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 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 im- munity. 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 demon- strated 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 enzo- otic 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 con- h of birth from cages containing up to 7-d-old dirty bedding.1 trolled study to evaluate the potential of the 4-drug medicated However neither paradigm was completely successful in Heli- diet to eradicate H. hepaticus and H. typhlonius from young, cobacter elimination, as evidenced by follow-up PCR testing of naturally infected nude and Rag1 mice lacking functional T cells cross-fostered offspring.1 Furthermore, isolation of H. hepaticus (Foxn1nu) or T and B cells (B6.129S7-Rag1tm1Mom/J), respectively. from the viscera of several late-stage C.B-17/Icr-Prkdcscid (SCID/ We also evaluated the physical effect of both Helicobacter infec- NCr) embryos belonging to an infected dam suggests that trans- tion and medicated diet in growing mice by weekly recordings placental transmission is possible in immunodeficient mice.30 of body weight and assessment of gross and histologic changes The administration of antibiotics to eliminate Helicobacter spp. between medicated and control diet in groups of infected and in rodents has evolved considerably since first used to prevent uninfected mice after an 8-wk treatment course and again 8 wk chronic active hepatitis and colitis in young male SCID/NCr after treatment completion. mice naturally infected with H. hepaticus.36 In that initial report, various combinations of amoxicillin, bismuth, metronidazole, Materials and Methods neomycin, and tetracycline were either added to the drinking Generation of Helicobacter-infected mice. Male and female 36 water or administered orally 3 times daily for 14 d. Although Foxn1nu (nude) and B6.129S7-Rag1tm1Mom/J (Rag1) mice (age, 6 36 clearly labor-intensive, the cited study determined that H. wk; Jackson Laboratory, Bar Harbor, ME) were housed for 4 wk hepaticus could be eliminated by using all treatment regimens in same-sex cohorts with unrelated C57BL/6J mice naturally that included amoxicillin. A similar dosing regimen using ei- infected with both H. hepaticus and H typhlonius. Prior to place- ther amoxicillin or tetracycline-based triple therapy was used ment, all C57BL/6J mice were screened by fecal PCR (Mouse 16 to eliminate H. hepaticus from infected A/JCr mice. The first Surveillance Plus PRIA panel, Charles River, Wilmington, MA) medicated diet formulation intended to eliminate infection in for commonly excluded pathogens as well as opportunistic mice was a triple therapy of amoxicillin, metronidazole, and agents including Corynebacterium bovis, Klebsiella spp., and 17 bismuth. When fed continuously for 2 wk to 6- to 10-mo-old Pasteurella pneumotropica; Helicobacter spp. and Staphylococcus DBA/J mice, the diet successfully eliminated chronic H. hepati- xylosus were the only agents accepted. One week after removal cus infection as confirmed by posttreatment culture and PCR of the C57BL/6J mouse, fresh fecal samples were collected from 17 testing 1 mo later. Despite this success, the same amoxicillin- each nude and Rag1 mouse and tested inhouse for Helicobacter based triple combination diet fed for an unspecified duration spp. by PCR assay. Positive mice were retested inhouse 2 wk failed to eliminate H. hepaticus from an immunodeficient later. Positive samples were sent to a commercial diagnostics −/− 16,51 breeding colony of Rag1 mice. When administered to a laboratory (IDEXX RADIL, Columbia, MO) for Helicobacter spe- scid tm1tyi SCID (Prkc /Tpr53 on a B6.129/Sv × C.B17 background) ciation. After confirmation of infection, breeding trios of both breeding colony severely affected by diarrhea associated with strains were established. Infection status of resulting offspring H. rodentium and H. bilis coinfection, the triple therapy reduced was tested by inhouse fecal PCR for generic Helicobacter spp. clinical illness during treatment period but did not eliminate at weaning (3 to 3.5 wk of age) and again at 6 to 6.5 wk of age 41 infection or persistent diarrhea. prior to study initiation. Positive samples were speciated by a

