DISEASES OF AQUATIC ORGANISMS Published July 30 Dis Aquat Org

Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. 11: Gyrodactylus sp.

J. L. Tojo*, M. T. Santamarina

Department of Microbiology and Parasitology, Laboratory of Parasitology, Faculty of Pharmacy, Universidad de Santiago de Compostela, E-15706Santiago de Compostela, Spain

ABSTRACT: A total of 24 drugs were evaluated as regards their efficacy for oral treatment of gyro- dactylosis in rainbow trout Oncorhj~nchusmykiss. In preliminary trials, all drugs were supplied to infected fish at 40 g per kg of feed for 10 d. Twenty-two of the drugs tested (aminosidine, amprolium, benznidazole, b~thionol,chloroquine, diethylcarbamazine, flubendazole, levamisole, mebendazole, n~etronidazole, mclosamide, nitroxynil, oxibendazole, parbendazole, piperazine, praziquantel, roni- dazole, secnidazole, tetramisole, thiophanate, toltrazuril and trichlorfon) were ineffective Triclabenda- zole and nitroscanate completely eliminated the infection. Triclabendazole was effective only at the screening dosage (40 g per kg of feed for 10 d), while nitroscanate was effective at dosages as low as 0.6 g per kg of feed for 1 d.

KEYWORDS: Gyrodactylosis . Rainbow trout Treatment. Drugs

INTRODUCTION to the hooks of the opisthohaptor or to ulceration as a result of feeding by the parasite. The latter is the most The monogenean genus Gyrodactylus is widespread, serious. though some individual species have a restricted distri- Transmission takes place largely as a result of direct bution. Gyrodactyloses affect numerous freshwater contact between live fishes, though other pathways species including salmonids, cyprinids and ornamen- (contact between a live fish and a dead fish, or with tal fishes, as well as marine fishes including gadids, free-living parasites present in the substrate, or with pleuronectids and gobiids. Outbreaks of gyrodacty- drifting parasites in the water) may also be important losis have been reported from farmed Pleuronectes (Bakke et al. 1992). pla tessa (McKenzie 1970). Various methods have been described for the control In natural environments, massive infections by Gyro- of gyrodactylosis, all based on pharmacological bath dactylus species are rare. A notable exception is G. treatments (Mehlhorn et al. 1988, Schmahl et al. 1989, salaris, which causes high mortality among salmon Santamarina et al. 1991, Tojo et al. 1992, 1993a, b, Tojo in Norwegian rivers (Heggberget & Johnsen 1982, 1993).The aim of the present study was to investigate Johnsen & Jensen 1992, MO 1994). Among farmed possible oral pharmacological treatments of gyrodac- fishes, gyrodactyloses are more common, and initially tylosis in rainbow trout. A total of 24 drugs were tested minor outbreaks can become a serious problem in a at high dosages (40 g per kg of feed, for 10 d). Oral short time. treatments have a number of advantages, including Gyrodactylus species are parasites of the fins, gills, ease of administration and the fact that there is no eyes and body. MO (1994) states that the epidermal need to handle the fish. Here we continue the screen- lesions arising in gyrodactyloses are chiefly due either ing study of possible oral pharmacological treatments of parasitoses in rainbow trout initiated with hexamito- sis (Tojo & Santamarina 1998).

Q Inter-Research 1998 Resale of full article not permitted 188 Dis Aquat Org 33: 187-193, 1998

