High Prevalence of Helicobacter Species Detected in Laboratory Mouse Strains by Multiplex PCR-Denaturing Gradient Gel Electrophoresis and Pyrosequencing

Abstract

Rodent models have been developed to study the pathogenesis of diseases caused by Helicobacter pylori, as well as by other gastric and intestinal Helicobacter spp., but some murine enteric Helicobacter spp. cause hepatobiliary and intestinal tract diseases in specific inbred strains of laboratory mice. To identify these murine Helicobacter spp., we developed an assay based on PCR-denaturing gradient gel electrophoresis and pyrosequencing. Nine strains of mice, maintained in four conventional laboratory animal houses, were assessed for Helicobacter sp. carriage. Tissue samples from the liver, stomach, and small intestine, as well as feces and blood, were collected; and all specimens (n ‫؍‬ 210) were screened by a Helicobacter genus-specific PCR. Positive samples were identified to the species level by multiplex denaturing gradient gel electrophoresis, pyrosequencing, and a H. ganmani-specific PCR assay. Histologic examination of 30 tissue samples from 18 animals was performed. All mice of eight of the nine strains tested were Helicobacter genus positive; H. bilis, H. hepaticus, H. typhlonius, H. ganmani, H. rodentium, and a Helicobacter sp. flexispira-like organism were identified. Helicobacter DNA was common in fecal (86%) and gastric tissue (55%) specimens, whereas samples of liver tissue (21%), small intestine tissue (17%), and blood (14%) were less commonly positive. Several mouse strains were colonized with more than one Helicobacter spp. Most tissue specimens analyzed showed no signs of inflammation; however, in one strain of mice, hepatitis was diagnosed in livers positive for H. hepaticus, and in another strain, gastric colonization by H. typhlonius was associated with gastritis. The diagnostic setup developed was efficient at identifying most murine Helicobacter spp.

Enterohepatic Helicobacter Species Are Prevalent in Mice from Commercial and Academic Institutions in Asia, Europe, and North America

“…of mercury and then filled with an 80:10:10 mixture of nitrogencarbon dioxide-hydrogen rather than anaerobic jars with gasgenerating packs, as described by Robertson et al (24). Also, the Helicobacter species present in these mice and the number of mice infected may be underestimated since PCR of DNA directly isolated from fecal samples is more sensitive than culture (9,20,25 Other countries in this study that have not previously published information on the prevalence of helicobacters in murine research colonies included Switzerland, England, The Netherlands, Scotland, France, and Canada. All were found to have colonies infected with Helicobacter spp.…”

“…Nilsson et al (20) reported that the only pathology seen in their study of 42 young adult mice was hepatitis associated with H. hepaticusmonoinfected C57BL/6 ApoE Ϫ/Ϫ mice and BALB/cA mice infected with multiple helicobacters (H. hepaticus, H. bilis, H. rodentium, and H. typhlonius). Goto et al (13) surveyed 820 mice 9 weeks of age or older and found that the only pathology observed was in 5/174 (2.8%) mice colonized with H. rodentium and H. rodentium-like strains.…”

Prevalence and Spread of Enterohepatic Helicobacter Species in Mice Reared in a Specific-Pathogen-Free Animal Facility

“…In samples that are positive in a group-specific PCR a further step is required to identify the Helicobacter species. DNA sequencing and restriction fragment length polymorphism (RFLP) analysis are commonly used for this purpose (2,27), although denaturing gradient gel electrophoresis has also been successfully used (25). Here, we used genusspecific PCR and consecutive DNA sequencing combined with RFLP analysis to evaluate the prevalence of Helicobacter infections and their spread in the SPF facility of our central animal laboratory.…”

“…Basically, two types of PCR assays were developed: speciesspecific PCR assays that allow the identification of single Helicobacter species (27,30) and Helicobacter group-specific PCR assays which can detect several Helicobacter species in a single assay (2,25,27). In samples that are positive in a group-specific PCR a further step is required to identify the Helicobacter species.…”

Low frequency of Helicobacter species in the stomachs of experimental rabbits

“…Likewise, H. muridarum, a natural inhabitant of the mouse intestine, is able to allocate itself a niche in the gastric mucosa of older animals, prompting severe inflammation (Lee et al 1992, Queiroz et al 1992. Some mouse strains are very susceptible to Helicobacter infections (Fox & Lee 1997, Nilsson et al 2004.…”