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Applied and Environmental Microbiology, November 2004, p. 6488-6494, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6488-6494.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

Linda D. Rhodes,* Alison M. Coady,{dagger} and Rebecca K. Deinhard{ddagger}

Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, United States Department of Commerce, Seattle, Washington

Received 5 March 2004/ Accepted 12 July 2004


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ABSTRACT
 
Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon (Oncorhynchus tshawytscha) by an average of 34% at doses of ≤105 cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 106 cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.


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INTRODUCTION
 
Bacterial kidney disease (BKD) is a persistent, debilitating disease in salmonid fishes. Renibacterium salmoninarum, the etiological agent of BKD, accumulates primarily in the kidney, causing edema, granuloma development, and membranous glomerulonephritis (36) and resulting in morbidity and mortality (17). The prevalence of chinook salmon (Oncorhynchus tshawytscha) infected with R. salmoninarum can be as high as 90% in hatcheries (41) and 91% in outmigrating river populations (27). BKD is a persistent infectious disease problem in salmon culture (26), affecting wildlife conservation and restoration efforts. Twenty-six stocks of Pacific salmon and steelhead have been designated threatened or endangered by the United States federal government (23), and restoration activities such as captive rearing are hampered by recurrent outbreaks of BKD.

Fish infected with R. salmoninarum mount an antibody response that is directed principally against a 57-kDa extracellular protein (5) called major soluble antigen (MSA). MSA has been implicated as a major pathogenicity determinant that is involved in host immunosuppression (6, 15, 19, 40, 45), leukocyte agglutination (43, 44), and virulence (7, 24, 37). MSA is encoded by two identical genes, msa1 and msa2 (25), and both genes are transcriptionally active (31). During a survey of R. salmoninarum isolates for mutations in the two msa genes, we discovered evidence of a third msa gene that was not present in all isolates. In this study we characterized the third msa gene and identified an associated virulence phenotype.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions.
A total of 26 isolates of R. salmoninarum were used in this study. The geographic origin and source of each isolate are shown in Table 1. Liquid cultures were grown in modified KDM2 broth (1.0% Bacto Peptone [Difco], 0.05% yeast extract, 0.05% L-cysteine; adjusted to pH 6.5) (12) at 15°C. Plate cultures were grown on modified KDM2 agar (1% Bacto Peptone, 0.05% yeast extract, 0.05% L-cysteine, 10% newborn calf serum, 5% R. salmoninarum-conditioned medium [13]; adjusted to pH 7.5) at 15°C. Culture purity was determined by Gram staining, and identities were confirmed by direct fluorescent antibody staining with an anti-R. salmoninarum polyclonal antibody (Kirkegaard and Perry). Cultures recovered from the pathogenicity challenges (see below) for species confirmation and genotyping were inoculated onto selective KDM2 (SKDM2) agar, which contained modified KDM2 agar as described above supplemented with cycloheximide (final concentration, 50 µg ml–1), D-cycloserine (final concentration, 12.5 µg ml–1), polymyxin B (final concentration, 25 µg ml–1), and oxolinic acid (final concentration, 2.5 µg ml–1) (1).


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  		TABLE 1. Geographic origins, years of isolation, and sources of R. salmoninarum isolates used in this study

Chromosomal DNA analysis.
Chromosomal DNA was prepared as previously described (32) and was analyzed by standard Southern blotting (35). Probes were labeled with digoxigenin-11-dUTP, and hybridized probes were detected by chemiluminescence according to the manufacturer's instructions (Boehringer Mannheim). The msa open reading frame (ORF) probe was synthesized by random prime labeling of a 1.06-kb BglII fragment from msa1 cloned into pCO2 (25). Probes specific for sequences flanking the msa1 ORF were synthesized by PCR with pCO2 and the following primers pairs: for the 5' flanking sequences, 5'-GAAGCTGAACTGACTGA-3' and 5'-AACAGGGCCACCAGATC-3'; and for the 3' flanking sequences, 5'-GAAAGCCACAAACCGTCTGCTG-3' and 5'-AACCAACTACAGAGACAGCGGTA-3'. Sequence analysis was performed by cycle sequencing with a Big Dye terminator kit (version 3.1; Applied Biosystems) and analysis with an ABI 3100 sequencer. The 5' portion of msa3 was derived by excising and eluting the 8.4-kb XhoI fragment from isolate MMMvir and amplifying a 1.0-kb product with primers 5'-GTTTCTGAGGGACGGGCCGC-3' and 5'-GTCTTAGGCTCGAGAGTTGCTGCTGT-3'; this product was subjected to sequence analysis. The 3' portion of msa3 was derived by excising and eluting the 4.5-kb XhoI fragment from isolate MMMvir and cloning it into vector pCR2.1 (Invitrogen) for subsequent sequence analysis.

