Fadel, A. EVMPSJ 2021; 17:20-34 Original Article Digenean Transversotrema haasi (Witenberg, 1944) infection in cultured Dicentrarchus labrax, and in vitro anthelmintic assays Amr Fadel* Abstract Laboratory of Fish A little knowledge still available for fish digenean, although Diseases, Aquaculture some of them have pathogenic and economic importance. Fish Division, National Institute morbidity and mortalities were reported in two fish farms culturing D. of Oceanography and labrax fingerling with the primarily isolated parasites belonged to fish Fisheries, Egypt. digenean trematodes. Therefore, this study was directed mainly to investigate the main causative digenean parasites. Additionally, the ecological water parameters were reported, and the anthelmintic *Corresponding author efficacy using levamisole was evaluated. The prevalent digenean Amr Fadel parasite was identified Digenean, Transversotrema haasi, an extra- Laboratory of Fish Diseases, intestinal digenean that was mainly isolated from gill and skin. The National Institute of infected D. labrax exhibited abnormal external, and internal lesions Oceanography and Fisheries, indicative of deteriorating health status. The recorded infection rates Alexandria, were 48.67 and 38.67%, parasitic intensities, 6.14 and 2.4 and Egypt.ElAnfoshy, Qyiet Bay mortalities rates 28 and 19.33% in Manzala, and Wadi-mariot Castle, Alexandria, Egypt. respectively. The recoded ecological parameters were ranged from NIOF, Zipcode: 21556 14.22 - 29.7 oC temperature, 7.49 - 8.36 pH, and 13.54 - 34.36 ppt for Telephone NIOF: +203 salinity. Besides, the in vitro therapeutic baths with levamisole were 4807138 & 4807140 dose-dependent, with the highest anthelmintic efficacy at higher doses Mobile no: 202-01008580037 effective against digenean T. hassi. Higher anthelmintic efficacy was [email protected] obtained/ at 70 µg/l levamisole concentration, the lowest mean viability [email protected] 0.33, the highest parasitic mortality rate 81.81% occurred at the lowest exposure time. Although the anthelmintic efficacy of the levamisole &orcid.org/0000-0002-8177-6825 bath has been proven, other friendly environmental agents of plant origin should be taken under consideration to avoiding potential Accepted: January environmental and human hazards. 2021 Keywords: Digenean; Outbreak; D. labrax; levamisole; Assays. INTRODUCTION productivity are out there, that requiring the more understanding of frequent problems Various significant challenges related to management and diseases problems against aquaculture development and (Islam et al., 2020, Abdel-aziz et al., 2020). 20 Fadel, A. EVMPSJ 2021; 17:20-34 Marine fishes are receiving widely sphaerovarium, and T. patialense (Erik commercial importance; however, it faces 2002; Morsy et al., 2013; Abou Zaid et al., many obstacles including infectious diseases, 2018). In Egypt, Derogenes varicus was that constrain the expansion of the identified from M. surmuletus (Amel et al., aquaculture industry worldwide (Bessat and 2009; Abou Zaid et al., 2018), Hemiurata Fadel, 2018; Fadel et al., 2020; Aly et al., suborder species (Al-Zubaidy and 2021). Parasitic disease outbreaks have Mhaisen, 2011), A. secundus, C. become a clear threat, stems directly from its serranis, and P. parupnai were isolated pathological effect, and indirectly through its from M. labrax (Heba and Walaa, 2012). impacts on fecundity, productivity, and Fish infected with trematodes economic situation (Przybyla et al., 2014; exhibited signs that include Eichner et al., 2018). scratching, excess mucus, lethargy, anoxia, Parasitic trematodes of monogenean and loss of appetite (Eissa et al., 2017; Aly and digenean orders are considered serious et al., 2020). In severe infection cases, pathological problems facing the aquaculture opacity, ulcers, hemorrhages, in addition to sector (Alvarez-Pellitero, et al., 2004). They histopathological alterations of focal affect fish of various habitats and hyperplasia, lamellar fusion, hemorrhages, environments including freshwater, marine, and inflammatory infiltration also appeared and brackish (Buchmann and Bresciani, in the infected gill tissues (GonzálezLanza 2006). Different species of fish trematodes et al., 1991; Woo 2006). have been isolated locally, and To improve the efficiency of parasite worldwide. Diplomonorchis treatment, coordinated treatment measures sphaerovarium was isolated from S. are recommended which have been testudineus (Fernandes et al., 2004), D. successfully applied in controlling Hopkins, and D. catarinensis from M. parasites (Bumgarner et al., 1986; Costello furnieri (Amato, 1982), D. leiostomi (Kohn 2004; Kelly et al., 2010). Nevertheless, the et al., 1982), and Boridia lack of understanding of the effective grossidens (Fernandes et al., 1985). Other treatment protocols has been implicated in species were also identified that include T. reduced management efficiency (Bravo et praeterita, P. panamense, D. sphaer al., 2014). Moreover, the extensive misusing ovarium, M. crassulata, D. and overdosing of chemotherapeutic agents 21 Fadel, A. EVMPSJ 2021; 17:20-34 resulted in the emergency of antimicrobial the Laboratory of Fish Diseases, National resistance phenomena in environment, Institute of Oceanography and Fisheries, human, and animal products (Cabello, Alexandria. The fish sampling procedures, 2006). handling, and examination were conducted Although the pathogenic importance according to the addressed legislation of of some fish digenean, a little knowledge still animal welfare guidelines and humane available regarding its identification, treatment (Ecpakfp 2006 and Branson, biological, and ecological related features, as 2008). well the efficient treatment aspects. Fish 2. Water basic analysis morbidity and mortalities were reported in Representative water samples from two fish farms culturing D. labrax fingerlings the same ponds were collected in plastic in two locations in Egypt. This study was bottles and kept in insulated coolers then directed mainly to investigate the main transported directly to the laboratory. The causative digenean parasites in Damietta physical and chemical basic parameters of Governorate and Alexandria Governorate. water quality parameters; water temperature, Moreover, the ecological water parameters oxygen, pH, and salinity were measured were reported, and the anthelmintic efficacy using a specific Electronic probe, and the using levamisole was evaluated. testing photometer (APHA 2005). MATERIALS AND METHODS 3. Parasitological examination 1.Fish sampling Gill and skin biopsies and scraping A total of 300 European Sea were prepared by dissection of the gill arch, bass Dicentrarchus labrax samples cultured covered with coverslips placed on a slide, in earthen ponds, with an average weight of submerged in water, and examined freshly 74.15±9.24 g. and length 17.54±2.04 cm. was under a light microscope and collected throughout June- December 2020. dissecting microscope (Optika). The isolated The first Farm A is located trematodes were carefully collected fixed in in Damietta, Manzala, 31 ° 24 ′ 32 ″ N, 31 ° buffered formal saline 3%, and preserved in 52 ′ 19, and the second Farm B is located 1:4 alcohol 70% and 5% glycerin Alexandria Governorate, Wadi-maiot 30° 50′ respectively. The fixed trematodes were then 56″ N, 29° 36′ 42″ E. Fish samples were then stained with Semichon's aceto-carmine and kept in ice boxes and transported quickly to dehydrated in graded concentrations of 22 Fadel, A. EVMPSJ 2021; 17:20-34 alcohol, cleared in clove oil and xylene, and substances were 20, 40, 50, 60, and 70, µg//L were finally mounted in Canada for 1 h. balsam (Manter, 1961; Cribb et al., 1992; 5.2. Experimental conduction Pirarat et al., 2011). The prevalence rate of A total of 60 infected D. the isolated parasites was labrax fingerlings were collected from Farm calculated (Reiczigel and Rózsa, 2005). The A, Manzala. They were anesthetized by TMS mean intensity of infection was represented (MS-222), before the experimental as a number of parasites per microscopical conduction, gills and skins swabs were examination field (Silan and Maillard, collected inspected, and checked for 1989). digenean presence, prevalences, and other 4. Clinical and Post Mortem (PM) infectious agents (Bush et al., 1997). investigation Then the fish were divided into seven Throughout the study period and the groups; G1 negative control, G2 positive experimental conditions, the fish were control, G3, G4, G5, G6, and G7 exposed to checked visually for behavior abnormalities, 20, 40, 50, 60, and 70 µg/ L of levamisole, external clinical signs such as erosions, respectively, for 1 hour. For each fish group; ulcesr, and the abnormal discoloration of the 5 ml of the corresponding prepared skin, and gills. Also, the internal post-mortem concentrations of levamisole were poured, changes, and the mortalities and morbidities then the branchial arch and skin scrapings were reported according to Schmidt (1992) of the naturally parasitized D. labrax was Woo (2006), Mahmoud, et al., (2014). placed and immersed. After submerging, the 5. In vitro assays of anthelminthic efficacy branchial arches of the different of levamisole against Digenean, T. haasi concentrations of anthelmintics were 5.1. Preparation of levamisole stock observed at 15‐min intervals to detect solutions parasite viability, activity, and mortality. The The initial concentration of the anthelmintic efficacy was recorded based commercial levamisole hydrochloride 10 % upon the time taken till