1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
Test cytokine antibodies against human, bovine, and porcine cytokines for cross-reactivity with Atlantic bottlenose dolphin tissue samples. A recent publication has shown that certain anti-human, anti-bovine, and anti-porcine cytokine antibodies cross-react with cetacean (Atlantic spotted dolphin, striped dolphins, and fin whales) cryopreserved tissues. We propose to test these antibodies on bottlenose dolphin tissues. This would be a low-risk method that could identify an antibody that could be used as part of an ELISA for measuring bottlenose dolphin cytokines. For each cytokine, a second reagent would still need to be developed as described below, but this would be a good starting point with potential time-saving benefits. Development of additional specific cytokine primers. Utilize sequence information to develop primers for measuring select pro-inflammatory and anti-inflammatory cytokines. While primer sets are already available for specific bottlenose dolphin cytokines, there is a need for a few additional key primer sets. For example, Il-1beta and Il-8 are two key proinflammatory mediators and these cytokines would be useful to monitor cetacean health. New reagent development. Recombinant cytokines would be produced using either a yeast expression or mammalian expression system. 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.
3. Progress Report:
We prepared a cDNA library from bottlenose dolphin splenic tissue total RNA. The dolphin spleen tissue sample was obtained from personnel with the US Navy Marine Mammal Program. The cDNA library was sent to Kingfisher Biotechnology, Inc. Target cytokine genes were then amplified from the cDNA library using the appropriate PCR primer sets. The amplified PCR products were cloned into an expression vector for the yeast Pichia pastoris, sequenced and expressed to produce the cytokine proteins as fusion product. To date, we have expressed and purified bottlenose dolphin interlekin (IL)-1a, tumor necrosis factor (TNF)-a, IL-8, IL-1B, and interferon (IFN)-y. We immunized rabbits with recombinant dolphin IL-8 and produced a rabbit anti-IL-8 polyclonal antibody. Recombinant cytokines were used for western blot analysis. For western blot analyses, cytokines (dolphin IL-8 or bovine IL-4) were diluted in loading buffer. Samples were heated at 95°C for 5 min. Samples and prestained protein ladder were loaded into gel and electrophoresed at 75V. Gels were transferred to nitrocellulose membrane using a blot transfer system. The membrane was incubated in blocking buffer for prior to addition of rabbit anti-dolphin IL-8 followed by HRP-conjugated anti-rabbit. The antibody reacted with dolphin IL-8, but not bovine IL-4. Immunohistochemical (IHC) assays have been performed on bottlenose dolphin frozen tissue sections using samples of dolphin lymph node, spleen or liver obtained as described above. Sections (5 vm) were cut on a cryostat and collected on clean poly-L-lysine coated glass slides and dried at room temperature. Sections were fixed in acetone/PBS for 15 min. Sections were blocked for 30 min with serum-free protein block to reduce non-specific binding. Primary antibodies against bovine (IFN-gamma and IL-10), human (transforming growth factor-beta), swine (IL-6) or dolphin (IL-8) cytokines (tested at several concentrations) were added in a staining chamber and incubated at 4oC overnight. The following day, appropriate secondary antibodies conjugated to fluorescent dyes were added. In some cases, sections were stained with DAPI to visualize the cell nucleus. We have found that anti-bovine IFN-g and IL-10, anti-swine IL-6, and anti-human TGF-g exhibit cross-reactivity with dolphin tissues. In addition, the anti-dolphin IL-8 works for IHC staining of dolphin spleen. An ELISA is in development using recombinant dolphin IL-8 as a standard and using the rabbit anti-IL-8 polyclonal Ab as capture and biotinylated anti-dolphin IL-8 as the detecting Ab.