Cronicon OPEN ACCESS EC CLINICAL and EXPERIMENTAL ANATOMY Research Article

Cronicon OPEN ACCESS EC CLINICAL AND EXPERIMENTAL ANATOMY Research Article

Occurrence of Metacercarial Cyst of Ascocotyle (Ascocotyle Sp.) in the Gills of Ruby-Red-Fin Shark (Rainbow Shark) Epalzeorhynchos frenatum (Teleostei: Cyprinidae): Light Microscopic Study

Sara Abdelhakiem1, Soha A Soliman2, Hanan H Abd-Elhafeez3*, Enas Abdel- Hafez4 and Rania S Zaki5

1Lecturer, Department of Zoology, Faculty of Science, Assuit University, Egypt 2Asistant Professor, Department of Histology, Faculty of Veterinary Medicine, South Valley, Qena, Egypt 3Assistant Professor, Department of Anatomy, Embryology and Histology, Faculty of Veterinary Medicine, Assuit University, Egypt 4Professor, Department of Anatomy, Embryology and Histology, Faculty of Veterinary Medicine, Assuit University, Egypt 5Lecturer, Department of Meat Hygiene, Faculty of Veterinary Medicine, New Valley University, Egypt

*Corresponding Author: Hanan H Abd-Elhafeez, Assistant Professor, Department of Anatomy, Embryology and Histology, Faculty of Veterinary Medicine, Assuit University, Egypt.

Received: May 07, 2019; Published: August 17, 2019

Abstract The aim of the current study was to estimate the metacercarial infection in Ruby-Red-Fin Shark (Rainbow Shark), Epalzeorhyn- chos frenatum

(Teleostei: Cyprinidae). Samples of fish were commercially purchased from an ornamental shop in Assuit city, Egypt benzocaine (4 mg/L). Fish were ranging from 10 to 12 cm in standard body length. Fish were deeply anesthetized with benzocaine and brought to the laboratory. Fish samples were used for light histological analysis. All fish measured and deeply anesthetized with Ascocotyle (Ascocotyle sp.). The meta-

(4 mg/L). Using Histological examination, we identified a heterophyid parasite metacercaria; Cartilage enlargement occurs as a result of invaginations of the perichondrium into the cartilage mass and kl. proliferation of the cercarial cyst was located inside the cartilaginous support of the gill arches which were surrounded by Thick fibrous perichondrium. - tilage was reported with formation of tunnels in the cartilage. Increase rodlet cells in the branchial epithelium and in the underlying chondrocytes progeny. As well as hyperplasia, hypertrophy, and fusion were present in the affected gill filaments. Erosion of the car

Lymphatic cells. Poor controls of diseases permit the transport of parasites between different countries and increase the prevalence connective tissue. The disappearance of the gill lamellae could affect respiratory efficiency. Lymphatic spaces were rich in numerous of infections.

Keywords: Ascocotyle; Ruby-Red Fin Shark; Chondrocytes; Proliferation

Introduction Trematodes of the family Heterophyidae are widely distributed in different piscivorous [1]. Ascocotylinae have been described as a subfamily of heterophyid species which include three subgenera, Ascocotyle, Phagicola and Leighia [2]. The metacercaria of the genus As- cocotyle

was found encyst in different organs of fish [3]. The adult worm is non-pathogenic intestinal parasites of fish-eating birds and - mammals, contrariwise metacercariae have a pathogenic effect on fish in the long term [4]. They are hermaphroditic and one parasite can produce a march with eggs that are passed into the water [5]. They have two intermediate hosts; first intermediate hosts are amnico

Citation: Hanan H Abd-Elhafeez., et al. “Occurrence of Metacercarial Cyst of Ascocotyle (Ascocotyle Sp.) in the Gills of Ruby-Red-Fin Shark (Rainbow Shark) Epalzeorhynchos frenatum (Teleostei: Cyprinidae): Light Microscopic Study”. EC Clinical and Experimental Anatomy 2.7 (2019): 296-304. Occurrence of Metacercarial Cyst of Ascocotyle (Ascocotyle Sp.) in the Gills of Ruby-Red-Fin Shark (Rainbow Shark) Epalzeorhynchos frenatum (Teleostei: Cyprinidae): Light Microscopic Study