239 Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014 commercial diagnostic laboratory (IDEXX RADIL). In addition, (thickness, 5 μm), and stained with hematoxylin and eosin for Helicobacter-negative breeding colonies of both strains were histopathologic examination. Additional sections of esophagus maintained to provide control offspring for this study. and stomach were examined by using Gram, Gomori methena- Housing. Mice were housed in an AAALAC-accredited facility mine silver, and periodic acid–Schiff stains. in compliance with the Guide for the Care and Use of Laboratory Histopathology. Histopathology was performed on the liver Animals25 and procedures were approved by the Johns Hopkins and gastrointestinal tract of all mice on study. The pathologist IACUC (Baltimore, MD). Mice were pair-housed with a single evaluating the tissues was blinded to the diet and infection cotton nesting square within individually ventilated cages (Al- status of the animals. Histopathologic changes were limited to lentown Caging Equipment, Allentown, NJ) assembled with the esophagus and forestomach and thus were the only tissues corncob bedding (Harlan Laboratories, Indianapolis, IN) and for which a scoring system was established. The following autoclaved as a unit. Irradiated feed was added to each cage groups were compared by strain and sex as independent groups: subsequently, in a filtered-air change station (Lab Products, infected–control diet for 8 or 16 wk; infected–medicated diet Seaford, DE) with the use of chlorine-dioxide–based disinfect- for 8 wk; infected–medicated diet for 8 wk followed by control ant (100 ppm, Vimoba, Quip Laboratories, Wilmington, DE). All diet for 8 wk; uninfected–medicated diet for 8 or 16 wk; and mice received reverse-osmosis–treated water by means of an in- uninfected–control diet 8 or 16 wk. Esophageal scoring was cage automated watering system (Rees Scientific, Trenton, NJ). performed on all mice within each group by using a scale of Cages changes occurred on a 2-wk cycle in filtered-air change 0 to 3 as follows: 0, normal keratinized squamous epithelial stations (Lab Products) with the use of chlorine-dioxide–based mucosa; 1, hyperkeratinization less than the width of the un- disinfectant (100 ppm, Vimoba) and precautionary measures, derlying mucosa; 2, hyperkeratinization greater than width of which included changing gloves between cages to prevent cross the underlying mucosa; and 3, hyperkeratinization greater than contamination and spraying disinfectant liberally on the change or equal to the width of underlying mucosa and with extension station, outside of cages, and gloved hands. into the forestomach. Individual scores for each group were Health surveillance. All mice on study as well as those used averaged to establish a final group score as follows: 0 to 0.5, for breeding were monitored through the use of soiled-bedding no significant esophageal pathology; greater than 0.5 to 1.5, sentinels, which remained free of Sendai virus, pneumonia minimal hyperkeratosis; greater than 1.5 to 2.5, mild to moder- virus of mice, mouse hepatitis virus, mouse minute virus, ate hyperkeratosis; and greater than 2.5, severe hyperkeratosis. mouse parvovirus 1 and 2, Theiler mouse encephalomyelitis Helicobacter testing. All mice on study were tested by inhouse virus, reovirus, epizootic diarrhea of infant mice, lymphocytic PCR within 1 wk of study initiation (time 0) and again at 4, 8, choriomeningitis virus, ectromelia virus, murine adenovirus, and 16 wk (when applicable). Fresh fecal samples were frozen at murine cytomegalovirus, Mycoplasma pulmonis, fur mites, and −20 °C prior to testing and analyzed by PCR assay as described pinworms. previously.2,9 Briefly, genus-specific primers ′(5 TAT GAC GGG Study groups. Helicobacter-positive nude and Rag1 mice (age, TAT CCG GC 3′ and 5′ ATT CCA CCT ACC TCT CCC A 3′) from 7 wk) were assigned randomly to either experimental or control regions of the 16S rRNA gene were synthesized by Integrated groups (6 to 8 male mice and 6 to 8 female mice in each group) DNA