Laboratory Animal Science Vol 48, No 5 Copyright 1998 October 1998 by the American Association for Laboratory Animal Science

Helicobacter bilis/ rodentium Co-Infection Associated with Diarrhea in a Colony of scid Mice

Nirah H. Shomer,* Charles A. Dangler, Robert P. Marini, and James G. Fox† Abstract ͉ 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 organ- ism 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 eradi- cate 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. Immunodeficient mice present special husbandry consid- Case History erations. Frequently, immunodeficient mice are affected Index case and initial examination: Irritated peria- clinically by organisms that are usually nonpathogenic in nal areas were observed in three mice housed in the same immunocompetent mice, and such mice are therefore cage. The mice had diarrhea, which caused a characteristic housed in sterile caging with limited exposure to environ- clumping of the bedding. Samples were submitted for fecal mental organisms. One example is the scid (Prkdcscid) flotation. Rectal swab specimens were submitted for culture. mouse. Etiologic diagnosis of disease in scid mice is com- Signalment: The index cases were Prkdcscid/Tpr53tm1tyj plicated by unavailability of diagnostic serologic tests in B homozygous knockout mice (scid/Trp53 -/-) on a C57BL/ cell-deficient mice; rapid progression of disease resulting 6,129/Sv X CB.17 background (bred-in-house). Mice were in death before effective diagnosis can be attempted; and 10-week-old male littermates that had been weaned at 3 clinical presentation patterns that may be quite different weeks of age. This phenotype develops thymic lymphoma by from the classical presentation in immunocompetent mice. 10 weeks of age, with mortality at or before 12 weeks of age. The husbandry difficulties in the scid mice are far out- History: The mice were part of a scid/Trp53 breeding weighed by their usefulness in immunologic research. In colony housed in a cubicle in a barrier room. Because of the addition, scid mice are often particularly sensitive to cer- early mortality in homozygous scid/Trp53 knockouts, most tain organisms, and reveal the pathogenic potential or the breeding mice were heterozygous, and the colony mice were pathogenic mechanism of infectious organisms that do not variably homozygous or heterozygous at the scid or Trp53 cause acute disease in immunocompetent mice. The follow- locus. The mice were housed under SCID 1 conditions (au- ing report is a description of the pathogenic potential of toclaved sterile Micro-IsolatorTM [Lab Products, Inc., two bacterial species, H. bilis and H. rodentium, that were Seaford, Del.] caging, Bed-o’cobs bedding [Bed-o’cobs, In- not previously associated with acute enteric disease. Since dustrial Products Division, Meehan, Ohio], feed [autoclav- this outbreak, we have discovered through experimental able rodent chow; PMI Feeds, Inc., St. Louis, Mo.]), and wa- and clinical investigations that Helicobacter species may ter, with all mouse manipulations performed in a biosafety be important pathogens in many species, including humans. hood. The cubicle was kept at a temperature of 21 to 22ЊC, with relative humidity between 40 and 70%, 10 to 15 air Division of Comparative Medicine, Massachusetts Institute of Technology, changes/h, and a 12-h day length. Mice were treated with Cambridge, Massachusetts trimethoprim/sulfamethoxazole in the water 4 days per *Present address: Section of Comparative Medicine, Yale University, P.O. Box 208016, New Haven, CT 06520. week to prevent caused by Pneumocystis carinii. †Reprint requests to Dr. James G. Fox, Division of Comparative Medi- The colony had been housed in this cubicle for approxi- cine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. mately 6 months. However, it was determined that, con-

