Edwardsiella ictaluri Strains - Applied and Environmental Microbiology

Vol. 59, No. 9

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1993, p. 2830-2836

0099-2240/93/092830-07$02.00/0 Copyright © 1993, American Society for Microbiology

Plasmid and Serological Differences between Edwardsiella ictaluri Strains CRAIG J. LOBB,* SEYED H. GHAFFARI, J. RUSSELL HAYMAN, AND DEXTER T. THOMPSON Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505 Received 25 January 1993/Accepted 11 June 1993

Several studies have shown that isolates of Edwardsiella ictaluri obtained from infected channel catfish in the southeastern United States harbor two cryptic plasmids, designated pCL1 (5.7 kb) and pCL2 (4.9 kb). These isolates appear to be serologically homogeneous. To extend these studies, we focused our analyses on two isolates of nonictalurid origin. Plasmid analyses of a danio isolate showed that it harbored plasmids which were similar if not identical to pCL1 and pCL2. This strain was also serologically indistinguishable from those isolated from channel catfish. In contrast, a green knife fish (GNF) isolate harbored four plasmids with relative mobilities of 6.0, 5.7, 4.1, and 3.1 kb. Southern blot analyses indicated that only the 5.7- and 4.1-kb plasmids strongly hybridized under high-stringency conditions to probes specific for pCL1 and pCL2, respectively. The GNF isolate showed minimal reactivity when reacted with polyclonal antiserum prepared against a channel catfish isolate. However, polyclonal antiserum to the GNF isolate strongly reacted with the GNF isolate in both surface fluorescence and agglutination reactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of cell lysates showed that the protein banding patterns of the strains compared were similar. However, Western blots of proteinase K-digested cell extracts showed that 0 antigen of the GNF isolate was antigenically distinct from the 0 antigen of the other isolates. These studies indicate that there are different serotypes of E. ictaluri and suggest that plasmid and serological analyses of future isolates ofE. ictaluri can be used to determine whether structurally distinct strains are emerging in major channel catfish aquaculture areas. United States. Since this report other investigators have confirmed these results (8, 10). Strains of E. ictaluri isolated from infected catfish have been shown to be homogeneous when analyzed in a battery of biochemical tests (14). In addition, serological analyses using monoclonal antibodies have indicated that different isolates of E. ictaluri appear to be serologically homogeneous (9). This general conclusion has led to studies directed toward vaccination approaches to control ESC in cultured channel catfish. However, there have now been several reports of E. ictaluri infecting other fish besides channel catfish. We focused our analyses on two of these isolates. The first isolate was from an infected danio (Danio devario) which was cultured in the southeastern United States (14). The second isolate was from an infected South American green knife fish (GNF) (Eigemannia virescens) that was maintained in laboratories in La Jolla, Calif. (4a). This strain was recovered in pure culture from the kidneys of fish dying from an acute gram-negative bacteremia. Earlier studies had indicated that the plasmid profile of the GNF isolate was apparently distinct, bearing plasmids of different relative mobilities. The plasmid profile of the danio isolate, however, was similar to the profiles of isolates recovered from infected channel catfish (8, 11). This apparent difference in plasmid profiles suggested that additional structural differences between E. ictalun strains might exist. These studies have led to this report, which reaffirms the value of plasmid analysis in characterizing E. ictaluri isolates and, equally important, establishes that there are different serotypes of E. ictaluri.

Edwardsiella ictaluri was first identified and characterized in 1981 (4) and can be readily distinguished from the more common member of the genus, E. tarda, by biochemical analyses (15). E. ictalun is the bacterial pathogen responsible for enteric septicemia of catfish (ESC) (3). ESC has been viewed as an acute septicemia that can progress rapidly in apparently healthy, fast-growing catfish and can result in extensive mortality. ESC has become a significant problem in the aquaculture of channel catfish in the southeastern United States. The incidence of disease-associated mortality occurs within the optimum temperature range for in vitro growth of the bacteria (22 to 28°C). When the bacterium is isolated from kidney, brain, blood, etc., it generally requires 48 h at 30°C to form typical colonies approximately 2 mm in diameter. This relatively slow rate of growth initiated our earlier studies to determine the plasmid profile of different isolates of E. ictaluri obtained from infected channel catfish raised in the southeastern United States (7). The analyses showed that two plasmids, designated pCL1 (5.7 kb) and pCL2 (4.9 kb), were both present in each of the E. ictaluri strains examined. The two plasmids were isolated and mapped with various restriction enzymes. These analyses indicated that the restriction maps of the two plasmids were distinct; this apparent lack of homology was further supported by the inability to visualize possible cointegrate structures when plasmid pools were analyzed by agarose electrophoresis. These analyses strongly suggest that plasmid analysis should be an effective method for rapidly identifying isolates of E. ictalun from channel catfish cultured in the southeastern