297 lid and hydrobiid snails in which eggs are developed to cercaria that leaves and swims out of the snail. The second intermediate hosts

areThe cyprinodontid, cercaria of Ascocotyle centrarchiid, mugilid and poeciliid fishes. - - penetrates the vasculature of the gills to enter the bloodstream and encyst in fish tissues [3,4]. In some spe cies, cercariae were encysted immediately in the gills by a multilayered hyaline around it [5]. The infected fishes were suffering increas gills to infection is considerable due to their external location contacted to water medium. ingly difficult time extracting dissolved oxygen from the liquid medium due to pathological changes of gill filaments [6]. Sustainability of -

The ornamental fishes are one of the species which have economic importance. Poor controls of diseases permit the transport of para Epalzeorhynchos frenatum is commonly known sites between different countries and increase the prevalence of infections [7]. As well the ornamental fish requires the highest level of sanitation and health quality control [8]. Ruby shark belongs to the family of Cyprinidae. Materialsas ruby shark, and red-finned Methods shark and rainbow shark which is distributed in Southeast Asia and indogenous to many countries. from each shop) in Assiut City, Egypt and brought to the laboratory. The standard length body for each sample was measured from the top Sample collection: Samples of 100 Ruby shark fish were commercially purchased from four different ornamental shops (25 samples of snout to caudal peduncle. Fish were deeply anesthetized with benzocaine (4 mg/L) for histological analysis.

Histological investigations

The whole five fish were immersed in a mixture of 20 mL of 2.5% glutaraldehyde and 80 mL 0.1 M Na-phosphate buffer pH 7.2- 7.4). Hoffman [11] keys. The histological sections were stained with Haematoxylin and eosin [12]. Combined Alcian blue Ph 2.5 and PAS stain Then Specimens were processed according to Abd-Elhafeez and Soliman [9]. The parasite was identified according to Scholz [10] and was used to differentiate between neutral and acidic polysaccharides. All staining was cited in Bancroft’s theory and practice of histological [13]. Stained sections were examined by Leitz Dialux 20 Microscope. Photos were taken using a Canon digital camera (Canon Power- shot A95).

Preparations of resin embedding samples processed according to description in paper of Abd-Elhafeez and Soliman [9]. Some of affected gill were separated and fixed in Karnovsky fixative [14] for preparations of resin embedding samples. Specimens were Results Ascocotyle (Ascocotyle sp.) Price (1935). Metacercarial cysts were located inside the cartilaginous support of the gill arches which were surrounded by thick The histological examinations were found metacercarial cysts that were identified as a heterophyid parasite; fibrousThe perichondrium (Figureof the cartilage 1A and 1B)was and enlargement within the as gill a filamentsresult of the(Figure proliferation 3A and 3B). of the chondrocytes progeny (Figure 1C). Cysts wereErosion widely of oval, the cartilagethin-walled, was 280 reported - 420 with(μm) formation long and 264 of tunnels - 365 (μm) in the wide. cartilage Genital (Figure pore is1D a andslit-like 1E). openingAs well as (Figure hyperplasia 1D). (Figures 2A and 2B), hypertrophy of pavement cells (Figures 2D). Fusions were present in the affected gill filaments (Figure 2G). Increase rodlet cells were observed in the connective tissues and in the lymph space between the epithelial cells (Figures 2C, 2H and 2I). in the epithelium and connective tissue (Figure 2B, 2E and 2F), disappearance of the gill lamellae. Massive infiltrations of lymphocytes

298 We demonstrated a Large number of granular stages of rodlet cells using the combined Alcian blue pH 2.5 and periodic acid sheiff

(Figure 3A-3C) in comparison to non-infected fish (Figure 3 D). Large numbers of Mucous cells with positive staining to Alcian blue pH 2.5 were observed (Figure 3A-3C). In the Semithin section, we identified the granular stages and transitional stages of rodlet cells around the gill blood vessels and gill filaments (Figure 3E and 3F).

