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Survey of Helicobacter Species in Laboratory Mice and Gerbils in Japan

Kazuo Goto

ICLAS Monitoring Center

Central Institute for Experimental Animals

Kanagawa, Japan

Since Helicobacter muridarum was first reported (Lee and others 1992), additional Helicobacter species have been isolated from intestinal tracts of rodents. In these subsequent studies, species such as Helicobacter hepaticus (Fox and others 1994), H. muridarum, Helicobacter bilis (Fox and others 1995), Helicobacter rodentium (Shen and others 1997) and “Flexispira rappini” (Schauer and others 1993) were surveyed using reverse transcription-nested polymerase chain reaction (PCR) to clarify the so-called current status of Helicobacter infection in laboratory mice and gerbils in Japan.

MATERIALS AND METHODS

For H. hepaticus detection, a total of 310 mice were sampled. The total included 116 mice from two breeding colonies, 194 mice from 19 research institutions, and 74 gerbils including 11 gerbils from two breeding facilities and 63 gerbils from six research institutions. RNA from the samples was transcribed to cDNA using Helicobacter genus-specific primers. Primers used for the first PCR were also Helicobacter genus specific. Primers for the second (nested) PCR were specific for H. hepaticus. H. hepaticus-specific primers were selected according to the previous report (Battles and others 1995).

For Helicobacter species detection, 149 mice from 17 facilities were used. The samples were different from those used in the H. hepaticus study described above. RNA was transcribed to cDNA using Helicobacter genus-specific primers, and primers for the first and second PCR were also Helicobacter genus specific. Using samples found to be positive with Helicobacter genus-specific



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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE Survey of Helicobacter Species in Laboratory Mice and Gerbils in Japan Kazuo Goto ICLAS Monitoring Center Central Institute for Experimental Animals Kanagawa, Japan Since Helicobacter muridarum was first reported (Lee and others 1992), additional Helicobacter species have been isolated from intestinal tracts of rodents. In these subsequent studies, species such as Helicobacter hepaticus (Fox and others 1994), H. muridarum, Helicobacter bilis (Fox and others 1995), Helicobacter rodentium (Shen and others 1997) and “Flexispira rappini” (Schauer and others 1993) were surveyed using reverse transcription-nested polymerase chain reaction (PCR) to clarify the so-called current status of Helicobacter infection in laboratory mice and gerbils in Japan. MATERIALS AND METHODS For H. hepaticus detection, a total of 310 mice were sampled. The total included 116 mice from two breeding colonies, 194 mice from 19 research institutions, and 74 gerbils including 11 gerbils from two breeding facilities and 63 gerbils from six research institutions. RNA from the samples was transcribed to cDNA using Helicobacter genus-specific primers. Primers used for the first PCR were also Helicobacter genus specific. Primers for the second (nested) PCR were specific for H. hepaticus. H. hepaticus-specific primers were selected according to the previous report (Battles and others 1995). For Helicobacter species detection, 149 mice from 17 facilities were used. The samples were different from those used in the H. hepaticus study described above. RNA was transcribed to cDNA using Helicobacter genus-specific primers, and primers for the first and second PCR were also Helicobacter genus specific. Using samples found to be positive with Helicobacter genus-specific