Protein analysis.
Equivalent numbers of R. salmoninarum cells in broth cultures from early to late logarithmic growth (optical densities at 525 nm between 0.12 and 1.35) were harvested by centrifugation for 20 min at 10,000 x g, and dry pellets were stored at –20°C until analysis. The volume of the harvested culture supernatant was adjusted on the basis of the optical density at 525 nm of the culture. Phenylmethylsulfonyl fluoride (Sigma) was added to a final concentration of 1.4 mM, and samples were stored at –20°C until analysis. Cells pellets were prepared for analysis by resuspending cells in 1x sample buffer (0.31 M Tris[pH 6.8], 2.5% glycerol, 0.5% sodium dodecyl sulfate,1.25% ß-mercaptoethanol). Supernatants were concentrated by using Centriprep-30 or Centricon-30 spin concentrators (Amicon) according to the manufacturer's instructions; in this procedure we made sure that samples from a given sampling time received equivalent concentrations. Sample buffer was added to the supernatants to obtain preparations containing 1x (final concentration) sample buffer. Samples were heat denatured at 99°C for 10 min and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the standard immunoblot method with alkaline phosphatase visualization (20). MSA was detected with monoclonal antibody 3H1 (43).

BKD ELISA.
The level of R. salmoninarum antigens in the kidney was determined by an enzyme-linked immunosorbent assay (ELISA) (28, 29) by using a goat antibody directed against whole R. salmoninarum (Kirkegaard and Perry). For detection we used horseradish peroxidase activity measured at 405 nm. The threshold value for negative controls was 0.0896.

Pathogenicity in salmon.
A cohort of juvenile fall chinook salmon (O. tshawytscha) was reared to smoltification and transferred to seawater approximately 6 months after hatching. The fish were reared for an additional 8 months in seawater to a mean weight of 15.3 g and to a mean length of 108.4 mm without significant morbidity or mortality. Prior to challenge, 10 fish were examined for the presence of R. salmoninarum in kidney tissue by a nested PCR assay (8), and all fish were negative. For the challenge, fish were inoculated intraperitoneally with 0.1 ml of peptone-saline (0.1% Bacto Peptone, 0.85% NaCl) containing no bacteria (vehicle control) or peptone-saline containing R. salmoninarum at a concentration of 104, 105, 106, or 107 cells ml–1. Twenty fish were used in the vehicle control groups. For each isolate and dose, 15 to 19 fish were inoculated. Cell concentrations were determined by a membrane fluorescent antibody technique (11), and viability was confirmed by culture on modified KDM2 agar. The fish were held in 3-foot-diameter, circular, flowthrough tanks receiving ambient, UV-treated seawater, and the temperature ranged from 8 to 11.5°C from the beginning to the end of the experimental period. The fish were fed 2-mm grower diet (Ewos) at a rate of 2% of body weight on alternate days. Dead fish were removed daily from the tanks, stored on ice, and necropsied within 48 h of removal. Bacteria were recovered from the kidney by plating on SKDM2 agar, and then the entire kidney was removed and stored at –20°C for subsequent BKD ELISA analysis. The identity of bacteria on SKDM2 agar was confirmed by fluorescent antibody microscopy with a polyclonal antibody directed against R. salmoninarum (Kirkegaard and Perry). Mortality was attributed to BKD if viable R. salmoninarum was recovered on SKDM2 agar or the BKD ELISA value was greater than or equal to 0.5. No BKD mortality was observed among fish inoculated with peptone-saline without bacteria. The duration of the experiment for each treatment group was as follows: 106 cells per fish, 85 days; 105 cells per fish, 92 days; 104 cells per fish, 102 days; and 103 cells per fish, 106 days. Kaplan-Meier survival analysis was used to estimate the median length of survival (in days) and the proportion of susceptible fish surviving at the end of the experimental period. Survival curves were compared by the log rank test.


