Identification of Isolated Salmonella enterica Serotype gallinarum Biotype Pullorum and Gallinarum by PCR-RFLP

Abstract

Background: Salmonella spp. is the major bacterial pathogen in poultry and is responsible for significant economic losses of the poultry industry in many parts of the world. Among Salmonella spp., Salmonella gallinarum and Salmonella. pullorum are the most common causative agents of chicken salmonellosis resulting in high mortality and morbidity.

Objectives: The aim of this study was to identify S. gallinarum and S. pullorum by using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Materials and methods: In this study, 13 samples of Salmonella, isolated from local poultry, were obtained from Razi Type Culture Collection (RTCC). For the PCR-RFLP method based on the fliC gene, extracted DNA was used as a template for amplifying of the fliC gene (197bp) using specific primers. PCR products were subjected to digestion using Hinp1I restriction endonuclease.

Results: For the PCR, 197 bp fliC fragment was amplified from all 13 isolates. Ten out of 13 were S. gallinarum and the other three were S. pullorum. As part of the PCR-RFLP, two fragments were obtained (82 bp and 115 bp) for all S. gallinarum, whereas no digestion was observed in S. pullorum, and 197 bp fragment was seen.

Conclusions: PCR-RFLP with fliC gene and Hinp1I endonuclease were successfully applied to differentiate the two biotypes. The results suggested that this technique could be effective in detecting S. gallinarum and S. pullorum.

Keywords: Hinp1I Endonuclease; RFLP; Salmonella gallinarum; Salmonella pullorum.

Figure 1.. Electrophoresis of fliC Gene amplicons From S. gallinarum and S. pullorum Samples

M: 100 bp marker plus DNA ladder (Fermentas Inc.). SP: lanes 1 to 3; SG: lanes 4 to 12, NC: negative control (S. enteritidis).

Figure 2.. Electrophoretic Analysis of the fliC Gene After Enzymatic Treatment With Hinp1I Restriction Enzyme

M: 100 bp marker plus DNA ladder (Fermentas Inc.); lane 1: S. pullorum positive control; lane 2: S. gallinarum positive control; lanes 3, 4, 5, 7,8,10 and 12: S. gallinarum isolates; lanes 6, 9, and 11: S. pullorum isolates.