Effects of medicated diet to eradicate Helicobacter spp. on growth, pathology, and infection status in Rag1-/- and nude mice.

Despite 2 decades of reports documenting Helicobacter-associated gastrointestinal disease in mice, infections continue to persist widely not only at academic institutions in the United States but also at commercial vendors in other regions of the world.1,3,31,44 Within the academic setting, health monitoring and exclusion policies vary markedly between universities and even within different animal facilities at a single institution. Often these policies are based on financial factors (the costs of screening by PCR and the resources required to rederive infected animals) in addition to the potential for Helicobacter infections to confound research. Attempting to predict the overall effect of infection on research can also be problematic. The development and severity of gastrointestinal pathology can vary considerably by mouse strain, species of Helicobacter, and disease model. Some strains of mice, including A/JCr, BALB/cAnNCr, C3H/HeNCr, and SJL/NCr, are particularly susceptible and develop chronic enterohepatic disease of considerable severity.19,21,30,49,50 In addition, several immunodeficient strains of mice develop severe disease after chronic infection. C.B-17/Icr-Prkdcscid (SCID/NCr) mice, which lack functional T and B cells, develop progressive hepatitis and proliferative typhlocolitis after natural infection with H. hepaticus.30 IL10−/− mice such as B6.129P2-IL-10tm1Cgn/J develop severe typhlocolitis after infection with several Helicobacter spp.51,52 In contrast to immunocompetent strains, immunodeficient mice may manifest clinical signs such as diarrhea, perianal bleeding and rectal prolapse of variable severity.18,29,51 The species of Helicobacter affects the severity of disease. Although H. hepaticus remains the most well-studied enterohepatic mouse species, other closely related Helicobacter spp. also result in gastrointestinal disease.15,31 Natural and experimental monoinfection with H. typhlonius led to typhlocolitis in C57BL/6J IL-10−/− and SCID/NCr mice.18,22,23 H. mastomyrinus infection led to granulomatous typhlocolitis (inflammatory bowel disease, IBD) in telomerase-deficient C57BL/6J mice during crucial early-senescence studies.15 Interestingly, gastrointestinal disease was significantly more severe in mice infected with H. mastomyrinus than in those infected with H. hepaticus.15 In addition, the research impact of Helicobacter infections varies with disease model in a complex dynamic resulting from interactions between host gastrointestinal immunity, microflora, diet, and environmental conditions. Mouse models of IBD highlight these complexities. Helicobacter spp. infection rather than genetic modification was found to be responsible for the susceptibility and pattern of IBD development in T cell receptor αβ mutant mice.6,10 Intentional inoculation has been used to study Helicobacter spp.- associated alterations in resident intestinal microflora and induction and severity of IBD in immunodeficient mice.51 Given the difficulties of predicting the research impact of Helicobacter spp. infection in mice, perhaps tolerance for enzootic infections should be reconsidered. Benefits to eradication include not only elimination of the agent as an experimental confounder but also as a means to improve welfare through reduced clinical disease.1 Complete exclusion of infected animals may serve as the least labor intensive and most cost effective strategy. A 10-y institution-wide exclusion policy that required all imported mice to be either rederived by embryo transfer or purchased from an approved Helicobacter-free vendor resulted in either complete elimination or significant reduction in 4 facilities tested in 1999 and again in 2009.31 Although effective, this method has the potential to interfere with the ability of individual investigators to receive mice from collaborating institutions that maintain facilities of unknown or positive infection status. In addition, this strategy would be ineffective for inhouse breeding colonies of genetically modified mice. In such cases, rederivation by in vitro fertilization, embryo transfer, and postpartum cross-fostering have all been proven successful. Although often effective, embryo transfer can be impeded by factors such as insufficient response to superovulation, unavailability of stud males, inadequate yield of fertilized eggs, unsuccessful embryo transfer, and the need for genotyping—all of which delay the initiation of research.47 Furthermore, the detection of H. typhlonius from sex organs of both female and male Hsd:Athymic Nude-Foxn1nu mice has demonstrated the potential for transmission from vasectomized male and recipient female mice.38 Rederivation by cross fostering has been used to eliminate enzootic Helicobacter spp. infections.1,46 This technique is less costly and labor