RESUMO
Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease.
Assuntos
Antígenos de Bactérias/metabolismo , Códon , Lactobacillus/genética , Mycobacterium avium subsp. paratuberculosis/genética , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Sequência de Bases , Bovinos , Lactobacillus/metabolismo , Dados de Sequência Molecular , Paratuberculose/microbiologia , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We describe a novel HRM-PCR (high-resolution melting) assay capable of the accurate identification of the G2576T point mutation in domain V of the 23S rRNA genes attributed to linezolid resistance in Staphylococcus epidermidis. This rapid method demonstrated 100% correlation with the previously established NheI restriction digest assay.
Assuntos
Acetamidas/farmacologia , Antibacterianos/farmacologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Oxazolidinonas/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genética , Temperatura de Transição , DNA Ribossômico/genética , Humanos , Linezolida , Testes de Sensibilidade Microbiana/métodos , Mutação Puntual , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Sensibilidade e EspecificidadeRESUMO
In this study, we describe a novel HRM-PCR assay that clearly differentiates the two main types of Mycobacterium avium subsp. paratuberculosis (cattle and sheep) based on the polymorphic variation of a previously described tandem repeat. This modern genotyping technique has several advantages over alternative methods, including cost, ease of use and rapidity.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/métodos , Doenças dos Ovinos/microbiologia , Animais , Técnicas de Tipagem Bacteriana/economia , Bovinos , Genótipo , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/genética , Reação em Cadeia da Polimerase/economia , Polimorfismo Genético , Ovinos , Sequências de Repetição em TandemRESUMO
BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic gastroenteritis affecting many species. Johne's disease is one of the most widespread and economically important disease of ruminants. Since 1992 and the opening of the European market, the exposure and the transmission of MAP in cattle herds considerably increased. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. In total, 290 cattle from seven Irish herds with either a history or a strong likelihood of paratuberculosis infection were selected by a veterinary team over 2 years. Faecal samples (290) were collected and screened for MAP by a conventional culture method and two PCR assays. In order to further evaluate the usefulness of molecular testing, a nested PCR was also assessed. RESULTS: M. paratuberculosis was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. A complete correlation (100%) was observed between the results of both molecular targets (IS900 and ISMAP02). Sensitivity was increased by ~10% with the inclusion of a nested PCR for ISMAP02 (29 further samples were positive). When culturing and PCR were retrospectively compared, every culture positive faecal sample also yielded a PCR positive result for both targets. Alternatively, however not every PCR positive sample (n = 105, 36%) produced a corresponding culture isolate. Interestingly though when analysed collectively at the herd level, the correlation between culture and PCR results was 100% (ie every herd which recorded at least 1 early PCR +ve result later yielded culture positive samples within that herd). CONCLUSION: PCR on bovine faecal samples is a fast reliable test and should be applied routinely when screening for MAP within herds suspected of paratuberculosis. Nested PCR increases the threshold limit of detection for MAP DNA by approximately 10% but proved to be problematic in this study. Although slow and impractical, culturing is still regarded as one of the most reliable methods for detecting MAP among infected cattle.