A new real-time PCR protocol for detection of avian haemosporidians

Abstract

BackgroundBirds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction.MethodsUsing available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR).ResultsThe real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ2 = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples.ConclusionsOur real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.

Climate variation influences host specificity in avian malaria parasites

“…; Bell et al . ). As evidence indicates that avian malaria lineages differing by one cyt‐ b nucleotide may be reproductively isolated entities (Bensch et al .…”

“…The majority of observations came from field studies led by the authors from the period of 2005-2016, with remaining observations extracted from published studies that took place in the study region (see for details). Protocols detailing reactions, reagents, primer names, cycling conditions and how lineages were determined can be found in Bensch et al 2009;Bell et al 2015). As evidence indicates that avian malaria lineages differing by one cyt-b nucleotide may be reproductively isolated entities , we use the standard practice of referring to each unique cyt-b lineage as a unique parasite.…”

An inverse latitudinal gradient in infection probability and phylogenetic diversity for Leucocytozoon blood parasites in New World birds

“…DNA was extracted from bird tissues using the Qiagen DNeasy 96 Blood and Tissue kit (Qiagen), following the Qiagen tissue protocol for both blood and tissue stored in 95% ethanol. For most samples, the protocols of Bell, Weckstein, Fecchio, and Tkach () were followed to both initially screen samples for haemosporidian DNA with real‐time PCR and then to amplify a 477‐bp region of the Leucocytozoon cytochrome b gene (cyt‐ b ) from positive samples using nested PCR. Samples from Alaska and Minnesota were screened and amplified following the protocols of Hellgren, Waldenström, and Bensch (), which amplifies the same 477‐bp region of cyt‐ b .…”

“…Blood and Tissue kit (Qiagen), following the Qiagen tissue protocol for both blood and tissue stored in 95% ethanol. For most samples, the protocols of Bell, Weckstein, Fecchio, and Tkach (2015) were followed to both initially screen samples for haemosporid- (Hall, 1999). Given the evidence indicating that avian haemosporidian haplotypes differing by one cyt-b nucleotide may be reproductively isolated entities (Bensch, Pérez-Tris, Waldenström, & Hellgren, 2004), we followed the conventional practice of referring to each unique cyt-b haplotype as a unique parasite lineage.…”

Avian migration and the distribution of malaria parasites in New World passerine birds

“…Blood samples were brought, under the appropriate USFWS and APHIS permits, from the field to the laboratory, where DNA was extracted. We screened each DNA sample for the presence of Plasmodium or Haemoproteus with primers that amplify a 154 base pair segment of the mitochondrial 16S rRNA gene (Fallon et al, 2003;Bell et al, 2015). For all positive samples, we sequenced a c. 500 base pair portion of the cytochrome b gene to establish the lineage identity of each infection (see Ricklefs et al, 2005b;Svensson-Coelho et al, 2013).…”