INVESTIGATION ON THE MECHANISMS OF HELICOBACTER TYPHLONIUS TRANSMISSION FAILURES IN SENTINEL MICE EXPOSED TO SOILED BEDDING
Open Access
- Author:
- Villano, Jason So
- Graduate Program:
- Laboratory Animal Medicine
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 07, 2010
- Committee Members:
- Dr Catherine Beckwith, Thesis Advisor/Co-Advisor
Catherine Beckwith, Thesis Advisor/Co-Advisor - Keywords:
- Helicobacter typhlonius
PCR
sentinel mice
western blot - Abstract:
- Helicobacter spp. are bacteria that colonize the gastrointestinal and hepatobiliary tracts of laboratory rodents and may potentially confound animal-based research. Helicobacter diagnosis and monitoring, primarily by fecal polymerase chain reaction (PCR), is often included in health surveillance programs for mouse colonies. These programs often involve transfer of soiled bedding from colony animals to expendable “sentinel” mice to facilitate disease diagnosis. However, recent studies indicate that transmission of Helicobacter to sentinel mice does not always occur. We investigated the mechanisms of transmission failure of H. typhlonius, one of the most recently discovered Helicobacter species. We transferred soiled bedding from female BALB/c mice experimentally infected with H. typhlonius to cages of pair-housed female Swiss Webster (SW) mice once weekly or every other week. After 20 wk, mice in only 3 out of 8 cages (38%) that were exposed weekly became Helicobacter fecal PCR-positive, while none of the mice from the group exposed every other week became PCR-positive. In contrast, 2 cages of 2 female Swiss mice housed in direct contact with an inoculated BALB/c mouse became PCR-positive within 4 wk of exposure. Some inoculated BALB/c mice remained Helicobacter PCR-positive for up to 31 wk post-inoculation (“consistently PCR-positive mice”) while others reverted to PCR-negative status after as few as 4 wk of PCR-positive results. Our qPCR results indicated that mice shed between 103 and 107.4 copies of H. typhlonius genome/ìg fecal DNA. To determine whether there was H. typhlonius transmission failure or elimination of infection in sentinel mice, western blot against H. typhlonius membrane antigens was performed. Helicobacter fecal PCR results correlate with western blot of mouse sera for detection of H. typhlonius colonization. All inoculated BALB/c mice had mild typhlocolitis but the consistently PCR-positive mice had quantitatively more severe pathology than the inconsistently positive mice. To test the colonization ability of our strain of H. typhlonius, male BALB/c mice and male and female SW mice were gavaged with fresh fecal suspensions from PCR-positive mice. Only 3 out of 15 mice (20%) became infected. We attempted to culture H. typhlonius from mouse feces at 24 hr intervals to determine how long H. typhlonius remains viable in feces. Although H. typhlonius could be isolated from 100% of fresh fecal samples (immediately after deposition), the isolation rate dropped to 25% for time points 24 and 48 hr. Our observations show that H. typhlonius transmission to sentinel mice is dependent on both its limited duration of viability in the environment and colonization ability. In conclusion, H. typhlonius is not reliably transmitted to sentinel mice by soiled bedding transfer. Accurate detection of H. typhlonius by fecal PCR requires that colony mice be tested directly. We postulate that H. typhlonius colonization in our experiments was impeded and controlled by the resident intestinal microbiota of the mice.