Table 1.Drugs used in the study, showing manufacturer, brand name and until respiration became weak. A mucus form of presentation. p.p.: pure product sample was then taken by gently scraping fins and body surfare. The sample was Drug Brand name Form Manufacturer mixed with 3 drops of water on a slide, - coverslipped and examined with a light Aminosidine Gabbrocol Sol, inject. Vetern S P.A. microscope (40x). Infestation intensity Amprolium Powder Iteve Prolsal was recorded on a 5-point scale, after Benznidazole P P. Powder Roche Bithionol P-P. Powder Syva examination of a sample area of 24 X Chloroquine P.P. Powder Cidan 32 mm, as follows: 'minimal' (+/-), only 1 Diethylcarbamazine P.P. Powder Cidan individual of Gyrodactylus sp. detected in Flubendazole P.P. Powder Esteve Ovejero the sample; 'low' (+), more than 1 individ- Levamisole P.P. Powder Mebendazole P.P. Powder Esteve ual of Gyrodacfylus sp. detected in the Metronidazole ~ia& Powder Rhone Merieux sample, the average number per micro- Niclosamlde Fugotenil Pills Uriach scope field being less than 10; 'moderate' Ovejero Nitroxynil P.P Powder (++), average number of individuals per Nltroscanate Lopat01500 Pills Ciba-Geigy microscope field 10 to 50; 'high' (+c+), Gxibendazole P.P- Powder Syva Parbendazole P.P. Powder Smith Kline average number of individuals per micro- Piperazine P.P. (sranulate Sobrino scope field >50; 'zero' (-), Gyrodactylus Praziquantel Droncit Pills Bayer sp. not detected in the sample. Sobrino Ronidazole P-P. Powder Drugs and assay design. The drugs Secnidazole P-P. Powder Rhone Merieux Tetramisole P-P- Powder Ovejero tested in the study are listed in Table 1. Thiophanate P.P. Powder Uriach Each druq was assayed in 20 infested fish Toltrazuril P.P. Powder Bayer maintained in an 80 1 tank with a conti- Trichlorfon Neguvon Powder Bayer nous flow (5 1 min-l). A simultaneous Triclabendazole P.P. Powder Ciba-Geigy control assav (also of 20 fish; identical l I A. treatment, but without drugs) was per- formed for each drug. Tank conditions MATERIALS AND METHODS (water source, flow, aeration, pH, temperature, light/dark cycle) were identical to those during the Fish. Rainbow trout Oncorhynchus mykiss Walbaum acclimatization period, except that water temperature (weighing 16 g at the start of the experiments) were dropped to as low as lZ°C on a few occasions in the obtained from a local fish farm (Piscifactonas Coruiie- secnidazole assay, and rose to as high as 18°C on a sas S.A., Carballo, A Coruna, Spain) and acclimatized few occasions in the bithionol assay. The treated fish for at least 36 h before assay in a 350 1 tank with aera- received feed containing 40 g of drug per kg of feed tion and continuous flow of water (15 * 1°C, pH 6.5 rt for 10 d. The weight of food supplied to each tank on 0.5) from a nearby spring. Fish were fed daily with a each day was equivalent to 2% of the total body commercial feed. weight of the fish in that tank. Note that this proce- Infestation. All fish used in the trials showed high- dure does not guarantee that all fish received the intensity infestation by Gyrodactylus sp., in some same dose of drug. Throughout the assay period the cases as a result of natural infections, and in other fish were monitored regularly to ensure that they cases following experimental infestation in the labora- were eating the food, and to check for signs of toxic- tory. Parasite-free fish were experimentally infected ity. Twenty-four hours after the end of the assay, fish by holding them for 15 to 20 d in a 350 1 tank that also were anaesthetized for determination of infestation contained fish showing high-intensity infestation (400 intensity as above. Drug found to be effective at the uninfected fish to 100 infected fish). Twenty fish were screening dosage were subsequently tested at lower then sampled at random for determination of dosages (see 'Results'). infestation intensity (see below). Since infestation intensity was high in only 10 of the 20 fish, the exper- imental infestation period was extended by 7 d, after RESULTS which time infestation intensity was again determined in 10 randomly sampled fish. At this time all 10 fish Of the 24 drugs tested, 22 were not effective (i.e. did showed high-intensity infestation, and the assays not completely eliminate infestation); the results for were thus commenced. these drugs are listed in Table 2. The results for the 2 Determination of infestation intensity. Fish were effective drugs (nitroscanate and tnclabendazole) are anaesthetized by bathing in MS-222 (Sandoz, 0.05 g I-') listed in Table 3; both eliminated infestation in all 20 Tojo & Santamarina: Tredtments for trout parasites. 11 189

Table 2. Results of the assays that proved ineffective.For each drug (in all cases administered at 40 g per kg of feed for 10 d), in- festatlon ~ntensity24 h post-treatment infection intensity (as evaluated by exdmination of body scrapings; see text) is shown for each of the 20 fish included in the assay for that drug. Infestation intens~ty+++, high, ++, moderate; +, low; +/A, minimal; -, zero (i.e.no Gyrodactilus sp detected In body scrapings);nd: not determined;D: fish died dunng expenment