MATERIALS AND METHODS *

Bacterial strains and culture conditions. The isolates of E. ictaluri recovered from infected channel catfish (Ictalurus

Corresponding author. 2830

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PLASMID AND SEROLOGICAL DIFFERENCES IN E. ICTALURI

punctatus) were identified and characterized earlier (7). The strains are designated S85-1377, S85-1381, AL 84-142, and AL 84-187. The type strain ATCC 33202, originally isolated in Georgia from an infected channel catfish, was obtained from the American Type Culture Collection, Rockville, Md. The danio and GNF isolates of E. ictaluri were obtained from James Bertolini and were designated 10.15 and 10.16, respectively, in an earlier study (1). Biochemical characteristics of the danio and GNF strains were reported (1), and separate analyses confirmed the identification of these strains as E. ictaluri. Characteristic negative biochemical reactions included indole, motility at 37°C, H2S production on triple sugar iron medium, malonate utilization, Simmons citrate, Voges-Proskauer, urease, lactose, adonitol, dulcitol, sorbitol, arabinose, and arginine dihydrolase. Prior to analysis, the cells were recovered from frozen stocks maintained at -70°C and cultured at 30°C in brain heart infusion medium (Difco Laboratories, Detroit, Mich.). Plasmid and hybridization analyses. The methods used to obtain plasmid pools from the E. ictaluri isolates used in this report, as well as the agarose gel electrophoresis conditions used, were identical to those reported earlier (7). For hybridization analyses, pCL1 and pCL2 were first resolved by agarose gel electrophoresis, and the supercoiled plasmids were separately recovered from individual gel slices by electroelution. To ensure purity, the plasmids were subjected to a second round of purification, in which the supercoiled plasmids were linearized with EcoRI, separated by electrophoresis, and subsequently electroeluted from agarose gels. The purified plasmids were radiolabeled to a specific activity of about 1 x 109 cpm/,ug by using a random hexamer commercial kit (Amersham, Arlington Heights, Ill.). The methods used for Southern blot analysis and the high-stringency hybridization conditions used followed procedures described in detail elsewhere (2). Antigenic analyses and immunoassays. Murine polyclonal antisera were prepared to isolate S85-1377 as well as to the GNF isolate of E. ictaluri by the following procedure. Bacteria were inoculated from frozen stocks into 5 ml of brain heart infusion medium and grown for 15 h at about 30°C. The pelleted cells were washed three times in sterile 0.135 M NaCl at 4°C, and the cell pellet was resuspended in 10 ml of the saline solution. Six BALB/c mice, obtained from a colony maintained at the Department of Microbiology of the University of Mississippi Medical Center, were each injected subcutaneously with a total of 0.2 ml of the cell suspension. The mice were boosted three times with freshly cultured cells prepared in the same manner at weekly intervals. Concomitant with the last injection the animals were injected with 0.5 ml of pristane (2,6,10,14-tetraethylpentadecane; Sigma Chemical Co., St. Louis, Mo.), and ascitic fluid was induced with the SP2/0 murine myeloma line as described previously (5). Ascitic fluid was clarified by centrifugation, and the supernatant was frozen at -20°C until used. The agglutination titer of each polyclonal antiserum was determined in 96-well microtiter plates (Flow Laboratories, McLean, Va.) by using freshly cultured bacteria. Briefly, 15-h cultures of E. ictaluri isolates were washed three times in phosphate-buffered saline (PBS) and standardized to an optical density at 550 nm between 0.95 and 1.0. The polyclonal antiserum was serially diluted twofold in the microtiter plates, and an equal amount of the washed bacteria was added. The plates were agitated and kept at 4°C overnight. Agglutination titers were expressed as the reciprocal of the

