MORPHOLOGICAL IDENTIFICATION AND PREVALENCE OF GASTROINTESTINAL HELMINTHS IN BACKYARD CHICKEN FROM SELECTED AREAS OF BANGLADESH

Received 10 March, 2021 Revised 20 April, 2021 Accepted 26 April, 2021 Online May, 2021 -----------------------


INTRODUCTION
Poultry, especially chicken is one of the most intensively reared domesticated species, and is the most profitable livestock production businesses (FAO, 1987). Its importance in developing countries through improving the nutritional status and income generation of many small farmers have been recognized by various scholars and rural development agencies in the last two decades (Eyinnaya, 1992). The purposes of backyard chicken production are for women empowerment and income generation through egg production and hatching, consumption, cultural and religious ceremonies (Moges et al., 2010). Like other countries, poultry production is a promising sector in Bangladesh which is increasing day by day contributing approximately 37% of total animal protein (Ahmed and Islam, 1990). However, Poultry diseases are the major hindrances for poultry production, resulting in decrease economic returns, and may therefore; negatively affect the development of the industry (Abebe et al., 1997). Among poultry diseases, helminthosis is considered to be the most important disease of backyard chicken, and are considered as the major cause of ill-health and loss of productivity in different parts of the world (Yimer et al., 2001). This problem is seen wherever poultry are raised, whether in large commercial operations or in small backyard flocks, economic losses due to the parasites can be significant (Fatihu et al., 1991). Gastrointestinal parasites which invade the host possess morphological and physiological features such as small thread like cylindrical body; hooks and hard body cuticle enhance their adaptation to long living and existence in their hosts. These parasites constitute a major factor limiting factor for the poultry industry (Soulsby, 1982).
Previous researches on gastrointestinal helminths were conducted to observe the prevalence by many authors in Bangladesh (Rabbi et al., 2006;Alam et al., 2014;Ferdushy et al., 2016). These researches had very limited morphological data regarding to the helminths. Identification of the helminths was based on the size and location of helminths. These could only identify the genus of a helminth. Therefore, a study was undertaken to determine the morphological identification and prevalence of gastrointestinal helminths in backyard chicken from selected areas of Bangladesh. The study may provide useful information on the prevalence of helminths parasites in backyard chicken and may aid in the identification of the helminths described here, and thereby, assist in designing effective control strategies.

Sampling areas
The study was carried out from April to July, 2018. The gastrointestinal tracts of the slaughtered chickens were collected from the rural areas of four districts of Bangladesh, namely Dhaka (36), Bandarban (24), Mymensingh (24) and Pabna (24). Finally, the identification was performed in the Microbiology & Parasitology Laboratory, Sher-e-Bangla Agricultural University, Dhaka.

Collection of samples
Around noon in each sampling day, a batch of 6 intestines was randomly picked up from a group of intestines, slaughtered in the rural market between early morning and at the time of collection. The intestines were placed in separate, labelled polythene bags and transported to the laboratory maintaining a cool chain protocol. Firstly, the whole intestines were separated into three parts, namely small intestine, large intestine and caecum. The entire length of each part of the intestine was incised longitudinally, and the contents were emptied into sieves placed on large clean plastic cups with labelling. Contents were washed in normal saline and examined under light. Larger helminths were collected directly by curved needle or forceps, and smaller ones were isolated through dropper. Worms were counted and grouped on the basis of gross morphology before being stored in plastic bottles containing 70% alcohol according to the method described by Permin and Hansen (1999).

Processing of cestodes and trematodes
In case of trematodes and cestodes, permanent slides were made after the staining of the specimens. For this purpose, trematodes and cestodes were collected from plastic bottles containing 70% alcohol. Then the specimens, preserved in 70% alcohol were flattened between two glass slides with slight pressure and fixed in 70% alcohol until future works. After flattening for a week, the specimens were dipped in 50% alcohol for one hour and then into distilled water for another one hour. Then the specimens were transferred in Haematoxylin-Carmine solution and kept overnight for staining. The excessive stain was removed by 3% HCl-Alcohol. The stained specimens were washed with ascending grades of

Processing of nematodes
The nematodes were washed well in water to remove the preservatives, dehydrated in 70-90 % alcohol depending on the thickness of the worm and cleared by submerging them in lactophenol. Then the nematodes were examined under microscope and photographs were taken from different body parts as an aid for identification (Anderson et al., 2009).

