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Open Access Research

Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR

Taneli Tirkkonen12*, Timo Nieminen1, Terhi Ali-Vehmas1, Olli AT Peltoniemi1, Gerard J Wellenberg3 and Jaakko Pakarinen4

Author Affiliations

1 Faculty of Veterinary Medicine, Department of Production Animal Medicine, University of Helsinki, POB 66, FIN 00014, Helsinki, Finland

2 Present address of T. Tirkkonen: A-Farmers Ltd, POB 173, FIN 65101, Vaasa, Finland

3 Animal Health Service (GD-Deventer), Arnsbergstraat 7, PO Box 9, Deventer, 7400 AA, The Netherlands

4 Faculty of Agriculture and Forestry, Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland

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Acta Veterinaria Scandinavica 2013, 55:26  doi:10.1186/1751-0147-55-26

Published: 22 March 2013

Abstract

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log105 to log107Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 104–107 mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.

Keywords:
Mycobacterium; Pig; Porcine; Real-time qPCR; Quantification