J. V. Kitzman; W. Austin; S. S. Rollin
Department of Basic and Applied Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS
Enteric: Septicemia of Catfish (ESC) is the most economically important bacterial disease in the commercial catfish industry. The organism responsible for producing the disease is Edwardsiella ictaluri, a gram negative rod in the family Enterobacteriacae. The disease is particularly devastating in catfish fingerlings (4-6 inch fish), often causing 5080% mortality in infected ponds. The disease is only manifested in the catfish when pond water temperatures are between 22-28°C. In Mississippi and the southeast, ESC causes morbidity and mortality for approximately 4-8 weeks in the late spring, and 4-8 weeks in the early fall during changes of the seasons. The economic impact of the disease is between 2-6 million dollars annually.
New broad-spectrum, orally absorbed antibiotics are urgently needed in the commercial catfish industry. Currently, only oxytetracycline and the potentiated sulfonamide Romet-30® are approved by the FDA for treatment of ESC and other bacterial diseases of catfish. Bacterial resistance, a long withdrawal period (21 days), and heat instability, are the major problems with the use of oxytetracycline. Field bacterial strains of Edwardsiella ictaluri have recently shown resistance to Romet-30®.
Ten references strains of Edwardsiella ictaluri, isolated and frozen from clinical cases of ESC in Mississippi, were utilized in this study. Ile clinical cases were presented to the Fish Diagnostic Laboratory at CVM/MSU, and the organisms were isolated from moribund fish from separate outbreaks of ESC.
The ten strains were prepared for inoculation by transferring the isolates into sterile Brain-Heart Infusion (BHI) broth. The broth was incubated at 27°C for 18 hours, centrifuged at 1100 x g for 10 minutes, decanted, and the bacteria were resuspended in an equal volume of sterile Dulbecco's phosphate buffered saline (PBS). The resultant suspension was quantitated for bacterial numbers by measurement of the optical density at 745 nm and by plate counts. The suspension was diluted to produce a final concentration of approximately 108 bacteria/ml.
Florfenicol was added to Muller-Hinton agar plates to yield antibiotic concentrations in a 2-fold dilution series from 0.0156 µg/ml to 128 µg/ml.
The bacterial strains were tested against each antibiotic concentration in triplicate. Using pipets, 100 µl of stock bacterial suspension were added to each agar plate. All plates were incubated at 27°C. After incubation, the plates were examined at 24 and 48 hours for evidence of bacterial growth. Plates were reported as having growth (+) or no growth (-) based on results of the triplicate plate inoculations.
Eighty-eight (88) channel catfish fingerlings (10 grams each) were randomly assigned to 4 identical 160 liter polyethylene circular tanks. A flow-through system providing aerated, dechlorinated water at 25°C was used in the tanks. The fish were acclimated 2 weeks prior to experimentation.
The fish were given a commercial sinking feed fortified with florfenicol (FF) to yield one of the following dosing rates: 0, 10, 20, or 40 milligrams per kilogram of fish weight. All diets were stored in polyethylene bags and were frozen until used in the study. Fish were fed twice daily at a rate of 2% of body weight per day (1% morning and evening).
An exposure bath containing Edwardsiella ictaluri was prepared from a field isolate submitted to the Aquatic Diagnostic Laboratory that had produced high mortality in a pond of fingerlings. The bacteria was isolated and identified as a pure culture, passed twice (105 bacteria injected IP into catfish fingerlings), and reisolated from the brain and posterior kidney. A culture was grown in a 1 liter bioreactor containing Brain-Heart Infusion broth incubated at 27°C. After 48 hours of incubation, the number of bacteria per milliliter was determined by the standard plate count method.
The catfish fingerlings were infected by immersing them for 45 seconds in the pure culture of Edwardsiella ictaluri according to the method of Baxa, et al. 1990. 1 The tanks of fish were infected in the following order: control (0 mg/kg FF), 40 mg/kg FF, 10 mg/kg FF, and 20 mg/kg FF. Following immersion in the exposure bath, the fish were immediately returned to their tanks. The infected fingerlings were fed either control or medicated feed beginning the day prior to infection and continuing for 5 days, and then all surviving fish were fed control feed for an additional 14 days. Fish that died during the study as well as all fish remaining at the end of the study were cultured for the presence of Edwardsiella ictaluri in the brain and posterior kidney. Other bacteria found were identified and reported.
