DISEASES OF AQUATIC ORGANISMS Vol. 13: 181-187, 1992 1 Published September 3 Dis, aquat. Org.

Isolation of Carnobacterium piscicola and an unidentified Gram-positive bacillus from sexually mature and post-spawning rainbow trout On corh ynch us m ykiss

Clifford E. Starliper', Emmett B. shotts2, John ~rown~

' National Fish Health Research Laboratory. Box 700. Kearneysville.West Virginia 25430. USA Dept of Medical Microbiology. College of Veterinary Medicine. University of Georgia, Athens. Georgia 30602, USA

ABSTRACT: Blochemica1 and electrophoretic studles were done on bacterial isolates collected from rainbow trout Oncorhynchus mykiss from 2 private hatcheries In the Pacific Northwest, USA. Five isolates onginated from post-spawning mortalities and morbidities; 2 other isolates were obtained from reproductively active 17 mo old rainbow trout. After 35S-methion~ne-labeledprotein lysates were prepared from each isolate, correlation coefficients of electrophoretic proflles were calculated for each pair of isolates using an automated m~crobiologicalidentification system (AMBIS) The Gram-pos~tive isolates appeared to represent 1 or possibly 2 genera: Carnobacterium and Five isolates were identified as C. piscicola and two (106891,106892) did not correspond to any previously described . Comparisons based upon correlation coefficients calculated for the 2 unidentified isolates and for biochemically similar strains, including C. piscicola (ATCC 15434), L. alimentarius (ATCC 29643) and L. homohiochi (ATCC 15434), resulted in values no greater than 0.792. This dissimilarity suggests that the 2 unidentified isolates represent a different blotype of C. plscicola or a previously undescribed species of Lactobacillus.

INTRODUCTION 1984, Collins et al. 1987), a comprehensive characteri- zation of the Gram-positive associated with For the past several years, 2 privately owned trout the disease was undertaken. facilities in Idaho have experienced a chronic disease problem in their adult fish. Although significantly high mortality has not occurred, low level mortality has MATERIALS AND METHODS been repeatedly observed ongoing in post-spawned fish and fish of spawning age, primarily among females. During a recent 16 mo period, bacterial isolates were Some mortality resulting from handling during the collected from rainbow trout Oncorhynchus mykiss at spawning process is not unusual; however, the consis- 2 facilities, one a brood stock station and the other a tent low level losses over a number of years generated rearing facility. Four isolates were obtained from mori- an interest to determine a cause. Health inspections bund post-spawning females, and 1 from a dead post- and post-mortem examinations implicated a Gram- spawning female. All of these fish were between 2 and positive bacillus as the etiological agent. Because of 5 yr of age. Two additional isolates came from the the hatcheries' geographical location in a region where rearing facility and involved 17 mo old reproductively Renibacterium salmoninarum, causative agent of active females that otherwise appeared to be healthy. bacterial kidney disease, is enzootic, and because The brood stock station was experiencing a chronic of an increased awareness of the diversity of the post-spawning mortality problem. Daily mortality Gram-positive associated with fish (Hiu et al. ranged from 1 to 6 fish (in a population of nearly 182 Dis. aquat. Org. 13: 181-187, 1992

