**2. Research methods**

#### **2.1** *Salmonella* **from domestic animals sources 2.1.1** *Salmonella* **from feedlot cattle**

One hundred and thirty eight (138) 1-year-old steers distributed in 24 pens (6 steers/pen) were used in this study (Tabe et al (2010a, 2010b). Cattle from various private farms were housed at the North Dakota State University feedlot facility in October 2006. From October 2006 to March 26, 2007 cattle were placed on growers diet and then on finishing diet from March 27 to June 2007. Cattle in different pens could not directly contact each other, and there was no sharing of feed or water sources between pens. Fecal samples were collected from cattle every three weeks from March 2007 to June 2007. During the first and second sampling periods (March and April 2007 respectively), one-hundred-thirty eight cattle were available for the study. At the third sampling period (May 2007), two unhealthy cattle were withdrawn from the study while at the last sampling period (June 2007), forty six cattle were available as the rest had been taken for slaughter.

Samples were collected in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) following a previously described protocol (Khaitsa et al., 2007a). The feces were put into sterile plastic cups and placed in iced-pack coolers before transport to the laboratory for processing. The sampling procedure was repeated every three weeks for the entire finishing period. For the isolation of *Salmonella*, fecal samples were cultured using conventional culture methods optimized for the detection of *Salmonella* (Khaitsa et al., 2007). Briefly, a sterile swab was loaded with fecal sample and pre-enriched in buffered peptone water (Difco, Becton Dickinson) at 37°C overnight followed by immunomagnetic beads separation specific for *Salmonella* species (Dynabeads anti-*Salmonella,* Dynal Biotech, Inc., Lake Success, N.Y.) according to the **manufacturer's** instructions. After the final wash, the beads were transferred to 10 ml of Rappaport Vassiliadis R10 (RV) broth (Becton Dickinson) and incubated (with constant gentle shaking) at 420C for 24 h. Following incubation, the RV cultures were streaked onto modified brilliant green agar (Becton Dickinson) and mannitol lysine crystal violet brilliant green agar (Oxoid, Basingstoke, UK). Colonies with typical *Salmonella*  characteristics (Fratamico et al., 2000) were stabbed in 10-ml triple sugar iron agar slants (Becton Dickinson), and the biochemical results read after 24-h incubation as described. Presumptive *Salmonella* isolates were stabbed into 2 ml tryptic soy agar (Difco, Becton Dickinson) slants and shipped to the National Veterinary Service Laboratories, Animal and Plant Health Inspection Services, US Department of Agriculture, Ames, Iowa, for serotyping. The detection sensitivity culture post immunomagnetic separation and enrichment using culture media for *Salmonella* was based on growth of bacteria of interest on the culture plates. Fifty eight (58) isolates of *Salmonella* were shipped to the *E. coli* reference center (University Park, PA) for PFGE.
