BackgroundStudies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cross-reactive terrestrial-specific antibodies in order to assess the changes in the immune cell populations of dolphins under human care and free-ranging dolphins. The blood and PBMC fraction of blood samples from human care and free-ranging dolphins were characterized by H&E staining of cytospin slides and flow cytometry using a panel of terrestrial-specific antibodies.ResultsIn this study, we show that out of 65 terrestrial-specific antibodies tested, 11 were cross-reactive and identified dolphin immune cell populations within their peripheral blood. Using these antibodies, we found significant differences in the absolute number of cells expressing specific markers within their lymphocyte and monocyte fractions. Interestingly, the peripheral blood mononuclear cell profile of free-ranging dolphins retained an additional population of cells that divided them into two groups showing a low (<27%) or high (>56%) percentage of smaller cells resembling granulocytes.ConclusionsWe found that the cross-reactive antibodies not only identified specific changes in the immune cells of free-ranging dolphins, but also opened the possibility to investigate the causal relationship between immunosuppression and mortality seen in free-ranging dolphins.

Marine mammals are ideal sentinel species for human health due to exposure to the same oceans and consumption of the same foods. There have been many studies which demonstrate that wild Atlantic Bottlenose Dolphins are exposed to high levels of contaminants which lead to a suppressed immune system and are therefore more susceptible to opportunistic infections, many of which are zoonotic diseases. However, nearly no research has been done on determining defects in the immune cell population of dolphins, especially DCs which are essential for initiating an immune response. We hypothesize phenotypic and functional differences in the PBMC, including DC precursors, of wild dolphins as compared to managed dolphins. Specifically in this study, we have used terrestrial-specific antibodies and growth factors to characterize immune cells in PBMC and to generate monocyte-derived DCs. We have identified cross-reactive terrestrial antibodies that could detect immune cell subsets within PBMC, including B cells, T cells, NK cells, monocytes and APCs. Interestingly, using these antibodies we found significant changes in immune cell subsets within PBMC of wild and managed dolphins. Finally among the terrestrial DC growth factors tested we found rat GM-CSF and IL-4 generated DCs expressing higher levels of CD11c, CD14, CD40, CD80, CD86, MHC I and MHC II. Our findings allow us to further study defects in the immune cells, especially DCs, in response to environmental contaminants.

In response to vaccines, DCs do not act in isolation but potentiate their efficiency by interacting with NK cells. The recognition of PAMPs by TLRs triggers DC maturation which is essential for their migration and T-cell priming. Maturing DCs release IL-12 which regulates the function of NK cells and drives the Th1 cells differentiation. In turn, NK cells which are activated by IL-12 and PAMPs through their own TLRs, provide IFN-γ necessary for enhancing stable IL-12 production by DCs and maintaining Th1 cell polarization. Studies using Balb/c strain showed that TLR agonist R848 induces NK cell recruitment into lymph nodes and augments Th1 polarization through unknown mechanisms. In this study we tested whether the genetic background will influence the NK cell recruitment and subsequent Th1 polarization in response to vaccine formulated with a panel of TLR agonists and alhydrogel. We found that immunization with OVA protein mixed with TLR agonist R848 among adjuvants tested, increased up to 3-fold recruitment of IFN-γ producing CD27+CD11b+ NK cell expressing CXCR3 and CD62L in lymph nodes and spleen of Balb/c but to a much lesser extent in B6 mice. Interestingly, however, the increased in NK cell recruitment had limited impact on Th1 polarization in Balb/c as compared to Th1-prone B6 mice. These data suggest that for optimal Th1 polarization, adjuvant or adjuvant combination should be selected based on their ability to both recruit and maximize DC-NK bidirectional signaling.