Finding a needle in a haystack: DNA Haemoproteus columbae enrichment using percoll density gradient and flow cytometry.

ABSTRACT

Isolation of genomic DNA of blood parasites in birds, reptiles, amphibians, and fishes is a challenging task, given that their red blood cells are nucleated; for that reason, parasite genomic DNA is only a fraction of the total extracted DNA, and it is challenging to obtain concentrated high-quality genetic material. Percoll Density Gradient (PDG) and flow cytometry are tools for separating and analyzing cell populations or even a single cell, and both represent potent approaches for isolating avian haemosporidians parasites. Our experimental design included several steps seeking to concentrate the parasite´s DNA. We used blood samples from a Rock pigeon infected with Haemoproteus columbae. After inducing parasite exflagellation and gametogenesis in vitro, we subjected the samples to a Percoll Density Gradient to separate the parasites from the rest of the blood cells. Following centrifugation, the layer containing extracellular parasites underwent a flow cytometry and cell sorting process, during which we selected two different subpopulations of cells for analysis. Based on qPCR analyses, we demonstrate parasite DNA enrichment in Percoll Density Gradient and flow cytometry samples; simultaneously, these samples showed the lowest concentration of Columba livia DNA. However, the concentration of parasite DNA was higher in the PDG than in the cell sorting sample. This study reports the concentration of the Haemoproteus parasite by flow cytometry without DNA-intercalating dyes, and this methodology can serve as a technique for DNA enrichment of blood parasites infecting nucleated red blood cells to improve techniques that allow obtaining complete genomes.