Technologies (Coralville, IA). PCR amplification was to receive 8 wk of medicated diet, 8 wk of control diet, 8 wk of done by using a Chromo4 real-time detector (Bio-Rad Labora- medicated diet followed by 8 wk of control diet, 16 wk of medi- tories, Hercules, CA) and the Quanti-Tect SYBRGreen PCR kit cated diet, or 16 wk of control diet. The same experimental and (SYBRGreen, Valencia, CA). Primers were used at 0.20 μM and control groups were established for Helicobacter-negative mice. MgCl2 at 2.5 mM. DNA was extracted from approximately 20 mg Diet and feeding protocol. Control diet consisted of a 5-g, ir- fecal material (equivalent to 50 to 600 ng/mL feces as tested by radiated and nutritionally complete grain-based, bacon-flavored spectrophotometer) by using the QIAamp DNA Stool Mini Kit tablet (product no. S05072, BioServ, Frenchtown, NJ). Medicated (Qiagen, Ocala, FL). Sample DNA was heat-denatured at 95 °C diet was the same diet with the addition of 3.0 mg amoxicil- for 10 min and then amplified during 40 cycles at 95° C for 15 s, lin, 0.5 mg clarithromycin, 1.0 mg metronidazole, and 0.02 mg 58 °C for 30 s, and 72 °C for 30 s. A standard curve was generated omeprazole to each 5-g tablet (product no. S05723, Rodent Heli- according to serial dilutions of DNA spiked into buffer. At the cobacter MD Four-Drug Combo, BioServ). Because the diets used completion of cycling, a melting curve analysis was performed on study were different from those in general use in the facility, on samples with late amplification to check for products other all mice were acclimated to the control diet fed ad libitum from than Helicobacter spp. Controls included DNA from a negative weaning (21 to 23 d) to study initiation (7 wk of age). fecal sample and Helicobacter DNA spiked into a negative fecal Body weight recording. Body weights were recorded weekly sample. To control for false negatives caused by PCR inhibi- (same day and time each week) for each mouse over the course tors in the feces, each sample was divided and tested by using of the 8- or 16-wk study period. primers for an arbitrarily chosen mouse gene during the same Gross pathology. Complete necropsies to assess gross pathol- PCR run. In addition to inhouse testing, paired samples were ogy were performed on all mice after the 8- or 16-wk study sent to a commercial diagnostics laboratory (IDEXX RADIL) to period. Liver, cecum, and total body weights were documented. corroborate inhouse test results and to speciate positive samples. Ceca were systematically excised in situ at the ileocecal junc- Statistical analysis. Statistical analyses were performed by tion (proximally) and proximal colon (distally) immediately on using Prism 6.0 (GraphPad, San Diego, CA). Weekly changes opening of the abdomen cavity, with minimal manipulation of in body weight between groups according to infection status luminal contents prior to each weight recording. Tissue collec- (infected and uninfected) and diet type (medicated and non- tion for histopathology was limited to the liver (a representative medicated) over the 16-wk study period were compared by section of the left lateral and median lobes), esophagus, stomach, using repeated measures 2-way ANOVA followed by posthoc and small and large intestines because these were the tissues Tukey multiple comparison tests. Cecum-to-body weight ratios most likely to be affected by Helicobacter infection or adminis- between diet types (medicated and nonmedicated) and study tration of the medicated diet. All collected tissues were fixed in duration (8 and 16 wk) were compared by using 2-way ANOVA 10% buffered formalin, embedded in paraffin blocks, sectioned followed by posthoc Tukey multiple comparison tests. Histo-

240 Medicated diet to eradicate Helicobacter spp. from immunodeficient mice

logic scores were analyzed by using the Kruskal–Wallis test for nonparametric data followed by the Dunn multiple comparison test. Helicobacter fecal PCR results were not compared statisti- cally because only 100% eradication was considered relevant. Results of analyses were considered statistically significant when the P value was less than or equal to 0.05.

