Trichomonas gallinae Identification and Histopathological Study in Pigeon (Columba livia domestica) in Baghdad City, Iraq

Trichomonas gallinae causes avian trichomoniasis, which is one of the most common protozoan infections in birds worldwide. Therefore, this study was conducted to investigate and identify the Trichomonas gallinae in domestic pigeons (Columba livia domestica) by microscopic examination (direct smear and Giemsa stain) and histopathological examination in Baghdad city, Iraq, during the period from beginning of October 2018 to end of March 2019. Giemsa-stained cytoplasm with light purple and nucleus with dark purple, clarification of flagella, nucleus, and cytoplasm very well. Histopathological findings of infected birds showed gross existence of yellowish white caseous necrotic material in the oral cavity and esophagus. The histopathological examination in the larynx, esophagus, trachea, crop, liver, and lung as infiltration of inflammatory cell mainly (heterophils); thickening of mucosa because of extensive infiltration of heterophils and disruption of esophageal gland; the thickness in bronchi wall of lung due to glandular hyperplasia and muscular fibroplasia, in liver focal necrosis of parenchyma with mononuclear cell (MNCs) infiltration and granuloma composed of MNCs and heterophils. The current study may contribute to determining the histopathological changes of esophagus, trachea, crop, and liver of trichomoniasisinfected pigeons. B *Correspondence: laylafadhil71@gmail.com Received: 23 June 2020 Accepted: 1 September 2020 Published: 28 December 2020 DOI: https://doi.org/10.30539/ijvm.v44i(E0).1022 This article is an open access distributed under the terms and conditions of the Creative Common Attribution License (CC BY 4.0)


INTRODUCTION
irds form part of the human diet consumption. Some are used for breeding, decorative or entertainment purposes, while other birds are used for scientific experiments or teaching the art of mummification (1). However, birds like any animals can suffer from diverse types of disease (viruses or infectious diseases). Pigeons, for example, may infect with a group of external parasites such as lice and ticks (2). Pigeons can be infected by nematodes and cestoda parasites, such as the species of Raillietina, Capillaria and Ascaridia, as well as protozoa parasites such as Eimeria spp., Cryptosporidium spp. and Trichomonas spp., that leads to trichomoniasis (3). Avian trichomoniasis is protozoan disease caused by Trichomonas gallinae, which is a mitochondrial anaerobic protozoan, a flagellated parasite belongs to the class Zoomastigophorea and order Trichomonadida (4). Trichomonas infects the upper digestive tract of birds and this parasite infects a widespread of birds, such as pigeons, chickens, turkeys, and

A B S T R A C T
Trichomonas gallinae causes avian trichomoniasis, which is one of the most common protozoan infections in birds worldwide. Therefore, this study was conducted to investigate and identify the Trichomonas gallinae in domestic pigeons (Columba livia domestica) by microscopic examination (direct smear and Giemsa stain) and histopathological examination in Baghdad city, Iraq, during the period from beginning of October 2018 to end of March 2019. Giemsa-stained cytoplasm with light purple and nucleus with dark purple, clarification of flagella, nucleus, and cytoplasm very well. Histopathological findings of infected birds showed gross existence of yellowish white caseous necrotic material in the oral cavity and esophagus. The histopathological examination in the larynx, esophagus, trachea, crop, liver, and lung as infiltration of inflammatory cell mainly (heterophils); thickening of mucosa because of extensive infiltration of heterophils and disruption of esophageal gland; the thickness in bronchi wall of lung due to glandular hyperplasia and muscular fibroplasia, in liver focal necrosis of parenchyma with mononuclear cell (MNCs) infiltration and granuloma composed of MNCs and heterophils. The current study may contribute to determining the histopathological changes of esophagus, trachea, crop, and liver of trichomoniasis-infected pigeons.
other poultry worldwide (5). Infection by Trichomonas gallinae (T. gallinae) may occur without signs while it may lead to death with intermediate symptoms including anorexia, vomiting, ruffled feathers, diarrhea, dysphagia, dyspnea, weight loss, and increased thirst (6,7). Several reports indicated that many large events of avian mortality have been associated with Trichomoniasis (8,9). The main host of T. gallinae is domestic pigeon (Columba livia domestica), this pigeon breeds a role in the spread of this disease. Young pigeons are frequently infected by these protozoa and can die from the infection, but adult birds' species may act as a carrier with no sign (10). Therefore, this study was designed to study the histopathological changes that occur in some organ tissue of pigeon that infected with T. gallinae in Baghdad, Iraq.

