DISEASES OF AQUATIC ORGANISMS Published October 4 Dis. aquat. Org. l

Comparative efficacies of commercially available benzimidazoles against Pseudodactylogyrus infestations in eels

' Department of Fish Diseases, Royal Veterinary and Agricultural University, 13 Biilowsvej, DK-1870 Frederiksberg C, Denmark Department of Pharmacy, Royal Veterinary and Agricultural University, 13 Biilowsvej. DK-1870 Frederiksberg C,Denmark

ABSTRACT: The antiparasitic efficacies of 9 benzimidazoles in commercially avalable formulations were tested (water bath treatments) on small pigmented eels Anguilla anguilla, expenmentally infected by 30 to 140 specimens of Pseudodactylogyrus spp. (Monogenea).Exposure time was 24 h and eels were examined 4 to 5 d post treatment. Mebendazole (Vermox; 1 mg 1-') eradicated all parasites, whereas luxabendazole (pure substance) and albendazole (Valbazen) were 100 % effective only at a concen- tration of 10 mg I-'. Flubendazole (Flubenol), fenbendazole (Panacur) and oxibendazole (Lodltac) (10 mg l-') caused a reduction of the infection level to a larger extent than did triclabendazole (Fasinex) and parbendazole (Helmatac).Thiabendazole (Equizole), even at a concentration as high as 100 mg l-', was without effect on Pseudodactylogyrus spp.

INTRODUCTION range of commercially available benzimidazole com- pounds. If drug resistance will develop under practical The broad spectrum anthelmintic drug mebendazoIe eel-farm conditions in the future, it is likely to be was reported as an efficacious compound against infes- recognized during treatments with commercially avail- tations of the European eel Anguilla anguilla with gill able drug formulations. Therefore this type of drug parasitic monogeneans of the genus Pseudodactylo- preparations were used in the present study. gyms (Szekely & Molnar 1987, Buchmann & Bjerre- gaard 1989, 1990, Mellergaard 1989). As benzimida- zoles share many molecular features and action mech- MATERIALS AND METHODS anisms (Vanden Bossche 1985), other representatives of this drug group are also likely to have an effect Eels. Batches, each comprising 10 specimens of small on these parasites. The present paper reports on trials pigmented eels Anguilla anguilla (total body length 8 with 9 different benzimidazoles for this host-parasite to 16 cm and body weight 0.7 to 5.5 g) were exposed to system. aerated drug solutions (7 1) at 24 to 25°C in plastic The increasing use of mebendazole in eel farms aquaria (volume 17 1). Eels were exposed for 24 h imposes a risk for selection of drug resistant parasites. whereafter they were transferred to clean aquaria with Side resistance, defined as resistance to another com- pure aerated tap water at the same temperature. After pound belonging to the same drug group (Waller 1985), 4 to 5 d they were examined for Pseudodactylogyrus to other benzimidazoles would then be expected to spp. as described by Buchmann (1988a). These eels occur, as this type of resistance has been recorded in had been kept in a parasite-free fish tank for 2 mo and parasitic nematodes (Martin 1985). However, side were infected before the experiments as described resistance could only be properly evaluated if trials below. Two batches of eels were incubated in pure tap with these benzimidazoles have been conducted on a water and kept as untreated controls. For each trial, the susceptible, anthelmintic-naive parasite population. body length and weight of examined eels were This trial also serves as a base-line study to determine recorded (Table 1) together with prevalence (percent- the susceptibility of such a parasite population to a age of hosts infected) and abundance (mean number of

Inter-Research/Printed in Germany 118 Dis. aquat. Org.