20000). A Gram-negative bacteremia was concur- with each sugar filter-sterilized and added to yield a rently detected in the population from which Isolate 1 % final concentration. Carbohydrates examined were 33587 1 originated. D-glucose, D-mannitol, D-ribose, cellobiose, lactose, Bacterial samples were collected from the kidney, D-sorbitol, esculin, salicin, sucrose, D-mannose, treha- spleen, swimbladder and intestine of moribund fish. lose, rhamnose, raffinose, melibiose, L-arabinose, fruc- Tissue samples were streaked onto blood agar and tose, glycerol, erythritol, inositol, dulcitol, L-sorbose, tryptic soy agar. Plates were incubated at 15 to 20 "C adonitol, D-xylose, D-galactose, maltose. D-melezitose, and periodically examined for growth. A list of the iso- and D-gluconic acid. Presence of lysine and ornithine lates obtained is given in Table 1. Reference strains decarboxylase and of arginine dehydrolase was deter- included Lactobacillus piscicola (ATCC 35586), L. ali- mined in Moeller basal medium with 1 % of the L-form mentarius (ATCC 29643), and L. homohiochi (ATCC amino acid. Tests previously described by Koneman et 15434). They were selected for the electrophoretic al. (1983) and MacFaddin (1980) were done to assess analysis because they were the most similar, bio- utilization of citrate (Simrnon's) and malonate as sole chemically, to the 2 isolates (106891, 106892) that did carbon sources, production of acetoin from glucose, not correspond to any described species. hydrolysis of starch, action in litmus milk medium, Bacterial strains were examined for purity prior to oxidation of gluconate to reducing compounds, methyl characterization by streaking onto 5 % bovine blood red reaction, and the presence of urease, phenyl- agar. Colonies were reinoculated onto blood agar and alanine dearninase and P-galactosidase. Production brain heart infusion agar plates to obtain bacterial of ammonia from arginine was detected using the growth for further biochemical and morphological method of Niven et al. (1942). Growth at 4, 15, 37 and studies. Unless otherwise indicated, incubation was at 42 "C was determined by inoculating test bacteria onto 30 "C, the temperature which produced the most blood agar plates. The ability of the isolates to grow in luxurious growth. The initial characteristics determined nutrient broth in the presence of various concentra- were those of diagnostic value for the genera Carno- tions (6.5, 7.0, 8.0, 9.0 and 10.0 %) of sodium chloride bacterium, Lactobacillus, Lactococcus and : was tested, as was their ability to grow on Macconkey the TSI reaction, Gram reaction, catalase and cyto- agar, tomato juice agar (TJA; Kulp & White 1932) and chrome oxidase production, hemolysis on blood agar, on selective lactobacillus isolation medium (SL; Rogosa nitrate reduction, and motility (by microscopic exami- et al. 1951). nation of a 24 h brain heart infusion broth culture) Analysis of the electrophoretic profiles of the isolates (Schleifer et al. 1985, Kandler & Weiss 1986, Collins et was accomplished using an automated microbiological al. 1987, 1989). identification system (AMBIS; Automated Micro- Determination of lactic acid production as a product biology Systems, Inc., San Diego, CA, USA). Use of the of hexose fermentation in homofermentative or hetero- AMBIS requires metabolic labeling of the bacterial fermentative proportions was accomplished on HHD proteins with a beta-emitting radioactive isotope such agar plates (McDonald et al. 1987). Individual colonies as 35Sor I4C. Since a detailed description of the AMBIS of homofermentative strains were blue as a result of is presented elsewhere (Hook et al. 1987, Tabaqchali the high concentrations of lactic acid produced from et al. 1987), the methodology is only briefly outlined fermentation of the hexose (fructose) present in HHD here. Bacterial strains were grown on brain heart agar. Colonies of heterofermentative strains remained infusion agar (BHIA; DIFCO) for 48 h. Several colonies white. were picked and used to inoculate 50 yl of methionine- Production of acid from a number of carbohydrates deficient amino acid assay medium (DIFCO) supple- was examined utilizing purple broth base medium mented with 10 pCi 35S-methionine (Amersham Corp., Arlington Heights, IL, USA). Following incubation for

Table 1.Lactobacillus/Carnobactenurn isolates from rainbow 48 h at 30 "C, whole cell lysates were prepared by trout Oncorhynchus mykiss originating from a broodstock adding 50 p1 of 2X concentrated sample lysis buffer. station or rearing facility in Idaho, USA The 100 p1 of volume was transferred to a 0.5 m1 rnicro- fuge tube filled with 0.15 mm zirconium oxide beads Isolate Facility Tissue Year and placed on a mini-beadbeater (Biospec Products, 33587 1 Broodstock Swimbladder 1987 Bartlesville, OK, USA) for 3 min. The sample was 00188 Rearing Kidney 1988 removed from the beads by centrifugation, boiled for 01488 Rearing Intestine 1988 2 min, and then subjected to sodium dodecyl sulphate- 27488 Broodstock Kidney 1988 polyacrylamide gel electrophoresis (SDS-PAGE) using 032891 Broodstock Kidney 1989 a 12.5 % gel. After electrophoresis, gels were dried 106891 Broodstock Kidney 1989 106892 Broodstock Kidney 1989 and scanned (AMBIS beta scanner). The protein band profile of each lane was filed in a computer as the Starliper et al.: Isolation of two Gram-positive bacteria from rainbow trout 183