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RESULTS
 
Identification of a third msa locus.
The occurrence of two copies of the msa gene with identical ORFs has been known for several years (25), and these two copies can be distinguished by Southern analysis of genomic DNA digested with BamHI (Fig. 1A). The msa ORF contains a single XhoI restriction site, located near the middle of the ORF (nucleotides 727 to 732 in the 1,677-nucleotide ORF), and Southern analysis of XhoI-digested DNA from type strain ATCC 33209T with an msa ORF probe revealed hybridizing bands at 2.7, 6.4, and >17 kb (Fig. 1A). Sequence analysis of the regions flanking the msa1 and msa2 ORFs and Southern analysis with copy-specific probes identified the 2.7-kb band as the 5' portion of msa2 (data not shown) and the 6.4-kb band as the 5' portion of msa1 (Fig. 1A). The hybridizing fragment larger than 17 kb actually consists of two bands (G. Wiens, personal communication), representing the 3' portions of both msa1 and msa2.



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  		FIG. 1. Demonstration of a third msa locus in some isolates of R. salmoninarum. Blot images were captured with an ImageStation 440CF by using the 1D Image Analysis software, version 3.5.3 (Kodak). (A) Southern blot analysis of 1 µg of genomic DNA from five isolates of R. salmoninarum. The blots on the left were hybridized to an msa ORF probe and then stripped and hybridized to a probe specific for sequences located 5' to the msa1 ORF (blots on the right). The DNA in the upper blots was digested with BamHI, and the DNA in the lower blots was digested with XhoI. Lane 1, ATCC 33209T; lane 2, Marion Forks; lane 3, MMMvir; lane 4, Bonneville-1; lane 5, Carson 5b. The restriction digestion used is indicated at the left. Lane m contained molecular mass marker VII (Boehringer Mannheim) in the upper blot and molecular mass marker II (Boehringer Mannheim) in the lower blot. Sizes (in kilobases) are indicated next to lane m. (B) Schematic diagram comparing the three msa loci based on sequence analysis (solid black lines) or Southern analysis (gray lines). Restriction sites are indicated as follows: Xho, XhoI; B, BamHI; H, HindIII; X, XbaI. The solid bars above msa1 show the positions (from left to right) of the msa1 5'-specific probe, the msa ORF probe, and the msa1 3'-specific probe.

Surprisingly, some isolates of R. salmoninarum displayed two additional hybridizing bands at 4.5 and 8.4 kb after XhoI digestion. Because Southern analysis of BamHI-digested DNA from these isolates revealed only two msa loci, we predicted that the third copy was a duplication of either msa1 or msa2. For isolates displaying the two additional XhoI bands, the msa ORF probe exhibited increased hybridization signal intensity for the 5.0-kb BamHI band corresponding to msa1, suggesting that there were more copies of msa1 than of msa2 (Fig. 1A). Region-specific probes for msa1 showed that the 4.5-kb band hybridized to the 3' portion of msa1 (data not shown) and the 8.4-kb band hybridized to the 5' fragment of msa1 (Fig. 1A). The 4.5-kb XhoI band was directly cloned and sequenced, which revealed that this fragment indeed contained 943 nucleotides of the msa ORF and the 3' flanking sequences unique to msa1. The isolated 8.4-kb XhoI fragment was used to generate a 1.0-kb PCR product encompassing 301 bp of the 5' flank and the first 729 bp of the msa ORF, including the XhoI site that was in the downstream primer. This PCR product was sequenced and was found to be identical to the corresponding region of msa1, confirming the Southern analysis observations. From these results, we concluded that the third copy of msa is a duplication of msa1. Because the context of this third copy differs from that of msa1, we provisionally designated the third copy msa3. A schematic diagram comparing the three loci is shown in Fig. 1B.

