Detection and molecular characterization of Trichomonas gallinae recovered from domestic pigeons in Egypt

Trichomonas gallinae is a protozoan parasite that causes canker in pigeons. Squabs (young pigeons) are frequently infected with T. gallinae and can die because of the infection, while adult pigeons can act as carriers showing no clinical signs. In the present study, 50 squabs, up to 1-month-old, were purchased from pigeon markets in different regions of the Giza governorate, Egypt. Direct wet mount preparations of the oral excretions of the squabs (mouth wash) and Giemsa staining revealed that 64% (32/50) were positive for T. gallinae. Experimental infection of ten squabs with 103 T. gallinae trophozoites/ml resulted in oral lesions on the mouth, tongue, and soft palate, with the presence of yellowish-white nodules (cheese-like) in the oral cavity on the sixth day post-infection in all squabs. A subset of five samples were cultured in modified Diamond’s media, their DNA was extracted, and a portion of the ribosomal internal transcribed spacer region (ITS1/5.8S/ITS2) was amplified by polymerase chain reaction (PCR) followed by sequencing. Phylogenetic analysis of the five isolates revealed 64–91% homology with some reference isolates circulating in Egypt and related countries.


Introduction
Avian trichomoniasis is a parasitic protozoan disease that affects pigeons, doves, chickens, turkeys, and raptors (Bulbul et al. 2018). The disease is called canker in pigeons (Saikia et al. 2021) and frounce in birds of prey. The causative agent, Trichomonas gallinae, is a flagellate belonging to the family Trichomonadidae, order Trichomonadida. Recently, two new species were recognized: T. stableri (Girard et al. 2014) and T. gypaetinii (Martínez-Díaz et al. 2015).
T. gallinae inhabits the upper digestive tract, mostly the esophagus and crop, but it can also infect the lungs, liver, internal lining of the body, air sacs, pancreas, bones, and skull sinuses. The disease is transmitted to birds through various routes, including crop milk, billing or feeding courtship rituals, aggregation at bird feeders or contaminated birdbaths, and the consumption of infected prey (Grunenwald et al. 2018).
The disease can be diagnosed in the laboratory by molecular identification of the organism and by clinical signs. Due to its low sensitivity, the wet mount method cannot distinguish strains of Trichomonas spp. Using molecular data  (Hochleithner and Hochleithner 2021). Low amounts of Trichomonas spp. can be detected due to the sensitivity of the polymerase chain reaction (PCR) to identify parasite DNA. Various DNA targets, including the internal transcribed spacer region (ITS), 18S rRNA, and iron hydrogenase, have proven effective for identifying trichomonads and for differentiating strains (McBurney et al. 2015). The present study aimed to investigate the isolated T. gallinae parasite by PCR, perform sequencing analyses targeting the ITS region, and compare the Egyptian T. gallinae sequences with those from other countries.

Collection of pigeon samples and study region
Fifty squabs, up to 1-month-old, showing signs of depression, weakness, anorexia, ruffled feathers, reluctance to fly, and caseated material in the oral cavity were purchased from Giza governorate, Egypt pigeon markets, from September-December 2021.

Microscopic examination for detection of T. gallinae trophozoites and staining method
In the direct wet mount method, Florin-Christensen and Schnittger (2018) sampled oral excretions (mouth wash) from squabs and checked for motile trophozoites within 30 minutes under a light microscope at 10x and 40x magnifications. They were identified by their motility and some morphological features, such as being pyriform to round, 7-11 μm in size, having four free flagellae, a well-developed undulating membrane, and an oval nucleus.
Then, the slides were Giemsa-stained and examined using an oil immersion lens (100x) according to Hamad and Hassan (2017).

Preparation of T. gallinae culture and experimental infection
Ten positive samples of T. gallinae were selected, and individually cultivated in modified Diamond's media (MD, trypticase yeast extract media) prepared according to Raza et al. (2018).
Motile trophozoites were counted using a hemocytometer (Neubauer Improved, Germany) at 40x magnification, according to Hamad and Hassan (2017), by the following equation: The media was inoculated with 2 × 10 5 trophozoites/ml, the inoculated tubes were tightly capped, incubated at 37 °C, and examined daily for 5 days. Only motile T. gallinae were estimated.
Each of the five positive samples was selected at random from the ten samples of T. gallinae cultivated in MD media, and was inoculated individually into ten healthy squabs for up to 1 month. The squabs had been collected from Giza governorate markets and subjected to parasitological examination to confirm they were infection-free.
They had been reared under entirely hygienic conditions and were infected orally with 1 ml of 10 3 T. gallinae trophozoites/ml using a dropper (Mohamed et al. 2009). Post-infection, the samples were collected from the oral cavity daily and examined via a direct wet smear.

