Blood agar base no 2

Individual fecal samples were collected from rectal ampulla from each piglet, on days -1, 1, 2, 3 and 4 to perform microbiological analysis and evaluate the challenger strain shedding. For each sample, 1 g of feces was homogenized with 1 ml of saline solution and incubated overnight at 37 °C on sheep blood agar plates 5% (Blood Agar Base No. 2-Oxoid) in order to examine the presence of hemolytic colonies. The total hemolytic bacteria count was performed by counting the number of colonies cultured from serial dilutions of each fecal sample in order to evaluate the presence of hemolytic E. coli in relation to the total bacteria population.

The strains used in this study are listed in Table S1. P. multocida isolates were cultured on heart infusion (HI) agar plates (Oxoid) or horse blood agar plates (Blood agar base no.2 from Oxoid, defibrinated horse blood from Australian Ethical Biologicals Pty Ltd.) or in HI broth for liquid cultures. P. multocida on solid media were grown at 37°C for 24 h to 48 h. Broth cultures were grown at 37°C for up to 24 h with shaking at 200 rpm.

All strains and plasmids used in this study are listed in Supplementary Table S2 (Supplementary Information). H. pullorum 6350-92 (CCUG 33838), isolated from a stool sample of a patient with gastroenteritis and hepatitis^{52 (link)}, was used as the wild type strain. Cells were routinely cultivated at 37 °C, in a microaerobic atmosphere (6% O₂, 7% CO₂, 3.5% H₂, and 83.5% N₂) generated by the Anoxomat system (Mart Microbiology), in horse blood agar (BA) composed of Blood Agar Base no. 2 (Oxoid) with 10% (v/v) defibrinated horse blood (Probiológica), supplemented with an antibiotic-antifungal mix composed by 6.3 g/L vancomycin (Roth), 3.1 g/L trimethoprim (Sigma), 2.5 g/L amphotericin B (Roth) and, when required, 20 µg/mL kanamycin or 5 µg/mL gentamicin. Bacteria were taken as fully grown when cultured in BA plates for 5 days, with two serial passages.
Unless otherwise indicated these cells, designated as fully grown H. pullorum, were used as the starting material in the following assays.
E. coli pre-cultures were grown overnight at 37 °C and 150 rpm in LB medium, supplemented when required with kanamycin, ampicillin, and isopropyl-1-thio-β-D-galactopyranoside (IPTG).

The human gastric epithelial MKN28 and AGS (ATCC CRL-1739) cell lines were maintained in RPMI 1640 and nutrient mixture F12 Ham medium, respectively, supplemented with 10% heat inactivated fetal calf serum and 2 mM L-glutamine (Invitrogen) at 37°C in a 5% CO₂ humidified atmosphere.
H pylori strains were cultured on Blood Agar Base No 2 containing 5% (vol/vol) horse blood (Oxoid, Cambridge, UK) at 37°C under microaerobic conditions. cagPAI+ H pylori strains 60190, 84183,25 (link) 26695, B128 7.13 and the cagPAI− strains SS126 (link) and Tx30a25 (link) were used, along with isogenic mutants deficient in vacA (60190ΔvacA, 84183ΔvacA), cagA (60190ΔcagA, 84183ΔcagA) and cagE (60190ΔcagE, 84183ΔcagE). A slt (HP0645) deletion mutant (26695Δslt) and 26695 parental strain were kindly donated by Dr Richard Ferrero, Monash University, Victoria, Australia.6 (link)

The H pylori strain GC026 (cagA+, cagE+, cagL+, cagT+, vacA s1 m1+, babA+, oipA+, dupA+ and sabA+) was isolated from a GC patient at the University Hospital of Malaysia, Kuala Lumpur [21] (link), [22] (link). The H. pylori strain 26695 (cagA+ and vacA s1m1+) was isolated from a patient with gastritis [23] (link) (ATCC code 700392). Both H. pylori strains were grown for two days on horse blood agar plates (Blood Agar Base No.2 supplemented with 6% sterile defibrinated horse blood (Oxoid, Heidelberg West, Vic., Australia) at 37°C in an anaerobic jar containing a gas generating kit (Oxoid) to provide a microaerobic atmosphere of 6% O₂, 10% CO₂ and 84% N₂.