Comparison of Lipopolysaccharide and Protein Profiles Between

Journal of Fish Diseases 2006, 29, 657–663

Comparison of lipopolysaccharide and protein proﬁles between Flavobacterium columnare strains from different genomovars

Y Zhang1, C R Arias1, C A Shoemaker2 and P H Klesius2

1 Department of Fisheries and Allied Aquacultures, Auburn University, Auburn, AL, USA 2 Aquatic Animal Health Research Laboratory, USDA, Agricultural Research Service, Auburn, AL, USA

Abstract Introduction Lipopolysaccharide (LPS) and total protein profiles Flavobacterium columnare is the causal agent of from four Flavobacterium columnare isolates were columnaris disease, one of the most important compar. These strains belonged to genetically dif- bacterial diseases of freshwater fish. This bacterium ferent groups and/or presented distinct virulence is distributed world wide in aquatic environments, properties. Flavobacterium columnare isolates ALG- affecting wild and cultured fish as well as orna- 00-530 and ARS-1 are highly virulent strains that mental fish (Austin & Austin 1999). Flavobacter- belong to different genomovars while F. columnare ium columnare is considered the second most FC-RR is an attenuated mutant used as a live vac- important bacterial pathogen in commercial cul- cine against F. columnare. Strain ALG-03-063 is tured channel catfish, Ictalurus punctatus (Rafin- included in the same genomovar group as FC-RR esque), in the southeastern USA, second only to and presents a similar genomic fingerprint. Elec- Edwardsiella ictaluri (Wagner, Wise, Khoo & trophoresis of LPS showed qualitative differences Terhune 2002). Direct losses due to F. columnare among the four strains. Further analysis of LPS by are estimated in excess of millions of dollars per immunoblotting revealed that the avirulent mutant year. Mortality rates of catfish populations in lacks the higher molecular bands in the LPS. Total ponds can reach 50–60% and can be as high as protein analysis displayed by immunoblotting 90% in tank-held catfish fingerlings (USDA showed differences between the strains analysed 2003a,b). although common bands were present in all the Columnaris disease usually begins as an external isolates. FC-RR lacked two distinct common bands infection of fins, body surface or gills. The fins (34 and 33 kDa) shared by the other three isolates. become necrotic with greyish to white margins, Based on the difference of LPS and total protein and initial skin lesions appear as discrete bluish profiles, it is possible to discriminate the attenuated areas that evolve into depigmented necrotic mutant FC-RR from other F. columnare strains. lesions. Skin lesions may have yellowish mucoid material accompanied by mild inflammation. Keywords: Flavobacterium columnare, genomovars, Lesions can develop exclusively on the gills, which lipopolysaccharide, modified live vaccine, virulence, usually results in subacute disease and mortality, whole protein profiles. as is typically the case in young fish (Plumb 1999). Due to the ubiquitous presence of F. columnare in aquatic environments, eradication of the disease in fish farms is not likely to occur. Control and treatment of columnaris have primarily been direc- Correspondence C R Arias, 203 Swingle Hall, Auburn University, Auburn, AL 36849, USA ted towards the use of improved water-management (e-mail: [email protected]) practices to reduce physiological and environmental