parasites per host - infected and uninfected) calculated After few hours eels turned sluggish and lay on their according to Margolis et al. (1982). Only surviving eels side or back on the bottom of the aquaria, until they were examined. were removed from the medicated water However the Parasites. A mixed population of Pseudodactylo- mortality during the 4-d observation period was low gyrus anguillae and P. bin] on larger eels (10 to 20 g (Table 1). The other drugs elicited no irnmediate body weight) tvere maintained in a 250 1 aquarium adverse reactions, but a high mortality (4 of 10) in the (25°C). This population of parasites has never been oxibendazole group (10 mg 1-') was noted by 4 d post- treated with drugs and an infection pressure occurs in treatment. the aquarium. The formerly uninfected eels were placed in small net cages (Buchmann 1988b) and sub- DISCUSSION merged in the aquarlum for 6 d, whereby all eels became infected (30 to 140 parasites eel-'). The trials With the exception of luxabendazole, the ben- were conducted 14 to 20 d post-infection. zimidazoles used in this investigation were the com- Drugs. The drug concentrations used are listed in mercially available preparations, including their vehi- Table !. With one exception (luxabendazo!e), where cles. -4s additions may influence to a greater or lesser pure substance was used with acetone as solvent, the extent, the suspension of drugs In water, it should be drug preparations tvere the commercially available stressed that the present comparative study is on the products: albendazoie (Vaibazen vei. suspension commercially available products and their vchic!cs. 19 mg ml-l), fenbendazole (Panacur suspension These can be ranked after decreasing potency on 100 mg ml-l), flubendazole (Flubenol 50 mg g-l), lux- Pseudodactylogyrus spp. It was found that meben- abendazole (pure substance), mebendazole (Vermox dazole (Vermox) was the most potent drug and our suspension 50 mg ml-l), oxibendazole (Loditac vet. 50 results confirm work of Szekely & Molnar (1987), Buch- mg parbendazole (Helmatac vet. 300 mg g-l), mann & Bjerregaard (1989, 1990) and Mellergaard thiabendazole (Equizole 330 mg g-') and tricla- (1989). However luxabendazole (with acetone as sol- bendazole (Fasinex suspension 100 mg ml-l). vent) and albendazole (Valbazen) also showed efficacy against infections with Pseudodactylogyrus spp. When fenbendazole, flubendazole and oxibendazole, in their RESULTS commercially available formulahons, were used in water bath treatments with 10 mg 1-' of the drug, only a Anthelmintic efficacy reduction of the infection level was seen. At this con- centration triclabendazole treatment only elicited a It was found that exposure to mebendazole (1 mg 1-l) slight reduction of the infection, and parbendazole for 24 h was 10O0/0 effective in parasite eradication. (Helmatac) could only reduce the infection level at a Luxabendazole was nearly as efficacious: only one concentration as high as 50 mg I-'. Thiabendazole had parasite was found in 8 eels after treatment with 1 mg no effect even at 100 mg 1-' and elicited acute toxic 1-l. Albendazole and flubendazole had some effect at symptoms in the eels. this concentration, but parasite eradication was only Because of the unknown influence of the vehicles in accomplished for albendazole, as for luxabendazole, at the different benzimidazole-products on drug suspen- 10 mg I-'. sion in, water and penetration of drug into the parasite, A reduction of the infection level, without total para- it is not possible to make clear conclusions about the siticidal effect, was found with exposure for 24 h with different pure compounds' action in the monogeneans. 10 mg 1-' of the compounds flubendazole, oxiben- However, it is noteworthy that the benzimidazoles with dazole and fenbendazole. A less convincing reduction the best effect on Pseudodactylogyrus spp, all have was seen after treatment with triclabendazole (10 mg substitutions with methyl carbamate in the 2-position 1-l) and parbendazole (5 mg I-'). The latter drug had a of the molecule. Thiabendazole and triclabendazole better, but still inadequate, effect at a concentration of are both without the carbamate moiety and have no or 50 mg 1-l. Even with a concentration of thiabendazole little effect on the parasites. The substitutions at the 5- as h~ghas 100 mg I-' no reduction of the infection level position differ markedly between the 3 most potent was found (Table 1). benzimidazoles, mebendazole, luxabendazole and albendazole, and future research should elucidate the importance, if any, of this part of the molecule on the Toxicity observations gill parasites. The major target for benzimidazoles' action seems to be the microtubule system (Behm & Thiabendazole (both 10 and 100 mg 1-l) elicited Bryant 1985, Vanden Bossche 1985, Roberson 1988). escape reactions in eels a few seconds after exposure. Triclabendazole, which differs structurally from other Buchmann & Bjerregaard: Benzimidazole efficacy in eels 119

