Introduction

At present, vaccines are not available for commercially raised channel catfish. In cases such as Motile Aeromonad Septicemia (MAS) epizootics, vaccination of catfish could prove to be efficacious since the success of antibiotic therapy is limited. Previous studies have demonstrated that monovalent, whole cell motile aeromonad bacterins confer protection to fish after challenge with a homologous strain but not heterologous strains (Post 1966; Acuigrup, 1980). Even polyvalent whole cell vaccines conferred protection to channel catfish only if one of the vaccine strains was used as the challenge organism (Thune and Plumb, 1982). Since whole cell bacterins do not protect fish against heterologous challenge, investigators have considered using bacterial components or virulence factors as antigens in vaccines. The S-layer protein of motile aeromonads is one such factor that has been correlated with virulence (Dooley and Trust, 1988; Thune, unpublished data) and is a prevalent virulence factor in clinical isolates from MAS epizootics. For these reasons, the S-layer protein was chosen for testing as a potential immunogen in channel catfish.

Characteristics of S-Layers

Houwink (1953) first reported "periodic macromolecular monolayers" as a component of the bacterial cell envelope. Since that time, Houwink's monolayers have been referred to as crystalline surface layers or simply, S-layers. The presence of S-layers as a component of the bacterial cell membrane is not as rare as once suggested. In fact, S-layers have been demonstrated as part of the bacterial cell structure for members of every taxonomic group of walled bacteria including gram negative bacteria, gram positive bacteria, Archaebacteria, Cyanobacteria and Chlamydia (Sleytr and Messner, 1988; Sleytr and Messner, 1983; Smit, 1987). Several fish bacterial fish pathogens are known to possess Slayers. Properties of the S-layer of Aeromonas salmonicida are well documented and recently S-layers have been reported in motile aeromonads as well as Vibrio salmonicida (Dooley and Trust, 1988; Dooley et al., 1989; Hjelmeland et al. 1988).

S-layers are composed of repeating units of protein or glycoprotein that are aligned in hexagonal of tetragonal arrays. These protein arrays compose the outer most layer of the bacterial cell structure. Often in gram negative bacteria, the carbohydrate chains of the lipopolysaccharide molecule will extend through the S-layer and come in contact with the outer environment. For example, A. salmonicida has been reported to have carbohydrate chains exposed at the outer surface of their S-layer as demonstrated by LPS-specific monoclonal antibody labeling (Chart et al., 1984).

Functions of Layers

The functions of S-layers are not well understood although several roles have been suggested. First, the S-layers may function as a physical barrier against infective agents or other foreign material. The S-layer may also serve as a barrier to protect the cell from bacteriocidal components of the infected host serum or secretions. Munn et al. (1982) demonstrated that S-layer positive strains of A. salmonicida were more resistant to complement activity than the S-layer negative strains. S-layer positive strains have also been shown to be more resistant to proteolytic enzymes (Trust et al., 1982). The maintenance of cell shape and structure has been suggested as a possible role of S-layers, however; this function is considered to be of little significance to gram positive and gram negative bacteria since isogeneic strains that are S-layer negative show no difference in morphology compared to the S-layer positive strains (Sleytr and Messner, 1988).

S-layers may play a role in cell adhesion and association with phagocytes. For example, S-layers alone or in conjunction with LPS molecules and/or pili are thought to induce autoaggregation of the bacterial cells indicating a decrease in surface hydrophilicity and enhanced association with phagocytes (Dooley et al., 1986; Jiwa, 1983). This theory is supported by evidence showing that Slayer positive A. salmonicida strains have an enhanced ability to associate with trout macrophages (Trust et al., 1983). Channel catfish neutrophils have also been reported to phagocytize a S-layer positive Aeromonas hydrophila strain more readily than S-layer negative strains (Finco-Kent, 1986).

S-Iayers of Motile Aeromonads

The S-layer of motile aeromonads is composed of -52 kd protein that is arranged tetragonally. Similar to other S-layer positive bacteria, motile aeromonads that possess a S-layer tend to autoaggregate in static cultures and resist bactericidal activity of normal fish serum (Dooley et al., 1986). The role of the S-layer protein in determining virulence of A. salmonicida strains is well documented. In fact the S-layer must be present in a particular strain for that strain to be pathogenic in fish (Kay et al., 1984). The role of the S-layer in the virulence of motile aeromonads is not as well understood, although researchers have reported that the more virulent motile aeromonads are those that are S­layer positive and have been isolated from diseased fish. S-layer negative motile aeromonads and strains isolated from water samples are not as virulent as the S-layer positive strains (Dooley et al., 1986; Thune et al., unpublished data).

