title: A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays.
abstract: Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays.
  
  
  
  Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.
authors.affiliation: Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia.
authors.forename: Hannah B
authors.initials: HB
authors.lastname: Pooley
authors.affiliation: Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia.
authors.forename: Kumudika
authors.initials: K
authors.lastname: de Silva
authors.affiliation: Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia.
authors.forename: Auriol C
authors.initials: AC
authors.lastname: Purdie
authors.affiliation: Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Begg
authors.affiliation: Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Whittington
authors.affiliation: Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW, Australia karren.plain@sydney.edu.au.
authors.forename: Karren M
authors.initials: KM
authors.lastname: Plain
mesh_terms: D000818:Animals; D058491:Bacterial Load / Q000379:methods*; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D050296:Microbial Viability; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification* / Q000502:physiology; D000067996:RAW 264.7 Cells; D013997:Time Factors