Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Microbiology and Antimicrobial Resistance Veterinary Research Institute Brno Czech Republic

Laboratory of Neurobiology and Pathological Physiology Institute of Animal Physiology and Genetics Czech Academy of Sciences Libechov Czech Republic

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Whittington RJ, Marshall DJ, Nicholls PJ, Marsh AB, Reddacliff LA. Survival and dormancy of Mycobacterium avium subsp paratuberculosis in the environment. Appl. Environ. Microb. 2004;70:2989–3004. doi: 10.1128/Aem.70.5.2989-3004.2004. PubMed DOI PMC

Cocito C, et al. Paratuberculosis. Clin. Microbiol. Rev. 1994;7:328–345. doi: 10.1128/CMR.7.3.328. PubMed DOI PMC

Whittington RJ, Sergeant ES. Progress towards understanding the spread, detection and control of Mycobacterium avium subsp paratuberculosis in animal populations. Aust. Vet. J. 2001;79:267–278. doi: 10.1111/j.1751-0813.2001.tb11980.x. PubMed DOI

Sweeney RW, Whitlock RH, Rosenberger AE. Mycobacterium-paratuberculosis isolated from fetuses of infected cows not manifesting signs of the disease. Am. J. Vet. Res. 1992;53:477–480. PubMed

Gill CO, Saucier L, Meadus WJ. Mycobacterium avium subsp paratuberculosis in dairy products, meat, and drinking water. J. Food Protect. 2011;74:480–499. doi: 10.4315/0362-028x.Jfp-10-301. PubMed DOI

Dundee, L., Grant, I. R., Ball, H. J. & Rowe, M. T. Comparative evaluation of four decontamination protocols for the isolation of Mycobacterium avium subsp paratuberculosis from milk. Lett. Appl. Microbiol.33, 173–177, 10.1046/j.1472-765x.2001.00979.x (2001). PubMed

Elguezabal N, et al. Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods. Appl. Environ. Microbiol. 2011;77:8615–8624. doi: 10.1128/AEM.05818-11. PubMed DOI PMC

Kralik P, Nocker A, Pavlik I. Mycobacterium avium subsp paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int. J. Food Microbiol. 2010;141:S80–S86. doi: 10.1016/j.ijfoodmicro.2010.03.018. PubMed DOI

Fittipaldi M, Nocker A, Codony F. Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. J. Microbiol. Methods. 2012;91:276–289. doi: 10.1016/j.mimet.2012.08.007. PubMed DOI

Soejima T, Iwatsuki K. Innovative use of palladium compounds to selectively detect live Enterobacteriaceae in milk by PCR. Appl. Environ. Microb. 2016;82:6930–6941. doi: 10.1128/Aem.01613-16. PubMed DOI PMC

Soejima T, Minami J, Xiao J, Abe F. Innovative use of platinum compounds to selectively detect live microorganisms by polymerase chain reaction. Biotechnol. Bioeng. 2016;113:301–310. doi: 10.1002/bit.25711. PubMed DOI

Fraisse A, et al. Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR. Int. J. Food Microbiol. 2018;269:64–74. doi: 10.1016/j.ijfoodmicro.2018.01.015. PubMed DOI

Randazzo, W., Vasquez-Garcia, A., Aznar, R. & Sanchez, G. Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids. Front. Microbiol.10.3389/fmicb.2018.01973 (2018). PubMed PMC

Cechova, M., Beinhauerova, M., Babak, V., Slana, I. & Kralik, P. A novel approach to the viability determination of Mycobacterium avium subsp. paratuberculosis using platinum compounds in combination with quantitative PCR. Front. Microbiol.12, 748337. 10.3389/fmicb.2021.748337 (2021). PubMed PMC

Sevilla IA, et al. Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay. J. Clin. Microbiol. 2015;53:930–940. doi: 10.1128/Jcm.03168-14. PubMed DOI PMC

Slana, I., Kralik, P., Kralova, A. & Pavlik, I. On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int. J. Food Microbiol.128, 250–257, doi:10.1016/j.ijfoodmicro.2008.08.013 (2008). PubMed

Macnevin WM, Kriege OH. Chelation of platinum group metals - spectrophotometric determination of palladium with ethylenediaminetetraacetic acid. Anal. Chem. 1954;26:1768–1770. doi: 10.1021/ac60095a024. DOI

Basallote MG, Vilaplana R, Gonzalezvilchez F. Synthesis and kinetic-study of palladium and platinum complexes with aminopolycarboxylate ligands. Polyhedron. 1994;13:1853–1858. doi: 10.1016/0277-5387(94)80007-3. DOI