Present Study

The objective of this study was to determine if the S-layer protein of motile aeromonads could serve as an immunogen and induce protection in channel catfish challenged with virulent A. hydrophila strains. Channel catfish were immunized with a variety of S-layer preparations with and without Freund's incomplete adjuvant (FICA). A heterologous aeromonad strain was determined by western blot analysis of the lipopolysaccharide antigens. Data indicated that catfish immunized with the crude, acid extracted preparation of the S-layer plus FICA were protected against challenge with either the homologous or heterologous strain of A. hydrophila. Since immunization with the S-layer induces protection in fish challenged with both the homologous and heterologous strains, the Slayer of motile aeromonads may serve as the common antigen needed for vaccine development.

References

1.  Acuigrup. 1980. Trial vaccination of rainbow trout against Aeromonas liquefaciens In: Ahne, W. (ed.), Fish Diseases Third COPRAQ Session. Springer-Verlag, Berlin. p.206-211.

2.  Chart, H., T.W. Pearson and T.J. Trust. 1984. Detection of specific fish antibodies using an inhibition enzyme linked immunosorbent assay (inhibition ELISA). J. Immunol. Methods 68: 19-24.

3.  Dooley, J.S.G. and T.J. Trust. 1988 Surface protein composition of Aeromonas hydrophila virulent for fish: identification of an S-layer protein. J. Bacteriol. 170: 499-506.

4.  Dooley, J.S.G., R. Lallier and T.J. Trust. 1986. Surface antigens of virulent strains of Aeromonas hydrophila. Vet. Immun. Immunopath. 12: 339-344.

5.  Finco-Kent, D. 1986. The in-vivo and in-vitro cellular response of channel catfish, Iccalurus punctatus, to strains of Aeromonas hydrophila. Master's thesis. Louisiana State University. Baton Rouge, LA.

6.  Hjelmeland, K., K. Stensvag, T. Jorgensen and S. Espelid. 1988. Isolation and characterization of a surface layer antigen from Vibrio salmonicida. J. Fish Dis. 11:197- 205.

7.  Houvink A.L. 1953. A macromolecular monolayer in the cell wall of Spirillum spec. Biochem. Biophys. Acta. 10: 360-366.

8.  Jiwa, S.F.H. 1983. Enterotoxigenicity, hemagglutination and cell surface hydrophobicity in Aeromonas hydrophila, A.sobria and A. salmonicida. Vet. Microbiol. 8: 17-34.

9.  Kay, W.W. , B.M. Phipps, E.E. Ishiguro, R.W. Olafson and T.J. Trust. 1984. Surface layer virulence A-proteins from Aeromonas salmonicida strains. J. Bacteriol. 164: 1332-1336.

10. Munn, C.B., E.E. Ishiguro, W.W. Kay and T.J. Trust. 1982. Role of surface components in serum resistance of virulent Aeromonas salmonicida. Infect. Immun. 36: 1069-1075.

11. Post, G. 1966. Response of rainbow trout (Salmo gairdneri) to antigens of Aeromonas hydrophila. J. Fish. Res. Bd. Canada 23: 1487-1494.

12. Sleytr, U.B. and P. Messner. 1988. Crystalline surface layers in procaryotes. J. Bacteriol. 170: 2891-2897.

13. Sleytr, U.B. and P. Messner. 1983. Crystalline surface layers on bacteria. Ann. Rev. Microbiol. 37: 311-339.

14. Smit, J. 1987. "Protein surface layers of bacteria" In: Bacterial Outer Membranes as Model Svstems. M. Inouye (ed. ) John Wiley and Sons, New York pp. 343 - 376.

15. Thune, R.L. and J.A. Plumb. 1982. Effect of delivery method and antigen preparation on the production of antibody against Aeromonas hydrophila in channel catfish. Prog. Fish-Cult. 44: 53-54.

16. Trust, T.J., W.V. Kay and E. E. Ishiguro. 1983. Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida. Curr. Microbiol. 9: 315318.

17. Trust, T.J., W.W. Kay, E.E. Ishiguro, J.T. Buckley and T.W. Pearson. 1982. Properties of a protein, a virulence factor on the surface of Aeromonas salmonicida. Dev. Comp. Immunol. Suppl. 2 pp. 175-180.