Cytochrome b lineages of Haemoproteus tinniculi in the endangered saker falcon (Falco cherrug) in Saudi Arabia

1 Introduction

Avian haemosporidian parasites are widely distributed across the globe. Three main genera, each consisting of numerous species, are recognised in birds: Haemoproteus, Plasmodium and Leucocytozoon (Atkinson and Van Riper III, 1991). They are transmitted by blood-sucking insects, including biting midges, louse flies, black flies and mosquitoes (Ritchie et al., 1994; Valkiunas, 2004). Haemoparasites are found in many raptors, and their incidence varies by host species and geography (Joseph, 1999). Because raptors are at the top of many food chains, their health can reflect the health of entire ecosystems (Cooper, 2002). Habitat loss and fragmentation have resulted in increased exposure of some raptor populations to parasitism, potentially altering host-parasite balances and increasing parasite pathogenicity.

While blood parasites are generally considered non-pathogenic, some evidence demonstrates that they can be harmful (Remple, 2004). Haemoparasites can cause increased thrombotic thrombocytopenic purpura (TPP) levels, leucocytosis, anaemia and even death (Garvin et al., 2003; Redig, 2003). Haemoproteus spp. are the most common blood parasites in raptors (Garvin et al., 2003). Prolonged recuperation time and higher mortality rates are observed in captive raptors with haemoproteid infections (Shihab et al., 2013). During severe infection, clinical signs may include poor flight performance, reduced flight distance, inappetence, weight loss, acute anaemia, dyspnea and lethargy (Butterworth and Harcourt-Brown, 1996; Remple, 2004). Rahim, Bakhiet, and Hussein (2013) found that increased eosinophil count and splenic size were closely associated with haemoproteid infection in captive saker falcons.

From a conservation perspective, the diagnosis and treatment of avian haemosporidians are important for controlling their cumulative effects in raptors (Ciloglu et al., 2016). Nested cytochrome b polymerase chain reaction (PCR) analysis is widely used to detect avian haemosporidian parasites in the peripheral blood of infected birds (Bukauskaitė et al., 2015; Hellgren et al., 2004). This molecular diagnostic tool provides greater sensitivity and accuracy than traditional microscopic examination methods. Moreover, the technique has made possible the discovery of a wide diversity of parasites within the traditional morphospecies (Bensch et al., 2009). Despite the popularity of birds of prey, knowledge of their haemosporidian diversity is limited (Valkiunas, 2004), possibly attributable to the difficulty of capturing adult raptors in the wild (Pérez-Rodríguez et al., 2013). The avian haemosporidian Haemoproteus tinnunculi has been previously reported in captive falcons in Saudi Arabia using only traditional microscopic examination of blood smears (Naldo and Samour, 2004; Rahim et al., 2013). Only one previously published study included molecular analyses of Haemoproteus tinnunculi in captive falcons in Saudi Arabia (Alfaleh et al., 2020). However, the reported prevalence of H. tinnunculi was quite low (3%) compared to an earlier study based only on microscopic examination (Rahim et al., 2013), possibly due to the study’s relatively small sample size. From a total of 100 falcons examined, only three birds were found infected with H. tinnunculi. Thus, a larger-scale study of this haemoparasite is needed to improve our understanding of phylogenetic relationships and genetic diversity of avian haemosporidian parasites from captive falcons in Saudi Arabia and more generally, the Middle East.

This study aimed to examine the genetic diversity of Haemoproteus tinnunculi obtained from captive saker falcons (Falco cherrug) in Saudi Arabia via cytochrome b gene sequence analysis.

2 Materials and methods

3 Results

A total of 1280 blood samples were collected from captive saker falcons (Falco cherrug) in Riyadh, Saudi Arabia, between December 2018 and February 2020. Microscopic examinations showed that 58 (4.53%) samples were infected by Haemoproteus tinnunculi (Fig. 1). All samples that were positive by microscopy were also positive by PCR. Details of the falcon samples infected by H. tinnunculi is summarised in Table 1.

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Fig. 1

Haemoproteus tinnunculi gametocytes in blood smear from saker falcon (modified wright-giemsa stain).

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The sequenced PCR products from all samples were edited and aligned, and the resulting alignment of 406 bp was analysed. The ML tree (Fig. 2), genetic distance matrix and heatmap plot (Table 2 and Fig. 3 respectively) clustered them into one distinct group with high confidence. The genetic relationships among these sequences indicated two distinct lineages of H. tinnunculi. In 54 samples, the lineages were 100% matches to previously deposited lineages (NCBI accessions MN780909 and MN780908), and all sequences obtained were uploaded into GenBank (MT571763–M571816). We identified a new H. tinnunculi lineage in four falcons (MZ571817–-MZ571820). One sequence is reported here for the first time.

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Fig. 2

Maximum likelihood phylogeny of Haemoproteus tinnunculi. Phylogenetic tree was done using 406 bp of cytochrome b gene. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei, 1993). The tree with the highest log likelihood (−1745.73) is shown. Tree is showed as traditional tree in straight view. Values at nodes represent bootstrap probabilities (bootstrap 10000). Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The sequences used in constructing the tree were obtained from GenBank. Red rectangle: subgroup composed of accessions MN780909, MN780908, MT571763-M571816 and MZ571817-MZ571820.

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Table 2 Genetic distance matrix (%). Estimates of Evolutionary Divergence between Sequences. The number of base substitutions per site from between sequences are shown. Analyses were conducted using the Tamura-Nei model (Tamura and Nei, 1993). This analysis involved 31 nucleotide sequences. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

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Fig. 3

Genetic distance (%) heatmap. Data from Table 2 were ploted as a heatmap using CLUSTVIS web tool (Metsalu and Vilo, 2015). A) Genetic distance of 31 accession. Red square: subgroup of 19 accessions re-plottered in B. B) Genetic distance of 19 accession. Red square: subgroup composed of accessions MN780909, MN780908, MT571763-M571816 and MZ571817-MZ571820.

