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Haemoproteosis and avian malaria in Columbidae and Corvidae from Iran

Abstract Avian malaria (Plasmodium) and related genera (Haemoproteus and Leucocytozoon) are diverse and widespread parasites. Despite the extent of knowledge on avian haemosporidian parasites, information about domestic and wild bird's blood parasites is overall insufficient in Iran. Prevalence of the haemosporidian parasites’ and phylogenetic relationship of lineages are studied by using molecular and morphological results of 152 examined hosts belonging to 17 species. Molecular analysis for haemosporidian detections demonstrated overall prevalence 22.36%. Inspected hosts mostly belonging to Common Pigeons (Columba livia) parasitized by Haemoproteus spp., and Hooded Crows (Corvus cornix) and Carrion Crow (C. corone) were identified as hosting Plasmodium spp. Detected lineages COLIV03, COQUI05, LINN01, ROFI04 and SGS01 are identified as new reports from Iran. We detected no evidence of Leucocytozoon lineages, while the high prevalence of H. columbae was found in Common Pigeons. Such investigation on avian blood parasites contributes to providing new information on the prevalence, epidemiology and geographical distribution of haemosporidian parasites circulating in domestic, pets and wild birds.


INTRODUCTION
Avian haemosporidian intracellular parasites are transmitted by bloodsucking arthropods. Plasmodium spp. and related genera (Haemoproteus and Leucocytozoon) are diverse and widespread (Valkiūnas, 2005) leading to avian malaria, haemoproteosis and leucocytozoonosis infrequently damaging or causing mortality in birds (Valkiūnas, 2005;Valkiūnas & Iezhova, 2017). On the other hand, serious negative effects of hemoparasites on metabolism' efficiency and avian fitness have been registered in domestic birds like poultries and pigeons (Tiwari et al., 2012) and wild birds resulting in progressive faintness and death This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd. (Dinhopl et al., 2015;Olias et al., 2011). In the last two decades using nested cytochrome b polymerase chain reaction (PCR) have uncovered greater genetic diversity of haemosporidian parasites in a wide range of birds than traditional morphologic examinations Hellgren et al., 2004;Križanauskienė et al., 2006). Different investigations have studied haemosporidian parasites detection by microscopic and molecular approaches in pet and captive birds across the world (Alley et al., 2008;Belo et al., 2009;Bensch et al., 2012;Brossy, 1992;Chagas et al., 2017;Dinhopl et al., 2011;Nakayima et al., 2019;Nourani et al., 2020a;Pacheco et al., 2011;Paperna & Martelli, 2008;Scaglione et al., 2016a;Schrenzel et al., 2003;). Despite the extent of Vet Med Sci. 2021;7:2043-2050.
wileyonlinelibrary.com/journal/vms3 2043 knowledge on the avian haemosporidian parasites, information about domestic and wild bird's blood parasites is overall insufficient in Iran (Nourani et al., 2017a;Nourani et al., 2017b, Nourani et al., 2018aNourani et al., 2018b;Nourani & Dinparast Djadid, 2019;Nourani et al., 2020a;Nourani et al., 2020b;Nourani et al., 2020c). To date there have been limited investigations documenting morphological and molecular results of blood intracellular parasites infection in pet and domestic birds (Chaechi-Nosrati et al., 2018;Mirzaei et al., 2016;Nematollahi et al., 2012). Haemoproteus (Hippoboscidae and Ceratopogonidae) and Plasmodium (Culicidae) parasites can be transmitted during blood meal of hematophagous dipteran (Valkiūnas, 2005) that their contribution in circulation parasites and distribution map remains unidentified in Iran (Nourani et al., 2020c). Parasites are of main hazards to bird populations; endangered or domestics (Bunbury et al., 2008) and birds may encounter multiple pathogens that contribute to the transmission of pathogens to other birds and humans (Reed et al., 2003). Thus, expanding our knowledge of distribution in novel hosts and less-studied geographical regions seems crucial (Al-Barwari & Saeed, 2012;Cray, 2011;Hunter et al., 1997;Jacob et al., 2011). Detection of parasites with a great impact on birds' health requires more attention in designing field surveillance and performing the control programs. Here we present results from blood parasites screening in the referred pet, domestic and wild birds to the veterinary clinic during a year. Our objectives were to assess haemosporidian prevalence infections by molecular and morphological methods, to determine novel host records and/or parasitic lineages in examined birds, and to study their phylogenetic relationship with known lineages in similar hosts.

Preparation of blood samples for morphological detection
Blood samples were collected of pets, domestic and wild injured birds referred to the veterinary clinic during October 2018 to September 2019 and some blood samples were collected by a veterinarian during visiting hosts in farms. Brachial vein blood was taken with a syringe (50-100 µL of whole blood). Two thin blood smears were prepared for each host, were immediately dried in the air, preserved in absolute methanol and subsequently stained with Giemsa 10% at pH 7.2 (60 minutes) for the morphological screening of haemosporidian parasites. Blood films were examined under 400× and 1,000× of the Olympus BH2 light microscope (Olympus Co, Japan) via oil immersion in 100 fields for 35-40 minutes for haemosporidian parasites gametocytes discovery (Martinsen et al., 2006;Valkiūnas, 2005;Valkiūnas & Iezhova, 2018 Leucocytozoon (Bensch et al., 2000;Hellgren et al., 2004). Positive PCR products of the previous investigation (Nourani et al., 2020b) and ultrapure water were utilised as positive and negative controls. Subsequently, 1% agarose gels were used for visualisation of amplified amplicons. Purification and sequencing of positive products were made by Codon Inc. (Codon Genetic Group, Tehran, Iran).