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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE primers, the first PCR products were amplified with five sets of species-specific primers to identify Helicobacter species. For samples that could not be identified using the primer sets, the first PCR products were sequenced. To avoid nonspecific bands, the restriction pattern was evaluated. RESULTS Results of H. hepaticus detection are shown in Table 1. For mice, all 116 samples from two breeding facilities were negative, but 35 samples from five research institutions of the 194 samples from 19 research institutions were positive. For gerbils, seven samples from one breeding facility of the 11 samples from two breeding facilities were positive, and 43 samples from four research institutions of the 63 samples from six research institutions were positive. Multiple pale to yellow foci were seen on the liver surface of almost 50% of H. hepaticus-PCR-positive mice and gerbils. Results of Helicobacter species detection are shown in Table 2. Seventy-nine of 149 samples were positive with Helicobacter genus-specific primers. Among the 79 samples, 20, 35, and 26 samples were identified as H. hepaticus, H. rodentium, and other species, respectively. Two samples from one facility were positive for both H. hepaticus and H. rodentium. No gross lesions were observed in the mice. According to the sequences of the first PCR products from the 26 samples classified as others, these samples were identified as Helicobacter species belonging to the same cluster as H. rodentium. CONCLUSION H. hepaticus was detected not only from mice but also from gerbils. In this study, it was suggested that the gerbil is one of the hosts of H. hepaticus infection. TABLE 1 Detection of Helicobacter hepaticus 16S rRNA in Laboratory Mice and Gerbils Using Polymerase Chain Reaction   No. Positive / No. Tested in Samples No. Positive / No. Tested in Facilities Mice   Breeding facilities 0 / 116 (0%) 0 / 2 (0%) Research institutions 35 / 194 (18.0%) 5 / 19 (26.3%) Gerbils   Breeding facilities 7 / 11 (63.6%) 1 / 2 (0%) Research institutions 43 / 63 (68.3%) 4 / 6 (66.7%)

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Microbial Status and Genetic Evaluation of Mice and Rats: PROCEEDINGS OF THE 1999 US/JAPAN CONFERENCE TABLE 2 Detection of Helicobacter Species 16S rRNA in Laboratory Mice Using Polymerase Chain Reaction Species No. Positive / No. Tested in Samples No. Positive / No. Tested in Facilities H. hepaticus 20 / 149 (13.4%) 1 / 17 (5.9%) H. muridarum 0 / 149 (0%) 0 / 17 (0%) H. bilis 0 / 149 (0%) 0 / 17 (0%) H. rodentium 35 / 149 (23.5%) 6 / 17 (35.3%) “F. rappini” 0 / 149 (0%) 0 / 17 (0%) Others 26 / 149 (17.4%) 3 / 17 (17.6%) Total 79 / 149 (53.0%) 10 / 17 (58.8%) Because gerbils can be infected with H. pylori (Hirayama and others 1996), they are widely used for H. pylori infection studies. These results indicate the necessity of checking H. hepaticus contamination in gerbils. H. hepaticus and H. rodentium were the most common Helicobacter species in mice in Japan, and H. muridarum, H. bilis, and “F. rappini” were not detected. REFERENCES Battles, J.K., J.C. Williamson, K.M. Pike, P.L. Gorelick, J.M. Ward, and M.A. Gonda. 1995. Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene. J. Clin. Microbiol. 33:1344-1347. Fox, J.G., F.E. Dewhirst, J.G. Tully, B.J. Paster, L. Yan, N.S. Taylor, M.J. Collins, Jr., P.L. Gorelick, and J.M. Ward. 1994. Helicobacter hepaticus sp. Nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. 32:1238-1245. Fox, J.G., L.L. Yan, F.E. Dewhirst, B.J. Paster, B. Shames, J.C. Murphy, A. Hayward, J.C. Belcher, and E.N. Mendes. 1995. Helicobacter bilis sp. Nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice. J. Clin. Microbiol. 33:445-454. Hirayama, F., S. Takagi, Y. Yokoyama, E. Iwao, and Y. Ikeda. 1996. Establishment of gastric Helicobacter pylori infection in Mongolian gerbils. J. Gastroenterol. 31:24-28. Lee, A., M.W. Phillips, J.L. O'Rourke, B.J. Paster, F.E. Dewhirst, G.J. Fraser, J.G. Fox, L.I. Sly, P.J. Romaniuk, T.J. Trust, and S. Kouprach. 1992. Helicobacter muridarum sp. Nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. Int. J. Syst. Bacteriol. 42:27-36. Shen, Z., J.G. Fox, F.E. Dewhirst, B.J. Paster, C.J. Foltz, L. Yan, B. Shames, and L. Perry. 1997. Helicobacter rodentium sp. Nov., a urease-negative Helicobacter species isolated from laboratory mice. Int. J. Syst. Bacteriol. 47:627-634. Schauer, D.B, N. Ghori, and S. Falkow. 1993. Isolation and characterization of “Flexispira rappini” from laboratory mice. J. Clin. Microbiol. 31:2709-2714.