A total of 4047 fecal samples were collected from the sites described above between 2013 and 2017 for isolation of Salmonella spp. Two sample types were collected: (i) sterile containers from fresh cows, swine, horses and wild animal feces that were deposited in the environment, and (ii) samples directly obtained from animals (horses, wildlife and birds) (Table 1). For the latter, rectal or cloacal samples were extracted using Cary Blair transport media (Copan Italia Spa, Brescia, Italy). All samples were collected under sterile conditions and transported to the laboratory at 4 °C for further processing.

The microbiology isolation method used in this study has been previously described [21]. Samples were cultured in buffered peptone water (Beckton-Dickinson, Franklin Lakes, NJ, USA) at 37 °C for 24 h. One-hundred microliters were transferred into Rappaport Vassiliadis (RV) media (Beckton-Dickinson) supplemented with novobiocin (20 mg/mL), and 1 mL was transferred into tetrathionate (TT) (Beckton-Dickinson) supplemented with iodine. Consequently, these samples were incubated at 42 °C for 24 h. Finally, a 100 uL aliquot of each selective enrichment broth was streaked into an XLT-4 agar plate (Beckton-Dickinson) and incubated at 37 °C for additional 24 h. Four presumptive Salmonella colonies were selected from each plate and transferred into a non-selective enrichment media tryptic soy agar (TSA) (Beckton-Dickinson). Subsequently, they were confirmed as S. enterica strains via polymerase chain reaction (PCR) of the invA gene using previously described primers [23]. Confirmed isolates were stored at −80 °C with 20% glycerol concentration, and confirmed as S. enterica strains via polymerase chain reaction (PCR) of the invA gene using previously described primers [23].

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