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).

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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

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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

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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 100% of digenean suspension was diluted in fresh seawater to were dead within an hour time span (Zhang the required concentrations (Székely & et al., 2014; Soares et al., 2017). Molnár 1987; Butcher 1993). The tested 6. Statistical analysis dilution concentrations of chemotherapeutic

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Statistical analysis was made with the test using the SAS package (SAS 2004). computer program Statistical Package for Levels of significance were set at (p <0.05) Social Sciences) SPSS. Differences among as indicated by an asterisk (*) in figures and groups were performed with a chi-squared tables. RESULTS The clinical signs and post-mortem The prevalence of Transversotrema examinations haasi in D. labrax The infected D. labrax showed Higher infection rates, parasitic abnormal swimming, debilitation, decreased intensities, and mortalities of 48.67, 6.14, and appetite, and sluggish behavior. Besides, skin 28 were recorded in Farm A, Manzala than darkness, gills paleness, excess mucous Farm B, Wadi-mariot of 38.67%, 2.4, and secretion, congestion, and hemorrhage may 19.33% respectively (Table 1). also be exhibited. The internal organs often The parasitological examination appeared anemic, with pale liver, and spleen, The isolated parasites were identified and congested kidney (Figure 2). as Transversotrema haasi (Witenberg, Physiochemical water parameters 1944a), belonged to, digenetic trematodes, The recorded water quality Digenea, Transversotrematidae, parameters were significantly differed (p ≤ Platyhelminthes. This is according to the 0.05) among the two locations throughout the distinct morphological features as the study period. Overall higher levels were following: the body of the digenetic recorded of water temperature 26.16 °C, trematode is elongated, flattened, transversal salinity 30.34 ppt, ammonia 0.46, pH 8.02, lancet with eye-spots, small ventral sucker, and lower oxygen tension 5.37 in Farm A, absence of oral sucker, lobed testis, and the Manzala. On the other hand, decreased presence of only a single ovary. The levels in the Farm B, Wadi-mariot were identified developmental stages were mostly reported of temperature 24.85 °C, salinity adult and often with the nearby smaller 14.51 ppt, ammonia 0.13, pH 7.45, and stages, and mainly recovered from the skin higher oxygen tension 7.61 (Table 2). and gill (Figure 1).

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Table 1: The prevalence of T. haasi in examined D. labrax in the farms under study (Farm A, Manzala, and Farm B, Wadi-mariot). No. of examined No. of the infected Prevalence Parasitic Cumulative mortalities Location fishes fish (%) intensity (%) Farm A 150 58 38.67 2.4 19.33

Farm B 150 73 * 48.67* 6.14* 28*

*: Averages in the same column having different superscripts were significantly different ( P≤0.05).

Figure 1: (A&B): showing non-stained digenean, Transversotrema haasi skin scraping from D. labrax (X10), (B) carmine stained T. haasi (X 200). The scale bar is 500 μM

Figure 2: D. labrax fingerlings infected with Transversotrema haasi, showing debilitation, erosions, and anemic discoloration of the internal organs. The scale bar is 1 cm. 25

Fadel, A. EVMPSJ 2021; 17:20-34

Table 2: The physiochemical parameters of the collected water samples in Farm A, Manzala, and Farm B, Wadi-mariot.

Temperature Salinity Ammonia Oxygen pH (°C) (ppt) (ppt) (mg l−1) Farm A 26.16 30.34 0.46 8.02 5.37

Farm B 24.85* 14.51* 0.13* 7.45* 7.61*

*: Averages in the same column having different superscripts were significantly different (P≤0.05).

