Parasitology Research

, Volume 118, Issue 1, pp 191–201 | Cite as

A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites

  • Arif CilogluEmail author
  • Vincenzo A. Ellis
  • Rasa Bernotienė
  • Gediminas Valkiūnas
  • Staffan Bensch
Genetics, Evolution, and Phylogeny - Original Paper


Accurate detection and identification are essential components for epidemiological, ecological, and evolutionary surveys of avian haemosporidian parasites. Microscopy has been used for more than 100 years to detect and identify these parasites; however, this technique requires considerable training and high-level expertise. Several PCR methods with highly sensitive and specific detection capabilities have now been developed in addition to microscopic examination. However, recent studies have shown that these molecular protocols are insufficient at detecting mixed infections of different haemosporidian parasite species and genetic lineages. In this study, we developed a simple, sensitive, and specific multiplex PCR assay for simultaneous detection and discrimination of parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon in single and mixed infections. Relative quantification of parasite DNA using qPCR showed that the multiplex PCR can amplify parasite DNA ranging in concentration over several orders of magnitude. The detection specificity and sensitivity of this new multiplex PCR assay were also tested in two different laboratories using previously screened natural single and mixed infections. These findings show that the multiplex PCR designed here is highly effective at identifying both single and mixed infections from all three genera of avian haemosporidian parasites. We predict that this one-step multiplex PCR assay, being convenient and inexpensive, will become a widely used method for molecular screening of avian haemosporidian parasites.


Plasmodium Haemoproteus Leucocytozoon Parasite detection Mixed infection Multiplex PCR 



We thank Xi Huang for assisting with quantitative PCR analyses in the laboratory and Tatjana A. Iezhova for microscopic identification of parasites.

Funding information

This work was supported by the Swedish Research Council (grant 621-2013-4839 to SB) and also partly funded by the Research Council of Lithuania (grant S-MIP-17-27 to RB). VAE was supported by a postdoctoral fellowship from the Carl Tryggers Foundation.

Compliance with ethical standards

Permission for taking blood samples from birds in Sweden was approved by the Malmö/Lund Committee for Animal Experiment Ethics (M45-14). Procedures with birds in Lithuania were performed by licensed researchers and were approved by the Ethical Commission of the Baltic Laboratory Animal Science Association, Lithuania; Lithuanian State Food and Veterinary Office and Environmental Protection Agency, Vilnius.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

436_2018_6153_MOESM1_ESM.docx (14 kb)
Supplementary Table 1 (DOCX 14 kb)
436_2018_6153_MOESM2_ESM.pdf (151 kb)
Supplementary Figure 1 (PDF 151 kb)