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stress in the fish. Recently, a modified live F. col- from diseased channel catfish at the Alabama Fish umnare vaccine has been developed using a rif- Farming Center, Greensboro, AL. The ARS-1 ampicin-resistant strategy (Shoemaker, Klesius & isolate was recovered from diseased channel catfish Evans 2005b). This methodology was previously at the Aquatic Animal Health Research Unit, used by Montaraz & Winter (1986) to generate a USDA-ARS, Auburn, AL. These two isolates have rough Brucella abortus strain, currently employed as been demonstrated to be virulent for channel catfish the official vaccine for cattle brucellosis in the USA. (Shoemaker, Klesius, Lim & Yildirim 2003a; The same strategy was used by Klesius & Shoe- Welker, Shoemaker, Arias & Klesius 2005). maker (1999) to create an Edwardsiella ictaluri Flavobacterium columnare ALG-03-063 was also rifampicin-resistant mutant patented as a modified included because it was isolated from diseased live vaccine against enteric septicaemia of catfish channel catfish and was the genetically most similar (ESC) (AQUAVAC-ESC; Intervet, Millsboro, strain to the avirulent mutant FC-RR present in our DE, USA). Characterization of B. abortus and collection. E. ictaluri rifampicin-resistant mutants revealed that lipopolysaccharide (LPS), a main virulence Genetic fingerprinting factor for Gram-negative bacteria, lacked the high molecular bands observed in virulent isolates (Kle- A previous study by Arias, Welker, Shoemaker, sius & Shoemaker 1999; Vemulapalli, McQuiston, Abernathy & Klesius (2004) divided F. columnare Schurig, Sriranganathan, Halling & Boyle 1999; strains into four well-defined genetic groups. All Arias, Shoemaker, Evans & Klesius 2003). catfish isolates were grouped into three different Flavobacterium columnare belongs to the Cyto- genogroups (I–III; genogroup IV included only phaga–Flavobacterium–Bacteroid group and is tilapia isolates). Strains ALG-00-530 and ARS-1 phylogenetically distant from the better studied belong to genogroups I and II, respectively; subclass Gammaproteobacteria, which contains while ALG-03-063 belongs to genogroup III. major human and animal pathogens (including The avirulent mutant FC-RR was not included Brucella and Edwardsiella). To date, virulence factors in that study but was fingerprinted in the present in F. columnare are poorly characterized. Specifically, work. Amplified fragment length polymorphism the role of LPS in columnaris pathogenicity has not (AFLP) patterns from the FC-RR mutant were been explored. The main objective of this study was obtained as described by Arias et al. (2004), added to investigate whether an attenuated F. columnare to the existing database and analysed. mutant originated through a rifampicin-resistance strategy presented a modified LPS and a different Production of polyclonal antiserum against total protein profile by comparison with virulent Flavobacterium columnare strains. Twelve channel catfish were randomly divided into three groups of four fish each. Two groups Materials and methods were used to produce antibodies (Ab) against the ALG-00-530 and FC-RR F. columnare strains Bacterial isolates and culturing (Ab-ALG-00-530 and Ab-FC-RR) while the third Four F. columnare strains, ALG-00-530, FC-RR, group served as a negative control. Antigen ARS-1 and ALG-03-063 were used in this study. preparation was performed according to Arias The FC-RR strain is a rifampicin-resistant mutant, et al. (2003) with the following modifications: avirulent for catfish (Shoemaker et al. 2005b). This cells were grown overnight in modified Shieh attenuated mutant has been licensed as a modified broth and control fish were also inoculated with live vaccine by Intervet, Inc. and it is now Shieh broth. Three weeks after booster immuniza- undergoing field trials (Patent no.: US tion, fish were bled and antiserum was used to 6 881 412B1). Unfortunately, the original parent probe Western blots (see below). In order to strain used to derive the avirulent mutant could not determine the relative titre of the sera, enzyme- be included in this work as it lost viability during linked immunosorbent assays (ELISA) were con- storage. Instead, other comparable (same geograph- ducted according to Shoemaker, Shelby & Klesius ical origin and source) virulent F. columnare strains (2003b). Serum from each individual fish was were analysed. Isolate ALG-00-530 was obtained tested against each antigen dilution.

