Enterohepatic Lesions in SCID Mice Infected with Helicobacter Bilis
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Laboratory Animal Science Vol 48, No 4 Copyright 1998 August 1998 by the American Association for Laboratory Animal Science Enterohepatic Lesions in SCID Mice Infected with Helicobacter bilis Craig L. Franklin, Lela K. Riley, Robert S. Livingston, Catherine S. Beckwith, Cynthia L. Besch-Williford, and Reuel R. Hook, Jr. Abstract _ 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 re- port inoculation of 6-week-old C.B-17 scid/scid mice by gastric gavage with approximately 107 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. The genus Helicobacter is a rapidly expanding genus volved in lesion development. Culture of specimens from currently containing 17 named species. Members of this mice confirmed intestinal colonization with H. hepaticus. Fox genus are microaerophilic, have curved to spiral rod mor- et al. reported enteric lesions in immunocompetent germ- phology, and are motile by flagella that vary in number free Swiss Webster mice infected with H. hepaticus (15), and and location among various species (1). All known Cahill et al. recently reported inflammatory bowel disease Helicobacter spp. live in animal hosts where colonization in C.B-17 scid/scid mice inoculated with H. hepaticus, fur- is restricted to the intestine or liver. The type species of ther strengthening the role of this bacterium as an etiologic the genus is Helicobacter pylori. In all likelihood, this or- agent of intestinal as well as liver disease (17). ganism was present in the stomach of humans for years Much less is known about H. bilis, which has been docu- but was not recognized as a pathogen until 1985, when mented to colonize the intestine and liver of several strains Marshall et al. established H. pylori as the causal agent of of mice (8). Hepatic colonization was associated with mild chronic gastritis and peptic ulcer disease in people (2–4). hepatitis in aged mice, and hepatomas have been identi- Several species of Helicobacter have been identified in fied in aged CBA/CA mice infected with H. bilis (8). Re- rodents, including H. hepaticus, H. bilis, H. muridarum, cently, H. bilis was reported to cause inflammatory bowel H. cinaedi, H. cholecystus. H. trogontum, H. rodentium, and disease in defined flora ICR-scid/scid mice inoculated in- the related bacterium, “Flexispira rappini” (5–12). Of these, traperitoneally with the bacterium; however, liver lesions H. hepaticus and H. bilis have received the most attention were not evident (18). Here, we describe proliferative typh- because they have been associated with disease and are litis and chronic active hepatitis in conventional flora C.B- the most prevalent species in laboratory animals. 17 scid/scid mice experimentally infected with H. bilis by Helicobacter hepaticus was first recognized in 1992, when gastric gavage. hepatitis was observed in untreated A/JNCr mice during a long-term carcinogenicity study (13, 14). Examination of Materials and Methods silver-stained sections of liver revealed slender, curved to Animals: The C.B-17-scid/scid (SCID) mice used in in- spiral-shaped bacteria associated with lesions. A bacterium oculation studies were obtained from Charles River Labo- subsequently cultivated from the intestinal tract and liver ratories (Raleigh, N.C.). Vendor surveillance indicated that was found to be a novel member of the genus Helicobacter mice were free of all known bacterial, viral, and parasitic and was named Helicobacter hepaticus. In experimental pathogens, including but not limited to the enteric patho- studies, Koch’s postulates were fulfilled, confirming the role gens, mouse hepatitis virus (MHV), Citrobacter rodentium, of this bacterium in disease pathogenesis (13–15). and Clostridium piliforme. Mice were also determined to In 1996, Ward et al. reported high incidence of rectal be Helicobacter free by generic Helicobacter fecal PCR ex- prolapse and chronic proliferative typhlocolitis and procti- amination (19). Mice were housed in polycarbonate tis in immunodeficient mice (16). Helical organisms con- microisolator cages containing Paperchip Laboratory Ani- sistent with H. hepaticus were seen within the lumen of mal Bedding (Canbrands International, Ltd., Moncton, New the affected colon, suggesting that these bacteria were in- Brunswick, Canada) and were allowed ad libitum access to Autoclavable Laboratory Rodent Diet 5010 (Purina Mills, Department of Veterinary Pathobiology, University of Missouri, Columbia, Inc., Richland, Ind.) and tap water. The animal room was Missouri 334 Helicobacter bilis Infection in SCID Mice maintained at 248C, 45% humidity, and Table 