The Host-specific Microbiota is Required for Diet-Specific Metabolic Homeostasis.

In complex mammals, the importance and host-specificity of microbial communities have been demonstrated through their positive effects on host immune fitness or performance. However, whether host metabolic physiology homeostasis depends on a specific bacterial community exclusive to the host remains unclear. Here, we show that the coevolved host-specific microbiota is required to maintain diet-specific flexible and sufficient metabolic homeostasis through a high colonization rate, modulating gut metabolites, and related targets. Using germ-free (GF) mice, we tested whether the fitness benefiting the host metabolic phenotype of microbiota was host-specific. We demonstrated that GF mice associated with exogenous microbiota (human microbiota (HM)), which exhibited different and reduced gut microbial species diversity, significantly elevated metabolic rate, and exhibited metabolic insufficiency, all characteristics of GF mice. Strikingly, the absence of the host-specific microbiome attenuated high-fat diet-specific metabolism features. Different diets caused different metabolic changes in only host-specific microbiota-associated mice, not the host-microbiota mismatched mice. While RNA sequencing revealed subtle changes in the expression of genes in the liver, GF mice and HM mice showed considerably altered expression of genes associated with metabolic physiology compared to GF mice associated with host-specific microbiota. The effect of diet outweighed microbiota in the liver transcriptome. These changes occurred in the setting of decreased luminal short-chain fatty acids (SCFAs) and the secondary bile acid (BAs) pool and downstream gut signaling targets in HM and GF mice, which affects whole-body metabolism. These data indicate that a foreign microbial community provides little metabolic benefit to the host when compared to a host-specific microbiome, due to the colonization selection pressure and microbiota-derived metabolites dysfunction. Overall, microbiome fitness effects on the host metabolic phenotype were host-specific. Understanding the impact of the host-specificity of the microbiome on metabolic homeostasis may provide important insights for building a better probiotic. Highlights: Microbiome fitness effects on the host metabolic phenotype were host-specific in mammals.Human microbiota-associated mice exhibited lower host metabolic fitness or performance, and similar functional costs in GF mice.Different diets cause different metabolic changes only in host-specific microbiota-associated mice, not the host-microbiota mismatched mice.The defective gut microbiota in host-specific microbiota, microbial metabolites and related targets likely drive the metabolic homeostasis.

Preference of Escaped Mice for Live Capture or Glue Traps and Relevance to Pest Control Programs.

Insects are potential disease vectors for research animals. Therefore, implementing an effective pest control program is an essential component of any animal care and use program. The Guide for the Care and Use of Laboratory Animals emphasizes the humane use of traps; however, insect traps commonly use glue that can entrap escaped research mice, leading to their potential distress and injury. This situation is challenging for research facilities attempting to identify insect populations. In an effort to improve pest control in animal facilities, we sought to characterize the behavioral interactions of mice with common vermin traps. Three experiments using different combinations of traps (glue trap, live mouse trap with a clear viewing window, and live mouse trap with a red-tinted viewing window) were used in multiple behavioral testing arenas to address these questions. Experiments 1 and 2 were performed in a small arena, and Experiment 3 was performed in a simulated mouse housing room. Dependent measures included exploration of the test environment, grooming behavior, time spent near each trap, and latency to capture. Results indicate that mice were captured significantly more quickly by live traps than by glue traps, and were far more likely to enter a live trap as compared with a glue trap. Mice did not appear to differentiate between clear or red-tinted window live traps. Taken together, the results indicate that deploying both a live trap and a glue trap will allow humane capture of escaped mice yet will also capture insects in the same environment.

Atypical behavioral and thermoregulatory circadian rhythms in mice lacking a microbiome.