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After blotting, one membrane was incubated with Lipopolysaccharides and total protein extraction anti-ALG-00-530 serum while the second mem- The four isolates were cultured in modified Shieh brane was incubated with anti-FC-RR serum. After broth for 24 h at 28 C. Three millilitres of broth blocking for 30 min, 1:500 diluted polyclonal was centrifuged at 3000 g for 15 min. Pelleted cells catfish serum was incubated with the membrane were resuspended in lysis buffer and proteins were overnight followed by 1-h incubations with a extracted according to Arias, Verdonck, Swings, monoclonal antibody (E-8) specific for channel Aznar & Garay (1997a). Crude LPS was extracted catfish IgM (1:10) (Klesius 1990), and labelled with following the phenol–water protocol described by conjugated goat antimouse Ig (1:5000), followed by Westphal & Jann (1965). After lyophilization, the detection with Opti-4CNTM (Bio-Rad). LPS extract was diluted in sample buffer at a final ) concentration of 1 mg mL 1. Aqueous and phenol LPS phases were stored at )80 C until use. Results Amplified fragment length polymorphism analysis Lipopolysaccharides and total protein analysis Figure 1 shows the results of the cluster analysis of Electrophoresis the AFLP patterns from the strains used in this Lipopolysaccharides from both phases were resus- study. Attenuated mutant FC-RR displayed a pended in sample treatment buffer and electro- unique AFLP fingerprint although it clustered phoresed by discontinuous sodium dodecyl among other genogroup III strains. The AFLP sulphate-polyacrylamide gel electrophoresis (SDS- profile of strain ALG-03-063 presented the highest PAGE) on a 4% stacking gel and a 20% separating per cent of similarity with that of the attenuated gel (Laemmli 1970). Gels were run at 15 mAmp for mutant. Based on this result, ALG-03-063 was 90 min. Gels were silver stained following the selected for further comparisons as the most manufacturer’s instructions for the Bio-Rad Silver genetically similar strain to FC-RR present in our Stain kit (Bio-Rad, Hercules, CA, USA). Total collection (that currently includes more than 150 protein electrophoresis followed the procedure F. columnare isolates; data not shown). described by Arias, Verdonck, Swings, Garay & Aznar (1997b). Protein concentration was measured ELISA analysis using the Quick Start Bradford protein assay (Bio- Rad) and approximately 15 lg of total proteins was All fish immunized with F. columnare yielded loaded per lane. Coomassie staining was used to positive titres. Control fish presented a very low visualize the protein bands following standard cross-reactivity with the sonicated cells. All immun- methods (Sambrook, Fritsch & Maniatis 1989). ized fish produced strong immunity against both antigens. Although antisera from immunized fish reacted with both ALG-00-530 and FC-RR anti- Western blotting gens, the corresponding antigen provided the Protein and LPS samples were analysed in dupli- highest titre (data not shown). Individual fish sera cate. First, samples were resolved on 12% SDS- showing the highest titre (‡1/800) were chosen for PAGE gel and then transferred to polyvinylidene immunoblotting analysis (fish no. 3 anti-ALG-00- difluoride membrane (Bio-Rad) at 100 V for 1 h. 530 and fish no. 1 anti-FC-RR, respectively).

% similarity 95 100 90 80 85 75

FC-RR Genogroup III AlG-03-063 ARS-1 Genogroup II AlG-00-530 Genogroup I

Figure 1 Dendrogram based on ampliﬁed fragment length polymorphism (AFLP) patterns of four strains of Flavobacterium columnare (ALG-00-530, FC-RR, ARS-1 and ALG-03-063). The tree was derived by UPGMA cluster analysis of the AFLP proﬁles.

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Lipopolysaccharide characterization (a) 1 2 3 4 5 6 7 8 9 The results of SDS-PAGE of LPS from F. column- 35.6 kD are are shown in Fig. 2. The characteristic ladder- 21 kD like pattern typical of Gram-negative pathogens was not observed. Only a few bands ranging from 3.5 to 6.9 kD