Table 1 Anguilla anguilla. Level of parasit.ization with Pseudodactjdogyrus spp. after exposure to various benzimidazoles. Only surv~vingeels were examined. Length and weight of eels presented

Drug Conc. No. Body -length (cm) Body weight (g) After treatment (mg 1-') eels X (SDI X (SD) Prevalence Abundance

(n*p) (SD)

Albendazole (Valbazen)

Fenbendazole (Panacur) Flubendazole (Flubenol) Luxabendazole (pure substance)

Mebendazole (Vermox) Oxibendazole (Loditac) Parbendazole (Helmatac) Thiabendazole (Equizole) Triclabendazole (Fasinex)

Control 1 Control 2

benzimidazoles, has been suggested to act on a However, based on the present investigations, para- number of targets in the liver fluke (Bennett & Kohler sites which are susceptible to mebendazole (Vermox) 1987). The cumulative effect could thus result in the will not be affected by thiabendazole (Equizole) and killing of the parasite. In addition, mitochondrial elec- triclabendazole (Fasinex). In addition only a lesser tron transport is also inhibited by thiabendazole effect was found with fenbendazole (Panacur), fluben- (Kohler & Bachmann 1987), and Sanval et al. (1989) dazole (Flubenol), oxibendazole (Loditac) and parben- found that enzymes of lipid metabolism are affected by dazole (Helmatac). The importance of the vehicles for thiabendazole and fenbendazole. Future research the anthelmintic effect of these preparations (drug sus- should elucidate the exact action mechanisms of ben- pension, parasite penetration) should be investigated. zimidazoles in Pseudodactylogyrus spp. Also it will be relevant to investigate if mebendazole- Mebendazole has proved to be a safe and useful resistant gill-parasites, if these will evolve, also have anthelmintic against pseudodactylogyrosis in intensive lost susceptibility to luxabendazole and albendazole. eel-farming enterprises based on recirculated water Finally it should be accentuated that before luxaben- (Buchmann & Bjerregaard 1989, 1990), and the drug is dazole and albendazole can be recommended for treat- now commonly used. However, resistance to anthel- ment of pseudodactylogyrosis in intensive eel-farming mintics in parasitic helminths is an increasing problem enterprises, controlled investigations on long-term tox- in livestock (for review see Anderson & Waller 1985), icity and the impact on biological and physico-chen~i- and the risk of selection of mebendazole-resistant cal parameters in the farms should be conducted, as monogeneans exists if indiscriminate use of this drug our results only were based on short term laboratory occurs in eel-farms. Side resistance to other ben- experiments. zimidazoles might then be expected. If the failing effect of other commercially available benzimidazoles is Acknowledgements. This investigation was supported by a recorded by eel-farmers after resistance has evolved, grant from the Danish Agricultural and Veterinary Research side resistance would then be a tempting explanation. Council. 120 Dis. aquat. Org. 9 117-120, 1990

