European Journal of Wildlife Research

, Volume 58, Issue 1, pp 335–344 | Cite as

Haemosporidian infections in skylarks (Alauda arvensis): a comparative PCR-based and microscopy study on the parasite diversity and prevalence in southern Italy and the Netherlands

  • Pavel Zehtindjiev
  • Asta Križanauskienė
  • Sergio Scebba
  • Dimitar Dimitrov
  • Gediminas Valkiūnas
  • Arne Hegemann
  • B. Irene Tieleman
  • Staffan Bensch
Original Paper


Changes in agricultural management have been identified as the most probable cause for the decline of Skylark (Alauda arvensis) populations in Europe. However, parasitic infections have not been considered as a possible factor influencing this process. Four hundred and thirty-four Skylarks from the Southern Italy and the Netherlands were screened for haemosporidian parasites (Haemosporida) using the microscopy and polymerase chain reaction (PCR)-based methods. The overall prevalence of infection was 19.5%; it was 41.8% in Italian birds and 8.3% in Dutch birds. The prevalence of Plasmodium spp. was 34.1% and 6.5% in Skylarks from Italy and Netherlands, respectively. Approximately 15% of all recorded haemosporidian infections were simultaneous infections both in Italian and Dutch populations. Six different mitochondrial cytochrome b (cyt b) lineages of Plasmodium spp. and three lineages of Haemoproteus tartakovskyi were found. The lineage SGS1 of Plasmodium relictum was the most prevalent at both study sites; it was recorded in 24.7% of birds in Italy and 5.5% in the Netherlands. The lineages SYAT05 of Plasmodium vaughani and GRW11 of P. relictum were also identified with a prevalence of <2% at both study sites. Two Plasmodium spp. lineages (SW2 and DELURB4) and three H. tartakovskyi lineages have been found only in Skylarks from Italy. Mitochondrial cyt b lineages SYAT05 are suggested for molecular identification of P. vaughani, a cosmopolitan malaria parasite of birds. This study reports the greatest overall prevalence of malaria infection in Skylarks during the last 100 years and shows that both Plasmodium and Haemoproteus spp. haemosporidian infections are expanding in Skylarks so it might contribute to a decrease of these bird populations in Europe.


Avian malaria Haemoproteus Plasmodium Microscopy PCR Mitochondrial DNA 



We thank A. Warren, the Natural History Museum, London, U.K. for providing the type and voucher material of P. vaughani. The authors are grateful to Vaidas Palinauskas for the help with images of parasites. Tatjana A. Iezhova is gratefully acknowledged for assistance during identification of parasites. Thanks are due to Najden Chukerov for participation during field studies. This study was partly funded by FP7 Capacities project WETLANET (PZ). The field work was supported by a grant from the Associazione dei Migratoristi Italiani per la Conservazione dell’Ambiente Naturale (ANUU). Laboratory work was supported by the Swedish Research Council (SB). The investigations described herein comply with the current laws of Italy and Netherlands.