1. Incidence of cecal (proliferative typhlitis) and hepatic (chronic active hepatitis) lesions in 14.5 air changes/h; a 14/10-h light/dark mice inoculated with Helicobacter bilis and in unmanipulated control mice Mice inoculated with Control mice cycle was used. Cages, water, and feed Weeks after Helicobacter bilis (Uninoculated) were autoclaved prior to use, and cage Tissue inoculation Male (n = 10)a Female (n = 9) Male (n = 10) Female (n = 2) changes were performed under a lami- Cecum 12 3/3* 3/3 0/4 0/2 nar flow hood. During the course of this 24 2/3 1/3 0/2 NTb 36 4/4* 3/3 0/4 NT study, sentinel mice housed in the ani- Total 9/10* 7/9 0/10 0/2 mal room were subjected to monthly se- Liver 12 3/3 0/3 0/4 0/2 rologic and parasitologic examinations. 24 3/3 2/3 0/2 NT 36 4/4* 1/3 0/4 NT Sentinel mice were consistently found Total 10/10* 3/9 0/10 0/2 free of MHV, Sendai virus, pneumonia aNumber of mice with lesions score $2/number of mice in the group virus of mice, Theiler’s murine encepha- bNT = not tested; female mice in the 24- and 36-week postinoculation groups were contaminated with lomyelitis virus, ectromelia, epizootic di- another Helicobacter species during the course of the study and were removed. Statistical analysis was not done on data from female groups at these time points. arrhea of infant mice virus, lymphocytic *Significantly different from control (P < 0.05); Fisher’s exact test choriomeningitis virus, mouse adenoviruses, polyoma virus, reovirus type 3, cilia-associ- lobe also were collected for H. bilis PCR analysis. ated respiratory bacillus, Encephalitozoon cuniculi, My- Histologic examination: Biopsy specimens of liver and coplasma pulmonis, Helicobacter hepaticus, Clostridium intestine were preserved in neutral-buffered 10% forma- piliforme, mouse parvoviruses, and endo- and ectoparasites. lin. The liver was sectioned through the medial left lobe, All animal use procedures were approved by the University the gallbladder, and the medial right lobe; the distal por- of Missouri Institutional Animal Health and Use Committee. tion of the colon was sectioned longitudinally; and the Bacteria: Helicobacter bilis, strain MU, was isolated ileocecocolic biopsy specimen was sectioned longitudinally from a colony of naturally infected BALB/c mice. Briefly, through the ileocecal junction, the cecocolic junction, most fecal pellets from mice were homogenized in phosphate- of the cecal tip, and the proximal portion of the colon. buffered saline (PBS) and incubated at room temperature Trimmed biopsy specimens were embedded in paraffin, for 30 min. Fecal slurries were filtered through a 0.80-mm and 3-mm sections were prepared, stained with hematoxy- filter, and a single droplet was placed on plates of trypticase lin and eosin, and examined. To objectively evaluate the soy agar that contained 5% sheep blood (blood agar). Cul- severity of proliferative typhlocolitis and chronic active tures were incubated in a microaerobic environment of 90% hepatitis, a lesion scoring system was developed (Tables N2, 5% CO2, and 5% H2. Pinpoint colonies that grew under 2 and 3), and two pathologists (CSB and RSL) assigned these conditions were tested for urease activity (rapid ure- lesion scores without prior knowledge of mouse infection ase test; Remel, Lenexa, Kans.) and motility. Motile ure- status (blind study). Discrepancies in lesion scores be- ase-positive isolates were subcultured on blood agar, and a tween the two pathologists were resolved by their view- first-passage subculture was used as inoculum. The isolate ing slides together and agreeing on a single score; the lat- was confirmed to be H. bilis by sequencing of a 374-bp poly- ter discussions were also conducted without prior knowl- merase chain reaction (PCR)-amplified fragment from the edge of mouse infection status. 16S rRNA gene. Sequence analysis revealed 100% simi- Generic Helicobacter and Helicobacter bilis PCR: larity with the type species of H. bilis (GenBank acces- Feces or frozen hepatic tissue were analyzed for H. bilis by sion no. U18766). PCR analysis as described (19, 20). Briefly, DNA was iso- Experimental design: Ten female and 10 male lated from feces or tissues, using a QIAmp Tissue Kit Helicobacter-free 4-week-old SCID mice were inoculated (Quiagen, Inc., Chatsworth, Calif.). The PCR analyses were with approximately 107 H. bilis colony-forming units in 0.5 performed, using Helicobacter genus-specific primers in a ml of sterile PBS by gastric gavage. Two weeks after inocu- Perkin-Elmer 2400 thermocycler. The PCR products were lation, fecal pellets were collected from cages harboring digested in separate reactions with Mbo I, Mae I, and Hha I inoculated SCID mice and were tested for H.