number of counts per minute of isotope decay for each liver, swirnbladder, musculature, and lower intestine. specific region of the lane scanned. Three lanes of The extent of clinical signs varied from none (there known molecular weight standards were included on were apparent carrier fish such as the one from which each gel to allow for molecular weight determination Isolate 01488 originated) to that of the post-spawned of a particular protein(s) of interest and also to serve as broodstock fish which displayed most or all of the gel standards which were normalized to a previously signs mentioned. Bacterial isolations were made from filed absolute standard. Sample lanes were then nor- several internal sites (including the swimbladder, malized to the gel-specific standard lanes. Normaliza- kidney, lower intestine, ascites fluid, liver and spleen) tion was done to reduce gel to gel variability. Normal- in those fish having the more severe manifestations of ized lanes were referred to as 'adjusted lanes'. clinical disease. Typically, a lane profile is 504 pixels in length. How- Two isolates (00188 and 01488) were recovered ever, only the region between 60 and 300 pixels (corre- during routine fish health examinations of 17 mo old sponding to a molecular weight range of 242 Da to fish culled from the rearing facility. Fish were trans- 14.8 kDa, respectively) was considered so as to elimi- ferred to the egg-taking station only after each lot had nate those regions of the gel containing unincor- passed numerous fish health examinations and were porated isotope. Qualitative and quantitative differ- found to be free of certifiable pathogens. Fish lots were ences in proteins for each pair of isolates were inspected periodically by examining samples of 30 evaluated using the Pearson product-moment correla- fish. The group of fish from which these 2 isolates tion coefficient algorithm (Sneath & Sokal 1973). The were obtained were classified as reproductively active similarity of any 2 isolates increased as the value for r and contained mature males and overripe females. approached 1. Hatchery records indicated no abnormal mortality in Pathogenicity of the fish isolates, including the refer- this population. Isolate 01488 was made from the lower ence strain of Lactobacillus pisicola (ATCC 35586), intestine, whereas Isolate 00188 came from a swollen was assessed by intraperitoneal (ip) injection and kidney. A bacteremia of minor significance due to a waterborne-exposure. Eighteen 20 g rainbow trout Gram-negative bacterium occurred in some fish in this were challenged in each experiment. Fish were main- group but was not present in the individuals that tained in tanks containing 35 1 of water with a flow of yielded Isolates 00188 and 01488. 1 1 min-' at 12 "C. Each isolate was grown in 1 1 BHIB None of the isolates grew at 4 or at 42 "C. All isolates for 24 h at 25 to 30 "C. For ip injections, fish were grew at 15 "C, and growth at 37 "C varied (Table 2). anesthetized and inoculated with 0.1 m1 of broth con- Normal atmospheric conditions were sufficient for taining 1.3 X 10' CFU ml-'. Waterborne-challenge was primary isolation, subsequent subculturing and bio- done by transferring the fish to 1 1 of broth culture chemical tests. Cells were Gram-positive, non-spore- (1.3 X 107 CFU ml-l) for 10 min and then returning forming rods, ca 0.5 pm by 1 to 1.5 pm. Good growth them to their tanks. Controls were exposed to sterile was obtained on culture media such as blood agar and BHIB using the same procedures. Seven days after brain heart infusion agar. After 48 h incubation on challenge, 5 fish were removed from each tank and blood agar plates, colonies were approximately 1 mm samples of their kidney tissue were streaked onto in diameter, white, rounded and smooth. Colonial con- BHIA plates. After 30 d, the remaining fish were sistency was creamy rather than friable or mucoid. removed; their kidneys were excised, homogenized, With 2 of the isolates (106891 and 106892), colony and diluted in sterile 0.85 % saline. Plate counts on morphology was pleomorphic. At 48 h incubation, 2 the dilutions were done to determine the number of colony sizes were apparent with each of these 2 CFU g-' of tissue. During the period of experimental isolates. One was typically 1 mm in diameter and the infection, isolations were made from mortalities by other, 2- to 3-fold larger. The colony pleomorphism in- streaking kidney tissue onto BHIA. creased with additional incubation time. Physiological and biochemical differences between the 2 colony sizes were not demonstrable, and mixed sizes were RESULTS present after repeated streaking of individual small or large colonies. External signs usually seen in the moribund females All isolates were a-hemolytic, cytochrome oxidase included lethargy, loss of appetite, a general darken- negative and non-motile. They failed to reduce nitrates ing of the body surface and an occasional blister-like and gave an A/A TSI reaction with no H2S being pro- lesion. Necropsy revealed the following internal signs: duced. Six isolates (335871, 01488, 106892, 00188, abnormally high amounts of ascitic fluid and in some 27488, 032891) were catalase positive. Species of the cases, bloody ascitic fluid; swollen kidney, liver and genera Carnobacterium, Lactococcus, Vagococcus and spleen; and varying degrees of hemorrhage in the most of the Lactobacillus spp. are catalase negative, 184 Dis. aquat. Org. 13: 181-187, 1992

