Urinary neopterin, a non-invasive marker of mammalian cellular immune activation, is highly stable under field conditions.

Michael Heistermann,James P Higham

doi:10.1038/srep16308

Abstract

Studying immunity and immune function in ecology and evolution requires field studies, but there has been a dearth of non-invasive markers of immune activation available for studying large wild mammals. Recently, we analytically and biologically validated the measurement of urinary neopterin (NEO), a biomarker of cellular immune activation, in captive macaques. However, applying this to free-ranging settings is complicated by issues involving sample collection, processing, storage, and transport. Here, we collected urine samples from captive macaques and undertook experiments simulating common field issues. We tested the effects on urinary NEO sample measurements following: dirt and faecal contamination; storage at room temperature; differences in processing and long-term storage methods (freezing, lyophilising, blotting onto filter paper); and freeze-thaw cycles. Our results show that concentrations of urinary NEO are highly stable – they are not affected by soil or faecal contamination, can be collected on filter paper and stored for many months frozen or lyophilised with minimal effect, and are resistant to multiple 24 hr freeze-thaws. With the addition of a biocidal preservative, concentrations are even stable at room temperature for long periods. Urinary NEO is remarkably resilient, and is highly suitable for non-invasive field studies of cellular immune responses in wild large mammals.

Highlights

Have included the measurement of faecal and urinary glucocorticoids and androgens[13,14,15,16]
We evaluated the potential utility of potentially informative non-invasive markers for monitoring immune activation and infection in macaques[29]
Our study aimed to test the likely utility of urinary NEO as a biomarker of cellular immune activation for use in wild and free-ranging settings, where there are many complications involved in sample collection, processing and storage

Summary

Introduction

Have included the measurement of faecal and urinary glucocorticoids and androgens[13,14,15,16]. These include: regular cross-contamination of faecal and urine samples when either sample type is collected in the field, and contamination of both matrices with dirt; storage issues, such as how to stabilize samples before they can be returned to a field station or freezer, and how to store samples in the field (e.g. on filter paper, by freezing); how samples might be transported and the effects of potential freeze-thaws on analyte concentrations; and the question of whether and how samples might be stored long-term (e.g. for months) without degrading before they can be analysed in a laboratory These types of problems have been addressed several times for the non-invasive measurement of steroid hormones[36,37,38,39], and for urinary C-peptide[22]. Contamination with faeces Collection onto filter paper 21 days storage at RT without preservative 21 days storage at RT with ProClin 200

Methods

Results

Conclusion