cages were positive and one was negative. No bacterial growth occurred for the control fecal samples at any time point. With regard to the gastrointestinal tract, we found hyperplasia of the glandular stomach in 17 of 81 (21%) exposed mice and in 11 of 82 (13%) controls, not a signiﬁcant difference (p = 0.22). Histopathologic examination of ceca revealed hyperplasia of GALT in 7–40% of mice, with no signiﬁcant differences in inci- dence between exposed and control mice. Proliferative typhlitis occurred predominately in the exposed mice (Table 1); incidence was highest at 12–54 weeks after exposure and was significantly greater than controls at 12, 24, and 36 weeks, with an apparent decrease thereafter. The incidence in all exposed mice at 12–36 weeks (21 of 40) was significantly greater than the incidence at > 54 weeks (0 of 6; p = 0.025). Conﬁrmation of lack of tumorigenic effec- tiveness of experimental infection with H. hepaticus in A/J, BALB/c, and C57BL/6 as weanlings. To conﬁrm the results noted above for A/J mice and to test whether other mouse strains might be more susceptible, we repeated the experiment and included BALB/c and C57BL/6 mice (Table 3). Small subsets of mice were euthanized at interim time points and all mice remaining at 1 year of age were maintained until natural morbidity. Fecal sam- ples throughout the course of the study con- firmed the presence of helical bacteria shed from all of the treated mice and from none of the controls. We observed few age-dependent differences. A summary of lesions for all mice in the study is presented in Table 3. We diagnosed chronic active hepatitis rarely in two exposed BALB/c mice and one exposed C57BL/6 mouse (Table 3). Liver tumors occurred in low incidence in old mice and were not signiﬁcantly different in treated versus con- trol groups. Among the experimentally infected mice presenting H. hepaticus–associated hepati- tis, we found one hepatocellular carcinoma in a BALB/c mouse at 778 days of age, and three adenomas in a C57BL/6 mouse at 720 days. Several histopathologic changes were sig- niﬁcantly associated with treatment in at least one of the mouse strains. We found hyperpla- sia of the nonglandular stomach in 6 of 30 (20%) infected A/J mice, compared with 0 of 29 controls (p = 0.024). Five of the cases were in old mice, and four were moderate or severe. In the other strains, gastric hyperplasia was uncommon and not associated with treat- ment. Table 3 shows other signiﬁcant altera- tions. In A/J mice, proliferative typhlitis was more common in ceca of infected versus con- trol mice at all end points, and these differ- ences were significant for all mice combined (Table 3). However, the incidence of prolifera- tive typhlitis was reduced to 7% in infected A/J mice at 2 years of age. Signiﬁcant differences in Environmental Health Perspectives • VOLUME 116 | NUMBER 10 | October 2008 these parameters were also observed for BALB/c mice; in this strain, frequency of hyperplasia of GALT was also higher in the mice infected with H. hepaticus: 15 of 23 (65%) versus 9 of 33 (27%) (p = 0.0066). Fecal samples confirmed that all three strains of experimentally infected mice, but no controls, were shedding H. hepaticus at all time points. Discussion The results of this study conﬁrm the ﬁndings of other investigations (Avenaud et al. 2003; Ihrig et al. 1999); we found that ig injection of weanling or young adult A/J mice with H. hepaticus reliably leads to early hepatic changes—in 40% of the mice in our study at 12 weeks after exposure—as well as marked increases in oxidative DNA damage, but liver tumors are rarely an outcome. With increasing time after exposure, fecal samples became less uniformly positive, a lower proportion of livers exhibited Helicobacter-related damage, and the elevated oxidative DNA damage relative to controls disappeared. We discovered few hepatitis-related tumors or preneoplastic foci, none in A/J mice. These results are in contrast to those reported for naturally infected mice; for example, Sipowicz et al. (1997) measured a steady