† Deceased
The authors have declared that no competing interests exist.
Conceived and designed the experiments: BAK KY RAB GKB. Performed the experiments: KY DK JPM MO. Analyzed the data: BAK KY GKB. Contributed reagents/materials/analysis tools: DK RAB JPM. Wrote the paper: BAK DK GKB.
Changes in body odor are known to be a consequence of many diseases. Much of the published work on disease-related and body odor changes has involved parasites and certain cancers. Much less studied have been viral diseases, possibly due to an absence of good animal model systems. Here we studied possible alteration of fecal odors in animals infected with avian influenza viruses (AIV). In a behavioral study, inbred C57BL/6 mice were trained in a standard Y-maze to discriminate odors emanating from feces collected from mallard ducks (
It has long been speculated that infections may cause odor changes in animals and humans
There is also convincing evidence that healthy individuals modify their social behaviors when exposed to infected conspecifics themselves or their body odors. Avoidance of individuals on the basis of odors associated with illness may serve to reduce the probability of disease spread
The hypothesis that immune activation may produce a meaningful alteration of body odor led us to study odor changes resulting from administration of vaccines in mice (unpublished data). Using biosensor panels of trained mice, we demonstrated that body odor was altered by immunization with either rabies or West Nile Virus vaccine. Based on this promising result, we conducted similar experiments with an avian influenza virus (AIV) to evaluate whether infection produces a distinctive odor change in a relevant species; mallard ducks (
We hypothesized that infection with a low pathogenic influenza virus (H5N2) would alter the volatile profile of feces. We first postulated that these changes would be discriminable by mice trained in a Y-maze to identify odors associated with AIV infection. The Y-maze has successfully been used to discriminate between many different sources of odor variation, including: fetal odortype
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching of the Federation of Animal Science Societies. Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the Monell Chemical Senses Center (#1123), USDA National Wildlife Research Center (#2021), and Colorado State University (#09-1317A).
Eight farm-raised mallards of mixed gender were used in this study. Six ducks were infected ocularly, intranasally and orally with 1 ml of brain heart infusion broth containing 1×106 pfu/ml of A/Mallard/MN/346250/00 (H5N2). Cloacal swabs were collected on days three and four following experimental infection. Infectivity was confirmed by real-time RT-PCR and inoculation into 10-day old embryonating chicken eggs.
Two pooled feces samples were collected from each duck. Feces were collected daily for seven days immediately preceding experimental infection and again for days three through 10 post-infection. Eight pre-treatment and eight post-treatment samples were stored frozen at −80°C. To facilitate safe transport and handling of feces, all 16 samples were subjected to 2.7 Mrad of cesium irradiation for 27 hours. Lack of infectivity was confirmed by inoculation into 10-day old embryonating chicken eggs.
Six female C57BL/6 in-bred mice (born and raised in our laboratory) were trained to discriminate between feces from non-infected and infected ducks in a Y-maze as previously described
In training sessions, fecal samples from three ducks (#3, 4, and 13;
Duck | Treatment | Biosensor Design |
1 | Control | Generalization |
2 | Control | Generalization |
3 | H5N2 | Training |
4 | H5N2 | Training |
5 | H5N2 | Generalization |
11 | H5N2 | Generalization |
12 | H5N2 | Generalization |
13 | H5N2 | Training |
Sessions | Pre-treatment Donor | Post-Treatment Donor |
1 thru 4 | Duck 3 | Duck 4 |
5 thru 7 | Duck 3 | Duck 13 |
8 | Duck 3 | Duck 3 |
9 | Duck 4 | Duck 4 |
10 | Duck 13 | Duck 13 |
11–13 | Duck 3Duck 13 | Duck 3Duck 13 |
Mice were rewarded for selection of the maze arm associated with feces collected after experimental AIV infection (post-treatment collections).
Sessions of unrewarded generalization trials were initiated when each biosensor demonstrated >80% concordance. Fecal samples from three infected ducks (#5, 11, and 12;
Cumulative responses of the full panel of trained mice were calculated for each generalization trial. Success rates (number of correct trials divided by the total number of generalization trials) were subjected to statistical tests of binomial proportion using the continuity correction for small numbers of observations
Headspace analyses were conducted with a HT3 dynamic headspace analyzer (Teledyne Tekmar, Mason, OH, USA) outfitted with Supelco Trap K Vocarb 3000 thermal desorption trap (Sigma-Aldrich Co., St. Louis, MO, USA) attached to a Thermo Trace GC-MS equipped with a single quadrapole mass spectrometer (Thermo Scientific, Waltham, MA, USA) and a 30 m×0.25 mm id Stabiliwax-DA fused-silica capillary column (Restek, Bellefonte, PA, USA). Fecal samples (0.5 g) were maintained at 50°C, swept with helium for 60 min (flow rate of 75 mL/min), and the volatiles collected on the thermal desorption trap. Trap contents were desorbed at 260°C. The GC oven program had an initial temperature of 40°C (held for 3.0 min) followed by a ramp of 7.0°C/min to a final temperature of 230°C (held for 6.0 min). The MS was used in scan mode from 41 to 400 m/z.
