Quantitation of Hp copy numbers in mice

Three groups of 4-6-week-old female C57BL/6 mice (five per group) were orally inoculated thrice in three consecutive days with different doses (3 × 10⁴, 3 × 10⁵, and 3 × 10⁶ CFU/mouse) with mouse-adapted Hp. Immunization was performed, as described previously[9], with minor modifications [10]. Briefly, mice were orally intubated with inoculums containing 10 µg of cholera toxin and 100 µg of recombinant Urease B subunit (UreB)[11], in 250 µl of gavage buffer (0.5% casein hydrolysate, 0.2 mol/L of sodium bicarbonate, and 0.5% glucose in PBS), thrice with two-week intervals. The sham-immunized control group received only the gavage buffer. Two weeks following the last immunization, both groups were inoculated with 3 × 10⁵ CFU of 24-hour old, liquid cultured Hp[12], and sacrificed two weeks thereafter. At sacrifice, mouse stomachs were longitudinally divided into two parts for: (1) RUT and (2) DNA extraction. RUT color development was documented at 4 h. Total DNA was extracted from snap-frozen tissue by a commercial kit (MN, Germany). Optimized concentrations were used for quantitation of the tissue- colonizing Hp by qPCR. Standard curves of PT and HpDNA were employed to calculate the Hp copy numbers per microgram of gastric tissue.