Table 2. Variable biochemical and physiological reactions of Lactobacillus/Carnobacteriurn isolates from rainbow trout. Results reported as: no. positive/no. tested; -, +;W (weakly positive); or NA (not available)

Published results Present study Lactobacillus Carnobacterium 00188 106891 piscicola piscicola 335871 106892 01488 27488 032891

Catalase Arginine dehydrolase Gluconate NH3 from argin~ne ONPG~ Acid from: D-mannitol Lactose D-sorbitol Raffinose Meliblose L-arabinose Glycerol Inositol D-galactose Melezitose

Growth: Tomato juice agar 37 "C 7 % NaCl 8 % NaCl 9 % NaCl

Wata for L. piscicola from Hiu et al. (1984) and for C. piscicola from Collins et al. (1987) o-nitrophenyl-P-D-galactopyranoside

although catalase positive reactions have been re- similarities of the biochemical and physiological reac- ported for L. plantarurn (Dacre & Sharpe 1956, Whit- tions with those reported by Hiu et al. (1984) and tenbury 1960), L. divergens (Holzapfel & Gerber 1983) Collins et al. (1987). Furthermore, these isolates pro- and L. mali (Carr & Davies 1970). All isolates fer- duced mainly lactic acid from hexoses (blue colonies mented D-glucose, sucrose, trehalose, fructose, salicin, on HHD medium) and were able to utilize pentoses, D-ribose, cellobiose, D-mannose, D-gluconic acid and characteristics which are consistent with facultatively maltose, reduced litmus or fermented lactose (white heterofermentative lactobacilli (Kandler & Weiss 1986). color) in litmus milk, were esculin positive, grew in Isolates 106891 and 106892 differed from Carnobac- nutrient broth containing 6.5 % NaC1, and produced terium piscicola in several of the biochemical tests sufficiently acidic conditions for a positive methyl red (Table 2). They also differed in their biochemical char- reaction. None of the isolates produced acid from acteristics from species of Lactococcus and Vagococcus rhamnose, erythritol, L-sorbose, adonitol, D-xylose, or including production of acid from various sugars, dulcitol and all were negative for the following tests: starch hydrolysis, ONPG, Voges-Proskauer, motility, lysine and ornithine decarboxylase, phenylalanine arginine dehydrolase, and growth in various con- deaminase, Simmons citrate, urea, malonate, Voges- centrations of NaCl (Schleifer et al. 1985, Collins et Proskauer, and starch hydrolysis. None of the iso- al. 1989, Wallbanks et al. 1990, Williams et al. 1990). lates grew on Lactobacillus-selective medium (acetate A comparison of the biochemical results for these 2 medium), Macconkey agar or in nutrient broth isolates with those of previously described Lacto- containing 10 % NaCl. bacillus spp. (Kandler & Weiss 1986) revealed L. ali- Five of the isolates (00188, 335871, 01488, 27488, mentarius and L, homohiochi as the most similar 032891) were identified as Carnobacterium piscicola organisms. To further examine similarities of these (formerly Lactobacillus piscicola) based upon the isolates, correlation coefficients (r values) of the Starliper et dl lsolat~onof two Gram-positive bacteria from rainbow trout 185

