The ideal vaccine is one that is non-toxic, easy to produce, cheap, stable and efficacious. To meet these objectives, over the last two decades the trend in bacterial vaccines for humans is to utilize the polysaccharide complexes of the outer cell wall of the bacteria as immunogens. The complexes may be the capsule, envelope, or portions of the surface structure of the cell Gram negative bacteria have received the most emphasis but recently this has been extended to selected gram positive pathogens.

Bacterial vaccines for marine mammals have been few and only in the experimental stage Extracts of Pasteurella multocida, Carter A, Type 111 for pinnipeds and Pseudomonas pseudomallei for cetaceans have been used with success The extracted complexes included amino sugars with some outer membrane proteins and did not contain KDO lipids, or nucleic acids IgG and IgM were produced in significant levels but no studies were done to determine the degree of hypersensitivity that might result from repeated injections of the protein nor was the specific antibody response to the polysaccharide portion of the immunogen determined.

In this preliminary report a purified polysaccharide was injected into 2 California sea lions (CSL), 2 Northern fur seals (NFS) and 1 Tursiops aduncus The IgG and IgM levels were determined and in one species (CSL) the bactericidal and opsonization activity of the antibodies were determined.

Materials and Methods

Antigen

The antigen used was a Group C Neisseria meningitidis vaccine polysaccharide consisting of polymers of alpha 2-9, N-acetylneuraminic acid (Kd. 0.45) (received through the courtesy of the Institute Merieux, Lyon France). The lyophilized material was reconstituted in pyrogen free sterile saline to a concentration of 500ug per ml. Each animal received one ml injected intramuscularly at times noted in the text.

Serology

Antibodies were measured using the Enzyme Immunoassay (EIA) as previously described.¹ Both IgG and IgM were isolated from the species under test and antisera produced in rabbits (IgG) or guinea pigs (IgM). The tests were run in triplicate with appropriate controls. The positive control consisted of rabbit anti-meningococcus Group C polysaccharide and negative controls were from animals non-immunized with the polysaccharide. The O.D values are corrected for background and in one experiment the dilution of serum that dropped to background was used.

Bactericidal and Opsonization Tests

The bactericidal activity of the serum of two CSL were determined by the method of Gold and Wyle.² Opsonization and intracellular killing were conducted by a modification of the procedure of van Furth and van Swet.³ Briefly, 1.0ml of bacterial dilution was added to a suspension of leukocytes isolated by Ficoll-Hypaque and after incubation at 15 or 120 min. (37C), the cells were washed 3X and 3.0ml of RPMI, 0.5ml of heat-inactivated serum, and 0.5ml of guinea pig complement were added to plastic tubes. An E coli filtrate was used to activate the complement. After washing the leukocytes and associated bacteria were incubated at 0 and 60min without rotation (37C) Aliquots were removed, lysed with distilled water containing 0.01% bovine albumin (lysing confirmed by phase microscopy) and viable bacteria determined in triplicate on Mueller-Hinton Blood Agar plates. The Leukocyte-Attachment-Engulfment Index was determined by subtracting the engulfed bacteria in RPMI buffer at time zero from the engulfed bacteria in immune serum at time zero (CFU/ml).

Results

Antibody Response

The IgG and IgM response of the Tursiops aduncus (J4) and one of the Northern fur seals are shown in Figures 1 and 2 respectively. The cetacean became pregnant during the trials and only one booster and three post-booster bleedings were possible. Both the IgG and IgM levels rose only slightly during the first 90 days following a single injection. There was a drop in titer 7 days post-booster but then a rise until the last bleeding at 30 days. A similar pattern was seen in the NFS after a single booster at 90 days and a second booster at 180 days. The IgG level remained stable post-booster but the IgM dropped by 30 days post-single booster or a second booster. In the CSL (one booster at 1 83days) the titers rose to 1:2000 by the time of the booster, dropped to 1:400, and then rose to 1:4000 at day 30 post-booster (data not shown).

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	Figure 1. Antibody response in Tursiops aduncus following immunization with Group C meningococcal polysaccharide.
	Figure 2. Antibody response in NFS following immunization with Group C meningococcal polysaccharide.
	Figure 3. Bactericidal activity against Neisseria meningitidis of California sea lion sera following immunization with Group C meningococcal polysaccharide.
	Figure 4. Opsonization and killing of Neisseria meningitidis in California sea lion sera following immunization with Group C meningococcal polysaccharide.

Bactericidal Response

As shown in Figure 3 the bactericidal response against the meningococcus was significant in the 14 day post-booster serum of one of the CSL. This bactericidal activity was specific since adsorption with the homologous meningococcus removed most of the bactericidal activity.

Opsonization

The ability of one of the CSL sera at 15 and 28 days post-booster to opsonize and kill the meningococci intracellularly is shown in Figure 4. After 15 or 120 minutes the bacteria were efficiently opsonize.

Discussion

Bacterial polysaccharide immunogens have the advantage of being more resistant to degradation in the tissues and being located on the bacterial cell that is accessible to antibodies. Their disadvantages are that they are often poor immunogens and have a poor immunologic memory induction. The bacterial polysaccharide used in this study does not induce antibodies in children <2yrs of age but are very good immunogens in adults.

In this study the observation that the meningococcal polysaccharide produced both IgG and IgM (although at low levels) is unique since this immunogen has been shown to not produce antibodies in any animal except humans and selected species of mice. In CSL the polysaccharide induced effective cidal and opsonic antibodies. The area of comparative immunology, the dosage of polysaccharide (in humans it is 50ug/adult), and the efficacy of a polysaccharide- protein complex for protection against gram negative bacteremias in marine mammals are under study and will be reported at a later date.

References

1.  Suer, L., N. Vedros, J.P. Schroeder, and L. Dunn. 1988. Erysipelothrix rhusiopathiae. Enzyme immunoassay of sera from wild and captive marine mammals. Dis. Aquat. Org. 5: 7-13.

2.  Gold, R. and F.A. Wyle. 1970. New classification of Neisseria meningitidis by means of bactericidal reactions. Infect. Immun. 1: 479-484.

3.  Van Furth, R. and T.L. van Zwet. 1973. In: Handbook of Experimental Immunology. D.Weir, ed. pp 36.1-36.24. Blackwell Scientific, Oxford.