Results Clinical health observations. None of the mice on study demonstrated any clinical signs associated with Helicobacter in- fection or with medicated diet treatment. A mild rectal prolapse occurred in 1 of the 8 uninfected male nude mice fed 8 wk of medicated diet. None of the other mice euthanized at either the 8- or 16-wk time point showed any evidence of rectal prolapse. Body weight. Nude mice. Statistical analyses were conducted separately for male and female mice. Because weight trends did not differ statistically, mean body weights and standard Figure 1. Medicated diet significantly (*, P < 0.02) reduced weight gain deviations reported in Figure 1 reflect the 2 sexes combined. Ir- in all nude mice by wk 7 of the 8-wk treatment. Medicated groups respective of initial infection status, male and female nude mice subsequently returned to control diet normalized in weight by wk 13. on medicated diet weighed significantly (P < 0.02) less than did Infection status and sex did not affect findings. Each group contained 16 male and 16 female mice (weeks 0 through 8) or 8 male and 8 female those on control diet by week 7 of the 8-wk treatment (Figure mice (weeks 9 through 16); data are given as mean ± 1 SD. 1). All mice subsequently fed 8 wk of control diet normalized in weight compared with their nonmedicated controls by week 11 (Figure 1). All groups that continued on medicated diet for an additional 8 wk (16 wk on medicated diet in total) remained significantly (P ≤ 0.02) lighter than those on control diet for the duration on the study (Figure 1). Rag1 mice. Statistical analyses were conducted separately for male and female mice. Because weight trends did not differ sta- tistically, mean body weights and standard deviations reported in Figure 2 reflect the 2 sexes combined. Irrespective of initial infection status, Rag1 mice fed medicated diet weighed signifi- cantly (male mice, P < 0.02; female mice, P < 0.04) less than did those on control diet by week 7 of the 8-wk treatment (Figure 2). All mice subsequently fed 8 wk of control diet normalized in weight compared with their nonmedicated controls by week 13. All groups that continued on medicated diet for an additional 8 wk (16 wk on medicated diet in total) remained significantly (P ≤ 0.02) lighter than those on control diet for the duration on the study (Figure 2). Helicobacter PCR. Coinfection with H. hepaticus and H. Figure 2. Medicated diet significantly reduced weight gain in all Rag1 typhlonius was confirmed within 1 wk of study initiation in mice by week 7 of the 8 wk treatment. *, Significant (P < 0.04). Medicat- ed groups subsequently returned to control diet normalized in weight all infected nude and Rag1 mice. All infected mice fed 8 wk by wk 13. Infection status and sex did not affect findings. Each group of medicated diet became negative for Helicobacter spp. when contained 16 male and 16 female mice (weeks 0 through 8) or 8 male tested by PCR of fecal samples at 4 wk and again at 8 wk by and 8 female mice (weeks 9 through 16); data are given as mean ± 1 SD. fecal PCR analysis inhouse and at a commercial laboratory. All treated mice remained Helicobacter-negative for 8 wk after sex, values presented in Figure 4 reflect the 2 sexes combined. returning to control diet. The infection status of positive and Cecum–to–body-weight findings in male Rag1 mice were con- negative controls did not change over the course of the study. sistent with those reported for both male and female nude mice Gross pathology. Nude mice. Statistical analyses were con- (P < 0.0001, Figure 4 B). In contrast to nude mice and Rag1 male ducted separately for male and female mice. Because findings mice, Rag1 female mice fed 8 wk of medicated diet followed by did not differ statistically by sex, values presented in Figure 3 8 wk of control diet still had significantly (P < 0.0001) elevated reflect the 2 sexes combined. Irrespective of initial infection sta- cecum–to–body-weight ratios compared with those of groups tus, mice euthanized directly after 8 or 16 wk of medicated diet fed 8 or 16 wk of control diet (Figure 4 A). Elevated ratios did had significantly (P < 0.0001) greater cecum–to–body-weight not differ statistically differ between female mice fed 8 or 16 wk ratios compared with groups fed 8 or 16 wk of control diet and of medicated diet and those fed 8 wk of medicated diet followed groups fed 8 wk of medicated diet followed by 8 wk of control by 8 wk of control diet. diet (Figure 3). Elevated ratios did not differ between groups fed Irrespective of strain, medicated groups exhibited markedly 8 or 16 wk of medicated diet. Normal cecum–to–body-weight distended ceca with luminal contents that were softer in con- ratios occurred in mice fed 8 or 16 wk of control diet and those sistency than those of nonmedicated infected and uninfected fed 8 wk of medicated diet followed by 8 wk of control diet. groups (Figure 5). Liver, gall bladder, esophagus, stomach, Rag1 mice. small intestines, and the remaining segments of large intestine Statistical analyses were conducted separately for male and appeared grossly normal in all mice necropsied. Liver weights female mice. Because findings did not differ statistically by