Samples were analyzed in triplicate. Chromatographic data were converted to NetCDF format for baseline correction, noise elimination, and peak alignment processing using Metalign
Semi-quantitative analyses of acetoin (3-hydroxy-2-butanone) and 1-octen-3-ol in fecal samples were conducted by subjecting 0.25–0.75 g fecal samples to extraction with 10.0 mL ethanol (Fisher Scientific, Pittsburg, PA) in 25-mL screw-cap culture tubes. Analytical standards were obtained from Sigma-Aldrich (Milwaukee, WI). The tubes and contents were placed in a vortex mixer (Fisher Multitube Vortexer, Fisher Scientific) for 30 minutes and centrifuged at 2000 rpm (Thermo IEC Centra CL2, Thermo Scientific, Waltham, MA). Non-volatile extractives (e.g. plant pigments from duck diets) were removed by passing green-colored extracts through graphitized carbon solid phase extraction (SPE) cartridges (Supelco, Bellefonte, PA). Sample eluates were colorless, indicating that plant pigments were removed from the solvent extracts.
Splitless injections (1 µl) were made into a Thermo Scientific ISQ GC-MS equipped with a single quadrapole mass spectrometer (Thermo Scientific) and a 30 m×0.25 mm id Stabiliwax-DA fused-silica capillary column (Restek, Bellefonte, PA, USA). The GC oven program had an initial temperature of 40°C (held for 1.0 min) followed by a ramp of 3.0°C/min to 112°C and a ramp of 25°C/min to 235°C (held for 3.0 min). The MS was used in scan mode from 33 to 400 m/following an 8.5 min solvent delay. Selection ion monitoring (SIM) chromatograms were produced from the sum of the m/z responses of 45 and 57. Peak area responses were normalized by dividing peak area by sample mass (g). Peak area ratios were also calculated by dividing the 1-octen-3-ol peak area response by the acetoin response. Peak and peak ratio responses were evaluated by t-test to determine if they were impacted by AIV infection. Because the pre-treatment fecal sample from duck 11 was exhausted during bioassays, peak responses from extracts of the post-infection sample were not included in statistical analyses.
Several experiments were conducted to evaluate method performance. A standard solution containing both acetoin (5.69 µg/mL) and 1-octen-3-ol (5.05 g/mL) was repeatedly passed through SPE columns to evaluate irreversible loss of the analytes. Replicate extractions were also conducted with the post-infection sample from one duck (duck 5) to evaluate repeatability.
Results of real-time RT-PCR and chicken egg inoculation confirmed infectivity in test mallards three days after experimental infection with the virus. Furthermore, the virus was no longer viable in the fecal samples following irradiation.
All six trained mice demonstrated greater than 80% concordance (choosing the maze arm associated with fecal odors from infected ducks) in 13 training sessions. These mice generalized the trained response correctly for all within-duck comparisons (pre- vs. post-infection for ducks #5, 11, and 12;
Duck | Treatment | % Correct | Sessions | Trials | ||
1 | Control | 57% |
1.03 | 0.152 | 2 | 56 |
2 | Control | 56% |
0.385 | 0.350 | 1 | 27 |
5 | H5N2 | 84% | 4.67 | <0.0001 | 2 | 50 |
11 | H5N2 | 72% | 3.20 | 0.0007 | 1 | 28 |
12 | H5N2 | 72% | 2.00 | 0.0228 | 1 | 25 |
In all cases, mice were trained to select the maze arm associated with the odor of feces collected after experimental infection with avian influenza.
Binomial test probability with null hypothesis % Correct = 50%.
Indicates rate of selection of post-sham infection feces sample.
