Abstract
BackgroundBovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation. In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. As mycobacteria tend to concentrate in granuloma and the infected tissue in early stages of infection does not necessarily show any visible lesions, it is likely that DNA extraction from only small tissue samples (20–40 mg) of a randomly chosen spot from the organ and following PCR testing may result in false negative results. In this study two DNA extraction methods were developed to process larger sample volumes to increase the detection sensitivity of mycobacterial DNA in animal tissue.The first extraction method is based on magnetic capture, in which specific capture oligonucleotides were utilized. These nucleotides are linked to magnetic particles and capture Mycobacterium-tuberculosis-complex (MTC) DNA released from 10 to 15 g of tissue material. In a second approach remaining sediments from the magnetic capture protocol were further processed with a less complex extraction protocol that can be used in daily routine diagnostics. A total number of 100 tissue samples from 34 cattle (n = 74) and 18 red deer (n = 26) were analyzed with the developed protocols and results were compared to the prescribed protocol.ResultsAll three extraction methods yield reliable results by the real-time PCR analysis. The use of larger sample volume led to a sensitivity increase of DNA detection which was shown by the decrease of Ct-values. Furthermore five samples which were tested negative or questionable by the official extraction protocol were detected positive by real time PCR when the alternative extraction methods were used. By calculating the kappa index, the three extraction protocols resulted in a moderate (0.52; protocol 1 vs 3) to almost perfect agreement (1.00; red deer sample testing with all protocols).ConclusionBoth new methods yielded increased detection rates for MTC DNA detection in large sample volumes and consequently improve the official diagnostic protocol.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0816-2) contains supplementary material, which is available to authorized users.
Highlights
Bovine tuberculosis, which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany
Culture material was used for DNA extraction and implemented as positive control showing average Ct-values of CtHeli = 18.30 (ß-actin + Heli) and CtIS 1081 = 17.53 (ß-actin + insertion sequence 1081 (IS 1081))
Establishing the magnetic capture protocol by utilizing artificially spiked lymph nodes Twenty samples were processed according to protocol 1 and tested negative for MTC DNA and only eight samples were MTC-positive
Summary
Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. With participation of interleukinproducing dendritic cells, macrophages, lymphocytes, neutrophils and epitheloid cells, the host immune system forms a tuberculoid granuloma [9] This small granuloma prevents further spread of the pathogen to surrounding tissues and it can stay arrested for a long time. In contrast to the typical granuloma formation mostly in lymphoid tissue, non-visible lesions (NVL) may be present in many individuals after infection with bTB These individuals show immune reactions in the tuberculin skin test in an early state of infection [9, 12]. Due to the localized manifestation of mycobacterial infection sometimes as only very small granuloma or tiny lesions, visual screening of carcasses after slaughter is challenging [13, 14]
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