17 kDa were present in all four strains. Phenol- (b) 1 2 3 4 5 6 7 8 9 phase extracts contained higher amounts of LPS 35.6 kD than the aqueous-extracted samples. Strain ALG- 00-530 showed a few bands in the phenol-phase 21 kD extract while only one weak band was visualized in 6.9 kD the aqueous phase. A similar pattern was observed with strain ARS-1 although band intensities were Figure 3 Immunoblot using anti-ALG-00-530 serum (a) and stronger. Attenuated mutant FC-RR shared a anti-FC-RR serum (b) with lipopolysaccharide (LPS) prepar- similar LPS pattern with ALG-03-063 although ation. Lane 1, molecular standard; lanes 2–5, LPS aqueous phase FC-RR bands were slightly smaller. from: ALG-00-530 (2), FC-RR (3), ARS-1 (4) and ALG-03-063 (5); lanes 6–9, LPS phenol phase from ALG-00-530 (6), FC-RR Bands from F. columnare LPS were visualized (7), ARS-1 (8) and ALG-03-063 (9). much more effectively in immunoblots. When catfish polyclonal antiserum was used for immunoblotting, important differences between the four Total protein analysis strains were revealed (Fig. 3). While all strains Western blots of total proteins were also analysed isolated from diseased channel catfish (ALG-00- with both sera (Fig. 4). The immunoblots indicated 530, ARS-1 and ALG-03-063) presented high that the four strains did contain different antigenic molecular bands (above 21 kDa) in both phenol proteins but also shared some common bands. and aqueous phases, the FC-RR mutant exhibited ARS-1 showed more distinct antigenic protein bands below that range. ALG-00-530 and ARS-1 bands than any other strain analysed (Fig. 4). again showed a similar LPS pattern regardless of Strains ALG-00-530, ARS-1 and ALG-03-063 antisera or extraction phase used. ALG-03-063 contained two very distinct bands around 35 kDa. presented the same size bands in the phenol phase The FC-RR mutant lacked these bands but instead as FC-RR but also exhibited at least one higher presented two additional bands in the 30 kDa molecular weight band that was absent in the range. Each strain had a very similar banding pattern mutant. Interestingly, these results were consistent with anti-ALG-00-530 and anti-FC-RR sera. When regardless of antisera used (anti-ALG-00-530 or ALG-530 serum was used, reactivity against low anti-FC-RR). However, significant differences molecular weight bands present in the three clinical between aqueous and phenol phase samples were strains was observed. FC-RR low molecular bands found in both immunoblots. did not react with anti-ALG-00-530 serum. On the contrary, when anti-FC-RR was used, low molecular weight proteins in FC-RR were revealed.

Discussion 192 3 4 5 6 7 8 Lipopolysaccharide is a major suprastructure of 29 kD 21 kD Gram-negative bacteria which contributes greatly to 6.9 kD the structural integrity of the bacteria, and protects them from host immune defences. The LPS has been described and well characterized as a virulence Figure 2 Silver stained sodium dodecyl sulphate-polyacrylamide factor in many bacterial species, e.g. Salmonella sp. gel electrophoresis proﬁles of an lipopolysaccharide (LPS) (Makela, Valtonen & Valtonen 1973), Escherichia preparation from Flavobacterium columnare strains used in this coli (Medearis, Camitta & Heath 1968), Shigella study. Lane 1, molecular standard; lanes 2–5, LPS aqueous phase from: ALG-00-530 (2), FC-RR (3), ARS-1 (4) and ALG-03-063 ﬂexneri (Rajakumar, Jost, Sasakawa, Okada, Yoshi- (5); lanes 6–9, LPS phenol phase from ALG-00-530 (6), FC-RR kawa & Adler 1994), Brucella abortus (Vemulapalli, (7), ARS-1 (8) and ALG-03-063 (9). He, Buccolo, Boyle, Sriranganathan & Schurig

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(a) (b) 1 2 3 4 5 1 2 3 4 5

210 kD 210 kD

125 kD 125 kD

101 kD 101 kD

56 kD 56 kD

35.6 kD 35.6 kD

21 kD 21 kD

6.9 kD 6.9 kD

Figure 4 Western blot analysis of total protein with anti-ALG-00-530 serum (a) and anti-FC-RR serum (b). Lane 1, molecular standard; lane 2, ALG-00-530; lane 3, FC-RR; lane 4, ARS-1; lane 5, ALG-03-063.

