Multilocus variable-number of tandem-repeats analysis of Salmonella enterica serotype Gallinarum and comparison with pulsed-field gel electrophoresis genotyping

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, a severe disease of poultry, responsible for heavy economic losses. Epidemiologic investigation of fowl typhoid significantly benefits from molecular typing tools, RAPD and PFGE have been proposed for this purpose. PFGE, a well established technique, is still the gold standard among typing methods for most bacteria, including salmonella. Nevertheless, it has some limitations regarding execution and reproducibility, in particular it is labour intensive and requires good technical expertise. Furthermore, it needs accurate standardization and results can be ambiguous to interpret. Such limitations can hamper reproducibility and transfer of results. As a possible alternative to PFGE, multilocus variable-number of tandem-repeats analysis (MLVA) has recently emerged as an effective genotyping method for many bacterial pathogens showing high discriminatory power associated to robustness. We developed a six-loci MLVA protocol for Salmonella Gallinarum and compared it to PFGE performed with SpeI, XbaI and NotI on fifty isolates. The proposed MLVA has a high discriminatory power, equivalent to that of the three-enzyme PFGE (Simpson's index 0.94 for MLVA, 0.93 for three-enzyme PFGE) but it is simpler to perform and straightforward in genotype identification, allowing unambiguous exchange of results. Stability of selected VNTR loci, assessed in vitro and in vivo, is good but not absolute, reflecting the sensitivity of MLVA to detect evolutionary changes of bacteria. Clustering of the isolates as determined by MLVA typing is substantially confirmed by PFGE.