Research Article

The Presence and Histopathology of Encysted Metacercaria of Clinostomum complanatum (Rudolphi, 1819) (Digenea:clinostomidae) in Garra rufa (Heckel, 1843)

Öznur Özil*, Öznur Diler, Mevlüt Nazıroğlu, Aşkın Atabay

Eğirdir Fisheries Faculty, Isparta Applied Sciences University, Isparta, Turkey.

Editor | Muhammad Imran Rashid, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.

Received | November 23, 2021; Accepted | August 22, 2022; Published | December 09, 2022

*Correspondence | Öznur Özil , Eğirdir Fisheries Faculty, Isparta Applied Sciences University, Isparta, Turkey; Email: [email protected]

Citation | Özil O, Diler O, Nazıroğlu M, Atabay A (2022). The presence and histopathology of encysted metacercaria of clinostomum complanatum (rudolphi, 1819) (digenea:clinostomidae) in garra rufa (heckel, 1843). J. Adv. Parasitol. 9(1): 1-7.

DOI | http://dx.doi.org/10.17582/journal.jap/2022/9.1.1.7

ISSN | 2311-4096

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Garra rufa (Heckel, 1843) commonly known as doctor fish, are freshwater cyprinid species that naturally inhabit river basins, muddy streems, ponds and lakes in Eurasia. They are widely used in the health and beauty industries at the spa center for ichthyotherapy. The spread areas of G. rufa were reported in Iraq, Iran, some coastal rivers in southern Turkey and northern Syria. G. rufa might poses a potential risk to customers or ornamental and native fish with zoonotic pathogens. Zoonotic agents could be transmitted not only directly from affected fish, but also indirectly through water (Bhattacharya, 2016).

Yellow spot disease is a common fish parasitosis. This trematode has a very economic importance because of damage to fish carcasses depending on white-yellowish nodules (Sutili et al., 2014). In addition to Clinostomum spp. is a fish-borne zoonotic parasite responsible for Halzoun syndrome in humans who consume raw or undercooked freshwater fish that infected by metacercariae (Kifune et al., 2000; Park et al., 2009; Sutili et al., 2014).

Adult forms of digenean family Clinostomidae Lühe, 1901 were found in the mouth, esophagus and pharynx of fish-eating birds, reptiles. The first intermediate hosts are freshwater snails, and the second definitive hosts are many species of fish (Kanev et al., 2002). They are known as yellow grubs due to their color and encyst in organs such as muscles, dermis and gills or in the body cavity (Olsen, 1974; Bullard and Overstreet, 2008).

Clinostomum complanatum is a cosmopolitan trematode and low host specificity. The metacercariae have been morphologically reported from fresh and brackish water fish species in Turkey (Çolak, 2013; Soylu, 2013, 2014; Ozturk and Ozer, 2015) and molecularly (Simsek et al., 2018). Due to risk of disease transmission to humans, the metacercariae of C. complanatum have zoonotic potential (Patimar et al., 2010).

Ichthyotherapy was adopted and commonly commercialized worldwide including Japan, Croatia, China, Netherlands, Malaysia, Singapore and Korea. In spite of these medical preferences, unfortunately there are a limited number of reports about the potential pathogens that might be carried by G. rufa (Majtàn et al., 2012). The infestation of parasites particularly trematodes as a serious zoonotic and threat to health were reported (WHO, 1995). Therefore, this is the first study to determine the histopathological effect of Clinostomum complanatum in G. rufa fish.

MATERIALS AND METHODS

G. rufa (9.5 - 10 cm total body length) was obtained from local aquarist in Antalya. Twenty five of these fish were immediately transported alive to the fish disease laboratory in suitable containers, and fish were dissected carefully for observed the presence of encysted metacercariae into the all organs. They were observed under a stereozoom microscope (Leica S6) for larval digeneans. The attached cysts visible to the naked eye were photographed by the present authors. Identification of C. complanatum metacercariae (Rudolphi, 1819) (Digenea:Clinostomidae) was made according to (Bychovskaya and Pavlovskaya, 1962; Yamaguti, 1958, Dawes, 1968; Markevic, 1951; Matthews and Cribb, 1998; Simsek et al., 2018; Cagatay et al., 2022). Prevalence, mean intensity and abundance of infection were measured following Bush et al. (1997).