455 Vol 48, No 5 Laboratory Animal Science October 1998 trary to established policies, the investigator had recently mination of no growth was made. introduced conventionally raised mice from another room Blood culture: Blood was obtained from euthanized into the scid cubicle. Several weeks before the outbreak, mice by cardiac puncture and was inoculated into BBL the investigator placed an unknown number of male and Septi-chek brain-heart infusion broth with sodium female Trp53 (-/-) (homozygous knockout) mice in cages with polyanethanelsulfonate and CO2 (Becton Dickinson Mi- scid mates for breeding purposes. crobiological Systems, Cockeysville, Md). Clinical course and time frame of the outbreak: Five Polymerase chain reaction (PCR): The DNA was pre- days after the initial examination, two of the index mice pared from on plates (1), feces (2), or ground cecal were euthanized by CO2 asphyxiation for necropsy (the tissue (3) as previously described. The PCR primers were third was found dead) because they were severely ill, with used to amplify Helicobacter species-specific sequences bloody, mucoid diarrhea. The colony was re-examined, and (primers C97 and C98 [4]), H. bilis-specific sequences (prim- four additional cages contained mice with diarrhea. Diar- ers C12 and C62, [1]), or H. rodentium-specific sequences rhea appeared to be confined to mice with the scid geno- (primers D86 and D87 [4]), using described methods. type; one severely affected scid/Trp53 (+/-) heterozygous Treatment: Mice were placed on a 2-week regimen of mouse was caged with a non-scid Trp53 (-/-) mouse that sulfamethoxazole food wafer treatment (1.5 mg of ampicil- was asymptomatic. The cubicle was quarantined. Person- lin, 0.69 mg of metronidazole, and 0.185 mg of bismuth/ nel were required to don protective garments (gloves, shoe mouse per day (Bio-serv, Frenchtown, N.J.). Trimethoprim/ covers, coveralls, mask, caps) to enter the cubicle, and to sulfamethoxazole treatment was stopped for the duration remove the garments upon exiting the cubicle. All hus- of the wafer-treatment period. bandry materials were bagged in biohazard bags and au- Humane animal care and use: All mice were involved toclaved before going through the cage washer. Cages with in protocols approved by the MIT Institutional Animal Care evidence of diarrhea were changed last. New sentinels and Use Committee. (Helicobacter-free adult female Swiss Webster mice; (Taconic Farms, Germantown, N.Y.) were placed in the room 1 week Results after onset of the outbreak and were exposed at each weekly Antemortem laboratory findings for index mice: cage changing to bedding from mice with diarrhea. A total Results of fecal flotation for the three index mice were nega- of 16 new cages having mice with diarrhea (of approximately tive for endoparasites. Cultures of rectal specimens from 50 cages containing approximately 150 mice) were identi- the initial examination were negative for Salmonella sp. fied over the 8-week course of the outbreak (one cage at week and Citrobacter rodentium. 0, four cages at week 1, three cages at week 3, four cages at Postmortem results for two index mice. Gross and week 7, and four cages at week 8). The colony was depopu- microscopic findings: The diameter of the colon from both lated a few months after the outbreak. euthanized mice was two to three times larger than nor- mal, and the colon was markedly thickened with bloody, Materials and Methods mucoid contents (Figure 1). The spleen of one mouse was Fecal flotation for identification of endoparasites: enlarged about three times (not shown). The colonic mu- Fresh fecal pellets were suspended in flotation medium cosa and tunica muscularis were markedly thick in the af- buffer in a Fecalyzer (EVSCO Pharmaceuticals, Buena, fected mice (Figure 2). The length of the colonic crypts was N.J.), and the coverslip was examined by light microscopy increased approximately fivefold, and the crypts were char- for the presence of endoparasite eggs. acterized by multifocal to diffuse hyperplasia and loss of Serologic testing: Serum was obtained from the cubicle goblet cells. The lamina propria was heavily infiltrated by sentinels at quarterly intervals up to 1 year past the onset mixed inflammatory cells with multifocal extension into of the outbreak. Serum was assayed by ELISA for antibod- the submucosa and muscular layers. Crypts had multifo- ies to Sendai virus, mouse virus, rotavirus, reovi- cal epithelial necrosis and focal erosions and contained sup- rus 3, pneumonia virus of mice, mouse encephalitis (GDVII purative exudate. Moderate multifocal mucosal hyperpla- strain), lymphocytic choriomeningitis virus, minute virus sia, epithelial necrosis, and inflammation were apparent of mice, ectromelia virus, K virus, polyoma virus, adenovi- in the cecum (Figure 3). Dilated cecal crypts were filled rus, cilia-associated respiratory bacillus, minute virus of with thin, curved to helical argyrophilic bacteria. There was mice, and Mycoplasma pulmonis. moderate multifocal necrosis, ulceration, and inflammation Microaerobic fecal/cecal culture: Fresh fecal pellets in the rectum. There was minimal to moderate multifocal were homogenized in phosphate-buffered saline, and the mixed cellular infiltrates in the portal and lobular areas of homogenate was either applied directly to blood agar plates the liver (not shown; the lesions were consistent with sep- containing trimethoprim, vancomycin, and polymixin B ticemia). The brain, lungs, spleen, kidneys, and hearts were (TVP plates, Remel) or filtered through a 0.45-␮m pore- unremarkable. size filter, then applied to blood agar plates. Specimens of Laboratory findings: Results of fecal flotation were nega- cecal tissue were obtained aseptically at necropsy, ground tive for endoparasites; examination of ileal scrapings was in a sterile glass tissue grinder, and plated on blood agar. negative for coccidian oocysts; and aerobic culture of rectal Plates were incubated under microaerobic conditions (37ЊC; swab specimens yielded negative results for Salmonella sp.,

