Untangling the actual infection status: detection of avian haemosporidian parasites of three Malagasy bird species using microscopy, multiplex PCR, and nested PCR methods

Abstract

The development of new molecular methods has significantly improved the detection and identification of avian haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) compared to microscopic examination. Very large numbers of previously hidden Haemosporida species of a wide range of avian hosts have thus been discovered in the last two decades. However, test parameters of the various detection methods remain largely unevaluated. In this study, the merits of microscopy, multiplex PCR, and nested PCR were compared to identify the infection status of three Malagasy bird species. A total of 414 blood samples of Hypsipetes madagascariensis, Foudia omissa and F. madagascariensis, as well as 147 blood smears, were examined for haemosporidian infection. Thirty-four lineages of haemosporidian parasites could be identified, of which six have been detected for the first time. Microscopy, multiplex and nested PCR showed differences in detection rate, most likely due to low parasitemia of chronically infected birds. The combination of both PCR methods yielded the best results. In particular, detection of multiple infections could be greatly improved and will enable more precise prevalence estimates of individual haemosporidian species in wild birds in the future.

Insights into the Biology of Leucocytozoon Species (Haemosporida, Leucocytozoidae): Why Is There Slow Research Progress on Agents of Leucocytozoonosis?

“…The application of CISH tools using parasite-specific probes opens new opportunities to link tissue stages found in wild birds with certain haemosporidian parasites [ 93 , 94 , 95 , 234 , 250 , 251 , 252 ]. This was difficult or impossible to do before the molecular era because co-infections of Apicomplexan parasites are common and even predominating in many avian hosts worldwide [ 4 , 7 , 9 , 10 , 23 , 38 , 40 , 42 , 58 , 105 , 110 , 118 , 253 , 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 , 265 , 266 , 267 , 268 , 269 , 270 , 271 , 272 , 273 , 274 ]. Recent molecular and microscopic studies show that the morphological diversity of haemosporidian exo-erythrocytic stages is unexpectedly diverse, particularly in Haemoproteus and Leucocytozoon parasites, which have historically been difficult or impossible to link to certain parasite species or lineages, even on genera levels, due to common co-infections […”

“…Second, the molecular characterization of Leucocytozoon species is often confused because of common co-infections of parasites belonging to the same genus [ 7 , 10 , 38 , 40 , 42 , 105 , 118 , 119 , 254 , 256 , 257 , 259 , 260 , 261 , 262 , 263 , 268 , 270 , 272 , 273 , 274 ]. Microscopic examination of blood films from many naturally infected birds shows that infection with a single species of Leucocytozoon is rare.…”

Haemosporidian parasite infections of Malagasy Philepittidae and Nectariniidae are driven by phylogeny rather than ecology

“…The absence of specialized haemosporidian lineages in the Philepittidae species is quite surprising. Among these lineages, lPHICAS01 appears to be the only one that might be specialized or even prefers to infect P. castanea , as it has been just sporadically detected in 2 other bird species (Musa et al ., 2022). Phylogenetic analysis revealed a very close relationship (with only 4 out of 479 base pairs differing) between lPHICAS01 and the generalist lineage lHYPMA02, which was also detected in both Philepittidae species.…”

Genotype–environment associations reveal genes potentially linked to avian malaria infection in populations of an endemic island bird

“…(2019) (samples collected in 2019 and onwards). Recent research into the comparative effectiveness of nested and multiplex PCR methods in diagnosing malaria suggests that they exhibit comparable sensitivity for Plasmodium and Haemoproteus (Ciloglu et al., 2019; Musa et al., 2022). However, the multiplex PCR assay outperforms the nested PCR in detecting co‐infections, involving more than one species or strain of pathogen.…”

“…based on mitochondrial genome sequences using either the nested PCR protocols described in Waldenström et al (2004) (samples collected prior to 2019) or the multiplex PCR assay described in Ciloglu et al (2019) (samples collected in 2019 and onwards). Recent research into the comparative effectiveness of nested and multiplex PCR methods in diagnosing malaria suggests that they exhibit comparable sensitivity for Plasmodium and Haemoproteus (Ciloglu et al, 2019;Musa et al, 2022).…”