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Applied and Environmental Microbiology, June 2009, p. 3896-3902, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.00294-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Optimization of a Phage Amplification Assay To Permit Accurate Enumeration of Viable Mycobacterium avium subsp. paratuberculosis Cells{triangledown}

Antonio Foddai, Christopher T. Elliott, and Irene R. Grant*

Institute of Agri-Food and Land Use, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, United Kingdom

Received 5 February 2009/ Accepted 21 April 2009

A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 ± 36.8 min at 37°C compared to 63 ± 17.5 min for M. smegmatis mc2 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37°C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc2 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

* Corresponding author. Mailing address: Institute of Agri-Food and Land Use, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, United Kingdom. Phone: 44 28 9097 2109. Fax: 44 28 9097 5877. E-mail: i.grant{at}qub.ac.uk

{triangledown} Published ahead of print on 24 April 2009.

Applied and Environmental Microbiology, June 2009, p. 3896-3902, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.00294-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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