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Obesity places a tremendous burden on individual health and the healthcare system. The gut microbiome (GM) influences host metabolism and behaviors affecting body weight (BW) such as feeding. The GM of mice varies between suppliers and significantly influences BW. We sought to determine whether GM-associated differences in BW are associated with differences in intake, fecal energy loss, or fetal growth. Pair-housed mice colonized with a low or high microbial richness GM were weighed, and the total and BW-adjusted intake were measured at weaning and adulthood. Pups were weighed at birth to determine the effects of the maternal microbiome on fetal growth. Fecal samples were collected to assess the fecal energy loss and to characterize differences in the microbiome. The results showed that supplier-origin microbiomes were associated with profound differences in fetal growth and excessive BW-adjusted differences in intake during adulthood, with no detected difference in fecal energy loss. Agreement between the features of the maternal microbiome associated with increased birth weight here and in recent human studies supports the value of this model to investigate the mechanisms by which the maternal microbiome regulates offspring growth and food intake.

Background Zebrafish used in research settings are often housed in recirculating aquaculture systems (RAS) which rely on the system microbiome, typically enriched in a biofiltration substrate, to remove the harmful ammonia generated by fish via oxidation. Commercial RAS must be allowed to equilibrate following installation, before fish can be introduced. There is little information available regarding the bacterial community structure in commercial zebrafish housing systems, or the time-point at which the system or biofilter reaches a microbiological equilibrium in RAS in general. Methods A zebrafish housing system was monitored at multiple different system sites including tank water in six different tanks, pre- and post-particulate filter water, the fluidized bed biofilter substrate, post-carbon filter water, and water leaving the ultra-violet (UV) disinfection unit and entering the tanks. All of these samples were collected in quadruplicate, from prior to population of the system with zebrafish through 18 weeks post-population, and analyzed using both 16S rRNA amplicon sequencing and culture using multiple agars and annotation of isolates via matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. Sequencing data were analyzed using traditional methods, network analyses of longitudinal data, and integration of culture and sequence data. Results The water microbiome, dominated by Cutibacterium and Staphylococcus spp., reached a relatively stable richness and composition by approximately three to four weeks post-population, but continued to evolve in composition throughout the study duration. The microbiomes of the fluidized bed biofilter and water leaving the UV disinfection unit were distinct from water at all other sites. Core taxa detected using molecular methods comprised 36 amplicon sequence variants, 15 of which represented Proteobacteria including multiple members of the families Burkholderiaceae and Sphingomonadaceae. Culture-based screening yielded 36 distinct isolates, and showed moderate agreement with sequencing data. Conclusions The microbiome of commercial RAS used for research zebrafish reaches a relatively stable state by four weeks post-population and would be expected to be suitable for experimental use following that time-point.

The mouse is the most commonly used model species in biomedical research. Just as human physical and mental health are influenced by the commensal gut bacteria, mouse models of disease are influenced by the fecal microbiome (FM). The source of mice represents one of the strongest influences on the FM and can influence the phenotype of disease models. The FM influences behavior in mice leading to the hypothesis that mice of the same genetic background from different vendors, will have different behavioral phenotypes. To test this hypothesis, colonies of CD-1 mice, rederived via embryo transfer into surrogate dams from four different suppliers, were subjected to phenotyping assays assessing behavior and physiological parameters. Significant differences in behavior, growth rate, metabolism, and hematological parameters were observed. Collectively, these findings show the profound influence of supplier-origin FMs on host behavior and physiology in healthy, genetically similar, wild-type mice maintained in identical environments., Ericsson et al. show that different vendors (suppliers of mouse strains) harbor distinct microbiomes, which drive distinct behavioral phenotypes when the genetics are fixed. They specifically focus on changes relating to exploratory and anxiety-related behavior, physiological phenotypic parameters, glucose metabolism, and blood leukocytes. They conclude by emphasizing that supplier-origin fecal microbiomes represent potential sources of poor experimental reproducibility and suggest means to optimize experimentation with mice and their microbiomes.

Background Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database. Results Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009). Conclusions Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.

Visual Abstract, Highlights • Little is understood regarding the connection between congenital heart disease, the intestinal Microbiome, and how this could influence systemic inflammation. • To date, no study has evaluated the microbiome in congenital heart disease and the effects of cardiopulmonary bypass. • There is significant dysbiosis in patients with congenital heart disease and this is exacerbated following cardiopulmonary bypass. • Cardiopulmonary bypass induces intestinal barrier dysfunction which was not seen in control surgeries. • This data improves our understanding of intestinal dysbiosis, intestinal barrier dysfunction, and intestinal inflammatory eicosanoids and their interactions with cardiopulmonary bypass., Summary There are no data evaluating the microbiome in congenital heart disease following cardiopulmonary bypass. The authors evaluated patients with congenital heart disease undergoing cardiopulmonary bypass and noncardiac patients undergoing surgery without bypass. Patients with congenital heart disease had differences in baseline microbiome compared with control subjects, and this was exacerbated following surgery with bypass. Markers of barrier dysfunction were similar for both groups at baseline, and surgery with bypass induced significant intestinal barrier dysfunction compared with control subjects. This study offers novel evidence of alterations of the microbiome in congenital heart disease and exacerbation along with intestinal barrier dysfunction following cardiopulmonary bypass.

