Abstract
Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10-0, 10-2, 10-4 and 10-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.
Highlights
Paratuberculosis, a chronic progressive enteritis of domestic and wild ruminants, is prevalent in all countries with intensive dairy, beef or sheep industry
volatile organic compounds (VOCs) not detectable in the media or VOCs with significant higher concentrations in the different cultures compared to the media blank (> 120% compared to media) were considered as potential biomarkers
More than 100 VOCs were detected in the headspace over Mycobacterium avium subsp. paratuberculosis cultures by means of HS-Solid Phase Micro Extraction (SPME)-GC-MS
Summary
Paratuberculosis (paraTB), a chronic progressive enteritis of domestic and wild ruminants, is prevalent in all countries with intensive dairy, beef or sheep industry. Control of paraTB in livestock is complicated by the lack of appropriate tests for the identification of animals in the early phase of the disease. This is due to the low sensitivity of established direct and indirect diagnostic methods [2]. Fecal shedding of MAP and formation of specific antibodies are not detectable in the first years after infection. A strong correlation between intensity of shedding and formation of specific antibodies has been noted and the highest proportion of antibody positive animals can be found in animals shedding high amounts of MAP [4]
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