I: Hexamita Salmonis

Home , Flubendazole, Nitroscanate, Oxibendazole

DISEASES OF AQUATIC ORGANISMS Vol. 33: 51-56,1998 Published May 14 Dis Aquat Org

Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. I: Hexamita salmonis

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

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

ABSTRACT: Various drugs were evaluated as regards efficacy for the treatment of Hexamita sahonis infection in rainbow trout. The results confirm the efficacy of nitroimidazoles: infection was completely eradicated not only by metronidazole (which has been recommended previously for the treatment of hexamtosis), but also by benznidazole, ronidazole and secnidazole, which have not been assayed previously. The non-nitroimidazoles albendazole, amnosidine, hethylcarbamazine and nitroscanate also completely eliminated infection. The remaining non-nitroin~idazoles tested (amproliun~,bithionol, febantel, flubendazole, levamisole, netobimin, niclosamide, nitroxynil, oxibendazole, parbendazole, piperazine, praziquentel, tetramisole, thiophanate, toltrazuril, trichlorfon and triclabendazole) were not effective

KEY WORDS. Hexamitosis . Ranbow trout. Treatment Drugs

INTRODUCTION Affected fish typically swim on their side, or with cork- screw movements (Lom & Dykova 1992, Woo 1994). Intestinal infection of fish by flagellates of the genus Populations affected by acute hexamitosis show high Hexamita is often associated with high mortality. Some mortality over a very short period, due to rapid multi- authors have suggested that pathological effects arise plication of the parasite and associated damage to the when the host is weakened by other factors such as intestinal epithelium. The chronic form of hexamitosis inadequate diet, a change in diet, low oxygen content is likewise common, and generally occurs between in the water, overcrowding, inappropriate handling, spring and autumn; mortality per unit time is only and/or keeping fish of different sizes together (Becker slightly higher than in healthy fish, but severe losses 1977, Vickerman 1989). In both salmonids and tropical may occur because the situation continues for a period aquarium fish, intestinal parasitoses are often patho- of weeks (Woo 1994). genic only when the number of parasites present is In trout and other salmonids infected with Hexamita very high (Post 1987 and Andrews et al. 1988, cited by salmonis, the effects commonly observed are anaemia, Woo & Poynton 1995).Gratzek (1988) suggests that fla- weight loss (Naich & Nilgees 1992), dark coloration, gellate parasites interfere with nutrition by competing enteritis, excessive body mucus, and yellowish intesti- for essential nutrients and/or by damaging the intestinal mucus (attributable to modified release of bile into nal epithelium. the digestive tract); in addition, intestinal haemor- Hexamitosis is probably the most frequent internal rhage and liver cell necrosis may also be observed flagellate parasitosis of fish, notably in young sal- (Woo & Poynton 1995). Nevertheless, fish that appear monids, though also in carp, aquarium species, and to be heavily infected (i.e. numerous parasites in the various marine fish. Heavily infected fish are weak, pyloric caecae and the intestine) may show no signs of listless, anorexic and emaciated, so that the head damage to the mucosa, and no evidence of invasion of appears large with respect to the body ('pinhead fish'). the epithelium by the parasite (Ferguson 1979). The only oral pharmacological treatments of Hexa- mita salmonis described to date have been drugs of the

