Helicobacter hepaticus infection induces chronic hepatitis and fibrosis in male BALB/c mice via the activation of NF-κB, Stat3, and MAPK signaling pathways.

BACKGROUND: It has been documented that Helicobacter hepaticus (H hepaticus) infection is linked to chronic hepatitis and liver cancer. However, our understanding of the molecular mechanisms underlying progression of the H hepaticus-induced hepatic inflammation to cellular hepatocarcinoma is still limited. MATERIALS AND METHODS: In our study, male BALB/c mice were infected by H hepaticus for 8, 12, 16, 20, and 24 weeks. Histopathology, H hepaticus colonization dynamics, select signaling pathways, and expression of key inflammatory cytokines in the liver were examined. RESULTS: We found that H hepaticus was detectible in feces of mice at 7 days postinfection (DPI) by PCR, but it was not detected in the livers by PCR until 8 weeks postinfection (WPI). In addition, abundance of colonic and hepatic H hepaticus was progressively increased over the infection duration. H hepaticus-induced hepatic inflammation and fibrosis were aggravated over the infection duration, and necrosis or cirrhosis developed in the infected liver at 24 WPI H hepaticus infection increased levels of alanine aminotransferase and aspartate aminotransferase. Moreover, mRNA levels of Il-6 and Tnf-α were significantly elevated in the livers of H hepaticus-infected mice compared to uninfected control from 8 WPI to 24 WPI. Furthermore, Stat3, nuclear factor-κB (p65), and MAPK (Erk1/2 and p38) were activated by H hepaticus infection. CONCLUSIONS: These data demonstrated that male BALB/c mice can be used as a new mouse model of H hepaticus-induced liver diseases and that the H hepaticus-induced liver injury is triggered by NF-κB, Jak-Stat, and MAPK signaling pathways.

Helicobacter hepaticus infection in primary hepatocellular carcinoma tissue.

INTRODUCTION: Helicobacter (H.) hepaticus infection causes chronic active hepatitis and induces hepatocellular tumours in A/JCr mice, but evidence of this in humans is scarce. This study aimed to demonstrate the correlation between H. hepaticus and human primary hepatocellular carcinoma (HCC). METHODS: The sera of 50 patients with primary HCC were tested for the presence of anti-H. pylori and anti-H. hepaticus immunoglobulin G (IgG) antibodies. The liver tissues of patients who tested positive for serum antibody were analysed for H. hepaticus-specific 16S rRNA, H. hepaticus cdtB, H. pylori cagA, H. pylori vacA and H. pylori ureC genes using polymerase chain reaction. RESULTS: After the anti-H. pylori antibodies in the serum samples were absorbed by H. pylori antigen, the anti-H. hepaticus IgG serum antibody detection rate was 50.0% in patients with primary HCC. This was significantly higher (p < 0.001) than the detection rate in the benign liver tumour (7.7%) and normal liver tissue (6.3%) groups. Of the 25 primary HCC samples that tested positive for anti-H. hepaticus IgG serum antibody, the H. hepaticus-specific 16S rRNA gene was detected in nine (36.0%) samples. Sequencing showed that the polymerase chain reaction-amplified product exhibited 95.5%-100% homology to the H. hepaticus-specific 16S rRNA gene. Among these nine primary HCC tissue samples, the H. hepaticus cdtB gene was detected in four (44.4%) samples, while no such expression was observed in the benign liver tumour or normal liver tissue groups. CONCLUSION: The present study identified the presence of H. hepaticus infection in patients with primary HCC using serological and molecular biological detection, suggesting that H. hepaticus infection may be involved in the progression of HCC.

Augmented cholesterol absorption and sarcolemmal sterol enrichment slow small intestinal transit in mice, contributing to cholesterol cholelithogenesis.