The tissue samples containing the encysted metacercariae were fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin wax to cut 5 µm thick sections using a rotatory microtome, for histology and stained with haematoxylin and eosin (H&E). The DPX mounted sections were observed under microscope and photographed.

RESULTS

In total, of 25 examined fish specimens 56% were infected with the metacercariae (yellow grubs) of C. complanatum including G. rufa. The mean intensity value of the infection was 27.14 cysts per host, varying between 1-75 cysts. The parasites were found encysted in the fins bases, muscle, inner wall of the operculum, lips, gill arches, palate, upper jaw and body cavity, forming small 2-3 mm diameter white-yellowish nodules. The operculum and mouth were the most common locations of the parasites than other tissue. In the fins, the parasites were frequent in dorsal, anal and pelvic fins, while only very rarely they were present in the caudal fin (Figure 1 A-F).

The prevalance and intensity of parasites in G. rufa were 0.56 and 27.14, respectively. The metacercariae were observed as encysts and excysted worms in the muscles, buccal cavity, sub operculum and pectoral fins bases. The highest prevalence of the metacercariae was in C. complanatum with 59.73% in gill tissue (Figure 1D).

The prevalence, mean of intensity and abundance of parasites in different microhabitats were as follows: in the muscle (22.10%, 6 and 3.36), in the gill (59.73%, 16.21 and 9.08), in the different fins (3.94%, 1.07 and 0.6) and and the other area of body (14.21%, 3.86 and 2.16) (Table 1).

 

Table 1: Infection intensity, prevalance, abundance on different microhabitats in G. rufa

Host Fish	Microhabitats in host fish	Total of parasites	Prevalance* (%)	Mean intensity**	Mean abundance***
Gara rufa (n=25) infected hosts=14	Muscle	84	22.10	6	3.36
	Gill	227	59.73	16.21	9.08
	Fin	15	3.94	1.07	0.6
	Other	54	14.21	3.86	2.16

*Prevalence: Number of parasites in per microhabitats divided by the number of parasites in total microhabitats expressed as a percentage.

**Mean intensity: Number of parasites in per microhabitats divided by the infected hosts examined.

***Abundance: Number of parasites in per microhabitats divided by the total fish examined.

 

The parasites were identified as C. complanatum metacercariae according to the described keys Matthews and Cribb (1998). The morphologic structures of C. complanatum metacercariae from the tissue of infected fishes were as follows: Oral collar visible. Ventral sucker larger than oral. Glandular structure present in forebody, anterior to ventral sucker. Intestinal ceca lateral to ventral sucker and genital complex, with prominent lateral pouches. Ovary irregular in shape, located dextrally in intertesticular space. Vitellaria not evident (Figure 2).

 

The histopathology of the infection of G. rufa by C. complanatum is described in this study. Gill lamellae surrounding the metacercariae were necrotic. Desquamation and thickness were seen in epithelium of gill tissue (Figure 3, 4). The parasites were determined encysted in the muscle (Figure 5). In all the locations the parasites were surrounded by a fibrous capsule consisting of a layer of dense connective tissue followed, in most of the cases, via a layer of loose connective tissue (Figures 5 and 6). The thickness of these layers varied with the location of the parasites. When the parasites encysted in the muscle tissue a slight degeneration of the muscle fibers was observed (Figure 6).

 

Table 2: Data from literature review studies about Clinostomum sp.

Fish Name	Species of Parasite	Country	References
Danio rerio	Clinostomum sp.	Brazil	Silveira et al., 2021
Trichogaster fasiatus	C. complanatum	India	Rizvi et al., 2020
Garra rufa	C. complanatum	Iranian	Meleki et al., 2018
Squalius cephalus	C. complanatum	Turkey	Şimsek et al., 2018
Lepomis macrochirus	Clinostomum sp.	United States of America	Calhoun et al., 2018
Myxocyprinus asiaticus	C. complanatum	China	Li et al., 2018
Hemibarbs labeo	C. complanatum	Taiwan	Wang et al., 2017
Synbranchus marmoratus	C. complanatum	Brazil	Acosta et al., 2016
Hoplias malabricus	C. marginatum	Brazil	Alcântara and Tavares-Dias, 2015
Capoeta damascina	C. complanatum	Israel	Caffara et al., 2014
Carassius carassius	C. schizothoraxi	India	Shah et al., 2013
Oreochromis niloticus	C. tilapiae	Kenya	Ochieng et al., 2012
Oreochromis niloticus	C. tilapiae	Nigeria	Echi et al., 2012
Etheostoma nigrum	C. detruncatum	Canada	Bonett et al., 2011
Rhamdia guatemalensis	Clinostomum sp.	Mexican	Pérez Ponce de León et al., 2009
Lepomis macrochirus	C. marginatum	North America	Zimmermann and Ingold., 2008
Capoeta capoeta	C. complanatum	Iranian	Malek and Mobedi, 2001
Acheilognathus rhombea	C. complanatum	Korea	Chung et al., 1995
Carassius carassius	C. complanatum	Japan	Aohagi et al., 1992
Perca fluviatilis	C. complanatum	Poland	Grabda-Kazubska, 1974
Alosa sapidissima	C. marginatum	United States of America	Hollis and Coker, 1948