To assess the distribution of msa3 in R. salmoninarum, we examined 26 isolates obtained from a variety of geographic locations and at different times (Table 1) by Southern analysis. The majority (19 of 26) of the isolates examined exhibited the three-locus genotype (Fig. 2). The origins of the isolates displaying the two-locus genotype varied and included locations in Oregon (Round Butte, Marion Forks, ATCC 33209T), on the Columbia River (Bonneville-5, W88B2), on Lake Michigan (GL64), and in Scotland (MT239). Because the probe used in the Southern analysis whose results are shown in Fig. 2 was derived from the msa ORF, which is identical at the three msa loci, the hybridization signal intensity was expected to be proportional to the number of copies for each locus. For isolates with the msa3 locus, the densities of the hybridization signal intensity for the 8.4-kb band (msa3), the 2.7-kb band (msa2), and the 6.4-kb band (msa1) were determined, and the density ratios of the msa3 and msa1 bands and of the msa2 and msa1 bands were calculated. For the 19 isolates, the average msa3/msa1 ratio was 1.42 with a coefficient of variation of 83.9%, while the average msa2/msa1 ratio for hybridization signal intensity was 1.14 with a coefficient of variation of 19.3%. Thus, while the average msa3/msa1 and msa2/msa1 ratios of signal intensities were similar, the coefficient of variation for the msa3/msa1 ratio was four times greater than that for the msa2/msa1 ratio. These results suggest that the msa3 copy number is not constant among the isolates.



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  		FIG. 2. Distribution of the msa3 locus in 26 isolates of R. salmoninarum: Southern blot analysis of 500 ng of XhoI-digested genomic DNA with an msa ORF probe. Lane 1, Little Goose; lane 2, D6; lane 3, 91-127; lane 4, Bonneville-1; lane 5, Lookingglass; lane 6, Round Butte; lane 7, LR-95; lane 8, Marion Forks; lane 9, Lake Billy Chinook; lane 10, Bonneville-5; lane 11, MK91; lane 12, ss-ChS-94-1; lane 13, Cow-ChS-94; lane 14, W88B2; lane 15, GL64; lane 16, Sawtooth91; lane 17, DWK91; lane 18, DWK90; lane 19, MT239; lane 20, BPA2001-6031; lane 21, BPA2001-6050; lane 22, Lost18; lane 23, MMMvir; lane 24, ATCC 33209T; lane 25, Carson5b; lane 26, Willamette. Lanes m1 contained molecular mass marker II (Boehringer Mannheim), and lanes m2 contained molecular mass marker VII (Boehringer Mannheim). Sizes (in kilobases) are indicated next to the gels. Blot images were captured with an ImageStation 440CF by using the 1D Image Analysis software (version 3.5.3; Kodak). The ratios of densitometric measurements for the 8.4-kb band to densitometric measurements for the 6.4-kb band (msa3:msa1) and the ratios of densitometric measurements for the 2.7-kb band to densitometric measurements for the 6.4-kb band (msa2:msa1) are shown beneath the lanes.

In vitro phenotype of isolates with msa3.
The presence of a third copy of msa may affect in vitro characteristics of R. salmoninarum, such as replication. The growth of two isolates with two msa loci (type strain ATCC 33209T and Marion Forks) and four isolates with three msa loci (MMMvir, Bonneville-1, Carson 5b, and 91-127 Idaho) was compared daily for up to 4 days by visible light spectrophotometry. No significant differences in the growth rate were observed for the two the genotypes (data not shown).

The protein encoded by msa, MSA, has been implicated as a virulence factor in the pathogenesis of BKD, and previous work has shown that msa1 and msa2 are expressed in vitro (31). If a protein is expressed from msa3, the levels of MSA may be greater in isolates bearing msa3 than in isolates bearing only msa1 and msa2. We compared two isolates with the two-locus genotype (ATCC 33209T and Marion Forks) with four isolates having the three-locus genotype (MMMvir, 91-127 Idaho, Bonneville-1, and Carson 5b). Broth cultures were harvested as the culture density increased, and total protein was isolated from the culture supernatants and the cell pellets. The MSA levels were assessed by Western blot analysis of equivalent cell amounts from each fraction (Fig. 3). No consistent, detectable differences were observed in any of the fractions at any time during culture growth for any of the isolates, demonstrating that the total in vitro MSA levels were not significantly affected by the presence of msa3.



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  		FIG. 3. Western blot analysis for MSA from six isolates of R. salmoninarum. Bacterial pellets and supernatants from broth cultures were harvested at the times indicated. Total proteins from equivalent numbers of cells were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blots were probed with monoclonal antibody 3H1. Lane 1, ATCC 33209T; lane 2, Marion Forks; lane 3, MMMvir; lane 4, 91-127; lane 5, Bonneville-1; lane 6, Carson5b. Lanes m contained protein size markers (Invitrogen), and the bands corresponding to 68 and 43 kDa are labeled. Blot images were captured with an ImageStation 440CF by using the 1D Image Analysis software (version 3.5.3; Kodak).