Cholesterol gallstones are associated with slow intestinal transit in humans as well as in animal models, but the molecular mechanism is unknown. We investigated in C57L/J mice whether the components of a lithogenic diet (LD; 1.0% cholesterol, 0.5% cholic acid and 17% triglycerides), as well as distal intestinal infection with Helicobacter hepaticus, influence small intestinal transit time. By quantifying the distribution of 3H-sitostanol along the length of the small intestine following intraduodenal instillation,we observed that, in both sexes, the geometric centre (dimensionless) was retarded significantly (P <0.05) by LD but not slowed further by helicobacter infection (males, 9.4±0.5 (uninfected), 9.6±0.5 (infected) on LD compared with 12.5±0.4 and 11.4±0.5 on chow). The effect of the LD was reproduced only by the binary combination of cholesterol and cholic acid. We inferred that the LD-induced cholesterol enrichment of the sarcolemmae of intestinal smooth muscle cells produced hypomotility from signal-transduction decoupling of cholecystokinin (CCK), a physiological agonist for small intestinal propulsion in mice. Treatment with ezetimibe in an amount sufficient to block intestinal cholesterol absorption caused small intestinal transit time to return to normal. In most cholesterol gallstone-prone humans, lithogenic bile carries large quantities of hepatic cholesterol into the upper small intestine continuously, thereby reproducing this dietary effect in mice. Intestinal hypomotility promotes cholelithogenesis by augmenting formation of deoxycholate, a pro-lithogenic secondary bile salt, and increasing the fraction of intestinal cholesterol absorbed.

Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation.

OBJECTIVES: To investigate whether Helicobacter spp. infection and the cagA of H. pylori are associated with hepatobiliary pathology, specifically biliary inflammation, cell proliferation and cholangiocarcinoma (CCA). METHODS: Helicobacter species including H. pylori, H. bilis and H. hepaticus were detected in the specimens using the polymerase chain reaction (PCR). Biliary inflammation of the liver and gallbladders was semi-quantitatively graded on hematoxylin and eosin (H&E)-stained slides. Biliary proliferation was evaluated by immunohistochemistry using the Ki-67-labelling index. RESULTS: Helicobacter pylori was found in 66.7%, 41.5% and 25.0% of the patients in the CCA, cholelithiasis and control groups (P < 0.05), respectively. By comparison, H. bilis was found in 14.9% and 9.4% of the patients with CCA and cholelithiasis, respectively (P > 0.05), and was absent in the control group. The cagA gene of H. pylori was detected in 36.2% and 9.1% of the patients with CCA and cholelithiasis, respectively (P < 0.05). Among patients with CCA, cell inflammation and proliferation in the liver and gallbladder were significantly higher among those DNA H. pylori positive than negative. CONCLUSIONS: The present findings suggest that H. pylori, especially the cagA-positive strains, may be involved in the pathogenesis of hepatobiliary diseases, especially CCA through enhanced biliary cell inflammation and proliferation.

Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period.

Viral, bacterial and parasitological infections in rats and mice used in biomedical research continue to occur despite improved housing and biosurveillance. The presence of disease in laboratory animals can lead to spurious results for research undertaken in universities, research institutes and the pharmaceutical industry. Here the authors report the results of serological, microbiological, parasitological and molecular tests done on mice and rats from Australasia submitted to a rodent health monitoring laboratory (Cerberus Sciences) from 2004 to 2009. In tested mice, norovirus was the most prevalent virus and ectromelia virus was the least prevalent virus. In tested rats, pneumonia virus of mice was the most prevalent virus and adenoviruses 1 and 2 were the least prevalent viruses. In mice, Helicobacter hepaticus was the most prevalent bacterium, and in rats, Proteus spp. were the most prevalent bacteria. The most common positive helminthological finding in mice and rats was the presence of all pinworms (including Aspicularis spp. and Syphacia spp.). The most common positive protozoan findings in mice and rats were Chilomastix spp. and Trichomonads.

Serodiagnosis of Helicobacter hepaticus infection in patients with liver and gastrointestinal diseases: western blot analysis and ELISA using a highly specific monoclonal antibody for H. hepaticus antigen.