Accumulating studies show that the host microbiome influences the development or progression of many diseases. The eukaryotic virome, as a key component of the microbiome, plays an important role in host health and disease in humans and animals, including research animals designed to model human disease. To date, the majority of research on the microbiome has focused on bacterial populations, while less attention has been paid to the viral component. Members of the eukaryotic virome interact with the commensal bacterial microbiome through trans-kingdom interactions, and influence host immunity and disease phenotypes as a collective microbial ecosystem. As such, differences in the virome may affect the reproducibility of animal models, and supplementation of the virome may enhance the translatability of animal models of human disease. However, there are minimal empirical data regarding differences in the virome of mice from different commercial sources. Our hypotheses were that the mice obtained from pet store sources and lab mice differ in their eukaryotic virome, and that lab mice from different sources would also have different viromes. To test this hypothesis, the ViroCap platform was used to characterize the eukaryotic virome in multiple tissues of mice from different sources including three sources of laboratory mice and two pet stores. As expected, pet store mice harbored a much greater diversity within the virome compared to lab mice. This included an ostensibly novel norovirus strain identified in one source of these mice. Viruses found in both laboratory and pet store populations included four strains of endogenous retroviruses and murine astrovirus with the latter being restricted to one source of lab mice. Considering the relatively high richness virome within different samples from healthy humans, these data suggest that mouse models from alternative sources may be more translational to the human condition. Moreover, these data demonstrate that, by characterizing the eukaryotic murine virome from different sources, novel viruses may be identified for use as field strains in biomedical research.

Simple Summary Horses are susceptible to a condition known as Equine Metabolic Syndrome (EMS), which is somewhat similar to metabolic syndrome and type II diabetes in people, and is characterized by elevated insulin levels and increased susceptibility to other adverse health outcomes. Common treatments, including change to an all-hay diet and a drug called metformin, may provide benefits through modulation of intestinal bacteria, collectively known as the gut microbiota. In the studies reported here, horses undergoing such a diet change, regardless of the presence of metformin, showed a significant expansion in their fecal microbiota of a specific, poorly characterized group of bacteria. Notably, this phylum is distantly related to the bacteria found to expand in the fecal microbiota of mice and humans following metformin administration, suggesting removal from pasture to reduce the caloric intake may exert effects on the equine microbiota similar to those seen in other host species treated with metformin. Abstract Common treatments for Equine Metabolic Syndrome (EMS) and associated conditions include removal from pasture and adoption of an all-hay diet. Pharmacological treatments for EMS include metformin, a biguanide antihyperglycemic agent also administered to people to help improve glucose tolerance and insulin sensitivity. Both treatments may work, at least partially, through the gut microbiota, yet little is known regarding these effects in the equine host. To determine the influence on the fecal microbiota of this diet change and administration of metformin, six healthy horses were removed from pasture and switched to an all-hay diet, with four of those horses also receiving oral metformin for seven days. Control horses (n = 24) remaining on pasture and receiving no metformin were sampled at the beginning and end of one week. All samples were subjected to 16S rRNA sequencing, and horses undergoing the diet change were subjected to an oral sugar test twice, one week apart. Characteristic changes in the microbiota following diet change included the significant expansion of the phylum Kiritimatiellaeota. As Kiritimatiellaeota are related to Verrucomicrobia, found to expand in the microbiota of mice and humans in response to metformin, this taxon may represent the cognate microbes in equine hosts.

Tissue-resident macrophages (TRMs) are heterogeneous populations originating either from monocytes or embryonic progenitors, and distribute in lymphoid and non-lymphoid tissues. TRMs play diverse roles in many physiological processes, including metabolic function, clearance of cellular debris, and tissue remodeling and defense. Macrophages can be polarized to different functional phenotypes depending on their origin and tissue microenvironment. Specific macrophage subpopulations are associated with disease progression. In studies of fate-mapping and single-cell RNA sequencing methodologies, several critical molecules have been identified to induce the change of macrophage function. These molecules are potential markers for diagnosis and selective targets for novel macrophage-mediated treatment. In this review, we discuss some of the recent findings regarding less-known molecules and new functions of well-known molecules. Understanding the mechanisms of these molecules in macrophages has the potential to yield new macrophage-mediated treatments or diagnostic approaches to disease.