Trillions of microbial oscillators reside throughout the mammalian body, yet their contributions toward fundamental features of host circadian rhythms (CRs) have not been characterized. Here, we demonstrate that the microbiome contributes to host CRs in activity and thermoregulation. Mice devoid of microbes (germ-free, GF) exhibited higher-amplitude CRs in a light-dark cycle and longer circadian periods in constant darkness. Circadian entrainment to food was greater in GF mice, but resetting responses to simulated jet-lag were unaffected. Microbial transplantation with cecal contents of conventionally-raised mice normalized CRs of GF mice, indicating that the concurrent activity of gut microbes modulates host circadian networks. Obesogenic effects of high-fat diet were absent in GF mice, but some circadian-disruptive effects persisted. Transkingdom (host-microbe) interactions affect circadian period and entrainment of CRs in diverse traits, and microbes alter interactions among light- and food-entrainable circadian processes in the face of environmental (light, diet) perturbations.

Identification and Control of an Ornithonyssus Bacoti Infestation in a Rodent Vivarium by Using Molecular Diagnostic Techniques.

Ornithonyssus bacoti, commonly known as the tropical rat mite, is a zoonotic ectoparasite that occasionally infests research rodent colonies. Most infestations have been attributed to wild rodents that harbor the mite and spread it to research animals, often during building construction or other activity that disrupts wild rodent populations. Although infestation may be clinically silent, severe outbreaks have been reported to cause pruritis, dermatitis, decreased reproductive performance, and anemia in rodents. In mid-2020, our institution experienced increased activity of wild mice, which were found to be infested with O. bacoti, diagnosed by microscopic exam and confirmed by fur swab PCR analysis. We elected to add O. bacoti to our quarterly health monitoring exhaust air dust (EAD) testing PCR panel, increase wild mouse control measures, and treat the environment with a sustained-release synthetic pyrethroid spray in an attempt to prevent colony animal infestation. Initial quarterly EAD health monitoring results in September of 2020 were negative for O. bacoti. However, in early 2021, multiple IVC racks tested positive for O. bacoti at quarterly testing. Treatment consisted of providing permethrin-soaked nesting material and surface spray treatment of the room and hallway with a sustained-release synthetic pyrethroid. Historically in the literature, O. bacoti outbreaks of research mice were not identified until mite burden was high enough to cause dermatitis on animal care workers. Due to modern molecular diagnostics and proactive PCR-based health monitoring surveillance, we were able to identify the outbreak earlier than would have otherwise been possible. To the best of our knowledge, this is the first report to successfully identify O. bacoti using environmental health monitoring PCR techniques. This outbreak demonstrates the importance of screening for O. bacoti in facilities with the potential for wild rodent infestation and highlights unique considerations when managing O. bacoti infestations. In addition, a novel permethrin-soaked enrichment item was developed for cage-level treatment.

Evaluation of Pain and Distress and Therapeutic Interventions for Rectal Prolapse in Mice to Reduce Early Study Removal.

Rectal prolapse (RP) is a common clinical condition in mice, that does not have a recognized or documented standard of care. At our institution, an average of 240 mice develop RP each year. Our practice has been to recommend euthanasia upon identifying a RP based on its appearance as a painful or distressful condition. This study aimed to assess treatment options that would maintain the RP mucosa and allow mice to reach their study endpoint, and to evaluate the perception of this condition as a painful or distressful event. This study used 120 mice with spontaneous RP, concurrently assigned to ongoing research protocols. Mice were randomly assigned to 1 of 3 treatment groups: petroleum jelly, lidocaine jelly, or no treatment. Fecal samples were collected for pathogen testing, and all mice received an initial base score, followed by weekly blind scores. Upon euthanasia, RP tissue was collected for histopathology. Of the 120 mice identified with RP, 47 mice were breeders; 28% successfully produced 22 additional litters after developing RP. Seventy-three were nonbreeders, with 92% reaching their research study endpoint. No statistically significant differences were detected between the 3 treatment groups based on gross mucosal health, pain and distress, or histopathology. In this study, none of the mice in any group were euthanized based on the RP endpoint scoring criteria. These findings demonstrate that treatment is unnecessary for RP, and mice with RP did not show signs of pain or distress. In adherence to the 3Rs, this study supports animal number reduction and clinical refinement, allowing mice with RPs to reach their intended research study endpoints or produce additional litters.

