Region of Difference 4 in Alpine Mycobacterium caprae Isolates Indicates Three Variants

Abstract

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...

Mycobacterial Pathogenomics and Evolution

“…Based on the distribution of the region of difference 9 (RD9) and the M. tuberculosis deleted region TbD1, three main lineages were defined (57). The lineage deleted for RD9 comprises M. africanum, prevalent in humans in West Africa, as well as a range of subspecies with animal reservoirs, such as Mycobacterium microti, a pathogen originally isolated from voles that also infects cats (61) and sporadically humans (62), Mycobacterium pinnipedii, which was isolated from fur seals and sea lions in different continents (63), Mycobacterium caprae, isolated from goats and deer (64,65), and M. bovis, the bovine tubercle bacillus, which can infect a wide range of wild and domestic animals (66). The spectrum of RD9-deleted tubercle bacilli was further enriched by the identification of Mycobacterium mungi (67), the dassie bacillus (68, 69), a chimpanzee isolate (70), and Mycobacterium orygis, which is found in antelopes but was also repeatedly isolated from human tuberculosis cases (40).…”

Bovine Tuberculosis in the Republic of Macedonia: Postmortem, Microbiological and Molecular Study in Slaughtered Reactor Cattle

“…In our approach using the SybrGreenPCR and melting curve analysis as described by Pinsky and Banaei (14), we identified 79 isolates with RD4-and 7 isolates with RD4+. During our research Domogalla et al (15) reported diversity in the RD4 in alpine M. caprae isolates and found three variants of the isolates: Allgau type (conserved RD4), Karwendel type (deletion of 5kb) and Lechtal type (deleted RD4). These variants were also confirmed in Bavarian and Austrian cattle and wildlife (red deer and fox) (31).…”

“…In order to identify whether the RD4-isolates were all M. bovis or the Macedonian M. caprae isolates have deleted RD4, all of the RD4-isolates were subjected to the protocol for detection of M. caprae specific SNPs in the lepA gene (15,16). From the 79 (RD4-) isolates, 8 isolates were identified as M. caprae.…”

“…During this study, Domogalla et al (15) detected RD4-isolates of M. caprae. Considering this, the isolates were further identified by the presence of specific single nucleotide polimorfisms (SNPs) in the lepA gene by the previously described protocols (15,16).…”

A Mycobacterium tuberculosis komplex archaikus DNS kimutatása emberi csontvázmaradványokból

“…fordul elő a környezetünkben, de a Mycobacterium tuberculosis komplex tagjai között található a legtöbb aerob, obligát patogén (Brites et al, 2015;Domogalla et al, 2013;Donoghue, 2008;Holloway et al, 2011;Mándi, 2013;. Az aktív tüdő-tbc talaján a megbetegedettek 3-5%-nál fejlődhet ki csonttuberkulózis, amely látható nyomot hagy a csontokon is.…”