Modified semisolid rappaport vassiliadis agar plates

Fecal shedding and organs colonization of STM^wt and STM^ΔznuABC were determined according to the ISO 6579: 2002/Amendment 1:2007 protocol. Briefly, samples were weighed and homogenized in nine parts of Buffered Peptone Water (BPW) (Oxoid Ltd., UK). This initial solution was then used to perform a decimal dilution series carried out by systematically transferring an aliquot of 0.5 ml of each successive dilution in 4.5 ml of BPW. All BPW-diluted samples were incubated at 37°C for 18 ± 3 h. 0.1 ml of cultures were subsequently seeded on Modified Semisolid Rappaport-Vassiliadis (MSRV) agar plates (Oxoid Ltd., UK) and incubated at 41.5°C for 24 h for the selective enrichment of Salmonella. A loopful of growth from each MSRV plate was streaked onto Xylose-Lysine-Desoxycholate Agar (Oxoid Ltd., UK) and Brilliant Green Agar (Oxoid Ltd., UK) plates and hence incubated at 37°C overnight. Suspect Salmonella colonies were subjected to biochemical identification by the BBL Enterotube II (BD Franklin Lakes, USA) and serological identification using Salmonella group-specific antisera (Remel, Lenexa, USA). This is a semi-quantitative approach that allows the quantification of Salmonella in a sample within a tenfold band (detection limit 1 CFU/g feces).

The microbiological analysis of fecal and organ samples were conducted according to the ISO 6579:2002/Amendment 1:2007 protocol. Briefly, samples were weighed and homogenized as 10% suspension in Buffered Peptone Water (BPW) (Oxoid Ltd., UK). This initial solution was then used to perform a decimal dilution series carried out by systematically transferring an aliquot of 1 ml of each successive dilution in 9 ml of BPW. All BPW-diluted samples were incubated at 37°C for 18 ± 3 h. Cultures (0.1 ml) were subsequently seeded on Modified Semisolid Rappaport-Vassiliadis (MSRV) agar plates (Oxoid Ltd., UK) and incubated at 41.5°C for 24 h for the selective enrichment of Salmonella. A loopful of growth from each MSRV plate was streaked onto Xylose-Lysine-Desoxycholate Agar (Oxoid Ltd., UK) and Brilliant Green Agar (Oxoid Ltd., UK) plates and hence incubated at 37°C overnight. Typical colonies were confirmed serologically as Salmonella by polyvalent antiserum (Salmonella Test Serum; Siemens Healthcare Diagnostics, Italy) and API rapid 20 E (Api Rapid 20E; Biomerieux, Italy). This is a semi-quantitative approach that allow the determination of the concentration of Salmonella in a sample within a tenfold band.