Long-Term Effects of Sulfadiazine-Trimethoprim Medicated Diet on Cardiac Function, Hematology, and Weight Gain in Hsd:ICR (CD1) and Tac:SW Mice.

With the alarming increase in heart disease and heart failure, the need for appropriate and ethical animal models of cardiac dysfunction continues to grow. Currently, many animal models of cardiomyopathy require either invasive procedures or genetic manipulation, both of which require extensive expertise, time, and cost. Serendipitous findings at our institution revealed a possible correlation between sulfadiazine-trimethoprim (SDZ-TMP) medicated diet and the development of cardiomyopathy in IcrTac:ICR mice. We hypothesized that mice fed SDZ-TMP medicated diet continuously for 3 to 6 mo would develop cardiomyocyte degeneration and fibrosis, eventually leading to dilated cardiomyopathy. A total of 44 mice (22 Hsd:ICR (CD1) and 22 Tac:SW) were enrolled in the study. Half of these 44 mice were fed standard rodent diet and the other half were fed SDZ-TMP medicated diet. Baseline samples, including weights, CBCs, select biochemistry parameters, and echocardiography were performed prior to the start of either diet. Weights were obtained monthly and all other parameters were measured at least once during the study, and again at its conclusion. After 42 wk, mice were euthanized, and heart, lung and bone marrow tissue were submitted for histopathologic evaluation. Histologically, hearts were scored for the degree of degeneration, fibrosis, inflammation, and vacuolation. The data showed that SDZ-TMP did not have a significant effect on cardiac function, RBC parameters, biochemistry parameters (ALT, AST, calcium, magnesium, creatine kinase, and creatinine), hematopoiesis, or histologic heart scores. In addition, mice fed the SDZ-TMP medicated diet gained less weight over time. In summary, we were unable to reproduce the previous findings and thus could not use this approach to develop a novel model of cardiomyopathy. However, these results indicate that SDZ-TMP medicated diet containing 1,365 ppm of SDZ and 275 ppm of TMP does not appear to have long-term detrimental effects in mice.

Unexpected Cardiomyopathy and Cardiac Dysfunction after Administration of Sulfadiazine-trimethoprim Medicated Diet to ICR mice (Mus musculus).

For many years, the University of Chicago administered sulfamethoxazole-trimethoprim sulfate (SMZ-TMP) oral suspension to select immunocompromised mouse colonies via the drinking water. In 2014, SMZ-TMP oral suspension was placed on back-order and medicated diet with a different sulfonamide, sulfadiazine-trimethoprim (SDZ-TMP) was used as a replacement. Months after this transition, sentinel mice from the same room as one of the remaining immunocompromised colonies on this diet were found dead or appeared sick. Necropsies revealed cardiomegaly, and histology confirmed myocardial fibrosis in the first 4 sentinel mice examined, consistent with cardiomyopathy. Subsequent sequential monitoring of 2 sentinel mice via echocardiography showed their progression toward decreased cardiac function. Investigation of the housing room revealed that the sentinel mice had been accidently placed on SDZ-TMP diet upon entering the colony housing room. This case report describes cardiomyopathy in 6 ICR mice after long term consumption of SDZ-TMP medicated feed.

Cost Comparison of Rodent Soiled Bedding Sentinel and Exhaust Air Dust Health-Monitoring Programs.