80% N2; 10% H2; 10% CO2) for up to 3 weeks before deter- Citrobacter rodentium, and hemolytic . The

456 H. bilis/H. rodentium Co-Infection

Figure 2. Photomicrograph of a section of colon from a scid mouse with severe proliferative colitis. The affected colon is characterized by marked crypt hyperplasia and cystic dilatation, loss of goblet cell differentiation, and inflammation. H&E stain; bar = 500 ␮m.

Figure 1. Marked, diffuse colonic hypertrophy in a scid mouse. The colon is thick and dilated with soft, unformed feces. The perineum is stained with feces. Enterococcus sp. septicemia in one index-case mouse was probably due to bacterial migration through the compro- mised mucosal barrier, and not an indication of primary infectious disease. Colon specimens were positive for Pro- Figure 3. Photomicrograph of a section of cecum from a scid mouse teus and Enterococcus sp. and Bacteroides vulgaris. The with proliferative typhlitis. The crypts are elongated and irregu- cecum of one mouse was culture positive for H. bilis; this lar, with marked epithelial hyperplasia. The deep crypts are was confirmed by results of PCR. heavily colonized by bacteria. An infiltrate composed chiefly of mononuclear inflammatory cells occupies the submucosa and ex- Additional colony mice. Clinical history and micro- tends into the lamina propria. H&E stain; bar = 200 ␮m. scopic findings: Four additional mature mice (three males, one female; age range, ten weeks to 5 months) and ten 2- these Helicobacter species were confirmed by use of PCR week-old pups were necropsied. The one female mouse was to be H. bilis and H. rodentium. To confirm the presence of a scid/Trp53 +/- dam with ten 2-week-old pups of mixed mixed Helicobacter spp. infection in the cubicle, feces from types from a heterozygote X heterozygote mating. The dam cages containing mice with diarrhea were pooled and sub- had a moderately severe rectal prolapse, which made as- mitted for microaerobic culture and PCR analysis; the sessment of diarrhea difficult. All pups had greasy, yellow pooled sample was positive by results of culture and PCR fur but did not appear to have diarrhea, thus the yellow for H. bilis and H. rodentium. coloring was probably due to staining with feces from the Treatment results: Mice with diarrhea were treated with dam. The male mice (all homozygous scid and Trp53 -/-, +/ antibiotic-impregnated feed wafers, using a dosage previ- -, or +/+) had similar findings of moderate to severe prolif- ously found to be effective in eradicating H. hepaticus in erative or ulcerative typhlocolitis, even though some of the mice (5). When the first treatment group was examined 4 mice did not have clinical signs of diarrhea. The dam had days after the start of therapy, diarrhea was not evident. proliferative colitis and proctitis; however, the pups did not Mice returned to their regular diet after 2 to 2.5 weeks of have microscopic lesions. treatment. However, pooled fecal samples were culture posi- Laboratory findings: Cecal specimens from the dam and tive for Helicobacter spp., even at the end of treatment. pups were culture positive for mixed Helicobacter infection; New and recurrent cases of diarrhea were observed in the

