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

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...

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Bibliographic Details
Published in:Veterinary parasitology 2024-06, Vol.328, p.110170, Article 110170
Main Authors: Gamboa-Suárez, Brayan Andrés, Lotta-Arévalo, Ingrid Astrid, Sarmiento-Salazar, Felipe, Matta, Nubia E.
Format: Article
Language:English
Subjects:
Animals
Bird Diseases - parasitology
Blood parasites
Centrifugation, Density Gradient - veterinary
Columbidae - parasitology
DNA enrichment
DNA, Protozoan
Exflagellation
Flow Cytometry - veterinary
Genomics
Haemosporida
Haemosporida - genetics
Haemosporida - isolation & purification
Organic Chemicals - chemistry
Povidone
Protozoan Infections, Animal - diagnosis
Protozoan Infections, Animal - parasitology
Silicon Dioxide
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Summary: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. •Gametogenesis is a pivotal process in the enrichment of parasitic DNA.•Enrichment parasitic DNA is ostensible after percoll gradient and flow cytometry.•Flow cytometry recognizes Haemoproteus parasites without DNA intercalating dyes.•Percoll density gradient obtaining a higher concentration of Haemoproteus DNA
ISSN:0304-4017
1873-2550
1873-2550
DOI:10.1016/j.vetpar.2024.110170