J.P. Hawke, MS; S.M. Plakas, PhD; R.V. Minton, MS; R.M. McPherson, DSc; TG
Snider, DVM, PhD; AM Guarino, PhD
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,
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,
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).
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
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