intensive and requires less expertise than embryo transfer.1,42,46 One study comparing 2 cross fostering paradigms to eliminate Helicobacter spp., murine norovirus, mouse hepatitis virus and Syphacia obvelata found success was dependent on both pup age at the time of transfer and bedding changing patterns.1 Pups transferred within 24 h of birth from cages that underwent bedding changes every 24 h tested negative more frequently than did pups transferred within 48 h of birth from cages containing up to 7-d-old dirty bedding.1 However neither paradigm was completely successful in Helicobacter elimination, as evidenced by follow-up PCR testing of cross-fostered offspring.1 Furthermore, isolation of H. hepaticus from the viscera of several late-stage C.B-17/Icr-Prkdcscid (SCID/NCr) embryos belonging to an infected dam suggests that transplacental transmission is possible in immunodeficient mice.30 The administration of antibiotics to eliminate Helicobacter spp. in rodents has evolved considerably since first used to prevent chronic active hepatitis and colitis in young male SCID/NCr mice naturally infected with H. hepaticus.36 In that initial report, various combinations of amoxicillin, bismuth, metronidazole, neomycin, and tetracycline were either added to the drinking water or administered orally 3 times daily for 14 d.36 Although clearly labor-intensive, the cited study36 determined that H. hepaticus could be eliminated by using all treatment regimens that included amoxicillin. A similar dosing regimen using either amoxicillin or tetracycline-based triple therapy was used to eliminate H. hepaticus from infected A/JCr mice.16 The first medicated diet formulation intended to eliminate infection in mice was a triple therapy of amoxicillin, metronidazole, and bismuth.17 When fed continuously for 2 wk to 6- to 10-mo-old DBA/J mice, the diet successfully eliminated chronic H. hepaticus infection as confirmed by posttreatment culture and PCR testing 1 mo later.17 Despite this success, the same amoxicillin-based triple combination diet fed for an unspecified duration failed to eliminate H. hepaticus from an immunodeficient breeding colony of Rag1 −/− mice.16,51 When administered to a SCID (Prkcscid/Tpr53tm1tyi on a B6.129/Sv × C.B17 background) breeding colony severely affected by diarrhea associated with H. rodentium and H. bilis coinfection, the triple therapy reduced clinical illness during treatment period but did not eliminate infection or persistent diarrhea.41 Although a newer medicated diet containing amoxicillin, clarithromycin, metronidazole, and omeprazole has been available for a decade, reports of efficacy are still quite limited. One study demonstrated successful eradication of H. hepaticus and H. bilis from a colony of 129 × 1/SvJ desmin-null and heterozygotic mice after 8 wk of continual treatment and 19 mo of PCR follow-up testing.28 In addition, the 4-drug therapy was successful in eliminating Helicobacter spp. from several genetically modified rat strains whereby infected male rats were medicated for 3-two week cycles and pregnant rat dams and offspring were fed continuously from day 7 of gestation through weaning.26 Posttreatment follow-up testing for 8 mo by fecal PCR confirmed that all treated rats remained negative.26 More recently, we have reported the successful eradication of H. hepaticus, H. bilis, and H. rodentium from 2 strains of mice with innate immune deficiencies.9 Questions remain, however, regarding the broad applicability of medicated diet for the elimination of Helicobacter spp., particularly with regard to strains with modifications of the immune system. The 4-drug medicated diet was unable to eradicate Helicobacter spp. from in B6.129P2-IL10tm1Cgn/J mice,40 and there is no information regarding efficacy in mice with deficiencies in adaptive immunity. Given the widespread use of these mice in gastrointestinal cancer and IBD research, evaluation of medicated diet to eradicate Helicobacter spp. from such strains is warranted. Although many immunodeficient inbred strains are available Helicobacter-free from commercial vendors, these strains often are genetically modified further, maintained, and imported from collaborating institutions with endemic Helicobacter spp. infections that need to be eliminated in a timely manner. We therefore conducted a prospective controlled study to evaluate the potential of the 4-drug medicated diet to eradicate H. hepaticus and H. typhlonius from young, naturally infected nude and Rag1 mice lacking functional T cells (Foxn1nu) or T and B cells (B6.129S7-Rag1tm1Mom/J), respectively. We also evaluated the physical effect of both Helicobacter infection and medicated diet in growing mice by weekly recordings of body weight and assessment of gross and histologic changes between medicated and control diet in groups of infected and uninfected mice after an 8-wk treatment course and again 8 wk after treatment completion.