Helicobacter bilis/Helicobacter rodentium co-infection associated with diarrhea in a colony of scid mice

An outbreak of diarrhea spanning 3 months occurred in a breeding colony of scid/Trp53 knockout mice. Approximately a third of the 150 mice were clinically affected, with signs ranging from mucoid or watery diarrhea to severe hemorrhagic diarrhea with mortality. Helicobacter bilis and the newly recognized urease-negative organism H. rodentium were isolated from microaerobic culture of feces or cecal specimens from affected mice. Dual infection with H. bilis and H. rodentium were confirmed by culture and polymerase chain reaction (PCR) in several animals. Both Helicobacter species rapidly colonized immunocompetent sentinel mice exposed to bedding from cages containing affected mice, but the sentinel remained asymptomatic. Mice with diarrhea had multifocal to segmental proliferative typhlitis, colitis, and proctitis. Several affected mice had multifocal mucosal necrosis with a few focal ulcers in the cecum, colon, and rectum. Mice with diarrhea were treated with antibiotic food wafers (1.5 mg of amoxicillin, 0.69 mg of metronidazole, and 0.185 mg of bismuth/mouse per day) previously shown to eradicate H. hepaticus in immunocompetent mice. Antibiotic treatment resulted in resolution of diarrhea, but not eradication of H. bilis and H. rodentium; mice continued to have positive PCR results after a 2-week treatment regimen, and clinical signs of diarrhea returned in some mice when treatment was suspended. To the authors' knowledge, this is the first report of natural infection with either H. bilis and/or H. rodentium causing acute diarrheal disease and suggests that H. bilis and/or H. rodentium can be an important pathogen for scid mice.     

Long-term colonization levels of Helicobacter hepaticus in the cecum of hepatitis-prone A/JCr mice are significantly lower than those in hepatitis-resistant C57BL/6 mice

Helicobacter hepaticus infection causes hepatitis in A/JCr mice but mild or no disease in C57BL/6 mice. Colonization of H. hepaticus in the cecum of experimentally infected A/JCr and C57BL/6 mice was quantified by use of real-time polymerase chain reaction (PCR) analysis with primers for the H. hepaticus cdtB gene and mouse 18srRNA. Eight-week-old mice were experimentally (n = 48) or sham (n = 24) infected with H. hepaticus, then were necropsied 6 months after infection. Liver specimens from experimentally infected mice had negative results of PCR analysis for H. hepaticus; thus, real-time quantification was not attempted. Quantitative PCR analysis of H. hepaticus in cecal specimens indicated that C57BL/6 mice were colonized to a greater extent than were A/JCr mice (P < 0.006). Appreciable typhlitis was not observed, but was consistent with that of previous reports; A/JCr mice developed more severe parenchymal necrosis, portal inflammation, and phlebitis in the liver (P < 0.0001), with mild disease observed in infected C57BL/6 mice. Thus, hepatitis in A/JCr mice caused by H. hepaticus infection is associated with significantly lower colonization levels of H. hepaticus in the cecum, compared with those of hepatitis-resistant C57BL/6 mice. Host responses of A/JCr mice that limit cecal colonization with H. hepaticus may have important roles in the pathogenesis of hepatic lesions.     

啮齿动物螺杆菌多重PCR检测方法的建立

目的建立一种可同时检测肝、胆汁、啮齿类三种螺杆菌的多重PCR方法。方法根据已公布的肝、胆汁、啮齿类三种螺杆菌16SrRNA基因序列设计三对特异性引物进行多重PCR并对反应条件进行优化。结果三对引物能分别扩增出特异性的417 bp、364 bp、324 bp目的条带。最佳退火温度为52℃,镁离子浓度为2.0mmol/L,dNTP浓度为200μmol/L,引物浓度为0.625μmol/L。在此条件下多重PCR同时检测的肝、胆汁、啮齿类三种螺杆菌敏感度均为10 fg。结论本实验建立的多重PCR是一种敏感、特异、高效的方法,为同时检测啮齿动物中肝、胆汁、啮齿类三种螺杆菌奠定了良好的基础。     

Helicobacter-induced inflammatory bowel disease in IL-10- and T cell-deficient mice

Inflammatory bowel disease (IBD) is thought to result from a dysregulated mucosal immune response to luminal microbial antigens, with T lymphocytes mediating the colonic pathology. Infection with Helicobacter spp has been reported to cause IBD in immunodeficient mice, some of which lack T lymphocytes. To further understand the role of T cells and microbial antigens in triggering IBD, we infected interleukin (IL)-10(-/-), recombinase-activating gene (Rag)1(-/-), T-cell receptor (TCR)-alpha(-/-), TCR-beta(-/-), and wild-type mice with Helicobacter hepaticus or Helicobacter bilis and compared the histopathological IBD phenotype. IL-10(-/-) mice developed severe diffuse IBD with either H. bilis or H. hepaticus, whereas Rag1(-/-), TCR-alpha(-/-), TCR-beta(-/-), and wild-type mice showed different susceptibilities to Helicobacter spp infection. Proinflammatory cytokine mRNA expression was increased in the colons of Helicobacter-infected IL-10(-/-) and TCR-alpha(-/-) mice with IBD. These results confirm and extend the role of Helicobacter as a useful tool for investigating microbial-induced IBD and show the importance, but not strict dependence, of T cells in the development of bacterial-induced IBD.     