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last log2 dilution of antiserum which yielded positive agglutination. Indirect fluorescent-antibody analysis of E. ictaluni isolates used both the anti-S85-1377 antiserum and the antiGNF isolate antiserum. Briefly, fresh cultures of bacteria were prepared as described above, and 100-,ul aliquots of washed cells were reacted with an equal amount of antiserum that had been diluted 10-fold in PBS. After a 20-min incubation at 4°C the cells were washed and reacted for an additional 20 min with fluorescein isothiocyanate (FITC)conjugated rabbit anti-mouse immunoglobulin G (IgG) (Accurate Chemical, Westbury, N.Y.). The cells were washed and examined microscopically, and the relative fluorescent intensity of the cells was determined qualitatively as contrasted with bacteria that had not been reacted with the primary antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by standard procedures as reported in detail elsewhere (6). Cell lysates of the E. ictaluni strains were prepared from fresh cultures as described above. Two milliliters of bacteria, standardized to an optical density at 600 nm of 0.3, was pelleted in a microcentrifuge tube and washed in Tris-buffered saline, and 200 ,ul of SDS-PAGE sample buffer was added. The cells were heated at 100°C for 2 min, and any insoluble matter was removed with a sterile toothpick. Samples of cell lysates were analyzed directly by SDS-PAGE or, alternatively, incubated for 1 h at 60°C with 50 ,ug of proteinase K (Sigma) prior to SDS-PAGE analysis. Western blotting (immunoblotting) of replicate lysates was performed by standard methods (13) using 1:100 dilutions of primary polyclonal antiserum and developed with alkaline phosphatase-conjugated goat antimouse

IgG (Promega, Madison, Wis.).

RESULTS E. ictaluri isolates. Plasmid of different Plasmid profile DNA was prepared from the GNF and danio isolates of E. ictaluni and compared with plasmid DNA prepared from representative isolates of E. ictaluri recovered from infected channel catfish (Fig. la, lanes A through G). Each of the channel catfish isolates had previously been shown to harbor both pCL1 (5.7 kb) and pCL2 (4.9 kb) (7). The plasmid profile of the danio isolate was identical to the plasmid profile of the channel catfish isolates; two plasmids, similar in size to pCLl and pCL2, were observed. The plasmid profile of the GNF isolate, however, was distinct. The GNF isolate contained four plasmids; the relative mobilities of these plasmids were 6.0, 5.7, 4.1, and 3.1 kb, as judged by interpolation from the reference plasmid ladder. Only the GNF 5.7-kb plasmid had a mobility similar to that of one of the known plasmids (pCL1). To determine the relationship of the plasmids in the GNF and danio isolates to pCL1 and pCL2, as well as to resolve questions regarding the potential similarity of pCL1 and pCL2, pCL1 and pCL2 were separately purified from agarose gels and radiolabeled to the same specific activity. The radiolabeled pCL1 and pCL2 probes were then hybridized separately to replicate Southern blots containing the plasmid DNA from the E. ictaluri isolates. In addition, plasmid pools of pCL1 and pCL2 derived from strain S85-1377 were restricted with enzymes predicted from the previously derived restriction maps to yield fragments for specific regions of both plasmids. Hybridization analyses showed that pCL1 or pCL2 did not cross-hybridize with one another, nor did the probes cross-hybridize with the bacterial chromosome

2832

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FIG. 1. Plasmid profile and Southern blot analyses of different strains of E. ictaluri hybridized with probes specific for pCL1 and pCL2. (a) Lane Ml, plasmid ladder (the sizes of the reference plasmids [in kilobase pairs] are indicated on the left); lanes A to G, plasmids detected in E. ictaluri ATCC 33202, S85-1381, S85-1377, AL84-142, and AL84-187, danio isolate 10.15, and GNF isolate 10.16, respectively; lanes H to J, plasmids of ATCC 33202 restricted with EcoRV plus BamHI (lane H), BglI (lane I), and EcoRI (lane J), respectively; lane M2, PstI digest of lambda DNA (the sizes of the standard linear fragments [in kilobase pairs] are indicated on the right). The gel was stained with ethidium bromide. (b) Replicate Southern blot of the gel depicted in panel a hybridized with pCL2. (c) Replicate Southern blot of the gel depicted in panel a hybridized with pCL1. The lanes in panels b and c contained the same preparations as the corresponding lanes in panel a. Southern blot hybridization was performed under high-stringency conditions (see Materials and Methods). (d and e) Restriction maps of pCL1 and pCL2, respectively, as determined previously by Lobb and Rhoades (7).