Birds and Study Area
The procedures used in this study were reviewed and approved by the scientific committee at the University of Baghdad's College of Veterinary Medicine in compliance with animal welfare ethical standards.
A total of 180 pigeons (Columba livia domestica) of different in ages and sexes were brought from local markets at Baghdad city, Iraq during the period from the beginning of October 2018 to the end of March 2019.

Microscopic Examination
Oropharyngeal swab samples were taken from the mouths and crop of pigeons randomly by using sterile, premoistened, cotton-tipped applicators which was confirmed by microscopic examination (wet mount method). The Trichomonas were identified if they motile and flagellated protozoa were observed under light microscope (11).

Staining
Swabs were taken from the oral cavity and crop of pigeons randomly. Swabs were blended with phosphate buffered saline, fixed with methanol, and dried at room temperature, then stained with Giemsa stain prepared by adding 1 part of Giemsa stain solution to 9 part of buffer solution (pH 7.2) (12). The slides were examined under the light microscope with oil immersion lens (100×). The parasite was identified according to (13).

Histopathological Examination
Samples were taken from the oral cavity, esophagus, crop, liver, larynx, trachea, and lung of 30 birds and placed in 10% neutral buffered formalin before being sent to the pathology laboratory at the College of Veterinary Medicine, University of Baghdad for histopathological examination.
Formalin fixed samples were sectioned at 5 µm thickness and stained with Hematoxylin and Eosin (14).

Gross Examination
The gross lesion in the infected birds showed to be white to yellowish nodule of varied sizes in the oropharyngeal cavity associated with inflammation and ulceration. The lesion extended sometimes into esophagus, crop and proventriculus blocking of the respiratory tract and led to the death of the birds (Figure1).

Figure1. Gross lesions of a domestic pigeon (Columba livia domestica)
naturally infected with Trichomonas gallinae shows white material to yellowish in the pharynx and oral cavity of the upper gastrointestinal tract (black arrow)

Microscopic Examination
Microscopic examinations were done to the direct wet mount that taken from oral cavity, pharynx, and crop swab of the domestic pigeon. The motile organism was seen under the microscope using x40, showed vibrant active movement of the parasite was called jerky movement. The parasite was identified according to characteristic morphological features. It had a pear-shaped or oval shape with four anterior flagella and one undulating membrane in addition to having prominent axostyle at the posterior end of parasite ( Figure 2).

Staining Examination
The primary purpose of staining was to optimize the visualization of key anatomic structures to facilitate accurate identification of an organism. In this study, it was used Giemsa stain. Microscopic structures of T. gallinae appeared when smears prepared and stained by Giemsa. Giemsa stained the cytoplasm with light purple and nucleus with dark purple color and visualizing the flagella and both of nucleus and cytoplasm were also very well ( Figure 3).  Multiple caseous necrotic lesions were recognized in lamina propria associated with focal epithelial sloughing, the laryngeal mucous glands showed various degree of sever degeneration with evidence of sloughed glandular epithelium with necrotic debris in lumen (Figure 4), the laryngeal was stratified epithelium exhibited focal keratinization with diffuse mononuclear cells (MNCS) infiltration and congestion in the lamina properia as well as slight lymphoid depletion recorded in sub mucosal layer. Cystic glandular dilation with flat epithelial lining and luminal debris were also noticed. In addition, laryngeal findings showed moderate to severe congestion of laryngeal muscular coated with evidence of vacuolation of muscle bundles, some laryngeal glands appeared lining with hyper plastic epithelium.

Trachea
The main histopathological finding of trachea was cystic dilation of major mucus glands with evidence of hyperactivity. This was associated with moderate hyperemia of tunica properia, severe degeneration of tracheal submucosal glandes with sub mucosal blood vessel congestion with no clear lesion in sub mucosal layer was noticed. Another section showed severe hyperatrophy of goblet cell with evidence of degeneration of some mucosal glands associated with vascular congestion of sub mucosal tissue ( Figure 5).

Esophagus
The stratified squamous epithelial of esophagus in many parts was necrotized and debris was seen in the lumen. Moderate to severe disruption of esophageal submucosal glands with cellular infiltration and blood vessel congestion were also noticed in (Figure 6). There was focal MNCS infiltration composed mainly of lymphocyte that was seen between muscular bundles (Figure 7). Intense MNCS infiltration between mucus gland was also seen (Figure 8).

