Diagnostic sequences that distinguish M. avium subspecies strains

Authors: Bannantine, John; Stabel, Judith; Bayles, Darrell; CONDES, CYRIL - Universite De Tours; BIET, FRANCK - Universite De Tours

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2020
Publication Date: 1/28/2021
Citation: Bannantine, J.P., Stabel, J.R., Bayles, D.O., Condes, C., Biet, F. 2021. Diagnostic sequences that distinguish M. avium subspecies strains. Frontiers in Veterinary Science. 7. https://doi.org/10.3389/fvets.2020.620094.
DOI: https://doi.org/10.3389/fvets.2020.620094

Interpretive Summary: Molecular diagnostics are revolutionizing veterinary infectious disease detection. Their effects will be significant in dairy herd settings where timely and accurate diagnostic tools are critical for treatment decisions and outcomes. PCR is the most developed molecular technique currently, and has a wide range of potential clinical applications, including specific pathogen detection and John's disease surveillance. Recognizing that not all Mycobacterium avium subspecies paratuberculosis diagnostic gene targets have been identified for Johne's disease detection, a primary goal of this study was to identify genetic targets that can distinguish M. avium subspecies from each other. To accomplish this, we initiated a comprehensive genome comparison and PCR combinatorial approach to identify 86 new Map genes with true diagnostic specificity. Genes specific to other M. avium subspecies strains were also identified. These newly identified sequences are also important to understanding the unique biology, evolution and niche adaptation of this subspecies.

Technical Abstract: Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were examined and tested by comparing partial genome sequences of K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in a partial list of 32 Map specific genes, not including repetitive elements, based on the dual genome comparison. There are now 29 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah and Map. This has enabled a more comprehensive comparison of these sequences using two independent multi-genomic comparison tools, PanOCT and Roary, to obtain 279 subspecies specific genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. BLAST similarity searching further reduced the number of diagnostic targets, which showed strong similarity to other mycobacterial species recently added to the database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 16 M. avium subspecies strains and confirmed 86 genes as Map-specific, 7 and Maa-specific and 3 as Mah-specific. A single-tube PCR reaction was tested as a proof of concept method to quickly distinguish M. avium subspecies strains. These novel sequences represent new targets for DNA-based diagnostics for Johne’s disease as well as nontuberculous mycobacterial diseases.