The anthelmintic efficacy viability were reported. Meanwhile, from 40 of levamisole against T. haasi. µg/L levamisole elevated efficacy, decreased Levamisole application exhibited an parasitic viabilities, higher parasitic exponential efficiency that correlated with its mortalities, and lower exposure time which increasing dosage rates. Generally, the higher were the best in 60 µg/L, and 70 µg/l doses led to the subsequent decreased levamisole. Accordingly, at 70 µg/L parasitic viability and increased parasitic levamisole concentration, the lowest mean mortality rates. Thus, in a lower viability 0.33, the highest parasitic mortality concentration of 20 µg/L levamisole and the rate 81.81% were reported (Table 3). highest exposure time, the decreased anthelmintic efficacy and higher parasitic Table 3: The effect of different concentrations of levamisole on the viability and mortality of T. Hassi in the infected D. labrax.

G2† G3 G4 G5 G6 G7

+ Control 20 µg/l 40 µg/l 50 µg/l 60 µg/l 70 µg/l Exposu ‡ § Viable Dead viable Dead Viable Dead Viable Dead Viable Dead re time Viable Dead 0 hr 9.67 0 8.67 7.6 10.33 3.2 10.33 9.68 10.67 15.62 11 18.18

15 min. 0 8.33 12 7 32.26 5 51.61 6 37.5 5 39.39 9.67 30 min. 0 8 27 5.67 45.16 4.67 58.06 3.33 46.88 4 48.48 9.67 45 min. 0 8 27 5.33 48.39 3.33 67.74 2 75 0.33 81.81 9.67 1 hr. 0 7.33 30.77 5 54.84 1 87.10 0.33 90.63 0 0 9.67 †: Mean skin and gill scrapings of 10 infected D. labrax, ‡: Mean parasitic intensities of T. Hassi, §: Mean parasitic mortality (%). 26

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DISCUSSION stages were mostly adult with the near Infectious parasites are of great smaller stages. Their adult and pathogenic, biological, ecological, and developmental stage are mostly pathogenic economic importance for marine for fish (Smith, 1972; Woo, 2006). The habitats (Cribb et al., 2002; Carvallho et prevalence of T. hassi in the examined D. al., 2014; Abou Zaid et labrax recorded higher infection rates (48.67, al., 2018). Numerous parasitic trematodes and 38.67%), parasitic intensities (6.14, and species were reported in a wide range of 2.4), and (28, and 19.33%) mortalities rates fresh, marine, and brackish water fish in in Farm A, Manzala, and Farm B, Wadi- Egypt, and worldwide, but the knowledge of mariot respectively. The dissemination of their pathogenic effects still little and digenean infection from one geographical questionable (Fernandes et al., 2004; Amel region to another could be allowed, et al., 2009; Al-Zubaidy and Mhaisen, translocated, and altered through human 2011; Heba and Walaa, 2012; Morsy et al., intervention, piscivorous bird trematodes 2013; Abou Zaid et al., 2018). Digenetic snails carried with cultured fish trematodes have indirect life cycles and their stock (Mitchell et al., 2002). outbreaks need other hosts that include Clinically, the infected D. crustaceans, snails, and birds (Scholtz and labrax exhibited bad health status, abnormal Salgado, 2000). Under optimum favorable behavior, and decreased growth rearing, ecological, stress conditions, the life performances. Besides, the skin exhibited cycle of digenetic trematodes, and their darkness, excess mucous secretion, and gills potential parasitic infection was paleness congestion, hemorrhagic, and initiated (Woo, 2006; Noga, 2010). oedematous may also be exhibited. These Morphologically, the prevalent serious abnormal external and internal digenean parasite was identified as postmortem changes indicated the harm, Transversotrema haasi (Witenberg, 1944a). inflammatory, and irritation reaction of the The taxonomical features have belonged to skin-inhabiting Transversotrema spp. Digenea, Transversotrematidae, Transversotrematidae is an ectoparasitic Platyhelminthes. The isolated parasite was inhabit under the scales of fish. They have extra-intestinal digenean, mainly isolated leaf-like, transversely elongated with their from gill and skin, and the developmental oral sucker is used for attachment to the host

