Absence of Gut Microbiota Is Associated with RPE/Choroid Transcriptomic Changes Related to Age-Related Macular Degeneration Pathobiology and Decreased Choroidal Neovascularization.

Studies have begun to reveal significant connections between the gut microbiome and various retinal diseases, including age-related macular degeneration (AMD). As critical supporting tissues of the retina, the retinal pigment epithelium (RPE) and underlying choroid play a critical role in retinal homeostasis and degeneration. However, the relationship between the microbiome and RPE/choroid remains poorly understood, particularly in animal models of AMD. In order to better elucidate this role, we performed high-throughput RNA sequencing of RPE/choroid tissue in germ-free (GF) and specific pathogen-free (SPF) mice. Furthermore, utilizing a specialized laser-induced choroidal neovascularization (CNV) model that we developed, we compared CNV size and inflammatory response between GF and SPF mice. After correction of raw data, 660 differentially expressed genes (DEGs) were identified, including those involved in angiogenesis regulation, scavenger and cytokine receptor activity, and inflammatory response-all of which have been implicated in AMD pathogenesis. Among lasered mice, the GF group showed significantly decreased CNV lesion size and microglial infiltration around CNV compared to the SPF group. Together, these findings provide evidence for a potential gut-RPE/choroidal axis as well as a correlation with neovascular features of AMD.

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.

Antibody-mediated immune control of a retrovirus does not require the microbiota.

Commensal microbes are often required to control viral infection by facilitating host immune defenses. However, we found that this does not hold true for retroviral infection. We report that retrovirus-resistant mice control the pathogen with virus-neutralizing antibodies independently of commensal microbiota. This is in contrast to orthomyxoviruses and arenaviruses, where resistance is ablated in animals depleted of microbiota. Clearly, when it comes to antiviral immunity, the role of the microbiota cannot be generalized.

Milky spots promote ovarian cancer metastatic colonization of peritoneal adipose in experimental models.

The goal of controlling ovarian cancer metastasis formation has elicited considerable interest in identifying the tissue microenvironments involved in cancer cell colonization of the omentum. Omental adipose is a site of prodigious metastasis in both ovarian cancer models and clinical disease. This tissue is unusual for its milky spots, comprised of immune cells, stromal cells, and structural elements surrounding glomerulus-like capillary beds. The present study shows the novel finding that milky spots and adipocytes play distinct and complementary roles in omental metastatic colonization. In vivo assays showed that ID8, CaOV3, HeyA8, and SKOV3ip.1 cancer cells preferentially lodge and grow within omental and splenoportal fat, which contain milky spots, rather than in peritoneal fat depots. Similarly, medium conditioned by milky spot-containing adipose tissue caused 75% more cell migration than did medium conditioned by milky spot-deficient adipose. Studies with immunodeficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Finally, consistent with the role of lipids as an energy source for cancer cell growth, in vivo time-course studies revealed an inverse relationship between metastatic burden and omental adipocyte content. Our findings support a two-step model in which both milky spots and adipose have specific roles in colonization of the omentum by ovarian cancer cells.

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.

Microbiota-dependent early life programming of gastrointestinal motility.

Gastrointestinal microbes modulate peristalsis and stimulate the enteric nervous system (ENS), whose development, as in the central nervous system (CNS), continues into the murine postweaning period. Given that adult CNS function depends on stimuli received during critical periods of postnatal development, we hypothesized that adult ENS function, namely motility, depends on microbial stimuli during similar critical periods. We gave fecal microbiota transplantation (FMT) to germ-free mice at weaning or as adults and found that only the mice given FMT at weaning recovered normal transit, while those given FMT as adults showed limited improvements. RNAseq of colonic muscularis propria revealed enrichments in neuron developmental pathways in mice exposed to gut microbes earlier in life, while mice exposed later - or not at all - showed exaggerated expression of inflammatory pathways. These findings highlight a microbiota-dependent sensitive period in ENS development, pointing to potential roles of the early life microbiome in later life dysmotility.

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.

Oral Metformin Inhibits Choroidal Neovascularization by Modulating the Gut-Retina Axis.

