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.

Keywords: Haemoproteus; Leucocytozoon; Mixed infection; Parasite detection; Plasmodium.

Fig. 1

Haemoproteus micronuclearis (RBQ11) from a Forest Fody (Foudia omissa, Ploceidae) sampled in the Maromizaha rainforest, Madagascar. Macrogametocyte in erythrocyte. Malaria pigment (hemozoin) is marked by a black arrow. Nucleus of host cell is marked by a white arrowhead. Giemsa stained blood smear. Scale bar = 10 µm

Fig. 2

Haemoproteus sanguinis (BUL2) from a Madagascar Bulbul (Hypsipetes madagascariensis, Pycnonotidae) sampled in the Maromizaha rainforest, Madagascar. Macrogametocyte in erythrocyte. Malaria pigment (hemozoin) is marked by a black arrow. Nucleus of host cell is marked by a white arrowhead. Giemsa stained blood smear. Scale bar = 10 µm

Fig. 3

Haemosporidian parasites detected in Giemsa stained blood smears of Malagasy birds. A Gametocytes of Plasmodium BUL07 in erythrocytes of Hypsipetes madagascariensis. B Microgametocyte (black arrowhead) and macrogametocyte (white arrowhead) of Haemoproteus FOUMAD02 in erythrocytes of Foudia omissa. C Gametocyte of Leucocytozoon FOMAD01 in roundish host cell of H. madagascariensis. D Gametocyte of Leucocytozoon HYPMA02 in roundish host cell of F. omissa. Scale bar = 10 µm

Fig. 4

Comparison of the percentage of individuals infected by haemosporidian parasites detected (P: Plasmodium, H: Haemoproteus, L: Leucocytozoon, neg: negative) detected using microscopy, multiplex PCR (Ciloglu et al. 2019), and nested PCR method (Bensch et al. ; Hellgren et al. 2004). The total number of individuals screened in this analysis was 147 (H. madagascariensis: n = 38; Foudia spp.: n = 109)

Fig. 5

Evolutionary relationship among haemosporidian cytochrome b lineages estimated using maximum likelihood approach implemented in MEGA. Bootstrap support is shown above branches. Bootstrap values below 70% are not reported. The blue branch represents Leucocytozoon, the orange branch Haemoproteus, and the green one Plasmodium lineages. Newly detected lineages are marked with an asterisk. Squares to the right of lineage names indicate the host species in which the lineage was recovered (H: Hypsipetes madagascariensis, Fo: Foudia omissa, Fm: F. madagascariensis, Hy: Foudia sp.). Numbers within the squares indicate the number of host individuals infected

Fig. 6

Number of Plasmodium and Haemoproteus detections in samples of H. madagascariensis (n = 113) and Foudia spp. (n = 301) using multiplex PCR (Ciloglu et al. 2019) and nested PCR method (Bensch et al. ; Hellgren et al. 2004) and combination of results