Molecular detection and genetic characterization of Trichomonas gallinae in falcons in Saudi Arabia

Avian trichomonosis is primarily caused by Trichomonas gallinae, a flagellated protozoan parasite that especially infects the upper digestive tract of columbid bird species and their avian predators. However, this parasite has recently been found to be distributed worldwide in various other avian species. This parasitic disease is common in captive falcons in Saudi Arabia and the Middle East. This study aimed to examine and identify the genetic variation of T. gallinae obtained from three species of falcons in Saudi Arabia via the sequencing analysis of the internal transcribed spacer (ITS) region. Swab samples from 97 saker falcons (Falco cherrug), 24 peregrine falcons (Falco peregrinus) and 37 gyrfalcons (Falco rusticolus) were cultured and analysed for infection between 2018 and 2019. The overall prevalence of infection by T. gallinae was 26.58% (n = 42), of which 35 (83.33%) were collected from Riyadh region and seven (16.67%) were collected from Qassim region. The results indicate the presence of four genotypes of T. gallinae in Saudi falcons: A, C, II, and KSA11. This study reports for the first time genetic diversity of T. gallinae in these falcons in Saudi Arabia.


Introduction
Avian trichomonosis is a parasitic disease caused by the flagellated protozoan parasite Trichomonas gallinae [1]. This parasite infects the upper digestive tract of domestic and wild birds, including the mouth, crop, oesophagus and pharynx, causing erosions and necrotic lesions, as well as necrotic foci within internal organs, such as the liver and lungs. Severe cases can be deadly in the early days following infection [2,3]. Columbid species are considered the principal reservoir hosts of T. gallinae [1]. In addition to Columbiformes, several other bird species worldwide are infected by this parasite, including raptors, gallinaceous birds and passerines [4][5][6][7][8].
The main route of transmission of T. gallinae between birds includes the feeding of squabs by an adult, normal feeding via courtship billing during nesting, sharing contaminated food or water sources, and via predation and necrophagy from raptors in the wild [9,10]. Trichomonas gallinae in captive falcons in Saudi Arabia and Middle East seem to be related to the traditional Arabian falconry practices of training falcons using live or freshly domestic pigeons [11,12]. Recently, T. gallinae was reported as an etiologic agent of epidemic outbreaks of disease affecting wild birds across Europe and Canada [2,13,14]. This disease can cause significant effects at the population level, especially for naïve populations. It is considered a major threat to the endangered pink pigeon (Columba mayeri) in Mauritius [15].
There are several studies from Europe and North America on the prevalence, virulence and genetic diversity of avian trichomonads, but reports from Asia and the Middle East are rare [16,17]. The genetic diversity of T. gallinae and its correlation with clinical presentations correlated to the sequencing of the internal transcribed spacer (ITS) region performed in Europe, the UK and the USA have revealed a relationship between the genetic variation and the presence of necrotic ingluvitis lesions [5,13,[18][19][20][21]. In a recent study, sequencing was performed on 15 distinct ITS genotypes within T. gallinae isolates from pigeons, common mynas (Acridotheres tristis), chickens and turkeys in Riyadh, Saudi Arabia [8].
This study aimed to examine the genetic diversity of T. gallinae obtained from different falcon species in Saudi Arabia via the sequence analysis of 5.8S ribosomal RNA (rRNA) and its flanking ITS regions 1 and 2 (ITS1 and ITS2).  (Table 1). "All procedures for the samples collection were carried out in strict accordance with the recommendations by the Research Ethics Sub-Committee (REC) of the College of Sciences at the King Saud University (KSU) in Riyadh, Kingdom of Saudi Arabia (KSA) (Ethics Reference No: KSU-SE-19-77)". The ethics committee has approved the current study. The sample collection procedures were conducted under direct veterinary supervision. Clinical signs and descriptions of any lesions were recorded for each examined bird. The falcons collected from the centers were anesthetised with isoflurane via a facemask before undergoing an endoscopic examination of the upper digestive tract to look for the presence of macroscopic lesions. The falcons collected from the Saudi Falcons and Hunting Exhibition were examined for the presence of oropharyngeal trichomonosis lesions by direct inspection. Veterinarians used sterile cotton swabs to gently swab live falcons over approximately 120˚to match the natural anatomical curvature of the oral cavity, oropharynx and crop. All samples were subsequently cultured at 37˚C in 5 ml of fresh trypticase-yeast extract-maltose (TYM) medium containing 10%

