Helicobacter spp. Infections

Helicobacter spp. have emerged as a major group of gastrointestinal commensals with pathogenic potential in the laboratory mouse. Mice serve as hosts to several named species and additional novel isolates that are yet to be formally named.

There is evidence that H. bilis infections in humans may be linked to gall bladder, biliary and liver disease, and associated cancers, and H. pullorum is a human pathogen. Aside from their direct pathogenic potential, particularly in immunodeficient mice, the association of Helicobacter spp. with promotion of hepatic neoplasia and the presence of invasive and dysplastic intestinal lesions in infected mice have led to claims that Helico­bacter is a carcinogen. Helicobacter hepaticus (and proba­bly other Helicobacter spp.) is associated with an increase in hepatocellular tumors in certain strains of infected mice, and has also been shown to promote experimental chemical hepatocarcinogenesis.

Epizootiology and Pathogenesis

Natural outbreaks of hyperplastic typhlocolitis in immu­nodeficient mice have been associated with multiple species of Helicobacter, or (often) mixed infections. Observations by the authors have confirmed that most Helicobacter spp. of the mouse are capable of induc­ing proliferative typhlocolitis in immunodeficient mice, but some isolates may be nonpathogenic commensals. These agents appear to be widespread in conventional colonies of laboratory mice, but as awareness has arisen,

there has been a concerted effort to eliminate Helico­bacter spp. from both commercial and research labora­tory settings. Helicobacter hepaticus and H. rodentium appear to be the most prevalent members of the group among laboratory mice.

Mice are likely exposed to infection through inges­tion of contaminated feces, and Helicobacter spp. are readily transmitted by contaminated bedding. Coloni­zation of the cecum with H.

In the liver, H. hepaticus and H. bilis localize and persist indefinitely within the bile canaliculi. Mouse genotype is pivotal in expression of hepatic disease when infected with H. hepaticus. A/JCr, SCID/NCr, BALB/cANCr, C3H/HeNCr, and SJL/NCr mice have been found to be susceptible to hepatitis, whereas B6 and B6C3F1 mice are hepatitis-resistant. A/J mice also develop an earlier onset and higher prevalence of hepa­tocellular tumors when infected with H. hepaticus. Studies indicate that multiple genes are involved in genetic sus­ceptibility and resistance to liver disease. Hepatic lesions are more common in male A/JCr mice than females, and the incidence of lesions is increased in mice 6 months of age or older. Other Helicobacter spp. may also be associated with hepatitis. For example, H. bilis has been associated with hepatitis in outbred Swiss Webster mice.

The pathogenesis of hepatitis and hepatocellular tumors is unknown, but hepatotoxins or autoimmunity to heat shock protein 70 are suspected. Colonization of the liver leads to induction of apoptosis and cellular prolifera­tion, which are linked to promotion of neoplasia. Patho­genesis of typhlocolitis, which has marked inflammatory and hyperplastic features, has been studied in SCID and nude mice as well as GEMs with selective immune defi­ciencies, including IL-2, IL-10, T-cell receptor (alpha, beta, delta), RAG, MHC class II, and other null mutations. It has been proposed that Helicobacter infection induces a highly

Th1-polarized mucosal immune response, with produc­tion of IL-12, gamma interferon, and tumor necrosis factor (TNF) alpha in the lamina propria, resulting in expression of the epithelial mitogen, keratinocyte growth factor. Adoptive transfer of CD4 T cells expressing high levels of CD45RB into immunodeficient mice infected with H. hepaticus accelerates inflammation and hyperplasia, resulting in the conclusion that bowel disease is an immune-mediated disease that is directed against “nor­mal” intestinal microflora.

Pathology

Infections in immunocompetent mice are usually inapparent. Clinical signs in immunodeficient mice include wasting (often complemented by pneumocys­tosis or other forms of pneumonia in immunodeficient mice) and mortality. Feces may be unformed, sticky, mucoid, or hemorrhagic. Rectal prolapse is a frequent, but nonspecific, sign of colitis in mice. The prolapsed mucosa is usually eroded and markedly inflamed and hyperplastic. These changes are nonspecific and due to the trauma of prolapse and may not be directly associ­ated with Helicobacter spp. Thus, other areas of bowel should be examined to confirm the diagnosis. Segmental areas of cecum and colon are grossly thickened and opaque. Affected mucosa is thickened due to varying degrees of crypt hyperplasia, with markedly immature and mitotically active enterocytes occupying the entire crypt column.

