Introduction

A cooperative project to restore the populations of striped bass (Morone saxatilis) in coastal Alabama and Mississippi has been underway since 1982. Striped bass are reared to juvenile size (0.3-0.7 g body weight) in closed systems at the Gulf Coast Research Laboratory (Ocean Springs, MS) and transported to the Claude Peteet Mariculture Center (CPMC, Gulf Shores, AL) for rearing in ponds to advanced fingerling size (over 20 g body weight). Fingerlings are harvested at approximately 200 days of culture at CPMC, marked with internal anchor tags and released into the coastal waters of Alabama and -Mississippi (Minton, 1983).

In October 1984 an outbreak of fish pasteurellosis occurred in the pond-cultured populations of striped bass. Mortality was estimated at 80% of the total stock. Although epizootics of fish pasteurellosis have been reported in natural populations of striped bass (Snieszko et at., 1964; Paperna and Zwerner, 1976; Robohm, 1979), this was the first documented occurrence of the disease in cultured fish in the U.S. (Hawke et al., 1987).

Materials and Methods

Striped bass are cultured in 0.08 ha earthen ponds at CPMC. Brackish water is pumped from the Gulf Intracoastal Waterway and filtered through a nylon encasement sleeve before entering the ponds. Fingerlings were stocked at three densities (62,500, 93,750 and 137,500 fish/ha) on 24 and 25 May 1984 and fed a commercial ration (Ziegler Bros. Inc., Gardners, PA) throughout the rearing period.

On 14 October 1984, diminished feeding activity and light mortality of striped bass were noted in two of twelve rearing ponds and in a reservoir pond. The following day, extensive mortality of striped bass in these three ponds had occurred. On 16 October, antibiotic treatment of fish in the other ten ponds was initiated. Oxytetracycline hydrochloride (92.5% active ingredient, Argent Chemical Co., Redmond, WA) was mixed in a vegetable oil base and blended into a commercially pelleted feed (Ziegler Bros.) to achieve a uniform coating. The target dosage was originally 50 mg/kg body weight per day, but was increased to 150 mg/kg/day three days into treatment.

Isolation and Characterization of the Bacterium

Tissue scrapings and grain-stained tissue smears from moribund striped bass collected during the initial epizootic were examined for parasitic and bacterial organisms. Using sterile techniques, samples of kidney, liver and spleen from moribund fish were streaked by loop onto 5% sheep blood agar plates (Biocon Inc., Gulf Breeze, FL) and incubated at 25°C. Isolated colonies were restreaked on 5% sheep blood agar and on tryptic soy agar (Difco Laboratories, Detroit, MI) supplemented with sodium chloride to a final concentration of 1.5%. Cultures were maintained for later testing on long term preservation medium (FDA, 1984).

Morphology of the bacterium was examined after gram-staining. Motility was determined by microscopical observation of wet mounts (1% sea salts) and by stabbing into glucose motility deeps (Walters and Plumb, 1978).

Standard biochemical tests were applied for characterization. Cultures of Pasteurella piscicida ATCC 17911 and ATCC 29687 (American Type Culture Collection, Rockville, MD) were tested simultaneously with the CPMC isolate. Additional biochemical characterization was performed using the API 20E system (Analytab Products, Plainview, NY) and the Minitek System (BBL, Cockeysville, Md) supplemented with 1% sodium chloride.

A confirmatory microtitre agglutination test with antiserum produced in rabbits against the P. piscicida strain isolated from striped bass by Robohm in 1977 (Robohm, 1979) -was conducted at the National Fish Health Research Laboratory, U.S. Fish and Wildlife Service (Kearneysville, WV). Vibriostat (0/129) sensitivity (Shewan et al., 1954) and confirmatory biochemical tests were performed at the Southeastern Cooperative Fish Disease Laboratory, Auburn University (Auburn, AL).

