IDENTIFICATION OF MURINE HELICOBACTERS BY PCR AND RESTRICTION ENZYME ANALYSES

Three murine helicobacter species have recently been identified: Helic obacter hepaticus, Helicobacter muridarum, and Helicobacter bills. Inf ections with H. hepaticus and H. bills have been associated with hepat itis and hepatic neoplasia. In this study, oligonucleotide primers wer e designed from regions of the 16S rRNA gene that are conserved among members of the He[icobacter genus. The assay amplified the expected 37 3-bp product from all three rodent Helicobacter species and was able t o detect as little as 5 pg of H. hepaticus. H. bills, or H. muridarum DNA. The specificity of the reaction was determined by testing cecal D NA from uninfected mice and mice with documented Helicobacter infectio ns and by testing DNA from other bacterial genera. A product of the ex pected size was generated with cecal DNA from Helicobacter-infected mi ce but not with DNA from uninfected mice, With the exception of that o f ''Flexispira rappini,'' which is closely related to the Helicobacter genus, DNA from other bacterial genera was not amplified with the Hel icobacter genus-specific primers. MboI, MaeI, and HhaI restriction enz yme analyses of the amplified product were able to differentiate among the murine Helicobacter species but could not differentiate H, bills from ''F. rappini. To distinguish H. bilis, a reverse primer based on H. bilis 16S rRNA sequence was designed, PCR with the H. bilis-specifi c reverse primer (Hbr) and the Helicobacter genus-specific forward pri mer (H276f) amplified H. bills DNA but not DNA from ''F. rappini'' or other rodent helicobacters. Examination of a large number of murine ce cal tissues with this combination of PCR assays and restriction enzyme analyses indicated that H. hepaticus and H. bills infections are wide spread in laboratory mouse and rat colonies.