FIG. 1.58. Colon from an immunodeficient mouse, depicting Helicobacter organisms within hyperplastic crypts (Steiner stain).

FIG. 1.59. Colon from a MRL-lpr (immunodeficient) mouse chronically infected with Helicobacter hepaticus. There is marked mucosal hyperplasia, with mononuclear cell infiltration in the lamina propria.

especially mucosa that has been aggravated by erosion or rectal prolapse, there is often focal crypt dysplasia and invasion of hyperplastic crypts into the underlying sub­mucosa, accompanied by cystic change with mucous retention. Depending upon the mouse genotype and stage of infection, there are varying degrees of mixed leukocytic infiltration in the lamina propria. In some strains, there may be marked lymphocytic infiltration (Fig. 1.59). Although there may be extensive lesions in the large intestine, hepatic lesions may be uncommon. An atypical enteric syndrome has been described in H. muridarum-infected mice with gastritis and gastric atrophy, but Helicobacter spp. are more typically associ­ated with lower bowel disease in mice.

Hepatic lesions are quite variable. If present, they consist of randomly scattered grossly visible white foci up to 4 mm in diameter. Early lesions may be confined to one or more lobes of the liver, with focal necrosis and mixed leukocytic infiltrates (Fig. 1.60). Within a few months, there is marked hypertrophy and hyperplasia

FIG. 1.61. Liver from a mouse chronically infected with Helicobacter hepaticus. Note the hepatocytomegaly, polykarya, Ito cell proliferation, and leukocytic infiltration.

of Kupffer, Ito, and oval cells, with increased mitotic activity among hepatocytes. There may be prominent bile ductule formation extending from the portal regions with apoptosis of individual hepatocytes. Cellular infil­trates consist primarily of lymphocytes and plasma cells (Fig. 1.61). In typical cases of chronic hepatitis, the elongated, helical microorganisms are best demon­strated within biliary canaliculi using the Steiner silver staining method (Fig. 1.62). Selected strains of mice, such as A/J mice, with Helicobacter-associated hepatitis develop foci of cellular alteration, including clear cell, vacuolated, and basophilic foci, with an increased inci­dence and earlier onset of hepatocellular tumors. C57L mice infected with H. bilis and fed a lithogenic diet develop a high frequency of gallstones.

FIG. 1.60. Liver from a mouse infected with Helicobacter hepaticus with focal necrotizing hepatitis.

FIG. 1.62. Liver from a mouse infected with Helicobacter hepaticus depicting characteristic Helicobacter organisms within bile canaliculi (Steiner stain).

Diagnosis

Diagnosis is based upon the liver or intestinal lesions, but other etiologic agents must be considered. Definitive diagnosis requires culture, PCR, or demonstration of typical organisms within bile canaliculi or crypts using silver stains. The major challenge to serologic or molec­ular methods of diagnosis is the antigenic and genetic diversity among the various species of mouse Helico­bacter. Major impediments to successful serology are the fact that there are no known or useful cross-species Helicobacter antigens. Furthermore, immunocompetent mice mount minimal antibody responses, and generally only late in infection. Relatively insensitive serologic assays are available that utilize species-specific mem­brane antigen extracts or recombinant proteins for detecting serum IgG and fecal IgA. PCR amplification of a conserved segment of 16S rRNA gene can be used to detect most Helicobacter spp., and embellished with restriction enzyme analysis to speciate Helicobacter. Mul­tiplex species-specific PCR primers have also been devel­oped for discrimination of several species. PCR is frequently used to test feces, but the authors have found that some species of mouse Helicobacter may not be consistently shed in feces, or only intermittently so. Optimal PCR detection (and culture) can be achieved by sampling cecal mucosa. Differential diagnosis of liver lesions includes infections with Salmonella, Proteus, C. piliforme, MHV, ectromelia virus, or nonspecific foci of hepatic necrosis and inflammation. Intestinal lesions must be differentiated from those induced by infection with E. coli, C. rodentium, enterotropic MHV, or the nonspecific lesions of rectal prolapse.