241 Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014

Figure 5. Significant (P < 0.0001) cecal enlargement after 8 wk of medi- cated diet (left) compared with 8 wk of control diet (right). This find- ing was present in all groups euthanized directly after 8 wk of treat- Figure 3. Ratios of cecum:body weight of nude mice fed medicated ment irrespective of strain, sex, or Helicobacter infection status. Cecal diet for 8 or 16 wk were significantly (P < 0.0001) greater than those size normalized in all medicated groups (except Rag1 female mice) of mice fed the control diet for the same periods of time. Ratios did that were returned to control diet for 8 wk. not differ between 8- and 16-wk medicated groups. Mice fed 8 wk of medicated diet followed by 8 wk of control diet had significantly (‡, P The following groups were compared independently by strain < 0.0001) reduced cecum:body weight ratios. Sex and infection status did not affect findings. Each group (8 or 16 wk) comprised 8 male or 8 and sex: infected–control diet for 8 or 16 wk; infected–medi- female mice; data are given as mean ± 1 SD. cated diet for 8 wk; infected–medicated diet for 8 wk followed by control diet for 8 wk; uninfected–medicated diet for 8 or 16 wk; and uninfected–control diet for 8 or 16 wk. All groups that received medicated diet for 8 or 16 wk had severe esophageal hyperkeratosis (score, 2.5 or greater) compared with all other groups (P < 0.0001, Figure 6 B). No significant difference was determined between groups medicated for 8 compared with 16 wk. Groups that received medicated diet for 8 wk followed by control diet for 8 wk exhibited mild to moderate esophageal hyperkeratosis (score, greater than or equal to 1.5 to 2.5) that was significantly (P < 0.0001) less extensive than that of groups with severe lesions but that was still significantly (P < 0.0001) more pronounced than that in control-diet groups, which exhibited no esophageal pathology (Figure 6 A). Special stains (Gram, Gomori methenamine silver, and periodic acid–Schiff) of the esophagus and stomach from the medicated-diet and control groups demonstrated no organisms. Infection status, strain, and sex had no effect on histologic findings. Ceca of medicated mice were histologically normal despite being markedly enlarged on gross examination. All remaining sections of the gastrointestinal tract and liver were likewise histologically unremarkable.

Discussion To our knowledge, this study is the first to demonstrate suc- cessful use of medicated diet for eradication of H. hepaticus and H. typhlonius in mice with adaptive immune deficiencies. Overall 100% of PCR tests remained negative 8 wk after medicated diet Figure 4. Ratios of cecum:body weight of Rag1 mice fed the medicated was discontinued, indicating eradication rather than temporary diet for 8 or 16 wk were significantly (P < 0.0001) greater than those suppression. In our study, all infected mice treated with medi- of mice fed the control diet for the same periods. (A) Medicated male cated diet were Helicobacter-negative by week 4 and remained mice returned to the control diet for 8 wk had a significantly (‡, P < negative through week 16. Although this pattern suggests that 0.0001) reduced cecum:body weight ratio. (B) The cecum:body weight duration of treatment might be reduced, the decision to shorten ratios of medicated female mice remained elevated compared with those of controls. Infection status did not affect findings. Each group the treatment period should be considered in light of the age, (8 or 16 wk) comprised 8 male or 8 female mice; data are given as background strain, genetic modification, immune status, and mean ± 1 SD. chronicity of infection. The relatively short period of infection prior to treatment in the current study is one conceivable ex- did not differ significantly between any of the experimental or planation for the timely clearance. In a previous study using tm1Gru control groups of either strain. the same diet, 24-wk-old Helicobacter-infected B6.129-Cd1 −/− Histopathology. Histopathology of the liver and gastrointesti- (Cd1 ) mice remained positive until week 8 of treatment, nal tract was performed on all mice. Because abnormalities were whereas 8- to 12-wk-old mice tested negative after only 2 wk 9 limited to the esophagus and, in severe cases, the forestomach, and remained negative for the duration of the study. However, 9 a histologic scoring system was devised only for these tissues. in the cited study, increased intestinal inflammation resulting

