IDENTIFICATION OF WIDESPREAD HELICOBACTER-HEPATICUS INFECTION IN FECES IN COMMERCIAL MOUSE COLONIES BY CULTURE AND PCR ASSAY

The identification of a new murine pathogen, Helicobacter hepaticus, a nd its association with chronic active hepatitis and liver tumors prom pted an evaluation of the prevalence of H, hepaticus in commercially a vailable mice. Of the 28 different strains or stocks, totaling 160 mic e from four major commercial vendors, cultured for H. hepaticus, 100% of mice from two outbred strains from one vendor were infected with H. hepaticus, whereas of 13 inbred mouse strains from another vendor wer e infected, This high prevalence of H, hepaticus established a need fo r a rapid and reproducible, noninvasive assay for the screening of col ony-maintained mice being used for biomedical research. The culturing of fecal material by using 0.45-mu m-pore-size filtration for H. hepat icus consistently yielded reproducible results but required extended p eriods of time (1 to 3 weeks) to obtain a definitive answer. Although it is rapid, the use of a direct PCR-based detection assay with fecal specimens is restricted by inhibitory agents. To circumvent these inhi bitory agents and to augment our H. hepaticus culture technique, we ha ve developed a novel PCR system in which the bacteria are isolated fro m fecal material in the presence of polyvinylpyropyrollidone and lysed by treatment with Chelex 100. The PCR is performed with Tth polymeras e supplemented with a polymerase enhancer. By this PCR method, 24 H. h epaticus culture-positive and 30 H. hepaticus culture-negative fecal s amples were correctly identified, Moreover, two samples which were PCR positive and culture negative initially were positive by both methods upon retesting of fresh material, Southern blot hybridizations and se quencing of PCR products showed them to be H, hepaticus specific. A co mparison of results obtained under identical conditions indicated a 10 0-fold increase in sensitivity with Tth polymerase over Tag polymerase . This PCR method can be used as a noninvasive means of rapidly screen ing large numbers of colony mice for H. hepaticus.