PASTEURELLA SP. FROM AN EPIZOOTIC OF WHITE PERCH ( ROCCUS AMERICANUS ) IN CHESAPEAKE BAY TIDEWATER AREAS

Abstract

and spread during the summer to widely separated locations. In addition to white perch (Roccus americanus), striped bass (R. saxatilis) were involved to a lesser degree. Carefully selected moribund specimens were collected and delivered to the laboratory. Mi-croscopy disclosed an abundance of polarly staining gram-negative rods in blood and organs of diseased fish. First isolations of these bacteria were carried out on media enriched with blood, because it was suspected that bacteria were fastidious; later, agar media with 1 to 3% sodium chloride, or half-strength seawater, were used. Thirty cultures of this bacterium were isolated from internal organs of 17 white perch and 5 striped bass. The bacterium was isolated in pure culture from perch, but a Vibrio, and other unidentified gram-negative rods, were also isolated from some of the bass. Since the polarly staining rod was implicated in the epizootic, owing to its presence in large numbers and in pure culture in the perch, its characteristics were determined in cultures grown at 20 and 30 C. No growth occurred at 37 C. The cells were gram-negative, nonmotile, polarly stained rods. From fish tissues, they measured 1.5 by 1.0 ,u. On agar media with salt, colonies were regular, convex, and viscid, and the cells were capsulated. Striking pleomorphism of cells was noted on several media, and was influenced by sodium chloride concentration. Growth was observed in nutrient broth with 0.5 to 5.0% sodium chloride. Levels above 5.0% were not investigated. The isolate was negative in respect to production of indole, hydrogen sulfide, gelatinase, urease, and amylase, and to reduction of nitrates and methyl red. A trace of acetyl-methyl-carbinol was detected (Page, J. Bacteriol. 84:772, 1962). The cytochrome oxidase test (Ewing and John-son, Intern. Bull. Bacteriol. Nomenclature Taxon. 10:223, 1960) was positive. From 17 carbohydrates tested, dextrose, maltose, fructose, and sucrose were anaerogenically fermented. Because of its strong polar-staining nature and many physiological reactions, this bacterium was placed in the genus Pasteurella. No attempt at speciation was made. So far as we know, this is the first time a representative of the genus Pasteurella was isolated from a fish epizootic. Pile fabrics are the most frequently used materials in replica plating techniques (Lederberg and Lederberg, J. Bacteriol. 63:399, 1952). However, they pick up moisture from agar surfaces, causing colonies to spread and merge. Replication of small, randomly distributed colonies isolated from natural environments then becomes very difficult to follow. Greene et al. (Appl. Microbiol. 10:567, 1962) used a "replicate floc" (Medical Products Division , Minnesota Mining & Mfg. Co., St. Paul, Minn.) consisting of a tough, flexible backing with translucent, flexible, bristle-like fibers 1 Contribution no. 164 from The Virginia Institute of Marine Science. attached to one surface. The fibers are bent and intermingled, rather than erect and separate, giving the surface a fuzzy, white appearance. In hope of finding a material which would give improved replication of small colonies, we tested floc against velveteen. Raw seawater, suitably diluted, was surface-plated on yeast-beef-peptone-seawater-agar (2 %) and incubated at 20 C for 7 days. Plates selected for replication showed 50 to 220 small colonies per plate. Replicates were made in ink-stamp fashion. Circles of floc or velveteen, autoclaved at 121 C for 15 min, were attached to the outer surface of a circular aluminum plate with double-faced Scotch taDe. Plates for replication were dried at room 1815 NOTES