  1. Atkinson CT, van Riper IIIC (1991) Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon, and Haemoproteus. In: Loye JE, Zuk M (eds) ird-parasite interactions: Ecology, evolution, and behaviour. Oxford University Press, Oxford, U.K., pp 19–48Google Scholar
  2. Atkinson CT, Lease JK, Drake BM, Shema NP (2001) Pathogenicity, serological responses, and diagnosis of experimental and natural malarial infections in native Hawaiian thrushes. Condor 103:209–218CrossRefGoogle Scholar
  3. Bennett GF, Whiteway M, Woodworth-Lynas C (1982) A host-parasite catalogue of the avian haematozoa. Occas Pap Biol, Memorial University of Newfoundland 5:1–243Google Scholar
  4. Bensch S, Stjenman M, Hasselquist D, Östman Ö, Hansson B, Westerdahl H, Torres-Pinheiro R (2000) Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. P Roy Soc Lond B Bio 276:1583–1589. doi: 10.1098/rspb.2000.1181 CrossRefGoogle Scholar
  5. Bensch S, Hellgren O, Pérez-Tris J (2009) MalAvi: a public database of malaria parasites and related pigment-forming haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Mol Ecol Resour 9:1353–1358. doi: 10.1111/j.1755-0998.2009.02692.x PubMedCrossRefGoogle Scholar
  6. BirdLife International (2004) Birds in Europe: population estimates, trends and conservation status. Cambridge, UK: BirdLife International. (BirdLife conservation series No. 12)Google Scholar
  7. Bishop MA, Bennett GF (1992) Host-parasite catalogue of the avian haematozoa: supplement 1, and bibliography of the avian blood-inhabiting haematozoa: supplement 2. Occas Pap Biol, Memorial University of Newfoundland 15:1–244Google Scholar
  8. Burtikashvili LP (1978) Blood parasites of wild birds in Georgia. Tbilisi, Metsniereba (in Russian)Google Scholar
  9. Chavatte JM, Gres V, Snounou G, Chabaud A, Landau I (2009) Plasmodium (Apicomplexa) of the skylark (Alauda arvensis). Zoosystema 31:369–383CrossRefGoogle Scholar
  10. Corradetti A, Scanga M (1973) The Plasmodium vaughani—complex. Exp Parasitol 34:344–349PubMedCrossRefGoogle Scholar
  11. Cramp S (1988) The birds of the Western Palearctic, vol 5. Oxford University Press, OxfordGoogle Scholar
  12. Donald PF (2004) The skylark. T&AD Poyser, LondonGoogle Scholar
  13. Donald PF, Sanderson FJ, Burfield IJ, van Bommel FPJ (2006) Further evidence of continent-wide impacts of agriculture intensification on European farmland birds, 1990–2000. Agr Ecosyst Environ 116:189–196. doi: 10.1016/j.agee.2006.02.007 CrossRefGoogle Scholar
  14. Garnham PCC (1966) Malaria parasites and other Haemosporidia. Blackwell, OxfordGoogle Scholar
  15. Godfrey RD, Fedynich AM, Pence DB (1987) Quantification of hematozoa in blood smears. J Wildlife Dis 23:558–565Google Scholar
  16. Greiner EC, Bennett GF, White EM, Coombs RF (1975) Distribution of the avian hematozoa of North America. Can J Zool 53:1762–1787PubMedCrossRefGoogle Scholar
  17. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor v. 5.0.9. Nucl Acid S 41:95–98Google Scholar
  18. Hellgren O, Waldenström J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon spp., Plasmodium spp. and Haemoproteus spp. from avian blood. J Parasitol 90:797–802PubMedCrossRefGoogle Scholar
  19. Hellgren O, Waldenström J, Pérez-Tris J, Szöll ÖE, Hasselquist D, Križanauskienė A, Ottosson U, Bensch S (2007) Detecting shifts of transmission area in avian blood parasites—a phylogenetic approach. Mol Ecol 16:1281–1290. doi: 10.1111/j.1365-294X.2007.03227.x PubMedCrossRefGoogle Scholar
  20. Knowles SCL, Palinauskas V, Sheldon BC (2010) Chronic malaria infections increase family inequalities and reduce parental fitness: experimental evidence from a wild bird population. J Evol Biol 23:557–569PubMedCrossRefGoogle Scholar
  21. Križanauskienė A, Hellgren O, Kosarev V, Sokolov LV, 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
  22. Križanauskienė A, Pérez-Tris J, Palinauskas V, Hellgren O, Bensch S, Valkiūnas G (2010) Molecular phylogenetic and morphological analysis of haemosporidian parasites (Haemosporida) in a naturally infected European songbird, the blackcap Sylvia atricapilla, with description of Haemoproteus pallidulus sp. nov. Parasitology 137:217–227PubMedCrossRefGoogle Scholar
  23. Krone O, Waldenström J, Valkiūnas G, Lessow O, Muller K, Iezhova TA, Fickel J, Bensch S (2008) Haemosporidian blood parasites in European birds of prey and owls. J Parasitol 94:709–715. doi: 10.1645/GE-1357.1 PubMedGoogle Scholar
  24. Levin II, Outlaw DC, Vargas FH, Parker PG (2009) Plasmodium blood parasite found in endangered galapagos penguins (Spheniscus mendiculus). Biol Conserv 142:3191–3195. doi: 10.1016/j.biocon.2009.06.017 CrossRefGoogle Scholar
  25. 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. Parasitol 136:713–722. doi: 10.1017/S0031182009006118 CrossRefGoogle Scholar
  26. 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. doi: 10.1016/j.ympev.2007.11.012 PubMedCrossRefGoogle Scholar