Morphological identification
Through the microscopic examination of all 108 samples, five helminths were confirmed. These include two species of nematodes (A. galli & H. gallinarum), two species of trematodes (E. revolutum & C. verrucosa) and only one species of cestode (R. tetragona).

Ascaridia galli
Adult worms, recovered from small intestines, were yellowish white in color and semitransparent. The males were generally 45 to 60 mm long, while females were longer, ranging from 70 to 90 mm. The oral opening was surrounded by three prominent lips. Two conspicuous papillae were observed on the dorsal lip and one on each of the subventral lips (Fig. 1a). The esophagus was filariform and the intestine was simple where the whole body was enclosed in a tough proteinaceous covering called cuticle. The cuticle was distinctly striated and cuticular alae are poorly developed (Fig. 1b). Morphologically, sexual dimorphism in ascarids characterized by ventrally coiled tail with precloacal sucker was found in males, and a blunt and rounded posterior end in females. The posterior portion of female also possessed a single large anal opening just before the extremity (Fig. 1c). The posterior end of male was more complex. There were two prominent apertures, anus towards the posterior end and precloacal or preanal sucker immediately anterior to the spicules. The precloacal sucker was supported by a sclerotized ring which serves the functions as aid to attach during copulation. The worms had two well-developed unequal spicules at the posterior end (Fig. 1d). All of these morphological characteristics correspond to the properties to the Genus Ascaridia, and the Species A. galli.

Heterakis gallinarum
The adult worms, collected from the caecum, differed in length. The females (8 to 13 mm) were generally longer than that of the males (7 to 11 mm). They were small, white in colour and had three well-defined lips with slightly curved head when collected (Fig. 2a). The esophagus was connected with a short narrow anterior portion (pharynx) and ended in a well-developed bulb (Fig. 2b). The tail end of female was elongated, gradually tapered. The anal opening was at the posterior part of body. The vulva of the female was located at the middle of the body. Male worms had stylet-like tail end that smoothly tapered posteriorly. The worms had two well-developed unequal spicules at the posterior end (Fig. 2c). The preanal sucker could be easily seen, which was round, well-developed and surrounded by a chitinized ring. Eggs in the uterus were ellipsoidal, with a thick, smooth shell, containing a single cell (Fig. 2d). Each of every morphological characteristic is identical to the Genus Heterakis, and Species H. gallinarum.

Reillietina tetragona
Multiple mature cestodes, measuring 12-25 cm long were isolated from small intestines. These cestodes were whitish in color, elongated, dorso-ventrally flattened. The body was divided into the head region called 'scolex', followed by an unsegmented 'neck', and then by highly segmented body called 'strobila' (Fig. 3a). Each scolex beared an apical rounded rostellum, which is medium and armed with many minute hammer-shaped hooks arranged in single row. The scolex was also surrounded by four ovoid suckers which are lined with several rows of spines (Fig. 3b). The strobila was composed of a series of body segments called proglottids, gradually increasing in size posteriorly. The mature segment was longer than broad and the common genital pores were single and being in front of the anterior 1/3 of the lateral margin of the mature segment. Each mature proglottid beared a set of male and female reproductive organ (Fig. 3c). Testes were located on both sides of the ovary and behind vitellarium. These features matched with the morphology of the Genus Reillietina, and Species R. tetragona.

Catatropis verrucosa
A large number of minute flukes were recovered from the caecum of the poultry intestine which was 1.5-2.0 cm long and upto 0.5 cm wide. The specimens had small muscular body which was dorsoventrally flattened and narrowing anteriorly. The pharynx was absent and the esophagus was very short (Fig. 4a). Cup-shaped oral sucker was terminal while there was no ventral sucker. Long caeca were bifurcated, smooth, extending posteriorly between the uterine loops and vitelline follicles, then passed through the testes and ovary, and finally terminated blindly at the level of excretory pore. Cirrus sac was elongated, containing prostatic cells and coiled seminal vesicle. Genital pore was median, closely posterior to the caecal bifurcation (Fig. 4b). There were two testes which were irregularly lobed, located on extracaecal field in posterior third of the body. Ovary was trilobed, situated at the testicular level (Fig. 4d). Uterus had a number of closely packed loops, overlapping cirrus sac, reaching anteriorly up to the level of Mehli's gland. Uterine loops were 18 in number (Fig. 4c). Vitellaria were arranged extracaecally but at some places it overlapped the caeca, which are the special morphological features of the Genus Catatropis, and Species C. verrucosa.