BACKGROUND: Helicobacter hepaticus infection might be associated with liver and biliary tract diseases. To investigate its pathogenic role, the properties of anti-H. hepaticus serum antibody in patients with liver and diseases were elucidated. METHODS: Serum samples were collected from 166 patients-69 with liver diseases, 38 with upper gastrointestinal diseases, 17 with lower gastrointestinal diseases, 26 with biliary tract diseases, and 16 with pancreas diseases; 30 control sera were obtained from 30 healthy blood donors. Serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) and western blot using the new monoclonal antibody HR II-51. RESULTS: Anti-H. hepaticus serum antibody concentrations in patients with liver disease (n = 69) were significantly increased compared with those in other disease groups (p = 0.014 to <0.001). Particularly, liver cirrhosis (n = 19) showed a significantly higher antibody level compared with other liver diseases (n = 50, p = 0.005) and healthy donors (n = 30, p = 0.0005), as well as a higher seroprevalence (68.4%) compared with other liver diseases (p = 0.05) and healthy donors (p = 0.004). Furthermore, the ELISA value in liver cirrhosis (n = 19) was significantly higher than that in patients with hepatitis B virus (HBV)-and/or hepatitis C virus (HCV)-infected chronic hepatitis (n = 15) (0.389 ± 0.084 vs. 0.350 ± 0.084, p = 0.029). However, there was no relationship between the total immunoglobulin concentration and the anti-H. hepaticus antibody level in each liver disease (Spearman's rank correlation coefficient [rs] < 0.225). CONCLUSIONS: H. hepaticus infection might play a role in the development of liver diseases; in particular, it might increase the risk of the development of HBV- and/or HCV-infected liver diseases.

Triplex PCR for the simultaneous detection of Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium.

The accurate and economical diagnosis of pathogenic bacteria is necessary for the microbiological control of laboratory animals. In this study, we developed a triplex PCR method for the direct detection of three common gastroenteric bacteria, Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium. Targets were specifically amplified by conventional PCR assay using a genomic fragment from P. aeruginosa, 16S ribosomal RNA from H. hepaticus, and the invA gene from S. typhimurium. To investigate the specificity of our primers, they were tested against purified DNA from many other bacterial species. There were no amplification products from other bacteria. Under optimized conditions, the triplex assay simultaneously yielded a 726-bp product from P. aeruginosa, a 417-bp product from H. hepaticus, and a 246-bp product from S. typhimurium. The detection limits of this assay in pure culture were 10 pg for P. aeruginosa, and 0.1 pg for H. hepaticus and S. typhimurium. All three bacteria were successfully detected in the liver, cecum, and feces of experimentally infected mice. This method is a useful and convenient assay that allows the simultaneous identification of bacterial pathogens in mice. Our triplex method will be used to improve quality control in the detection of pathogenic bacterial infections in laboratory animal facilities.

Detection of Helicobacter hepaticus in human bile samples of patients with biliary disease.

BACKGROUND: Since the discovery of Helicobacter pylori, various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown. MATERIALS AND METHODS: We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis. RESULTS: Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher (p = .029) prevalence of H. hepaticus infection than samples from patients with other diseases. CONCLUSION: Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.

[Cloning and bioinformatics analysis of recombinant methyl-accepting chemotaxis signal transduction protein of Helicobacter hepaticus].

OBJECTIVE: To clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods. METHODS: With the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method. RESULTS: A 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes. CONCLUSION: The prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.

Characterization and application of a new monoclonal antibody with high specificity for Helicobacter hepaticus.

BACKGROUND AND AIMS: Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria. METHODS: The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate. RESULTS: HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus-antigen-based ELISA. CONCLUSION: Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans.

Study of Helicobacter hepaticus in gallbladders with cholelithiasis and its sensitivity pattern.

BACKGROUND: Cholelithiasis is one of the most common digestive surgical disorders. The natural history of gallstone development is unknown. Bacteria are found in high concentration in bile and stone. It is difficult to ascertain whether bacterial infection of the bile arose before stone formation or vice versa. MATERIALS & METHODS: Prospective study was carried out in 30 cholecystectomy specimens of patients with cholelithiasis collected from 15th January 2005 to 24th July 2005 in Department of Pathology, Kathmandu Medical College Teaching Hospital. The samples were collected in a sterile vial containing 0.5% saline. A small piece of gallbladder was taken in Blood agar and Chocolate agar media for micro anaerobic culture at 370 degrees Celsius. The growth of the organism was observed after 48 hours of incubation. Gram stain and various biochemical tests were performed for the confirmation of Helicobacter hepaticus and its sensitivity pattern was studied. Histologically Helicobacter hepaticus was demonstrated in formalin fixed tissue sections using Warthin Starry Silver stain. RESULTS: A total of 76.66% cases showed growth positivity. Nitrofurantoin was found to be the most sensitive drug (95.7%) for Helicobacter hepaticus followed by Ciprofloxacin (91.3%), Cephalaxin (91.3%), Certriaxone (91.3%),Ofloxacin (82.6%), Amikacin (65.2%) and Norfloxacin (60.9%). Helicobacter hepaticus was least sensitive to Amoxycillin (57%). CONCLUSION: From this study, it is quite apparent that Helicobacter hepaticus infection is commonly associated (76.66%) with cholelithiasis. If the patients are treated with the sensitive antibiotics regularly and the infection, that is the number one cause for the precipitation of bile leading to the formation of gallstones, can be controlled, the incidence of cholelithiasis can be reduced so that the health care expenditure related to gallbladder disease can be reduced markedly.

