Simple quantification of blood and plasma antimicrobial capacity using spectrophotometry

Abstract

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. 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. 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 1·5 μL), 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. 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. © 2009 British Ecological Society.