Volatile metabolomic signatures of rabies immunization in two mesocarnivore species

Rabies is a zoonotic disease caused by infection with rabies virus, which circulates naturally in several wild carnivore and bat reservoirs in the United States (US). The most important reservoir in the US from an animal and public health perspective is the raccoon (Procyon lotor). To prevent the westward expansion of a significant raccoon rabies epizootic along the eastern seaboard, an operational control program implementing oral rabies vaccination (ORV) has existed in the US since the 1990s. Recently, two vaccine efficacy studies conducted with raccoons and striped skunks (Mephitis mephitis) provided the opportunity to determine if volatile fecal metabolites might be used to non-invasively monitor ORV programs and/or predict virus protection for these species. The volatile metabolome is a rich source of information that may significantly contribute to our understanding of disease and infection. Fecal samples were collected at multiple time points from raccoons and striped skunks subjected to oral treatment with rabies vaccine (or sham). Intramuscular challenge with a lethal dose of rabies virus was used to determine protection status at six (raccoons) and 11 (skunks) months post-vaccination. In addition to fecal samples, blood was collected at various time points to permit quantitative assessment of rabies antibody responses arising from immunization. Feces were analyzed by headspace gas chromatography with mass spectrometric detection and the chromatographic responses were grouped according to cluster analysis. Cluster scores were subjected to multivariate analyses of variance (MANOVA) to determine if fecal volatiles may hold a signal of immunization status. Multiple regression was then used to build models of the measured immune responses based on the metabolomic data. MANOVA results identified one cluster associated with protective status of skunks and one cluster associated with protective status of raccoons. Regression models demonstrated considerably greater success in predicting rabies antibody responses in both species. This is the first study to link volatile compounds with measures of adaptive immunity and provides further evidence that the volatile metabolome holds great promise for contributing to our understanding of disease and infections. The volatile metabolome may be an important resource for monitoring rabies immunization in raccoons and striped skunks.