2000) and including fish pathogens such as Vibrio 29 kDa while FC-RR exhibited a unique band vulnificus (Amaro, Fouz, Biosca, Marco-Noales & under 21 kDa. Our results suggest that the induc- Collado 1997), E. ictaluri (Arias et al. 2003) and tion of rifampicin-resistant mutation(s) in F. col- F. psychrophilum (MacLean, Vinogradov, Crump, umnare results in loss of the high molecular weight Perry & Kay 2001). Attempts to characterize the bands displayed by virulent isolates. Similar results structure of the LPS in F. columnare were made by were reported in B. abortus and E. ictaluri (Arias MacLean, Perry, Crump & Kay (2003) and by et al. 2003) when LPS from rifampicin-resistant Vinogradov, Perry & Kay (2003). Unfortunately, mutants was analysed. Rifampicin is a potent and the strain used by these authors (ATCC 43622) had broad-spectrum antibiotic well known as a DNA- been originally misidentified as F. columnare but directed RNA polymerase inhibitor. Mutations in actually belongs to the species Flavobacterium the RNA polymerase gene that conferred resistance johnsoniae (Darwish, Ismaiel, Newton & Tang against rifampicin have been widely documented 2004; Shoemaker, Arias, Klesius & Welker 2005a). (Jin & Gross 1988). On the other hand, the Therefore, no information on F. columnare LPS mechanism by which rifampicin induces mutations composition or immunogenic role was available. that affect LPS structure are relatively poorly In this study, catfish polyclonal antisera against known. It is remarkable how such different two isolates, ALG-00-530 and FC-RR (attenuated pathogens (Brucella, Edwardsiella and Flavobacter- mutant) were generated and used to determine LPS ium) behave similarly under the same stressor immunogenic properties between genetically differ- suggesting that short O-chain LPS will favour cell ent F. columnare strains. Differences in LPS survival. It has been postulated (Kirschbaum & between four isolates of F. columnare were evident Gotte 1993) that rifampicin, like other hydropho- after electrophoresis and silver staining, but were bic molecules, enters the cell via simple diffusion even more obvious after Western blot analysis. through the outer membrane with LPS being the Although a typical LPS ladder has been reported in main obstacle. A structural change in the LPS might other Flavobacterium species (MacLean et al. 2001), enhance the barrier effect protecting the cell against our silver staining of F. columnare LPS showed only rifampicin. Vemulapalli et al. (1999) suggested that a few bands. multiple genes involved in LPS biosynthesis are Immunogenic bands present in the LPS were disrupted in B. abortus and this results in the revealed by immunoblots. However, there were attenuated phenotype. differences in band molecular weights between Our results indicate that the LPS in F. columnare strains isolated from diseased channel catfish and may play an important role in columnaris patho- the attenuated strain FC-RR. The three isolates genesis as a lack of LPS high molecular weight from diseased fish (ALG-00-530, ARS-1 and ALG- bands seems to be correlated with total lack of 03-063) presented LPS bands between 21 and virulence in other bacterial species (Kimura &

