Tetrathionate tt

Alongside positive (Salmonella Typhimurium ATCC 14028) and negative (E. coli ATCC 25922) controls, feces and sponge swab washings were each dispensed into Tetrathionate (TT; BD Canada, Mississauga, ON, Canada) and Rappaport-Vassiliadis (RV; EMD Millipore, Etobicoke, ON, Canada) broths and incubated at 42 °C for 20–24 h. Tetrathionate enrichments were streaked onto Xylose Lysine Tergitol 4 agar (XLT-4; BD Canada, Mississauga, ON, Canada) and RV enrichments were transferred to modified semisolid RV (MSRV) agar plates and incubated at 37 °C for 24 h. A loop of MSRV culture from the edge of opaque zones was streaked onto modified Brilliant Green agar (MBGA; Hardy Diagnostics, Santa Maria, CA, USA) and incubated overnight at 37 °C for 24 h. Up to three colonies per XLT-4 and MBGA plate were sub-cultured to fresh XLT-4 plates and incubated at 37 °C for 24 h. Presumptive Salmonella isolates were tested for agglutination with Poly A-I + Vi antiserum (BD Canada, Mississauga, ON, Canada).

Thirty grams of litter from each sample were weighed out and combined with 270 mL of buffered peptone water (BPW; BD, Franklin Lakes, NJ) in a sterile Whirl-Pak® bag (Fig 1); sample weights were not adjusted according to moisture content. Each bag was vigorously hand-shaken/massaged for one min. Bags were transferred to a shaking incubator set to 37°C and 50 rpm. After 24 h incubation, 1 mL was transferred from the Whirl-Pak® bag (without filter) into 9 mL of BPW, which was returned to the shaking incubator for an additional 20 h at 37°C and 50 RPM. After 20 h, 100 μL was transferred into tubes containing 10 mL Rappaport-Vassiliadis (RV; BD, Franklin Lakes, NJ) broth and 1000 μL was transferred into 10 mL Tetrathionate (TT; BD, Franklin Lakes, NJ) broth. Tubes were incubated at 42°C for 48 h (RV) or 24 h (TT). After incubation, 10 μL broth was streaked onto a Xylose Lysine Tergitol-4 (XLT-4; BD, Franklin Lakes, NJ) plate and incubated at 37°C for 24–48 h. Up to four presumptive positive colonies (black, or red with a black center) were re-streaked onto individual XLT-4 plates and incubated an additional 24–48 h prior to PCR confirmation.