Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite Vaidas Palinauskas ⇑ , Rita Z ˇ iegyte ˙ , Tatjana A. Iezhova, Mikas Ilg  unas, Rasa Bernotiene ˙ , Gediminas Valki  unas Nature Research Centre, Akademijos 2, LT-08412 Vilnius, Lithuania article info Article history: Received 15 March 2016 Received in revised form 9 May 2016 Accepted 17 May 2016 Available online 25 June 2016 Keywords: Avian haemosporidian Plasmodium elongatum pERIRUB01 Experimental infection Virulence Syncytium abstract Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and mor- phological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in widespread Culex quinquefasciatus and Culex pipiens pipiens form molestus mosquitoes. This experimental study provides information on virulence and within species lineage diversity in a single pathogenic species of haemosporidian parasite. Ó 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved. 1. Introduction Avian malaria parasites (Plasmodiidae, Haemosporida) are broadly distributed all over the world (Garnham, 1966; Valki  unas, 2005). More than 50 avian malaria parasite species have been described. They have different life history traits and specifici- ties to the vertebrate hosts and vectors. Some species of avian malaria are specialists and infect birds of one species or genus, but some of them are generalists and are able to infect broad ranges of avian hosts (Waldenström et al., 2002; Valki  unas, 2005; Ishtiaq et al., 2007; Beadell et al., 2009; Dimitrov et al., 2010). One of the most pathogenic avian malaria agents is Plasmodium elongatum. This species was first described more than 80 years ago and attributed to the subgenus Huffia (Garnham, 1966). Since then P. elongatum has been recorded by many authors on all continents (except Antarctica) in birds of several orders (Anseriformes, Falconiformes, Columbiformes, Sphenisciformes, Strigiformes, Passeriformes and some others) (Fleischman et al., 1968; Nayar et al., 1998; Valki  unas, 2005; Beadell et al., 2009; Dimitrov et al., 2010; Baillie and Brunton, 2011; Howe et al., 2012; Clark et al., 2014; Vanstreels et al., 2014). According to the MalAvi database this is the most generalist species among avian malaria agents after Plasmodium relictum which infects more than 300 bird species of 10 orders. Plasmodium elongatum is of particular importance due to its pathogenicity in both wild and captive birds (Atkinson et al., http://dx.doi.org/10.1016/j.ijpara.2016.05.005 0020-7519/Ó 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Fax: +370 5 2729352. E-mail addresses: vaidas@ekoi.lt, palinauskas@gmail.com (V. Palinauskas). International Journal for Parasitology 46 (2016) 697–707 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara