Abstract

Piscine mycobacteriosis is a chronic progressive bacterial disease that leads to systemic infections and often death. This is a disease of virtually all species of fish, and is often caused by Mycobacterium marinum.¹ While the immune responses against aquatic Mycobacterium sp. have been partly characterized,^2,3 an effective vaccine against mycobacteriosis has not been developed. A DNA vaccine was chosen as an appropriate construct for a M. marinum vaccine because of its potential to induce protective Th1/cell-mediated immune responses against this intracellular pathogen.^4,5 In addition, the Mycobacterium sp. 85A antigen (Ag85A) was utilized because it has been shown to induce protective immune responses in mammals.⁴

The hypothesis for this study was as follows: a DNA vaccine encoding the M. marinum Ag85A will induce strong and protective humoral and cell-mediated immune responses. Furthermore, higher vaccination doses and the intramuscular route of administration will stimulate greater immune responses and confer protection against live bacterial challenge with M. marinum. The long-term goal of this project was to create a safe, effective vaccine that would improve the health and immunocompetence of economically important aquarium fish and cultured foodfish.

The vaccine was assembled by cloning the M. marinum Ag85A gene in the pcDNA 3.1 eukaryotic expression vector (Invitrogen, San Diego, CA). The resulting pCMV-85A construct expresses the Ag85A under a cytomegalovirus (CMV) promoter after injection into fish tissue. Hybrid striped bass (Morone saxatilis x M. chrysops), a proven M. marinum-susceptible fish species,⁶ were used as models to test the vaccine. The pCMV-85A construct was injected on Days 0 and 14 at doses of 5, 25 or 50 µg intramuscularly (i.m.) or 25 µg intraperitoneally (i.p.), along with control groups injected with an empty plasmid (pCMV-0) or saline alone. Each group consisted of 80 fish. Every two weeks, blood and tissue samples were obtained to evaluate the humoral and cell-mediated responses by ELISA, cytokine assay, lymphoproliferative assay, lysozyme assay, and macrophage stimulation test. On Day 90, the fish were challenged with 8 x 10⁵ cfu live M. marinum/g body weight fish, a dose known to cause the onset of mortalities in approximately 15 days. Survival patterns and splenic bacterial counts were then evaluated.

All of the pCMV-85A vaccine groups exhibited specific antibody and cellular responses to the Ag85A that increased over time. The group that received the i.p. injections and the 5 µg i.m. injections developed slight short-term responses. The fish that received the 25 and 50 µg i.m. injections exhibited significant long-term responses. However, all groups showed equivocal non-specific immune responses. Through day 35 post-challenge, the groups receiving the 25 and 50 µg i.m. injections showed later onset of mortality, better survival rates, and lower splenic bacterial counts than the other groups. Evaluation of the survival rates indicated approximately 80 percent and 90 percent protection conferred by the 25 and 50 µg i.m. vaccination, respectively. Therefore, this study demonstrated that substantial specific immune responses could be generated with the DNA vaccine and that the vaccine conferred a significant level of protection.

References

1.  Austin B, DA Austin. 1993. Mycobacterium spp. In: Austin, B., and D.A. Austin (eds.). Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. Ellis Horwood, New York, New York, Pp. 45-50.

2.  Bartos JM, CV Sommer. 1981. In vivo cell mediated immune response to M. tuberculosis and M. salmoniphilum in rainbow trout (Salmo gairdneri). Developmental and Comparative Immunology 5:75-83.

3.  Chen SC, T Yoshida, A Adams, KD Thompson, RH Richards. 1996. Immune response of rainbow trout to extracellular products of Mycobacterium spp. Journal of Aquatic Animal Health 8:216-222.

4.  Denis O, A Tanghe K Palfliet F Jurion, TP van den Berg, A Vanonckelen, J Ooms, E Saman, JB Ulmer, J Content, K Huygen. 1998. Vaccination with plasmid DNA encoding mycobacterial antigen 85A stimulates CD4+ and CD8+ T-cell epitopic repertoire broader than that stimulated by Mycobacterium tuberculosis H37Rv infection. Infection and Immunity 66:1527-1533.

5.  Heppell J, HL Davis. 2000. Application of DNA vaccine technology to aquaculture. Advanced Drug Delivery Reviews 43:29-43.

6.  Wolf JC, SA Smith. 1999. Comparative severity of experimentally induced mycobacteriosis in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp. Diseases of Aquatic Organisms 38:191-200.