Infection to Sentinel Mice by Contaminated Bedding
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Laboratory Animal Science Vol 48, No 3 Copyright 1998 June 1998 by the American Association for Laboratory Animal Science Transmission of Helicobacter hepaticus Infection to Sentinel Mice by Contaminated Bedding Robert S. Livingston,* Lela K. Riley, Cynthia L. Besch-Williford, Reuel R. Hook, Jr., and Craig L. Franklin Helicobacter hepaticus is a newly recognized gram-nega- mouse hepatitis virus, Sendai virus, pneumonia virus of tive, helical, microaerobic, pathogenic bacterium of mice mice, reovirus 3, Theiler’s murine encephalomyelitis virus, (1). Helicobacter hepaticus has been associated with chronic ectromelia virus, polyoma virus, lymphocytic choriomen- active hepatitis in a variety of mouse strains (1–5), with ingitis virus, mouse adenovirus, minute virus of mice, hepatocellular tumors in A/JCr mice (2, 4), and with in- mouse parvovirus, and Mycoplasma pulmonis. Helicobacter flammatory large bowel disease in immunodeficient (6) and hepaticus-contaminated bedding was collected from a cage germ-free mice (5). Identification of H. hepaticus-infected of eight DBA/2 mice of an in-house colony that was infected mice is essential because H. hepaticus can confound or in- with H. hepaticus. Fecal shedding of H. hepaticus by DBA/2 validate research data obtained from infected mice. mice was confirmed by PCR testing of individual fecal pel- Samples for diagnostic evaluation are routinely collected lets collected from the cage. The DBA/2 mice were from a from colony mice or sentinel mice to survey mouse colonies colony that was free of ectoparasites, endoparasites, and for pathogenic agents. Sentinel mice are immunocompe- known pathogenic bacteria except H. hepaticus, and were tent mice housed in direct or indirect contact with the popu- seronegative for mouse hepatitis virus, Sendai virus, pneu- lation to be surveyed so that they will be exposed to infec- monia virus of mice, reovirus 3, Theiler’s murine encepha- tive agents harbored by colony mice. Indirect contact of lomyelitis virus, ectromelia virus, lymphocytic choriomen- sentinel mice is commonly used to expose sentinel animals ingitis virus, mouse rotavirus, minute virus of mice, mouse to potential infective agents, and is achieved by regular parvovirus, and Mycoplasma pulmonis. transfer of soiled bedding from the population to be sur- All mice were kept in the same animal room with a veyed (7). Sentinel mice provide a readily available source macroenvironment of 248C, 45% humidity, and 14.5 air of animals for health monitoring, avoiding the use of valu- changes/h. Fluorescent lighting was provided on a 14/10-h able colony mice for this purpose. Sentinel mice are also light/dark cycle. Each experimental group of mice was essential when serologic assays are used to monitor colo- housed in a separate sterile MicroBarrier cage (Allentown nies of immunodeficient mice. Caging, Allentown, Pa.) containing approximately 1,500 cc The purpose of the study reported here was to determine of Paper Chip bedding (Canbrands International Ltd., whether sentinel mice could be used to detect H. hepaticus Moncton, New Brunswick, Canada) and was provided with infections. To this end, we exposed mice free of Helicobacter Purina 5001 rodent diet (Lab Chows, Purina Mills, St. infection to soiled bedding from H. hepaticus-infected mice. Louis, Mo.) and water ad libitum. Mouse cages and bed- Exposed mice were monitored for H. hepaticus coloniza- ding were changed weekly under sterile conditions in a tion by use of polymerase chain reaction (PCR) assay and laminar flow hood. Mice were maintained in accordance for development of H. hepaticus-specific serum antibodies with the standards set forth in the Guide for the Care and by use of enzyme-linked immunosorbent assay (ELISA). Use of Laboratory Animals, and the University of Missouri Our data indicate that sentinel mice can be used to iden- Institutional Animal Care and Use Committee approved tify H. hepaticus-infected mice. all animal experiments. Four-week-old female C57BL/6NCr mice documented to Soiled bedding (50 cc) was transferred twice a week for be free of Helicobacter infections by results of fecal PCR 8 weeks from the cage of infected DBA/2 mice to a cage were obtained (Fredrick Cancer Research and Development containing nine C57BL/6NCr mice. Five C57BL/6NCr mice Center, Fredrick, Md.) and were used as sentinel and con- not exposed to soiled bedding served as controls. Every 2 trol mice. The C57BL/6NCr mice were from a colony that weeks, a fecal pellet was collected from each experimen- was documented to be free of ectoparasites, endoparasites, tal and control mouse by placing mice individually in a known bacterial pathogens, and relevant histologic lesions sterile, empty MicroBarrier cage until the mouse def- of the gastrointestinal tract, liver, lungs, and kidneys. Mice ecated. The fecal pellets were analyzed for the presence from the colony were also consistently seronegative for of H. hepaticus DNA by use of a restriction digest-based PCR (8, 9). The PCR reactions that generated the ex- University of Missouri, Department of Veterinary Pathobiology, Columbia, pected 374-base pair fragment and yielded the expected Missouri *Address correspondence to Dr. Robert S. Livingston, University of Mis- restriction enzyme digest map were considered positive souri, College of Veterinary Medicine, Department of Veterinary for H. hepaticus. Pathobiology, 1600 E. Rollins Road, Columbia, MO 65211. 