Background Gut microbiota (GM) contribute to obesity and insulin resistance (IR). Obstructive sleep apnea (OSA), characterized by intermittent hypoxia (IH), promotes IR and alters GM. Since circulating exosomes are implicated in IR, we examined the effects of IH and physical activity (PA) in mice on GM, colonic epithelium permeability, systemic IR, and plasma exosome cargo, and exosome effects on visceral white adipose tissues (vWAT) IR. Methods C57BL/6 mice were exposed to IH or room air (RA) for 6 weeks with and without PA (n = 12/group), and GM and systemic IR changes were assessed, as well as the effects of plasma exosomes on naive adipocyte insulin sensitivity. Fecal microbiota transfers (FMT) were performed in naive mice (n = 5/group), followed by fecal 16S rRNA sequencing, and systemic IR and exosome-induced effects on adipocyte insulin sensitivity were evaluated. Findings Principal coordinate analysis (PCoA) ordinates revealed B-diversity among IH and FMT recipients that accounted for 64% principal component 1 (PC1) and 12.5% (PC2) of total variance. Dominant microbiota families and genera in IH-exposed and FMT-treated were preserved, and IH-exposed GM and IH-FMT induced increased gut permeability. Plasma exosomes from IH-exposed and IH-FMT mice decreased pAKT/AKT responses to exogenous insulin in adipocytes vs. IH+PA or RA FMT-treated mice (p = 0.001). Interpretation IH exposures mimicking OSA induce changes in GM, increase gut permeability, and alter plasma exosome cargo, the latter inducing adipocyte dysfunction (increased IR). Furthermore, these alterations improved with PA. Thus, IH leads to perturbations of a singular GM-circulating exosome pathway that disrupts adipocyte homeostasis resulting in metabolic dysfunction, as reflected by IR. Funding This study was supported by grants from the National Institutes of Health grants HL130984 and HL140548 and University of Missouri Tier 2 grant. The study has not received any funding or grants from pharmaceutical or other industrial corporations.

Biomedical research relies on the use of animal models, and the animals used in those models receive medical care, including antibiotics for brief periods of time to treat conditions such as dermatitis, fight wounds, and suspected bacterial pathogens of unknown etiology. As many mouse model phenotypes are sensitive to changes in the gut microbiota, our goal was to examine the effect of antibiotics commonly administered to mice. Therefore, four treatment groups (subcutaneous enrofloxacin for 7 days, oral enrofloxacin for 14 days, oral trimethoprim-sulfamethoxazole for 14 days, and topical triple antibiotic ointment for 14 days) alongside a fifth control group receiving no treatment (n = 12/group) were included in our study. Fecal samples were collected prior to treatment, immediately after two weeks of exposure, and four weeks after cessation of treatment, and subjected to 16S rRNA library sequencing. The entire experimental design was replicated in mice from two different suppliers. As expected, several treatments including enrofloxacin and triple antibiotic ointment substantially decreased the amount of DNA recovered from fecal material, as well as the microbial richness. Notably, many of these effects were long-lasting with diminished gut microbiota (GM) richness four weeks following exposure, in both substrains of mice. Trimethoprim-sulfamethoxazole induced minimal to no discernible changes in the taxonomic composition beyond that seen in control mice. Collectively, these data highlight the need to consider the impact on GM of brief and seemingly routine use of antibiotics in the clinical care of research animals.

The microbial communities colonizing the gastrointestinal tract of vertebrate hosts exist in symbiosis with their host, providing a wide array of functions that confer benefit to the collective superorganism. Intuitively, disruptions in microbial community structure can lead to deleterious host effects, and multiple associations have been made between gut inflammatory conditions and dysbiosis. Considering the myriad mechanisms of bi-directional communication between the gut microbiota and host autonomic nervous system, it is not surprising that the gut microbiota is now implicated as a factor in several behavioral, cognitive, and neuro-psychological conditions affecting people. While gut microbiomes have co-evolved with (and are very specific for) their cognate hosts, the functions provided by the communities are highly conserved, suggesting that the same microbiome-induced effects observed in people may also affect companion animals like horses. This presentation will show data generated in a range of host species including zebrafish, rodents, and horses, demonstrating the influence of the gut-brain axis on behavior, and physical and mental health. The objective is to provide listeners a new appreciation for the physiological, and perhaps clinical, influence of the gut microbiota, and implications for clinical practices such as antibiotic usage and feeding changes.

Antibiotic-resistant microorganisms are drawing a lot of attention due to their severe and irreversible consequences on human health. The animal industry is considered responsible in part because of the enormous volume of antibiotics used annually. In the current research, veterinary antibiotic (VA) degradation, finding the threshold of removal and recognizing the joint effects of chlortetracycline (CTC) and Tylosin combination on the digestion process were studied. Laboratory scale anaerobic digesters were utilized to investigate potential mitigation of VA in swine manure. The digesters had a working volume of 1.38 L (in 1.89-L glass jar), with a hydraulic retention time (HRT) of 21 days and a loading rate of 1.0 g-VS L&minus, 1 d&minus, 1. Digesters were kept at 39 &plusmn, 2 &deg, C in incubators and loaded every two days, produced biogas every 4 days and digester pH were measured weekly. The anaerobic digestion (AD) process was allowed 1.5 to 2 HRT to stabilize before adding the VAs. Tests were conducted to compare the effects of VAs onto manure nutrients, volatile solid removal, VA degradation, and biogas production. Concentrations of VA added to the manure samples were 263 to 298 mg/L of CTC, and 88 to 263 mg/L of Tylosin, respectively. Analysis of VA concentrations before and after the AD process was conducted to determine the VA degradation. Additional tests were also conducted to confirm the degradation of both VAs dissolved in water under room temperature and digester temperature. Some fluctuations of biogas production and operating variables were observed because of the VA addition. All CTC was found degraded even only after 6 days of storage in water solution, thus, there was no baseline to estimate the effects of AD. As for Tylosin, 100% degradation was observed due to the AD (removal was 100%, compared with 24&ndash, 40% degradation observed in the 12-day water solution storage). Besides, complete Tylosin degradation was also observed in the digestate samples treated with a mixture of the two VAs. Lastly, amplicon sequencing was performed on each group by using the 50 most variable operational taxonomic units (OTUs)s and perfect discriminations were detected between groups. The effect of administration period and dosage of VAs on Phyla Firmicutes Proteobacteria, Synergistetes and Phylum Bacteroides was investigated. These biomarkers' abundance can be employed to predict the sample&rsquo, s treatment group.