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Fadel, A. EVMPSJ 2021; 17:20-34 skin (Angel, 1969; Mills, 1979; Woo, 2006; where, higher water temperature 26.16 °C, Abou Zaid et al., 2018). Moreover, the salinity 30.34 ppt, ammonia 0.46, pH 8.02, metabolic function and nutrition of digeneans and lower oxygen tension 5.37 were depend on the plasma of blood capillaries, recorded. The higher water temperature and ingestion of blood cells, and in particular, ammonia levels cause decreased oxygen transversotrematidae, their feeding habits consumption, and subsequent delayed innate were digestion of the host epidermis (Lester, immunity to equilibrate these higher levels 1980; Woo, 2006). (Meyers et al., 1994; Noga, 2010). The ecological parameters recorded Moreover, the higher ammonia values in the throughout the study period could consider second Farm A, could refected the other predisposing factors for outbreaks of occurrence of the poor sanitary conditions, digenean infections. Throughout different and microbial load (Santhosh and Singh, seasons, we recorded water temperature 2007). Further, the recorded water ranges from 14.22 - 29.7 oC, 7.49 - 8.36, and parameters were suitable for the growth of salinity 13.54 ppt- 34.36 ppt. Accordingly, trematode and their propagation between the highest prevalence specifically in Farm A their definitive and intermediate indicates the availability of the ecological, hosts (Syobodova and Kolarova, 2004; environmental, and biological factors that Noga, 2010). favor the growth of the primary and the Importantly, the integrated principles definitive host that assist the survival and of pest management advocate the limitation spread of trematodes in different of pesticide usage to avoid the development localities (Syobodova and Kolarova, of resistance and minimize environmental 2004). For example, the snail is a major residues (Brooks, 2009; Burridge et al., intermediate host, its growth needs water 2010). Most chemical and husbandry temperature over 17 ° C (Noga, 2010). practices were used to maintain the parasitic Given, the ecological stresses have infestation levels at their lowest permits negative influences on fish homeostasis and following each country's could inhibit the fish resistance to infectious authorities (Helgesen et al., pathogens (Small and Bilodeau, 2015). However, these managing tasks are 2005). Particularly in Manzala farm, a greatly complicated by the limited higher infection rate of 48.67% was reported, licensed anthelmintic drugs (Morales‐Serna

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Fadel, A. EVMPSJ 2021; 17:20-34 et al., 2018; Zuskova et al., 2018). In our CONCLUSION study, we reported the efficient lowest The pathological importance of the concentration of levamisole at which digenean Transversotrema hassi was efficient control was achieved in the form of indicated in the fish morbidities, mortalities, decreased parasitic load and increased fish and clinical lesions. Also, the digenean health status. It was observed that the infection was greatly influenced by the increasing concentration dose of levamisole, associated ecological factors that favor the was corresponding to increased anthelmintic growth and outbreaks between hosts. efficacy of the therapeutic agent. Thus, the Besides, in vitro therapeutic baths with higher anthelmintic efficacy was obtained at levamisole were dose and exposure time- 70 µg/L levamisole concentration, wherein, dependent, with the highest anthelmintic the lowest mean viability 0.33, the highest efficacy was recorded at higher doses. parasitic mortality rate 81.81% has been Nevertheless, other friendly environmental recorded. Higher concentrations of 100 and natural agents of plant origin should be taken 125 mg/L levamisole were used by Alves et under consideration to avoid environmental al., (2018), which cause parasites inactivity and human hazards. and mortality just after 20 min exposure time, REFERENCES and 88.2% efficacy. Levamisole can block Abdel-Aziz, M.; Bessat, M.; Fadel, A. and the neuromuscular junctions and stimulated Elblehi, S. (2020). Responses of dietary both sympathetic and parasympathetic supplementation of probiotic effective ganglia causing detachment of the parasites microorganisms (EMs) in Oreochromis from the host gills (Martins et al., niloticus on growth, hematological, 2017). Similarly, Santamarina et al., (1991) intestinal histopathological, and reported the efficacy of 100 mg/L antiparasitic activities. Aquacult. Int. J. of levamisole, but at 3-hour baths pp.1-17. against Gyrodactylus sp. In O. Mykiss, a Abou Zaid, A. A., Bazh, E. K., Desouky, A. lower concentration of 20–50 Y., Abo-Rawash, A. A. 2018. Metazoan parasite fauna of wild sea mg/L levamisole in 10–18 min baths was bass; Dicentrarchus labrax (Linnaeus, effective against U. 1758) in Egypt. Life Sci. J. 15,6. vistulensis in Silurus glanis (Szekely and Alvarez-Pellitero, P. 2004. Report about fish parasitic diseases. In: Alvarez Pellitero Molnar, 1990). P, Barja JL, Basurco B, Berthe F, 29