242 Medicated diet to eradicate Helicobacter spp. from immunodeficient mice

forestomach. There were no significant differences in severity between the 8- and 16-wk groups, suggesting that prolonged treatment did not exacerbate the condition. Medicated groups subsequently fed 8 wk of control diet showed mild to moderate hyperkeratosis, indicating that the condition was reversible. The weight disparity in medicated mice was not sufficient for them to appear obviously underweight (although formal body condition scoring was not done), and signs associated with anorexia such as lethargy were not apparent, although the pos- sibility for subtle behavioral changes exists. Volume of stomach and intestinal contents varied from mouse to mouse, but there were no appreciable differences between medicated and non- medicated groups at the time of necropsy. Although esophageal cultures to rule out opportunistic bacterial and fungal etiologies of hyperkeratosis were not performed at the time of necropsy, these differentials seemed unlikely given the reversibility of the condition after the return to control diet, the lack of pathology in control diet groups, and the absence of inflammation (mice were either T-cell–deficient or T- and B-cell–deficient but still had functional macrophages and innate immunity). In addition, special histologic stains of the esophagus and stomach revealed no organisms in any of control or medicated-diet treatment groups. Medicated-diet-induced hyperkeratosis independent of body weight is another possibility, but esophageal pathology associated with these drugs has not been reported in mice. An unexplained increased incidence of liver portal fibrosis was found in medicated male Cd1 −/− mice in a prior study9 but not in the current study, suggesting that the specific genetic makeup of the mice may play a role in some of the pathologic changes. Alternative hypotheses for reduced weight gain are more Figure 6. (A) Histologic appearance of normal esophagus of nude complex but may involve interactions between intestinal mi- and Rag1 mice fed control diet. (B) Severe hyperkeratosis (*) of the croenvironment, energy balance, gene expression, and immune esophageal mucosa as found in all mice fed 8 or 16 wk of medicated function. Antibiotics have the potential to disrupt biologic diet. Severity did not differ between 8- and 16-wk medicated groups. processes and reduce overall health.8 Changes in body weight Hematoxylin and eosin stain; bar, 50 µm. and composition after antibiotic administration have been an area of interest in both animals and humans.5,7 Although the from chronic infection may have led to reduced drug penetra- incorporation of low doses of antibiotics into livestock feed tion and absorption and thus the need for a longer treatment as a growth-promoting strategy is still a common practice in 9 period. Because intestinal inflammation is largely facilitated the United States, similar subtherapeutic antibiotic treatment by T cells, the rapid Helicobacter clearance in the current study protocols in mice have not been able to fully replicate this phe- may have been facilitated by a lack of T-cell–mediated intestinal nomenon.7 Early research using Swiss mice to study the effect inflammation. Therefore, treatment duration and efficacy should of antibiotics and dietary formulations on microflora11,13,14,39 be considered in light of strain susceptibility to IBD and the documented decreased weight gain after antibiotic adminis- potential for Helicobacter spp. to exacerbate the disease process. tration,12 but there have been profound changes in rodent diet, Administration of medicated diet led to a reduced growth genetics, and infection status in laboratory mice since these rate compared with that of mice fed control diet in both nude previous studies were published, making direct comparisons and Rag1 mice irrespective of initial Helicobacter infection status. with current studies difficult. These differences in weight disappeared in mice that returned More recently, immunocompetent inbred strains have been to the control diet halfway through the 16-wk study, but the used to demonstrate that decreases in total gut microbial density differences persisted in mice that