(Fig. lb and c). The supercoiled and linearized plasmids, as well as the restriction fragments derived from either pCLl and pCL2, hybridized only with probes derived from the respective plasmid. In addition, pCLl and pCL2 probes hybridized to the 5.7and 4.9-kb plasmids, respectively, of the danio isolate. Because these danio plasmids have the same relative mobility as pCL1 and pCL2 and hybridized with probes specific for pCL1 and pCL2, respectively, under stringent hybridization conditions, it seems highly probably that these plasmids are very similar to pCL1 and pCL2. The GNF isolate, which contained four plasmids of different mobilities, was also found to have two plasmids which shared similarity to pCLl

and pCL2. The GNF 5.7-kb plasmid hybridized to probes derived from pCL1, and the GNF 4.1-kb plasmid hybridized to probes derived from pCL2. These results are consistent with the conclusion that there is a high degree of similarity between pCL1 and the GNF 5.7-kb plasmid, as well as a high degree of similarity between pCL2 and the GNF 4.1-kb plasmid. The GNF 6.0- and 3.1-kb plasmids did not strongly hybridize to probes derived from either pCL1 or pCL2. The GNF isolate harbors two other plasmids besides those similar to pCL1 and pCL2. It was important to determine whether the 6.0- and 3.1-kb GNF plasmids, which did not strongly hybridize with probes derived from either pCL1 or pCL2, were probably different from each other. Plasmid


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TABLE 1. Agglutination titers and relative fluorescent intensities of isolates of E. ictaluri reacted with two antisera E. ictaluri isolate

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FIG. 2. Restriction analysis of the plasmids in the GNF isolate of E. ictalun. Lane M, PstI digest of lambda DNA (the sizes of the standard fragments [in kilobase pairs] are indicated on the right); lane A, undigested plasmid DNA (the locations of the four plasmids are indicated on the left with arrows); lanes A to G, plasmid DNA following digestion with restriction endonucleases HindIll, SalIl, SstII, XhoI, BamHI, EcoRI, and ClaI, respectively.

pools of the GNF isolate were restricted either with enzymes which did not have sites in pCL1 or pCL2 or with enzymes which cut these reference plasmids only once. From these analyses, it appears that the GNF 6.0- and 3.1-kb plasmids are distinct from one another. Of the seven enzymes used for restriction analyses shown in Fig. 2, Sall was the only enzyme which restricted the GNF 3.1-kb plasmid. However, the same seven enzymes yielded a different restriction profile for the 6.0-kb plasmid. This plasmid did not contain a SalI restriction site but had single sites for EcoRI and ClaI and at least two restriction sites for BamHI. Therefore, there are at least five differences in the restriction sites of the 6.0and 3.1-kb plasmids. These results suggest that the GNF 6.0and 3.1-kb plasmids are probably distinct from each other. These results, coupled with the hybridization results described above, indicate that E. ictaluni isolates may harbor additional small plasmids which are not similar to pCL1 and pCL2. Serological analysis. Polyclonal antiserum to the GNF isolate as well as polyclonal antiserum to a channel catfish isolate (S85-1377) were prepared to determine whether these isolates were serologically different. Comparative analyses using either microtiter agglutination assays or fluorescentantibody analyses indicated that the GNF isolate appeared to be antigenically distinct from these other E. ictaluni strains (Table 1). The anti-GNF isolate antiserum reacted strongly only against the GNF isolate; the serological reactions with the other isolates were marginal. In contrast, the antiserum made to isolate S85-1377 strongly cross-reacted with the type strain ATCC 33202, as well as other E. ictaluri