457 Vol 48, No 5 Laboratory Animal Science October 1998 colony over a pe- Table 1. Necropsy features of homozygous scid mice with Helicobacter species infections riod of 3 months, Mouse Trp53 Helicobacter spp. including several ID no. Sex Age genotype Clinical signs Histopathologic findings identified mice previously 2288 (index) M 10 wk -/- Diarrhea Proliferative typhlocolitis H. bilis 2289 (index) M 10 wk -/- Diarrhea Proliferative typhlocolitis NAa treated with anti- 2373 F Adult +/- Diarrhea/prolapse Proliferative colitis/proctitis H. bilis, H. rodentium biotic wafers. Be- 2374 (pups) M/F 2 wk +/+, +/-, -/- None None H. bilis, H. rodentium 2459 M 5 mo +/- or +/+b Relapse of diarrhea Proliferative typhlocolitis H. bilis cause no mice 9623 M 10 wk +/- None Proliferative typhlocolitis NAa died of diarrhea 9624 M 10 wk +/- or ++b None Proliferative typhlocolitis NAa while on treat- aSamples unavailable ment, antibiotic bExact genotype unavailable from investigator treatment was in- stituted when diarrhea was observed. Although the index normal mice, and there have been no reports of asso- cases and a few subsequent cases had severe diarrhea, most ciation with enteric disease. mice had diarrhea for approximately 1 week (time from Helicobacter bilis and H. rodentium are prevalent in con- identification of diarrhea in cage to start of wafer treat- ventionally raised colony mice in our facility and in mice ment) with no significant morbidity and no mortality. from commercial vendors (Fox, unpublished observations). Source of Helicobacter sp.: We sought to determine The organisms are transmitted directly or indirectly via whether the infection was enzootic in the scid cubicle, and contaminated bedding. Because these organisms were as- whether it was likely introduced into the scid cubicle via sociated with severe typhlocolitis, control and eradication the Trp53 mice from the conventional room. Pooled fecal strategies should be instituted. In this instance, antibiotic samples were collected for culture and PCR from therapy was not successful in eradicating the infection. It Helicobacter-free sentinel mice exposed to dirty bedding for is not clear whether this was because of the antibiotic sus- 2 weeks, unaffected colony mice in the scid cubicle, mice ceptibilities of the bacteria, or because the mice were im- with diarrhea in the scid cubicle, and unaffected colony mice munodeficient. It is possible that the diarrhea would have from the conventional room (the source of the Trp53 breeder resolved naturally without treatment; however, the index mice). All samples were culture and PCR positive for H. cases (which were also Trp53 knockouts) did not resolve (one bilis and H. rodentium. died, two became moribund), suggesting that antibiotic treat- ment was able to reduce mortality attributable to diarrhea. Discussion Antibiotic treatment may be of value in maintaining a colony Helicobacter spp. were implicated as possible pathogens of valuable animals until they can be re-derived. by identifying them in affected scid mice and by ruling out Strain, sex, and age have been documented to affect se- other viral, bacterial, or protozoal causes in these mice. No verity of disease in infections with some Helicobacter spe- pathogens were identified by serologic testing of the senti- cies (11–13). In this outbreak, five of six mice with clinical nels from this room (testing was not done on the scid mice). signs or histologic lesions that were necropsied were males. No endoparasites, coccidia oocysts, Salmonella sp., C. However, we cannot determine from this number whether rodentium, or hemolytic E. coli were identified in the af- there was a true sex predilection; mice of both sexes were fected scid mice. Helicobacter rodentium was readily trans- observed to have diarrhea. Helicobacter bilis causes more missible to sentinel mice. Although the helicobacters ap- severe disease in male than in female nu/nu rats (8), and parently became enzootic in the mice housed in the cubicle, H. hepaticus causes more severe disease in male than in the outbreak had the characteristics of an epizootic, and female A/J and B6C3F1 mice (11, 14). Mice ranging from one or both organisms may indeed have been introduced 10 weeks to at least 5 months of age were clinically af- with the conventionally raised breeding mice. In this out- fected. Interestingly, H. bilis and H. rodentium were iso- break, H. bilis and H. rodentium co-infection was associ- lated from clinically normal 2-week-old pups of a dam with ated with diarrhea and proliferative typhlocolitis in im- diarrhea and a combined H. bilis/H. rodentium infection, munodeficient mice. Neither organism had previously been indicating that the infection was acquired at an early documented to be an intestinal pathogen, although we (6) (preweaning) age. The pups were clinically and histologi- and others (7) have induced intestinal disease in scid mice cally normal, possibly because they had not been infected and nu/nu rats (8) experimentally infected with H. bilis. sufficiently long to cause disease. Alternatively, it may be Helicobacter bilis is a urease-positive helical bacterium that the microenvironment and microflora in an infant found in the liver and intestines of mice (1), rats (8, 9), mouse are sufficiently different from those in a mouse eat- dogs (10), cats (Fox, unpublished observations), and ger- ing solid food to prevent diarrheal disease in infected pups. bils (Fox, unpublished observations). It is a fastidious mi- We cannot rule out the possibility that the strain of these croaerophilic organism, which is why it was not isolated by mice (a cross of C.B-17, 129, and C57BL/6) or absence of one use of initial diagnostic screening tests. Helicobacter or both copies of the Trp53 tumor suppressor gene influenced rodentium is a recently named, fastidious microaero- severity of disease during this outbreak; however, experi- philic urease-negative helical bacterium found in the mentally induced infection of outbred ICR scid mice (wildtype ceca and colons of mice (4). Helicobacter rodentium was for Trp53) with H. bilis resulted in proliferative typhlocolitis, initially isolated from the ceca and colons of clinically suggesting that the scid genotype is sufficient to allow de-

