Parasitology Research

, Volume 102, Issue 6, pp 1185–1193 | Cite as

Polymerase chain reaction-based identification of Plasmodium (Huffia) elongatum, with remarks on species identity of haemosporidian lineages deposited in GenBank

  • Gediminas Valkiūnas
  • Pavel Zehtindjiev
  • Dimitar Dimitrov
  • Asta Križanauskienė
  • Tatjana A. Iezhova
  • Staffan Bensch
Original Paper


Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.


Blood Film Blood Stage Blood Parasite Infected Bird Great Reed Warbler 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This article benefited from comments made by John R. Baker. The authors are grateful to Alan Warren, Natural History Museum, London for providing the type material of P. elongatum, and Vaidas Palinauskas for assistance during the preparation of Fig. 1. The study was supported in part by the Swedish Research Council, Carl Tryggers Foundation, the Lithuanian State Science and Studies Foundation, and SYNTHESYS. The experiments described herein comply with the current laws of Sweden, Bulgaria and Lithuania.


  1. Beadell JS, Ishtiaq F, Covas R, Melo M, Warren BH, Atkinson CT, Bensch S, Graves GR, Jhala YV, Peirce MA, Rahmani AR, Fonseca DM, Fleischer RC (2006) Global phylogeographic limits of Hawaii’s avian malaria. Proc R Soc Lond, B Biol Sci 273:2935–2944CrossRefGoogle Scholar
  2. Beier JC, Stoskopf MK (1980) The epidemiology of avian malaria in black-footed penguins (Spheniscus demersus). J Zoo Anim Med 11:99–105Google Scholar
  3. Bennett GF, Whiteway M, Woodworth-Lynas C (1982) A host–parasite catalogue of the avian haematozoa. Meml Univ Newfoundl Occas Pap Biol 5:1–243Google Scholar
  4. Bensch S, Stjernman M, Hasselquist D, Ostman O, Hansson B, Westerdahl H, Pinheiro RT (2000) Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proc R Soc London B 267:1583–1589CrossRefGoogle Scholar
  5. Bensch S, Pérez-Tris J, Waldensrtöm J, Hellgren O (2004) Linkage between nuclear and mitochondrial DNA sequences in avian malaria parasites: multiple cases of cryptic speciation? Evolution 58:1617–1621PubMedGoogle Scholar
  6. Bensch S, Waldenström J, Jonzén N, Westerdahl H, Hansson B, Sejberg D, Hasselquist D (2007) Temporal dynamics and diversity of avian malaria parasites in a single host species. J Anim Ecol 76:112–122PubMedCrossRefGoogle Scholar
  7. Cranfield MR, Shaw M, Beall F, Skjoldager M, Ialeggio D (1990) A review and update of avian malaria in the African penguin (Spheniscus demersus). Proc Am Ass Zoo Vet, 243–248Google Scholar
  8. Escalante AA, Freeland DE, Collins WE, Lal AA (1998) The evolution of primate malaria parasites based on the gene encoding cytochrome b from the linear mitochondrial genome. Proc Natl Acad Sci U S A 95:8124–8129PubMedCrossRefGoogle Scholar
  9. Fallon S, Bermingham E, Ricklefs E (2003) Island and taxon effects in parasitism revisited: avian malaria in the Lesser Antilles. Evolution 57:606–615PubMedGoogle Scholar
  10. Garnham PCC (1966) Malaria parasites and other Haemosporidia. Blackwell Scientific, OxfordGoogle Scholar
  11. Godfrey RD, Fedynich AM, Pence DB (1987) Quantification of hematozoa in blood smears. J Wildl Dis 23:558–565PubMedGoogle Scholar
  12. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. ver. 6.0.6. Nucleic Acids Symp Ser 41:95–98Google Scholar
  13. Hellgren O, Križanauskienė A, Valkiūnas G, Bensch S (2007) Diversity and phylogeny of mitochondrial cytochrome b lineages from six morphospecies of avian Haemoproteus (Haemosporida, Haemoproteidae). J Parasitol 93:889–896PubMedCrossRefGoogle Scholar
  14. Hellgren O, Waldenström J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol 90:797–802PubMedCrossRefGoogle Scholar
  15. Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755PubMedCrossRefGoogle Scholar
  16. Iezhova TA, Valkiūnas G, Barlein F (2005) Vertebrate host specificity of two avian malaria parasites of subgenus Novyella: Plasmodium nucleophilum and Plasmodium vaughani. J Parasitol 91:472–474PubMedCrossRefGoogle Scholar