Drug Trout number 12 6 7 8 9 10 11 12 13 14 15 16

Aminosidine +++ Amprolium ++ Benznidazole +++ Bithionol ++ Chloroquine + Diethylcarbamazine ++ Flubendazole +++ Levarnisole + Mebendazole + Metron~dazole +++ Niclo~arnide~~~+ Nitroxynil + Oxibendazole ++ Parbendazole + Piperazine +++ Praziquantel ++ Ronidazole +++ Secnidazole +++ Tetranlisole ++ Thiophanate +++ Toltrazuril +++ Trichlorfon +++

"Most observed monogeneans were dead or mmobile; "drug reduced food consumption

fish included in each assay. Triclabendazole was effec- DISCUSSION tive at doses as low as 40 g per kg feed for 10 d, and nitroscanate at doses as low as 0.6 per kg feed for 1 d. The benzimidazole group is that group which has Neither of the 2 effective drugs had evident toxic been most extensively investigated for the treatment effects. of fish helminthioses. We tested 5 drugs from this

Table 3. Results of the assays that proved effective. For each drug, infestation intensity 24 h post-treatment infection intensity (as evaluated by examination of body scrapings and of trout; see text) is shown for each of the 20 fish included in the assay for that drug. Results of control assays are also shown. Infection intensity: +++,high, ++, moderate; +, low; +/-, minimal;,7cro (i.e.no Gyrodactylus spp. detected in body scrapings),nd not determined;D: fish died during experiment

Drugs Dose Days Trout number (g kg-') 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