Enteric lesions in SCID mice infected with "Helicobacter typhlonicus," a novel urease-negative Helicobacter species.

BACKGROUND AND PURPOSE: Several rodent helicobacters have been associated with chronic active hepatitis or inflammatory bowel disease. Severe combined immunodeficient (SCID) mice appear to be inherently susceptible to disease attributable to these emerging pathogens. With the advent of polymerase chain reaction (PCR) analysis, it has become clear that several as yet unidentified Helicobacter species may also colonize rodents, but their capacity to cause disease is unknown. METHODS: A Helicobacter species isolated from feces of a BALB/c mouse and provisionally named "H. typhlonicus" was used to inoculate helicobacter-free 4-week-old SCID mice (n = 11 males and 11 females). At various weeks after inoculation, mice were sacrificed and liver and intestinal specimens were collected for histologic examination and PCR analyses. RESULTS: The C.B-17 scid/scid mice inoculated with "H. typhlonicus" developed moderate to severe proliferative typhlocolitis, similar to that seen in SCID mice infected with H. hepaticus or H. bilis. However, in contrast to mice infected with H. hepaticus or H. bilis, lesions of chronic active hepatitis were not detected in mice inoculated with "H. typhlonicus." A similar disease syndrome developed in SCID mice cohabitated with B6D2F1 mice naturally infected with a novel Helicobacter species that was genetically identical to "H. typhlonicus." CONCLUSION: "Helicobacter typhlonicus" joins a growing list of helicobacters that are capable of inducing enteric disease in immunodeficient mice.     

Induction of differential immune reactivity to members of the flora of gnotobiotic mice following colonization with Helicobacter bilis or Brachyspira hyodysenteriae

Aberrant host immune responses to bacterial components of the resident microflora may initiate and perpetuate gastrointestinal inflammation. To investigate how microbial perturbation promotes host immunological responsiveness to commensal bacteria and contributes to the development of typhlocolitis, we selectively colonized defined (altered Schaedler) flora C3H mice with either Helicobacter bilis or Brachyspira hyodysenteriae. Following selective colonization, tissues were analyzed for gross/histopathologic lesions and bacterial antigen-specific B- and T-cell responses. Gnotobiotic mice colonized with H. bilis or B. hyodysenteriae developed typhlocolitis of varying severity, with the most severe gross and histopathogical lesions observed in B. hyodysenteriae-colonized mice. Antigen-specific IgG1 and IgG2a responses to the resident microflora were increased in both H. bilis-and B. hyodysenteriae-colonized mice. The greater antibody responses were associated with less severe cecal inflammation in H. bilis-colonized mice. Altered Schaedler flora (ASF)-stimulated mesenteric lymphocytes from B. hyodysenteriae-colonized mice produced higher levels of interferon-gamma and interleukin (IL)-4 than did lymphocytes from H. bilis-colonized mice. However, ASF-stimulated mesenteric and splenic lymphocytes from both H. bilis and B. hyodysenteriae-colonized mice secreted higher amounts of IL-10 compared to similarly stimulated lymphocytes recovered from control mice. These results indicate that microbial perturbation may induce differential immune responses to nonpathogenic resident bacteria that can lead to intestinal inflammation.     

Helicobacter hepaticus HHGI1 is a pathogenicity island associated with typhlocolitis in B6.129-IL10 tm1Cgn mice

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P<0.001), HhPAId1 did not cause typhlocolitis and hyperplasia in IL10-/- mice. Colonization levels of HhPAId1 were significantly higher in the cecum (P<0.007) and similar in the colon (P=0.27) when compared to H. hepaticus by 13 or 16 weeks post inoculation (WPI). The magnitude of the Th1-associated IgG2c response against HhPAId1 was less than that against H. hepaticus (P<0.004). There was no significant difference in Th2-associated IgG1 responses against these two strains. Cecal and colonic mRNA levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-17a in the HhPAId1-infected mice were significantly lower than those in the H. hepaticus-infected mice (P<0.05) at 13 WPI. These results demonstrate that genes in the HHGI1 contribute to the pathogenicity of H. hepaticus, at least in part via up-regulation of proinflammatory mediators IFN-gamma, TNF-alpha and IL-17a.     

