Label-free enrichment of avian Leucocytozoon using flow cytometric sorting

Abstract

The group of haemosporidian parasites is of general interest to basic and applied science, since several species infect mammals, leading to malaria and associated disease symptoms. Although the great majority of haemosporidian parasites appear in bird hosts, as in the case of Leucocytozoon buteonis, there is little genomic information about genetic aspects of their co-evolution with hosts. Consequently, there is a high need for parasite-enrichment strategies enabling further analyses of the genomes, namely without exposure to DNA-intercalating dyes. Here, we used flow cytometry without an additional labelling step to enrich L. buteonis from infected buzzard blood. A specific, defined area of two-dimensional scattergramms was sorted and the fraction was further analysed. The successful enrichment of L. buteonis in the sorted fraction was demonstrated by Giemsa-staining and qPCR revealing a clear increase of parasite-specific genes, while host-specific genes were significantly decreased. This is the first report describing a labelling-free enrichment approach of L. buteonis from infected buzzard blood. The enrichment of parasites presented here is free of nucleic acid-intercalating dyes which may interfere with fluorescence-based methods or subsequent sequencing approaches.

Malaria's Many Mates: Past, Present, and Future of the Systematics of the Order Haemosporida

“…Although it is possible to use gradient centrifugation to isolate parasite DNA, the volume of starting material needed to utilize these methods is typically more than the total volume of blood in the host, so it is not often feasible, particularly when infections are quite low in parasitemia. There are 3 potential ''work-arounds'' for these challenges that could be used, namely cell-sorting methods (Omori et al, 2010;Chakarov et al, 2012), utilizing exflagellated parasites to obtain purified parasites , and focusing on transcriptomics, relying on the parasites being more transcriptionally active than their hosts (Hellgren et al, 2013).…”

Finding a Needle in a Haystack: DNA Haemoproteus Columbae Enrichment Using Percoll Density Gradient and Flow Cytometry

“…Haemoproteus (Haemoproteus) columbae is a widely distributed parasite that infects rock pigeons (Columba livia) and other columbiforms and is transmitted by the bite of louse ies (Pseudolynchia canariensis) (Aragão, 1908;Klei, 1971;Cepeda et al, 2019). Despite signi cant advances in molecular diagnostic tools and sequencing, there is still an information gap for haemosporidians infecting wildlife (Chakarov et al, 2012). This is because: i) the erythrocytes of birds, reptiles and amphibians are nucleated, so a large amount of host DNA dilutes the parasite DNA, making it di cult to obtain the necessary amount of genetic material for sequencing; ii) parasitemia in wildlife is usually low; iii) the genome size of vertebrate hosts is 50 to 100 times larger than that of parasites; iv) they are obligate intracellular parasites in their vertebrate host (Omori et al, 2010;Chakarov et al, 2012).…”

“…The size of the cell can be interpreted as the intensity of the forward scattered light (FSC), while the granularity or complexity of the cell is obtained from the 90° side scatter (SSC). Infected cells are selected based on their morphological characteristics or by using uorescent markers to signal parasite nucleic acids (Omori et al, 2010;Chakarov et al, 2012). The aim of this study was to standardise a methodology that allows DNA enrichment of the Haemoproteus columbae parasite from infected Columba livia blood samples.…”

Apparent vector‐mediated parent‐to‐offspring transmission in an avian malaria‐like parasite

“…, ; Chakarov et al . ), making it possible to obtain nuclear genomic parasite sequences from which rapidly evolving markers such as microsatellites may be developed. Microsatellites are particularly useful due to their high levels of polymorphism and can be successfully PCR amplified from blood extracts even when parasite DNA concentrations are low.…”

“…However, with only a tiny percentage of nucleated cells being infected (<5%) and the parasite genome being approximately 150 times smaller than that of the host (approximately 20 Mbp versus approximately 3 Gbp, respectively), shotgun sequencing of blood samples would be expected to generate negligible amounts of parasite sequence data. Fortunately, recent developments allow the enrichment of parasite cells (Henry & Dick 1978;Omori et al 2010;Palinauskas et al 2010Palinauskas et al , 2013Chakarov et al 2012), making it possible to obtain nuclear genomic parasite sequences from which rapidly evolving markers such as microsatellites may be developed. Microsatellites are particularly useful due to their high levels of polymorphism and can be successfully PCR amplified from blood extracts even when parasite DNA concentrations are low.…”