aMost of the observed monogeneans were dead or immobile I 190 Dis Aquat Org

group (flubendazole, mebendazole, oxibendazole, par- We tested one probenzimidazole, thiophanate. Drugs bendazole and triclabendazole). The benzimidazole of this group have been little studied as regards effec- most widely used against monogeneans is mebenda- tiveness against fish helrninthioses. The 3 drugs from zole, generally administered by immersion (Szekely & this group which have been studied to date (netobimin, Molnar 1987, Buchmann & Bjerregaard 1990, Szekely febantel and thiophanate) have all proved ineffective, & Molnar 1990, Mellergaard 1991, Tojo 1993). Immer- regardless of mode of administration (Sanmartin et al. sion treatment with mebendazole is also widely used 1989, Santamarina et al. 1991, Tojo 1993).Thiophanate against cestodes (Boonyaratpalin & Rogers 1984, is ineffective against gyrodactylosis both in bath treat- Alarcon & Castro 1988, Sanmartin et al. 1989) and ment (Santamarina et al. 1991, Tojo 1.993) and when nematodes (Taraschewski et al. 1988). Despite their administered orally at 40 g per kg of feed for 10 d (pre- widespread use, however, mebendazole treatments sent results). are often less than 100% effective. Toxicity is rarely We tested 2 phenol derivatives, bithionol and nitrox- observed: in bath treatment, low concentrations and/or ynil. The phenol derivatives niclofolan and nitroxynil brief exposures are well tolerated, while oral treatment have previously been shown to be effective for bath with doses of 150 mg kg-' have effects of varying treatment of gyrodactylosis in rainbow trout at 0.2 mg severity, depending on species (Taraschewski et al. 1 for 3 h and at 50 mg 1-' for 3 h respectively; however, 1988, Sanmartin et al. 1989). Other drugs from this both drugs also had severe toxic effects (Santamarina group, such as albendazole, fenbendazole, flubenda- et al. 1991, Tojo et al. 1993a). Bithionol was tested by zole, oxfendazole, oxibendazole, parbendazole and Buchmann et al. (1992) against Pseudodactylogyrus thiabendazole, have been tested for bath treatment spp. and has been recommended by Herwig et al. against Gyrodactylus sp. infections of Oncorhynchus (1979) against Bothriocephalus spp. and Echinorhyn- mykiss; of these drugs, only fenbendazole was 100% chus spp. We have previously tested bithionol for bath effective, while the others were ineffective or only par- treatment of gyrodactylosis in rainbow trout, finding it tially effective; toxic effects varied between the differ- effective and without evident toxic effects when expo- ent drugs (Tojo et al. 1992, Tojo 1993). In the present sure was to 20 mg 1-' for 3 h; however, higher con- study, neither flubendazole, mebendazole, oxibenda- centrations had clear toxic effects (Santamarina et al. zole nor parbendazole, administered orally at 40 g per 1991). In the present study, neither bithionol nor kg of feed for 10 d, were 100% effective against nitroxynil were effective when administered orally at the Gyrodactylus sp. infection. None of these drugs 40 g per kg of feed for 10 d. In neither case did we showed signs of being toxic to the fish. However, tri- observe signs of toxicity; this is in accordance with a clabendazole was 100 % effective. Previous stu&es have previous study in which bithiono1 was administered found that immersion exposure to triclabendazole at at the same dose to rainbow trout infected with 25 mg 1-' for 12 h is 100% effective and does not have Ichthyophthinus multifillis (Tojo 1993) evident toxic effects (Tojo et al. 1992, Tojo 1993). In the Two imidathiazoles were tested in the present study, present study, triclabendazole was 100% effective, levamisole and tetramisole. As with the phenol deriva- and likewise lacked side-effects, when administered tives, the effectiveness of these drugs is often condi- orally at 40 g per kg of feed for 10 d. Treatment at the tioned by toxicity. Levamisole has been used success- same dosage for 5 d was not sufficient to eliminate in- fully, without evident toxicity, against nematodes, such fection, though most of the parasites showed reduced as AnguillicoIa crassus in the eel (1 mg 1-' for 24 h) mobility. This result highlights the importance of a suf- (Taraschewski et al. 1988). This drug has also been ficient treatment period when using benzimidazoles. reported to be effective against monogeneans, includ- A possible explanation for our findings with tn- ing Diplozoon paradoxurn and Gyrodactylus aculeati clabendazole is that the drug in fact acts externally, (Schmahl & Taraschewski 1987, Schmahl et al. 1989), having passed into the water through the faeces of and several species of the genus Pseudodactylogyrus the treated fish. However, this possibility can almost (Buchmann et al. 1990b). However, we have previously certainly be ruled out, since the concentration of the found that levamisole concentrations effective against drug in the tank would have been at most 0.16 mg I-' Gyrodactylus species (37 mg 1-' for 3 h) are toxic to (even without considering replacement of the water); a rainbow trout (Santamarina et al. 1991, Tojo 1993). We previous study has indicated that exposure to at least also found that tetramisole at 100 mg 1-' for 3 h was 25 mg 1-' for at least 12 h is necessary for elimination less effective than levamisole, and likewise had toxic of parasites (Tojo et al. 1992). effects. In the present study, oral administration of Despite the above results, oral treatment with tri- these drugs at 40 g per kg of feed for 10 d had no evi- clabendazole is unlikely to be an economically viable dent toxic effects, but was not effective. option, since the required dosage (40 g per kg of feed Piperazine and its derivatives are widely used for the for 10 d) implies use of large amounts of the drug. treatment of helminthioses in livestock, and to date Tojo & Santamarina: Treatments for trout parasites. 11 191

