We have assessed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification (Bruker) of nontuberculous mycobacteria from newly positive liquid cultures of respiratory samples. Twelve (22%) of 54 isolates were identified directly from liquid medium. After subculture and with manual laser operation, this rose to 49/54 isolates (91%). MALDI-TOF MS is less promising than previously suggested. N ontuberculous mycobacteria (NTM) are increasingly recognized as mostly opportunistic pathogens of humans. The most frequent disease manifestation is a chronic pulmonary infection (1). Since NTM are environmental microorganisms, their presence in pulmonary samples need not indicate disease per se. The clinical relevance of NTM isolation differs strongly by species (2). Hence, correct identification is of paramount importance (3).Identification of NTM is done mostly by using molecular tools. While accurate, these require a good laboratory infrastructure and trained personnel and are costly. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized identification in general bacteriology and has also been tried for NTM (3). Most studies of MALDI-TOF MS identification of NTM have applied it to pure cultures from strain collections (4) rather than new positive cultures from clinical samples from nonsterile sites.We assessed MALDI-TOF MS identification of NTM directly from new positive liquid cultures of respiratory samples in a mycobacterial disease reference clinic.Primary cultures of respiratory samples were performed with the Mycobacterium Growth Indicator Tube (MGIT) system (BD Bioscience, Erembodegem, Belgium), as well as on Löwenstein-Jensen slants, both at 37°C, after decontamination by the 1% Nacetyl-L-cysteine-sodium hydroxide method (3).All of the positive cultures that we obtained during the MarchApril 2014 and January-February 2015 periods that tested negative in the TBc-ID immunochromatography assay (BD Bioscience) were studied.We performed MALDI-TOF MS identification with the MALDI Biotyper (Bruker Daltonics, Bremen, Germany) platform. Protein extraction from liquid MGIT medium for MALDI-TOF MS analysis was performed by the manufacturer's MycoEx protocol. In short, we collected biomass by aspirating 1.2 ml of liquid medium from the bottom of the MGIT tube and transferring it to a reaction tube, which was then centrifuged for 2 min at 13,000 rpm (8,124 ϫ g; 43-mm rotor radius); the supernatant was pipetted off, the pellet was resuspended in 300 l of high-performance liquid chromatography grade water, and cells were then inactivated by boiling for 30 min. A 900-l volume of pure cold ethanol was then added, and the tube was vortexed and centrifuged for 2 min at 13,000 rpm. The supernatant was pipetted off. The last two steps were repeated once. The pellet was dried at room temperature before the addition of zirconium beads and 10 to 50 l of pure acetonitrile, depending on the pellet size. After 1 min of vortexing, we adde...

Docking the tails of lambs in long-tailed sheep breeds is a common practice worldwide. But this practice is associated with pain. Breeding for a shorter tail could offer an alternative. Therefore, this study aimed to analyze the natural tail length variation in the Merinolandschaf and to identify causal alleles for the short tail phenotype segregating within long-tailed breeds. We used SNP-based association analysis and haplotype-based mapping in 362 genotyped (Illumina OvineSNP50) and phenotyped Merinolandschaf lambs. Genome-wide significant regions were capture sequenced in 48 lambs and comparatively analyzed in various long and short-tailed sheep breeds and wild sheep subspecies. Here we show a SNP located in the first exon of HOXB13 and a SINE element located in the promotor of HOXB13 as promising candidates. These results enable more precise breeding towards shorter tails, improve animal welfare by amplification of ancestral alleles and contribute to a better understanding of differential embryonic development.

Background: Docking the tails of young lambs in long-tailed sheep breeds is a common practice worldwide. This practice is associated with pain, suffering and damage to the affected animals. Breeding for a shorter tail in long-tailed sheep breeds could offer one of the alternatives. This study aimed to analyze the natural tail length variation in the most common German Merino variety, and to identify possible causal alleles for the short tail phenotype segregating within a typical long-tailed breed.Results: Haplotype-based mapping in 362 genotyped (Illumina OvineSNP50) and pehenotyped Merinolandschaf lambs resulted in a genome-wide significant mapping at position 37,111,462 bp on sheep chromosome 11 and on chromosome 2 at position 94,538,115 bp (Oar_v4.0). Targeted capture sequencing of these regions in 48 selected sheep and comparative analyses of WGS data of various long and short-tailed sheep breeds as well as wild sheep subspecies identified a SNP and a SINE element as the promising candidates. The PCR genotyping of these candidates revealed complete linkage of both the candidate variants. The SINE element is located in the promotor region of HOXB13, while the SNP was located in the first exon of HOXB13 and predicted to result in a nonsynonymous mutation. Conclusions: Our approach successfully identified HOXB13 as candidate genes and the likely causal variants for tail length segregating within a typical long-tailed Merino breed. This would enable more precise breeding towards shorter tails, improve animal welfare by amplification of ancestral alleles and contribute to a better understanding of differential embryonic development.

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