increase in 8-oxo-dG at 10, 12, and 18 months of age. Such mice had high incidence (92%) of liver tumors at 18 months of age (Ward et al. 1994b). Together, the results are consistent with host immune response successfully suppressing H. hepaticus in mice infected after weaning. In the A/J and BALB/c mouse strains, infection with H. hepaticus resulted in several hepatic and cecal changes, including prolifera- tive typhlitis, as observed in other studies. We observed no effects in C57BL/6 mice, con- ﬁrming the resistance of this strain to hepatic effects of the bacteria (Ihrig et al. 1999; Ward et al. 1994a, 1994b), despite the greater con- centration of bacteria in the ceca of C57BL/6 than in A/J mice (Whary et al. 2001). In contrast, perinatal infection of A/J mice by ip injection of their mothers with Perinatal H. hepaticus and liver tumors H. hepaticus before breeding led to a high incidence of progressive hepatitis and signiﬁ- cant numbers of multiple liver tumors, includ- ing hepatocellular carcinomas, in their male offspring. This conﬁrms the previous ﬁnding of Rogers et al. (2004). The present study par- tially reproduces the high incidence of liver tumors in naturally infected A/J males and provides an experimental paradigm for gener- ating H. hepaticus–associated liver tumors. We observed lesser effects in male offspring of mothers treated with H. hepaticus ig before conception. Intragastric treatment might be expected to be less effective for establishing a systemic infection with H. hepaticus than ip treatment. We conﬁrmed this by the presence of chronic inflammation in the livers of ip- treated mothers but not ig-treated mothers. In a study with H. pylori and a novel Helicobacter species, McCathey et al. (1999) found that ip-administered bacteria, but not ig adminis- tration at the same titer, resulted in hepatitis and elicited a much greater immune response. Thus, the likelihood of transmission of pro- gressive disease to offspring may be a function of degree of infection of the mother, and the very high incidences of hepatitis and tumors in male offspring of naturally infected moth- ers, seen in our original population of infected mice, may reﬂect the level of bacterial burden accumulated over generations. However, the possibility of attenuation of bacterial virulence with serial passaging cannot be ruled out at present. The bacteria might pass from mother to offspring transplacentally, through milk, or through feces. Li et al. (1998) were able to culture H. hepaticus from about 20% of fetal viscera in severe combined immunodeﬁciency (SCID) mice, but not from any of 14 A/J fetuses. This indicates that transplacental transmission is at least possible. However, Singletary et al. (2003) eliminated mother-to- pup transmission of H. hepaticus for C57BL/6 females experimentally infected ig with this bacterium by foster nursing the newborns within 24 hr after birth. Thus, under these conditions, at least, transmission was not Proliferative typhlitis 11 (37)* 3 (10) 7 (30)* 1 (3) 1 (3) 3 (11) 1355 Table 3. Summary of lesions in livers and ceca of mice of different strains experimentally infected as weanlings with H. hepaticus and in controls. Strain A/J BALB/c C57BL/6 Group Infected Control Infected Control Infected Control No. 30 29 23 33 33 28 Hepatitis 0 0 2 (9) 0 1 (3) 0 Liver tumor 0 1a 1 (4)b 3 (9)c 6 (18)d 8 (29)e Values shown are number (%). aHepatocellular adenoma, mouse 462 days of age. bHepatocellular adenoma, mouse 778 days of age, grade 2 hepatitis; also eosinophilic foci. cTwo hepatocellular adenomas in mice 637 and 683 days of age, and one carcinoma in mouse 610 days of age; all without hepatitis.d Three mice with single adenomas, one mouse with three adenomas and hepatitis (neg- ative for presence of Helicobacter with Steiner’s stain), and one mouse each with hepatoblastoma or with hepatocholan- giocarcinoma. eFour mice with single hepatic adenoma, two mice each with two adenomas, and two mice each with an adenoma and a carcinoma. *p < 0.05 compared with control mice of that strain.