  1. Altshuler ML (2006) PCR troubleshooting: the essential guide. Caister Academic Press, NorfolkGoogle Scholar
  2. Asghar M, Hasselquist D, Bensch S (2011) Are chronic avian haemosporidian infections costly in wild birds? J Avian Biol 42:530–537. CrossRefGoogle Scholar
  3. Beadell JS, Gering E, Austin J, Dumbacher JP, Peirce MA, Pratt TK, Atkinson CT, Fleischer RC (2004) Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region. Mol Ecol 13:3829–3844. CrossRefGoogle Scholar
  4. Bensch S, Stjernman M, Hasselquist D, Östman Ö, 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 B Biol Sci 267:1583–1589. CrossRefGoogle Scholar
  5. Bensch S, Hellgren O, Pérez-Tris J (2009) MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Mol Ecol Resour 9:1353–1358. CrossRefGoogle Scholar
  6. Bensch S, Hellgren O, Križanauskienė A, Palinauskas V, Valkiūnas G, Outlaw D, Ricklefs RE (2013) How can we determine the molecular clock of malaria parasites? Trends Parasitol 29:363–369. CrossRefGoogle Scholar
  7. Bensch S, Canbäck B, DeBarry JD, Johansson T, Hellgren O, Kissinger JC, Palinauskas V, Videvall E, Valkiūnas G (2016) The genome of Haemoproteus tartakovskyi and its relationship to human malaria parasites. Genome Biol Evol 8:1361–1373. CrossRefGoogle Scholar
  8. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2005) GenBank. Nucleic Acids Res 33:D34–D38. CrossRefGoogle Scholar
  9. Bernotienė R, Palinauskas V, Iezhova T, Murauskaitė D, Valkiūnas G (2016) Avian haemosporidian parasites (Haemosporida): a comparative analysis of different polymerase chain reaction assays in detection of mixed infections. Exp Parasitol 163:31–37. CrossRefGoogle Scholar
  10. Cannell BL, Krasnec KV, Campbell K, Jones HI, Miller RD, Stephens N (2013) The pathology and pathogenicity of a novel Haemoproteus spp. infection in wild little penguins (Eudyptula minor). Vet Parasitol 197:74–84. CrossRefGoogle Scholar
  11. Ciloglu A, Yildirim A, Duzlu O, Onder Z, Dogan Z, Inci A (2016) Investigation of avian haemosporidian parasites from raptor birds in Turkey, with molecular characterisation and microscopic confirmation. Folia Parasitol 63:023. CrossRefGoogle Scholar
  12. Clark NJ, Clegg SM, Lima MR (2014) A review of global diversity in avian haemosporidians (Plasmodium and Haemoproteus: Haemosporida): new insights from molecular data. Int J Parasitol 44:329–338. CrossRefGoogle Scholar
  13. Dieffenbach CW, Lowe TM, Dveksler GS (1993) General concepts for PCR primer design. Genome Res 3:S30–S37. CrossRefGoogle Scholar
  14. Dimitrov D, Valkiūnas G, Zehtindjiev P, Ilieva M, Bensch S (2013) Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacilla flava), with description of in vitro ookinetes of Haemoproteus motacillae. Zootaxa 3666:369–381. CrossRefGoogle Scholar
  15. Ellis VA, Kunkel MR, Ricklefs RE (2014) The ecology of host immune responses to chronic avian haemosporidian infection. Oecologia 176:729–737. CrossRefGoogle Scholar
  16. 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 95:8124–8129. CrossRefGoogle Scholar
  17. Fallon SM, Ricklefs RE (2008) Parasitemia in PCR-detected Plasmodium and Haemoproteus infections in birds. J Avian Biol 39:514–522. CrossRefGoogle Scholar
  18. Fallon SM, Ricklefs RE, Swanson BL, Bermingham E (2003) Detecting avian malaria: an improved polymerase chain reaction diagnostic. J Parasitol 89:1044–1047. CrossRefGoogle Scholar
  19. Feldman RA, Freed LA, Cann RL (1995) A PCR test for avian malaria in Hawaiian birds. Mol Ecol 4:663–674. CrossRefGoogle Scholar
  20. Freund D, Wheeler SS, Townsend AK, Boyce WM, Ernest HB, Cicero C, Sehgal RNM (2016) Genetic sequence data reveals widespread sharing of Leucocytozoon lineages in corvids. Parasitol Res 115:3557–3565. CrossRefGoogle Scholar
  21. Garamszegi LZ (2010) The sensitivity of microscopy and PCR-based detection methods affecting estimates of prevalence of blood parasites in birds. J Parasitol 96:1197–1203. CrossRefGoogle Scholar
  22. Hellgren O, Waldenström J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol 90:797–802. CrossRefGoogle Scholar
  23. Huang X, Hansson R, Palinauskas V, Valkiūnas G, Hellgren O, Bensch S (2018) The success of sequence capture in relation to phylogenetic distance from a reference genome: a case study of avian haemosporidian parasites. Int J Parasitol 48:947–954. CrossRefGoogle Scholar
  24. Ishtiaq F, Rao M, Huang X, Bensch S (2017) Estimating prevalence of avian haemosporidians in natural populations: a comparative study on screening protocols. Parasit Vectors 10:127. CrossRefGoogle Scholar
