Identification of novel Helicobacter species in pig stomachs by PCR and partial sequencing

Evidence of infection with Helicobacter species in pig stomach was investigated by the use of a PCR with Helicobacter genus-specific primers. Forty pig stomachs, each of four different ulcer lesion grades, 0, 1, 2, and 3 in the pars esophagea area, were collected from a slaughterhouse in Minnesota. Of 160 stomach samples examined, 102 (63.8%) were positive by the PCR assay. The 40 samples each of lesion grades 0, 1, 2, and 3 showed 22.5, 52.5, 85.0, and 95.0% PCR-positive results, respectively. There was a significant trend (P ≤ 0.01) in the proportions of PCR-positive cases relative to severity of the lesion. About 80% of the 16S rRNA gene was amplified, and PCR-restriction fragment length polymorphism (RFLP) patterns were analyzed. Of 102 PCR-positive samples, the PCR-RFLP patterns resulted in four different types, 32 samples being classified into type MN 1, 16 samples into type MN 2, 43 samples into type MN 3, and 11 samples into type MN 4. When the sequences of each RFLP type were compared to those reported in databases by using BLAST software, types MN 1, MN 2, MN 3, and MN 4 showed homologies of 97.3, 95.4, 96.7, and 99.5% with the 16S ribosomal DNA of Helicobacter flexispira taxon 3, Helicobacter sp. strains MIT 94-022 and MZ 640285, and Helicobacter suis, respectively. None of the 102 samples positive for the Helicobacter genus were positive with a primer set specific for Helicobacter pylori. Attempts to culture the organisms from selected stomachs in vitro were unsuccessful.