Date Published: June 29, 2017
Publisher: Public Library of Science
Author(s): Brian S. Dugovich, Melanie J. Peel, Amy L. Palmer, Ryszard A. Zielke, Aleksandra E. Sikora, Brianna R. Beechler, Anna E. Jolles, Clinton W. Epps, Brian P. Dolan, Sanjay B Jadhao.
Ecoimmunology is a burgeoning field of ecology which studies immune responses in wildlife by utilizing general immune assays such as the detection of natural antibody. Unlike adaptive antibodies, natural antibodies are important in innate immune responses and often recognized conserved epitopes present in pathogens. Here, we describe a procedure for measuring natural antibodies reactive to bacterial antigens that may be applicable to a variety of organisms. IgM from desert bighorn sheep plasma samples was tested for reactivity to outer membrane proteins from Vibrio coralliilyticus, a marine bacterium to which sheep would have not been exposed. Immunoblotting demonstrated bighorn sheep IgM could bind to a variety of bacterial cell envelope proteins while ELISA analysis allowed for rapid determination of natural antibody levels in hundreds of individual animals. Natural antibody levels were correlated with the ability of plasma to kill laboratory strains of E. coli bacteria. Finally, we demonstrate that natural antibody levels varied in two distinct populations of desert bighorn sheep. These data demonstrate a novel and specific measure of natural antibody function and show that this varies in ecologically relevant ways.
Ecoimmunology seeks to explain variation in immunity within and among hosts by examining immunity in the context of the host’s ecology and life history. Because immune responses involve a complex network of protein and cellular signals and effectors, a central conundrum in the field of ecoimmunology is “what to measure” . Moreover, eco-immunological assays must reliably quantify immune responses in non-model species, including wildlife. In designing eco-immunological assays, the challenge is thus two-fold: First, to identify immune components that are relevant, by demonstrating that their level is indicative of functional immune responses, and second, to design robust assays that can capture variability in the immune component among hosts and populations of non-model animal species.
The field of ecoimmunology relies on a limited number of immune assays to document variation in immunity within and among wild animals. Experiments conducted with laboratory animals can utilize many different techniques to study natural antibody biology, including but not limited to, tracking specific B cell populations, analyzing germ-free or transgenic animals, expanding clonal B cells, and experimentally infecting animals. In wild animals, however, these techniques are not possible to implement. Two methods have commonly been used to measure nAb levels in a variety of wild animal systems: assessing the ability of nAbs to agglutinate red blood cells or to bind to a specific foreign protein, such as KHL or ovalbumin. It is widely held that nAbs are responsible for binding to foreign blood groups, however, not all nAbs may recognize foreign blood groups to the same extent that they recognize pathogenic bacteria signatures. In an study from Baxendale et al , natural antibody clones bearing non-mutated yet distinct heavy chains were able to bind to Pneumococcal capsular polysaccharides and ABO blood antigens with different affinities. Therefore, measuring erythrocyte agglutination may reflect the presence of natural antibodies with specificity for blood group antigens as opposed to bacterial products. KLH and ovalbumin are both glycoproteins and nAbs reactive to particular carbohydrate structures may bind to such targets. A vast heterogeneity of glycans is present on KLH , although ovalbumin tends to have fewer such structures . However, without exact knowledge about which moieties are specifically recognized by nAbs, it is difficult to know the degree to which these glycans will be present on the foreign protein chosen for the assay. Furthermore, bacterial glycan structures are very diverse, using many monosaccharides with a variety of branch-structures , suggesting that bacterial-specific nAbs which react to specific polysaccharide epitopes may not react with KLH or ovalbumin. Additionally, nAbs can also react to lipid moieties, such as phosphocholine , which will not be detected by measuring antibody interactions with glycoproteins. We therefore chose to isolate bacterial cell envelopes, which would contain several components that could serve as potential targets for nAbs including bacterial-glycoproteins and lipids, as the antigen for nAb-binding in our assays.