Pregnant Mongolian gerbils were infected experimentally with H. pylori. The stomachs of the litters were isolated and assessed for H. pylori transmission during pregnancy and at parturition day. Their mother was also evaluated for the H. pylori infectious status. The vertical transmission of H. pylori was assessed using a bacterial culture assay and polymerase chain reaction (PCR).

Specific pathogen-free (SPF) 3-month-old male and female Mongolian gerbils (Meriones unguiculatus) were obtained from the SPF Animal Facilities of College of Medicine, Seoul National University, Korea. All the animals were allowed one week to acclimatize in the inspection facility. Thereafter, the gerbils were kept in an isolated SPF barrier room with a regulated temperature (23 ± 1℃), humidity (50 ± 5%) and light/dark cycle (12/12 h). The animals were fed a pellet diet sterilized with 2 k Gy radiation (Purina, Korea) and sterilized water ad libitum. This study was performed in accordance with the Guide for Animal Experimentation by Seoul National University and approved by the Institutional Animal Care and Use Committee of Seoul National University (Seoul, Korea). All efforts were made to minimize pain or discomfort to the animals.

H. pylori ATCC 43504 was incubated in a brain-heart infusion broth containing 10% fetal bovine serum at 37℃ overnight under a microaerophilic atmosphere, and allowed to grow to a density of ~2.0 × 10⁹ colony-forming units (CFU) per 1 ml of culture broth. The animals were inoculated twice at 3-day intervals by the oral administration of 1.0 × 10⁹ CFU of H. pylori suspended in 0.5 ml of the brain-heart infusion broth. The challenged animals were confirmed to be H. pylori-positive by PCR of their fecal samples, as described previously [7]. The H. pylori-negative animals were excluded from the following study.

One week after H. pylori challenge, the infected females and males were transferred to each separate cage for mating. As soon as the female was confirmed to be pregnant, it was separated from the group and cared for until delivery. Six infected pregnant females were used to determine the vertical transmission. Three mothers were sacrificed 2 weeks after pregnancy. The gastric samples of the mother and fetuses were isolated and analyzed for H. pylori infection. In addition, another three pregnant gerbils were cared for until delivery. The stomachs of the mother and litters were isolated and assessed for the presence of H. pylori at the parturition day. For the negative control, an uninfected female and her litter was sacrificed at 2 weeks after pregnancy and at the parturition day, respectively. Thereafter, their gastric samples were submitted in order to determine the presence of H. pylori infection.

Aliquots of the homogenate were cultured on M-BHM pylori agar medium plates and incubated under the previous described conditions [7]. In order to confirm H. pylori infection, the remaining homogenate was used for the following PCR procedure.

The bacterial DNA was extracted from the above homogenate using the bead beater-phenol extraction method [7]. Each sample homogenate was suspended in 200 µl of Tris-EDTA-NaCl buffer (10 mM Tris-HCl, 1 mM EDTA, and 100 mM NaCl, pH 8.0). A bacterial suspension was placed in a 2.0-ml screw-cap microcentrifuge tube filled with 100 µl (packed volume) of glass beads (diameter, 0.1 mm; Biospec Products, USA) and 100 µl of phenol-chloroformisoamyl alcohol (25 : 24 : 1) (Sigma, USA). The tube was oscillated on a Mini-Bead Beater (Biospec Products, USA) for 1 min and centrifuged (12,000 × g, 5 min) to separate the phases. The aqueous phase was transferred into another clean tube, into which 10 µl of 3 M sodium acetate and 250 µl of ice-cold absolute ethanol were added. The DNA was precipitated by storing the mixture at 20℃ for 10 min. The harvested DNA pellets were dissolved in 60 µl of a Tris-EDTA buffer (10 mM Tris-HCl and 1 mM EDTA , pH 8.0) and used as the template DNA for PCR. A set of primers (HF, 5'-ACTTTAAACGCATGAAGATAT-3'; and HR, 5'-ATATTTTGACCTTCTGGGGT-3') was used to detect the specific nucleic acid of H. pylori [7]. The template DNA (50 ng) and 20 pmol of each primer were added to the PCR mixture tube (Bioneer, Korea) containing 1 U of Taq DNA polymerase, 250 µM each deoxynucleoside triphosphate, 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 1.5 mM MgCl₂, and the gel loading dye. The volume was adjusted to 20 µl with distilled water. The reaction mixture was subjected to 30 amplification cycles (5 min at 95℃, 30 s at 94℃, 30 s at 52℃, 45 s at 72℃, and 5 min at 75℃) followed by a 5 min extension at 72℃ (Thermocycler; Perkin-Elmer, USA). The PCR products were electrophoresed on 1.2% (wt/vol) agarose gel.

The gastric mucosal specimens of animals were collected aseptically and placed in a rapid urease test (Asan Pharma, Korea) to detect the presence of H. pylori urease. The test was performed according to the manufacturer's instructions and the results were interpreted after 24 h. A positive result was reported if a color change from yellow to pink was observed within 24 h of incubation in a 37℃ incubator.