Lactose broth

Totally, 824 fecal samples of diarrheic calves were collected randomly during January 2010 to January 2011. Geographical distribution (Isfahan, Chaharmahal, Fars and Khuzestan provinces) (Figure 1), season of samples collection (spring, summer, autumn and winter) and age of diarrheic calves (2 to 30 days) were recorded during sampling. Fecal samples were taken using sterile rectal swabs. All swab samples were placed into tubes containing Stuart medium (Merck, Germany). Samples were immediately transferred to the Microbiology and Infectious Diseases Research Center of the Islamic Azad University of Shahrekord. All samples were diluted in phosphate buffered saline (PBS, Merck, Germany). Then samples were cultured on MacConkey’s agar (MC, Merck, Germany) (24 h at 37°C). Lactose positive colonies were cultured on Eosin Methylene Blue (EMB, Merck, Germany) (24 h at 37°C). Metallic green colonies were considered as E. coli. Several biochemical tests including Triple Sugar Iron Agar (TSI), Indole, Citrate utilization, Voges-Proskauer, urease and Methyl red tests were used for E. coli confirmation. All isolates were stored at -70°C in lactose broth (Merck, Germany) restraining 20% glycerol for further description.Figure 1

Geographical areas of sample collection.

Rinse fluid was transferred into a 50 mL falcon tube and centrifuged at 4000 rpm for 10 minutes. Precipitates were suspended in 9 mL of lactose broth (Merck, Germany) and incubated at 37°C for 24 h. Amount of 1 mL of each re-enriched sample was transferred into 9 mL of selenite cystine broth (Bio-Rad) and incubated at 37°C for 24 h. Following incubation, a loopful of each culture was streaked onto brilliant green agar (Bio-Rad) which was incubated at 37°C for 24 h. Presumptive Salmonella colonies were confirmed biochemically using triple sugar iron (TSI), citrate, lysine decarboxylase, urease and indole tests (15 (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} .