Bacteriophage-Based Methods for Detection of Viable Mycobacterium avium subsp. paratuberculosis and Their Potential for Diagnosis of Johne's Disease

Abstract

Bacteriophage-based methods for detecting Mycobacterium avium subsp. paratuberculosis (MAP) are a potential new approach for diagnosis of Johne's disease (JD). The basis of these tests is a mycobacteriophage (D29) with a lytic lifecycle that is able to infect a range of Mycobacterium spp., not just MAP. When added to a test sample, the phages will bind to and infect mycobacterial cells present. If the host mycobacterial cells are viable, the phages will take over the metabolic machinery of the cells to replicate and produce multiple copies of themselves (phage amplification), before weakening the host cell walls by enzyme action and causing cell lysis. Cell lysis releases the host cell contents, which will include ATP, various enzymes, mycobacterial host DNA and progeny D29 phages; all of which can become the target of subsequent endpoint detection methods. For MAP detection the released host DNA and progeny phages have principally been targeted. As only viable mycobacterial cells will support phage amplification, if progeny phages or host DNA are detected in the test sample (by plaque assay/phage ELISA or qPCR, respectively) then viable mycobacteria were present. This mini-review will seek to: clearly explain the basis of the phage-based tests in order to aid understanding; catalog modifications made to the original plaque assay-based phage amplification assay (FASTPlaqueTB™) over the years; and summarize the available evidence pertaining to the performance of the various phage assays for testing veterinary specimens (bovine milk, blood and feces), relative to current JD diagnostic methods (culture, fecal PCR, and blood-ELISA).

A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

“…Furthermore, mycobacteriophage D29, a lytic phage, is commonly used in phage amplification studies in order to infect the viable MAP cells and liberate DNAs from the recovered cells. Although, D29 is not a specific phage for only MAP 28 , 31 , 32 , it specifically expresses its DNA in its viable mycobacterial hosts 33 .…”

A Review on Mycobacteriophages: From Classification to Applications

“…The capability of mycobacteriophages in crossing the mycobacterial barriers has turned them into practical tools for diagnosing pathogenic and non-pathogenic mycobacteria. Some of the most-known phage-based diagnostic techniques are as follows: shuttle plasmids [ 7 , 19 , 72 , 162 ] and transduction of fluorescent or non-fluorescent foreign DNA into the mycobacterial genome and distinguishing the antibiotic resistance or viability of mycobacteria through fluorescent emission or formation of turbid lysogenic plaques [ 19 , 20 , 163 ]; phage amplification and detection of the viability of mycobacteria [ 24 , 25 , 164 , 165 ]; capture of viable target mycobacteria using mycobacteriophage proteins [ 166 ] or whole phages as ligands [ 79 , 87 , 88 ].…”

“…D29 was initially applied in the FASTPlaqueTB phage assay to detect the viability of M. tuberculosis complex in human sputum specimens taken from patients suspected of having tuberculosis in 2005 [ 164 ]. Between 2007 and 2021, D29 was exploited to determine the viability of MAP in dairy products [ 24 ], blood, and feces [ 25 ] within an adapted FASTPlaqueTB [ 24 ] assay, Actiphage core 2-day assay, or modified phage amplification techniques. From 2013 to 2020, phage amplification via D29 underwent modifications, including changes in the protocol of DNA extraction from lysed cells that were indicated with plaques (i.e., heating agar plaques [ 24 ]; purification of the extracted DNA from excised plaques via Zymoclean DNA Clean and Concentrator columns [ 181 , 182 ]); selective capture and concentrating MAP cells in samples via magnetic beads coated with MAP-specific complementary peptides (i.e., aMp3 and aMptD) and consequent magnetic separation.…”

“…Today, mycobacteriophages in isolation/cocktail [ 15 ] or within a synergistic therapy along with antibiotics opened new horizons in the treatment of infections caused by pathogenic mycobacteria [ 16 , 17 , 18 ]. Furthermore, mycobacteriophages have been exploited in diagnostic approaches of slow-/fast-growing mycobacteria through shuttle plasmids that express resistance against antibiotics and induce the production of lysogenic plaques in target mycobacteria [ 19 ]; transduction of nanoluciferase reporter gene cassettes that discriminate viable drug-resistant and -sensitive strains [ 20 ]; phage amplification for detection of antimicrobial resistance species [ 21 , 22 , 23 ]; and viability assessment of target mycobacteria in various samples (i.e., dairy products, blood, stool [ 24 , 25 ]). This review aimed to look back on some of the most used mycobacteriophages in research, elucidating their applications in the diagnosis and treatment of drug-resistant pathogenic mycobacteria with a focus on three pathogenic mycobacterial species, M. tuberculosis , MAP, and M. abscessus, from past to now.…”

Mycobacterium avium subsp. paratuberculosis Proteome Changes Profoundly in Milk

“…MAP can either be shed directly into milk by infected cows or be introduced via fecal contamination [ 8 , 9 , 10 , 11 ]. Viable MAP were found in pasteurized milk, cheese, and even in dried milk products such as infant formula [ 12 , 13 , 14 ]. This might have been caused by post-processing contamination, but there is also evidence of MAP being able to survive the pasteurization process [ 15 , 16 , 17 , 18 , 19 ].…”