Primary liver cancer is one of the leading causes of cancer death worldwide. Surgical and non-surgical treatments are optional for liver cancer therapy based on the cancer stage. Accumulating studies show that the gut–liver axis influences the progression of liver diseases, including liver inflammation, fibrosis, cirrhosis, and cancer. However, the role of gut microbiota and their derived components and metabolites in liver cancer remains to be further clarified. In this review, we discuss the roles of gut microbiota and specific bacterial species in HCC and the strategies to modulate gut microbiota to improve antitumor therapy. Given the limitation of current treatments, gut microbiota-mediated therapy is a potential option for HCC treatment, including fiber diet and vegetable diet, antimicrobials, probiotics, and pharmaceutical inhibitors. Also, gut microbiota can be used as a marker for early diagnosis of HCC. HCC occurs dependent on various environmental and genetic factors, including diet and sex. Furthermore, gut microbiota impacts the immunotherapy of HCC treatment. Therefore, a better understanding of the role of the gut–liver axis in liver cancer is critically important to improve therapeutic efficacy.

Next-generation sequencing (NGS) methods have been used to identify a diverse ocular surface (OS) microbiota in humans. These results have highlighted limitations in microbial detection via traditional culture-based techniques. The OS has mechanisms such as tear film and mechanical blinking, which may aid in preventing adherence and colonization of microbes, suggesting that only low populations of microbes may reside on the OS. Additionally, closely related tissues to the OS are exposed to a similar array of microbes, but demonstrate different defense mechanisms. Information regarding concordance of microbial communities of the OS and nearby tissues is lacking. Our study purposes were to (1) characterize the conjunctival microbiota of healthy dogs, (2) compare the conjunctival microbiota to the periocular haired skin and distal nose, and (3) compare the bacteria identified by culture to NGS of the healthy canine conjunctiva. Here, NGS was used to evaluate samples from 25 healthy adult dogs of the conjunctiva, periocular haired skin, and distal nose. Additional samples were collected from each dog for traditional conjunctival culture. The 16S rRNA gene amplicon libraries were evaluated for coverage, relative abundance, richness, and diversity. Site-dependent similarities evaluated using principal coordinate analysis (PCoA) and PERMANOVA demonstrated relatedness in community compositions between sites. The conjunctiva of healthy dogs yielded a rich and diverse microbiota based on NGS. While some regional continuity was noted, microbial communities of the conjunctiva, periocular haired skin, and nose were significantly different from each other. Comparatively, traditional culture markedly underestimated the number of bacterial taxa present on the healthy canine OS. Findings suggest similarities in nasal and conjunctival microbial communities, which may be a result of similarities in mucosal immunity and anatomic connection via the nasolacrimal system. Further investigation using NGS into changes of the composition of bacterial communities in disease is warranted.

Countless studies have identified differences between the gut microbiota of humans affected with myriad conditions and healthy individuals, and animal models are commonly used to determine whether those differences are causative or correlative. Recently, concerns have arisen regarding the reproducibility of animal models between institutions and across time. To determine the influence of three common husbandry-associated factors that vary between institutions, groups of weanling mice were placed in either static or ventilated microisolator caging, with either aspen or paperchip bedding, and with one of three commonly used rodent chows, in a fully crossed study design. After thirteen weeks, samples were collected from multiple regions of the gastrointestinal tract and characterized using culture-independent sequencing methods. Results demonstrated that seemingly benign husbandry factors can interact to induce profound changes in the composition of the microbiota present in certain regions of the gut, most notably the cecum, and that those changes are muted during colonic transit. These findings indicate that differences in factors such as caging and bedding can interact to modulate the gut microbiota that in turn may affect reproducibility of some animal models, and that cecal samples might be optimal when screening environmental effects on the gut microbiota.