[Isolation and characterization of the first strain of Helicobacter hepaticus in China].

Helicobacter hepaticus is nongastric helicobacter that can reside in the hepatobiliary and intestinal systems of many animal hosts, leading to proliferative hepatitis, hepatocellular carcinoma, typhlitis, and colonitis. In this study, the intestinal mucosa was isolated from BALB/c mice to prepare tissue homogenate and spread onto selective C jejuni blood agar plates for incubation in the presence of trimethoprim, vancomycin, and polymyxin at 37 degrees Celsius; under microaerobic conditions in vented jars containing 5% O2, 10%CO2, and 85% N2. The bacteria were identified morphologically and biochemically. Gene sequence analysis of the 16s rRNA confirmed the presence of Helicobacter hepaticus, and the success in isolating this bacteria may have significant implications for studies of nongastric helicobacter.

Specific and quantitative detection of PCR products from Clostridium piliforme, Helicobacter bilis, H. hepaticus, and mouse hepatitis virus infected mouse samples using a newly developed electrochemical DNA chip.

We developed a microfabricated electrochemical DNA chip for detection of polymerase chain reaction (PCR) products from 16S rRNA sequences of Clostridium piliforme (Cp), Helicobacter bilis (Hb) and Helicobacter hepaticus (Hh), and the nucleocapsid protein gene of mouse hepatitis virus (MHV). This chip does not require DNA labeling, and the hybridization signal can be detected as an anodic current. The average anodic currents of 9 (Cp), 5 (Hb), 8 (Hh) and 7 (MHV) PCR positive samples derived from feces of spontaneously infected mice (Cp, Hb and Hh) and MHV-contaminated tumor cells were 27.9+/-7.2, 31.9+/-8.1, 29.3+/-10.1, and 27.6+/-3.0 nA, respectively. On the other hand, the average anodic currents of 19 (Cp), 27 (Hb), 18 (Hh), and 13 (MHV) PCR negative samples were 0.3+/-2.9, 3.7+/-2.4, -1.0+/-1.7, and -2.3+/-2.7 nA, respectively. The anodic current increased with increasing concentrations of pathogens. For experimentally infected samples, the results of PCR/electrophoresis were in complete accord with those of this system when anodic currents of 6.1 (Cp), 8.5 (Hb), 2.4 (Hh), and 3.1 nA (MHV) were taken as the cut-off value. The results suggested that the electrochemical DNA chip system is useful for specific and quantitative detection of PCR products.

Occurrence of Helicobacter species other than H. hepaticus in laboratory mice and rats in Sweden.

The aim of this study was to determine which Helicobacter species other than H. hepaticus colonize laboratory mice and rats in Sweden. We analyzed 63 intestinal samples from mice and 42 intestinal samples from rats by partial 16S rDNA sequence analysis. Previously these samples had been found positive for Helicobacter species but negative for H. hepaticus in a polymerase chain reaction screening assay at the National Veterinary Institute in Sweden. H. ganmani, H. typhlonius, H. rodentium, an uncharacterized Helicobacter species ('hamster B'), and a possibly novel species were detected in mice. The possibly novel species was most closely related to H. apodemus strain YMRC 000216 (98.3% sequence similarity). Two different Helicobacter species were detected in rats: H. ganmani and H. rodentium. H. ganmani colonization of rats has not previously been reported.

[Study on Helicobacter infection in liver tissue from hepatocellular carcinoma].

OBJECTIVE: To investigate the relationship between Helicobacter species and hepatocellular carcinoma(HCC). METHODS: Liver samples resected during operation from 34 patients with HCC diagnosed by histopathology and 20 without primary liver carcinoma as controls were studied. The two groups of sample were cranked out pathologic slice for in situ hybridization of Helicobacter, Helicobacter pylori and Helicobacter hepaticus. Qualitative and quantitative studies were used to assess the correlation of liver tissue Helicobacter infection with HCC. RESULTS: 64.71% (22/34). of the samples of HCC showed positive for Helicobacter specific 16S rRNA-mRNA gene by in situ hybridization, while none was positive in controls (P < 0.01). CONCLUSION: Helicobacter pylori were found in the liver of patients with HCC.