Results from between-duck generalizations were not as definitive. As would be predicted from mice trained to identify the odor of feces from AIV-infected ducks, mice did not discriminate between feces samples collected from two ducks collected during the pre-infection period (duck 12 versus duck 1;
Comparison | % Correct | Sessions | Trials | |||
1 - Pre | 12 - Pre | 42% |
0.588 | 0.278 | 1 | 26 |
2 - Post | 5 - Post | 71% |
3.074 | 0.0011 | 2 | 56 |
1 - Post | 5 - Post | 45% |
0.858 | 0.195 | 3 | 87 |
In all cases, mice were trained to select the maze arm associated with the odor of feces collected after experimental AIV infection. “Pre” indicates feces collected prior to experimental infection; “Post” indicates collection after infection. Ducks 5 and 12 were experimentally infected with avian influenza. Ducks 1 and 2 received a sham treatment. Responses were subjected to tests of binomial proportions equal to 50%.
Indicates rate of selection of 12 - Pre sample.
Indicates rate of selection of 5 - Post sample.
Data processing of headspace data identified >1900 significant mass spectral responses for each sample. Reconstruction of a composite chromatogram from these responses indicated that 96 individual chromatographic peaks were present in the headspace of the fecal samples. Principal Components Analysis (PCA) of headspace data identified two mass spectral responses that adequately segregated feces samples on the basis of AIV infection status. These responses were tentatively identified as acetoin (m/z = 88 at scan 2141) and 1-octen-3-ol (m/z = 57 at scan 2876).
Peak area ratios (1-octen-3-ol:acetoin) obtained from solvent extraction ranged from 0 to 0.88 across all ducks and collection periods (
Non-infected Duck Samples | Infected Duck Samples | ||||||||
Duck | Treatment | Period | Acetoin | 1-Octen -3-ol | Ratio | Period | Acetoin | 1-Octen -3-ol | Ratio |
1 | Control | Pre | 71 | 31.1 | 0.44 | ||||
2 | Control | Pre | 159 | 33.2 | 0.21 | ||||
1 | Control | Post | 29 | 25.6 | 0.87 | ||||
2 | Control | Post | 47 | 29.5 | 0.62 | ||||
3 | H5N2 | Pre | 77 | 44.6 | 0.58 | Post | 476 | 34.9 | 0.07 |
4 | H5N2 | Pre | 63 | 51.6 | 0.81 | Post | 179 | 24.3 | 0.14 |
5 | H5N2 | Pre | 47 | 41.2 | 0.88 | Post | 109 | 21.0 | 0.19 |
12 | H5N2 | Pre | 103 | 20.1 | 0.19 | Post | 646 | 21.3 | 0.03 |
13 | H5N2 | Pre | 92 | 18.8 | 0.20 | Post | 1307 | 13.2 | 0.01 |
Mean | 76.5 | 32.9 | 0.535 | 543 | 22.9 | 0.089 | |||
S.D. | 38.5 | 11.1 | 0.29 | 480 | 7.9 | 0.076 | |||
p-value | 0.095 | 0.10 | 0.0014 |
Responses of individual compounds are normalized for sample mass. Peak ratio equals 1-octen-3-ol response divided by acetoin response and is thus independent of sample mass. P-values correspond to differences of non-infected and infected ducks for the three different responses.
Trained mice readily discriminated feces of non-infected and infected mallards on the basis of volatile metabolites during training and generalized this response to novel fecal samples differing in infection status (
These compounds (1-octen-3-ol and acetoin) have been identified as potential biomarkers for diagnosing gastrointestinal diseases in humans
Two test systems (i.e. trained biosensors and chromatographic analysis) independently confirmed that alterations of fecal volatiles corresponded with AIV infection. However, there is no evidence that acetoin and 1-octen-3-ol represent the very same cues learned by the biosensors when they were trained to discriminate feces on the basis of infection status. Fecal odors associated with infection may involve many aspects of disease pathology and likely result in a myriad of qualitative and quantitative alterations. It is also unclear whether other infectious agents may produce similar or different changes in fecal volatiles. However, pathogen specificity is not a prerequisite for communicating health status to conspecifics. Odor changes in response to infection may be as general as fluctuation of body temperature, yet detectible changes could alert members of the brood to the presence of a potentially transmissible pathogen.
Chemical communication of infection can be adaptive to either the host population (causing individuals in the brood to avoid infected conspecifics) or the pathogen (making infected individuals attractive to other conspecifics). Avoidance of urine odors associated with influenza has been demonstrated in mice
All the authors read and approved the final manuscript, with the exception of Dr. Yamazaki who passed away suddenly on 11 April 2013. Most importantly, our friend and colleague Dr. Yamazaki actively contributed to the design and realization of this work. Mention of specific products does not constitute endorsement by the United States Department of Agriculture, Colorado State University, or Monell Chemical Senses Center.