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter Infections

Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter -infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals.
Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus , and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns.
Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H . bilis , in 91% for H. hepaticus, and in 82% for H. ganmani . The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium .
Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents.     

Enterohepatic lesions in SCID mice infected with Helicobacter bilis

Helicobacter bilis is a recently identified species that colonizes the intestine and liver of mice. In immunocompetent mice, infections have been associated with mild hepatitis, and in immunocompromised mice, inflammatory bowel disease has been induced by intraperitoneal inoculation of the organism. We report inoculation of 6-week-old C.B-17 scid/scid mice by gastric gavage with approximately 10(7) H. bilis colony-forming units. Groups of mice were euthanized and necropsied 12, 24, and 36 weeks after inoculation. Mild to moderate proliferative typhlitis was evident in all mice at 12 and 36 weeks after inoculation and in most mice 24 weeks after inoculation. Mild to severe chronic active hepatitis was detected in 10 of 10 male mice and 3 of 10 female mice. These results indicate that H. bilis can cause moderate to severe enterohepatic disease in immunocompromised mice.     

Respiratory syncytial virus G and/or SH glycoproteins modify CC and CXC chemokine mRNA expression in the BALB/c mouse

Chemokine mRNA expression by pulmonary leukocytes following infection of BALB/c mice with two strains of respiratory syncytial virus (RSV) and one strain of parainfluenza virus type 3 (PIV-3) was determined. The results suggest that RSV G and/or SH proteins inhibit early MIP-1alpha, MIP-1beta, MIP-2, MCP-1, and IP-10 mRNA expression. TCA-3 mRNA expression was found to be increased during PIV-3 infection.     

Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.     

Use of the P167 recombinant antigen for serodiagnosis of Helicobacter bilis

Helicobacter bilis is widespread among research mouse colonies. Serodiagnosis of Helicobacter infections involves use of bacterial lysates or membrane antigen preparations that lack specificity, necessitating the need to identify a specific and sensitive antigen. A previously reported recombinant protein (P167) was evaluated for use as an H. bilis-specific antigen for serologic testing. Seventy-six mice naturally infected with Helicobacter spp. were identified from commercially bred or sentinel mice. Infection was confirmed and speciated by use of cecal specimen culture and fecal polymerase chain reaction (PCR) analysis, followed by restriction enzyme digest of the amplicon. Forty-one mice were determined to be monoinfected with H. bilis, 27 mice were determined to be monoinfected with H. hepaticus, and eight mice were infected with another species of Helicobacter. Serum was diluted 1:100 to evaluate the immunoreactivity to enzyme-linked immunosorbent assay preparations of H. bilis membrane extract and the immunodominant C and D fragments of the p167 gene. The sensitivity was greatest for the membrane extract preparation (76%), whereas sensitivity to the P167C and D recombinants was lower (62 and 51%, respectively). However, the specificity of the membrane extract preparation was low (87%), compared with the much improved specificity of the recombinant P167C and D fragments (96 and 96%, respectively). These findings suggest that the recombinant P167C and D fragments of the p167 gene product from H. bilis can be used as specific reagents in the serodiagnosis of H. bilis infection in mice.     

Pathogenesis of mouse hepatitis virus infection in gamma interferon-deficient mice is modulated by co-infection with Helicobacter hepaticus

Gamma interferon-deficient (IFN-gamma KO) mice developed a wasting syndrome and were found to be co-infected with Helicobacter sp., and a new isolate of mouse hepatitis virus (MHV) designated MHV-G. The disease was characterized by pleuritis, peritonitis, hepatitis, pneumonia, and meningitis. Initial experiments used a cecal homogenate inoculum from the clinical cases that contained H. hepaticus and MHV-G to reproduce the development of peritonitis and pleuritis in IFN-gamma KO mice. In contrast, immunocompetent mice given the same inoculum developed an acute, self-limiting infection and remained clinically normal. This result confirmed the importance of IFN-gamma in preventing chronic infection and limiting viral dissemination. To understand the role of both agents in the development of peritonitis and pleuritis, IFN-gamma KO mice were infected with either agent or were co-infected with H. hepaticus and MHV-G. Infection with MHV-G induced a multisystemic infection similar to that described in the original cases, with multifocal hepatic necrosis, acute necrotizing and inflammatory lesions of the gastrointestinal tract, and acute peritonitis and pleuritis with adhesions on the serosal surfaces of the viscera. However, mice given H. hepaticus alone had minimal pathologic changes even though the organism was consistently detected in the cecum or feces. Although co-infection with H. hepaticus and MHV-G induced lesions similar to those associated with MHV-G alone, the pathogenesis of the MHV infection was modified. Helicobacter hepaticus appeared to reduce the severity of MHV-induced lesions during the acute phase of infection, and exacerbated hepatitis and meningitis at the later time point. We conclude that infection of IFN-gamma KO mice with MHV-G results in multisystemic infection with peritonitis, pleuritis, and adhesions due to the aberrant immune response in these mice. In addition, co-infection of these mice with H. hepaticus results in alterations in the pathogenesis of MHV-G infection.     