PLOS ONE
Trichomonas gallinae in Saudi falcons inactivated horse serum (Sigma-Aldrich, St. Louis, Missouri, USA), 100X antibiotic-antimycotic (10,000 units/mL penicillin + 10,000 μg/mL streptomycin + 25 μg/mL amphotericin B; UFC Biotech, KSA) at pH 7.2. All samples were examined under a microscope at 10X magnification at 24 hours post-incubation and thereafter at 24-hour intervals for up to five days to monitor for the motility of T. gallinae. The infection status of the samples was determined to be positive if motile T. gallinae were found in any of the culture tests for an individual bird, and the samples were designated as negative if no parasites were detected after five days of incubation. The positive samples were stored with 5% DMSO (Sigma-Aldrich) at −80˚C for any additional work.

DNA extraction
DNA was extracted from the positive culture isolates using DNAzol1 reagent (Invitrogen, UK) under the following conditions. The TYM medium containing T. gallinae was transferred to a 1-ml Eppendorf tube and centrifuged at 12,000 rpm for 5 min; the supernatant was removed and the pellet retained; 500 μl of DNAzol1 was added to each sample and briefly pipetted to rapidly lyse the cells and the samples were centrifuged at 10,000 rpm for 2 min at 4˚C. The supernatant was transferred to a fresh Eppendorf tube, the pellets discarded, and 500 μl of 100% ethanol added to the supernatant and vortexed, which was then centrifuged for 5 min at 10,000 rpm at 4˚C to precipitate the DNA. The supernatant was discarded, and the DNA pellet was washed in 70% ethanol and centrifuged at 10,000 rpm for 3 min. The new supernatant was removed and the DNA pellet air-dried for 10 min before adding 100 μL of nuclease-free water to dissolve the DNA. The extracted DNA was stored at −20˚C for future work if necessary. The DNA concentrations for each isolate were measured using spectrophotometry with an absorbance of 260 nm. The extracted DNA was electrophoresed through a 1% agarose gel stained with ethidium bromide and visualised using UV transillumination.

PCR amplification of the ITS1/5.8S/ITS2 fragment
The PRC analysis was carried out using forward primer TFR1 (5 0 -TGCTTCAGTTCAGCGG GTCTTCC-3 0 ) and reverse primer TFR2 (5 0 -CGGTAGGTGAACCTGCCGTTGG-3 0 ) [22,23]. The reaction was conducted in a 25-μL total volume comprising 8.5 μL Green Master Mix (2X, USA), 3 μL each of forward and reverse primers, 8.5 μL ddH 2 O and 2 μL of genomic DNA. The cycling parameters consisted of an initial denaturation at 98˚C for 5 min, 45 cycles at 98˚C for 30 s, annealing at 61˚C for 30 s with an extension at 72˚C for 1 min, and a final extension step at 72˚C for 5 min. The PCR products were electrophoresed in 1% agarose gel.

Sequence analysis and phylogenetic trees
The evolutionary relationships among the sequences of T. gallinae were compared by building a phylogenetic tree using MEGA, Version 7, [24] and CLUSTALX, Version 2.1, software [25]. All sequence data for the ITS regions were inspected and refined using the MEGA trace data file viewer/editor, and then aligned using the forward and reverse complement of the reverse primer. All sequences obtained in this study were uploaded in GenBank under the accession number MT300158-MT300161 and MW114451-MW114485. A number of reference sequences were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database for comparison with the ITS regions. The phylogenetic trees of the datasets obtained from GenBank and those identified in this study were constructed separately using the neighbour-joining method with genetic distance and Tamura-Nei models. These were used to analyse the relationships between taxa via nucleotide sequences [24,25]. Felsenstein's bootstrap method was used to calculate the associated taxa clustered in the bootstrap test (1,000 replicates).