0 Inter-Research 1998 Resale of full article not permtted 5 2 Dis Aquat Org 33: 51-56, 1998

nitroimidazole group [Herwig et al. 1979, Reinhold'et after examination of a sample area of 24 X 32 mm, as al. 1983 (cited by Woo 1984). Ghittino 1985, Lazaro- follows: 'zero' (-), Hexamita salmonis not detected in Chavez 1985, Imamovic 1986, Schmahl et al. 19891, the sample; 'minimal' (+/-), only 1 individual of H. which show activity against various protozoan groups, salmonis detected in the sample; 'low' (+), more than 1 including flagellates and ciliates. The current treat- individual of H. salrnonis detected in the sample, the ment of choice is dimetridazole or metronidazole in the average number per microscope field being less than feed (Stoskopf 1993, Woo 1994, Woo & Poynton 1995). 10; 'moderate' (++), average number of individuals per There have been few studies of the possible anti- microscope field 10 to 50; 'high' (+++), average number Hexamita activity of drugs of other groups. Here, we of individuals per microscope field >50. report a screening study of the possible efficacy of 21 Drugs and assay design. The drugs tested in the non-nitroimidazole drugs at high doses (40 g per kg of study are listed in Table 1. Each drug was assayed in feed for 10 d) for treatment of rainbow trout infected an 80 1 tank containing 20 infected fish. A simultane- with Hexamita salmonis. As positive controls, we ous control assay (also of 20 fish; identical treatment, treated some fish with 1 of 5 nitroimidazoles, including but without drug) was performed for each drug. Tank metronidazole, as recommended for this parasitosis by conditions (water source, flow, aeration, pH, tempera- Herwig et al. (1979) and Stoskopf (1993). ture, light/dark cycle) were identical to those during the acclimatization period. The treated fish received either (1) feed containing 40 g kg-' of drug (non- MATERIALS AND METHODS nitroimidazoles) for the 10 d, or (2) feed containing 2 or 5 g kg-' of drug (nitroimidazoles) for 2 d. Unamended Fish. Rainbow trout Oncorhynchus mykiss, Walbaum feed was administered to fish of control group. In all (weighing 16 g at the start of the experiments) were cases feed was supplied at 2% of total body weight per obtained from a local fish farm (Piscifactonas Corune- day. Throughout the assay period the fish were moni- sas S.A., Carballo, A Corufia) and acclimatized for at tored regularly to ensure that they were eating the least 36 h before assay in a 350 1tank with aeration and food, and to check for signs of toxicity. Twenty-four continuous flow of water (15 rt 3"C, pH 6.5 + 0.5) from hours after the end of the assay, fish were anaes- a nearby spring. Fish were fed daily with a commercial thetized for determination of infection intensity as feed. above. Infection. All fish used in the trials showed high-intensity infection by Hexamita salmo- Table 1. Drugs used in the study, showing manufacturer, brand and form nis, in some cases as a result of natural infec- of presentation. p.p. = pure product. 'Nitroimidazoles tions, and in other cases following experimental infection in the laboratory Parasite- Brand name Presentation Manufacturer free fish were experimentally infected by L Ovejero holdlng them for 15 to 20 d in a 350 l tank that Albendazole P.P. Powder Aminosidine Gabbrocol lnlectable Vetern S.P.A. also contained fish showing high-intensity Amproliurn Prolsal Powder Iteve infection (400 uninfected fish to 100 infected Benznidazole P?. Powder Roche fish). Twenty fish were then sampled at ran- Bithionol P.P. Powder Syva dom for determination of infection intensity Diethylcarbamacine p.p. Powder Cidan Febantel P.P. Powder Bayer (see below). Since infection intensity was Flubendazole P.P. Powder Esteve high In only 10 of the 20 fish, the experimen- Levarnlsole P.P. Powder Ovejero tal infection period was extended by 7 d, after Metronidazole ' Flagyl Powder Rhone Merieux which infection intensity was again deter- Netobimin P.P. Powder Ovejero mined in 10 randomly sampled fish. At this Niclosamide Fugotenil Pills Uriach Nitroscanate Lopat01500 Pills Ciba-Geigy time all 10 fish showed high-intensity infec- Nitroxynil P.P. Powder Ovejero tion, and the assays were thus commenced. Oxi bendazole P.P. Powder Syva Determination of infection intensity. Fish Parbendazole P.P. Powder Smith kline were anaesthetized by bathing in MS-222 Piperazine Piperso 1 Granules Sobrino Praziquentel Droncit Pills Rayer (Sandoz; 0.05 g 1-') until respiration became Ronidazole ' P.P. Powder Sobrino weak. A faecal sample was then obtained by Secnidazole ' P.P. Powder Rhone Merieux gently pressing the abdomen. The sample Tetramisole P.P. Powder Ovejero was mixed with 3 drops of water on a slide, Tiophanate P.P. Powder Uriach Bayer coverslipped and examined with a light Toltrazuril P.P. Powder Trichlorfon Neguvon Powder Bayer microscope (400~).Estimation of infection Triclabendazole P.P. Powder Ctba-Geigy intensity was recorded on a 5-point scale, Tojo & Santamarina: Treatments for trout parasites

Table 2. Results of the assays with non-nitroirnidazoles that proved ineffective. For each drug (in all cases administered at 40 g per kg feed for 10 d), 24 h post-treatment infection intensity (as determined by faecal examination; see test) is shown for each of the 20 fish included in the assay for that drug. Infection intensity: +++, high; ++, moderate; +, low; +I-, minimal; -, zero (i.e. no Hexamita salmonis detected in faeces). nd: not determined; D: fish died during experiment

Drug Trout number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17- 18 19 20

