Project Number: 3625-32000-106-13
Project Type: Specific Cooperative Agreement

Start Date: Jun 15, 2012
End Date: Nov 30, 2012

Objective:
We propose to develop cytokine assays for Atlantic bottlenose dolphins. This will require expression of recombinant proteins to be used in producing antibodies specific for each cytokine and for use as standards in enzyme-linked immunosorbent assay (ELISA) development. We will then utilize these reagents to compare the cyoktine responses of dolphins to respiratory viruses similar to those isolated from cattle with bovine respiratory disease complex.

Approach:
To begin with, proinflammatory molecules (IL-1beta, IL-8, TNF-alpha, IL-12), and type I (IFN-gamma), as well as type II (e.g., IL-10, IL-13) cytokine reagents will be proposed for development. The reasons for this selected set is as follows. As the name implies, pro-inflammatory cytokines play a critical role in mediating inflammatory processes. Type I cytokines are critical for cell-mediated immune responses to intracellular pathogens. Type II responses facilitate the production of specific classes of antibody. These cytokines would be expressed as recombinant proteins as follows: Mature proteins would be expressed using an expression vector with its own signal sequence and transfected into yeast (Pichia pastoris). Expressed proteins will be purified by high performance liquid chromatography (HPLC). A collaborator has expertise with this system. Bioassays will be used for testing the bioactivity of recombinant cytokines. Several of these assays have been successfully used for measuring bioactivity of cytokines from a wide variety of species (e.g., cattle, swine, poultry, and fish). Once bioactivity of each cytokine has been confirmed, polyclonal antibodies (pAb) to select recombinant cytokines will be produced in rabbits. Monoclonal antibodies (mAb) to specific cytokines (e.g., IFN-gamma and IL-13) would be produced in mice. Development of ELISA assays. ELISAs will be developed using two pAb, two mAb, or a mAb and a pAb along with the appropriate recombinant cytokine. Standard curves will be used to quantify the levels of cytokines in blood or in cultures of cetacean leukocytes. Development of ELISAs will allow for the evaluation of functional changes in the T. truncatus immune system in response to environmental insults, infectious diseases, and/or vaccination. This will be of substantial benefit to monitoring bottlenose dolphin health.