RESUMO
Repetitive elements in DNA sequences are a hallmark of Apicomplexan protozoa. A genome-wide screening for Tandem Repeats was conducted in Toxoplasma gondii and related Coccidian parasites with a novel strategy to assess compositional bias. A conserved pattern of GC skew and purine-pyrimidine bias was observed. Compositional bias was also present at the protein level. Glutamic acid was the most abundant amino acid in the purine (GA) rich cluster, while Serine prevailed in pyrimidine (CT) rich cluster. Purine rich repeats, and consequently glutamic acid abundance, correlated with high scores for intrinsically disordered protein regions/domains. Finally, variability was established for repetitive regions within a well-known rhoptry antigen (ROP1) and an uncharacterized hypothetical protein with similar features. The approach we present could be useful to identify potential antigens bearing repetitive elements.
Assuntos
Proteínas de Protozoários , Sequências de Repetição em Tandem , Toxoplasma , Toxoplasma/genética , Sequências de Repetição em Tandem/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Genoma de Protozoário , Composição de BasesRESUMO
In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
Assuntos
Animais , Humanos , Brucella/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas Bacteriológicas/métodos , Brucella/genética , Brucelose/diagnóstico , Primers do DNA/genética , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/diagnóstico , Fatores de TempoRESUMO
Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.
Assuntos
Animais , Bovinos , Variação Genética , Repetições Minissatélites , Tipagem Molecular/métodos , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/microbiologia , Argentina/epidemiologia , Doenças dos Bovinos/microbiologia , Genótipo , Cabras , Doenças das Cabras/microbiologia , Epidemiologia Molecular , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Ovinos , Doenças dos Ovinos/microbiologiaRESUMO
Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.