Isolation and Characterization of Novel Trichomonas gallinae Ribotypes Infecting Domestic and Wild Birds in Riyadh, Saudi Arabia

SUMMARY. Trichomonas gallinae, a single-celled protozoan parasite, is a causative agent of the disease trichomonosis, which is distributed worldwide and has recently been highlighted as a pandemic threat to several wild bird species. The aim of this study was to determine the prevalence and genotypic diversity of T. gallinae in Riyadh, Saudi Arabia. For this purpose, 273 oral swab samples from different bird species (feral pigeon Columba livia, common mynah Acridotheres tristis, chicken Gallus gallus domesticus, turkey Meleagris gallopavo, and ducks Anatidae) were collected and tested for T. gallinae infection with InPouch™ TV culture kits. The results showed that the overall prevalence of T. gallinae in these samples was 26.4% (n = 72). The PCRs were used to detect the internal transcribed spacer (ITS) region of T. gallinae, and the results of the sequence analysis indicated genetic variation. Among 48 sequences, we found 15 different ribotypes, of which 12 were novel. Three had been previously described as ribotypes A, C, and II. To our knowledge, this study demonstrated the presence of T. gallinae strain diversity in Saudi Arabian birds for the first time and revealed that ribotypes A and C are predominant among Riyadh birds.

The poultry market of Riyadh, Saudi Arabia. Some of these bird species, such as chickens (Gallus gallus domesticus), ducks (Anatidae), and pigeons, derive from local poultry farms; however, some ornamental birds, such as parrots (Psittaciformes), and wild migrating birds, such as falcons (Falco spp.), are imported from outside the country. Such birds are likely to have been exposed to a variety of parasitic diseases, including trichomonosis, and thus pose a spillover threat to endemic wild species. This disease can also be transmitted to local poultry farms, causing significant economic losses (16).
The infection can be transmitted directly via feeding of squabs or when adult males and females exchange food as breeding behavior or indirectly via sharing of food or water (8,17). Trichomonosis can be directly diagnosed through symptoms, such as lesions in the digestive and upper respiratory tracts and swelling in the throat, nostrils, and eyes (18,19). However, infected adult birds might not show signs of the disease because they can develop tolerance to the infection (17). Death occurs from breathing difficulties, starvation, or both (20,21,22).
Trichomonosis has threatened several rare wild bird species around the world. For example, it was introduced to Mauritius with exotic bird species and transmitted to the endemic bird species on the island, including the echo parakeet (Melopsittacus undulatus) and pink pigeon (Columba mayeri) (8,17). As a result, the populations of these two avian species have drastically declined in number in Mauritius (8,17). In addition to wildlife threats, there are increasing concerns that this disease could negatively affect the economic sectors and recreational values of certain countries (23).
Several T. gallinae strains with differing virulence have been identified. For example, Sansano-Maestre et al. (13) used the 5.8S ribosomal RNA (rRNA) region and restriction fragment length A These authors contributed equally to this work. B Corresponding author. E-mail: afrefaei@ksu.edu.sa AVIAN DISEASES 64:130-134, 2020 polymorphism markers to identify the strains of T. gallinae in wild or domestic pigeons and birds of prey in eastern Spain. They found two genotypes in isolates from the Columbiformes and raptors. In these isolates, the genotypes isolated from the Columbiformes were more prevalent in the Columbiformes, and the genotypes isolated from the raptors were more prevalent in the raptors, displaying lesions. Additionally, a comparison of the sequences of the 5.8S ribosomal DNA region and internal transcribed spacer (ITS) region of T. gallinae in Mauritian birds, including the pink pigeon (Columba mayeri) and Madagascar turtle-dove (Streptopelia picturata) (24) showed no variations between the isolates from these species.
In Saudi Arabia, investigations of T. gallinae in endemic and commensal birds are rare, and most research has been conducted on the birds of the family Falconidae (25,26). Here, we investigated the prevalence and genotypes of T. gallinae in avian species at the poultry market in Riyadh compared with its prevalence in the wild bird samples obtained locally. We provide evidence that the poultry market in Riyadh might be a source of infection in the area. The strength of this study is its focus on multiple species in a limited geographic area, a clear advantage over similar studies performed in this region of the world previously.

MATERIALS AND METHODS
Oral swab samples from a total of 273 birds were tested for Trichomonas infections. Of these, 183 samples were obtained from the live adult birds of four domestic species collected from the Al-Azizia poultry market in Riyadh. An additional 90 samples were obtained from adult wild birds collected from different locations on the south and southwestern sides of Wadi-Hanifa near Riyadh. Wild birds were caught by mist-net captures and released after sampling. All samples were collected between March and December 2018 ( Table 1). All birds were examined grossly for the presence of oropharyngeal trichomonosis lesions by direct inspection. Procedures for the sample collection were carried out in strict accordance with the recommendations by the Research Ethics Committee of the King Saud University, Riyadh, Saudi Arabia (Ethic Reference KSU-SE-19-77).
