engleski

RESTRICTION ENZYME FINGERPRINTING IN STUDY OF SALMONELLOSIS IN MAN AND POULTRY

Salmonella infections appear to be one of the most typical examples of an enteric disease that is transmitted from animals to humans. Biochemical, serological and phage typing diagnostic methods of Salmonella infections in man and animals are traditional. Unfortunately they are not enough reliable to establish differences among isolates and can not satisfactory serve in epidemiological study.Genotyping analysis of the Salmonella by plasmids and chromosome DNA restriction profiles (RFLP) has been widely used in recent years.Altogether 63 different strains of Salmonella spp. were analysed regarding plasmid content and restriction pattern of the ones containing plasmid. Bacterial strains were biochemically confirmed by using the API20E system (Bio-Merieux, France) and were serologically characterised by the slide agglutination test with Salmonella polyvalent O and group H antisera (Institute of Immunology, Zagreb, Croatia). The most strains were identified as S. enteritidis (20 human and 20 chicken isolates), while the reaming 23 strains were S. virchow, S. lichfield, S. hadar, S. typhimurium and S. gallinarum-pulorum. Plasmids harboured by Salmonella were extracted by the method of Birnboim and Doly. They were separated in horizontal 0.8% (wt/vol.) agarose gels in 1xTAE buffer at 80V for 1h and visualised by staining with 0.5 mg of ethidium bromide per ml. Total plasmid DNA was digested with EcoRI (Boehringer Mannheim Biochemicals) according to the manufacturer's recommendations. There was no differences obtained pattern for human S. enteritidis strains. So restriction enzyme digests of several strains were coprecipitated (0.3 M Na-acetate, 2V EtOH, incubated 30 min at –70 C, centrifuged at 13000g, 30 min at +4 C) and subsequently electrophoresed in a 0.8% (wt/vol.) agarose gel in 1xTAE buffer at 80V for 1h. They were visualised by staining with 0.5 mg of ethidium bromide per ml. The same was used for S. enteritidis isolated from chicken.Analysing plasmid profile of all strains investigated, 9 did not have any plasmid, 51 strains had only one of 25 kpb, and 3 had an additional plasmid of 2.5 kpb. Plasmid profile of human S. enteritidis strains compared to chicken ones did not reveal significant differences. Restriction pattern of S. enteritidis either human or chicken origin digested with EcoRI showed differences according to origin. Plasmid analysis could not be used as sole tool for epidemiological study, probably because of plasmid instability that can be loosed during manipulation in the laboratory. Plasmid restriction analysis shows differences between strains of different origin. To be able to determine relationship between the strains more precisely restriction pattern analysis of total DNA is now being used. The sensitivity of this method is increased with the number of enzymes used in the analysis.

Restriction enzyme fingerprinting; salmonellosis; man; poultry

nije evidentirano