The ten strains of Edwardsiella ictaluri were positively identified through biochemical testing, colonial morphology, and staining characteristics. All reference strains were verified to be free from contamination prior to and during use.
Results of the MIC study are presented in Table 1. Pluses (+) indicate detectable growth and minuses (-) indicate no growth. The overall minimum inhibitory concentration was calculated to be 0.19 ± 0.067 µg/ml (mean ± SD).
Table 1. MIC determination for Edwardsiella ictaluri on Muller-Hinton agar plates fortified with florfenicol (FF)
The number of fish that died in the four treatment tanks is summarized in Table 2. No fish died on the first day on medicated feed (the day prior to bath immersion) and no fish died on the day of immersion. During the next 3 days (days 3-5 of medicated feed), more than 90% of the fish in the control (0 mg/kg) and low dose (10 mg/kg) treatment groups died, whereas only 23% of fish in the medium dose (20 mg/kg) and only 8% of the high dose (40 mg/kg) group died.
Table 2. Mortality rate of channel catfish fingerlings infected with Edwardsiella ictaluri. Fish infected on day 0.
Table 2. Number of Dead Fish
After the seventh day of the study, all fish in the control and low dose treatment groups had died. At the completion of the study (5 days of medicated feed and 14 days of control feed), 27% of fish in the medium treatment group and 29% in the high dose treatment group survived.
The calculated MIC of florfenicol against Edwardsiella ictaluri in this study, 0.19 UG/ML, was consistent with the MIC of the antibiotic against other fish pathogens including Pasteurella piscicida (0.2 µg/ml)2, Vibrio anguillarum (0.4-0.8 µg/ml)3, Edwardsiella tarda (0.8µg/ml) 3, Aeromonas hydrophila (0.4 µg/ml) 3, Aeromonas salmonicida (0.4 µg/ml) 3, Streptococcus sp. (3.1µg/ml) 3, and Pasteurella piscicida (0.4 µg/ml) 3.
The bacterial exposure was effective in producing ESC in the catfish fingerlings. Bath exposure to the full strength bacterial culture may have been too severe to permit adequate discrimination among the treatment groups, as evidenced by 100% mortality in the control and low dose treatment groups 5 days after exposure. Florfenicol was capable of protecting the fish in the medium and high dose groups from ESC for a longer period of time, and prevented clinical signs of disease and death in about 30% of the fish in the medium and high dose groups. The medium and high dose FF treatments produced a significant change in the course of the disease in this study. Since all fish received the same infective dose, the marked improvement in survival appeared to be due to the presence of the florfenicol in the feed. Beginning on the ninth day after exposure, the medium and high dose FF treatment groups experienced mortality that could probably be explained by the severity of the infection and the course of treatment with medicated feed.
In conclusion, florfenicol at 20 mg/kg and 40 mg/kg in feed produced markedly better survival rates compared to control or 10 mg/kg in the bath treatment technique used. Fewer fish died in the medium and high dose groups, and Edwardsiella ictaluri was not routinely cultured from surviving fish in these groups.
1. Baxa DV, Groff JM, Wishkovsky A, et al. Susceptibility of nonictalurid fishes to experimental infection with Edwardisella ictaluri. Diseases of Aquatic Organisms 1990; 8:113-117.
2. Yasunaga N, Yasumoto S. Therapeutic effect of florfenicol on experimentally induced pseudotuberculosis in yellowtail. Fish Pathology 1988; 23 (1):1-5.
3. Fukui H. Fujihara Y, Kano T. In vitro and in vivo antibacterial activities of florfenicol, a new fluorinated analog of, against fish pathogens. Fish Pathology 1987: 22(4):201-207.