Table 3. Relationships among fish isolates and reference cultures based on electrophoretic profiles released from cells on lysis. Adjusted lane profile correlation coeff~cient (r) for each pair of isolates using the Pearson product-moment method Mean (SE) calculated for 3 gels. ATCC 35586: C~rnobactenump~sc~cola;ATCC 29643: Lactobacjllus alirnentrius; ATCC 15434: L. homohiochl

Isolate" ATCC ATCC ATCC 35586 29643 15434 106891 106892 335871 00188 01488 032891 --p - --p ATCC 35586 1 0.53 1 0.760 0.678 0.471 0.924 ' 0.877 ' 0.933 ' 0.952 ' (0.234) (0.213) (0.065) (0.063) (0.031) (0.042) (0.028) ATCC 29643 1 0.497 0.745 0.792 0.662 0.571 0.635 0.629 (0.334) (0.1 16) (0.164) (0.221) (0.149) (0.202)

ATCC 15434~ 1 0.556 0.315 0.752 0.695 0.617 0.740

106891 1 0.803 0.778 0.684 0.707 0.688 (0.155) (0.054) (0.183) (0.119) (0.180) 106892 1 0.559 0.463 0.568 0.530 (0.108) (0.016) (0.166) (0.143) 33587 1 1 0.930' 0.931 ' 0.925' (0.032) (0.044) (0.037) 00188 1 0.895' 0.868 (0.040) (0.014) 01488 1 0.948' (0.009) 032891 1

" Isolate 27488 d~dnot incorporate isotope; h only 1 value for this isolate; ' significant association, p < 0.05

35S-methionine protein profiles using the AMBIS were Seven days following challenge, 5 fish from each obtained (Table 3). Correlation coefficients (rvalues) challenge group were sacrificed and their kidneys of Isolate 106891 were 0.745 and 0.556 for L. alimenta- cultured for the challenge organism. Bacteria were not rius and L, homohjochi respectively, and r values for isolated from the group waterborne-challenged with Isolate 106892 were 0.792 and 0.315 respectively. For Isolate 00188 nor from the groups waterborne- or replicates of 3, isolates are considered highIy related at ip-challenged with Isolates 106891 and 106892. Of the the a = 0.05 significance level, if r 2 0.87 (Fisher & remaining groups of fish (i.e. those challenged with Yates 1949). Neither isolate (106891, 106892) had an Isolates 01488, 00188, 032891, 27488, 335871 and r > 0.678 with Carnobacterium piscjcola. ATCC 35586), bacteria were recovered from all One mortality occurred as a result of the bacterial groups: 56 % of the fish from the waterborne- challenges, on Day 4 in the ip-injected group with challenges and 97 % from the ip-injected groups Isolate 27488. Reisolation of the bacterium from the yielded the challenge bacteria. Results of reisolation kidney was successful. attempts from the fish remaining at the end of the experiment are given in Table 4.

Table 4. Reisolation of Lactobacillus/Carnobacterium spp. 30 d after challenge. Values shown are number of fish from which the bacterium was reisolated and, in parentheses, DISCUSSION mean CFU g-' kidney tissue. 13 fish per group

7 Lactobacillus spp. have been isolated from a variety Isolate Waterborne Ip-injection of seemingly healthy fish species (Kvasnikov et al. 1977, Knochel 1981, Humphrey et al. 1987). Studying 01488 0 - 7 (6 5 X 102) 350 fish including 336 of several trout species in waters 00188 0 - 8 (2X 102) 032891 3 (3 X 102) 8 (2.67 X 102) around Ontario, Canada, Evelyn & McDermott (1961) 27488 2 (2 X 102) 10 (2.53 X 103) showed that 19 % of the fish yielded Lactobacillus. Of 335871 3 (6 X 102) 7 (5 X 102) the fish examined, 89 % appeared normal and healthy. 106891 0 - 0 - Other reports demonstrate that Lactobacillus is often 106892 4 (4.5 X 102) 0 - associated with disease in fishes. Rucker et al. (1953) ATCC 35586 0 - 8 (6.75 X 102) Control 0 - 0 - reported a chronic disease resulting in mortality in coho salmon Oncherhynus kisutch in the Pacific north- 186 Dis. aquat. Org. 13: 181-187, 1992