Rodent vivaria have traditionally used soiled bedding sentinel (SBS) health-monitoring programs to detect and exclude adventitious pathogens that could affect research results. Given the limitations of SBS, a likely reduction in animal usage, and a decrease in animal care staff labor, exhaust air dust (EAD) health monitoring has been evaluated by several groups for its efficacy in detecting pathogens when used as a complete replacement for traditional SBS health-monitoring programs. Compared with SBS, EAD has also been shown to provide increased sensitivity for the detection of multiple pathogens. After implementing EAD at our institution, we conducted an analysis to compare the annual costs of the 2 health-monitoring programs. The EAD program was found to be 26% less expensive than SBS. In addition to these cost savings, EAD decreased the amount of time spent by the staff on heath-monitoring activities. For veterinary technicians, this decrease in time was calculated as a savings of 150 h annually, almost 3 h each week. Finally, the EAD program replaced the use of live sentinel animals, decreasing the associated yearly usage from 1,676 animals to zero.

Artificial microbiome heterogeneity spurs six practical action themes and examples to increase study power-driven reproducibility.

With >70,000 yearly publications using mouse data, mouse models represent the best engrained research system to address numerous biological questions across all fields of science. Concerns of poor study and microbiome reproducibility also abound in the literature. Despite the well-known, negative-effects of data clustering on interpretation and study power, it is unclear why scientists often house >4 mice/cage during experiments, instead of ≤2. We hypothesized that this high animal-cage-density  practice abounds in published literature because more mice/cage could be perceived as a strategy to reduce housing costs. Among other sources of 'artificial' confounding, including cyclical oscillations of the 'dirty-cage/excrement microbiome', we ranked by priority the heterogeneity of modern husbandry practices/perceptions across three professional organizations that we surveyed in the USA. Data integration (scoping-reviews, professional-surveys, expert-opinion, and 'implementability-score-statistics') identified Six-Actionable Recommendation Themes (SART) as a framework to re-launch emerging protocols and intuitive statistical strategies to use/increase study power. 'Cost-vs-science' discordance was a major aspect explaining heterogeneity, and scientists' reluctance to change. With a 'housing-density cost-calculator-simulator' and fully-annotated statistical examples/code, this themed-framework streamlines the rapid analysis of cage-clustered-data and promotes the use of 'study-power-statistics' to self-monitor the success/reproducibility of basic and translational research. Examples are provided to help scientists document analysis for study power-based sample size estimations using preclinical mouse data to support translational clinical trials, as requested in NIH/similar grants or publications.

Detection of Lactate Dehydrogenase Elevating Virus in a Mouse Vivarium Using an Exhaust Air Dust Health Monitoring Program.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.

Microbial Colonization of Germ-Free Mice Restores Neointimal Hyperplasia Development After Arterial Injury.

Background The potential role of the gut microbiome in cardiovascular diseases is increasingly evident. Arterial restenosis attributable to neointimal hyperplasia after cardiovascular procedures such as balloon angioplasty, stenting, and bypass surgery is a common cause of treatment failure, yet whether gut microbiota participate in the development of neointimal hyperplasia remains largely unknown. Methods and Results We performed fecal microbial transplantation from conventionally raised male C57BL/6 mice to age-, sex-, and strain-matched germ-free mice. Five weeks after inoculation, all mice underwent unilateral carotid ligation. Neointimal hyperplasia development was quantified after 4 weeks. Conventionally raised and germ-free cohorts served as comparison groups. Conclusions Germ-free mice have significantly attenuated neointimal hyperplasia development compared with conventionally raised mice. The arterial remodeling response is restored by fecal transplantation. Our results describe a causative role of gut microbiota in contributing to the pathogenesis of neointimal hyperplasia.

Comparing Mouse Health Monitoring Between Soiled-bedding Sentinel and Exhaust Air Dust Surveillance Programs.