isolates recovered from infected channel catfish. In addition, this antiserum strongly reacted against the danio isolate. However, the anti-S85-1377 antiserum gave only a marginal reaction when reacted with the GNF isolate. These analyses indicate that the GNF isolate appears serologically distinct from these other E. ictalun isolates. Structural and antigenic analysis of E. ictaluri isolates. To determine whether the serological difference observed between the GNF isolate and the other E. ictaluri strains could be defined, SDS-solubilized cell lysates were analyzed by SDS-PAGE. Cell lysates stained with Comassie brilliant blue showed that the channel catfish isolates as well as the danio isolate had similar protein profiles. The positions of the stained bands as well as their relative intensities were very similar. The GNF isolate also had an essentially similar protein profile, with only a few minor structural differences apparent (Fig. 3A). Identical aliquots of the lysates used above were also analyzed in Western blots using the two different anti-E. ictaluni antisera (Fig. 3B and C). Western blots developed with the anti-S85-1377 antiserum showed that the antigenic profile of the danio lysate was very similar to that observed with the other two E. ictaluri strains obtained from infected channel catfish. In contrast, the antigenic profile of the GNF lysate which reacted with this antiserum was somewhat different from that of these other strains. The anti-S85-1377 antiserum did not detect the same number of bands, nor did these bands exhibit the same relative staining intensity as those derived from the danio or channel catfish isolates. The general conclusion that the antigenic profile of the GNF isolate was different from that of these other isolates was supported when lysates from these strains were reacted with the antiserum derived to the GNF isolate. With this antiserum multiple bands were observed in the lysate of the GNF isolate, but many of the bands in the other strains either were not reactive or reacted with less staining intensity than those from the GNF isolate. There were, however, some proteins which appeared to be antigenically similar in each of these strains. As judged by the relative intensity of the staining reaction with both antisera, as well as the identical relative mobility of the reactive proteins, three proteins, exhibiting relative molecular masses of 97, 43, and 37 kDa, appear to be antigenically similar in each of these isolates. Proteinase K-treated lysates were also examined in Western blots using these two different antisera (Fig. 3D and E).

2834

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FIG. 3. SDS-PAGE and Western blot analyses of four strains of E. ictalun. (A) Commassie brilliant blue-stained SDS-PAGE gel of reduced cell lysates. Lane M contained molecular weight standards (a mixture of myosin, P-galactosidase, phosphorylase b, bovine albumin, egg albumin, and carbonic anhydrase). The masses of these standards (in kilodaltons) are indicated on the left. In all panels lanes 1 through 4 contained cell lysates of E. ictalun GNF strain 10.16, danio strain 10.15, strain S85-1377, and type strain ATCC 33202, respectively. (B and C) Western blots of cell lysates of E. ictaluri strains reacted with anti-GNF strain 10.16 and anti-S85-1377 antisera, respectively. (D and E) Western blots of proteinase K-treated cell lysates of E. ictaluri strains reacted with anti-GNF strain 10.16 and anti-S85-1377 antisera, respectively.

These reactions clearly distinguished the GNF isolate from the other strains. The anti-GNF E. ictaluni antiserum reacted with the lipopolysaccharide (LPS) ladder of the GNF isolate and not with the LPS ladder of the other strains. The converse reaction was observed when these strains were reacted with the anti-S85-1377 antiserum. These results are consistent with the conclusion that the 0 antigen of the GNF isolate is antigenically distinct from the 0 antigen of these other isolates and hence probably accounts for the major serological differences observed in the agglutination and fluorescent-antibody analyses. DISCUSSION The GNF isolate of E. ictaluri has a different plasmid profile and is serologically distinct from strains of E. ictaluni routinely isolated from infected channel catfish. It was previously shown that E. ictaluri strains isolated from infected catfish in the southeastern United States contained two different small plasmids, designated pCL1 and pCL2. Comparisons of the restriction maps of these plasmids, as well as the inability to detect possible cointegrate structures in plasmid pools, suggested that partial homology of these plasmids was unlikely. Thus, on the basis of the consistency of detecting both plasmids in channel catfish isolates, it was suggested that these plasmids should be valuable tools in the presumptive identification of this bacterium from infected channel catfish. These findings have been confirmed by other investigators (8, 10). The use of hybridization analyses in which plasmidspecific probes are used to detect E. ictaluri has obvious potential, as related earlier (7). Because of this potential, it