  17. Križanauskienė A, Hellgren O, Kosarev V, Sokolov L, Bensch S, Valkiūnas G (2006) Variation in host specificity between species of avian haemosporidian parasites: evidence from parasite morphology and cytochrome b gene sequences. J Parasitol 92:1319–1324PubMedCrossRefGoogle Scholar
  18. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  19. Martinsen ES, Paperna I, Schall JJ (2006) Morphological versus molecular identification of avian Haemosporidia: an exploration of three species concepts. Parasitology 133:279–288PubMedCrossRefGoogle Scholar
  20. Martinsen ES, Waite JL, Schall JJ (2007) Morphologically defined subgenera of Plasmodium from avian hosts: test of monophyly by phylogenetic analysis of two mitochondrial genes. Parasitology 134:483–490PubMedCrossRefGoogle Scholar
  21. Nylander JAA (2004) mrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. Software available at: <>
  22. Palinauskas V, Kosarev V, Shapoval A, Bensch S, Valkiūnas G (2007) Comparison of mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites of the subgenera Haemamoeba and Giovannolaia (Haemosporida: Plasmodiidae). Zootaxa 1626:39–50Google Scholar
  23. Pérez-Tris J, Bensch S (2005) Diagnosing genetically diverse avian malaria infections using mixed-sequence analysis and TA-cloning. Parasitology 131:1–9CrossRefGoogle Scholar
  24. Perkins SL (2000) Species concepts and malaria parasites: detecting a cryptic species of Plasmodium. Proc R Soc Lond B 267:2345–2350CrossRefGoogle Scholar
  25. Perkins SL, Sarkar IN, Carter R (2007) The phylogeny of rodent malaria parasites: simultaneous analysis across three genomes. Infect Genet Evol 7:74–83PubMedCrossRefGoogle Scholar
  26. Rambaut A, Drummond A (2003) TRACER: a program for analysing results from Bayesian MCMC programs such as BEAST & MrBayes. Available at <>
  27. Ricklefs RE, Fallon SM, Bermingham E (2004) Evolutionary relationships, cospeciation, and host switching in avian malaria parasites. Syst Biol 53:111–119PubMedCrossRefGoogle Scholar
  28. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  29. Sambrook J, Fritch FJ, Maniatis T (2002) Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, NYGoogle Scholar
  30. Sehgal RNM, Hull AC, Anderson NL, Valkiūnas G, Markovets MJ, Kawamura S, Tell LA (2006) Evidence for cryptic speciation of Leucocytozoon spp. (Haemosporida, Leucocytozoidae) in diurnal raptors. J Parasitol 92:375–379PubMedCrossRefGoogle Scholar
  31. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  32. Valkiūnas G (2005) Avian malaria parasites and other haemosporidia. CRC, Boca RatonGoogle Scholar
  33. Valkiūnas G, Bensch S, Iezhova TA, Križanauskienė A, Hellgren O, Bolshakov CV (2006) Nested cytochrome b PCR diagnostics underestimate mixed infections of avian blood hemosporidian parasites: microscopy is still essential. J Parasitol 92:418–422PubMedCrossRefGoogle Scholar
  34. Valkiūnas G, Križanauskienė A, Iezhova TA, Hellgren O, Bensch S (2007a) Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida, Haemoproteidae) of sylviid birds, with description of Haemoproteus parabelopolskyi sp., nov. J Parasitol 93:680–687PubMedCrossRefGoogle Scholar
  35. Valkiūnas G, Zehtindjiev P, Hellgren O, Ilieva M, Iezhova TA, Bensch S (2007b) Linkage between mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites, with a description of Plasmodium (Novyella) ashfordi sp. nov. Parasitol Res 100:1311–1322PubMedCrossRefGoogle Scholar
  36. Wilgenbusch JC, Warren DL, Swofford DL (2004) AWTY: a system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. Available at

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Gediminas Valkiūnas
    • 1
  • Pavel Zehtindjiev
    • 2
  • Dimitar Dimitrov
    • 2
  • Asta Križanauskienė
    • 1
  • Tatjana A. Iezhova
    • 1
  • Staffan Bensch
    • 3
  1. 1.Institute of EcologyVilnius UniversityVilnius LTLithuania
  2. 2.Institute of ZoologyBulgarian Academy of SciencesSofiaBulgaria
  3. 3.Department of Animal Ecology, Ecology BuildingLund UniversityLundSweden

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