Amprolium +++ ++ ++ ++ ++ + + + + +l- +l- +l------Bithionol ++ + ------nd nd Febantel +++ +++ +++ ++ ++ + + + +l------Flubendazole +++ + + + +/- +l- +l------Levamisole +++ +++ +++ +++ ++ ++ +++ + + + +l------Netobimin +++++++++ + + + + + p ------Niclosamide +++ ++ ++ ++ +a + + + +/- - - - nd nd nd nd nd nd Nitroxynll +++ +++ ++ ++ + + ------nd D Oxibendazole ++ ------Parbendazole + ------W - - - Piperazine +++ +++ +++ + ++/------ndDD Praziquentel +++ +++ ++ ------nd nd nd nd Tetramisole +++ +++ +++ +++ ++ ++++ + + + + + +/- . ----- Tiophanate +++ + + ------Toltrazuril +++ +++ +++ ++ ++ ++++ ++ ++ + + + + ,, ---- Trichlorfon +l- +l------Triclabendazole +l------nd nd nd

RESULTS even at the lower dose of 2 g per kg feed for 2 d (Table 4). These results confirm the efficacy of these Of the 21 non-nitroimidazoles, 17 were not effective drugs when administered for a shorter period and at (i.e. did not completely eliminate infection); the results much lower doses than the other drugs tested. for these drugs are listed in Table 2. The results for the 4 effective non-nitroimidazoles (albendazole, aminosidine, diethylcarbamazine, nitroscanate) are listed in DISCUSSION Table 3; all 4 eliminated infection in all 20 fish included in each assay. None of the 4 effective non-nitroimida- In the present study we considered a wide range of zoles had evident toxic effects. anti-parasitic drugs, including 3 that have been recom- The 4 nitroimidazoles likewise completely elimi- mended for the treatment of protozoan parasitoses of nated infection at 5 g per kg feed for 2 d (Table 4). All fish, namely amprolium (Awakura & Kurahashi 1967, nitroimidazoles except metronidazole were effective Taylor et al. 1973, Herwig et al. 1979, Schmahl et al.

Table 3. Results of the assays wlth non-nitroimidazoles that proved effective. For each drug-treated group (drug administered at 40 g per kg feed for 2 d) and the corresponding control group, 24 h post-treatment infection intensity (as determined by faecal examination; see text) is shown for each of the 20 fish included in that assay. Infection intensity: +++, high; ++,moderate; +, low; +I-, minimal; -, zero (i.e. no Hexamita salmonis detected in faeces). nd: not determined; D: fish died during experiment

Drug Trout number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Abendazole Control +++ +++ ++++++ ++++++ +++ +++ ++++++ ++ ++ + + - - - - - D Aminosidine nd Control +++ +++ +++ +++ ++ ++ ++ ++ ++ + + + + nd Diethylcarbamaclne ------Control +++ ++ ++ ++ ++++ + + + + +/- +/- +l- - - - Nitroscanate - nd nd Control +++ ++++++ +++ +++ +++ ++++ ------nd Dis Aquat Org 33: 51-56, 1998

Table 4. Results of the assays with the 4 nitroimidazoles. For each drug-treated group (drug administered at 2 or 5 g per kg feed, for the first 2 d of the 10 d experimental period1 and the corresponding control group, post-treatment infection intensity (as determined by faecal examination; see text) is shown for each of the 20 fish included in that assay. Infection intensity: +++, high; ++, moderdtc,. +, low; +/-, IIIIILIIII~~;-, zero (~.e,no Hexarnlta salmonls detected in faeces), nd: not determined

Drug Trout number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Benznidazole 5 ------2 Control - +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + ------Metronidazole 5 ------2 +/------. / Control - ++++++-c+++++ ++ rr ++ ++ r- + + T nd

Ronidazole 5 ------2 - Control - +++ +++ +++ +++ t++ +++ ++ ++ ++ t+ + - -

Secnidazole 5 ------A------2 ------Control - +++ +++ +++ +++ +++ +++ ++ ++ +++t + ------

