Screening of Microbial Volatile Organic Compounds for Detection of Disease in Cattle: Development of Lab-scale Method

Abstract

The primary hurdle for diagnosis of some diseases is the long incubation required to culture and confirm the presence of bacteria. The concept of using microbial VOCs as “signature markers” could provide a faster and noninvasive diagnosis. Finding biomarkers is challenging due to the specificity required in complex matrices. The objectives of this study were to (1) build/test a lab-scale platform for screening of microbial VOCs and (2) apply it to Mycobacterium avium paratuberculosis ; the vaccine strain of M . bovis Bacillus Calmette-Guérin; and M . kansasii to demonstrate detection times greater those typically required for culture. SPME-GC-MS was used for sampling, sample preparation, and analyses. For objective (1), a testing platform was built for headspace sampling of bacterial cultures grown in standard culture flasks via a biosecure closed-loop circulating airflow system. For (2), results show that the suites of VOCs produced by Mycobacteria ssp. change over time and that individual strains produce different VOCs. The developed method was successful in discriminating between strains using a pooled multi-group analysis, and in timepoint-specific multi- and pair-wise comparisons. The developed testing platform can be useful for minimally invasive and biosecure collection of biomarkers associated with human, wildlife and livestock diseases for development of diagnostic point-of-care and field surveillance.

Application of Volatilome Analysis to the Diagnosis of Mycobacteria Infection in Livestock

“…The diagnosis of mycobacterial infection is currently at the center of attention because, although well-established and reliable, it has its own limitations. Apart from being a tedious process, special consideration must be given to the lack of an optimal diagnostic sensitivity (Table 2) and the different variables which may interfere with the methods and techniques in use (2,21). Therefore, an accurate and reliable diagnostic methodology of the infection by mycobacteria, or a combination of various strategies, is the cornerstone of their control (23).…”

“…Although IST and IFN-γ assay have reasonable sensitivity and good specificity (Table 2), both techniques require a minimum of 48-72 h to obtain a result (21, 65) besides presenting other disadvantages and limitations. On the one hand, IST requires visiting the farm and restraint of the animals twice, and a delicate and difficult administration and interpretation of skin results, which may vary due to differences in tuberculin doses, site of application (Table 2), and interpretation schemes (25, 27, 66); on the other hand, IFN-γ assay implies a complex laboratory methodology (18), a considerably more expensive price than a skin test (65,67), and suffers from cross-reactivity with other related mycobacteria resulting in false-positive results (2). In addition, performance of these tests can be compromised by factors associated with the immune response and health status of the animal leading to a misinterpretation of the results.…”

“…Analysis of volatile organic compounds (VOCs) is an emerging research area in both human and veterinary medicine (1), which allows a non-invasive, fast, and economic diagnosis as well as identification of new biomarkers as alternative to current diagnostic techniques (2). VOCs are defined as a sub-category of small molecular mass substances within metabolites, which are characterized by its low boiling point and high vapor pressure (3,4).…”

Detection of Mycobacterium avium ssp. paratuberculosis in Cultures From Fecal and Tissue Samples Using VOC Analysis and Machine Learning Tools

“…A variety of VOCs has been identified over mycobacterial cultures, especially M. tuberculosis (Mtb) and M. bovis strains. Most of the substances described in our study were already reported previously ( 56 ). However, the knowledge about the origin and fate of VOCs within the metabolism of MAP is still limited.…”

Review on Volatolomic Studies as a Frontier Approach in Animal Research

“…paratuberculosis , Mycobacterium bovis ( M. bovis ) Bacillus Calmette‐Guérin and Mycobacterium kansasii bacteria from cattle cultured in bottles were collected with an SPME fiber for 1 h at 37 °C. [ 62 ] For obtaining the dynamic headspace, air at an average flow rate of 162 mL min –1 passed through each culture bottle in ≈13 mL closed loops made from PTFE Teflon tubing that went from the pump to the culture bottle, then to the sampling bulb, and back to the pump. The inlet and outlet fittings on each culture bottle included 0.22 µm filters to prevent the introduction of mycobacteria into the closed loop system.…”