Samples Collection

The focus of the current research was on domestic pigeons in Basrah province, Southern Iraq. This study spanned from May 2022 to October 2022. We selected Alzubir, Al- Hartha, and Abo Alkasib, regions which are famous for pigeon raising and have a significant number of pigeon breeders. After the initial screening, fifteen domestic pigeons from veterinary clinics located in in selected area ( Five birds from each clinic) were transported to the Department of Veterinary Pathology and Poultry Diseases at the Faculty of Veterinary Medicine, University of Basrah-Iraq. Six pigeons that showed severe signs of cankers were euthanized and examined individually to record a postmortem change according to [7].  The lesions were scrapped using a sterile surgical blade from the mouth and pharynx and were transferred into a sterile container containing 500 µL of 50% glycerol phosphate buffer solution and stored at -20 C for subsequent DNA isolation [9]. The current study was performed under the permission of the ethical committee in the Faculty of Veterinary Medicine, University of Basrah (Ref. No. 75/2022).

Microscopic Examination

The validation of oropharyngeal swab samples collected from suspected pigeons utilized the wet mount technique in microscopy (microscopic inspection). Swabs were blended with Phosphate Buffered Saline (PBS) and applied to sterile cotton-tipped applicators for observation under a light microscope at 40x magnification, enabling the identification of Trichomonas [10].

DNA Extraction and Polymerase Chain Reaction (PCR)

The DNA extraction from all samples was carried out using the gSYNCTM DNA Extraction kit's rapid procedure, specifically from the caseous masses in the mouth (Geneaid, Korea). The NanoDrop system was employed to determine the quantity of purified DNA. Subsequently, confirmation of all extracted DNA samples was achieved through PCR. The forward primer used was 5'-AACTAATACCAACTTCTTTT-3', while the reverse primer was 5'-TATTCGCGTAGAATAAGAAT-3'. These primers were utilized to amplify a 263 bp segment of the ITS rDNA region for each T. gallinae DNA sample using PCR. The PCR mixture included 25 μl Gotaq master mix (Promega, USA), 1.5 μl of each primer , 150 ng of DNA template, and nuclease free water up to 50 μl per each PCR reaction.  The PCR was done in Simpliamp thermos cycler – Applied bio system, USA. The PCR  program included 94 ̊C initial denaturation for 5 min, followed by 35 cycles at ( 94 ̊C for 30 sec, 46 ̊C for 30 sec and 72 ̊C for 30 sec), which were followed by a final 5 min extension at 72 ̊C to complete the elongation of products [11]. The PCR run was followed by gel electrophoresis of 5μl from each PCR sample on 1.5% agarose gel. After visualization of 5μl of each PCR product on 1.5% agarose gel, 30μl of the  positive PCR amplicons were sent for sequencing at Macrogen company , Korea using the same forward and reverse primers of the PCR in the sequencing process.

Sequence and Bioinformatics Analysis

After visualization of 5 µL of each PCR product on agarose gel, the positive PCR samples were sent for sequencing (forward and reverse direction using the same forward and reverse primers used in the PCR amplification of the ITS rDNA region). The PCR samples were processed and shipped according to the instructions provided by the sequencing provider (Macrogen Inc., South Korea). Sequences of trichomonas were edited, aligned, and evaluated using MEGA 6.06 software. . The chromatograph files containing the genetic data were meticulously inspected for quality. The annotation and variances were arranged in clean chromatographs that show the quality of sequencing. This step aimed to ensure the quality and accuracy of genetic information. The comparison also included contrasting the detected nucleic acid sequences of the samples with the retrieved reference sequences from the gene bank database to determine variation in those ITS rDNA region sequences.

Cladogram trees were constructed using the Maximum Likelihood method and the Tamura-Nei model were employed for tree construction, allowing a nuanced analysis of taxonomic relationships through nucleotide inspection [12]. The robustness of the cladogram trees was assessed using Felsenstein’s bootstrap test. This statistical method involved resampling the dataset multiple times (2,000 times) to calculate bootstrap values. These values provided insights into the reliability and support for taxa clusters identified in the phylogenetic trees [13].

Results

During the study period, many pigeons were examined in the targeted clinics, but only those that were clinically positive were targeted. The severity of the disease varied in between the examined pigeons. All pigeons  had lesions, whereas 6 of 15 birds exhibited more severe characteristic oral lesions.  Most of the affected birds showed identical typical signs such as opening mouth and complex repeated swallowing movements. The oropharyngeal cavity of the affected birds displayed a whitish to yellowish nodule of varying sizes, accompanied by ulceration and inflammation as depicted in (Figure 1). 

The severity and spreading of lesions were more sever in six birds which were noticed in the esophagus, proventriculus, and crop; this led to more severe clinical signs, as specific signs of illness, including weight loss, lethargy, reluctance to eat, and greenish diarrhea. These pigeons exhibited nasal discharge and neurological symptoms such as head tremors and reduced flying ability. In addition, dehydration, dull eyes, and respiratory distress, with complex and open-mouthed breathing blocking the airways and causing the birds to pass away.

Microscopic Examination

The distinctive jerky movement of the parasite was observed in the motile organism under a microscope with 40x magnification, revealing vibrant and dynamic activity. Identification of the parasite was established by discerning its unique physical attributes. The parasite exhibited a pear- or oval-shaped structure with a single undulating membrane, four anterior flagella, and a prominent axostyle at the posterior end, as illustrated in (Figure 2).

Detection of DNA by PCR

The DNA extracted from T. gallinae samples was subjected to amplification through PCR, employing both forward and reverse primers. The samples that underwent PCR analysis are depicted in Figure 3. Following analysis on an agarose gel, DNA bands of 263 base pairs in length were identified. Proper-sized DNA bands were observed in six samples.

Sequence and Bioinformatics Analysis

The PCR results of six samples were sequenced using ITS-rDNA-specific primers in both directions. These sequenced samples were then submitted to the GenBank database through the NCBI website, with accession numbers OP268446.1, OP268447.1, OP268448.1, OP268449.1, OP268451.1, and OP268452.1. In Figure 4, the phylogenetic analysis of the current study's samples is presented alongside sequences of Trichomonas spp., including genotypes A, B, and C of T. gallinae from different countries. All samples from the present study were grouped with genotype B of T. gallinae from China, Iran, Iraq, Austria, and Saudi Arabia.

Figure 5 illustrates a haplotype network analysis of T. gallinae genotype B. Our findings revealed four haplotypes associated with T. gallinae genotype B. Specifically, present study samples 3, 4, 5, and 6 were clustered with sequences of haplotype 1 (H_1), which also included NCBI reference sequences such as T. gallinae (MK418243.1, Iraq), T. gallinae (MK418241.1, Iraq), T. gallinae (MK418242.1, Iraq), T. gallinae (MK418257.1, Iraq), T. gallinae (MK418258.1, Iraq), T. gallinae (MK418239.1, Iraq), T. gallinae (KT869150.1, Iran), T. gallinae (KT869151.1, Iran), T. gallinae (KT869153.1, Iran), T. gallinae (KT869154.1, Iran), T. gallinae (KT869156.1, Iran), T. gallinae (MK765029.1, Saudi Arabia), T. gallinae (EU215364.1, USA), and T. gallinae (MH733816.1, China). However, present study samples 1 and 2 were separated in a single haplotype (H_2), while H_3 and H_4 only contained NCBI reference sequences.

Figures & Tables

Discussion