Rappaport vassiliadis broth

Bacteriological analyses were performed according to the EN/ISO 6579–2002/Amd1:2007 protocol for Salmonella detection in food and animal feedstuffs. 25 g of samples (droppings, feed, liver, caeca, neck skin) were individually pre-enriched with 225 mL of buffered peptone water broth (PWB) (Fluka, Sigma Aldrich, France). The swabs were individually placed in 10 mL PWB, while 100 mL of drinking and carcass rinsing water was individually mixed with 100 mL of double strength PWB for pre-enrichment according to NF U 47–101 Standard (2005) [17 ]. All samples were incubated at 37 °C for 18–20 h. From each pre-enrichment solution, 1 mL and 0.1 mL were respectively transferred into 10 mL of enrichment Muller-Kauffmann tetrathionate/novobiocin broth (AES Chemunex Combourg, France) and 10 mL of Rappaport Vassiliadis broth (Merck Darmstadt, Germany), incubated respectively at 37 °C and 42 °C for 24 h. Both enriched samples were then streaked on XLD (Fluka analytical Steinheim, Switzerland) and Hektoen agars (Pasteur Institute of Algeria) and incubated at 37 °C for 24 h [18 ]. Suspected colonies were first identified with the API 20E System (bioMérieux, France), then with MALDI-TOF (Bruker Daltonics GmbH, Germany) [19 (link)]. Confirmed Salmonella isolates were serotyped according to Kauffmann-White-Le Minor’s scheme [20 (link)].

The liver, spleen, mesenteric lymph nodes, the content of ileum and cecum and about 2 gr of feces were weighed and transferred into stomacher bags along with 9 times sterile phosphate buffer saline (PBS, pH: 7.2). They were then homogenized in a stomacher (Seward Stomacher 400 Blender BA6021, UK) for 2 min and then tenfold serial dilution was made in PBS. The dilutions were then surface plated on BG agar and incubated at 37 °C for 48 h. This process was duplicated for all the cultured samples. The Salmonella enumeration was expressed as CFU/gr^{5 (link),46} . Also, on the first day of sampling, one gr of liver, spleen and mesenteric lymph nodes were separately crushed using a scalpel and added into 9 ml lactose broth (Merck, Germany) and incubated at 37 °C for 24 h. After incubation, 1 ml of that was added to 10 ml Selenite Cystine broth (Merck, Germany) and in parallel, 0.1 ml was added to 10 ml Rappaport Vassiliadis broth (Merck, Germany). They were incubated at 37 °C and 42 °C, respectively, for 24 h. Then a loop of the selective enrichment broth was cultured on XLD agar and BG agar. The plates were incubated at 37 °C for 24 h and checked for Salmonella colonies^{14 (link),45} .