important to ensure that pCL1 and pCL2 did not cross-hybridize with each other. Another study indicated that these two plasmids cross-hybridized (11), although a subsequent study by this group indicated that pCL1 did not cross-hybridize with pCL2 (10). To resolve this question, pCL1 and pCL2 were twice purified from agarose gels and used as probes in hybridization analyses. These studies clearly showed that these probes do not cross-hybridize with each other and do not cross-hybridize with the bacterial chromosome. Plasmid analysis of the isolates of E. ictalun from the danio and the GNF support the evidence of Newton et al. (8) and Reid and Boyle (10) that the GNF isolate contained plasmids which are different in size from those found in either the danio isolate or the E. ictalun strains routinely isolated from channel catfish. Both of these earlier investigations showed that the GNF isolate contained three plasmids which had molecular sizes of about 5.6, 4.0, and 3.0 kb. Reid and Boyle (10) showed that the 5.6-kb plasmid crosshybridized with probes derived from pCL1 and the 4.0-kb plasmid hybridized with probes derived from both pCL1 and pCL2. Plasmid profiles of the GNF isolate in the studies of both Reid and Boyle and Newton et al., however, did not appear to reflect the 6.0-kb plasmid which was identified in this study. This difference might reflect several possibilities, including comigration of the 6.0- and 5.7-kb plasmids in these earlier studies, the loss of the 6.0-kb plasmid from these cultures, or even integration of the 6.0-kb plasmid into the chromosome. It is not clear which of these possibilities might be correct. However, this study shows that the GNF strain contains two plasmids in addition to plasmids similar to pCL1 and pCL2. The 6.0- and 3.1-kb GNF plasmids have

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different restriction patterns, and neither strongly hybridizes with probes derived from either pCL1 or pCL2. In contrast, the GNF 5.7-kb plasmid hybridized with probes derived from pCL1, whereas the GNF 4.1-kb plasmid hybridized with probes derived from pCL2. Because high-stringency hybridization conditions were used in these analyses, these plasmids share considerable sequence similarity with pCL1 and pCL2, respectively. The possibility that strains of E. ictaluri may lose plasmids during cell culture is of potential concern, although earlier in vitro attempts to cure pCL1 and pCL2 from E. ictaluri proved difficult (7). Studies have shown that a strain of E. ictalun isolated in Maryland from white catfish had a 4.0-kb plasmid which, by hybridization analysis, was probably similar to pCL2. However, this strain apparently lacked a 5.7-kb (pCL1-like) plasmid, and furthermore, hybridization analyses suggested that this plasmid was not integrated into the chromosome (10). Whether this strain may have lost a plasmid similar to pCL1 is not known, but these observations seem to caution that plasmid analyses should be conducted with strains before extensive cell passage. Earlier studies had shown that different isolates of E. ictalun appear to be biochemically homogeneous (15). Serological studies have also concluded that different strains of E. ictaluri appear to be serologically homogeneous. Plumb and Klesius (9) derived seven hybridoma cell lines from mice immunized with E. ictaluni type strain ATCC 33202. Their analyses suggested that although not all monoclonal antibodies reacted uniformly against all isolates, there were conserved antigens in all of the strains analyzed. Bertolini et al. (1) have also done serological assessments on different strains of E. ictaluri. In their analyses they compared an antiserum raised against a channel catfish isolate with an antiserum raised against the GNF isolate. In both cases, antiserum was prepared from rabbits that were immunized with formalin-killed cells. Bertolini et al. showed that there were antigens which were apparently specific to the GNF isolate but concluded that there was insufficient evidence to suggest that the GNF isolate represented a different serotype. In the present study murine polyclonal antisera obtained from mice immunized with live E. ictaluri from a channel catfish isolate or the GNF isolate were compared. By both agglutination reactions and the fluorescent-antibody techniques, the GNF isolate was judged to be serologically distinct. Comparative SDS-PAGE analyses of cell lysates indicate that the channel catfish isolates and the danio and GNF isolates have similar protein profiles, as judged by the relative staining pattern of similar-sized bands. However, when replicate Western blots were reacted with these two different antisera, the antigenic profiles were different. It is also interesting that there were several proteins which were judged to be similar in their antigenic reactions with both antisera. Although at this point little is known regarding the structural proteins of E. ictaluri, proteins with relative molecular masses of 97, 43, and 37 kDa appeared to stain with equal intensity with both antisera. This observation suggests that these proteins are major antigenic determinants when live whole cells are used for immunization. Western blots of proteinase K-treated lysates indicate that the 0 antigen of the GNF isolate is antigenically different from the 0 antigen of the other strains of E. ictaluri examined in this report. Studies with the more common member of the genus, E. tarda, have shown that there are different 0-specific chains in different isolates. This finding has resulted in the serological classification of different

