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

Abstract

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

A Review on Mycobacteriophages: From Classification to Applications

“…D29 is a lytic phage [ 83 ] that has a close genomic affinity to mycobacteriophage L5 [ 83 ] and Che12 [ 81 ]. D29 has predominantly been used in the assessment of MAP viability in various sample types, such as milk [ 84 ], blood [ 85 ], tissue [ 86 ], feces [ 86 ], and direct capture of MAP prior to viability assessment in milk samples [ 87 , 88 ]. D29 is stable at pH between 9 to 10.…”

“…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 ].…”

“…This assay was called phagomagnetic separation-qPCR assay [ 87 ]. On another level, recovered viable MAP cells were resuspended in Middlebrook (MB) 7H9 supplemented with 10% Oleic Albumin Dextrose Catalase (OADC) and 2 mM CaCl 2 , and that was followed by the incubation of suspensions at 37 °C/2 h and a thermal shock at 55 °C/1–2 min [ 87 , 88 ]. Later, the presence of MAP DNA in lysates ( Figure 4 ) was assessed by qPCR IS 900 analysis [ 87 ].…”

“…Later, the presence of MAP DNA in lysates ( Figure 4 ) was assessed by qPCR IS 900 analysis [ 87 ]. The modified phage assay introduced a meaningful sensitivity along with speed to the procedure of MAP viability assessment, in which the length of diagnosis decreased from 48 h (PMS-phage assay) to almost 7 h with a LOD of 10 MAP cells per 50 mL [ 87 ] or 10 mL [ 88 ] milk. Although any viable mycobacterial species that could be infected with D29 might be recovered, and their DNA would be transferred to the lysate, this is the qPCR analysis that influences the specificity of the assay remarkably.…”

“…Furthermore, the recent two prophages carry genes that contribute to the production of virus-like particles [96]. Lytic (Clear plaques) [26] Yes [27] L5/Siphoviridae [97] Superinfection-stable lysogens, transformation of slow-growing mycobacteria, immobilized tail protein (Gp6) [40][41][42], three tRNA genes [27] A/A2 [40][41][42] Isolated from lysogenic strain of M. smegmatis [98] 63.2 [98] M. smegmatis mc 2 155, M. tuberculosis [44] Temperate [99] Yes [98] PDRPv/Siphoviridae [100] Antimicrobial profiles [100], circular permuted dsDNA [ M. smegmatis mc 2 155, M. tuberculosis [83], MAP, M. bovis, M. fortitum [88] Lytic [83] Yes [83] BPs/Siphoviridae [57] Ultra-small genetic elements G [57,103] Soil 66.6 [57] M. smegmatis mc 2 155 [57], M. tuberculosis [57] Temperate [57] Yes [57] Angel/Siphoviridae [57] Ultra-small genetic elements G [57,103] Soil 66.6 [57] M. smegmatis mc 2 155 [57], M. tuberculosis [57] Temperate [57] Yes [57] Halo/Siphoviridae [57] Ultra-small genetic elements G [57,103] Soil 66.7 [61] M. smegmatis mc 2 155 [57], M. tuberculosis [57] Temperate [57] Yes [57] ZoeJ/Siphovirid...…”