Valkiūnas et al. Malar J (2018) 17:184 https://doi.org/10.1186/s12936-018-2325-2 Malaria Journal RESEARCH Open Access Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria Gediminas Valkiūnas1* , Mikas Ilgūnas1, Dovilė Bukauskaitė1, Karin Fragner2, Herbert Weissenböck2, Carter T. Atkinson3 and Tatjana A. Iezhova1 Abstract Background: Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confrmed this conclusion and identifed several mtDNA lineages, suggesting the existence of signifcant intra-species genetic variation or cryptic speciation. Most identifed lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of diferent lineages of this pathogen was reviewed, suggesting issues for future research. Methods: The new lineage pPHCOL01 was detected in Common chifchaf Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria. Organs of infected birds were examined using histology and chro- mogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra fnch Taeniopygia guttata, Budgeri- gar Melopsittacus undulatus and European goldfnch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identifed the same phylogenetic relationships among diferent, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was com- pared using fxed and stained blood smears, and biological properties of these parasites were reviewed. Results: Common canary and European goldfnch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra fnch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. Conclusion: Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, diferent lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop diferently in diferent bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in diferent isolates of the same lineage might be greater than the variation in diferent lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, feld observations, microscopic and molecular diagnostics is essential for understanding the role of diferent P. relictum lineages in bird health. Keywords: Plasmodium relictum, Birds, Morphological and molecular characterization, Review Background and even mortality during malaria infection [5, 6], but Plasmodium relictum is an invasive blood parasite, which some bird species and their populations appear to be causes malaria in many species of birds from all over the relatively resistant and can tolerate this infection [7–11]. word [1–4]. Naive birds often experience severe disease Tis parasite was the frst recognized and described agent of avian malaria [12], likely due to its high preva- lence in a wide range of diferent avian hosts and because *Correspondence: [email protected] morphological characteristics of its mature blood stages 1 Nature Research Centre, Akademijos 2, LT‑08412 Vilnius, Lithuania Full list of author information is available at the end of the article are so distinctive in blood flms. Mature stages typically © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Valkiūnas et al. Malar J (2018) 17:184 Page 2 of 21 possess prominent nuclei and cytoplasm, numerous pig- Twenty-three Common chifchafs Phylloscopus collybita ment granules and markedly infuence the position of the were caught with mist nets and large stationary traps. nuclei of their host erythrocytes, causing lateral shifts Te blood was taken by puncturing the brachial vein. in their position. Numerous synonymous names of this Tree blood flms were prepared immediately after with- organism exist [7, 13]. Tese names were suggested for drawal of the blood, air-dried using a battery-operated distinguishing the morphologically similar or even iden- fan, fxed in absolute methanol and stained with Giemsa. tical blood stages, which were reported in diferent avian About 30 μl of whole blood was taken in heparinized hosts and/or diferent geographical areas [13–15]. Micro- microcapillaries and stored in SET bufer (0.05 M Tris, scopic examination of blood flms, the main avian malaria 0.15 M NaCl, 0.5 M EDTA, pH 8.0) at ambient tempera- diagnostic tool used in the 20th Century, has identifed P. ture while in the feld and then maintained at − 20 °C in relictum as the most common agent of avian malaria with the laboratory. reports from over 300 species of birds belonging to 11 To detect and isolate the Plasmodium parasite strain, orders from all over the world [1, 7, 16, 17]. Recent molec- the blood flms from each captured bird were quickly ular studies have supported this conclusion and uncov- examined microscopically in the feld, as previously ered signifcant genetic diversity among diferent isolates described [35]. One naturally infected Common chif- of P. relictum, suggesting existence of intra-species genetic chaf was detected, with parasitaemia of 0.1%. Ten blood variation or even cryptic speciation [2, 18–22]. flms were prepared for microscopic examination from Partial sequences of mitochondrial cytochrome b gene this bird. Additionally, blood was also collected in hep- (cytb) have been successfully used in distinguishing difer- arinized microcapillaries and used to expose two unin- ent lineages of avian malaria parasites, and they are excellent fected domestic Common canaries Serinus canaria molecular markers for disease diagnostics [2, 4, 19, 23–25]. forma domestica by sub-inoculation into their pectoral Over 100 closely related cytb lineages of avian Plasmodium muscle of about 250 μl of a freshly prepared mixture of were deposited in GenBank and MalAvi database (http:// infected blood, 3.7% sodium citrate (anticoagulant) and mbio-serv2.mbioekol.lu.se/Malavi) and many of them 0.9% saline (4:1:5) [36]. may belong to P. relictum. However, few of these molecu- Parasitaemia developed in both exposed canaries, and lar lineages are supported by microscopic examination of blood of these birds was passaged as described above in well-fxed and stained blood smears and the small genetic three additional canaries. Two Zebra fnches Taeniopy- diference in cytb sequences alone cannot be considered as gia guttate, one Budgerigar Melopsittacus undulatus fnal proof that closely related lineages belong to the same and two European goldfnches Carduelis carduelis were morpho-species. For example, some morphologically dis- also exposed, as described above. Six uninfected canar- tinct haemosporidian species difer in their partial cytb ies were used as controls. Blood of all control and experi- sequences just by a few nucleotide bases [26, 27]. Currently, mental birds was tested by microscopic examination and only four lineages (pSGS1, pGRW4, pGRW11, pLZFUS01) PCR-based methods (see description below) twice before have been linked to P. relictum based on morphological the experiment to ensure that they were uninfected with characters of their blood stages, and these data are helpful malaria parasites. for distinguishing this infection in blood flms [28–30]. Two canaries were observed for 57 and 94 days post During the past 15 years, much data have been col- exposure (dpe) and then euthanized for histology and lected about host, geographical distribution, vectors, chromogenic in situ hybridization research. Two Euro- virulence, and other biological characters of P. relictum pean goldfnches were observed for 127 dpe. All remain- based on cytb lineages [2, 3, 8, 19, 30–34]. Tis provides ing birds were observed for 131 dpe. Post-exposure blood opportunities to examine patterns in the biology and samples were taken for microscopic examination and pathology of avian Plasmodium infection at the level of PCR-based testing as described above once every 4 days these specifc lineages. Tis study characterizes a new during the frst post-exposure month, once every week cytb lineage of P. relictum (pPHCOL01), makes com- during the second month and once every 1–2 weeks dur- prehensive comparisons of morphological characters of ing the remaining experiment time. All experimental and blood stages of all known lineages of this parasite, and control birds were kept indoors in a vector-free room reviews their biological features to help identify some under