western United States. High mortality in female brood- Slight differences are noted between results for stock due to a Lactobacillus was reported to have Carnobacteriunl piscicola isolates of our study and occurred on more than one occasion at the Hot Creek those reported for C. piscicola by Collins et al. (1987). Hatchery, California, USA, (Ross & Toth 1974). Be- Those differences include acid production from sor- cause the mortality due to Lactobacillus appeared to bit01 and gluconic acid by our C. piscicola isolates, the be related to the stress of spawning, Ross & Toth (1974) production of catalase, and negative Voges-Proskauer attempted to produce the disease experimentally in test after 3 wk incubation (Barritt 1936). In another post-spawned rainbow trout. Herman et al. (1985) also study, Herman et al. (1985) reported mortality due to a attempted to reproduce the disease in juvenile rain- Lactobacillus sp. in brood stock rainbow trout follow- bow trout. Both groups of workers were unable to ing spawning. This bacterium was also sorbitol positive reproduce the disease or mortality following intra- and Voges-Proskauer negative. peritonea1 injection. Other reports describe the isola- Six of the isolates in this study were shown to pro- tion of Lactobacillus from broodstock fish following the duce catalase. Two apparently distinct H202-splitting handling and stress of spawning (Cone 1982, Hiu et al. activities occur within the (Whitten- 1984, Herman et al. 1985). The present study, along bury 1960, Johnson & Delwiche 1962). One is a heme- with these previous reports, indicate that disease due or hematin-containing catalase activity which utilizes to this group of organisms is apparently a result of an the iron-protoporphyrin complex as a coenzyme and opportunistic organism of low virulence because most results in active catalase ('classical catalase'). The isolations have been made from fish experiencing other does not require hematin and has been termed stress due to spawning. These results agree with those pseudocatalase activity (Whittenbury 1964). Although of Michel et al. (1986) for isolates from Europe who also it is established that 2 types of catalases exist within showed the strains to possess low virulence. the lactic acid bacteria, there are conflicting reports The term pseudo-kidney disease has been used for as to whether both activities may be possessed by lactobacillosis and was used to distinguish the disease the same organism (Whittenbury 1964, Johnson & from bacterial kidney disease, due to Renibactenum Delwiche 1965). The isolates in our study which were salmoninarum which causes high mortality in all ages catalase positive (335871, 01488, 106892, 00188, of salmonid fishes. Although both diseases are caused 27488, 032891) were demonstrated to be of the by Gram-positive rods, R. salmoninarum is much more hematin-requiring type of catalase activity assessed by fastidious, has an absolute requirement of cysteine for the method of Whittenbury (1964). Catalase activity growth and grows optirnally at 15 to 18 'C, whereas was also demonstrated by Lactobacillus piscicola Lactobacillus spp. from fish grow on many routine ATCC 35586 and shown to be a hematin-requiring nonselective media and appear to grow best at 30 "C. enzyme. When grown on 0.5 or 1 % glucose nutrient In our studies, more luxurious growth occurred when medium, all strains were catalase negative, indicating glucose or fructose (about 1 5% w/v) was used to a lack of pseudocatalase activity. However, when they supplement a medium not containing a hexose. This were grown on heated blood agar which contained may be a consideration when attempts to isolate the 1 % glucose, catalase activity was present. With regard organism are to be made. to the latter medium, the catalase activity of the red The bacterial pathogen causing the disease lacto- blood cells is destroyed by defibrination and sub- bacillosis in salmonids was first named Lactobacillus sequent heating, but the necessary coenzyme, piscicola by Hiu et al. (1984). On the basis of a com- hematin, is retained. The foregoing results indicate parative study involving certain Lactobac~llus spp, fre- that in reporting catalase results for this group of quently seen in poultry and also including L. piscicola, bacteria it is important to specify the medium used Collins et al. (1987) proposed the new generic name in the test. Carn~bacfe~urnfor this group of bacteria, which they To conclude, our studies confirmed that Carnobac- considered atypical Lactobacillus. Two characteristics terium piscicola (formerly Lactobacillus piscicola) was for which L. piscicola was considered atypical of the the organism most frequently (5 of 7 isolates) recov- genus included the lack of growth on the selection ered from sexually mature and post-spawning trout, medium and the production of oleic acid instead of some of which showed signs of a disease that has vaccenic acid as a major cellular fatty acid. Both fatty been referred to in the past as pseudo-kidney disease acids are 18 carbon and mono-unsaturated, but differ or lactobacillosis. In addition, and consistent with the in the unsaturated position. The phylogenetic distinc- experience of others, the organism showed low viru- tion made between these 2 genera (Lactobacillus and lence in challenge tests with rainbow trout, once again Carnobacterium) has recently been confirmed follow- suggesting that it is likely only to cause problems in lng comparisons of the homogeneity of the 16s rRNA severely stressed fish. Two of the 7 isolates collected in sequences in the isolates (Wallbanks et al. 1990). the study also proved to be Gram-positive bacilli of low Starliper et al.. Isolation of two Gran? -positive bacteria from rainbow trout 187