291 Vol 48, No 3 Laboratory Animal Science June 1998 o.d. 405 nm Number of positive mice Weeks Weeks Figure 1. Number of C57BL/6NCr sentinel mice positive for Figure 2. Serum antibody response of C57BL/6NCr sentinel mice Helicobacter hepaticus infection by polymerase chain reaction during biweekly exposure to H. hepaticus-contaminated bedding. (PCR) assay or ELISA during biweekly exposure to H. hepaticus- Control mice were not exposed to contaminated bedding. Each contaminated bedding. Nine mice were tested at each time point. time point represents the mean of nine serum samples. Error bars represent SEM. At the same 2-week intervals, mice were anesthetized of H. hepaticus organisms occurred 2 to 4 weeks after ex- with methoxyflurane, and 200 ml of blood was collected by posure to contaminated bedding. Also, at that time suffi- retro-orbital sinus puncture. Individual serum specimens cient numbers of organisms were present to initiate a hu- were tested in duplicate for the presence of H. hepaticus- moral immune response so that H. hepaticus-specific serum specific antibodies by use of an ELISA containing an outer antibody was detected 4 to 6 weeks after contaminated bed- membrane protein preparation of H. hepaticus as antigen ding exposure. (10). Serum samples with mean optical density value Helicobacter hepaticus infection was rapidly transferred >0.238 were considered positive for H. hepaticus-specific from infected mice to uninfected sentinel mice by exposure to antibodies (10). contaminated bedding. This indicates that H. hepaticus can Sentinel mice exposed to H. hepaticus contaminated bed- be readily transmitted between mice and helps explain why ding rapidly became infected with the bacterium. the prevalence of H. hepaticus infections in mouse colonies is Helicobacter hepaticus-specific DNA was detected in the high (12, 13). Helicobacter hepaticus is thought to be trans- feces of 66% of exposed mice 2 weeks after the start of bed- mitted via the fecal-oral route. We suspect that the sentinel ding transfer, and all mice were positive by fecal PCR as- mice most likely became infected by ingesting infective or- say 4 weeks after the start of the study (Figure 1). Sentinel ganisms contained in the feces of the transferred bedding. mice also rapidly developed H. hepaticus-specific serum an- In other studies in our laboratory, in which similar experi- tibodies. None of the sentinel mice were positive by ELISA mental procedures were used, we have not been successful in at 2 weeks. However, serum antibodies to H. hepaticus were using sentinel mice to detect H. bilis-infected mouse colonies detected in 66% of exposed mice by 4 weeks after the start (data not shown). The reason for this has not been elucidated; of bedding transfer, and all mice were seropositive by 6 however, it suggests that H. bilis infections are not as easily weeks (Figures 1 and 2). All control mice were negative for transmitted to uninfected mice by exposure to contaminated H. hepaticus by PCR and ELISA throughout the study. bedding. Additional studies are needed to determine whether These data indicate that sentinel mice exposed to sentinel mice can be used to monitor mouse colonies for infec- H. hepaticus-contaminated bedding were rapidly colonized tion with Helicobacter species other than H. hepaticus. by the bacterium and that this infection could be detected The results of this study indicate that H. hepaticus is readily by PCR and serologic testing. It was not surprising that, transmitted via contaminated bedding and that sentinel mice for six of nine mice, PCR detection of H. hepaticus infec- provide a valuable means of detecting H. hepaticus infections tion preceded seroconversion. The fecal PCR assay detects in rodent colonies. Infections in sentinel mice can be readily DNA sequences specific for the 16S rRNA gene of identified by use of standard diagnostic techniques, such as Helicobacter sp. Bacterial DNA should be present in the PCR of feces or serum ELISA feces concurrent with or shortly after intestinal coloni- zation with H. hepaticus. The ELISA, however, detects H. hepaticus-specific immunoglobulins in the sera of in- References fected mice. Generation of serum antibodies requires an 1. Fox, J. G., F. E. Dewhirst, J. G. Tully, et al. 1994. Helicobacter adaptive immune response that usually occurs 5 to 10 days hepaticus sp. nov., a microaerophilic bacterium isolated from liv- after exposure to a substantial dose of antigen (11). Thus, ers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. it appears that intestinal colonization with fecal shedding 32:1238–1245. 292 Note 2.