Antibiotic sensitivity of isolates was determined in vitro by the disk diffusion method (Barry and Thornsberry, 1980), but modified by the addition of 1.5% sodium chloride to the Mueller-Hinton agar.

Minimum Inhibitory Concentration of Oxytetracycline

The minimum inhibitory concentration (MIC) of oxytetracycline was deter-mined according to the microdilution procedure of Cavan and Barry (1980). The minimum bactericidal concentration (MBC) was estimated by the method of Nusbaum and Shotts (1981).

Oxytetracycline Residue in Tissues

Fish were sampled every 3-4 days from ponds in which feeding activity was noted and, for comparison, from ponds in which feeding activity had ceased prior to or soon after treatment was initiated. As a control, tissues from striped bass of similar size which had never been offered medicated feed were analyzed.

The fluorometric assay developed by Argauer and Gilliam (1974) for oxytetracycline was adapted for use with fish tissues (Hawke et al., 1987).

Histopathological Examination of Striped Bass

Tissues were removed from moribund striped bass and fixed in 10% buffered format. The tissues were then processed using standard methods, sectioned at 4-6 um and stained with Meyer's hematoxylin and eosin or by a Gran method (Brown and Hopps, 1973).

Results

Approximately 49,000 fingerling striped bass (30-40 g body weight) died during a three week period beginning 15 October 1984. Moribund fish had no obvious external lesions; however, most fish displayed abnormal skin pigmentation. Slight infestations of Amyloodinium ocellatum and Ambiphrya sp. were present on the gills of some specimens. Dissection of moribund fish often revealed an enlarged spleen and kidney. In rare instances, multiple whitish areas (0.5-1.0 mm diameter) in the spleen were observed.

Large numbers of a non-motile, rod-shaped bacterium were observed in wet mounts of spleen spears. Gram-staining revealed a gram-negative, bipolar-staining, pleomorphic rod which was approximately I um in length. Individual colonies (1 mm diameter) were clearly visible on blood agar plates after 48 hours incubation at 25°C. The colonies were raised and slightly viscid.

Based on the morphological and biochemical characteristics, the bacterium was identified as Pasteurella piscicida (Janssen and Surgalla, 1968). Identification was confirmed serologically by a positive agglutination titer (1:64) with specific antiserum to P. piscicida (G.L. Bullock, personal communication, 1985).

The bacterium was sensitive to 0/129 vibriostat, sulfadimethoxine/ormetoprim, streptomycin, chloramphenicol, gentamycin, tetracycline, chlortetracycline and oxytetracycline, while resistant to sulfadiazine. The MIC of oxytetracycline was 0.25 ppm for the CPMC isolate, 0.50 ppm for ATCC 17911 and 0.12 ppm for ATCC 29687. The MBC for all three isolates was greater than 32 ppm, the highest concentration tested (Hawke et al., 1987).

Mean muscle concentrations of oxytetracycline were less than 0.25 ppm (limit of assay sensitivity) while mean liver concentrations ranged from 0.54 to 1.97 ppm. The oxytetracycline concentrations of the gastrointestinal (GO tract contents on 19 and 26 October were approximately 150-200 times greater than the respective liver values. Oxytetracycline was not detectable in fish from ponds in which feeding activity had ceased prior to or soon after therapy was initiated.

Histopathological changes in the spleen and liver were as follows: extensive, acute multifocal necrosis of the splenic lymphoid tissue characterized by a loss of cells, coagulation necrosis, karyorrhexis and large colonies of bacteria; and a moderate, acute multifocal necrosis of the hepatocytes with prominent karyorrhexis. Rod-shaped bacteria were visible in the sinusoids and within the hepatic vessels. This was accompanied by acute multifocal necrosis of the pancreatic tissue with reduced size and loss of zymogen granules. Inflammatory cellular accumulations were not present. Relatively large areas of the liver exhibited hyperplasia of the reticuloendothelial cells lining the hepatic sinusoides (Hawke et al., 1987).

References

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