have not been found to be effective against mono- ministered orally at 40 g per kg of feed for 10 d. None genean infections of fishes. In previous studies, keto- of the 4 drugs showed evident toxicity. conazole, diethylcarbamazine, piperazine citrate and Aminosidine and chloroquine are antiprotozoals that piperazine dihydrochloride all proved ineffective in have been tested previously for bath treatment of gyro- bath treatment at 200 mg 1-' for 3 h (Santamarina et al. dactylosis at 200 rng 1.' for 3 h. In no case was activity 1991, Tojo et al. 1993a, b). In the present study, both detected (Tojo et al. 199313). Similarly, in the present diethylcarbamazine and piperazine dihydrochloride study neither of these drugs was effective. were ineffective. These results thus confirm that piper- Amprolium is a thiamine with antiprotozoal activity, azine and its derivatives are not of value for treatment though in a previous study no anti-monogenean of gyrodactylosis. Furthermore, piperazine dihydro- activity was detected (Tojo et al. 199313).In the present chloride appears to have reduced food consumption. study, amprolium was ineffective. One salicylanilide was tested in the present study, Nitroscanate has previously been reported to be niclosamide. The drugs in this group vary in their effective in immersion treatment against Gyrodactylus effectiveness against fish helminthioses, but are invan- sp. in trout (Santamarina et al. 1991). In the present ably toxic to fish. Rafoxanide is not active against Pseu- study, oral administration of this drug at 40 mg per kg dodactylogyrus or Gyrodactylus species (Buchmann of feed for 10 d was likewise 100% effective. Sub- et al. 1990a, Tojo et al. 1993a), while toxicity varies sequent trials revealed that 100% effectiveness was depending on the fish species. Bath treatment with maintained with only 0.63 g per kg of feed for 1 d only. closantel at 0.25 mg 1-' for 3 h is effective against Gyro- Furthermore, no signs of toxicity were observed. This dactylus species, but toxic for rainbow trout (Santa- drug would therefore seem to be highly effective for marina et al. 1991). Orally administered niclosamide the treatment of gyrodactylosis. has been recommended against various cestodes, Praziquantel has been extensively tested against fish namely Bothriocephalus, Proteocephalus, Coralloboth- helminthioses, and in many cases has proved effective. rium and Khawia sinensis (Herwig et al. 1979, Moore It has been tested against monogeneans (Schmahl & et al. 1984, Sanmartin et al. 1989, Schmahl et al. 1989); Mehlhorn 1985, Moser et al. 1986, Buchmann 1987, in none of these studies was toxicity observed. This Schmahl & Taraschewski 1987, Schmahl et al. 1989, drug is also effective in immersion treatments for Buchmann et al. 1990b, Szekely & Molnar 1990, Santa- combating monogeneans, including various species marina et al. 1991), trematodes (Bylund & Sumari 1981, of Gyrodactylus (Schmahl & Taraschewski 1987, Moser et al. 1986, Schmahl et al. 1989, Lorio et al. Schmahl et al. 1989) and Pseudodactylogyrus (Buch- 1990), cestodes (Moser et al. 1986, Sanmartin et al. mann et al. 1990a), although in the latter case lethal 1989, Schmahl et al. 1989, Flores-Crespo et al. 1994) toxicity was observed. We have previously found that and nematodes (Moser et al. 1986). We have previously bath treatment with niclosamide at 1.5 mg 1-' for 3 h is tested praziquantel for bath treatment of gyrodactylo- 100% effective against gyrodactylosis in rainbow sis in rainbow trout, and were unable to eliminate the trout, and has no evident toxic effects, whereas higher infection at concentrations of 10 mg 1-I for 3 h, with concentrations are toxic (Tojo et al. 1993a). In the pre- higher concentrations proving lethally toxic (Santa- sent study, oral administration of niclosamide at 40 g marina et al. 1991). This drug was likewise ineffective per kg of feed for 10 d showed sings of toxicity and in the present study. was not l00 % effective, although a large proportion of Toltrazuril has been tested previously against mono- parasites were killed. Furthermore, niclosamide ap- genean infections of fishes (Schmahl & Melhorn 1988, pears to have reduced food consumption, leading to Schmahl et al. 1989). In bath treatment of gyrodacty- accumulation of large amounts of feed at the bottom of losis in rainbow trout, exposure to 200 mg 1-' for 3 h the tank. was not effective (Tojo et al. 1993b). Likewise, in the Four nitroimidazoles (benznidazole, metronidazole, present study oral treatment was ineffective; indeed, ronidazole and secnidazole) were tested in the present infection intensities remained very high at the end of study. The drugs of this group are typically more effec- the assay. tive against protozoa than helminths. The only previ- Trichlorfon (Neguvon) has been extensively studied ous study of effectiveness against monogeneans in fish as regards the treatment of fish helrninthioses, includ- is that of Tojo et al. (1993b),in which we tested dimetri- ing infections by Pseudodactylogyrus spp. (Imada & dazole, carnidazole, metronidazole, ronidazole and Muroga 1979, Buchmann et al. 1992) and Anguillicola benznidazole in bath treatment against gyrodactylosis crassus (Taraschewski et al. 1988). Its effectiveness in rainbow trout. None of these drugs proved effective. varies widely. Goven et al. (1980) found that bath treat- In the present study, benznidazole, metronidazole, ment at 25 mg 1-' for 72 h was necessary to eradicate ronidazole and secnidazole (not tested previously gyrodactylosis, and treatments for shorter periods against Gyrodactylus sp.) were ineffective when ad- were ineffective even when higher doses were used 192 Dis Aquat Org 33: 187-193, 1998