In vivo phenotype of isolates with msa3.
Although the level of MSA in vitro was not affected by the presence of msa3, it is possible that the msa3 locus has an in vivo effect. This possibility was tested by challenging juvenile chinook salmon (O. tshawytscha) with isolates bearing msa3 (MMMvir, Bonneville-1, and Carson 5b) or lacking msa3 (type strain ATCC 33209T and Marion Forks). In addition to a mock-challenged group, the following four challenge doses for each isolate were used: 106, 105, 104, and 103 bacteria per fish. Mortality due to R. salmoninarum was confirmed by both bacterial culture and BKD ELISA analysis of the kidney, and no BKD-associated mortality was observed in the mock-challenged fish.

At the highest dose (106 bacteria per fish), there was no statistical difference among the survival curves (P = 0.6470, as determined by the log rank test; df = 3), and the median survival times ranged from 30.0 to 36.0 days (Table 2). For the three lower doses, significantly lower levels of survival for fish challenged with isolates bearing msa3 were observed. The median survival time was reduced by at least 16.5 days at a dose of 105 bacteria per fish, by 22.5 days at a dose of 104 bacteria per fish, and by 25.0 days at a dose of 103 bacteria per fish (Table 2). For these three treatments, the median level of survival was reduced by an average of 34.0% (range, 32.1 to 35.7%) for the fish challenged with isolates bearing msa3 compared to the fish challenged with isolates lacking msa3. The proportions of fish surviving at the end of the experiment were also different for the isolates with and without msa3. Few or no fish challenged with isolates bearing msa3 survived to the end of the experimental period, regardless of the challenge dose. In contrast, substantial proportions of the fish challenged with isolates lacking msa3 survived to the end of the experimental period in the group that received the lowest dose (Table 2). At each of the three lower doses, there was no significant difference among the survival curves for the three msa3-bearing isolates (P ≥ 0.0680, as determined by the log rank test for each comparison; df = 2). However, the survival curves for type strain ATCC 33209T and Marion Forks differed significantly from those for the msa3-bearing isolates for each of the three dose groups (P ≤ 0.0042, as determined by the log rank test for each comparison; df = 4).


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  		TABLE 2. Median survival times and proportions of susceptible fish surviving at the end of the experimental challenges with five isolates of R. salmoninaruma

To assess the genotypes of the recovered bacteria, Southern blot analysis of chromosomal DNA from three to five recovery cultures for each isolate was performed. R. salmoninarum recovered from fish challenged with type strain ATCC 33209T or Marion Forks lacked the msa3 locus, while bacteria recovered from MMMvir, Bonneville-1, and Carson5b possessed msa3 (data not shown). Thus, the msa3 genotypes of the challenge bacteria and the bacteria recovered appeared to be identical.


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DISCUSSION
 
The discovery of another msa locus in R. salmoninarum is an intriguing finding for this salmonid pathogen, because two functional copies of the msa gene have been identified previously. Our characterization indicates that msa3 is within a 5-kb BamHI region that is a copy of the region containing msa1. In the msa1 locus, the BamHI sites are located within inverted, flanking IS994 elements (32). Because all isolates possess msa1 but not all isolates possess msa3, it is likely that msa3 was duplicated from msa1. The mechanism for this kind of duplication in R. salmoninarum is unknown, but the flanking IS994 elements may be involved (33, 34).

The physical context of the msa3 locus (i.e., chromosomal or extrachromosomal) was not determined in this study. The apparent stoichiometry of msa3 to msa1 varied widely among the isolates examined (Fig. 2), indicating that the msa3 copy number may vary. The msa3 locus could be located within an amplified chromosomal region or within mobile DNA, such as a plasmid or a bacteriophage. Chromosomal amplifications can occur spontaneously (34) or can result from selective pressures, such as metabolic demands (22, 30). If MSA is essential for survival, it is possible that amplification producing the msa3 locus may compensate for defects in msa1 or msa2. Because the genotype of the recovered bacteria was the same as that of the starting challenge bacteria, the msa3 locus does not appear to be a result of selective pressure during disease progression. Attempts to isolate bacteriophages, repetitive elements, or pathogenicity islands in R. salmoninarum have not been reported yet, although there has been a single anecdotal report of a bacteriophage associated with R. salmoninarum (16). An effort to isolate plasmid DNA from R. salmoninarum was unsuccessful (39), but this attempt involved type strain ATCC 33209T, which lacks msa3. Analysis of sequences flanking the msa3 locus in the 4.5-kb XhoI fragment resulted in identification of ORFs with significant homology to plasmid partition proteins (our observations), suggesting that in future investigations workers should address the possibility of a plasmid context.