The lungs of people and companion animals are now recognized to harbor diverse, low biomass bacterial communities. While these communities are difficult to characterize using culture-based approaches, targeted molecular methods such as 16S rRNA amplicon sequencing can do so using DNA extracted from samples such as bronchoalveolar lavage fluid (BALF). Previous studies identified a surprisingly uniform composition of the microbiota in the lungs of healthy research dogs living in a controlled environment, however there are no reports of the lung microbiota of client-owned dogs. Moreover, compositional changes in the lung microbiota depending on disease status have been reported in people, suggesting that similar events may occur in dogs, a species subject to several respiratory disease mechanisms analogous to those seen in people. To address these knowledge gaps, BALF samples from client-owned dogs presenting to the University of Missouri Veterinary Health Center for respiratory signs between 2014 and 2017 were processed for and subjected to 16S rRNA sequencing. Based on specific diagnostic criteria, dogs were categorized as Chronic Bronchitis (CB, n = 53) or non-CB (n = 11). Community structure was compared between groups, as well as to historical data from healthy research dogs (n = 16) of a uniform breed and environment. The lung microbiota detected in all client-owned dogs was markedly different in composition from that previously detected in research dogs and contained increased relative abundance of multiple canine fecal and environmental bacteria, likely due to aspiration associated with their clinical signs. While inter-sample diversity differed significantly between samples from CB and non-CB dogs, the variability within both groups made it difficult to discern reproducible bacterial classifiers of disease. During subsequent analyses to identify other sources of variability within the data however, population-wide temporal dynamics in community structure were observed, with substantial changes occurring in late 2015 and again in early 2017. A review of regional climate data indicated that the first change occurred during a historically warm and wet period, suggesting that changes in environmental conditions may be associated with changes in the respiratory microbiota in the context of respiratory disease. As the lung microbiota in humans and other animals is believed to result from repetitive micro-aspirations during health and in certain disease states associated with dyspnea and laryngeal dysfunction, these data support the increased colonization of the lower airways during compromised airway function, and the potential for temporal effects due to putative factors such as climate.

Vasoactive intestinal peptide (VIP) is crucial for gastrointestinal tract (GIT) health. VIP sustains GIT homeostasis through maintenance of the intestinal epithelial barrier and acts as a potent anti-inflammatory mediator that contributes to gut bacterial tolerance. Based on these biological functions by VIP, we hypothesized that its deficiency would alter gut microbial ecology. To this end, fecal samples from male and female VIP+/+, VIP+/–, and VIP–/– littermates (n = 47) were collected and 16S rRNA sequencing was conducted. Our data revealed significant changes in bacterial composition, biodiversity, and weight loss from VIP–/– mice compared to VIP+/+ and VIP+/– littermates, irrespective of sex. The gut bacteria compositional changes observed in VIP–/– mice was consistent with gut microbial structure changes reported for certain inflammatory and autoimmune disorders. Moreover, predicted functional changes by PICRUSt software suggested an energy surplus within the altered microbiota from VIP–/– mice. These data support that VIP plays an important role in maintaining microbiota balance, biodiversity, and GIT function, and its genetic removal results in significant gut microbiota restructuring and weight loss.

It is unknown how the respiratory microbiome influences and is influenced by bacterial pneumonia in dogs, as culture of lung samples and not microbial sequencing guides clinical practice. While accurate identification of pathogens are essential for treatment, not all bacteria are cultivable and the impact of respiratory dysbiosis on development of pneumonia is unclear. The study purposes were to (1) characterize the lung microbiome in canine bacterial pneumonia and compare deviations in dominant microbial populations with historical healthy controls, (2) compare bacteria identified by culture vs. 16S rDNA sequencing from bronchoalveolar lavage fluid (BALF) culture-, and (3) evaluate similarities in lung and oropharyngeal (OP) microbial communities in community-acquired and secondary bacterial pneumonia. Twenty BALF samples from 15 client-owned dogs diagnosed with bacterial pneumonia were enrolled. From a subset of dogs, OP swabs were collected. Extracted DNA underwent PCR of the 16S rRNA gene. Relative abundance of operational taxonomic units (OTUs) were determined. The relative abundance of bacterial community members found in health was decreased in dogs with pneumonia. Taxa identified via culture were not always the dominant phylotype identified with sequencing. Dogs with community-acquired pneumonia were more likely to have overgrowth of a single organism suggesting loss of dominant species associated with health. Dogs with secondary bacterial pneumonia had a greater regional continuity between the upper and lower airways. Collectively, these data suggest that dysbiosis occurs in canine bacterial pneumonia, and culture-independent techniques may provide greater depth of understanding of the changes in bacterial community composition that occur in disease.

The physicochemical properties of antimicrobial peptides (AMPs) including size, net charge, amphipathic structure, hydrophobicity, and mode-of-action together determine their broad-spectrum activities against bacteria, fungi, protozoa, and viruses. Recent studies show that some AMPs have both antimicrobial and anticancer activities, suggesting a new strategy for cancer therapy. Hepatocellular carcinoma (HCC), the primary liver cancer, is a leading cause of cancer mortality worldwide, and lacks effective treatment. Anticancer peptides (ACPs) derived from AMPs or natural resources could be applied to combat HCC directly or as a synergistic treatment. However, the number of known ACPs is low compared to the number of antibacterial and antifungal peptides, and very few of them can be applied clinically for HCC treatment. In this review, we first summarize recent studies related to ACPs for HCC, followed by a description of potential modes-of-action including direct killing, anti-inflammation, immune modulation, and enhanced wound healing. We then describe the structures of AMPs and methods to design and modify these peptides to improve their anticancer efficacy. Finally, we explore the potential application of ACPs as vaccines or nanoparticles for HCC treatment. Overall, ACPs display several attractive properties as therapeutic agents, including broad-spectrum anticancer activity, ease-of-design and modification, and low production costs. As this is an emerging and novel area of cancer therapy, additional studies are needed to identify existing candidate AMPs with ACP activity, and assess their anticancer activity and specificity, and immunomodulatory effects, using in vitro, in silico, and in vivo approaches.

Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a serious pest of maize (Zea mays L.) in North America and parts of Europe. With most of its life cycle spent in the soil feeding on maize root tissues, this insect is likely to encounter and interact with a wide range of soil and rhizosphere microbes. Our knowledge of the role of microbes in pest management and plant health remains woefully incomplete, yet that knowledge could play an important role in effective pest management strategies. For this study, insects were reared on maize in soils from different locations. Insects from two different laboratory colonies (a diapausing and a non-diapausing colony) were sampled at each life stage to determine the possible core bacteriome. Additionally, soil was sampled at each life stage and resulting bacteria were identified to determine the possible contribution of soil to the rootworm bacteriome, if any. We analyzed the V4 hypervariable region of bacterial 16S rRNA genes with Illumina MiSeq to survey the different species of bacteria associated with the insects and the soils. The bacterial community associated with insects was significantly different from that in the soil. Some differences appear to exist between insects from non-diapausing and diapausing colonies while no significant differences in community composition existed between the insects reared on different soils. Despite differences in the bacteria present in immature stages and in male and female adults, there is a possible core bacteriome of approximately 16 operational taxonomic units (i.e., present across all life stages). This research may provide insights into Bt resistance development, improved nutrition in artificial rearing systems, and new management strategies.

Murine norovirus (MNV) is an RNA virus that can prove lethal in mice with impaired innate immunity. We found that MNV-4 infection of Stat1(−/−) mice was not lethal, but produced a 100% penetrant, previously undescribed lymphatic phenotype characterized by chronic-active lymphangitis with hepatitis, splenitis, and chronic cecal and colonic inflammation. Lesion pathogenesis progressed from early ileal enteritis and regional dilated lymphatics to lymphangitis, granulomatous changes in the liver and spleen, and, ultimately, typhlocolitis. Lesion development was neither affected by antibiotics nor reproduced by infection with another enteric RNA virus, rotavirus. MNV-4 infection in Stat1(−/−) mice decreased expression of vascular endothelial growth factor (Vegf) receptor 3, Vegf-c, and Vegf-d and increased interferon (Ifn)-γ, tumor necrosis factor-α, and inducible nitric oxide synthase. However, anti–IFN-γ and anti–tumor necrosis factor-α antibody treatment did not attenuate the histologic lesions. Studies in Ifnαβγr(−/−) mice suggested that canonical signaling via interferon receptors did not cause MNV-4–induced disease. Infected Stat1(−/−) mice had increased STAT3 phosphorylation and expressed many STAT3-regulated genes, consistent with our findings of increased myeloid cell subsets and serum granulocyte colony-stimulating factor, which are also associated with increased STAT3 activity. In conclusion, in Stat1(−/−) mice, MNV-4 induces lymphatic lesions similar to those seen in Crohn disease as well as hepatitis, splenitis, and typhlocolitis. MNV-4–infected Stat1(−/−) mice may be a useful model to study mechanistic associations between viral infections, lymphatic dysfunction, and intestinal inflammation in a genetically susceptible host.

Using animal models, the gut microbiota has been shown to play a critical role in the health and disease of many organ systems. Unfortunately, animal model studies often lack reproducibility when performed at different institutions. Previous studies in our laboratory have shown that the gut microbiota of mice can vary with a number of husbandry factors leading us to speculate that differing environments may alter gut microbiota, which in turn may influence animal model phenotypes. As an extension of these studies, we hypothesized that the shipping of mice from a mouse producer to an institution will result in changes in the type, relative abundance, and functional composition of the gut microbiota. Furthermore, we hypothesized that mice will develop a microbiota unique to the institution and facility in which they are housed. To test these hypotheses, mice of two strains (C57BL/6J and BALB/cJ), two age groups (4 week and 8 week old), and originating from two types of housing (research animal facility under conventional housing and production facilities under maximum barrier housing) were obtained from The Jackson Laboratory. Fecal samples were collected the day prior to shipping, immediately upon arrival, and then on days 2, 5, 7, and weeks 2, 4, and 9 post-arrival. Following the first post-arrival fecal collection, mice were separated into 2 groups and housed at different facilities at our institution while keeping their caging, diet, and husbandry practices the same. DNA was extracted from the collected fecal pellets and 16S rRNA amplicons were sequenced in order to characterize the type and relative abundance of gut bacteria. Principal component analysis (PCA) and permutational multivariate analysis of variance (PERMANOVA) demonstrated that both the shipping and the institution and facility in which mice were housed altered the gut microbiota. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) predicted differences in functional composition in the gut microbiota of mice based on time of acclimation.