458 H. bilis/H. rodentium Co-Infection

velopment of Helicobacter-related large bowel disease (6). 2. Shames, B., J. G. Fox, F. E. Dewhirst, et al. 1995. Identifi- Helicobacter bilis and H. rodentium co-infection may be cation of widespread H. hepaticus infection in feces in com- mercial mouse colonies by culture and PCR assay. J. Clin. an important cause of morbidity and mortality in immuno- Microbiol. 33:2968–2972. deficient mice. In one study, Koch’s postulates were fulfilled, 3. Fox, J. G., L. Yan, B. Shames, et al. 1996. Persistent hepa- indicating that H. bilis alone is sufficient to cause prolif- titis and enterocolitis in germfree mice infected with erative typhlocolitis in scid mice (6). We have also observed . Infect. Immun. 64:3673–3681. mild typhlocolitis in A/J mice caused by experimental in- 4. Shen, Z., J. G. Fox, F. E. Dewhirst, et al. 1997. Helicobacter rodentium sp. nov., a urease negative Helicobacter species iso- fection with either H. bilis or H. rodentium, and severe lated from laboratory mice. Int. J. Syst. Bacteriol. 47:627–634. typhlocolitis and hemorrhagic diarrhea in scid mice experi- 5. Foltz, C., J. G. Fox, L. Yan, et al. 1995. Evaluation of antibi- mentally infected with a novel urease-negative Helicobacter otic therapies for the eradication of Helicobacter hepaticus. species similar to H. rodentium (unpublished results). Antimicrob. Agents Chemother. 36:1292–1294. 6. Shomer, N. H., C. A. Dangler, M. D. Schrenzel, et al. 1997. Helicobacter hepaticus, another intestinal Helicobacter spe- Helicobacter-bilis-induced inflammatory bowel disease in scid cies of mice that has been associated with liver lesions and mice with defined flora. Infect. Immun. 65:4858–4864. , also has been documented ex- 7. Franklin, C., L. Riley, R. Hunziker, et al. 1998. perimentally to cause large bowel lesions in scid (15, 16) Enterohepatic lesions in SCID mice infected with Helicobacter and immunocompetent (12) mice. and bilis. Lab. Anim. Sci. 48:334–339. 8. Haines, D. C., P. L. Gorelick, J. K. Battles, et al. 1998. H. fenelliae (previously classified as campylobacters) are Natural and experimental inflammatory large bowel disease intestinal Helicobacter species associated with proctitis, in immunodeficient rats infected with Helicobacter bilis. Vet. colitis, diarrhea, and bacteremia in humans (17, 18), and Pathol. 35:202–208. these organisms have been documented experimentally to 9. Riley, L. K., C. L. Franklin, R. R. Hook, Jr., et al. 1996. Identification of murine Helicobacters by PCR and restric- colonize and induce diarrhea and bacteremia in macaques tion enzyme analyses. J. Clin. Microbiol. 34:942–994. (19). It is possible that the ability to promote inflammation 10. Eaton, K. A., F. E. Dewhirst, B. J. Paster, et al. 1996. Preva- and hyperplasia of gastrointestinal tract epithelium is one lence and varieties of Helicobacter species in dogs from ran- shared by many members of the Helicobacter genus. dom sources and pet dogs: animal and public health implica- Helicobacter spp. are likely to be enzootic in many ro- tions. J. Clin. Microbiol. 34:3165–3170. 