Simple quantification of blood and plasma antimicrobial capacity using spectrophotometry

Abstract

Summary 1.Ecologists are becoming increasingly interested in characterizing immunological variation among and within species. However, many of the techniques available to immunologists are not viable for use in non-model species, which limits the resolution and progress of ecoimmunological research. Recently, an assay was developed to quantify the capacity of blood or plasma to kill microorganisms in vitro. Such assays are ideal for ecologists because they provide holistic, integrated measures of immune function with minimal sample size and equipment. 2. Although informative, the assay suffers from several shortcomings in its current form. Specifically, intra-assay variation is often prohibitive, quantification of results is time consuming, sample volumes required are somewhat high (especially for small-bodied species), and materials (e.g. Petri dishes for microbe culturing) are costly and generate excessive waste. 3. Here, we describe a modified version of the assay for use against Gram-negative, Gram-positive, and fungal microbes using spectrophotometry. This assay decreases sample processing time, reduces blood ⁄ plasma volume (to 1AE5 lL), and eliminates the need to score results manually. This technique was 3· more reliable than the alternative method when using house sparrow Passer domesticus plasma and Escherichia coli. 4. We expect this assay will enable quantification of immune defence in a variety of contexts previously difficult to study (e.g. endangered species) because of the small volumes needed and the simplicity of the protocol. We also provide a list of pathogens that affect passerine populations as potential candidates in future studies. Anti-microbial activity in other animal species should also be easy to characterize using our approach and the quantification of antimicrobial capacity of tissue and other body fluids should also be possible with only minor modifications of the assay.

Flight performance of western sandpipers Calidris mauri remains uncompromised when mounting an acute phase immune response

“…We assessed the antimicrobial capacity of plasma using spectrophotometry (Liebl and Martin, 2009 , and plasma was diluted 1:23 (1.5μl plasma in 34.5μl PBS). One-third of the sample volume of the solution (12.5μl) containing the E. coli was added to diluted plasma, vortexed and incubated at 37°C for 30 min.…”

Relationships among plumage coloration, blood selenium concentrations, and immune responses of adult and nestling tree swallows

“…We examined one aspect of the innate immune response of nestling and adult tree swallows in June and July 2012 by evaluating the bactericidal capacity of plasma (following Liebl and Martin, 2009). This bacteria killing assay (BKA) reflects the ability of complement enzymes and lysozyme to destroy cell walls and lyse cell membranes of bacteria (reviewed in Matson et al, 2006).…”

Does immune suppression during stress occur to promote physical performance?

“…In vitro BKA of blood was assessed following modified laboratory protocols (Liebl and Martin, 2009a). From each blood sample, and within 30s of sampling, a 1.5l aliquot was added to 34.5l of CO 2 -independent medium in four different microcentrifuge tubes: three to assess anti-E. coli (Gram-negative bacteria) activity and the fourth as a blank in spectrophotometric measurements.…”

“…Specifically, we asked whether after 6weeks in captivity (1) baseline CORT was elevated, and (2) constitutive [in vitro bacterial killing ability (Liebl and Martin, 2009b)] and induced (oxidative burst responses) innate immune responses (Sild and Horak, 2010) were reduced more so than the size of the major flight muscle and the ability of birds to perform hovering flight (Veasey et al, 1998). Although the re-allocation hypothesis was proposed to explain (among other things) the effects of glucocorticoids on immune functions over very short periods (e.g.…”