Identification of Corynebacterium pseudotuberculosis from sheep by PCR-restriction analysis using the RNA polymerase ß-subunit gene (rpoB).

Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, has a high prevalence in many regions of the world, including Argentina and Brazil. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase ß-subunit gene (rpoB). All available CorynebacteriumrpoB sequences were analyzed by computer-assisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and StuI clearly differentiated C. pseudotuberculosis from sixty-one other Corynebacterium species. This method was successfully applied to identify twelve wild C. pseudotuberculosis ovine isolates and one caprine isolate. It was also used to differentiate C. pseudotuberculosis from Arcanobacterium pyogenes, an ovine pathogen with similar clinical characteristics. These results indicate that this new molecular method can be used for the reliable identification of the pathogen, essential for the timely detection of infected animals and for epidemiological studies.

[Molecular study of Argentine strains of Bacillus anthracis]. / Estudio molecular de cepas argentinas de Bacillus anthracis.

Bacillus anthracis is one of the most monomorphic bacteria known and epidemiological studies of this microorganism have been hampered by the lack of molecular markers. For the genotyping of fourteen Argentine field strains and the vaccine strain Steme 34F2 the presence or absence of the virulence plasmids as well as vrrA locus containing a variable-number tandem repeat (VNTR) and presenting a polymorphism involving five variants, were analyzed. Strains were isolated from cows, sheep and pigs during outbreaks occurred in Buenos Aires, Entre Ríos, Santa Fe and La Pampa in the past fifty years. All of the field strains presented plasmids pXO1 and pXO2, except for a strain isolated from pig that only presented plasmid pXO2. All the strains and the vaccine strain belonged to the same VNTR variant that was defined by sequencing the vrrA locus from three of the isolates and the strain 34F2. These sequences were completely identical and corresponded to the variant VNTR4. Thus, the fourteen Argentine B. anthracis strains studied showed great uniformity at molecular level even though they had been isolated from different mammal species within a wide time period and covering an extensive geographical area.

[Molecular characterization of Brucella abortus strain 19 and its application for controlling biologics]. / Tipificación molecular de Brucella abortus cepa 19 y su aplicación al control de biológicos.

Brucella abortus is the etiological agent of bovine brucellosis. The strain 19 used in vaccine elaboration can be identified through a deletion in the eri region associated with its susceptibility to erythritol. We optimized a PCR assay for specific characterization of this strain. The method described here is a rapid procedure that enables identification of B. abortus, and simultaneous differentiation of the strain 19 from other B. abortus biovar 1 strains. We applied the assay to detect the strain 19 in vaccines against B. abortus produced in Argentina. Thq results show this method could be used to follow vaccine seed cultures of this strain. The methodology could also contribute to reduce the risk of a laboratory-acquired infection and could be of great help as a routine test for confirmation of B. abortus in non related vaccines.

Tipificación molecular de Brucella abortus cepa 19 y su aplicación al control de biológicos / Molecular characterization of Brucella abortus strain 19 and its application for controlling biologics

Brucella abortus es el agente etiológico de la brucelosis bovina. La cepa 19, utilizada en la elaboración de vacunas, puede ser identificada a través de una deleción en la región eri asociada con la sensibilidad al eritritol. Se optimizó un ensayo de PCR para caracterizar específicamente esta cepa. El método que describimos es un procedimiento rápido para identificar B. abortus y simultáneamente diferenciar la cepa 19 de otras cepas de B. abortus biovar 1. Hemos aplicado este ensayo para la detección de la cepa 19 en vacunas contra la brucelosis bovina elaboradas en Argentina. Los resultados indican que este método podría ser útil para el seguimiento de las cepas madres y semillas utilizadas en la producción industrial de esta vacuna. Esta metodología también contribuiría a la reducción del riesgo de la infección adquirida en el laboratorio y podría aplicarse como prueba de rutina para confirmar la presencia de B. abortus en vacunas no relacionadas.

Brucella abortus is the etiological agent of bovine brucellosis. The strain 19 used in vaccine elaboration can be identified through a deletion in the eri region associated with its susceptibility to erythritol. We optimized a PCR assay for specific characterization of this strain. The method described here is a rapid procedure that enables identification of B. abortus, and simultaneous differentiation of the strain 19 from other B. abortus biovar 1 strains. We applied the assay to detect the strain 19 in vaccines against B. abortus produced in Argentina. The results show this method could be used to follow vaccine seed cultures of this strain. The methodology could also contribute to reduce the risk of a laboratory-acquired infection and could be of great help as a routine test for confirmation of B. abortus in non related vaccines.

