Detection and molecular characterization of a Haemoproteus lineage in a Tawny Owl (Strix aluco) in Turkey

Abstract

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b (mt-cytb) gene of the avian haemosporidian species of Plasmodium, Haemoproteus, Fallisia and Leucocytozoon, offers a new tool to review the parasite specificity and status. The material of this study was a Tawny Owl (Strix aluco) which brought to an animal hospital with broken wings in Mugla province. Meanwhile blood smears were prepared from peripheral blood sample which was taken from the wing vein. Blood sample for genetic analyses was obtained by brachial venipuncture and genomic DNA extraction was conducted. The extracted genomic DNA was analyzed by Nested Polymerase Chain Reaction (PCR) for the amplification of partial avian haemosporidian mt-cytb gene. The final PCR product was gel purified and sequenced. The obtained isolate was deposited in GenBank International Nucleotide Sequence Database with the accession number JQ768232. Intraerytrocytic stages of Haemoproteus sp. were detected in the examination of the blood smears. The phylogenetic analyses of the amplified sequence confirmed that the owl was infected with Haemoproteus sp. According to the phylogenetic comparisons the Haemoproteus lineage showed the highest identity (99.8%) with the “H-STAL2” lineage isolated from an owl (Strix aluco) in Germany among the Haemoproteus lineages available in GenBank. In conclusion, this study reports the first microscopic and molecular detection of Haemoproteus infection in an owl in Turkey. The lineage characteristics and phylogenetic relationships among several Haemoproteus lineages were also evaluated in this study

Keywords

• Avian
• Haemoproteus
• haemosporidian
• owl
• Turkey

Türkiye’de bir Alaca Baykuş’ta (Strix aluco) Haemoproteus türünün saptanması ve molekülerkarakterizasyonu

Abstract

Kanatlı kan parazitlerinin taksonomik yeri ve konak spesifiteleri hakkında çok sınırlı bilgiler bulunmakta ve teşhis genellikle morfolojik metotlar ile yapılmaktadır. Ancak günümüzde, Plasmodium, Haemoproteus, Fallisia ve Leucocytozoon gibi kanatlı haemosporidian türlerinin özellikle mitochondrial cytochrome b (mt-cytb) gen bölgelerinin sekans analizlerinin yapılması, parazitlerin özgüllüğü ve durumları hakkında daha net bilgiler sunmaktadır. Bu çalışmanın materyalini, kanatları kırık vaziyette Muğla’daki bir hayvan hastanesine getirilen bir alaca baykuş (Strix aluco) oluşturmuştur. Baykuşun kanat altı venasından perifer kan örneği alınarak sürme preparatlar hazırlanmıştır. Genetik analizler için brachial damardan kan örneği alınmış ve genomik DNA ekstraksiyonu yapılmıştır. Elde edilen genomik DNA ekstraktı, avian haemosporidian mitokondrial (mt) DNA’sının parsiyel cyt-b gen bölgesinin amplifikasyonu için Nested PCR analizine tabii tutulmuştur. Son PCR ürünü jelden pürifiye edilerek sekanslanmıştır. Elde edilen izolat JQ768232 aksesyon numarası ile GenBank Uluslararası Nükleotid Sekans Veritabanı’na kaydedilmiştir. Kan preparatlarının mikroskobik incelemesinde Haemoproteus sp.’nin intraeritrositik formları saptanmıştır. Amplifiye edilmiş sekansın filogenetik analizi, baykuşun Haemoproteus sp. ile enfekte olduğunu doğrulamıştır. Filogenetik kıyaslamalara göre saptanan Haemoproteus izolatı GenBank’a kayıtlı diğer Haemoproteus izolatları arasında, en yüksek identikliği (%99,8) Almanya’da bir baykuştan (Strix aluco) izole edilen “H-STAL2” izolatı ile göstermiştir. Sonuç olarak bu çalışma, Türkiye’de bir baykuştaki Haemoproteus enfeksiyonunun mikroskobik ve moleküler teşhisini ilk kez rapor etmektedir. Ayrıca bu çalışmada, çeşitli Haemoproteus izolatları arasındaki soy özellikleri ve filogenetik ilişkiler de değerlendirmeye alınmıştır

