© Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2016, 63: 023 doi: 10.14411/fp.2016.023 http://folia.paru.cas.cz Research Article Investigation of avian haemosporidian parasites from raptor birds in Turkey, with molecular characterisation and [ Arif Ciloglu1, Alparslan Yildirim1, Onder Duzlu1, Zuhal Onder1, Zafer Dogan2 and Abdullah Inci1 1 Department of Parasitology, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey; 2 Department of Surgery, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey Abstract: Avian haemosporidians are common vector-borne blood parasites that have been reported in birds all over the world. Investi- gations of avian haemosporidian parasites are conducted mainly on passerine birds. However, studies that focus on non-passerine avian [ In the present study, blood samples from a total of 22 raptor birds belonging to two orders, two families and six species from the Central Anatolia Region of Turkey were investigated for three genera of avian haemosporidians (Plasmodium Marchiafava et Celli, 1885, Hae- moproteus Kruse, 1890 and Leucocytozoon!"[ PCR targeting the parasite mitochondrial cytochrome b gene (cyt-b!#"[ of Plasmodium or Leucocytozoon$%[[ parasite cyt-b[&Plasmodium haplotype is linked to a corre- sponding morphospecies (P-TURDUS1, \ Kikuth, 1931). All haplotypes were clearly distinguishable in phy- &$'&* Turkey, this study provides important new information on the phylogenetic relationships and genetic diversity of avian haemosporidian %["$ attention to the need for microscopy to detect parasite sexual development stages in surveys of avian haemosporidians. Keywords: Plasmodium, Haemoproteus, Leucocytozoon, Accipitriformes, Strigiformes, cytochrome b gene, blood parasites Avian haemosporidians (Apicomplexa: Haemosporida) [ belonging to the families Haemoproteidae, Plasmodiidae parasites in raptor birds, particularly since raptor hosts are and Leucocytozoidae are vector-borne parasites that are highly diverse and can support cryptic speciation of avian found in birds all over the world. Avian haemosporidian haemosporidians (Sehgal et al. 2006, Pérez-Rodríguez et \= al. 2013). Moreover, while the blood parasites are general- \>?G=<NQ!WNQ- ly thought to be harmless, there is evidence that infections cies of avian haemosporidians have been described and can be harmful (Remple 2004). Studies have suggested named according to morphological characteristics of para- that avian haemosporidian infection can cause reduced N> \ [ loss, anemia, airsacculitis and arthritis in hosts (Remple ?G=<NQXN'=NY!X- 1981, 2004, Dawson and Bortolotti 2000, Merino et al. cause these blood parasites are abundant and diverse, they 2000). Thus, from a conservation perspective, the diagno- constitute excellent model organisms for the study of wild- sis and treatment of avian haemosporidian infections are life parasitology and have been the focus of research efforts important for controlling the cumulative effects of blood ?G=<NQXNZ! parasites in raptor birds. Raptor birds (Accipitriformes and Strigiformes) are lo- Turkey is home to a high diversity of birds and is con- cated at the top of the food chain and can play very im- sidered one of the most important regions in Europe and portant roles in the ecosystem. However, most studies of the Middle East for migratory birds that travel through Af- avian haemosporidian parasites have been conducted on rica, Europe and Asia (Yigit et al. 2008). However, there passerines (Passeriformes), with very little focus on raptor are very few studies on avian haemosporidians and their hosts (Krone et al. 2008, Clark et al. 2014). More stud- vectors in this geographic region (Inci et al. 2012, 2013). ies are crucial to improve our understanding of the true Moreover, most avian haemosporidian investigations Address for correspondence: A. Ciloglu, Department of Parasitology, Faculty of Veterinary Medicine, Erciyes University, 38039, Kayseri, Turkey. Phone: +903522076666/29943; Fax: +903523372740; E-mail: [email protected] This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2016.023 Ciloglu et al.: Haemosporidians from raptor birds in Turkey Table 1.