Helicobacter hepaticus infection triggers inflammatory bowel disease in T cell receptor alphabeta mutant mice

The T cell receptor alpha chain-deficient (TCR alpha-/-) and TCR beta chain-deficient (TCR beta-/-) mice develop chronic intestinal inflammation that resembles inflammatory bowel disease by 3 to 4 months of age. The objective of the study reported here was to determine the role of infection with the bacterial pathogen Helicobacter hepaticus in the pathogenesis of disease in TCR alphabeta mutant mice. The H. hepaticus-infected TCR alphabeta mutant mice were rederived by use of embryo transfer to produce Helicobacter-free animals. Helicobacter-free TCR alpha-/-, TCR beta-/-, and TCR alpha-/- beta-/- mice were inoculated with H. hepaticus. Experimentally infected mice and uninfected control mice were examined for intestinal lesions at 3, 6, and 9 months after inoculation. The TCR alphabeta mutant mice inoculated with H. hepaticus developed intestinal epithelial cell hyperplasia and mucosal inflammation. By 6 months after inoculation, infected animals had moderate cecal and colonic lesions. Helicobacter-free TCR alpha-/- mice, but not TCR beta-/- or TCR alpha-/- x beta-/- mice, also developed H. hepaticus-independent colitis by 9 months after inoculation. Infection with H. hepaticus is sufficient to cause chronic proliferative intestinal inflammation in TCR alphabeta mutant mice. However, H. hepaticus infection is not necessary for intestinal disease in TCR alpha-/- mice.     

Liver-infiltrating T lymphocytes are attracted selectively by IFN-inducible protein-10

We have demonstrated that interferon-inducible protein-10 (IP-10) is produced in hepatocytes surrounded by infiltrative mononuclear cells in chronic hepatitis. To clarify the role of IP-10 in hepatitis, we examined the chemoattractive activity of IP-10 on liver-infiltrating lymphocytes in experimental animal models of hepatitis. IP-10 was specifically induced in the livers of mice treated intravenously (i.v.) with Con A, while monocyte chemotactic protein-1 (MCP-1) showed a much lower level of induction and neither RANTES nor macrophage inflammatory protein-1alpha (MIP-1alpha) was detected. The liver-infiltrating lymphocytes in Con A-induced hepatitis were attracted only by IP-10, and not by other chemokines such as RANTES, MCP-1 and MIP-1alpha. The chemoattractive effect of IP-10 was dose-dependent and was neutralized by monoclonal antibodies to IP-10. The specific effect of IP-10 on liver-infiltrating lymphocytes was also seen on those obtained from rat livers with fulminant hepatitis induced by sequential treatment with killed Propionibacterium acnes (P. acnes) and LPS. Peripheral blood lymphocytes were slightly attracted by IP-10 as well as RANTES and MIP-1alpha, while hepatic resident lymphocytes were not. On the other hand, thioglycolate-elicited peritoneal macrophages did not respond to IP-10, although they did show a response to RANTES, MCP-1 and MIP-1alpha. These results indicated that IP-10 is a specific chemoattractant for T lymphocytes in the inflammatory liver tissues and may play a specific role in the development of hepatitis.     

Evaluation of Helicobacter hepaticus bacterial shedding in fostered and sex-segregated C57BL/6 mice

Neonatal fostering has been evaluated as a means of eliminating Helicobacter hepaticus infection in laboratory mouse colonies. The purpose of the present study was to evaluate cross-fostering of neonatal C57BL/6 pups from experimentally infected dams after male-absent parturition and to determine the effects of sex and housing strategy on H. hepaticus populations. Approximately 20 C57BL/6 mice (age, 1 to 4 days) were fostered daily. In all fostered mice, fecal samples collected at 21 and 42 days of age and cecal samples collected at 42 days of age tested negative for H. hepaticus by polymerase chain reaction analysis. Our results demonstrate that removal of the male prior to parturition extends the fostering period to yield Helicobacter-free mice. In a second experiment, the effects of time of infection, housing strategy, and sex on fecal H. hepaticus shedding and cecal colonization were evaluated. Neither time nor housing strategy affected bacterial shedding. In contrast, fecal and cecal bacterial loads were higher in male mice versus female mice. A novel predictive algorithm was developed to predict cecal bacterial colonization levels in light of fecal bacterial loads. Our findings likely will prove useful in Helicobacter eradication efforts and in studies designed to further elucidate the role of H. hepaticus in disease.     

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.