Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.The most significant mycobacterial diseases of free-living, captive, and farmed red deer (Cervus elaphus) are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp. paratuberculosis, and avian tuberculosis, caused principally by M. avium subsp. avium (20).Differential diagnoses of infections by M. bovis, M. avium subsp. paratuberculosis, and M. avium subsp. avium are essential because the epidemiology, zoonotic concern, and prevention and control methods are different for each of these mycobacterial infections (20). In deer, tuberculous lesions are not pathognomonic of M. bovis infection and are actually indistinguishable from lesions due to M. avium subsp. paratuberculosis or M. avium subsp. avium infection because of their gross and microscopic similarities. Moreover, the actual differential diagnosis of these mycobacterial infections is difficult due to similarities in the clinical expression and pathology associated with these infections. Antigenic similarity ...
Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis. The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factor lef [from pXO1], and the capsule-biosynthesis-related capA [from pXO2]) and a conserved sequence in the adenylate cyclase gene (adk) for the Bacillus cereus group. B. anthracis-specific RPA assays were tested first with purified genomic DNAs (n = 60), including 11 representatives of B. anthracis, and then with soil (n = 8) and white powder (n = 8) samples spiked with inactivated B. anthracis spores and/or other biological agents. The RPA assays were also tested in another laboratory facility, which blindly provided DNA and lysate samples (n = 30, including 20 B. anthracis strains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 102 genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 and adk RPA assays were assessed under field conditions with a series of surface swabs (n = 13, including 11 swabs contaminated with B. thuringiensis spores) that were blindly brought to the field laboratory by a chemical, biological, radiological, and nuclear (CBRN) sampling team. None of the 13 samples, except the control, tested positive for B. anthracis, and all samples that had been harvested from spore-contaminated surfaces tested positive with the adk RPA assay. All three B. anthracis-specific RPA assays proved suitable for rapid and reliable identification of B. anthracis and therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release of B. anthracis spores and a hoax attack involving white powder.IMPORTANCE In recent decades, particularly following the 11 September 2001 and Amerithrax attacks, the world has experienced attempts to sow panic and chaos in society through thousands of white-powder copycats using household powders to mimic real bioterrorism attacks. In such circumstances, field-deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test the samples directly using a fast and reliable assay for detection of the presence of B. anthracis. While this would not preclude further confirmatory tests from being performed in reference laboratories, it would bring useful, timely, and relevant information to local crisis managers and help them make appropriate decisions without having to wait for quantitative PCR results (with turnaround times of a few hours) or phenotypic identification and sequencing (with turnaround times of a few days). In the current investigation, we developed a set of isothermal RPA assays for the rapid screening and identification of B. anthracis in powders and soil samples, with the purpose of discriminating a deliberate release of B. anthracis spores from a hoax attack involving white powder; th...
Motivation: Converting a pyrosequencing signal into a nucleotide sequence appears highly challenging when signal intensities are low (unitary peak heights ) or when complex signals are produced by several target amplicons. In these cases, the pyrosequencing software fails to provide correct nucleotide sequences. Accordingly, the objective was to develop the AdvISER-PYRO algorithm, performing an automated, fast and reliable analysis of pyrosequencing signals that circumvents those limitations.Results: In the current mycobacterial amplicon genotyping application, AdvISER-PYRO performed much better than the pyrosequencing software in the following two situations: when converting Single Amplicon Sample (SAS) signals into a correct single sequence (97.2% versus 56.5%), and when translating Multiple Amplicon Sample (MAS) signals into the correct sequence pair (74.5%).Availability: AdvISER-PYRO is implemented in an R package (http://sites.uclouvain.be/md-ctma/index.php/softwares) and can be used in broad range of clinical applications including multiplex pyrosequencing and oncogene re-sequencing in heterogeneous tumor cell samples.Contact: jerome.ambroise@uclouvain.be or jean-luc.gala@uclouvain.be
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