*"[}'*X= numbers of parasite haplotypes are given in parentheses. Positive in microscopic examination Positive in PCR screening No. No. Raptor species No. HLPco-infected Total H L P co-infected Total Parasite haplotypes with tested (H, L, P) (L, P) GenBank Accession Nos. Accipiter nisus (Linnaeus) 1 - - 1 - 1 - - 1 - 1 P-BUTBUT021 (KP883280) Asio otus (Linnaeus) 1 - - - - - - 1 - - 1 L-CIAE02 (KP000840) P-BUTBUT021 (KP883279), P-TURDUS1 (KP000842), Buteo buteo (Linnaeus) 5-11 1 3 -11 1 3L-BUTBUT011 (KP000841), L-CIAE02 (KC962152) [ Cretzschmar 13 - 1 - - 1 - 1 - - 1 L-CIAE02 (KC962151) Neophron percnopterus (Linnaeus) 1 --- - - --- - - - Linnaeus 1 - 1 - - 1 - 1 - - 1 L-STAL51 (KC876042) Total 22 - 3 2 1 6 - 4 2 1 7 - H – Haemoproteus sp.; L – Leucocytozoon sp.; P – Plasmodium sp.; 1 newly described haplotypes. that have been carried out in Turkey have relied entirely were brought to the Surgery Department of Veterinary Education on traditional microscopic examinations (Özmen et al. Research and Practice Hospital of Erciyes University, Kayseri 2005, 2009). According to our knowledge, there are only for treatment and rehabilitation between February 2013 and June two published molecular surveys of avian haemosporidi- 2014. Blood samples were taken from vena cutanea ulnaris of ans in Turkey, one that focused on the Krüper’s nuthatch 20 accipitriform and 2 strigiform hosts using insulin syringes for (Sitta krueperi Pelzeln) and another that sampled a tawny investigating the presence of haemosporidian parasites during owl ( Linnaeus) (Marzal and Albayrak 2012, the hospitalisation process (Table 1). About 50–100 μl of blood Yildirim et al. 2013). was taken and stored in EDTA (ethylenediaminetetraacetic acid) The ability to detect avian haemosporidians in verte- tubes and immediately placed at -20 °C for DNA extractions. Im- brate hosts has been improved recently with the advent of [ $>?XN were prepared on clean glass slides from each bird and air-dried Ricklefs and Fallon 2002, Beadell et al. 2004). In addition, QMX[" our understanding of the genetic diversity and host speci- 100% methanol and stained with 10% Giemsa as described by [ G=<?N! through molecular surveys (Hellgren et al. 2009, Ventim et Microscopic examinations were carried out to distinguish be- al. 2012) and these methods have facilitated the descrip- tween parasite sexual or asexual blood stages, a determination \]&> }'*[~[ deposited in GenBank and the MalAvi databases (Ricklefs carefully examined using an Olympus BX43 light microscope et al. 2004, Waldenström et al. 2004, Bensch et al. 2009, ?=!>\}Z MalAvi 2015). Nevertheless, although PCR-based meth- &["Y&& ods have very low detection thresholds and are considered [ ZMYQ W [ more sensitive than traditional parasitological methods, G=<?NQ! microscopic analyses remain essential for understanding Prior to DNA extraction, 10–15 μl of blood from each sample [ - was diluted with 200 μl of PBS buffer (0.01 M phosphate buff- ?G=<N'=NQ!# er, 0.0027 M KCl, 0.137 M NaCl, pH 7.4). Total genomic DNA therefore important to combine microscopic and molecular (gDNA) was extracted from blood samples using Blood Genom- surveys for characterising avian haemosporidians in wild ic DNA Kits (AP-MN-BL-GDNA-250, Axygen Biosciences, ?G=<N'=NY! Tewksbury, MA, USA) and eluted in 50 μl elution buffer fol- In the present study, we aim to investigate avian haemo- lowing the manufacturer’s instructions. The isolated gDNA was sporidian parasites in accipitriform and strigiform host stored at -20 °C until molecular analysis. DNA concentrations of samples from the Central Anatolia Region of Turkey. We samples were measured using the Nano Drop Spectrophotometer combine both traditional microscopy and molecular-based (ASP-3700, ACT Gene, Piscataway, NJ, USA) prior to molecular >[ analyses to standardise the amount of gDNA used in PCR ampli- of the phylogenetic relationships, haplotype diversity and [ [ Genomic DNA from blood samples was analysed by nested birds. }'*[b gene (cyt-b) of avian haemosporidian mitochondrial DNA (mtDNA). For the MATERIALS AND METHODS [ [ ]# ]*Z We sampled injured and/or sick individual accipitriform and which were designed to detect DNA from species of Leucocy- strigiform birds that were found in Kayseri Province or near cit- tozoon Sambon, 1908, Hae moproteus Kruse, 1890 and Plasmo- ies in the Central Anatolia Region of Turkey. All sampled birds dium Marchiafava et Celli, 1885 (see Hellgren et al. 2004). For Folia Parasitologica 2016, 63: 023 Page 2 of 8 doi: 10.14411/fp.2016.023 Ciloglu et al.: Haemosporidians from raptor birds in Turkey the second PCR round, HAEMF-HAEMR2 primers (Bensch et ible Model including invariable sites and variation among sites al. 2000) and HaemFL-HaemR2L primers (Hellgren et al. 2004) (GTR + I + G) was utilised according to the results of the Akaike’s [Haemoproteus/Plas- Information Criterion in jModelTest version 0.1.1 (Posada 2008). modium and Leucocytozoon[}'* Two Markov Chain Monte Carlo (MCMC) simulations were run NQ)NQ) simultaneously for 10 million generations with sampling every