A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays

Abstract

Background Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories. Methods Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays. Results This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium , Haemoproteus and Leucocytozoon with minimum infection quantities of 10 −5 (i.e. one parasite copy in 10 5 host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence ( R 2 = 0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians. Conclusions We developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories.

Prevalence and genetic diversity of avian haemosporidian parasites in wild bird species of the order Columbiformes

“…Despite a rather high overall prevalence, the infection intensity (parasitemia) was in general rather low. Studies on parasitemia are still limited despite their importance (Huang et al 2020 ). The damage produced on the host species greatly depends on the infection intensity with the harmful effects being most pronounced when parasitemia is very high (Sol et al 2003 ).…”

Avian haemosporidian parasites of accipitriform raptors

“…Clade L202 features five lineages in four sub-clades. L202a contains lACCOP01, exclusively detected in Accipiter cooperi (11) in the USA [9]. L202b contains lAC-CGEN01 (11) and lACCGEN03 (1), exclusively found in Accipiter gentilis from Czechia [12] and Austria.…”

“…The MalAvi database currently features a collection of about 4,000 unique CytB lineages recorded in over 35,000 birds. Only 279 records originate from accipitriform raptors (Accipitriformes), two-thirds of which were published in the following eight publications: Pérez-Rodríguez et al [8], Sehgal et al [9], Jasper et al [10], Huang et al [11], Hanel et al [12], Poharkar et al [13], Krone et al [14], and Ciloglu et al [15]. Avian raptors are under-represented in haemosporidian studies because they have low population densities and are usually not caught in mist nets like many passeriform birds.…”

Assessment of Associations between Malaria Parasites and Avian Hosts—A Combination of Classic System and Modern Molecular Approach

“…Although qPCR has enhanced the quantification of infection intensities to a great extent, the results largely rely on laboratory-based standard samples, which may degrade over time. Due to the constraint of standard samples, a comparison of infection intensity assessed in different laboratories or across many years is still inaccessible [79].…”

“…Although just introduced to avian haemosporidian parasite studies, ddPCR has already exhibit strong advantages in absolute quantification of infection intensities independent of standard samples. With the identical reaction system (i.e., same samples and same primers), ddPCR represented higher sensitivity and repeatability than other molecular methods, especially when infection intensity was low [79].…”