  27. McClure HE, Poonswad P, Greiner EC, Laird M (1978) Haematozoa in the birds of Eastern and Southern Asia. Memorial University of Newfoundland, St. John’s, p 296Google Scholar
  28. Newton I (2004) The recent declines of farmland bird populations in Britain: an appraisal of causal factors and conservation actions. IBIS 146:579–600. doi: 10.1111/j.1474-919X.2004.00375.x CrossRefGoogle Scholar
  29. Nylander JAA (2004) MrModeltest v2. Evolutionary Biology Centre, Uppsala, Program distributed by the authorGoogle Scholar
  30. Olias P, Wegelin M, Freter S, Gruber AD, Klopfleischer R (2011) Avian malaria deaths in parrots, Europe. Emerg Infect Dis 17:950–952PubMedGoogle Scholar
  31. 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
  32. Palinauskas V, Valkiūnas G, Križanauskienė A, Bensch S, Bolshakov CV (2009) Plasmodium relictum (lineage P-SGS1): further observation of effects on experimentally infected passeriform birds, with remarks on the treatment with MalaroneTM. Exp Parasitol 123:134–139PubMedCrossRefGoogle Scholar
  33. Parker PG, Whiteman NK, Miller E (2006) Conservation medicine on Galápagos Islands: partnership among behavioral, population, and veterinary scientists. Auk 123:625–638CrossRefGoogle Scholar
  34. Peirce MA (1981) Distribution and host-parasite check-list of the haematozoa of birds in Western Europe. J Nat Hist 15:419–458CrossRefGoogle Scholar
  35. Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574. doi: 10.1093/bioinformatics/btg180 PubMedCrossRefGoogle Scholar
  36. Sambrook J, Fritch FJ, Maniatis T (2002) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYorkGoogle Scholar
  37. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092 PubMedCrossRefGoogle Scholar
  38. Valkiūnas G (1986) Haemoproteus tartakovskyi sp. n. (Haemosporidia, Haemoproteidae) from crossbill. Parasitologia 20:307–310, In RussianGoogle Scholar
  39. Valkiūnas G (2005) Avian malaria parasites and other haemosporidia. CRC Press, Boca Raton, 934 pGoogle Scholar
  40. Valkiūnas G (2011) Haemosporidian vector research: marriage of molecular and microscopical approaches is essential. Mol Ecol 20:3084–3086PubMedCrossRefGoogle Scholar
  41. Valkiūnas G, Liutkevičius G (2002) Complete development of three species of Haemoproteus (Haemosporida, Haemoproteidae) in the biting midge Culicoides impunctatus (Diptera, Ceratopogonidae). J Parasitol 88:864–868. doi: 10.1645/0022-3395(2002)088[0864:CDOTSO]2.0.CO;2
  42. Valkiūnas G, Iezhova TA, Shapoval AP (2003) High prevalence of blood parasites in hawfinch Coccothraustes coccothraustes. J Nat Hist 37:2647–2652CrossRefGoogle Scholar
  43. 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 92:418–422. doi: 10.1645/GE-3547RN.1 PubMedCrossRefGoogle Scholar
  44. Valkiūnas G, Atkinson CT, Bensch S, Sehgal RNM, Ricklefs RE (2008a) Parasite misidentifications in GenBank: how to minimize their number? Tends Parasitol 24:247–248CrossRefGoogle Scholar
  45. Valkiūnas G, Iezhova TA, Križanauskienė A, Palinauskas V, Bensch S (2008b) A comparative analysis of microscopy and PCR-based detection methods for blood parasites. J Parasitol 94:1395–1401PubMedCrossRefGoogle Scholar
  46. Waldenström J, Bensch S, Kiboi S, Hasselquist D, Ottosson U (2002) Cross-species infection of blood parasites between resident and migratory songbirds in Africa. Mol Ecol 11:1545–1554. doi: 10.1046/j.1365-294X.2002.01523.x PubMedCrossRefGoogle Scholar
  47. Waldenström J, Hasselquist D, Bensch S, Östman Ö (2004) A new nested polymerase chain reaction method very efficient in detecting Haemoproteus and Plasmodium infections from avian blood. J Parasitol 90:191–194. doi: 10.1645/GE-3221RN PubMedCrossRefGoogle Scholar
  48. Yakunin MP, Zhazyltaev TA (1977) The blood parasite fauna of wild and domestic birds from Kazakhstan Trudi Instituta Zoooogii. Akademii Nauk Kazakskoi SSR 37:124–148 (in Russian)Google Scholar
  49. Zehtindjiev P, Ilieva M, Westerdahl H, Hansson B, Valkiūnas G, Bensch S (2008) Dynamics of parasitemia of malaria parasites in a naturally and experimentally infected migratory songbird, the great reed warbler Acrocephalus arundinaceus. Exp Parasitol 119:99–110. doi: 10.1016/j.exppara.2007.12.018 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Pavel Zehtindjiev
    • 1
  • Asta Križanauskienė
    • 2
  • Sergio Scebba
    • 3
  • Dimitar Dimitrov
    • 1
  • Gediminas Valkiūnas
    • 2
  • Arne Hegemann
    • 4
  • B. Irene Tieleman
    • 4
  • Staffan Bensch
    • 5
  1. 1.Institute of Biodiversity and Ecosystem ResearchBulgarian Academy of SciencesSofiaBulgaria
  2. 2.Nature Research CentreVilniusLithuania
  3. 3.Gruppo Inanellamento LimicoliPozzuoliItaly
  4. 4.Animal Ecology Group, Centre for Ecological and Evolutionary StudiesUniversity of GroningenHarenThe Netherlands
  5. 5.Department of EcologyLund UniversityLundSweden

Personalised recommendations