Crop
Crop lesion was characterized by focal surface mucosal desquamation with focal moderate necrotic lesion of the mucosal epithelia appeared with sloughed epithelium and focal MNCs with heterophils aggregation was also seen ( Figure 9).  Focal necrotic foci with congestion were observed in lamina propria accompanied with loss of sub mucosal papillae, as well as sub mucosal shortening of mucosal folds, together with severe fragmentation of muscle bundles and separation with intra-muscular edema and blood vessel congestion ( Figure 11).

Lung
Pulmonary parenchyma showed multiple necrotic foci were surrounded by various type of MNCS forming granulomatous like lesion with giant cell formation ( Figure  12) associated with severe destruction in adjacent parenchyma. The primary bronchi also showed moderate thickness of its wall due to glandular hyperplasia and muscular fibroplasia. Severe pulmonary congestion was detected mainly in arterial tissue (Figure13) while slight changes were detected in para bronchi as well as diffuse

Liver
Multiple MNCS and heterophils aggregation were observed in liver tissue mainly around blood vessels with evidence of sinusoidal dilation, while other finding showed granulomatous like lesion composed mainly of MNCS. Severe vacuolation of hepatocytes were also observed with evidence of hepatic steatosis, per vascular MNCS aggregation accompanied with appearance cytoplasmic fat droplets in adjacent hepatocytes, large MNCS aggregation was noticed in liver parenchyma with cellular swelling of adjacent hepatocytes. While other sections showed focal necrosis of parenchyma with MNCS and heterophils infiltration accompanied with individualization and atrophy of adjacent hepatocytes ( Figure 14).
Portal MNCS infiltration with heterophils also recorded together with evidence of portal fibrosis and ductal dilation. Severe congestion and dilation of hepatic sinusoids resulted in disruption of hepatic cords ( Figure 15) accompanied with severe congestion and dilation of central vein were also reported in many sections accompanied with focus MNCS infiltration, moderate portal enlargement due to marked MNCs infiltration and portal vein congestion, with evidence of portal fibrosis and focal perivascular infiltration. Other hepatic manifestations showed moderate number of apoptosis mainly adjacent to the central vein and granuloma was observed composed of MNCS and heterophils (Figure16). There was massive hemorrhage and sinusoidal congestion resulting in severe atrophy of hepatic cords.

DISCUSSION
Occluded mouth and esophagus of pigeons with cheesy white to yellow-colored materials were also described previously by (14).
Histopathological examination of tissues in domestic pigeon that infected naturally with T. gallinae in the present study revealed multiple caseous necrosis were mainly seen in larynx, esophagus, and lung and diffuse MNCS infiltration and congestion in lamina propria. Severe degeneration in mucosal gland, cystic glandular dilation with luminal debris in the larynx, trachea revealed cystic dilation mucus glands hyperactivity with moderate hyperemia of tunica propria and severe degeneration of tracheal glandes with sub mucosal blood vessel congestion were similar to observation seen with El-khatam (14) in Egypt when it was recorded in trachea and larynx severe inflammatory cell infiltration. Abd El-Rahman (15) revealed existence of necrosis and infiltration of inflammatory cells mainly eosinophil in the mucosa of esophagus and these results agreed with our study that revealed necrosis of epithelial cell, focal MNCS infiltration especially lymphocyte between muscular bundles, infiltration of inflammatory cell between mucus glands. The crop and proventriculus revealed desquamation and sloughed in epithelial cell, focal MNCs with heterophils aggregation, infiltration of inflammatory cells mainly heterophils, necrosis in sub mucosa, focal disruption of periventricular glandular tissue with diffuse sub mucosal MNCS infiltration, edema, and congestion in the proventriculus. The above results were in agreement with the results of Abd El-Rahman et al. (15) in Egypt, Begum et al. (16) in Bangladesh, Al-Sadi and Hamodi (17) in Mosul, and Jaafar (18) in Baghdad in which they recorded necrosis in the sub mucosa of crop and desquamation in the epithelial cells of proventriculus. lung showed multiple necrotic foci surrounded with various type of MNCS forming granulomatous like lesion with giant cell formation and congestion in pulmonary tissue, moderate thickness in bronchi wall due to glandular hyperplasia and muscular fibroplasia, and liver tissue showed multiple MNCs and heterophils aggregation necrosis in parenchyma, congestion and dilation in hepatic sinusoids, granuloma composed of MNCs and heterophils. The above results agreed with studies of (8,14). The current study may contribute to determining the histopathological changes in the esophagus, trachea, crop, liver, and liver of pigeons that infected with trichomoniasis.