Shedding of Mycobacterium caprae by wild red deer (Cervus elaphus) in the Bavarian alpine regions, Germany.

The number of natural infections with Mycobacterium caprae in wildlife and in cattle in the Bavarian and Austrian alpine regions has increased over the last decade. Red deer (Cervus elaphus) have been recognized as maintenance reservoir; however, the transmission routes of M. caprae among and from naturally infected red deer are unknown. The unexpected high prevalence in some hot spot regions might suggest an effective indirect transmission of infection. Therefore, this study was undertaken to diagnose the occurrence of M. caprae in faeces and secretions of red deer in their natural habitat. A total of 2,806 red deer hunted in this region during 2014-2016 were included in this study. After pathological examination, organs (lymph nodes, lung, heart), excretions and secretions (faeces, urine, saliva and tonsil swabs) were further investigated by qPCR specific for Mycobacterium tuberculosis complex (MTC), M. bovis and M. caprae. Samples tested positive by qPCR were processed for culturing of mycobacteria. In total, 55 (2.0%) animals were confirmed positive for M. caprae by pathological examination, PCR and culturing of the affected organ material. With the exception of one sample, all of the secretion and excretion samples were negative for mycobacteria of the Mycobacterium tuberculosis complex (MTC). From one red deer, M. caprae could be isolated from the heart sac as well as from the faeces. Whole-genome sequencing confirmed that both strains were clonally related. This is the first confirmation that M. caprae can be shed with the faeces of a naturally infected red deer. However, further studies focusing on a higher number of infected animals, sample standardization and coordinated multiple sampling are necessary to improve the understanding of transmission routes under natural conditions.

Matrixlysis, an improved sample preparation method for recovery of Mycobacteria from animal tissue material.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, is the main causative agent of bovine tuberculosis in alpine regions. Bacterial culture is the gold standard in bovine tuberculosis diagnostic but takes up to twelve weeks. This increases the time and costs for stocks affected with bovine tuberculosis. Hence this study focused on the implementation of a fast and precise mycobacterial detection method and compared it with currently used methods. Matrix lysis is a chemical lysis using high concentrations of urea to solubilize bovine and red deer tissue and was used to detect even smallest amounts or non-visible lesions of mycobacteria. A total of 64 samples collected from 44 animals (37 red deer and 7 cattle) were tested by Matrix lysis. Forty-three of these samples were used for Mycobacterium tuberculosis complex detection by quantitative PCR and other 21 for subtyping the genetically different variants of M. caprae. Furthermore, three Matrix lysis samples were used for Next Generation Sequencing. Our results confirm that Matrix lysis is a fast and precise method for detecting Mycobacterium tuberculosis complex in native tissue samples. However, at the moment it reaches its limits when the samples were analyzed by Next Generation Sequencing and RD4 subtyping.