received medicated diet for and shifts in microbial composition can alter metabolism and 16 wk. A similar finding was reported in a previous study lead to reduced growth.33 Young diet-induced obese C57BL/6J tm1Gru −/− in which young male B6.129-Cd1 mice (Cd1 ) treated mice given oral vancomycin daily for 8 wk had marked altera- with medicated diet gained less weight than did untreated tions in the proportions of 3 dominant bacterial phyla.34 These 9 controls. Reduced feed consumption due to poor palatability alterations led to a reduced growth rate compared with those of is a likely explanation for the reduced growth rates that we controls despite equivalent cumulative food intake.34 Common noted in medicated mice. Although feed consumption was not features of fecal metabolites postantibiotic treatment include measured, hyperkeratosis of the esophagus and forestomach decreased levels of short-chain fatty acids, amino acids, and mucosa was noted histologically in all medicated groups. primary bile acids.8 These changes indicate suppression of bac- Keratinized esophageal and forestomach epithelium is normal terial and protein degradation,8,54 likely due to a in mice (unlike humans and most carnivores), and hyperkera- reduction in anaerobic from the phylum Bacteroidetes, tosis of this tissue is associated often with anorexia and food the predominant flora in conventional mice.34,54 Bacteroides spp. 32,35,45 restriction. Irrespective of strain, groups of mice that are rich in enzymes that facilitate carbohydrate transport and received the medicated diet for either 8 or 16 wk developed protein metabolism, and they contain vitamin and cofactor severe esophageal hyperkeratosis, which often extended into the enzymes important for diet digestion.27 Broad-spectrum anti-

243 Vol 53, No 3 Journal of the American Association for Laboratory Animal Science May 2014 biotics significantly reduce their density and instead support clarithromycin, metronidazole, and omeprazole. Treatment the colonization of antibiotic-resistant facultatively anaerobic sequelae included a reduction in growth rate, gastroesophageal bacteria and low-frequency commensals.24,33,53,54 Therefore, hyperkeratosis, and cecal enlargement, but these changes were the reduced weight gain seen in the current study could have largely reversed after a return to normal diet. This result sug- resulted from similar changes in gut flora, leading to reduced gests a potential application for widespread dietary treatment absorption of nutrients and alterations in metabolism. to eliminate Helicobacter infections from mice with adaptive No appreciable hepatic or intestinal pathology associated with immune deficiencies. Helicobacter infection was noted in infected nude or Rag1 mice. The presence and severity of lesion development is, however, Acknowledgments highly dependent on strain susceptibility to a particular species This study was supported by NIH Training Grant NIH R25OD010913, of Helicobacter, immune status, and duration of infection. Most and additional support was provided by Dr Robert J Adams and Johns immunocompetent Helicobacter-resistant strains of mice do not Hopkins Research Animal Resources. We extend our gratitude to all develop intestinal pathology, and hepatic lesions are even less of those who helped with colony management, sample collection, and common.49,52 The inbred Rag1 mice in the current study were on processing. We thank Dr Karen M Froberg-Fejko (BioServ, Frenchtown, a background (C57BL/6J) known to be resistant to Helicobacter- NJ) for donation of the treatment and control diets. This project was not influenced by BioServ or any of its members, other than by follow- induced pathology. Rag1-deficient mice on this background ing manufacturer recommendations for the duration of the treatment. that were followed for approximately 9 mo after infection with either H. hepaticus or H. bilis demonstrated no evidence of clini- cal disease or histologic lesions associated with IBD.4 The nude References mice we used were an inbred strain derived from a spontane- 1. Artwohl JE, Purcell JE, Fortman JD. 2008. The use of cross-foster rederivation to eliminate murine norovirus, Helicobacter spp., and ous mutation (Foxn1nu) in an outbred stock at NIH. Outbred murine hepatitis virus from a mouse colony. J Am Assoc Lab Anim Swiss mice naturally infected with H. bilis develop multifocal Sci 47:19–24. 20 hepatitis, but this pathology occurs primarily in aged animals. 2. Beckwith CS, Franklin CL, Hook RR Jr, Besch-Williford CL, Riley Even among susceptible immunocompetent strains, time until LK. 1997. 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