Purpose: Emerging data indicate that metformin may prevent the development of age-related macular degeneration (AMD). Whereas the underlying mechanisms of metformin's anti-aging properties remain undetermined, one proposed avenue is the gut microbiome. Using the laser-induced choroidal neovascularization (CNV) model, we investigate the effects of oral metformin on CNV, retinal pigment epithelium (RPE)/choroid transcriptome, and gut microbiota. Methods: Specific pathogen free (SPF) male mice were treated via daily oral gavage of metformin 300 mg/kg or vehicle. Male mice were selected to minimize sex-specific differences to laser induction and response to metformin. Laser-induced CNV size and macrophage/microglial infiltration were assessed by isolectin and Iba1 immunostaining. High-throughput RNA-seq of the RPE/choroid was performed using Illumina. Fecal pellets were analyzed for gut microbiota composition/pathways with 16S rRNA sequencing/shotgun metagenomics, as well as microbial-derived metabolites, including small-chain fatty acids and bile acids. Investigation was repeated in metformin-treated germ-free (GF) mice and antibiotic-treated/GF mice receiving fecal microbiota transplantation (FMT) from metformin-treated SPF mice. Results: Metformin treatment reduced CNV size (P < 0.01) and decreased Iba1+ macrophage/microglial infiltration (P < 0.005). One hundred forty-five differentially expressed genes were identified in the metformin-treated group (P < 0.05) with a downregulation in pro-angiogenic genes Tie1, Pgf, and Gata2. Furthermore, metformin altered the gut microbiome in favor of Bifidobacterium and Akkermansia, with a significant increase in fecal levels of butyrate, succinate, and cholic acid. Metformin did not suppress CNV in GF mice but colonization of microbiome-depleted mice with metformin-derived FMT suppressed CNV. Conclusions: These data suggest that oral metformin suppresses CNV, the hallmark lesion of advanced neovascular AMD, via gut microbiome modulation.

High-Fat Diet Alters the Retinal Pigment Epithelium and Choroidal Transcriptome in the Absence of Gut Microbiota.

Relationships between retinal disease, diet, and the gut microbiome have started to emerge. In particular, high-fat diets (HFDs) are associated with the prevalence and progression of several retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy (DR). These effects are thought to be partly mediated by the gut microbiome, which modulates interactions between diet and host homeostasis. Nevertheless, the effects of HFDs on the retina and adjacent retinal pigment epithelium (RPE) and choroid at the transcriptional level, independent of gut microbiota, are not well-understood. In this study, we performed the high-throughput RNA-sequencing of germ-free (GF) mice to explore the transcriptional changes induced by HFD in the RPE/choroid. After filtering and cleaning the data, 649 differentially expressed genes (DEGs) were identified, with 616 genes transcriptionally upregulated and 33 genes downregulated by HFD compared to a normal diet (ND). Enrichment analysis for gene ontology (GO) using the DEGs was performed to analyze over-represented biological processes in the RPE/choroid of GF-HFD mice relative to GF-ND mice. GO analysis revealed the upregulation of processes related to angiogenesis, immune response, and the inflammatory response. Additionally, molecular functions that were altered involved extracellular matrix (ECM) binding, ECM structural constituents, and heparin binding. This study demonstrates novel data showing that HFDs can alter RPE/choroid tissue transcription in the absence of the gut microbiome.

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.

Host-versus-commensal immune responses participate in the rejection of colonized solid organ transplants.

Solid organ transplantation is the preferred treatment for end-stage organ failure. Although transplant recipients take life-long immunosuppressive drugs, a substantial percentage of them still reject their allografts. Strikingly, barrier organs colonized with microbiota have significantly shorter half-lives than non-barrier transplanted organs, even in immunosuppressed hosts. We previously demonstrated that skin allografts monocolonized with the common human commensal Staphylococcus epidermidis (S.epi) are rejected faster than germ-free (GF) allografts in mice because the presence of S.epi augments the effector alloimmune response locally in the graft. Here, we tested whether host immune responses against graft-resident commensal microbes, including S.epi, can damage colonized grafts independently from the alloresponse. Naive hosts mounted an anticommensal T cell response to colonized, but not GF, syngeneic skin grafts. Whereas naive antigraft commensal T cells modestly damaged colonized syngeneic skin grafts, hosts with prior anticommensal T cell memory mounted a post-transplant immune response against graft-resident commensals that significantly damaged colonized, syngeneic skin grafts. Importantly, allograft recipients harboring this host-versus-commensal immune response resisted immunosuppression. The dual effects of host-versus-commensal and host-versus-allograft responses may partially explain why colonized organs have poorer outcomes than sterile organs in the clinic.

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.

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.

Gnotobiotic Operations and Assembly for Development of Germ-Free Animal Model of Laser-Induced Choroidal Neovascularization.

Purpose: Compelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD. Methods: We developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase-polymerase chain reaction. Results: The experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment. Conclusions: We established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure. Translational Relevance: To our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host-microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.

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.

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.

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.

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.

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.