2835

0-antigens in this species (12). The present study would indicate that E. ictaluri may also be classified into different serotypes. Whether the diversity of 0 antigens represented in E. tarda will be reflected as more serological analyses are performed with additional isolates of E. ictaluni must await further studies. In conclusion, this study has identified a strain of E. ictaluri isolated from an infected GNF which has a different plasmid profile and is antigenically distinct from isolates of E. ictaluni recovered from channel catfish in the southeastern United States. Of the four plasmids identified in the GNF strain, only the 5.7-kb plasmid was similar in size to one of the characterized plasmids (pCL1). Hybridization analyses, however, showed that the 5.7-kb plasmid and the 4.1-kb plasmid shared significant similarity to pCL1 and pCL2, respectively. The serological differences observed between the GNF isolate and the other E. ictaluni isolates examined were determined by Western blotting to reflect significant differences in the 0 antigens of these two serological groups. These combined results suggest that isolates of E. ictaluri which have plasmid profiles different from those strains typically isolated from channel catfish should be examined to determine whether they are serological different from known isolates. It may be possible that the cryptic plasmids of E. ictaluri may mediate changes in the 0 antigen. If new serotypes of E. ictaluri prove to be virulent in challenge experiments with channel catfish, these strains may become of significant economic concern if they are introduced into major channel catfish aquaculture areas. ACKNOWLEDGMENTS Our appreciation is extended to Jim Bertolini for providing the danio and GNF isolates of E. ictaluri used in this study. This investigation was supported by Public Health Service grant AI23052 from the National Institutes of Health. REFERENCES 1. Bertolini, J. M., R. C. Cipriano, S. W. Pyle, and J. J. A. McLaughlin. 1990. Serological investigation of the fish pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish. J. Wild. Dis. 26:246-252. 2. Ghaffari, S. H., and C. J. Lobb. 1989. Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains. J. Immunol.

143:2730-2739.

3. Hawke, J. P. 1979. A bacterium associated with disease of pond cultured channel catfish, Ictalurus punctatus. J. Fish. Res. Board Can. 36:1508-1512. 4. Hawke, J. P., A. C. McWhorter, A. G. Steigerwalt, and D. J. Brenner. 1981. Edwardsiella ictaluri sp. nov., the causative agent of enteric septicemia of catfish. Int. J. Syst. Bacteriol. 31:396-400. 4a.Kent, M. L., and J. M. Lyons. 1982. Report. Fish Health News

2:ii. 5. Lobb, C. J., and L. W. Clem. 1982. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev. Comp. Immunol. 6:473-479. 6. Lobb, C. J., and L. W. Clem. 1983. Distinctive subpopulations of catfish serum antibody and immunoglobulin. Mol. Immunol.

20:811-818.

7. Lobb, C. J., and M. Rhoades. 1987. Rapid plasmid analysis for the identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus). Appl. Environ. Microbiol. 53: 1267-1272. 8. Newton, J. C., R. C. Bird, W. T. Blevins, G. R. Wilt, and L. G. Wilt. 1988. Isolation, characterization, and molecular cloning of cryptic plasmids from Edwardsiella ictalui. Am. J. Vet. Res. 49:1856-1860. 9. Plumb, J. A., and P. Klesius. 1988. An assessment of the

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10. 11. 12. 13.

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