Briefly, oral swab samples were obtained from the area between the oral cavity and the crop with sterile cotton swabs moistened with distilled water. The swab was gently moved four or five times in a figureeight motion around the oral cavity and crop mucosa to ensure that a sufficient sample of mucosal and trichomonad cells was obtained. The swabs were then immediately inoculated individually into InPouche TV culture kits (BioMed Diagnostics, White City, OR), according to the manufacturer's instructions. The samples were transferred to the laboratory and kept at 36 C in a sanitized incubator. All culture samples were examined microscopically 24 hr postincubation and thereafter at 24-hr intervals for up to 7 days to check for T. gallinae parasite growth. All culture-positive samples were stored with 5% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) at À80 C for future work. During swabbing, the birds were inspected for signs of T. gallinae infection, including lesions around the oral cavity and eyes. However, a diagnosis of T. gallinae infection was confirmed only when physical symptoms were validated by microscopic examination.
DNA was extracted from the culture-positive samples by DNAzolt Reagent (Invitrogen, Paisley, UK), according to the manufacturer's instructions. The InPouch TV cultures containing T. gallinae were transferred to 1000-ll Eppendorf tubes and then centrifuged at 7245 3 g for 3 min. The supernatant was removed, and the cell pellet was mixed with 500 lL DNAzol with gentle pipetting to lyse the cells. Subsequently, the cell lysates were centrifuged at 2795 3 g for 4 min at 4 C, and the resulting supernatant was transferred to a new Eppendorf tube. To precipitate DNA, 500 ll of 100% ethanol was added to each tube, followed by mixing through inversion and 3 min of centrifugation at 1789 3 g at 4 C. The supernatant was removed, and the DNA pellet was washed in 75% ethanol. The DNA pellets were left to air dry for 4 min before adding 100 ll of nuclease-free water to resuspend the DNA. The extracted DNA was stored at À20 C for subsequent work. The quantity of DNA for each isolate was estimated by using spectrophotometry based on an absorbance reading at 260 nm. The extracted DNA was confirmed visually on a 1% agarose gel stained with ethidium bromide under an ultraviolet transilluminator.
A PCR was conducted to amplify the ITS region with the forward primer (TFR1; 5 0 -TGCTTCAGTTCAGCGGGTCTTCC-3 0 ) and the reverse primer (TFR2; 5 0 -CGGTAGGTGAACCTGCCGTTGG-3 0 ) (24,27) according to the method described by Robinson et al. (28). The PCR amplification was carried out in a 25-ll reaction volume containing 15 ll of Green Master Mix (23; Thermo Fisher Scientific, Waltham, MA), 3 ll each of forward (TFR1) and reverse (TFR2) primers (Eurofins Genomics, Anzinger, Germany), 3 ll of doubly distilled water, and 1 ll of template DNA. Each PCR was run with a negative control containing no template DNA and a positive control containing the T. gallinae DNA.
The PCR was performed with the following temperature cycle: initial denaturation at 94 C for 15 min, followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 65 C for 30 sec, and extension at 72 C for 1 min, and a final extension at 72 C for 5 min.
We confirmed PCR amplification under ultraviolet light with a 1% agarose gel stained with ethidium bromide. A band of approximately 350 bp in size was confirmed with a Ready-Loade 100-bp DNA ladder (Promega, Madison, WI). The positive PCR products were submitted for sequencing to Macrogen Inc. (Seoul, Republic of Korea).
The molecular and phylogenetic relationship of the sequences of trichomonad parasites were determined by software programs molecular evolutionary genetics analysis (MEGA) version 7 (29) and CLUSTAL X version 2.1 (30). All sequence data were aligned with the forward and reverse complement of the reverse primer by the MEGA software. The T. gallinae sequences available in the National Center for Biotechnology Information (NCBI) GenBank database were used to compare the ITS regions. The phylogenetic trees of the datasets obtained from the ITS1/ 5.8S and rRNA/ITS2 regions were constructed separately by the neighbor-joining and the Tamura-Nei models and were used to analyze the relationships between taxa by nucleotide sequence analysis (29,30). We used Felsenstein's bootstrap testing to calculate the associated taxa clustered in the bootstrap values (1000 times) (31). The final dataset from the ITS ribotype included a total of 239 positions.