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Fig. 3

Genetic distance (%) heatmap. Data from Table 2 were ploted as a heatmap using CLUSTVIS web tool (Metsalu and Vilo, 2015). A) Genetic distance of 31 accession. Red square: subgroup of 19 accessions re-plottered in B. B) Genetic distance of 19 accession. Red square: subgroup composed of accessions MN780909, MN780908, MT571763-M571816 and MZ571817-MZ571820.

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4 Discussion

In this study, cytochrome b gene was used to compare the genetic variation of Haemoproteus tinnunculi that was collected from captive saker falcons in Saudi Arabia. We found a new lineage of H. tinnunculi that is capable of infecting falcons in Saudi Arabia. This data is crucial for providing a better understanding of the genetic variation of falcon parasites their phylogenetic relationships.

The cytochrome b gene is one of the most common markers used for molecular detection and genetic characterisation of avian haemosporidians (Staffan Bensch et al., 2009; Sehgal, 2015; Valkiūnas et al., 2020). Parallel analyses of mtDNA cytochrome b provide the best evidence for species lineages, demonstrating consistent gene-trees and apparent absence of recombination for several closely related lineages (Bensch et al., 2004; Hellgren et al., 2007; Pérez-Tris et al., 2007). We revealed two distinct lineages of H. tinnunculi in saker falcons based on cytochrome b analysis. One of these lineages had not been previously registered in GenBank. The cytochrome b gene was shown to be useful in building well-supported phylogenies for H. tinnunculi species.

Phylogenetic analysis revealed two distinct sequences that were clearly split into multiple branches depending on the cytochrome b gene (Fig. 1). Sequence alignment indicated that 54 H. tinnunculi samples were 100% identical to a previously identified H. tinnunculi sequence (GenBank MN780909) found in a saker falcon in Saudi Arabia (Alfaleh et al., 2020). One new detected lineage was found in four falcons, which had not been described previously. This sequence shared 99.75% identity with previously sequenced H. tinnunculi (GenBank MN780908) (Alfaleh et al., 2020).

Haemoproteus parasites are well-known avian haematozoa that can cause decreased productivity and increased mortality in birds (Elahi et al., 2014). Blood-sucking dipteran insects transmit avian haemoproteids (Friend et al., 1999; Ritchie et al., 1994; Valkiunas, 2004). Microscopic examination of a Giemsa-stained peripheral blood smear is usually used to diagnose Haemoproteus infection. Although the parasites resemble Plasmodium, the pigment inside the intra-erythrocytic gametocytes is more scattered, and there are no schizonts visible in peripheral blood smears. The pigment granules are formed by the digestion of haemoglobin in the host’s RBCs and appear as refractile yellow to brown granules inside the RBCs (Friend et al., 1999). Haemoproteus tinnunculi was the most prevalent hematozoan parasite found in captive falcons in Saudi Arabia and the Middle East ((Naldo and Samour, 2004; Samour and Silvanose, 2000). According to Rahim et al. (2013), haemoproteid infection is more common at the end of the winter season, which lasts from January to April and usually coincides with the rainy season; this could be due to increased density and activity of Culicoides spp. and other biting insects that could serve as vectors for Haemoproteus spp. Molecular detection of H. tinnunculi has included extracted DNA amplification by PCR as an extremely sensitive and specific tool for avian haemosporidians. However, there is no common naming of lineages, thus making it difficult to find and compare information (Valkiūnas et al., 2019). There have been minimal molecular investigation of H. tinnunculi in falcons. Although a smear-based diagnosis is necessary, it is often inadequate or inaccurate in identifying and diagnosing Haemoproteus species (Ortiz-Catedral et al., 2019; Valkiūnas et al., 2020). Previous investigations have shown that PCR has a comparably high sensitivity to microscopy in diagnosing avian Haemoproteus (Tavassoli et al., 2018; Valkiūnas et al., 2019).

In this study, we only examined H. tinnunculi that infected saker falcons. Rahim et al. (2013) and Alfaleh et al. (2020) reported that this falcon species experienced a greater risk of infection with H. tinnunculi. This species has also been reported in peregrine falcons (F. peregrinus) and gyrfalcons (F. rusticolus) in Kuwait and F. sparverius from Pennsylvania, USA (Apanius and Kirkpatrick, 1988; Tarello, 2007). These results indicate that F. cherrug may experience greater exposure than other falcons or that there are host species variations in H. tinnunculi infection. Meixell et al. (2016) reported that several variables, including vector exposure, host body size (a larger size possibly attracting more insects), and plumage color may influence host-specific vectors. Further analyses of genetic diversity of haemoproteid parasites and exploration of relevant clinical and subclinical effects and possible pathogenicity in other falcon species are needed.

Valkiūnas et al. (2019) established the first genetic markers for identifying haemoproteids parasitising falconiform birds overwintering in Africa. They described three new Haemoproteus species using morphological and molecular markers. Phylogenetic study of haemoproteid lineages obtained from falcons, as well as available morphological data, show that H. tinnunculi and H. brachiatus have similar cytochrome b gene sequences.

This study will be valuable for researchers interested in the evolutionary history of H. tinnunculi parasites and to falconry managers, clinicians, and microbiologists. Our results will inform avian ecology and contribute to the study of an important molecular toolbox in examining a disease of increasing importance.