virulence; they appeared to be identical to each other characteristics of the catalases of Lactobacillaceae. J. Bac- but could not be identified. The existence of these teriol. 90: 347-351 unidentified isolates in post-spawned fish suggests Kandler, 0.. Weiss, N. (1986). Regular. nonsporing gram- positive rods. In: Sneath, P. H. A., Mair. N. S.. Sharpe, M. that Gram-positive bacilli other than Carnobacterium E., Holt, J. G. (eds.) Bergey's manual" of systematic piscicolamay also be involved in causing rnortalities in bacteriology, Vol. 2. Williams and Wilkins, Baltimore, p. highly stressed sallnonids and indicates that further 1208-1260 work on such organisms is needed. Koneman, E. W, Allen, S. D., Dowell, V R Jr, Sommers, H. M., (eds ) (1983). Color atlas and textbook of diagnostic microb~ology. 2nd edn. J. P. Lippincott Co., Philadelphia Knochel, S. (1981). Occurrence and some characteristics LITERATURE CITED of Lactobacillus spp. isolated from fish. J. appl. Bacteriol. 51: X Barritt, M. M. (1936). The intensification of Voges-Proskauer Kulp, W. L., White, V. (1932). A modified medium for plating reaction by the addition of a-napthol. J. Path. Bact. 42: Lactobacillus acidophilus. Science 66: 17-18 441-454 Kvasnikov, E. I.Kovalenko, N. K.,Mater~nskaya, L. G. (1977) Carr, J. G., Davies, P A. (1970). Homofermentative Lacto- Lactic acid bacteria of freshwater fish Microbiology 46: bacilli of ciders including Lactobacillus mall nov spec. J 619-624 appl. Bact. 33: 768-774 MacFaddin, J. F. (ed.) (1980).Biochemical tests for identifica- Collins, M. D., Ash, C., Farrow, J. A. E., Wallbanks, S., tion of medicdl bacteria, 2nd edn. Williams and Wilkins, Williams, A. M. (1989). 16s Ribosomal ribonucleic acid Baltimore sequence analyses of Lactococci and related taxa. De- McDonald, L. C., McFeeters, R. F., Daeschel. M. A., Fleming. scription of Vagococcus fluvialis gen. nov.. sp. nov. J. appl. H. P. (1987). A differential medium for the enumeration Bact. 67: 453-460 of homofermentative and heterofermentative lactic acid Collins, M. D., Farrow, J. A. E., Phillips, B. A.. Ferusu, S., bacteria. Appl. environ. Microbiol. 53: 1382-1384 Jones, D. (1987). Classification of Lactobacillus diver- Michel, C., Faivre, B., Kerouault, B. (1986). Biochemical gens, Lactobacillus piscicola, and some catalase- identification of Lactobacillus piscicola strains from negative, asporogenous, rod-shaped bacteria from poultry France and Belgium. Dis. aquat. Org. 2: 27-30 in a new genus, Carnobacterium. Int. J. Syst. Bact. 37: Niven, C. F. Jr, Smiley, K. L., Sherman, J M. (1942). The 310-316 hydrolysis of arginine by streptococcl. J. Bacteriol. 43: Cone, D. K. (1982). A Lactobacillus sp. from diseased female 651-660 rainbow trout, Salmo gairdnen Richardson, in Newfound- Rogosa, M., Mitchell, J. A., Wiseman, R. F. (1951). A selective land, Canada. J. 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Responsible Subject Editor: T Evelyn, Nanaimo, B.C., Canada Manuscript first received: January 16, 1991 Revised version accepted: May 1, 1992