Discussion
Trichomonas gallinae infects captive and wild bird species worldwide and is the causative agent of avian trichomonosis. This disease is important because it affects a number of different bird species, is characterised by great variation between different T. gallinae isolates, and may have conservation implications for at-risk falcon species, particularly those facing multiple threats. In this study, the ITS1/5.8S rRNA/ITS2 region was used to analyse genetic variation of T. gallinae isolated from three different species of falcon in Saudi Arabia. To the best of my knowledge, this is the first study at the genetic level to characterizing the genotypes of T. gallinae isolated from different falcon species in Saudi Arabia.
The four types of sequences detected in the current study correspond to four genotypes previously reported (Fig 1). These sequences, A, C, II and KSA11, are distinct lineages of T. gallinae, and all the sequences are commonly found in many species of birds, including columbids, raptors, chickens and finches, in various European countries, such as the UK, the USA, Canada, Brazil and Saudi Arabia [5,6,8,13,18,19,23,28,29]. However, the most important finding is that sequence II, originally reported in a racing pigeon in Austria (GenBank: FN433474) [26], was the most prevalent in the Saudi falcons. The results indicate that within the group of bird with lesions (6 of 39; 15%), genotype A is the most frequent (3 of 6; 50%). This data is consistent with the results reported by Robinson et al. (2010) [9], who found genotype A most often in birds with pathognomonic lesions.
These data support a wide range of hosts for T. gallinae, including birds of prey that feed on columbids, such as the feral pigeon (Columba livia). Albeshr and Alrefaei (2020) [8] reported that sequence analyses of T. gallinae obtained from different species of birds in Riyadh showed at least 15 unique sequences, which were clearly divided into different branches depending on the ITS region sequence. However, all the sequences found in the current study (i.e. A, C, II and KSA11) were previously found only in pigeons within Saudi Arabia.
These results are consistent with those published by Albeshr and Alrefaei (2020) [8], who reported that infected samples of T. gallinae sequence C isolated from pigeons and chickens  [11,30], and trichomonosis is often linked to the practice of feeding falcons live or freshly killed pigeons, in particular domestic pigeons (Columba livia) [11,12]. In Saudi Arabia, falconers typically buy such pigeons from a poultry market or catch them on farms. Albeshr and Alrefaei (2020) [8] recently reported that in Riyadh, more than 63% of feral pigeons for sale in Al-Azizia's poultry market and 17% of wild pigeons caught on the south and south-western sides of nearby Wadi-Hanifa were infected by T. gallinae. Many studies have reported that populations of Columba livia, as well as other members of the order Columbiformes, are important reservoirs of the protozoan T. gallinae [5,9,19,[31][32][33]. There is a need to extend screening to additional falcon species worldwide to evaluate the genetic diversity of trichomonas in both falcons and their prey and investigate related clinical and subclinical impacts and potential pathogenicity. Phylogenetic tree based on ITS region indicating the relationship of Trichomonas gallinae genotypes using the NJ method. References to GenBank accession numbers are as follows: GQ150752 [13]; EU215368, EU215362, EU215358, EU215360, EU215365, EU215367, EU215366 [18]; FN433474 and FN433473 [26]; MK771125, MK771126, MK771127, MK771128, MK771129, MK771130, MK771131, MK771132, MK771133, MK771134, MK771135, and MK765029 [8]. Tritrichomonas foetus GenBank accession number DQ243911 [27] was included in the present study as an outgroup. The sequences identified in this study are shown in green.