In the 72 T. gallinae-positive samples, the ITS region was successfully amplified through PCR by ITS region-specific primers, resulting in a fragment of approximately 300 bp (Table 1). Of these positive PCR samples, 48 were sequenced (Table S1). The phylogenetic tree for the ITS region contained 12 novel lineages, in addition to three previously described ribotypes, A (29 samples), C (six samples), and II (one sample) (Fig. 1). Most samples clustered in lineage A (32) (GenBank GQ150752), which was found in 17 samples, including columbids (feral pigeon), passerines (common mynah), and galliforms (chicken, turkey). Eighteen samples of columbids (feral pigeon) were predominantly infected by lineage C (33) (GenBank EU215362). One isolate of T. gallinae obtained from feral pigeons clustered in the same clade as the sequence of the T. gallinae ITS region lineage II obtained from a racing pigeon (34) (GenBank FN433474). Interestingly, a comparison of the complete ITS region of all novel sequences determined in this study revealed 12 different sequence lineages (KSA1-KSA12), and all obtained sequences were uploaded to GenBank (accession MK771125-MK771135 and MK765029). These lineages demonstrated a degree of sequence divergence between each other, and each formed a separate branch. All of these occurred in columbids (feral pigeon). To our knowledge, these sequences types (ITS; KSA1-KSA12) are novel and reported for the first time in the present investigation in Riyadh.

DISCUSSION
Trichomonosis is an avian disease caused by the protozoan T. gallinae and characterized by a great variation in strain virulence and pathogenicity, affecting different bird species worldwide. In the last few years, interest in the investigation of T. gallinae strains has increased and spurred the introduction of new molecular techniques. In this paper, we studied the prevalence and genotypes of T. gallinae strains in avian species in Riyadh, including birds found at the poultry market and those that were caught in the wild. We demonstrated, to our knowledge for the first time, strains of T. gallinae by comparing the ITS ribotype isolated from different bird species in Riyadh.
We found T. gallinae to be present in four of the five bird species examined, confirming the cases in Saudi Arabia, with an overall prevalence rate of 26.4%. The prevalence of T. gallinae strains found in the poultry market in Riyadh was much higher in domestic pigeons than in other bird species. This result was predictable because feral pigeons have been considered the primary host of T. gallinae strains (18,35). However, this parasite can also affect chickens [34]. This study is among the few that have reported infection with T. gallinae in the common mynah (36) and turkey (37). Although all screened samples of duck in this study were negative for T. gallinae, infection with this parasite in the Anatidae family has been confirmed in a few studies (38). The prevalence rates were found to be higher in birds from the poultry market, especially in pigeons, compared with those caught in the wild, which might implicate the pigeons from the poultry market as a mode of transmission of T. gallinae among and within bird species. Although signs of infection have been found to be low in adult birds (39), we found a relatively high observation of lesions in both pigeons and chickens (43.46% and 33.33% of positive screened birds, respectively). Interestingly, 9 of 11 (81.82%) of the new strains of T. gallinae reported here were isolated from the infected birds with lesions. These birds might not have yet developed any form of tolerance or resistance to these strains of the parasite.
Trichomonas gallinae genotypes were divergent between different bird species, although our sample size was too small to be conclusive. Results revealed the existence of several T. gallinae strains circulating in Saudi Arabian avifauna. In this study, a phylogenetic analysis was used to identify 15 unique sequences, which were clearly divided into different branches depending on the ITS ribotype (Fig. 1). Of these 15 sequences, 12 were novel, and three have been previously described. In this study, we further presented data on the genetic diversity of T. gallinae found in Saudi Arabian birds, with different nomenclatures to differentiate between them. Of the 15 genetic lineages found in this study, three genotypes have been described in previous studies: genotypes A (32), C (33), and II (34). Regarding the previously described lineages and NCBI, KSA1-KSA12 might be the newly detected lineages because they have not been described in previous studies. Furthermore, these lineages appear to be distinct from lineages A/B and C/D/E (32,33); thus, they might not be as common or widespread as lineages A, B, C, and II (33,32,40) because they were found only in pigeons in Saudi Arabia. However, judging by the overall occurrence of these three genotypes, our findings propose a widespread distribution of these genotypes among different bird species (32,36,40,41,42,43).
In this study, we found that most birds were infected by T. gallinae ribotypes A and C, which is consistent with previous studies that found that the pathogenicity of genotype A was strong compared with genotype C, as demonstrated by the dramatic decline in the U.K. finch population in 2007 (28, 41). The C genotype isolated in our study was identified in pigeons and chickens with lesions and was an apparently pathogenic lineage. We also found this symptom in birds infected with a novel genotype, as described in this study. However, most of the birds infected with genotype A appeared to be healthy. Several pigeons and chickens with ulcerations in the oral cavity were also observed to be infected with genotype C or a novel genotype. In 2009, Sansano-Maestre et al.
(13) observed in Spanish samples that genotype C tended to be less virulent than genotype A, commonly associated with macroscopic lesions. Since then, a virulent clonal isolate of genotype A has been associated with both pigeon and passerine pathology and mortality (41). However, because virulence is likely a rapidly evolved trait (44), different strains within distinct genetic lineages will vary markedly in virulence. In support of this postulation, our study seems to suggest that it is genotype C rather than a genotype A with which the pathology of T. gallinae infection is primarily associated. Further research on this avian parasitic species, its genetic diversity, and its association with pathogenicity on a global scale will elucidate to what extent, if any, virulent traits can be ascribed to genetic lineages of this parasite. Additionally, further analyses using more isolates and a multilocus sequencing approach are required.