Feline immunodeficiency virus (FIV) is a naturally-occurring retrovirus that infects domestic and non-domestic feline species, producing progressive immune depletion that results in an acquired immunodeficiency syndrome (AIDS). Much has been learned about FIV since it was first described in 1987, particularly in regard to its application as a model to study the closely related lentivirus, human immunodeficiency virus (HIV). In particular, FIV and HIV share remarkable structure and sequence organization, utilize parallel modes of receptor-mediated entry, and result in a similar spectrum of immunodeficiency-related diseases due to analogous modes of immune dysfunction. This review summarizes current knowledge of FIV infection kinetics and mechanisms of immune dysfunction in relation to opportunistic disease, specifically in regard to studying HIV pathogenesis. Furthermore, we present data which highlight changes in the oral microbiota and oral immune system during FIV infection, and outline the potential for the feline model of oral AIDS manifestations to elucidate pathogenic mechanisms of HIV-induced oral disease. Finally, we discuss advances in molecular biology, vaccine development, neurologic dysfunction, and the ability to apply pharmacologic interventions and sophisticated imaging technologies to study experimental and naturally occurring FIV, which provide an excellent, but often overlooked resource for advancing therapies and management of HIV/AIDS.

It is estimated that 1.4 million people in the United States suffer from Inflammatory Bowel Disease (IBD), with an overall annual health care cost of more than $1.7 billion. Although the exact etiology of this disease remains unknown, research suggests that it is a multifactorial disease associated with aberrant gastrointestinal microbial populations (dysbiosis). The C57BL/6 and C3H/HeJBir mouse strains with targeted mutations in the IL-10 gene are commonly used models to study IBD. However, anecdotally, disease phenotype can vary in severity from lab to lab. Moreover, studies using germfree and monocolonized mice have suggested that gut microbiota (GM) are critical to disease induction in these models. With recent studies suggesting variation in naturally occurring GM composition and complexity among mouse producers, we hypothesized that differences in these naturally occurring complex GM profiles may modulate disease severity in the IL-10-/- mouse model. To test this hypothesis, we use a technique referred to as complex microbiota targeted rederivation (CMTR) to transfer genetically identical C57BL/6 IL-10-/- and C3H/HeJBir IL-10-/- embryos into surrogate CD-1 or C57BL/6 dams from different commercial producers with varying microbiota complexity and composition. We found that disease severity significantly and reproducibly differed among mice in both IL-10-/- strains, dependent on differing maternally inherited GM. Furthermore, disease severity was associated with alterations in relative abundance of several physiologically relevant bacterial species. These findings suggest that the composition of the resident GM is a primary determinant of disease severity in IBD and provide proof-of-concept that CMTR can be used to investigate the contribution of contemporary complex GM on disease phenotype and reproducibility.

Several associations have been made between characteristics of the resident gut microbiota and human health and disease susceptibility. Animal models provide the means to test these correlations prospectively and evaluate causality. Experimental fecal microbiota transfer, or the intentional transplantation of gut microbes into recipient mice depleted of their autochthonous microbes with antibiotics, is a commonly used method of testing these relationships. The true completeness of microbial transfer through such procedures is poorly documented in the literature, particularly in the context of reciprocal transfer of microbes between recipient and donor mice harboring microbial populations of differing richness and diversity. Moreover, it is unclear whether the use of frozen fecal contents or cecal contents would confer any difference in the outcomes of transfer. Herein, groups of mice colonized with distinct gut microbiota of differing richness and composition were used in a reciprocal fecal microbiota transfer study, with different groups receiving transfer of material prepared from fresh cecal contents, fresh feces, or frozen feces. Targeted 16S rRNA gene amplicon sequencing was used at intervals throughout the study to characterize the microbiota. Notably, despite comparable depletion of the microbiota in recipient mice prior to transfer, donor-specific taxa reliably colonized recipients only when relatively rich donor material was transferred to mice originally colonized with a simpler microbiota. It is unclear whether these differences were due to differences in the endogenous recipient microbiota or host factors induced in early life by microbial factors. These findings are of practical import for researchers using fecal microbiota transfer to prospectively assess the influence of the gut microbiota in mouse models, and to those studying host-microbial interactions and their influence on gut barrier function.

