gel-purified amplicons were prepared using the Ovation Ultralow System library kit (NuGEN Technologies) and sequenced using MiSeq. Sequence reads were assembled using CLC Genomics WorkBench, and Geneious programs, to map to a reference C. cayetanensis mitochondrial genome.
Results: This approach allowed us to sequence complete mitochondria genomes from produce samples seeded with C. cayetanensis oocysts, and from stool samples. SNP profiles of 30 stool samples, from a 2014 C. cayetanensis outbreak, exhibited discriminatory power based on geographical metadata. Isolates from different states grouped together in an evolutionary tree, suggesting that genomic analyses of mitochondria sequences may help to link outbreak cases to the source.
Significance: The described method will facilitate the application of genomics tools to epidemiologically link C. cayetanensis identified in clinical and food samples during outbreak investigations.