Abstract

Starch gel electrophoresis was used to study the relatedness of a group of spreading YPB. Thirty five enzymes were assayed. From these data, the isolates were placed into 5 groups by cluster analysis. A comparison was made of these groups with an analysis of these same isolates based on biochemical and enzymatic studies.

One group (3) was homogeneous and contained 21 isolates of Flexibacter columnaris. This agreed 100% with the biochemical and enzymatic data. A single isolate (isolated from a salmonid in Oregon) was unique and formed a group (5). Groups 1 and 4 correlated well with the biochemical and enzymatic groupings. Both were in 86% agreement. The final group (2) showed less agreement.

Introduction

The term YPB is a name given by Drs. E. B. Shotts, Jr. and T. C. Hsu to a group of bacteria producing yellow to yellow-orange pigmented colonial growth (pers. comm.). Included in this group of bacteria are gram positive and negative organisms as well as spreading and nonspreading organisms isolated from fish or aquatic environments. There are three genera included in the YPB: Flexibacter spp., Cytophaga spp., and Flavobacterium spp. Of theYPB, there are 2 recognized fish pathogens. One is F. columnaris, which causes columnaris disease. The other is T. aurantiacus (Cytophaga psychrophilia) which causes cold water disease. Additionally, a number of poorly described YPB have been isolated from both healthy and diseased fish (1,2,3). The taxonomy of the YPB is not clearly understood (4). From biochemical and enzymatic studies of 157 isolates of YPB, Drs. Shotts and Hsu have grouped the organisms into four major groups. The third group is made up of organisms with the ability to spread on an agar surface, which includes F. columnaris and F. aurantiacus. This group was further divided into -5 subgroups. Study was done using starch gel electrophoresis to study the similarities and/or diversity in the isozymes of 53 isolates of group 3, including 21 isolates of F. columnaris isolated from Oregon, Georgia, and Taiwan. A cluster analysis was done on the data in a similar method to the cluster analysis of the biochemical and enzymatic data and a comparison was made between the two.

Starch gel electrophoresis was first developed by Smithies (5). Hunter and Markert (6) applied histochemical staining techniques to demonstrate enzymes separated by electrophoresis. Starch gel electrophoresis has been used occasionally as a method of classification of bacteria (7,8,9,10,11).

Materials and Methods

The isolates used in this study were grown, harvested, and prepared for starch gel electrophoresis using the method described by Schill et al. (7), except for the medium used to grow the isolates. The medium consisted of 0.2% tryptone, 0.05% yeast extract, and 0.2% gelatin (Hsu and Shotts, unpublished data). F. columnaris isolates were grown at 30°C in a broth medium. The remaining isolates were grown in petri plates at 25°C using the medium containing 1.5% agar.

Starch gel electrophoresis was conducted by the method of Utter et al. (12). Hydrolyzed starch (Connaught Laboratories Limited, Ontario, Canada) was used at a concentration of 12.5%. Two buffer systems were used. One was described by Ridgway et al. (13), the other by Clayton and Tretiak. (14)

The following enzymes were assayed: mannose-6-phosphate isomerase, aconitase, malate dehydrogenase, phosphoglucomutase, adenosine deaminase, glucose dehydrogenase, glucose phosphate isomerase, phosphogluconate dehydrogenase, aldolase, malic enzyme, glyceraldehyde-phosphate dehydrogenase, isocitrate dehydrogenase, adenylate kinase, sorbitol dehydrogenase, alcohol dehydrogenase, creatine kinase, aspartate aminotransferase, lactate dehydrogenase, hexokinase, superoxide dismutase, nucleoside phosphorylase, fumarate hydratase, xanthine dehydrogenase, glycerol-3­phosphate dehydrogenase, fructose diphosphatase, beta-glucuronidase (GUS) , glucose-6­phosphate dehydrogenase, peptidase-glycylleucine, peptidase phonylalanylproline, diaphorase, glycerol dehydrogenase, triosephosphate isomerase, beta-N-acetylglucosaminidase (HEX), alanine dehydrogenase, and peptidase-glycylleucine. The methods for the enzyme stains have been previously described. (7,15,16,17)

The enzyme activity was scored as the distance of the band (site of enzyme activity) from the origin in mm. A cluster analysis was done on the data to group the isolates based on the number of differences and/or similarities in their isozyme profiles.

Results

Of the 35 enzymes assayed, 2 (GUS and HEX) showed no detectable activity. Based on the data from the isozyme cluster analysis, the isolates were grouped into 5 groups (the same number of groups as the biochemical data). Table 1 shows the number of isolates in each group as well as a comparison of results of the cluster analyses from the isozyme and biochemical studies.

The 21 isolates of F. columnaris form group 3. Only isolates previously identified as F. columnaris fell into this group. Furthermore, this group agreed 100% with the biochemical group 3. one isolate, designated the western isolate, was isolated from a salmonid in Oregon, It was unique and formed a group (5). Groups 1 and 4 each showed an 86% agreement with the biochemical data. Also, 6 of 8 isolates of F. aurantiacus, which were isolated in New York state from salmonids showing signs of cold water disease, formed part of group 1. Isozyme group 2 was not in good agreement (23%) with that of the biochemical data.

Table 1. Comparison of cluster analyses data of isozyme and biochemical and enzymatic studies of selected YPB

Discussion

The F. columnaris isolates displayed little variation even though there was a wide range of geographical areas of isolation. They formed a very homogeneous group within what is considered a taxonomically diverse group of bacteria (YPB).

As mentioned previously, 8 of the isolates were isolated from New York State. Six of these isolates fell into one group (1). Whereas, the lone isolate (non F. columnaris) isolated from the westcoast was unique. There could possibly be some geographical differences in the non F. columnaris isolates. Also, with the exception of group 2, there was good-agreement, overall, between the isozyme and the biochemical and enzymatic data.

Further isozyme studies are in progress to look at more isolates of YPB, both spreaders and non-spreaders.

References

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