Julian C. Thornton

An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon (Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCEs) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCEs dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCEs in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.

Molecular cloning of a phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Sequence homologies with lecithin-cholesterol acyltransferase and other lipases

We have determined the nucleotide sequence of a gene encoding Aeromonas hydrophila phospholipid-cholesterol acyltransferase, an enzyme which shares many properties with mammalian lecithin:cholesterol acyltransferase. The derived amino acid sequence of the protein contains two regions which are homologous to the proposed active sites and binding sites of the plasma acyltransferase and to similar sequences in other interfacially acting lipolytic enzymes. The amino terminus is preceded by a typical 18 amino acid signal sequence. The protein, which is released into the culture supernatant by Aeromonas hydrophila, is confined to the periplasm of Escherichia coli.

Surface-disorganized, attenuated mutants of Aeromonas salmonicida as furunculosis live vaccines.

A slow-growing, aminoglycoside-resistant mutant and a rapidly-growing pseudo-revertant were isolated from Aeromonas salmonicida, the causative agent of salmonid furunculosis. These mutants continued to elicit a variety of classical virulence factors associated with A. salmonicida pathogenesis. They differed morphologically from the wild-type and from one another with respect to A-layer organization, membrane antagonist sensitivity and particularly to aerobic metabolism. Both mutants were drastically altered in the architecture of the 2D crystalline surface array (A-layer), although both were similar to wild-type with respect to cell surface composition. The slow-growing, antibiotic-resistant mutant differed significantly from the wild-type by the apparent loss of virtually all aerobic metabolism; the pseudo-revertant had partially recovered the ability to aerobically metabolize certain carbon sources. Both mutants were avirulent and incapable of tissue persistence. The rapidly-growing, antibiotic-sensitive pseudo-revertant, when administered either intraperitoneally or by immersion, effectively protected salmonid fish from challenge by a heterologous virulent stain suggesting its candidature as a live, attenuated furunculosis vaccine.

Structure — Function Aspects of the Aeromonas salmonicida S-Layer

S-layers are important, proteinaceous, supramolecular assemblies common to a large number of bacterial species. When present they can account for up to 20% of the total cellular protein (Messner and Sleytr, 1992). In spite of their ubiquity, for the most part their specific biological functions have resisted elucidation. However, it is intuitively understood that their main roles must either be protective, aggressive, structural or some combination of these.

Does the S-Layer of Aeromonas salmonicida Exist in More Than One Functional Organizational State?

The S-layer of the fish pathogen Aeromonas salmonicida, the causative agent of furunculosis in salmonids, is probably the best characterized S-layer in regards to function. Not only does it constitute an important determinant in pathogenesis (Ishiguro et al., 1981), but it has also been assigned specific virulence functions (reviewed by Kay and Trust, 1991). We have recently demonstrated that the S-layer of A. salmonicida (or A-layer) possesses significant flexibility and plasticity. It normally exists in a single type of lattice capable of adopting different conformations (Garduno and Kay, 1992a), but under calcium depletion it forms two novel structural patterns as a consequence of structural rearrangements within the layer (Garduno et al., 1992). It is the objective of this present work to explore the functional relevance of one of these novel structural patterns, the “big squares” pattern.