Figure 1: Metacercarial cyst in gill arch. Hematoxylin and eosin staining on paraffin-embedded section (5-7 µm thickness). A and B: Low and high magnification of Metacercarial cyst (Metacercarial cyst, double arrowheads). A: the hyperplasia (three arrowheads). B: Metacercarial cyst was surrounded by Thick fibrous perichondrium (Thick perichondrium, double arrowheads). C: Cartilage enlargement occurs as a result of invaginations (arrow) of the perichondrium into the cartilage mass and proliferation of the chondrocytes progeny (arrowheads). D: Cysts pyriform shaped (pyriform body, arrowhead) surrounded by thin-walled (thin wall, arrowhead), were widely 280-420 (μm) long and 264-365 (μm) wide. Note: (Genotyl, arrowhead) appear as slit-like opening close to anterior margin of ventral sucker (vs) and excretory bladder (bladder, arrowhead) Y –shaped with short arms. E and F: invaginations of the perichondrium into the cartilage mass (arrows). Note the fibroblast within the thickened connective tissue (arrowhead).

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Figure 2: Changes of tissue responding to Metacercarial cyst. Hematoxylin and eosin staining on paraffin-embedded sections (5-7 µm thickness) showing the A Low and B: higher magnification of gill arches showing hyperplasia (double arrowheads), Increase number of granular stages of rodlet cells (blue arrows) and a low number of mucous cells (arrowheads), Note the rupture stages of rodlet cells (red arrows).C: Lymphocytic infiltrations (arrowheads). D: Hypertrophy of pavement cells (pv, arrowheads). F: a Large number of transitional stage of rodlet cells (arrowheads) and extravasation of lymphocytes around the blood vessels (E) and cartilage (F). Note Higher magnification of selected squares showing a group of rodlet cells. G: hyperplasia of gill epithelium and fusion were present in the affected gill filaments (double arrowheads). Note congestion of blood vessels (long, arrowhead) and necrosis of gill epithelium (red arrow). H: low magnification and I: Higher magnification showing massive invasion of lymphocytes (arrowheads). Note: In I the congestion of blood vessels (double arrowheads).

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Figures 3: Metacercarial cyst in gill filaments. A, B, C and D: Alcian Blue-Pas staining on paraffin-embedded section (5-7 µm thickness). Showing the affinity of granular stages of rodlet cells and mucous cells for a combination of AB/PAS. A and B: Low and higher magnification showing metacercarial cyst was located inside gill filaments (double arrows). Note, Increased granular stages of rodlet cells (blue arrows, a mix of blue and magenta stained) and mucous cells blue stained (Acid mucous) double arrowheads, (C) Example of infected gill and (D) healthy gill demonstrating the difference in numbers of granular stages of rodlet cells (blue arrows). Note the mucous cells with positive staining to alcian blue pH2.5 (red arrows). E and F: Semithin section (1µm thickness) and stained with toluidine blue showing the granular stages of rodlet cells around the blood vessels (red arrows) and attached to the gill filaments (blue arrows).