Bioinformatics and phylogenetic analysis
BioEdit v.7.1.7 was used for cleaning up, edition and alignments of raw sequences by ClustalW (Hall, 1999). The single base mutation was considered as a criterion for the novelty of detected lineages in comparison with the homologs sequences in Nucleotide BLAST analysis, accessible at NCBI and MalAvi sites Pérez-Tris & Bensch, 2005). Seventeen amplified sequences in the current study are deposited in GenBank (Accession number: MT802174-MT802190).
Phylogenetic relationship of lineages was studied by Bayesian analysis that performed with two concurrent Markov Chain Monte Carlo searches of 10 million generations using MrBayes v3.2 and sampling was done for 1 of 1000 trees (Ronquist & Huelsenbeck, 2003) under the evolutionary model achieved from MODELTEST v3.7 program (Posada & Crandall, 1998  Number of examined hosts (n), morphological (Morph) and molecular (Mol) detection of haemosporidian parasites are summarised. Birds are brought by their host to clinic (C), visiting in the farm (F), or found in the street and referred to the clinic (S). ISD, iron storage disease.

Haemosporidian parasites detection and diversity of lineages
One hundred fifty-two birds belonging to 17 species were screened for the detection of haemosporidian parasites (

DISCUSSION
The key result of this study is the application of nested PCR sequencing along with morphological methods to identify new records of haemosporidian parasites in birds of Iran. Six Haemoproteus and Plasmodium known lineages were registered as new host records that were previously discovered in passerine and non-passerine hosts. Similar to previous researches in wild passerines across Iran, Haemoproteus was the prevalent parasitic genus among birds (Nourani et al., 2017a;Nourani et al., 2017b, Nourani et al., 2018aNourani et al 2018b;Nourani & Dinparast Djadid, 2019;Nourani et al., 2020a;Nourani et al., 2020b).
Nested PCR assay and morphological examinations did not detect any record of Leucocytozoon spp. 2008) has been recorded in more than 100 wintering and resident bird species belonging to different families around the world (MalAvi 2020) Palinauskas et al., 2008;Palinauskas et al., 2009).
In this study, this lineage is recorded for the first time in a passerine bird in Iran (Corvus corone). Contrary to our study, molecular and histological examinations on Corvus corone tissues demonstrated the higher infections to Leucocytozoon (97.9%) and Haemoproteus or Plasmodium spp.
Pigeons' health as pets and cultural symbols or laboratory animals should be considered, which can be influenced by their growth, production and vulnerability to diseases (Dranzoa et al., 1999). In this study, the most infected host species was common pigeon, of which 17.76% were parasitised with HAECOL1 (H. columbae). To date, eight Haemoproteus, two Plasmodium and eight Leucocytozoon lineages have been detected in Columba livia, deposited in MalAvi ).
One-third of our samples was related to poultry, and none of them was infected with Plasmodium or Haemoproteus spp. Some species of haemosporidian parasites have a major impact on the poultry industry; high mortality up to 90% is observed in domestic chickens (Springer, 1991). Previous studies have informed absence or relative infections of haemosporidian parasites in the Phasianidae family (Castle et al., 1988;Christensen et al., 1983;Huchzermeyer, 1993;Laird, 1978;Mohammad et al., 2001;Otranto et al., 2010), possibly due to their specific activities avoiding encounters with vectors (Alavi et al., 2003;Loiseau et al., 2012). Nevertheless, some other investigations declared the haemosporidian infections in Phasianidae, white eared-pheasant (Crossoptilon crossoptilon) parasitised by P. juxtancleare (Murata et al., 2008), Ammoperdix griseogularis and Alectoris graeca infected by Haemoproteus and Ammoperdix griseogularis infected by Plasmodium spp. (Mohammad et al., 2001). Microscopic screening and PCR assays have been utilised to identify blood parasites in free-ranging domestic birds: chickens, ducks, turkeys and guinea fowl in Uganda. Their results demonstrated the infections to Plasmodium, Haemoproteus and Leucocytozoon, which provide information for parasite management in poultries to hinder other species (Nakayima et al., 2019). More epidemiological studies are necessary to be performed to detect haemosporidian parasites, as a prevalent infection in poultry, which prevents the spread of lineages to farm birds and other susceptible birds. The discovery of Haemoproteus and Plasmodium species and also coinfections for domestic and pet birds are also significant to prevent the potential transmission of these parasites within colonies and among wild birds in rural or urban areas (Scaglione et al., 2015). with long-term risk of diseases, epidemiologic investigations provide management practices to protect these inestimable animals (Camacho et al., 2016;Brown & O'brien, 2011;Lozano-Fuentes et al., 2011).
In this study, we reported the detection of haemosporidian parasites by using molecular and microscopic overview in pets, domestic and wild birds referred to a veterinary clinic in Iran. Forthcoming investigations will shed light on host-parasites relationships and parasites distribution that are necessary for veterinary surveillance and control of vector-borne parasites.

ACKNOWLEDGMENTS
We express our sincere appreciation to MVRG members for sharing their pearls of wisdom with us during this investigation. This research was supported by a Pasteur Institute of Iran grant to NDD.

ETHICS STATEMENT
This study was achieved in accordance with the guidelines and protocols permitted by the ethics committee for the care and use of animals for scientific purposes of the Pasteur Institute of Iran (No: IR.PII.REC.1395.96).

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.

DATA AVAILABILITY STATEMENT
Amplified sequences in the current study are deposited in GenBank (Accession number: MT802174-MT802190).