 

DISCUSSION

Ichthyotherapy is becoming on increasingly popular for skin disorder. There are reports that G. rufa fish are helpful in the treatment of psoriasis and atopic dermatitis (Bhattacharya, 2016). These fish, feding on dead skin on the body. However, there is a risk of transferring the zoonotic agent from water to human. Freshwater fish species are known to harbour C. complanatum metacerceria (Table 2). They are embedded in the muscle or under the skin. The World Health Organization (WHO, 1995) has estimated that the number of people infected with fish-borne trematodes. Many cases of human infection of C. complanatum have been reported from various regions of the World (Hung et al., 2013).

In this study, the prevalance and intensity of parasites in G. rufa were 56% and 27.14, respectively. The highest prevalence of the metacercariae was in C. complanatum with 59.73% in gill tissue. Also, Malek and Mobadi (2001) examined the C. complanatum in Capoeta capoeta gracilis from Shiroud River. They found the prevalence and abundance of parasites were significantly higher under the mouth to behind the opercule. The prevalence of infection was the highest in the 7-11 cm length group. In another study, Aohagi and Shibahara (1994) and Li et al. (2018) found that the main habitat of C. complanatum in Carassius spp. was the muscles around the gills and in the head region of Monochamus asiaticus, with lower levels of infection in the posterior part of the body, respectively. Maleki et al. (2018) determined the metacercariae (yellow grubs) of C. complanatum in the muscles, buccal cavity, sub operculum and pectoral fins bases in fish species.

The metacercariae of C. complanatum were known to cause considerable damage to the viscera and musculature of fish species (Menconi et al., 2020). The infected fish tissue revealed heavy infiltration of immune cells at the site of cyst wall (Shareef and Abidi, 2012). Lo et al. (1992) determined that when a large amount of cercariae of C. complanatum penetrate the fish (Misgurnus anguillicaudatus) body at the same time they may cause irritation and lesions to fish tissue, which may induce mortality of small fish. In this study, C. complanatum were found encysted in gill arches, fins, muscle, inner wall of the operculum and body cavity and metacercariae were caused necrotic and fibrotic muscle tissues lesions in G. rufa. These results are in agreement with the previous reports.

G. rufa are widely used in aestetic practice such as fish pedicure. However,  ichtyotherapy can be potentially dangerous procedure in immunocompromised persons (Pastorino et al., 2016). There is the a need for more studies assesing this procedure. The results of this study is supported the requirements of sanitary and legal aspects and pathogen screening procedure on the use of G. rufa for human health.

ACKNOWLEDGMENTS

Authors express sincere gratitude to Dr. Y. Ö. Boyacı for his contributions. Author Mevlüt NAZIROĞLU is a PhD scholar at 100/2000 the Council of Higher Education (CoHE) in department of Aquaculture.

CONFLICTS OF INTEREST

The authors have declared no conflict of interest.

novelty statement

Trematodes, which constitute an important part of parasites, are zoonotic and pose a threat to consumer health. Therefore, our study is the first to determine the presence and histopathological effect of Clinostomum complanatum parasite in G. rufa fish. In this study, it was determined that it is very important to monitor G. rufa fish in terms of disease before using them in ichtyotherapy.

authors contribution

Conceptualization, Ö.Ö. and Ö.D.; methodology, Ö.Ö., Ö.D. and M.N.; investigation, Ö.Ö. and A.A.; resources, A.A.; writing-original draft preparation, Ö.Ö. and Ö.D.; writing-review and editing, Ö.Ö., Ö.D. and M.N. All authors have read and agreed to the published version of the manuscript.

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