And Nude Mice

Journal of the American Association for Laboratory Animal Science Vol 53, No 3 Copyright 2014 May 2014 by the American Association for Laboratory Animal Science Pages 238–245 Effects of Medicated Diet to Eradicate Helicobacter spp. on Growth, Pathology, and Infection Status in Rag1–/– and Nude Mice Caroline M Garrett,* Dillon Muth, and Julie Watson The use of a commercial 4-drug diet has been shown to eradicate Helicobacter spp. from immunocompetent mice and those with innate immunodeficiencies. However the efficacy of this diet has not been confirmed in mice with altered adaptive immunity. We hypothesized that an 8-wk treatment with medicated diet would 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 evaluated helicobacter status, body weight, and gross and histologic changes between medicated and control diet in groups of infected and uninfected mice throughout treatment and at 8 wk after treatment completion. Initial infection status was confirmed by fecal PCR at weaning and 3 wk later, with study initiation in 7-wk-old mice. PCR testing demonstrated that independent of strain and sex, all treated mice tested negative for Helicobacter spp. after 4 wk of treatment and remained negative for the duration of the study. Irrespective of infection status, nude and Rag1 mice fed 8 wk of medicated diet gained less weight than did their untreated controls. Both strains normalized body weight while on control diet for the 8 wk after treatment. Mice fed medicated diet developed severe gastroesophageal hyperkeratosis, suggestive of reduced feed consumption, and enlarged ceca. These conditions improved or resolved after the return to control diet. This report is the first to demonstrate the efficacy and physical effects of providing medicated diet for the eradication ofHelicobacter spp. from mice with adaptive immune deficiencies. Abbreviations: IBD, inflammatory bowel disease; NU/J, Foxn1nu 1; Rag1tm1Mom, B6.129S7- Rag1tm1Mom/J. Despite 2 decades of reports documenting Helicobacter- may manifest clinical signs such as diarrhea, perianal bleeding associated gastrointestinal disease in mice, infections continue and rectal prolapse of variable severity.18,29,51 to persist widely not only at academic institutions in the United The species of Helicobacter affects the severity of disease. Al- States but also at commercial vendors in other regions of the though H. hepaticus remains the most well-studied enterohepatic world.1,3,31,44 Within the academic setting, health monitoring mouse species, other closely related Helicobacter spp. also result and exclusion policies vary markedly between universities and in gastrointestinal disease.15,31 Natural and experimental mo- even within different animal facilities at a single institution. noinfection with H. typhlonius led to typhlocolitis in C57BL/6J Often these policies are based on financial factors (the costs of IL-10−/− and SCID/NCr mice.18,22,23 H. mastomyrinus infec- screening by PCR and the resources required to rederive infected tion led to granulomatous typhlocolitis (inflammatory bowel animals) in addition to the potential for Helicobacter infections disease, IBD) in telomerase-deficient C57BL/6J mice during to confound research. crucial early-senescence studies.15 Interestingly, gastrointestinal Attempting to predict the overall effect of infection on research disease was significantly more severe in mice infected with H. can also be problematic. The development and severity of gas- mastomyrinus than in those infected with H. hepaticus.15 trointestinal pathology can vary considerably by mouse strain, In addition, the research impact of Helicobacter infections species of Helicobacter, and disease model. Some strains of mice, varies with disease model in a complex dynamic resulting including A/JCr, BALB/cAnNCr, C3H/HeNCr, and SJL/NCr, from interactions between host gastrointestinal immunity, are particularly susceptible and develop chronic enterohepatic microflora, diet, and environmental conditions. Mouse models disease of considerable severity.19,21,30,49,50 In addition, several of IBD highlight these complexities. Helicobacter spp. infection immunodeficient strains of mice develop severe disease after rather than genetic modification was found to be responsible chronic infection. C.B-17/Icr-Prkdcscid (SCID/NCr) mice, which for the susceptibility and pattern of IBD development in T cell lack functional T and B cells, develop progressive hepatitis and receptor αβ mutant mice.6,10 Intentional inoculation has been proliferative typhlocolitis after natural infection with H. hepati- used to study Helicobacter spp.