Keywords

• Kanatlı
• Haemoproteus
• haemosporidian
• baykuş
• Türkiye

References

1. Atkinson CT, van Riper III C (1991): Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon, and Haemoproteus. 19-48. In: JE Loye, M Zuk (Eds), Bird-Parasite Interactions. Ecology, Evolution, and Behavior. Oxford University Press, New York.
2. Atkinson CT (1991): Vectors, epizootiology, and pathogenicity of avian species of Haemoproteus (Haemosporina: Haemoproteidae). Bull Soc Vector Ecol, 16, 109-126.
3. Atkinson CT (2008): Haemoproteus. 13-34. In: CT Atkinson, NJ Thomas, DB Hunter (Eds), Parasitic Diseases of Wild Birds. Wiley-Blackwell, USA.
4. Bennett GF (1987): Hematozoa. 120. EW Burr (Ed), Companion Bird Medicine. Iowa State University Press, Ames, Iowa.
5. Bennett, GF, Peirce MA, Ashford RW (1993): Avian haematozoa: mortality and pathogenicity. J Nat Hist 27, 993-1001.
6. 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 Biol Sci, 267, 1583-1589.
7. Campbell TW (1994): Hematology. 176-191. In: BW Ritchie, GT Harrison, LR Harrison (Eds), Avian Medicine: Principles and Application. Wingers Publishing, Lake Worth FL.
8. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2013): Geneious v5.5, Available from http://www.geneious.com. Erişim Tarihi: 05.02.2013.

9. 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.
10. Gıcık Y, Arslan MÖ (2001): Blood parasites of wild pigeons in Ankara district. Turk J Vet Anim Sci, 25, 169- 172.
11. 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.
12. Inci A, Yildirim A, Njabo KY, Duzlu O, Biskin Z, Ciloglu A (2012): Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey. Vet Parasitol, 188, 179-184.
13. Ishtiaq F, Guıllaumot L, Clegg SM, Phillimore AB, Black RA, Owens IP, Mundy NI, Sheldon BC (2008): Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands. Mol Ecol, 17, 4545-4555.
14. 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.
15. Mimioğlu M, Göksu K, Sayın F (1969): Veteriner ve Tıbbi Protozooloji II. Ankara Üniv Basımevi, Ankara.
16. Njabo KY, Cornel AJ, Bonneaud C, Toffelmier E, Sehgal RN, Valkiunas G, Russell AF, Smith TB (2011): Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest. Mol Ecol, 20, 1049-1061.
17. Ortego J, Calabuig G, Cordero PJ, Aparicio JM (2007): Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, Falco naumanni. Parasitol Res, 101, 1153-1156.
18. Özmen Ö, Halıgür M, Yukarı BA (2005): A study on the presence of Leucocytozoonosis in wild bird of Burdur district. Turk J Vet Anim Sci, 29, 1273-1278.
19. Özmen Ö, Halıgür M, Adanır R (2009): Identification of different protozoa species from a common buzzard (Buteo buteo). Turk J Vet Anim Sci, 33, 257-260.
20. Santiago-Alarcon D, Outlaw DC, Ricklefs RE, Parker PG (2010): Phylogenetic relationships of haemosporidian parasites in New World Columbiformes, with emphasis on the endemic Galapagos dove. Int J Parasitol, 40, 463-470.
21. Tanigawa M, Sato Y, Ejiri H, Imura T, Chiba R, Yamamoto H, Kawaguchi M, Tsuda Y, Murata K, Yukawa M (2012): Molecular identification of avian Haemosporidia in Wild Birds and mosquitoes on Tsushima Island, Japan. J Vet Med Sci, [Epub ahead of print].
22. Tolgay N (1972): Çeşitli kanatlıların Plasmodium, Haemoproteus ve Leucocytozoon enfeksiyonları üzerinde araştırmalar. Ankara Univ Vet Fak, 19, 271-286.
23. Valkiunas G (2005): Avian Malaria Parasites and Other Haemosporidia. CRC Press, Boca Raton, FL.
24. Waldenström J, Bensch S, Hasselquist D, Ostman O (2004): A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. J Parasitol, 90,191-194.