1989, Stoskopf 1993), bithionol (Tojo 1993) and previous studies, none of which has demonstrated anti- toltrazuril (Melhorn et al. 1988, Schmahl & Melhorn protozoan activity of these drugs in fish (Obermeier 1989a, b, Schmahl et al. 2989, Schmahl et al. 1990, 1974, Gr1ffi.n 1989, Rydlo 1989, Tojo et al. 1994a, b). Yokoyama et al. 1990, Schmahl et al. 1991, Stoskopf The only non-nitroimidazole drugs that completely 1993). eliminated infection were albendazole, aminosidine, Of these, toltrazuril is that which has been most diethylcarbamazine and nitroscanate. Of these, the widely used for the treatment of parasitoses of fish, and only one previously recommended for the treatment of indeed this drug has been recommended for the treat- infection by Hexamita salmonis is aminosidine (15 g ment of various microsporidian and myxosporidian per kg feed for 3 consecutive days) (Herwig et al. infections (Schmahl & Melhorn 1989a, b). This drug is 1979). Nitroscanate appears to have a rather broad not, however, effective for the treatment of infestation activity spectrum, since it has been shown to be effec- by the ectoparasitic ciliate Ichthyophthirius multifilils tive for bath treatment of Gyrodactylus (Santamarina (From et al. 1992, Tojo et al. 1994~). et al. 1991). Neither albendazole nor diethylcarba- Of these drugs (amprolium, bithionol and toltrazuril), mazine has previously been shown to be effective for the only one previously found to be effective after oral treatment of protozoan parasitoses of fishes (see Griffin administration is amprolium; ind.eed, its administration 1989). by this route is recommended for myxosporidiosis. Four nitroimidazoles were used in the present study Bithionol and toltrazuril have been shown to be effec- as positive controls. Nitroimidazoles are currently the tive only in bath treatments. In a study of efficacy for only drugs recommended for oral treatment of Hexa- the treatment of infestation by the flagellate Ichthy- mita salmonis infection. We used doses of 2 or 5 g per obodo necator, complete el~minationof infestation In kg feed for 2 d. Previous reports have recommended all fish assayed was achieved after bathing with dimetndazole at 1.5 g per kg feed for 3 d (Herwig et al. bithionol at 25 mg 1-' for 3 h on 2 consecutive days, but 1979), or at 15 g per kg feed for 4 to 7 d (Schmahl et al. not after bathing with amprolium or toltrazuril (Tojo et 1989). Metronidazole has been recommended at doses al. 1994a.). of 0.5 mg per kg feed for 2 d (Imamovic 1986), 20 mg In the present study, none of these drugs was effec- per kg feed for 2 d (Reinhold et al. 1983, cited by Woo tive for oral treatment of Hexamita salmonis infection, 1994), or 1.5 g per kg feed for 3 d (Lazaro-Chavez despite the fact that the dose used was much higher 1985). Bath treatment with metronidazole (at 4 mg I-') than in simj.lar previous studies. Equally ineffective has been recommended for infections with Trichodina, were febantel, flubendazole, levamisole, netobimin, Ambiphyra and Chilodonella (Moore et al. 1984). Di- niclosamide, nitroxynil, oxibendazole, parbendazole, metridazole and metronidazole have also been tested piperazine, praziquentel, tetramisole, thiophanate, tri- against other protozoan parasitoses (bath treatment chlorfon and triclabendazole; this is in accordance with against Ichthyobodo necator; oral and bath treatment Tojo & Santamarina: Treat :merits for trout parasites 55

against I. multifiliis; Tojo 1993), with negative results. Imamovic V (1986) Parasites and parasitoses In fish in some As far as we are aware, the other nitroimidazoles salmonid hatcheries in Bosnia-Hercegovina, I. Ichthyo- tested in the previous study (benznidazole, ronidazole bodo and Hexamita infections. Veterinaria (Sarajevo) 35(1):47-66 and secnidazole) have never been used for the treat- Kent bIL, Ellis J, Fournie JW, Dawe SC, Bagshaw JW, ment of H. salmonis infection Whitaker DJ (1992) Systemic hexamitid (Protozoa: Diplo- Dimetridazole was not included in our assays, since monadida) infection in seawater pen-reared chinook we have previously found it to be toxic in bath treat- salmon Oncorhynchus tshawytscha. Dis Aquat Org 14: 81-89 ment (Tojo 1993). Oral treatment of trout with some Kulda J,Lom J (1964) Remarks on the diplomastigine flagel- nitroimidazoles has been reported to affect behaviour lates from the intestine of fishes. Parasitol 54:753-762 (Tojo 1993). The nitroimidazoles used here were Ldzaro-chavez E (1985) Sustanclas, desinfectantes y drogas selected in view of the absence of evident toxic effects de utilidad en las piscifactonas. Manual de usos. AGT Edi- tores S.A., Mejico when administered orally (Tojo 1993). Likewise, none Lom J, Dykova I (1992) Developments in aquaculture and of these drugs showed negative effects (signs of tox- fisheries science, Vol 26. Protozoan parasites of fishes. icity, behavioural effects including anomalous swim- Elsevier, Amsterdam ming movements, rejection of food) in the present Mehlhorn H, Schn~ahlG, Haberkorn A (1988) Toltrazunl study, suggesting that all 4 nitroimidazoles assayed are effective against a broad spectrum of protozoan parasites. Parasltol Res 15:64-66 viable options for the treatment of infection of Moore BR, Mitchell AJ, Grlffin BR, Hoffman GL (1984) 14. salmonids by Hexamita salmonis. This is of particular Parasites and diseases of pond fishes. 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Editorial responsibility: Wolfgang Korting, Submitted: January 6, 1998;Accepted: February 16, 1998 Hannover, Germany Proofs received from author(s): April 6,1998