RESUMEN
This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.
Asunto(s)
Infecciones por Actinomycetales/microbiología , Actinomycetales/clasificación , Actinomycetales/genética , Dermatitis/microbiología , Electroforesis en Gel de Campo Pulsado/métodos , Técnica del ADN Polimorfo Amplificado Aleatorio/métodos , Infecciones por Actinomycetales/veterinaria , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Ciervos , Desoxirribonucleasas de Localización Especificada Tipo II , Dermatitis/veterinaria , Electroforesis en Gel de Campo Pulsado/normas , Genes Bacterianos , Caballos , Técnica del ADN Polimorfo Amplificado Aleatorio/normas , Reproducibilidad de los Resultados , Mapeo Restrictivo , Ovinos , Enfermedades de las Ovejas/microbiologíaRESUMEN
A random amplified polymorphic DNA (RAPD) procedure was used to identify a specific 0.6 kb DNA fragment unique to Dermatophilus congolensis. This 0.6 kb fragment was evaluated as a specific DNA probe and used to design oligonucleotide primers for polymerase chain reaction (PCR) amplification. The nucleotide sequences adjacent to this DNA fragment were determined by inverse PCR allowing the identification of a 4.1 kb sequence. Analysis of this revealed a complete open reading frame (ORF) with a high similarity to an alkaline ceramidase from Pseudomonas aeruginosa. The molecular weight of the enzyme derived from the predicted amino acid sequence is 74,662 Da, its pI is 9.81. The predicted N-terminal sequence of the enzyme contains a signal sequence indicating that the enzyme is exported by the bacterium. Since ceramides have important protective and cell regulatory roles in the epidermis we suggest that this ceramidase may have a role in the pathogenesis of dermatophilosis. It is the first completely sequenced gene described for D. congolensis.
Asunto(s)
Actinobacteria/enzimología , Infecciones por Actinomycetales/veterinaria , Amidohidrolasas/genética , Proteínas Bacterianas/genética , Actinobacteria/genética , Infecciones por Actinomycetales/microbiología , Amidohidrolasas/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Secuencia de Bases , Southern Blotting , Ceramidasas , ADN Bacteriano/química , ADN Bacteriano/genética , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Técnica del ADN Polimorfo Amplificado Aleatorio , Alineación de SecuenciaRESUMEN
A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.
