Purpose S. aureus is the principal agent of bovine mastitis and it is known that the clinical outcome can vary in relation to the strains involved in the mammary infection. The aim of this work, funded by the Region Lombardia as "Mastfield" project, was to develop a diagnostic protocol for the identification of genetic subtypes of S. aureus responsible for bovine mastitis, with particular pathogenicity and infectivity. Methods S. aureus strains associated with intra-mammary infections were isolated from eight "case" (> 28% of infected cows) and eight "control" (< 4% of infected cows) herds. A total of 2015 cows were included in the study, milk samples were cultured with standard methods and the DNA was extracted from S. aureus strains using a protocol described in literature. A total of 650 strains isolated from single infected quarters were analyzed, 24 and 626 from "control" and "case" herds, respectively. All samples were analyzed both by a method based on the amplification of the 16S-23S rRNA intergenic spacer region (RS-PCR) and by multiplex PCR, in order to verify the presence of genes encoding for enterotoxins and other virulence genes. A subset of 55 strains representing the isolates from "control" and from "case" herds was also analyzed by ribotyping (automatic Riboprinter) and by protein profiling using MALDI-TOF MS. Results The RS-PCR analysis revealed 25 different profiles. In "case" herds, the analysis showed the presence of S. aureus belonging to one genetic profile spreading in the group. In 3 "case" herds the analysis showed more than one profile (maximum three different) one of which was in any case absolutely predominant. Moreover, in five out of the eight "case" herds, the RS-PCR analysis identified the genotype indicated in literature as characteristic of highly diffusive and pathogenic strains. Seven out of the eight "control" herds, revealed only one S. aureus profile, while in the eighth herd two different profiles circulated. Except for four strains, ribotyping gave a response similar to RS-PCR analysis in a cluster analysis. And more, MALDI-TOF analysis, proved to be able to group the S. aureus isolates with results close to RS-PCR. Finally, all the isolates from "case" herds possessed genes encoding for enterotoxins and showed genetic polymorphisms characteristic of pathogenic strains, while isolates from "control" herds were non-enterotoxigenic. Conclusions Our results demonstrated the validity of RS-PCR as a rapid tests for molecular typing of S. aureus strains isolated from bovine mastitis. The preliminary results of the ribotyping analisis and MALDI-TOF techniques showed that they could also used in molecular profiling of the strains, increasing the specificity of the analysis. Theese data could be usefull for the selection of a restricted panel of markers of virulence and diffusibility of the S. aureus strains isolated in the field.
Bovine mastitis: innovative molecular systems for Staphylococcus aureus genetic subtypes identification
Paola Cremonesi;Emanuele Capra;
2013
Abstract
Purpose S. aureus is the principal agent of bovine mastitis and it is known that the clinical outcome can vary in relation to the strains involved in the mammary infection. The aim of this work, funded by the Region Lombardia as "Mastfield" project, was to develop a diagnostic protocol for the identification of genetic subtypes of S. aureus responsible for bovine mastitis, with particular pathogenicity and infectivity. Methods S. aureus strains associated with intra-mammary infections were isolated from eight "case" (> 28% of infected cows) and eight "control" (< 4% of infected cows) herds. A total of 2015 cows were included in the study, milk samples were cultured with standard methods and the DNA was extracted from S. aureus strains using a protocol described in literature. A total of 650 strains isolated from single infected quarters were analyzed, 24 and 626 from "control" and "case" herds, respectively. All samples were analyzed both by a method based on the amplification of the 16S-23S rRNA intergenic spacer region (RS-PCR) and by multiplex PCR, in order to verify the presence of genes encoding for enterotoxins and other virulence genes. A subset of 55 strains representing the isolates from "control" and from "case" herds was also analyzed by ribotyping (automatic Riboprinter) and by protein profiling using MALDI-TOF MS. Results The RS-PCR analysis revealed 25 different profiles. In "case" herds, the analysis showed the presence of S. aureus belonging to one genetic profile spreading in the group. In 3 "case" herds the analysis showed more than one profile (maximum three different) one of which was in any case absolutely predominant. Moreover, in five out of the eight "case" herds, the RS-PCR analysis identified the genotype indicated in literature as characteristic of highly diffusive and pathogenic strains. Seven out of the eight "control" herds, revealed only one S. aureus profile, while in the eighth herd two different profiles circulated. Except for four strains, ribotyping gave a response similar to RS-PCR analysis in a cluster analysis. And more, MALDI-TOF analysis, proved to be able to group the S. aureus isolates with results close to RS-PCR. Finally, all the isolates from "case" herds possessed genes encoding for enterotoxins and showed genetic polymorphisms characteristic of pathogenic strains, while isolates from "control" herds were non-enterotoxigenic. Conclusions Our results demonstrated the validity of RS-PCR as a rapid tests for molecular typing of S. aureus strains isolated from bovine mastitis. The preliminary results of the ribotyping analisis and MALDI-TOF techniques showed that they could also used in molecular profiling of the strains, increasing the specificity of the analysis. Theese data could be usefull for the selection of a restricted panel of markers of virulence and diffusibility of the S. aureus strains isolated in the field.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