- associated alterations in resident cus.30 IL10−/− mice such as B6.129P2-IL-10tm1Cgn/J develop severe intestinal microflora and induction and severity of IBD in im- typhlocolitis after infection with several Helicobacter spp.51,52 In munodeficient mice.51 contrast to immunocompetent strains, immunodeficient mice 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 Received: 23 Sep 2013. Revision requested: 18 Oct 2013. Accepted: 30 Oct 2013. confounder but also as a means to improve welfare through re- Department of Molecular and Comparative Pathobiology, Johns Hopkins University duced clinical disease.1 Complete exclusion of infected animals School of Medicine, Baltimore, Maryland. may serve as the least labor intensive and most cost effective *Corresponding author. Email: [email protected] 238 Medicated diet to eradicate Helicobacter spp. from immunodeficient mice strategy. A 10-y institution-wide exclusion policy that required Although a newer medicated diet containing amoxicillin, all imported mice to be either rederived by embryo transfer or clarithromycin, metronidazole, and omeprazole has been purchased from an approved Helicobacter-free vendor resulted available for a decade, reports of efficacy are still quite limited. in either complete elimination or significant reduction in 4 One study demonstrated successful eradication of H. hepaticus facilities tested in 1999 and again in 2009.31 Although effec- and H. bilis from a colony of 129 × 1/SvJ desmin-null and tive, this method has the potential to interfere with the ability heterozygotic mice after 8 wk of continual treatment and 19 of individual investigators to receive mice from collaborating mo of PCR follow-up testing.28 In addition, the 4-drug therapy institutions that maintain facilities of unknown or positive was successful in eliminating Helicobacter spp. from several infection status. In addition, this strategy would be ineffective genetically modified rat strains whereby infected male rats for inhouse breeding colonies of genetically modified mice. In were medicated for 3-two week cycles and pregnant rat dams such cases, rederivation by in vitro fertilization, embryo transfer, and offspring were fed continuously from day 7 of gestation and postpartum cross-fostering have all been proven successful. through weaning.26 Posttreatment follow-up testing for 8 mo by Although often effective, embryo transfer can be impeded by fecal PCR confirmed that all treated rats remained negative.26 factors such as insufficient response to superovulation, una- More recently, we have reported the successful eradication of H. vailability of stud males, inadequate yield of fertilized eggs, hepaticus, H. bilis, and H. rodentium from 2 strains of mice with unsuccessful embryo transfer, and the need for genotyping—all innate immune deficiencies.9 of which delay the initiation of research.47 Furthermore, the Questions remain, however, regarding the broad appli- detection of H. typhlonius from sex organs of both female and cability of medicated diet for the elimination of Helicobacter male Hsd:Athymic Nude-Foxn1nu mice has demonstrated the spp., particularly with regard to strains with modifications of potential for transmission from vasectomized male and recipi- the immune system. The 4-drug medicated diet was unable ent female mice.38 to eradicate Helicobacter spp. from in B6.129P2-IL10tm1Cgn/J Rederivation by cross fostering has been used to eliminate mice,40 and there is no information regarding efficacy in mice enzootic Helicobacter spp. infections.1,46 This technique is less with deficiencies in adaptive immunity. Given the widespread costly and labor intensive and requires less expertise than use of these mice in gastrointestinal cancer and IBD research, embryo transfer.1,42,46 One study comparing 2 cross fostering evaluation of medicated diet to eradicate Helicobacter spp. from paradigms to eliminate Helicobacter spp., murine norovirus, such strains is warranted. Although many immunodeficient mouse hepatitis virus and Syphacia obvelata found success was inbred strains are available Helicobacter-free from commercial dependent on both pup age at the time of transfer and bed- vendors, these strains often are genetically modified further, ding changing patterns.1 Pups transferred within 24 h of birth maintained, and imported from collaborating institutions with from cages that underwent bedding changes every 24 h tested endemic Helicobacter spp. infections that need to be eliminated negative more frequently than did pups transferred within 48 in a timely manner. We therefore conducted a prospective

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