Because msa1 and msa2 are transcribed at a range of cell densities under in vitro conditions (31), it was expected that msa3 would contribute to MSA levels. Although sequence analysis revealed that the promoter region and ORF of msa3 are identical to those of msa1, the total level of MSA protein in cultured cells was not elevated in isolates possessing the msa3 locus. This unexpected result may have been due to posttranscriptional regulation of total MSA levels, which would mask contributions from msa3. The MSA protein can be involved in host immune cell interactions, such as leukocyte adhesion (42, 44), and a role for MSA in pathogenicity has been implicated (7, 24, 37). Close regulation of an important pathogenicity protein could be critical for appropriate function.

While in vitro MSA levels were not affected by the presence of the msa3 locus, a distinct difference in virulence was observed between isolates possessing msa3 and isolates lacking msa3. Mortality occurred more rapidly in fish challenged with isolates possessing the msa3 locus, and nearly all of the fish challenged with msa3-bearing isolates died before the end of the experimental period (Table 2). Notably, no significant difference in survival was observed among all of the isolates at the highest challenge dose (106 cells per fish), indicating that the effect is negligible above a threshold inoculum. If protein encoded by msa3 contributes to increased virulence, MSA expression must be differentially regulated under in vivo conditions. However, it is possible that the increased virulence is associated with sequences flanking the msa3 locus rather than with the msa3 gene itself.

Although the ATCC 33209T and Marion Forks isolates both lacked msa3, ATCC 33209T exhibited lower virulence than the Marion Forks isolate. The relatively lower pathogenicity of ATCC 33209T has been observed previously. This isolate has greatly reduced virulence in chinook and coho salmon (O. tshawytscha and Oncorhynchus kisutch) compared to the virulence of other isolates (38), and it has been reported that ATCC 33209T does not produce BKD symptoms in rainbow trout (21). Furthermore, ATCC 33209T is unable to infect the Epithelioma papillosum cell line, whereas more virulent isolates of R. salmoninarum can invade and multiply in this fish cell line (21). Nonetheless, the ATCC 33209T strain exhibits biochemical and morphological properties similar to those of more virulent isolates (3, 4, 9, 14, 18), including a capsule (10), and it survives within macrophages as well as more virulent strains (2). The Marion Forks strain was isolated in 1994 and has been subjected to few laboratory passages. In contrast, the ATCC 33209T strain was isolated in 1974 and has undergone extensive laboratory culture, which may have resulted in a mutation(s) or gene loss(es) that contributes to its relatively reduced virulence. Southern hybridization with a probe located 3' of the msa3 locus (i.e., flanking the BamHI site in the 4.5-kb XhoI fragment) hybridized to genomic DNA from the Marion Forks strain, but not to genomic DNA from the ATCC 33209T strain (data not shown). While the absence of the msa3 locus is at least partially responsible for the lower virulence of these two isolates, it is likely that there are additional factors not present in ATCC 33209T that are responsible for its lower-virulence phenotype.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge Paul Plesha for the fish culture isolation facilities and Frank Sommers for the chinook salmon. Gregory Wiens was instrumental in initiating this study and provided a critical review of the manuscript. Monoclonal antibody 3H1 was a gift from Gregory Wiens, James Winton, and Ronald Pascho.

A.M.C was supported by CSREES grant 00-35204-9225 from the U.S. Department of Agriculture. National Marine Fisheries Service (National Oceanic and Atmospheric Administration, U.S. Department of Commerce) provided substantial support.

Mention of trade names or commercial products in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Commerce.


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FOOTNOTES
 
* Corresponding author. Mailing address: Northwest Fisheries ScienceCenter, REUT Division, 2725 Montlake Blvd. East, Seattle, WA 98112. Phone: (206) 860-3279. Fax: (206) 860-3467. E-mail: Linda.Rhodes{at}noaa.gov. Back

{dagger} Present address: Peace Corps/Zambia, Kasama, Zambia. Back

{ddagger} Present address: Pacific States Marine Fisheries Commission, Portland, OR 97202. Back


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Applied and Environmental Microbiology, November 2004, p. 6488-6494, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6488-6494.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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