  25. Ivanova K, Zehtindjiev P, Mariaux J, Dimitrov D, Georgiev BB (2018) Avian haemosporidians from rain forests in Madagascar: molecular and morphological data of the genera Plasmodium, Haemoproteus and Leucocytozoon. Infect Genet Evol 58:115–124. CrossRefGoogle Scholar
  26. Karadjian G, Hassanin A, Saintpierre B, Gembu Tungaluna GC, Ariey F, Ayala FJ, Landau I, Duval L (2016) Highly rearranged mitochondrial genome in Nycteria parasites (Haemosporidia) from bats. Proc Natl Acad Sci U S A 113:9834–9839. CrossRefGoogle Scholar
  27. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. CrossRefGoogle Scholar
  28. Levin II, Valkiūnas G, Iezhova TA, O’Brien SL, Parker PG (2012) Novel Haemoproteus species (Haemosporida: Haemoproteidae) from the swallow-tailed gull (Lariidae), with remarks on the host range of hippoboscid-transmitted avian hemoproteids. J Parasitol 98:847–854. CrossRefGoogle Scholar
  29. Levin II, Zwiers P, Deem SL, Geest EA, Higashiguchi JM, Iezhova TA, Jiménez-Uzcátegui G, Kim DH, Morton JP, Perlut NG, Renfrew RB, Sari EH, Valkiūnas G, Parker PG (2013) Multiple lineages of avian malaria parasites (Plasmodium) in the Galapagos Islands and evidence for arrival via migratory birds. Conserv Biol 27:1366–1377. CrossRefGoogle Scholar
  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. CrossRefGoogle Scholar
  31. Lotta IA, Pacheco MA, Escalante AA, González AD, Mantilla JS, Moncada LI, Adler PH, Matta NE (2016) Leucocytozoon diversity and possible vectors in the Neotropical highlands of Colombia. Protist 167:185–204. CrossRefGoogle Scholar
  32. Lutz HL, Hochachka WM, Engel JI, Bell JA, Tkach VV, Bates JM, Hackett SJ, Weckstein JD (2015) Parasite prevalence corresponds to host life history in a diverse assemblage of afrotropical birds and haemosporidian parasites. PLoS One 10:e0121254. CrossRefGoogle Scholar
  33. Martínez J, Martínez-De La Puente J, Herrero J, Del Cerro S, Lobato E, Rivero-DE Aguilar J, Vásquez RA, Merino S (2009) A restriction site to differentiate Plasmodium and Haemoproteus infections in birds: on the inefficiency of general primers for detection of mixed infections. Parasitology 136:713–722. CrossRefGoogle Scholar
  34. Martinsen ES, Perkins SL, Schall JJ (2008) A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): evolution of life-history traits and host switches. Mol Phylogenet Evol 47:261–273. CrossRefGoogle Scholar
  35. Marzal A, Bensch S, Reviriego M, Balbontin J, De Lope F (2008) Effects of malaria double infection in birds: one plus one is not two. J Evol Biol 21:979–987. CrossRefGoogle Scholar
  36. Mata VA, da Silva LP, Lopes RJ, Drovetski SV (2015) The Strait of Gibraltar poses an effective barrier to host-specialised but not to host-generalised lineages of avian Haemosporidia. Int J Parasitol 45:711–719. CrossRefGoogle Scholar
  37. Matthews AE, Ellis VA, Hanson AA, Roberts JR, Ricklefs RE, Collins MD (2016) Avian haemosporidian prevalence and its relationship to host life histories in eastern Tennessee. J Ornithol 157:533–548. CrossRefGoogle Scholar
  38. Merino S, Moreno J, Sanz JJ, Arriero E (2000) Are avian blood parasites pathogenic in the wild? A medication experiment in blue tits (Parus caeruleus). Proc R Soc B Biol Sci 267:2507–2510CrossRefGoogle Scholar
  39. Mukhin A, Palinauskas V, Platonova E, Kobylkov D, Vakoliuk I, Valkiūnas G (2016) The strategy to survive primary malaria infection: an experimental study on behavioural changes in parasitized birds. PLoS One 11:e0159216. CrossRefGoogle Scholar
  40. Pacheco MA, Matta NE, Valkiūnas G, Parker PG, Mello B, Stanley CE Jr, Lentino M, Garcia-Amado MA, Cranfield M, Kosakovsky Pond SL, Escalante AA (2018a) Mode and rate of evolution of haemosporidian mitochondrial genomes: timing the radiation of avian parasites. Mol Biol Evol 35:383–403. CrossRefGoogle Scholar
  41. Pacheco MA, Cepeda AS, Bernotienė R, Lotta IA, Matta NE, Valkiūnas G, Escalante AA (2018b) Primers targeting mitochondrial genes of avian haemosporidians: PCR detection and differential DNA amplification of parasites belonging to different genera. Int J Parasitol 48:657–670. CrossRefGoogle Scholar
  42. Palinauskas V, Valkiūnas G, Bolshakov CV, Bensch S (2008) Plasmodium relictum (lineage P-SGS1): effects on experimentally infected passerine birds. Exp Parasitol 120:372–380. CrossRefGoogle Scholar
  43. Palinauskas V, Dolnik OV, Valkiūnas G, Bensch S (2010) Laser microdissection microscopy and single cell PCR of avian hemosporidians. J Parasitol 96:420–424. CrossRefGoogle Scholar