Echinostoma revulotum
The echinostomes were recovered from the large intestine of chicken. The body was muscular, dorsoventrally flattened and 'C' shaped in appearance. Pharynx was absent and the esophagus was very short. All flukes were 6-8 mm long and 1.5-2 mm wide and had a well-developed head collar bearing 37 spines (5 angle spines, 6 lateral spines on each side and 15 dorsal spines) (Fig. 5b). Adult flukes had a short forebody and the ventral sucker was large (Fig. 5a). There are two testes, arranged in a tandem position, located at the posterior part of the body. Testes were elongated with smooth margin and slightly separated from each other. The anterior testis was shorter and wider than the posterior testis. The ovary was oval, median, and transversely located between the posterior end of the uterus, and cranial margin of the anterior testis (Fig. 5c). The cirrus sac was oval and located transversely between the level of caecal bifurcation and anterior border of ventral sucker. Both intestinal caeca ran upto the posterior end of the fluke (Figure 5d). Multiple eggs were located in the uterus. The eggs were oval, large, thin shelled, operculated and contained unsegmented ovum (Fig.  5d) which is the special morphological characteristics of the Genus Echinostoma, and Species E. revolutum.

Prevalence
The study was carried out in a total of 108 gastrointestinal tract of backyard chicken. All examined intestine were found infected with various species of gastrointestinal helminths, comprising two species of nematodes, two species of trematodes and only one species of cestode. These parasites were found in different locations of the gastrointestinal tracts of backyard chicken.

Overall prevalence of helminths in backyard chicken
The encountered nematodes were Ascaridia galli and Heterakis gallinarum, of which A. galli (43.51%) was the most prevalent followed by H. gallinarum (28.70%). The recovered trematodes were included Catartropis verrucosa and Echinostoma revolutum where C. verrucosa (21.29%) was the most prevalent followed by E. revolutum (6.48%). The cestode encountered was only one species, Raillietina tetragona (67.59%). The results also showed that the huge number of the helminths prefer to colonize the small intestine than the large intestine. Some of the helminths were found in the caecum. No helminth was recovered in the crop and gizzard. The overall prevalence with their predilection site is given in Table 1.

Prevalence of helminths in different geographies
The samples were collected from Dhaka (n=36), Mymensingh (n=24), Pabna (n=24) and Bandarban (n=24). Helminths were recovered from different geography were given in the Table 2. Total five species of helminths were collected from Dhaka and Pabna. From the samples of Mymensingh, E. revulotum was not found whereas no trematode was from Bandarban.

Single and mixed type of infection
Examined gastrointestinal tract of chicken were infected by one or more species of helminth parasites (Table 3). Among the 108 intestines, 32 were infected with single species of helminths (29.62%) and rest 86 were infected with multiple species of helminths. In case of the mixed infection, 47 chicken were infected with two species of helminths (43.51%) and 39 chicken were infected with more than two species of helminths (36.11%).

DISCUSSION
In Bangladesh, there are very limited morphological data regarding precise identification of the helminths in backyard chicken. Only the gross morphology and the predilection site in host had been used as keys for identification of the helminths. Therefore, detailed morphological observations have been conducted in this study to provide a basis for identification of the helminths in backyard chicken. Both male and female A. galli possesses all the salient features of the ascarid nematodes described by Kung (1949 Whitish, highly elongated, dorso-ventrally flattened cestodes were identified as R. tetragona isolated from small intestine of poultry. The detailed moephological properties of these cestodes were identical to the findings of many authors (Soulsby, 1982;Ramadan and Zanda, 1991;Ahmed and Nabila, 2004). The very small trematode, C. verrucosa, recovered from the caecum of the poultry, were dorsoventrally flattened, attenuated anteriorly and broadly rounded posteriorly, containing 18 uterine loops. The shape and orientation of testes and ovary along with the other morphological characteristics of this fluke were very similar to the previous reports (Odening, 1966;Dvorjadkin, 1987;Kanev et al., 1994). Morphological identification of Echinostoma species in the 37-collar-spined 'revolutum' group was difficult due to the contradictory data from different hosts and geographical locations. In this study, the identification of E. revolutum was done by description of different authors (Kanev, 1994