The urinary bladder in healthy dogs has dogmatically been considered free of bacteria. This study used culture independent techniques to characterize the healthy canine urinary microbiota. Urine samples collected by antepubic cystocentesis from dogs without urinary infection were used for DNA extraction. Genital tract and rectal samples were collected simultaneously from the same dogs. The V4 hypervariable region of the 16S rRNA bacterial gene was amplified and compared against Greengenes database for OTU assignment and relative abundance for urine, genital, and rectal samples. After excluding 4 dogs with cultivable bacteria, samples from 10 male (M; 1 intact) and 10 female (F) spayed dogs remained. All samples provided adequate genetic material for analysis. Four taxa (Pseudomonas sp., Acinetobacter sp., Sphingobium sp. and Bradyrhizobiaceae) dominated the urinary microbiota in all dogs of both sexes. These taxa were also detected in the genital swabs of both sexes, while the rectal microbiota differed substantially from the other sample sites. Principal component (PC) analysis of PC1 through PC3 showed overlap of urinary and genital microbiota and a clear separation of rectal swabs from the other sample sites along PC1, which explained 44.94% variation. Surprisingly, the urinary microbiota (mean # OTU 92.6 F, 90.2 M) was significantly richer than the genital (67.8 F, 66.6 M) or rectal microbiota (68.3 F, 71.2 M) (p < 0.0001), with no difference between sexes at any sample site. The canine urinary bladder is not a sterile environment and possesses its own unique and diverse microbiota compared to the rectal and genital microbiota. There was no difference between the sexes at any microbiota sample site (urine, genital, and rectal). The predominant bacterial genus for either sex in the urine and genital tracts was Pseudomonas sp.

The consumption of probiotics has become increasingly popular as a means to try to improve health and well-being. Not only are probiotics considered beneficial to digestive health, but increasing evidence suggests direct and indirect interactions between gut microbiota (GM) and the central nervous system (CNS). Here, adult zebrafish were supplemented with Lactobacillus plantarum to determine the effects of probiotic treatment on structural and functional changes of the GM, as well as host neurological and behavioral changes. L. plantarum administration altered the β-diversity of the GM while leaving the major core architecture intact. These minor structural changes were accompanied by significant enrichment of several predicted metabolic pathways. In addition to GM modifications, L. plantarum treatment also significantly reduced anxiety-related behavior and altered GABAergic and serotonergic signaling in the brain. Lastly, L. plantarum supplementation provided protection against stress-induced dysbiosis of the GM. These results underscore the influence commensal microbes have on physiological function in the host and demonstrate bidirectional communication between the GM and the host.

Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a “failure to thrive” phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.

Background: Well over a quarter of human breast cancers are ErbB2-driven and constitute a distinct subtype with substantially poorer prognosis. Yet, there are substantial gaps in our understanding of how ErbB2 tyrosine kinase activity unleashes a coordinated program of cellular and extracellular alterations that culminate in aggressive breast cancers. Cellular models that exhibit ErbB2 kinase dependency and can induce metastatic breast cancer in immune competent hosts are likely to help bridge this gap. Materials and Methods: Here, we derived and characterized a cell line model obtained from a transgenic ErbB2/Neu-driven mouse mammary adenocarcinoma. Results: The MPPS1 cell line produces metastatic breast cancers when implanted in the mammary fat pads of immune-compromised as well as syngeneic immune-competent hosts. MPPS1 cells maintain high ErbB2 overexpression when propagated in DFCI-1 or related media, and their growth is ErbB2-dependent, as demonstrated by concentration-dependent inhibition of proliferation with the ErbB kinase inhibitor Lapatinib. When grown in 3-dimensional (3-D) culture on Matrigel, MPPS1 cells predominantly form large irregular cystic and solid structures. Remarkably, low concentrations of Lapatinib led to a switch to regular acinar growth on Matrigel. Immunofluorescence staining of control vs. Lapatinib-treated acini for markers of epithelial polarity revealed that inhibition of ErbB2 signaling led to rapid resumption of normal mammary epithelium-like cell polarity. Conclusions: The strict dependence of the MPPS1 cell system on ErbB2 signals for proliferation and alterations in cell polarity should allow its use to dissect ErbB2 kinase-dependent signaling pathways that promote loss of cell polarity, a key component of the epithelial mesenchymal transition and aggressiveness of ErbB2-driven breast cancers.

Helicobacter bilis-infected Smad3-/- mice represent an attractive model of inflammation-associated colon cancer. Most infected mice develop mucinous adenocarcinoma (MUC) by 6 weeks post inoculation (PI); however, approximately one third do not progress to MUC. The ability to predict the development of MUC in mice used in therapeutic studies would confer a considerable saving of time and money. In addition, the inadvertent use of mice without MUC may confound therapeutic studies by making treatments seem falsely efficacious. We assessed both magnetic resonance imaging (MRI) and fecal biomarkers in Helicobacter- and sham-inoculated mice as methods of noninvasively detecting MUC before the predicted onset of disease. Non-contrast-enhanced MRI was able to detect lesions in 58% of mice with histologically confirmed MUC; however, serial imaging sessions produced inconsistent results. MRI was also a labor- and time-intensive technique requiring anesthesia. Alternatively, inflammatory biomarkers isolated from feces at early time points were correlated to later histologic lesions. Fecal expression of interleukin 1β, macrophage inflammatory protein 1α, and regulated on activation, normal T-cell expressed, and secreted at 3 weeks PI correlated significantly with lesion severity at 9 weeks PI. For each biomarker, receiver-operator characteristic curves were also generated, and all three biomarkers performed well at 1 to 3 weeks PI, indicating that the development of MUC can be predicted based on the early expression of certain inflammatory mediators in feces.