Submitted to: Aquaculture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/12/2004
Publication Date: 8/1/2004
Citation: Shelby, R.A., Shoemaker, C.A., Klesius, P.H. 2004. Development of an ELISA to measure the humoral immune response of hybrid striped bass Morone chrysops X M. saxatilis to Streptococcus iniae. Aquaculture Research. 35(10):997-1001. Interpretive Summary: This paper describes a new method by which the immune response of hybrid striped bass to a specific bacterial disease can be measured using an enzyme-linked immunosorbent assay (ELISA). Streptococcus iniae causes significant losses in this important aquaculture species, so the ability to measure immune response to this pathogen can be used to monitor a disease outbreak or conversely, the effectiveness of a vaccination regimen. This test was made possible by the use of a previously made monoclonal antibody, 6E1, which is specific for the heavy chain of the immunoglobulin of hybrid striped bass (HSB). To demonstrate the accuracy of the assay, bass were immunized with killed S. iniae cells, and then challenged with live bacterium. This resulted in 100% mortality in non-vaccinated HSB, while vaccinated and non-challenged fish had 7% and 13% mortality, respectively. Humoral antibody titers were shown by ELISA to increase only after vaccination, and not in the other treatment groups. ELISA titers were also shown to be correlated with antibody titers measured by agglutination of bacterial cells. The target antigen of the bacterium was shown to be polysaccharide in nature by western blotting using immune HSB serum and monoclonal 6E1.
Technical Abstract: Hybrid striped bass Morone chrysops x M. saxatilis were immunized with formalin killed cells of Streptococcus iniae mixed with Freund¿s complete adjuvant (FCA). After boosting with cells mixed with Freund¿s incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non-immunized, immunized, and a non-challenged fish, respectively. Live S. iniae were recovered only from moribund non-immunized challenged fish, indicating that the immunization conferred protection to S. iniae infection. Serum samples were taken at weekly intervals and anti-S. iniae antibody was measured by both agglutination and indirect ELISA. Titers of anti-S. iniae antibody increased only in the immunized group. Sera taken from all surviving fish on the final bleeding at 42 days also showed an increase in serum complement in the immunized group. These data indicate that immunity to infection resulted from elevated anti-S. iniae immunoglobulin as a result of immunization and that the ELISA method for measuring antibodies is a reliable indicator of a protective immune response.