VOC breath profile in spontaneously breathing awake swine during Influenza A infection

Abstract

Influenza is one of the most common causes of virus diseases worldwide. Virus detection requires determination of Influenza RNA in the upper respiratory tract. Efficient screening is not possible in this way. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive and fast monitoring of disease progression. Breath VOC profiles of 14 (3 controls and 11 infected animals) swine were repeatedly analyzed during a complete infection cycle of Influenza A under high safety conditions. Breath VOCs were pre-concentrated by means of needle trap micro-extraction and analysed by gas chromatography mass spectrometry before infection, during virus presence in the nasal cavity, and after recovery. Six VOCs could be related to disease progression: acetaldehyde, propanal, n-propyl acetate, methyl methacrylate, styrene and 1,1-dipropoxypropane. As early as on day four after inoculation, when animals were tested positive for Influenza A, differentiation between control and infected animals was possible. VOC based information on virus infection could enable early detection of Influenza A. As VOC analysis is completely non-invasive it has potential for large scale screening purposes. In a perspective, breath analysis may offer a novel tool for Influenza monitoring in human medicine, animal health control or border protection.

Figure 1

Heatmap: Compound target response of each swine normalized to the individual maximum on day 0, 2, 4, 7, and 14 of measurement.

Figure 2

Principal Component Analysis: Scores (1.1, 2.1) and loadings (1.2, 2.2) of VOC emissions. (A) Control group (blue) compared to infected group (red), and room air (yellow) on day 4. (B) VOC emissions of the infected group on each day of breath measurement.

Figure 3

Exhaled concentration of compounds on day 0, 2, 4, 7, and 14 in control (blue) and infected group (red). Significant differences between control group and infected group on each day and significant differences in the infected group between each day *p < 0.001, **p = 0.001-0.01, ***p > 0.01.

Figure 4

(A) Intranasal inoculation process in swine. Animals were inoculated intranasally at seven weeks of age by MAD with 2 ml By09. (B) Sample protocol of breath gas analysis and determination of intranasal virus load. Breath samples and nasal swabs for virus determination were taken from each animal on day 0, prior to infection. After intranasal inoculation of 11 animals (infected group) with Influenza A on day 0, breath samples and virus load of the infected animals and the three control animals was determined by virus titration on MDCK II cells.

Figure 5

Breath sampling under high safety conditions. (1) breath sampling device, (2) breath sampling from swine in canvas sling, and (3) bi-directional NTD-sampling. A = breathing mask, B = virus-bacteria filter, C = T-piece, D = capnometer, E = three-way cock, F = 50 ml glass syringe, G = female/female adapter, H = IN stopper, I = Needle-trap, J = 1 ml syringe.