Tipificación molecular de Brucella abortus cepa 19 y su aplicación al control de biológicos. / [Molecular characterization of Brucella abortus strain 19 and its application for controlling biologics]

Brucella abortus is the etiological agent of bovine brucellosis. The strain 19 used in vaccine elaboration can be identified through a deletion in the eri region associated with its susceptibility to erythritol. We optimized a PCR assay for specific characterization of this strain. The method described here is a rapid procedure that enables identification of B. abortus, and simultaneous differentiation of the strain 19 from other B. abortus biovar 1 strains. We applied the assay to detect the strain 19 in vaccines against B. abortus produced in Argentina. Thq results show this method could be used to follow vaccine seed cultures of this strain. The methodology could also contribute to reduce the risk of a laboratory-acquired infection and could be of great help as a routine test for confirmation of B. abortus in non related vaccines.

Aeromonas salmonicida subsp. pectinolytica subsp. nov., a new pectinase-positive subspecies isolated from a heavily polluted river.

Aeromonas strains which phenotypically and genetically belong to the Aeromonas salmonicida species but that according to their phenotypic properties constitute a new subspecies have been isolated from the water of a heavily polluted river, the Matanza river, situated near the central district of Buenos Aires city. These strains were ascribed to the A. salmonicida species by using 65 biochemical tests and by DNA-DNA hybridization. They produce acid from -sorbitol, an unusual biochemical property found in a few members of the A. salmonicida species. They also utilize urocanic acid and do not ferment L-rhamnose or utilize LD-lactate, and are elastase- and gluconate-negative. The DNA relatedness was over 70%, the current limit accepted for the phylogenetic definition of a species, to the described A. salmonicida subspecies and nearly 100% within the new group of Aeromonas strains. Phenotypic differentiation from other A. salmonicida subspecies was readily achieved using the following characteristics: growth at 37 degrees C, melanin production, indole and Voges-Proskauer assays, growth on KCN broth, mannitol and sucrose fermentation and gas from glucose. A remarkable property of the strains of the new group was their ability to degrade polypectate, an unusual feature among Aeromonas species in general. The complete 16S rRNA gene of one strain of the new group was sequenced. Comparison with rDNA sequences of Aeromonas members available in databases revealed a close relationship between this strain and strains belonging to A. salmonicida subsp. salmonicida, masoucida and achromogenes, in agreement with the biochemical data. Since the new A. salmonicida strains constitute a tight genomic group that can be identified by phenotypic properties it was concluded that they represent a new subspecies for which the name Aeromonas salmonicida subsp. pectinolytica is proposed. The type strain of A. salmonicida subsp. pectinolytica is 34melT (= DSM 12609T).

Coprothermobacter platensis sp. nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge.

A new anaerobic, proteolytic, moderately thermophilic bacterium, strain 3RT, was isolated from a methanogenic mesophilic reactor treating protein-rich wastewater. The cells were Gram-negative, non-spore-forming, non-motile rods. The DNA base composition was 43 mol% G + C. The optimum pH and temperature for growth were 7.0 and 55 degrees C respectively. The bacterium fermented gelatin, casein, bovine albumin, peptone and yeast extract. Glucose, fructose, sucrose, maltose and starch were poorly fermented. The major fermentation products from glucose were acetate, CO2 and H2 and, from gelatin, propionate was also detected. Growth on glucose was stimulated by thiosulfate, which was reduced to sulfide. Sulfate and nitrate were not reduced. 16S rRNA gene analysis revealed that the isolated bacterial strain was phylogenetically related to Coprothermobacter proteolyticus (96.3% sequence similarity), the only known species within the genus. DNA-DNA hybridization analysis demonstrated a very low level of homology, indicating that the isolated strain and C. proteolyticus were not related at species level. Therefore, it is proposed to classify the described strain in the genus Coprothermobacter as a new species, Coprothermobacter platensis. The type strain of C. platensis is strain 3RT (= DSM 11748T).