Diagnosing genetically diverse avian malarial infections using mixed-sequence analysis and TA-cloning

Abstract

Birds harbouring several malarial parasites are common in the wild, and resolving such multiple infections is important for our understanding of host-parasite relationships. We propose a simple and reasonably accurate method for detecting and resolving multiple infections, based on the analysis of parasite cytochrome b DNA sequences: genetically mixed infections are first identified by double nucleotide peaks on sequence electropherograms, and later retrieved by TA-cloning. We applied this method to wild birds, and to experimentally created mixes with varying proportion of two parasites (Plasmodium spp. and Haemoproteus spp.). In general, the method was very efficient in detecting and resolving multiple infections, but some problems were encountered. Several multiple infections were erroneously scored as simple, either because one of the parasite lineages was a better target for the primers used, or because it was much more abundant in the mix. On the other hand, single nucleotide substitutions and template switching during PCR produced artificial sequences in some clones. We discuss the utility of the method, and propose a framework for its use when screening for genetically diverse avian malarial parasites.

High prevalence of Leucocytozoon parasites in fresh water breeding gulls

“…Representatives of all lineages were also sequenced with the reverse primer. Multiple infections were resolved by examining if the most common lineage could be part of the multiple infection and from this comparison we subtracted the other sequence from the sites with double base calling (Pérez-Tris and Bensch 2005).…”

The genetic structure of Borrelia afzelii varies with geographic but not ecological sampling scale

“…ospC and IGS alleles were named according to Bunikis et al (2004). Samples where sequences from any of the three loci had double peaks in the chromatogram at any nucleotide position were treated as multiple infections (Perez-Tris & Bensch, 2005) and excluded from further analyses. Mean genetic distance (P-distance) was calculated for the different loci using MEGA 4 (Tamura et al, 2007).…”

Genetic diversity of avian blood parasites in SE Europe: Cytochrome b lineages of the genera Plasmodium and Haemoproteus (Haemosporida) from Bulgaria

“…During the last ten years, the amplification of a specific part of the mitochondrial cytochrome b (cyt b) gene (Bensch et al 2000, Perkins and Schall 2002) has provided new opportunities in the studies of specificity, diversity, distribution, ecology and various aspects of phylogeny and evolution of avian haemosporidian parasites (Bensch et al 2000, Waldenström et al 2002, Beadell et al 2004, Fallon et al 2005. Recent papers have demonstrated that the diversity of cyt b lineages is much greater than that of the described morphospecies (Perkins and Schall 2002;Ricklefs and Fallon 2002;Waldenström et al 2002;Beadell et al 2004Beadell et al , 2006Bensch et al 2004;Pérez-Tris and Bensch 2005;Szymanski and Lovette 2005;Valkiūnas 2005). These studies suggest that many of the genetic lineages may represent distinct evolutionary entities .…”