Co-Infection Associated with Diarrhea in a Colony of <I>Scid

Laboratory Animal Science Vol 48, No 5 Copyright 1998 October 1998 by the American Association for Laboratory Animal Science Helicobacter bilis/Helicobacter 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 228C, 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 pneumonia 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 bacteria 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

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