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Hansen 1986; Rajakumar et al. 1994; Amaro et al. antisera. Thanks also to Dr Richard Shelby (Aqua- 1997; Arias et al. 2003). Although a thorough tic Animal Health Research Lab ARS/USDA), and antigenic characterization in F. columnare has not Dr Oscar Olivares-Fuster (Department of Fisheries yet been attempted, some serological diversity has and Allied Aquacultures, Auburn University) for been reported between isolates (Anacker & Ordal critical reading of the manuscript. This research was 1959; Sanders, Holt & Fryer 1976). This antigenic funded by the USDA/ARS-Auburn University variation can be justified by the genetic variability Specific Collaborative Agreement ÔFish Health: observed in this species (Arias et al. 2003; Thomas- Bacterial Genomics Research for Vaccine Develop- Jinu & Goodwin 2004). In fact, we observed some mentÕ no. 6420-32000-019-03S. serological differences in LPS within the virulent strains. While ALG-00-530 and ARS-1 both References showed a very similar pattern denoting common antigens, ALG-03-063 presented at least one Amaro C., Fouz B., Biosca E.G., Marco-Noales E. & Collado R. additional LPS band regardless of extraction phase (1997) The lipopolysaccharide O side chain of Vibrio vulnificus Serogroup E is a virulence determinant for eels. Infection and antisera used. Interestingly, these three strains and Immunity 65, 2475–2479. belong to different genogroups and present very Anacker R.L. & Ordal E.J. (1959) Studies on the myxobacter- distinct fingerprinting profiles; however, ALG-00- ium Chondrococcus columnaris I. Serological typing. Journal of 530 and ARS-1 showed very similar results with Bacteriology 78, 25–32. LPS immunoblots. These two strains have been Arias C.R., Verdonck L., Swings J., Aznar R. & Garay E. used by our group in virulence studies and although (1997a) A polyphasic approach to study the intraspecific both are able to cause columnaris disease in catfish diversity amongst Vibrio vulnificus isolates. Systematic and by immersion, ALG-00-530 has consistently exhib- Applied Microbiology 20, 622–633. ited a higher mortality rate than ARS-1 (C. D. Arias C.R., Verdonck L., Swings J., Garay E. & Aznar R. Shoemaker, unpublished data). (1997b) Intraspecific differentiation of Vibrio vulnificus biotypes by amplified fragment length polymorphism and When we compared the antigenic variability ribotyping. Applied and Environmental Microbiology 63, exhibited by the total protein profiles, all four 2600–2606. strains showed a different pattern. Differences in Arias C.R., Shoemaker C.A., Evans J.J. & Klesius P.H. (2003) A protein composition between isolates were expected comparative study of Edwardsiella ictaluri parent (EILO) and as they presented differences in their genome. E. ictaluri rifampicin-mutant (RE-33) isolates using lipopoly- However, there was a large set of common bands saccharides, outer membrane proteins, fatty acids, Biolog, API 20E and genomic analyses. Journal of Fish Diseases 26, 415–421. shared by all strains and revealed by both antisera. This indicates that the FC-RR mutant presents a Arias C.R., Welker T.L., Shoemaker C.A., Abernathy J.W. & Klesius P.H. (2004) Genetic fingerprinting of Flavobacterium number of common epitopes with other F. column- columnare isolates from cultured fish. Journal of Applied Mi- are isolates and could explain why this strain used as crobiology 97, 421–428. a modified live vaccine is able to confer protection Austin B. & Austin D.A. (1999) Bacterial Fish Pathogens: Diseases against different F. columnare isolates. of Farmed and Wild Fish. Springer, New York. In conclusion, we have shown that the F. col- Darwish A.M., Ismaiel A.A., Newton J.C. & Tang J. (2004) umnare FC-RR isolate lacks the high molecular Identification of Flavobacterium columnare by a species-specific weight components of LPS present in other virulent polymerase chain reaction and renaming of ATCC 43622 isolates. Differences in LPS and immunogenic strain to Flavobacterium johnsoniae. Molecular and Cell Probes 18, 421–427. proteins were observed between genetically different Jin D.J. & Gross C.A. (1988) Mapping and sequencing of F. columnare isolates. A secondary outcome of this mutations in the Escherichia coli rpoB gene that lead to study was the genetic fingerprinting of FC-RR. The rifampicin resistance. Journal of Molecular Biology 202, 45–58. attenuated mutant presents a unique AFLP profile, Kimura A. & Hansen E.J. 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Cold Spring Harbor Laborat- Journal of Aquatic Animal Health 14, 263–272. ory Press, Cold Spring Harbor, NY. Welker T.L., Shoemaker C.A., Arias C.R. & Klesius P.H. (2005) Sanders J.E., Holt R.A. & Fryer J.L. (1976) Serological Transmission and detection of Flavobacterium columnare comparison of Flexibacter columnaris isolates using rabbit in channel catfish Ictalurus punctatus. Diseases of Aquatic and rainbow trout (Salmo gairdneri) antisera. Journal of the Organisms 63, 129–138. Fisheries Research Board of Canada 33, 1386–1388. Westphal O. & Jann K. (1965) Bacterial lipopolysaccharide Shoemaker C.A., Klesius P.H., Lim C. & Yildirim M. (2003a) extraction with phenol-water and further application of Feed deprivation of channel catfish, Ictalurus punctatus the procedure. Methods in Carbohydrate Chemistry 5, 83–96. (Rafinesque), influences organosomatic indices, chemical Received: 20 March 2006 composition and susceptibility to Flavobacterium columnare. Revision received: 3 July 2006 Journal of Fish Diseases 26, 553–561. Accepted: 26 July 2006 Shoemaker C.A., Shelby R. & Klesius P.H. (2003b) Develop- ment of an indirect ELISA to detect humoral response to

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