To monitor rodent colony health in research facilities, soiled-bedding sentinel (SBS) animals have traditionally been used. SBS can be tested by various methods, which may include serology, PCR analysis, and necropsy. Several pathogens are unreliably detected by using SBS or transmitted poorly through soiled bedding, and collection and evaluation of SBS samples can be time-intensive. Recently, exhaust air dust (EAD) testing through PCR analysis has emerged as an adjunct or replacement method for rodent colony health monitoring. EAD monitoring may provide a more efficient, sensitive, and humane method for monitoring health status. Using both EAD and SBS health monitoring, we evaluated colony health over the course of 1 y in 3 research barrier rooms in which mice were housed exclusively on IVC racks. Three pathogens-Helicobacter spp., Rodentibacter spp. (previously Pasteurella pneumotropica), and murine norovirus (MNV)-were not excluded in 2 of the rooms, and we expected that these mice would test positive with some regularity. EAD monitoring was significantly more sensitive than SBS for detection of the bacterial agents. SBS failed to detect Helicobacter spp. at time points when EAD had 100% detection in the rooms that did not exclude the bacteria. The detection of MNV did not differ between health monitoring systems at any time point. The findings suggest that EAD is especially valuable in detecting bacteria poorly transmitted through soiled bedding. In addition, the corresponding results with MNV detection suggest that EAD surveillance can reliably be implemented as an alternative to SBS monitoring in a facility in which mice are housed exclusively on IVC racks.

Microbiota control acute arterial inflammation and neointimal hyperplasia development after arterial injury.

BACKGROUND: The microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice. METHODS: Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods. RESULTS: All GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson's trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-γ-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response. CONCLUSIONS: GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.

Mutual reinforcement of pathophysiological host-microbe interactions in intestinal stasis models.

Chronic diseases arise when there is mutual reinforcement of pathophysiological processes that cause an aberrant steady state. Such a sequence of events may underlie chronic constipation, which has been associated with dysbiosis of the gut. In this study we hypothesized that assemblage of microbial communities, directed by slow gastrointestinal transit, affects host function in a way that reinforces constipation and further maintains selection on microbial communities. In our study, we used two models - an opioid-induced constipation model in mice, and a humanized mouse model where germ-free mice were colonized with stool from a patient with constipation-predominant irritable bowel syndrome (IBS-C) in humans. We examined the impact of pharmacologically (loperamide)-induced constipation (PIC) and IBS-C on the structural and functional profile of the gut microbiota. Germ-free (GF) mice were colonized with microbiota from PIC donor mice and IBS-C patients to determine how the microbiota affects the host. PIC and IBS-C promoted changes in the gut microbiota, characterized by increased relative abundance of Bacteroides ovatus and Parabacteroides distasonis in both models. PIC mice exhibited decreased luminal concentrations of butyrate in the cecum and altered metabolic profiles of the gut microbiota. Colonization of GF mice with PIC-associated mice cecal or human IBS-C fecal microbiota significantly increased GI transit time when compared to control microbiota recipients. IBS-C-associated gut microbiota also impacted colonic contractile properties. Our findings support the concept that constipation is characterized by disease-associated steady states caused by reinforcement of pathophysiological factors in host-microbe interactions.

Commensal bacteria protect against food allergen sensitization.

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.

Reversible medetomidine/ketamine anesthesia in captive capuchin monkeys (Cebus apella).

BACKGROUND: Medetomidine, an alpha(2)-adrenergic receptor agonist administered with ketamine, induces sedation/anesthesia in non-human primates in a dose-dependent, species-specific manner. METHODS: Two adult male capuchin monkeys enrolled in a study of kinematics of mastication were administered intramuscular (IM) injections of 150, 200 and 400 microg/kg medetomidine or medetomidine (microg/kg)/ketamine (mg/kg) at 200/2, 150/4. Medetomidine was reversed with atipamezole. Animals were then fed for 9-75 minutes. A second anesthesia reversal episode allowed return to home cage. RESULTS: Medetomidine (150 microg/kg)/ketamine (4 mg/kg) provided anesthesia for handling and restraint within 10 minutes of administration. Atipamezole (750 microg/kg) IM provided reversal sufficient for the animal to eat by 22 minutes or perch by 31 minutes. CONCLUSIONS: Medetomidine/ketamine administered IM in Cebus apella results in rapid, reproducible and safe anesthesia which is reversible with atipamezole. Nausea, vomiting or regurgitation were not observed during anesthesia reversal episodes in either the fasted or fed state.