(Goven & Armend 1982, Santamarina et al. 1.991). Herwig N, Garibaldi L, Wolke RE (1979) Handbook of drugs Trichlorfon was not effective in the present study. and chemicals used in the treatment of fish diseases. In conclusion, the results of the present study indicate Thomas CC, Springfield, 1L Irnada R, Muroga K (1979) Pseudodactylogyrus rnicrorch~s that both triclabendazole and nitroscanate are effective (Monogenea) on the gills of cultured eels - 111 experimen- as oral treatments for gyrodactylosis in farmed rainbow tal control by trichlorfon. Bull Jpn Soc Sci Fisheries 45(1): trout. Nitroscanate is effective at a relatively low 25-29 dosage (0.6 g per kg of feed for 1 d). However, this Johnsen BO. Jensen AJ (1992) Infection of Atlantic salmon Salmo salar, by Gyrodactylus salaris, Malmberg 1957, in dosage is likely to be prohibitively expensive. Gyro- the River Lakselva, Misvaer In the northern Norway. dactylosis can be effectively treated by bath treatment J Fish Biol 40:433-444 with nitroscanate at only 0.7 mg 1-l. Oral treatment with Lorio WJ, Houser R, Powell RV (1990) Experimental control nitroscanate may be administered in situations in which of yellow grob in channel catfish. Aquac Mag 16(5):57-59 bath treatment is not possible (floating tanks, natural McKenzie K (1970) Gyrodactylus unicopula Glukhon, 1955, from young plaice Pleuronectes platessa L. with notes on ponds, etc.). the ecology of the parasite. J Fish Biol2:23-24 Mehlhorn H, Schmahl G, Haberkorn A (1988) Toltrazunl effective against a broad spectrum of protozoan parasites. Pdrdsitoi Res 15:64-66 LITERATURE CITED Mellergaard S (1991) Mebendazole treatment against Pseudo- dactylogyrus infections in eel (Anguilla anguilla). Aqua- Alarcon C, Castro JL (1988) Tratarniento experimental con culture 91~15-21 mebendazol para botriocefalosis en Carassius carassius. 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Tojo JL (1993) Tratamiento farmacologico de ectoparasitosis Gyrodactylus sp. Infecting ra~nbowtrout Oncorhynchus en trucha (Oncorhynchus mykiss): Gyrodactylus sp., mykiss. Dis Aquat Org 12:185-189 Ichthyobodo necator e Ichthyophthirius multifiliis. Doc- Tojo JL. SantamarinaMT. Ubeira FM, Estevez J, Leiro J. San- toral thes~s,Univers~ty of Santiago de Compostela, Spain martin ML (1993a)Efficacy of anthelmintic drugs against Tojo JL,Santamarina MT (1998) Oral pharmacological treat- gyrodactylosis In ra~nbowtrout (Oncorhynchus rnykiss). ments for parasitic d~seasesof rainbow trout O~COI-~~JI-Bull Eur Assoc Fish Pathol 13(2):45-58 chus mykiss. I: Hexamita salmonis. Dis Aqi~atOrg 33: Tojo JL, Santamarina MT, Ubeira FM, Leiro J, Sanmartin ML 51-56 (1993b)Efficacy of antiprotozoaldrugs against gyrodacty- To10 JL, Santamarina MT. Ubeira FM. Estevez J, Sanmartin losis in rainbow trout (Oncorhynchus mykiss). Bull Eur ML (1992)Anthelmintic activity of benzimidazoles agalnst Assoc F~shPathol 13(3):79-82

Editorial responsibility: WolfgangKorting, Submitted: February IG,1998; Accepted. May 5, 1998 Hannover, Germany Proofs received from author(s):July 15, 1998