disinfection, 2) on chick arrival day, 3) during rearing period (weekly) and 4) on slaughter day. Regarding the first step, after cleaning and disinfection, swabs from an area of 1 square meter of floor and wall of the broiler house were collected. For pan feeder and water nipple, pooled swab samples from 5 pieces were combined to constitute one sample. Water, litter (after disinfection), and pests were taken to assess Salmonella persistence after cleaning and disinfection. In the second step, on chick arrival day, meconium on box-liners was collected to identify Salmonella status of new chicks. Additionally, samples from boot swabs, wall swabs, new feed in hoppers, feed in pan feeders (5 pans per samples), water nipple swabs, water and pests were taken to assess Salmonella contamination in the broiler house before placing the new chicks. In the third step, during rearing period, samples were collected weekly until the birds were slaughtered on week 6. Either pooled fecal samples (for the first sampling period) or cloacal swabs and boot swabs (for the second and third sampling period) were collected to monitor Salmonella status of the broiler flocks. In addition, samples from new feed in hoppers, feed in pan feeders (5 pans per sample), water from inlet, and water from nipples (10 nipples per sample) and pests were taken to evaluate and investigate the source of Salmonella contamination in the broiler flocks. As the last step, on slaughter day, water (before spraying the chicken), cage swabs, hands of workers (before work) and 1 square meter of the truck floor were collected to investigate the source of Salmonella contamination before the slaughtering.
Sample collection in slaughterhouse: Feathers around cloaca (for the first sampling period) or cloacal swabs (for the second and third period) were collected at the holding area of the slaughterhouse to assess contamination status of arriving birds. Whole carcass rinse samples were collected at the end of the process for final product evaluation.
Salmonella isolation and identification: Salmonella isolation was performed according to the ISO 6579:2002/Amd 1:2007 (Annex D) standard method (International Organization for Standardization, 2007). For feces, litter, and feed samples, 25 g of the sample were mixed with 225 ml of buffered peptone water (BPW, Merck KGaA, Darmstadt, Germany) and incubated at 37°C for 24 h for pre-enrichment. For swab samples, the swabs were placed in small amount of BPW (5-30 ml depending on size and number of swabs). For egg and lizard samples, the whole content was mixed with 225 ml and 100 ml of BPW, respectively. For water samples, 100 ml of the water was mixed with 100 ml of double-strength BPW for pre-enrichment. After incubation, 0.1 ml of the pre-enriched broth was inoculated into modified semi-solid Rappaport-Vassiliadis medium (MSRV, Merck KGaA, Darmstadt, Germany) and 10 ml of the Rappaport-Vassiliadis with soya broth (RVS, Merck KGaA, Darmstadt, Germany). In addition, 1 ml of the culture was inoculated into Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn, Merck KGaA, Darmstadt, Germany). After the incubation of the MSRV and RVS at 42°C for 24 h and MKTTn at 37°C for 24 h, the cultures were streaked on xylose lysine deoxycholate agar plate (XLD, Merck KGaA, Darmstadt, Germany) and selective chromagar Salmonella medium (Microbiology, Paris, France). After incubating at 37°C for 24 h, approximately five suspected colonies were selected for biochemical testing: Triple sugar iron agar (TSI, Merck KGaA, Darmstadt, Germany), Lysine iron agar (LIA, Merck KGaA, Darmstadt, Germany), and Sulfide-Indole Motility medium (SIM, Merck KGaA, Darmstadt, Germany) as described by Ewing (Ewing, 1986). Typical colonies were further serotyped by slide agglutination test according to the Kauffmann-White-Le-Minor scheme (Grimont and Weill, 2007) at the WHO National Salmonella and Shigella Center, Bangkok, Thailand.