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الملخص العربي

انتشار اإلصابة بتريماتودا ترانسفيرسوتريما هازى فيأسماك القاروص األوروبي المستزرع وتقييم معملي للمكافحة الطفيلية

Amr Fadel

معمل امراض االسماك- شعبة تربية االحياء المائية- المعهد القومي لعلوم البحار والمصايد- مصر

على الرغم من االهمية المرضية لبعض انواع ديدان التريماتودا التي تصيب االسماك اال انه مازال القليل المعروف عن الصفات المرضية والبيولوجية لها. وقد سجلنا اصابه طفيلية الصبعيات االسماك القاروص األوروبي واوضح الفحص الطفيلي االولى احد انواع تريماودا الدايجينيا. وعليه، فقد ركزت تلك الدراسة على معرفة نسبة الحدوث الطفيلي، التأثير اإلكلينيكي، وكذلك التصنيف الباراسيتولوجى لديدان الدايجينيا وما يرتبط بها من العوامل االيكولوجية بموقعين مختلفين في المنزلة بمحافظة دمياط، ووادى مريوط باإلسكندرية. واألهم وسيلة مكافحته، وذلك بتقييم الكفاءة العالجية الحد المواد المعمول بها دوليا وهى الليفاميزول تحث تركيزات مختلفة 20، 40، 50، 60، 70 مليجرام/ لتر. ثم اختبار مقاومتها للطفيل معمليا في اطباق تحوى مسحات خيشومية وجلدية من األسماك المصابة مغموسة في المحلول وتحديد االعلى كفاءة عالجية.

وقد اوضحت النتائج ان االسماك تحوى طفيل تريماتودا يصنف الى نوع تران س فيرسوتريما هازى Transversotrema haasi. وهو نوع من التريماتودا غير المعوية يتطفل على الجلد والخياشيم ويؤدى الى حدوث اعراض سلوكية واكلينيكية غير طبيعية من هيجان في الجلد والخياشيم، التهابات ، وافرازات كثيفة للمخاط، ضعف االقبال على الغذاء، ضعف النمو، وتدهور الوضع الصحي لألسماك. وكانت نسبة االصابة تتراوح بين 48.67 و38.67%، الكثافة الطفيلية 6.14 و2.4، ونسبة النفوق في االسماك 28 و19.33% في كال من المنزلة ووادي مريوط على الترتيب. وسجلت معايير جودة المياه اعالها في موقع المنزلة والتي تراوحت خالل فترة الدراسة من درجة الحرارة 14.22-29.7 درجة سيليزية، األس الهيدروجيني 7.49-8.36، ودرجة الملوحة 13.54-34.36 جزء في االلف. وقد وجد ان كفاءة الليفاميزول العالجية تتزايد بزيادة التركيز الذى يصل الى احسن كفاءة عند 70 مليجرام / لتر، وفيه اقل كثافة طفيلية 0.33 ، واعلى نسبة نفوق للطفيل 81.81%. وعلى الرغم من كفاءته العالجية، اال انه من الضروري البحث عن الوسائل العالجية الصديقة للبيئة من المواد الطبيعية لتجنب مخاطر المقاومة الطفيلية، واثرها على البيئة واالنسان.

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