Abstract

In this report, we describe the evaluation of the potentiated sulfonamide Ro5-0037 (Hoffman-LaRoche, Inc., Nutley, NJ) for treatment of bacterial infection in channel catfish. We describe the studies in progress designed to generate data for submission to the Food and Drug Administration to gain approval for the use of this drug in the commercial production setting. The studies are being conducted as one investigation under the Interregional Research Project #4 (IR-4), a Federal/State program designed to facilitate the approval of minor use animal drugs.

Introduction

An urgent need of the catfish industry, and other industries involved in producing human food from "minor species", is to have an adequate arsenal of legal drugs and chemicals for disease prevention and control. The key to this statement is "minor species", which not only encompasses all of the aquaculture industries but also includes rabbits, ducks, pheasants, goats, sheep, etc. The Food and Drug Administration (FDA) lists cattle, swine, chickens, turkeys, and horses as major species; all other food animals, birds, and fish are considered minor species. Sheep are considered a major species from the standpoint of human safety requirements, but minor with regard to other FDA requirements.

Several things have happened this past year that hopefully will improve our chances for legally obtaining now drugs. Since 1963, the USDA has been cooperating with state agricultural experiment stations, industry groups, manufacturers, the FDA, and the Environmental Protection Agency (EPA) in successfully obtaining specialty pesticide approvals. This effort has been coordinated through the USDA Interregional Research Project #4 (IR-4). In late 1982, a revision of this project included an objective to obtain minor and specialty animal drug approvals. In March 1983, a workshop was held to define the drug needs and set priorities for all minor species. Essentially, all of the aquaculture industries were represented, and there are 4 approved I efforts in various stages of development currently underway. These include one each in the trout, lobster, alligator, and catfish industries.

Several years ago, a now antibiotic formulation, Ro5-0037 'Hoffman-LaRoche, Inc., Nutley, NJ), was proposed for use in food fish. Research began in trout and most of the necessary data were generated by the Fish and Wildlife Service at several locations (1,2,3). Upon request, the sponsor company began efforts to expand the label claim to include catfish. With the initiation of minor use regulations by the FDA and the IR-4 program, the necessary data for final approval of the drug are well underway, Essentially, all of the laboratory research for target animal safety, dose titrations, and toxicity tests are nearing completion. A major portion of the environmental impact research is complete and field trials are in progress.

Materials and Methods

Research underway at the College of Veterinary Medicine, Mississippi State University and the Delta Branch Experiment Station, Stoneville, Mississippi are designed to generate data on Ro5-0037 in channel catfish in the following areas: (1) efficacy and dose titration, (2) palatability and toxicity, (3) field efficacy, and (4) environmental residues. Since this work is currently in progress, the experimental procedures will be discussed in general terms.

Efficacy and dose titrations were conducted in channel catfish fingerlings held in 20 gal flow-through aquaria. The drug was incorporated into the feed which was then repelletized. A typical range of drug treatments were 0, 25, 50, 75, and 100 mg drug/kg fish per day for five days. Drug dose was verified by the manufacturer. An experiment consisted of five replicate aquaria (20 fish per aquarium) per treatment. Fish were acclimated in the aquaria for seven days (days -7 to -1), fed the appropriate drug treatment (days 0 to 4), challenged with the pathogenic bacterium Edwardsielle ictaluri (day 1), and fed a non-medicated ration (days 5 to 20). Mortalities were monitored throughout the study. At day 10, five fish per treatment were randomly sampled and bacterial cultures were taken from the posterior kidneys and streaked an blood agar. At day 20, ten percent of the remaining fish were sampled for similar bacterial isolations. Cultures were incubated for 10 days at 25 C and any growth was identified.

Palatability and toxicity of the drug were determined in channel catfish fingerlings held in 20 gal flow-through aquaria. Fish were fed high levels of Ro5-0037 and monitored for feeding activity and any adverse clinical signs. The drug was fed at 0, 50, 100, 200, and 400 mg drug/kg fish per day for 14 days. This was followed by seven days during which a non-medicated feed was fed. Fish were weighed at the initiation and termination of the study. Each treatment was applied to ten fish in each of three replicate aquaria. A second trial consisted of 0 and 1250 mg/kg treatments which were fed for one day. All other experimental conditions were the same, as previously described.

Field trials and environmental residue trials were combined and performed in earthen ponds. Catfish culture ponds with diagnosed bacterial infections were selected for field studies. Net enclosures were placed in the ponds and sample channel catfish were placed in the net enclosures. Fish remaining free in the pond and one group of netted fish received an Ro5-0037 medicated ration (50 mg/kg per day for 5 days), while the second group of fish received a non-medicated ration. Following the drug dosing, fish were fed a non-medicated ration for an additional 14 days. At the termination of the study, all fish in each net enclosure were inventoried. Samples for environmental residue analysis were taken from a number of randomly selected locations within the pond during the course of field trials. These were forwarded to the manufacturer for analysis of drug content.

Results and Discussion

As the study is presently underway, the results and discussion will deal primarily with various aspects of the experimental design.

An attempt was made to complete the research under conditions which would duplicate any potential industry use of the compound. An early decision was made regarding the incorporation of the drug in the ration. Channel catfish currently are treated with Terramycin (Pfizer, New York, NY) as a feed additive. This is the only legal means of drug delivery to a population of food fish held in a 20 acre pond. Terramycin is incorporated into the ration at the feed mill and the ration is pelleted. In laboratory studies, we followed a similar procedure using a laboratory scale California pellet mill. The amount of feed required for a pond treatment, necessitated the formulation of the feed at a commercial plant.

An oral drug treatment requires the fish to be actively feeding in order to be effective. In the laboratory, it was possible to observe fish consuming the drug-treated ration. We did, however, find it necessary to take care to reduce activity in the wet laboratory which might disturb feeding activity. We also found it helpful to reduce the level of artificial light in the tab. No such capabilities exist in the field trials. One must make the assumption that enough fish will obtain the proper dose of drug to provide disease control within the fish population in the pond.

Early dose titrations were performed with the facultative pathogen Aeromones hydrophila. This organism becomes a problem when fish are in a condition of stress.

Reproducible "standard challenges" were difficult to achieve with this pathogen, as the organism appeared to loose virulence rapidly upon successive culture in the laboratory.

Edwardsiella ictaluri was later selected as a challenge organism for more recent laboratory dose titrations. This obligate pathogen gave more consistent results in producing a standard challenge". Edwardsiella ictaluri appears to be a pathogen in the 22-28º C temperature range. Therefore, field trials are being conducted within this temperature "window".

At this writing, many of the laboratory studies for target animal safety, dose titration, and toxicity are nearing completion and a major portion of the environmental impact research is complete. Although a considerable amount of work remains to be done, we are encouraged by the results we have obtained to date.

References

1. Bullock, G.L.; Stuckey, H.M.; Collis, D.; Herman, R.L.; and Maestrone, G. In vitro and in vivo efficacy of a potentiated sulfonamide in control of furunculosis in salmonida. J. Fish. Res. Board Can. 31: 75-83 (1974).
2. Bullock, G.L.; Maestrone, G.; Starliper, C; and Schill, B. Furunculosis: Results of field trials for therapy with Ro5-0037, a potentiated sulfonamide. Progr. Fish-Cult 45: 51-53 (1983).
3. ullock, G.L; Maestrone, G.; Starliper, C.; and Schill, B. Potentiated sulfonamide therapy of enteric redmouth disease. Can. J. Fish. Aquat. Sci. 40: 101-102 (1983).