NOTE ADDED IN PROOF of pseudodactylogyrosis in an Intensive eel-culture system. Aquaculture 86. 139-153 In further studies with pure drug substances and Kohler, P., Bachmann, R. (1978). The effects of the anti- parasitic drugs levamisole, thiabendazole, praziquantel. formic acid as solvent, it was found that luxabendazole and chloroquine on mitochondrial electron transport in and flubendazole (1 mg 1-l) eradicated the parasites muscle tissue from Ascaris suum. Molec. Pharmac. 14: from lightly infected small eels [abundance (SD): 7.6 155-163 (7.2)].In addition it was confirmed that albendazole Margolis. L.. Esch. G. W., Holmes, J. C., Kuris, A. M.. Schad, G. A. (1982). The use of ecological terms in parasitology only had 100 O/O efficacy in a concentration of 10 mg 1-l. (report of an ad hoc comrnitee of the American Society of Parasitologists). J. Parasitol. 68: 131-133 Martin, P. J. (1985). Nematode control schemes and anthel- LITERATURE CITED mintic resistance In: Anderson, N., Waller, P. J. (eds.). Resistance in nematodes to anthelrnintic drugs CSIRO Anderson, N., Waller, P. J. (Eds.) (1985). Resistance in Division of Animal Health, Glebe, Australia Nematodes to anthelmintic drugs. CSIRO Division of Mellergaard, S. (1989). Mebendazole treatment against Animal Health, Glebe, Australia Pseudodactyloqyrus infections in eel. In: Fagerholm, M. P Behm, C. A., Bryant, C. (1985). The modes of action of some (ed.) Proc. 14th Scand. Symp. Parasit. Elsinore, Denmark, modern anthelmintics In: Anderson, N., Waller, P. J. (eds.). August 2-4, 1989, Aabo, Finland Resistance in Nematodes to anthelmintic drugs. CSIRO Roberson, E. L. (1988). Antinematodal drugs. In: Booth, N. H., Division of Animal Health, Glebe, Australia McDonald, L. E. (eds.). Veterinary pharmacology and Bennett, J. L,Kohler, P. (1987) Tasciola "iepatica. actioi; in therapeutics, 5th edn, :ows S+,a,, t- IT-'..-.u.LL.L,s,,) <+. ,DT- .,ss, P,.m,cs. vitro of triciabendazole on lmmature and adult stages. Expl Ephrata, Pa Parasit. 63: 49-67 Sarwal, R., Sanyai, S. N., Khera, S. (1989). In vitro effect of Buchmann, K. (1988a). Spatial distribution of Pseudodactylo- benzimidazole drugs on the enzymes of lipid metabolism gyrus ang~~illaeand P. bin1 (Monogenea) on the gills of the in Trichuris globulosa. J. Parasitol. 75: 808-810 European eel, Anguilla angullla. J. Fish Biol. 32: 801-802 Szekely, CS., Molnar, K. (1987).Mebendazole is an efficacious Buchmann, K. (1988b). Epidemiology of pseudodactylogyrosis drug against pseudodactylogyrosis in the European eel in an intensive eel-culture system. Dis. aquat. Org. 5: (Anguilla angullla). J. appl. Ichthyol. 3: 183-186 81-85 Vanden Bossche, H. (1985). Pharmacology of anthelmintics. Buchmann, K., Bjerregaard, J (1989). Effects of mebendazole In Vanden Bossche, H., Thienpont, D., Janssens, P. G. on Pseudodactylogyrus spp. from Anguilla anguilla and on (eds.) Chemotherapy of gastrointestinal helminths. the microflora and microfauna in an intensive eel-culture Springer Verlag. Berlin system. In: Fagerholm, M. P. (ed.)Proc. 14th Scand Symp Waller, P J. (1985). Resistance to anthelmintics and the impli- Parasit. Elsinore Denmark, August 2-4, 1989. Aabo, cahons for animal production. In: Anderson, N , Waller, P Finland J. (eds.) Resistance in nematodes to anthelmintic drugs. Buchmann, K., Bjerregaard, J. (1990).Mebendazole-treatment CSIRO Division of Animal Health, Glebe, Australia

Responsible Subject Editor: Professor W Korting, Hannover, Manuscript first received: January 11, 1990 Germany Revised version accepted: June 19, 1990