It is becoming increasingly apparent that immune responses play an important role in an organism's physiological, biochemical, and behavioral responses to its environment and thus have the potential to shape the evolution of life history strategies (Boughton et al., 2011
). “Immunocompetence”, an individual's capacity to mount an appropriate immune response following exposure to a pathogen, is a critical aspect of disease resistance and thus survival (Graham et al., 2011
). Therefore, biologists from a wide range of ecological disciplines are increasingly interested in assessing immunocompetence in their study organisms. However, one of the major challenges to researchers is determining what measures to use in a given experiment (Demas et al., 2011
). Further there are limitations depending on study species, requirements for specific antibodies, and relevance of the methodology to the study organism.
The microbiocidal assay historically referred to as the bacterial killing assay, measures the capacity to fresh whole blood or plasma to kill microbes ex vivo
(Millet et al., 2007
; Tieleman et al., 2005
). However, the utility of this method goes beyond measuring bacterial killing to many different types of microbes and we will therefore refer to it heretofore as the microbiocidal assay. One of the primary benefits of using the microbiocidal assay instead of other measures of immune function is that it determines the ability of an organism to remove a pathogen that could be encountered in the wild. This provides an environmentally-relevant immune response. Additionally, several immune components are measured in this immune challenge. Phagocytes (e.g., macrophages, heterophils, and thrombocytes), opsonizing proteins (complement and acute phase proteins), and natural antibodies (predominantly immunoglobulins M and A, IgM and IgA) can be assessed, depending on the type of microbe and whether whole blood or plasma is used. Consequently, a major advantage to this method of immune function is that a variety of different microbes can be used to test functional responses of different specific immune components. For example, unlike many other immune measures, such as total hemolytic complement activity, the killing of the bacteria Escherichia coli
also relies on the presence of natural antibodies and phagocytes, providing a more integrative measure of immunity while also providing an indication of complement activity. These benefits are in contrast to many other assays that only assess isolated immune components (e.g., lymphocyte proliferation) or responses to relatively artificial antigens and/or mitogens, (e.g., phytoheamagglutinin).
Further advantages to this method are that no specific antibodies are required for this procedure. Therefore, the microbiocidal assay is very adaptable, not species specific, and can be used in a number of species. For example, in the current paper we have validated this assay on non-traditional amphibian (rough skinned newts, Taricha granulosa), reptilian (garter snakes, Thamnophis elegans), avian (house finches, Carpodacus mexicanus), and mammalian (coyotes, Canis latrans) species. The selection of this wide range of taxa, with different life histories, from a variety of environments, and with varying blood volumes, helps to demonstrate the applicability of the microbiocidal assay across a range of different taxa.
Additional advantages to the microbiocidal assay are its simplicity, short duration, small sample volume requirements, and that it requires only a minimal amount of specialized equipment to perform. Ideally, a sterile laminar flow hood is used; however a relatively aseptic enclosure has been effectively used. This assay requires an incubator, plate absorbance reader (with standard filters), and a limited amount of disposables.
The traditional bacterial killing assay procedure involved growing a microbe either exposed or not exposed to sample (containing killing elements) on agar plates (Buehler et al., 2008
; Matson et al., 2006
; Rubenstein et al., 2008
; Ruiz et al., 2010
). In general, the method typically requires a sample diluted in media or phosphate buffered saline added to a known number of live microbes. In short, the microbes and sample are incubated for a brief period and then added to agar plates. After a longer incubation period, microbe growth is quantified by counting the number of colonies for each sample. By comparing the sample plates to the reference plates, which have only microbes and no sample, the degree of microbial killing is determined. While fresh whole blood is preferable, field work often necessitates the use of frozen plasma. If the frozen samples are used, however, the microbiocidal capability greatly decreases with both freeze-thaws and long periods of storage (over 20 days) (Liebl and Martin, 2009
It is also critical to note that this measure of immune function varies significantly between species and even individuals in the same population, depending upon a variety of factors (such as sex, age, and parasite load). While this variation allows for considerable comparison across different organisms in different contexts, it is necessary to optimize dilutions of the sample and microbe strain prior to conducting the full assay (Buehler et al., 2008
; Matson et al., 2006
; Rubenstein et al., 2008
; Ruiz et al., 2010
). The plating of samples on agar plates and manually counting microbial colonies, while standard in immunological research, is time consuming, requires comparatively large amounts of samples, and can be less reliable. In response to these challenges, Liebl and Martin introduced a new method that quantifies microbial colonies using a nanodrop spectrophotometer (Thermoscientific; Wilmington, DE) (Liebl and Martin, 2009
). This new approach significantly reduced variation among samples and reduced the amount of necessary sample used in the assay. However, access to nanodrop spectrophotometers is limited at some institutions making it difficult to perform the assay, and the correlation between nanodrop and the traditional agar plate analysis is not ideal (i.e., r
0.458), limiting its utility as a proxy for actual bacterial killing (Liebl and Martin, 2009
). Here we introduce a new variation, the microbiocidal assay that is adapted from Liebl and Martin for use on a microplate reader and will enable researchers across disciplines to effectively employ this method to accurately quantify microbial killing ability, using readily available microplate absorbance readers (Liebl and Martin, 2009