Sampling T. gallinae for PCR and DNA extraction
T. gallinae trophozoites were collected from experimentally infected squabs, counted and adjusted to 2 × 10 5 cells/ml, and cultured in MD media at 37 °C for 48 h until the count of motile trophozoites equaled 1.49 × 10 6 .
The cultures were centrifuged at 1500 × g for 10 min (Echenique et al. 2020) to obtain the five isolates, the supernatants were discarded, and the pellets were re-suspended in phosphate-buffered saline, and stored at − 20 °C until their DNA was extracted.

PCR amplification of the ITS1/5.8S/ITS2 fragment
Genomic DNA was extracted from the five isolates using the EasyPure® Genomic DNA Kit (China), according to the manufacturer's instructions. The concentration of the extracted DNA was measured on the Nanodrop 2000 microvolume UV-VIS spectrophotometer (Thermo Scientific, USA).

Sequencing the ITS1/5.8S/ITS2 amplicons
Positive PCR products were sent to Macrogen® Company for double-strand sequencing using an ABI 3730xl DNA Sequencer. The sequencing data were analyzed using NCBI Blast (Altschul et al. 1990), assembled, edited, and chromate graphed using the Jalview software version 1.8.3-1.2.9-JAL.
No. of motile trophozoites∕ml = no. of counted trophozoites × 10 4 The phylogenetic tree was created using the MegAlign module. Neighbor-joining phylogenetic analyses were performed in MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms (Kumar et al. 2018). The tree was rooted in the outgroup, Tetratrichomonas spp.

Results and discussion
Of 50 squabs, 32 tested positive for T. gallinae, as assessed by direct wet mount preparations. T. gallinae possesses four unequal, anterior, transparent flagella that may be single or in groups, a well-developed fin-like undulating membrane, and an oval nucleus. Giemsa-stained the nucleus and flagella light purple while the cytoplasm was stained dark purple. In the present study, the microscopic structures of T. gallinae resembled those described by Heinz (2016).
Cultivation of T. gallinae in MD media, with an initial inoculum of 2 × 10 5 cells/ml, produced the maximum growth with hyperactivity in movement at 48 hours post-incubation, when the trophozoite count reached 1.49 × 10 6 trophozoites/ ml. This agrees nearly with the estimation of 1.325 × 10 6 cells/ml reported by Hamad and Hassan (2017). Diamond's medium has been the most widely used diagnostic standard culture medium for the identification and propagation of T. gallinae. Fresh inactivated horse serum, a rich source of amino acids, fatty acids, and some trace elements, is an essential additive for the growth of Trichomonas species in culture (Raza et al. 2018).
Experimentally infected squabs showed oral lesions on the mouth, tongue, and soft palate, and yellowish-white nodules (cheese-like) in the oral cavity on the sixth day post-infection ( Fig. 1). The oral lesions began as small, white caseous nodules, which subsequently grew into large yellowish-white caseous nodules. Similar observations were reported by Fadhil et al. (2020).
The ITS1/5.8S/ITS2 sequences of the five examined T. gallinae isolates were submitted to GenBank under accession numbers OM688823, OM688824, OM679421, OM679422, and OM688825. The constructed phylogenetic tree (Fig. 2), as well as nucleotide identity analysis (Table 1), revealed different extents of homology between these isolates and other reference isolates circulating in Egypt and related countries.
The isolates OM688823, OM688824, OM679421, OM679422, and OM688825 were 88%, 90%, 64%, 64%, and 91% identical to another Egyptian strain,    The Egyptian T. gallinae isolates in this study appear to be similar to European (French and German) and Asian (Saudi Arabian and Turkish) isolates, probably due to some European Columbidae species (turtle dove) migrating long distances to Africa, passing through Italy, Malta, Tunisia, and through the Balkan countries, Egypt, and the Middle East. Turtle doves that breed in European Russia and Ukraine migrate mainly to Eastern Africa via Turkey and the Middle East (Marx et al. 2017).