  44. Palinauskas V, Žiegytė R, Ilgūnas M, Iezhova TA, Bernotienė R, Bolshakov C, Valkiūnas G (2015) Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes. Int J Parasitol 45:51–62. CrossRefGoogle Scholar
  45. Pérez-Tris J, Bensch S (2005) Diagnosing genetically diverse avian malarial infections using mixed-sequence analysis and TA-cloning. Parasitology 131:15–23. CrossRefGoogle Scholar
  46. R Core Team (2017) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. URL Google Scholar
  47. Richard FA, Sehgal RNM, Jones HI, Smith TB (2002) A comparative analysis of PCR-based detection methods for avian malaria. J Parasitol 88:819–822. CrossRefGoogle Scholar
  48. Richardson DS, Jury FL, Blaakmeer K, Komdeur J, Burke T (2001) Parentage assignment and extra-group paternity in a cooperative breeder: the Seychelles warbler (Acrocephalus sechellensis). Mol Ecol 10:2263–2273. CrossRefGoogle Scholar
  49. Ricklefs RE, Fallon SM (2002) Diversification and host switching in avian malaria parasites. Proc R Soc B Biol Sci 269:885–892. CrossRefGoogle Scholar
  50. Ricklefs RE, Swanson BL, Fallon SM, Martínez-Abraín A, Scheuerlein A, Gray J, Latta SC (2005) Community relationships of avian malaria parasites in southern Missouri. Ecol Monogr 75:543–559. CrossRefGoogle Scholar
  51. Rozen S, Skaletsky H (2000) Primer3 on the www for general users and for biologist programmers. In: Bioinformatics methods and protocols. Humana Press, Totowa, NJ, pp 365–386Google Scholar
  52. Sehgal RNM (2015) Manifold habitat effects on the prevalence and diversity of avian blood parasites. Int J Parasitol Parasites Wildl 4:421–430. CrossRefGoogle Scholar
  53. Valkiūnas G (2005) Avian malaria parasites and other haemosporidia. CRC Press, Boca RatonGoogle Scholar
  54. Valkiūnas G, Iezhova TA, Shapoval AP (2003) High prevalence of blood parasites in hawfinch Coccothraustes coccothraustes. J Nat Hist 37:2647–2652. CrossRefGoogle Scholar
  55. Valkiūnas G, Bensch S, Iezhova TA, Križanauskienė A, Hellgren O, Bolshakov CV (2006) Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential. J Parasitol 92:418–422. CrossRefGoogle Scholar
  56. Valkiūnas G, Iezhova TA, Križanauskienė A, Palinauskas V, Sehgal RNM, Bensch S (2008) A comparative analysis of microscopy and PCR-based detection methods for blood parasites. J Parasitol 94:1395–1401CrossRefGoogle Scholar
  57. Valkiūnas G, Kazlauskienė R, Bernotienė R, Bukauskaitė D, Palinauskas V, Iezhova TA (2014a) Haemoproteus infections (Haemosporida, Haemoproteidae) kill bird-biting mosquitoes. Parasitol Res 113:1011–1018. CrossRefGoogle Scholar
  58. Valkiūnas G, Palinauskas V, Ilgūnas M, Bukauskaitė D, Dimitrov D, Bernotienė R, Zehtindjiev P, Ilieva M, Iezhova TA (2014b) Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife. Parasitol Res 113:2251–2263. CrossRefGoogle Scholar
  59. Valkiūnas G, Žiegytė R, Palinauskas V, Bernotienė R, Bukauskaitė D, Ilgūnas M, Dimitrov D, Iezhova TA (2015) Complete sporogony of Plasmodium relictum (lineage pGRW4) in mosquitoes Culex pipiens pipiens, with implications on avian malaria epidemiology. Parasitol Res 114:3075–3085. CrossRefGoogle Scholar
  60. van Riper C, van Riper SG, Goff ML, Laird M (1986) The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecol Monogr 56:327–344. CrossRefGoogle Scholar
  61. Waldenström J, Bensch S, Hasselquist D, Östman Ö (2004) A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. J Parasitol 90:191–194. CrossRefGoogle Scholar
  62. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinf 13:134. CrossRefGoogle Scholar
  63. Zehtindjiev P, Križanauskienė A, Bensch S, Palinauskas V, Asghar M, Dimitrov D, Scebba S, Valkiūnas G (2012) A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction–based protocols for amplification of the cytochrome b gene. J Parasitol 98:657–665. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Arif Ciloglu
    • 1
    • 2
    • 3
    Email author
  • Vincenzo A. Ellis
    • 2
  • Rasa Bernotienė
    • 4
  • Gediminas Valkiūnas
    • 4
  • Staffan Bensch
    • 2
  1. 1.Department of Parasitology, Faculty of Veterinary MedicineErciyes UniversityKayseriTurkey
  2. 2.Molecular Ecology and Evolution Laboratory, Department of BiologyLund UniversityLundSweden
  3. 3.Vectors and Vector-Borne Diseases Implementation and Research CenterErciyes UniversityKayseriTurkey
  4. 4.Nature Research CentreVilniusLithuania

Personalised recommendations