11. Fox, J. G., X. Li, L. Yan, et al. 1996. Chronic proliferative dent colonies, but are identified only by use of special cul- hepatitis in A/JCr mice associated with persistent H. hepaticus ture techniques or species-specific PCR. Although many infection: a model of Helicobacter induced carcinogenesis. In- institutions and commercial vendors now screen for H. fect. Immun. 64:1548–1558. hepaticus, these screening protocols may not detect H. bilis 12. Whary, M. T., T. J. Morgan, C. A. Dangler, et al. 1998. Chronic active hepatitis induced by Helicobacter hepaticus in and H. rodentium. Also, H. bilis has a broad host range and the A/JCr mouse is associated with a Th1 cell-mediated im- may be of zoonotic importance; it has been identified in mune response. Infect. Immun. 66:3142–3148. rats (8) and several non-murine species, including cats (Fox, 13. Ward, J. M., M. R. Anver, D. C. Haines, et al. 1994. Chronic unpublished observations), dogs (10), gerbils (Fox, unpub- active hepatitis in mice caused by Helicobacter hepaticus. Am. lished observations), and humans (20). Recently, H. bilis J. Pathol. 145:959–968. 14. Fox, J. G., J. A. MacGregor, Z. Shen, et al. 1998. Compari- 16S sequences have been identified by PCR in the gallblad- son of methods identifying Helicobacter hepaticus B6C3F1 der and bile of humans with chronic cholecystitis who are mice used in a carcinogenesis bioassay mice. J. Clin. Microbiol. at high risk of developing gallbladder cancer (20). This out- 36:1382–1387. break underscores the importance of use of immunodefi- 15. Cahill, R. J., C. J. Foltz, J. G. Fox, et al. 1997. Inflamma- tory bowel disease: an immune mediated condition triggered cient mice in recognizing new pathogens, and the continu- by bacterial infection. Infect. Immun. 65:3126–3131. ing and evolving importance of the Helicobacter genus. 16. Li, X., J. G. Fox, M. T. Whary, et al. SCID/NCr mice natu- rally infected with Helicobacter hepaticus develop progres- sive hepatitis, proliferative typhlitis, and colitis. Infect. Immun. in press. Acknowledgements 17. Totten, P. A., C. L. Fennel, and F. C. Tenover. 1985. We thank Dr. Melanie Ihrig for helpful discussions of epide- Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae miology. (sp. nov.): two new Campylobacter species associated with en- This work was supported in part by grant nos. R01CA67529, teric disease in homosexual men. J. Infect. Dis. 151:131–139. R01DK52413, and RR01046 from the National Institutes of Health. 18. Skirrow, M. B., D. M. Jones, E. Sutcliffe, et al. 1993. Campylobacter bacteraemia in England and Wales, 1981–91. Epidemiol. Infect. 110:567–573. References 19. Flores, B. M., C. L. Fennell, and L. Kuller. 1990. Experi- 1. Fox, J. G., L. Yan, F. E. Dewhirst, et al. 1995. Helicobacter mental infection of pig-tailed macaques (Macaca nemertina) bilis sp. nov., a novel Helicobacter isolated from bile, livers, with Campylobacter cinaedi and Campylobacter fennelliae. and intestines of aged, inbred mouse strains. J. Clin. Microbiol. Infect. Immun. 58:3947–3953. 33:445–454. 20. Fox, J. G., F. E. Dewhirst, Z. Shen, et al. 1998. Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology 114:755–763.

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