188 



FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 



discoloration. Strain 3 was nonhemolytic. Blood- 

 agar and nutrient-agar cultures had a strong odor 

 similar to that detected in the incised lesion from 

 the infected fish. Colonies on eosin-methylene- 

 blue and MacConkey's agars appeared glistening 

 and colorless. On agar slants, growth was abun- 

 dant, filiform, glistening, butyrous, and colorless. 

 The appearance of the mediimi remained un- 

 changed. In nutrient broth a pellicle was 

 formed; there was dense clouding and a scant 

 flocculent sediment. A loopful of a 48-hour broth 

 cultm-e inoculated into nutrient broth resulted 

 in visible growth in 4 hom-s at 37° C. 



At 20° C, growth in gelatin was best at the 

 top, with subsequent stratiform liquefaction. At 

 37° C, liquefaction was complete in 24 hours. 

 Nitrates were rapidly reduced to nitrites. The 

 methyl-red test was negative. Acetylmethylcar- 

 binol and indole were produced by strains 1 and 2. 

 Strain 3 produced acetylmethylcarbinol but failed 

 to form indole. Growth occiu-red on Simmon's 

 citrate agar. In Koser's citrate broth, strain 1 

 was negative, strains 2 and 3 positive, after 3 

 days. Hydrolysis of cornstarch was complete in 

 24 hours (no color with iodine). Digestion of 

 egg albumin and Loeffler's serum slants began in 

 48 hom-s and was practically complete in 96 hom-s. 

 H2S was not produced in Pb acetate medium or in 

 Kligler's iron agar. The m-ease test was negative. 

 There was slight acid production (pH 6) with the 

 formation of a small amomit of precipitate in 

 bromo.cresol-pm'ple milk. Peptonization was evi- 

 dent in 48 hours and practically completed in 120 

 hours. Litmus milk was reduced in 24 hours. 



Various sugars, alcohols, and glucosides were 

 sterilized by filtration through a porcelain filter 

 and incorporated into standard basal medium in 

 1-percent concentrations. On original isolation, 

 strains 1 and 2 produced acid in lactose after 21 

 days, and culture 3 after 27 days. This conforms 

 to the behavior of the paracolon type of micro- 

 organisms which are described as slow lactose 

 fermenters in Bergey's Manual (Breed, Mm-ray, 

 and Hitchens, 1948). After several serial transfers 

 in lactose broth, the time of acid formation in 

 lactose was reduced by 6 to 11 days, depending on 

 the strain. Readings made during a 4-week period 

 showed that acid and gas were produced in 24 



hours at 25° C. and at 37° C. in L-arabinose 

 (weak), glucose, D-fructose, D-mannose, sucrose, 

 maltose, trehalose, soluble starch, dextrin, glyco- 

 gen, and mannitol. A faint acid reaction and 

 trace of gas appeared in salicin after 4 to 6 days' 

 incubation at 37° C. At 25° C, salicin was fer- 

 mented by all 3 strains in 24 hom-s. Strain 3 

 differed from strains 1 and 2 in that it produced 

 acid and gas in raffinose but not in sucrose at 

 either temperature. No acid or gas was'produced 

 in D-xylose, L-rhamnose, cellobiose, mellibiose, 

 melizitose, inulin, glycerol, erythritol, adonitol, 

 dulcitol, D-sorbitol, inositol, and esculin, at 25° 

 C. or 37° C. 



Preliminary antigenic analysis indicated that 

 strains 1 and 2 were antigenically diverse from any 

 of the paracolon types described by Stuart et al. 

 (1943). Strain 1 proved to possess somatic com- 

 ponents XXX and XL of the Salmonella group, 

 while all tests with flagellar antisera were negative. 

 Strain 2 was rough and consequently could not 

 be typed. Strain 3 was negative for somatic 

 components I to XXXVIII and for all flagellar 

 antigens. 



PATHOGENICITY 



Nine goldfish {Carassius auratus) and 27 adult 

 white mice were inoculated intraperitoneally with 

 0.2 ml. of 24-hour broth cultures of the 3 strains, 

 and in 19 hoiu-s all were dead. In every instance, 

 the organisms were reisolated in almost pure cul- 

 ture from the fluid present in the body cavity. 

 Similar tests were made with 0.2-ml. suspensions 

 of heat-killed bacteria and filtrates from the same 

 24-hom- nutrient broth cultures. All fish and mice 

 proved refractory. Strain 3 also proved to be 

 pathogenic for guinea pigs. 



Further experiments were carried out at the 

 Microbiological Laboratory, Kearneysville, W. 

 Va., in which 60 fingcrling trout were used. Ten 

 trout of each of the following species were inocu- 

 lated intraperitoneally with 0.2 ml. of a 24-hour 

 broth culture: Rainbow trout {Salmo gairdnerii), 

 eastern brook trout (Salvclinus jontinalis), and 

 brown trout {Salmo trutta). As controls, 10 fish 

 of each of these species were inoculated with 

 sterile broth. The temperature of the water in the 

 troughs was approximately 14° C. Some deaths 