Visualization of Helicobacter species within the murine cecal mucosa using specific fluorescence in situ hybridization.

BACKGROUND: Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species. MATERIALS AND METHODS: Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections. RESULTS: Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts. CONCLUSION: We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.

Sex influence on chronic intestinal inflammation in Helicobacter hepaticus-infected A/JCr mice.

Helicobacter hepaticus is a bacterial pathogen of mice that has been reported to cause chronic intestinal inflammation in A/JCr, germfree Swiss Webster, and immunodeficient mice. To the authors' knowledge, the influence of sex on development of chronic intestinal inflammation in H. hepaticus-infected mice has not been investigated. The purposes of the study reported here were to determine whether severity of intestinal inflammation differs between male and female A/JCr mice chronically infected with H. hepaticus and to characterize the mucosal immune response in these mice. The cecum of male and female A/JCr mice infected with H. hepaticus for 1 month and 3 months was objectively evaluated histologically for intestinal disease. Also, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was done to measure interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 4 (IL-4), IL-10, macrophage inflammatory protein-1alpha (MIP-1alpha), interferon-inducible protein of 10 kDa (IP-10), and monokine induced by gamma interferon (MIG) mRNA values in the cecal tissue of these mice. Significant differences in cecal lesion scores were not present at 1 month after infection. However, infected female mice had significantly up-regulated expression of cecal IL-10, MIP-1alpha, IP-10, and MIG mRNA compared with that in uninfected females, and expression of IL-10 and MIP-1alpha was significantly greater than that detected in infected male mice (P < or = 0.05). At 3 months after infection, cecal lesion scores were significantly (P < or = 0.05) increased in female and male mice compared with uninfected controls, and infected female mice had significantly (P < or = 0.05) higher cecal lesion scores than did infected male mice. In addition, infected females had significant (P < or = 0.05) increases in cecal IFN-gamma, TNF-alpha, IL-10, MIP-1alpha, IP-10, and MIG mRNA values compared with values in uninfected females and infected males, and male mice had significant (P < or = 0.05) increases in cecal TNF-alpha and IL-10 mRNA values compared with those for male control mice. These data indicate that, in H. hepaticus-infected A/JCr mice, females develop more severe intestinal inflammation than do males, and the chronic mucosal inflammation is polarized toward a Th1 response that is not down-regulated by increased activity of IL-10. We propose that H. hepaticus-infected A/JCr mice will serve as a good animal model with which to study the influence of sex on bacterial-induced mucosal inflammation.

Optimal age at fostering for derivation of Helicobacter hepaticus-free mice.

Helicobacter hepaticus is well established as an unwanted variable in laboratory rodent colonies. Historically, cesarean section and embryo transfer have been used to derive Helicobacter-free mouse colonies. Neonatal transfer of newborn mice onto Helicobacter-free foster dams was recently reported as an alternative method of deriving Helicobacter-free mice, but until now, the age by which pups must be fostered to remain Helicobacter-free was unknown. The purpose of the study reported here was to determine the age by which mouse pups must be fostered to remain free of H. hepaticus. Beginning on the day of birth, 20 C57BL/6 mice were fostered from H. hepaticus-positive parents onto Helicobacter-free BALB/c dams each day for 14 days for a total of 280 pups fostered. Fecal specimens collected at weaning, and fecal, liver, and cecal specimens collected at euthanasia were analyzed by use of polymerase chain reaction (PCR) analysis. No pup fostered within 24 h of birth became infected with H. hepaticus; however, many of those fostered after 24 h became infected. These results were supported by those of a large field trial, in which 201 litters representing 71 strains of mice were fostered within 24 h of birth. Follow-up fecal PCR analysis was performed on 52 mice or their progeny that were randomly sampled from the 201 fostered litters. All mice tested remained free of H. hepaticus approximately 100 days after fostering. The results indicate that mouse pups must be fostered within 24 h of birth to remain free of H. hepaticus. In addition, cecal and fecal PCR analyses detected more infections, than did liver PCR analysis, thus indicating that those specimens are preferred for detection of H. hepaticus infection.