The lack of complete genome sequence information for Mycobacterium caprae complicates a robust differentiation of M. caprae and Mycobacterium bovis. In this study, the presence or absence of M. caprae-specific single nucleotide polymorphisms in lepA and gyrB genes was assessed. The region of difference 4 (RD4) was analyzed for the identification and characterization of M. caprae. Molecular characteristics were evaluated in 12 recent M. caprae isolates from livestock and wildlife collected over a 3-year period in Bavaria, Germany. Conventional PCR strategies, sequence analysis of PCR fragments, and data from a next-generation sequencing approach together with variable-number tandem-repeat genotyping were utilized. Single nucleotide polymorphisms in the lepA and gyrB genes indicating the presence of M. caprae were detected in all the isolates. At least three different RD4 variants were found for Alpine M. caprae isolates. The results demonstrate that the RD4 region is rather heterogeneous in M. caprae genomes. As assumed by others, the presence of RD4 is critical for PCR-based differentiation of M. caprae from M. bovis, but in addition, the observed variability of RD4 allows the identification of M. caprae genotypes and may be indicative of a geographical-type appearance.M ycobacterium caprae was first described in samples from goats in Spain (1) and was established in 2003 as a unique species in the Mycobacterium tuberculosis complex (MTC) (2) based on the presence or absence of regions of difference (RDs) and single nucleotide polymorphisms (SNPs) (3). M. bovis and M. caprae are the most relevant pathogens for livestock and wildlife large animal hosts. In some countries bovine tuberculosis (TB) is still a problem, particularly when a wildlife reservoir exists and TB transmission from wildlife to cattle and cattle to wildlife on the pasture is a permanent danger (4). Three wildlife reservoirs of TB caused by M. bovis have been described extensively (4-7). To date a wildlife reservoir harboring M. caprae has not been described. However, there have been several reports about M. caprae infections in red deer (Cervus elaphus) (8-12) and wild boars (Sus scrofa) (8, 9, 13) from continental European countries, mainly from Spain (8, 9) and from countries with Alpine wildlife habitats (10-13).During the years 2007 and 2012 in the Bavarian Alpine "Allgäu" region, an area with plenty of cattle summer pastures also habituated by red deer, infections in cattle have been found to be mainly caused by M. caprae, whereas M. bovis was more frequent among animals found in the northern part of Germany, according to international unpublished findings of the national animal infectious disease reporting system, TierSeuchenNachrichten 3.0 (TSN 3.0).The majority of molecular strategies for M. caprae differentiation refer to sporadically examined M. caprae isolates (3,14,15). In addition, a complete genome sequence of M. caprae is not available in the NCBI GenBank so far, in contrast to genome sequences of M. tuberculosis and M. bovis. Co...

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