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Discussion and algae [15]. The occurrence of Ascocotyle (Ascocotyle Sp.) metacercaria encysted in the musculature of the gill arches is remarkable. Ruby shark is a freshwater fish which one of the ornamental species distributed in Assiut City, Egypt. It fed on decaying organic matter The present study revealed that the occurrence of heterophyid species of Ascocotyle (Ascocotyle sp.) in the gills of ruby shark, Epalzeo- rhynchos frenatum. These results agreed with Salgado-Maldonado., et al. [16] who found metacercariae of Ascocotyle (Ascocotyle sp.) in cichlidThe fishdistribution from Mexico. of Ascocotyle Ascocotyle species was now known in Africa, Egypt [17,18]. Prevalence of heterophyid infection in Tilapia fish rates from one study to another is related to local conditions which endure the parasite life cycle. Salem., et al. [17] reported that Asco- was 32% [17]. The epidemiological data about the prevalence of species in Egypt were varied. This variation in fish infection cotyle - sp. was obtained only from fresh water with 9.3 % recovery rate from Tilapia species. The risk of infection with trematode parasites is higher in wild-caught compared with farmed fish [19] in Egypt, the highest rate of infection with heterophyid parasites have been dem onstratedAscocotyle in fresh compared to brackish water fish [17]. among Egyptian dogs (Asmaa., et al. 2016), poor sanitary conditions and disposal of waste in water bodies permit the transmission of species were found in Egypt by Writenberg (1929) in infected dogs and cats fed on fishes. It recorded with percentage 4% fish-borne infections in different localities of Egypt [20,21]. Heterphid eggs are probably discharged in draining water uploaded with feces of infected dogs and cats as domestic animals and fish-eating birds which act as definitive hosts [22]. The difference in the pattern Ascocotyle species [5], Despite these parasites did of distribution may be attributed to encysted metacercariae species-species predilection site in the fish or the level and extent of water not cause acute disease, but in long-term, they appear economical loss due to damaging of gills with metacercariae. Lesions of the gills as pollution [23]. The infection, specific host and the site of infection are characterized to a result of the presence of parasitic infection were varied with the agent, host, and density of infection (Robert 2001). The structure of the normal gill was demonstrated by many authors [24,25].

In the current study, gills respond to the parasitic infestation by attracting and activation of several types of immune cells including lymphocytes and rodlet cells. A considerable number of the lymphocytes were observed in the lymph space between the epithelial cells and in the underlying connective tissue. Granular stages of rodlet cells have been observed as demonstrated by Abd-Elhafeez and Soliman [9]. The predominance of the acidic polysaccharides stained by alcian blue and diminishing of the neutral polysaccharides is remarkable features of infestation by the metacercarial cyst. This may indicate that the acidic components affect the stability of the metacercarial Fried and Johnson [26] who described that breaching of the metacercarial cyst occurs in the acid-pepsin medium. The infection of the gills in the present study with trematode metacercariae caused hyperplasia, hypertrophy, and a fusion of gill cyst. This was confirmed by

- filaments, with the proliferation of gill cartilage. Those results were demonstrated by Olson and Pierce [24]. The disappearance of the gill lamellae could affect respiratory efficiency. A similar conclusion was described in cichlids and carps by Paperna [27]. The extensive reac Ascocotyle (Ascocotyle tion of cartilage proliferation around metacercariae was elucidated in four species of aquarium fish and in two experimentally infected as heart, gill arches and mesentery [29]. Ascocotyle metacercariae were also reported encystment in different organs as gills, heart, kidney species [28]. Furthermore, metacercariae of sp.) were found in eight families and encysted in several organs of fish and muscle [30].

The introduction of some diseases via imported ornamental fish is not worthy of significant attention in developing countries. Further more, rearing of ornamental fish without a doubt unrecognized infections will become essential for spreading infected fish around the world. So, the present study fills some gaps in the knowledge of liability of Red-finned Shark to parasitic infections [31-33].

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Conclusion

As a conclusion, we recommend that Veterinary organization should get specific arrangements for sanitation and control of health Authors’quality and Contributions guidelines of fishes. Abdel-Hakeem and Abdel-Hafez designed and coordinated the study, shared in sampling and wrote the manuscript. Soliman assisted in work, histological analysis, manuscript writing and data analysis, Zaki participated in samples collection, data analysis and revising the manuscript. AbdelHafez helped in data analysis and reviewing the final manuscript. All authors discussed the results, commented on the Competingmanuscript and Interests gave final approval of the final version to be submitted. All authors read and approved the final manuscript. The authors declare that they have no competing interests.

Availability of Data and Materials All the data regarding this study are demonstrated in the manuscript.

Consent for Publication Not applicable.

Ethics Approval The method of consent and the animal work were approved by the ethics committee of Assiut University, Egypt.

Funding This research did not receive any research fund.

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