Mycobacterium strains may cause serious infections in animals and humans. Large economic losses are caused by many mycobacterial species. A high risk of transmission of infection from animal to human exists. The knowledge of the exact pathogen concentration in mycobacterial contaminated pork might be an important parameter to give information on the infection risk for humans. A low mycobacterial porcine tissue content may be indicative for an environmental contamination. A high concentration of Mycobacterium cells in porcine tissue may indicate a higher risk for humans to become infected after consumption of infected pork and may therefore represent a higher zoonotic risk. Earlier authors have described qPCR and serological results based on experimental M. avium subspecies porcine infections [11, 18]. Miranda et al.  found M. avium subsp. paratuberculosis in four out of fifty examined tissue samples with PCR whereas Klanicova et al.  examined various purchased meat products for M. avium subspecies by qPCR. However to the best of our knowledge this is the first publication to report the quantification of mycobacterial content in naturally infected porcine tissue by qPCR and verifying it with another quantification method.
The problems connected to cultivation have increased the interest in culture-independent methods. Different microscopical methods have been applied to detect mycobacterial cells in animal and environmental originating samples . The tendency of mycobacterial cells to clump may hinder accurate microscopic as well as cultivation based quantification of Mycobacterium cells. The specificity and sensitivity of these cultivation/microscopy methods need to be significantly improved before they can be applied to the analysis and quantification of Mycobacterium cells from animal samples.
Therefore, new methods, such as quantitative PCR methods in combination with reliable DNA extraction methods, are required. In general, a variety of methods can be used for DNA isolation from animal samples, from boiling the sample in distilled water, autoclaving, disruption by glass beads or sonication, to the use of different enzymes and surfactants. However, isolation of nucleic acids from Mycobacterium cells is more difficult than from other microorganisms because of the thick peptidoglycan layer characteristic to the mycobacterial cell wall, which makes it resistant to a number of lysis buffers. Moreover, certain samples of animal origin may contain various inhibitors of PCR amplification .
A number of published protocols and commercial kits are available for the extraction of DNA from mycobacterial isolates. However, most of these cannot be used to the determination of mycobacterial DNA from porcine tissues. Commercial kits show an excellent correlation with 16S rDNA sequencing results representing rapid, specific and versatile species identification of the most prevalent NTM-species from cultures . Recently several novel qPCR methods have been developed for the detection of Mycobacterium strains from human, animal and environmental originating samples [10, 18]. However, less laborous and complex methods are needed.
Our DNA extraction and real-time quantitative PCR protocol is a simple and effective method for the detection and quantification of Mycobacterium strains in porcine tissues. The DNA extraction method was found to be efficient in extracting different amounts of M. avium subspecies spiked into healthy pig liver and is also suitable to detect M. avium subspecies in porcine tissue samples. The qPCR method was shown to provide reliable quantitative results when M. avium concentrations ranged from log105 to log107 (Figure 1). These tissue concentrations can be regarded as relatively high, but to the best of our knowledge no exact information regarding the smallest zoonotic infection dose for M. avium subspecies is available. The total mycobacterial count using the developed extraction method for tissue lesions was as high as 107 cells per gram, indicating an infection.