Alterations in common marmoset gut microbiome associated with duodenal strictures

Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets (Callithrix jacchus). Despite standardized housing, diet and husbandry, a recently described gastrointestinal syndrome characterized by duodenal ulcers and strictures was observed in a subset of marmosets sourced from the New England Primate Research Center. As changes in the gut microbiome have been associated with GI diseases, the gut microbiome of 52 healthy, non-stricture marmosets (153 samples) were compared to the gut microbiome of 21 captive marmosets diagnosed with a duodenal ulcer/stricture (57 samples). No significant changes were observed using alpha diversity metrics, and while the community structure was significantly different when comparing beta diversity between healthy and stricture cases, the results were inconclusive due to differences observed in the dispersion of both datasets. Differences in the abundance of individual taxa using ANCOM, as stricture-associated dysbiosis was characterized by Anaerobiospirillum loss and Clostridium perfringens increases. To identify microbial and serum biomarkers that could help classify stricture cases, we developed models using machine learning algorithms (random forest, classification and regression trees, support vector machines and k-nearest neighbors) to classify microbiome, serum chemistry or complete blood count (CBC) data. Random forest (RF) models were the most accurate models and correctly classified strictures using either 9 ASVs (amplicon sequence variants), 4 serum chemistry tests or 6 CBC tests. Based on the RF model and ANCOM results, C. perfringens was identified as a potential causative agent associated with the development of strictures. Clostridium perfringens was also isolated by microbiological culture in 4 of 9 duodenum samples from marmosets with histologically confirmed strictures. Due to the enrichment of C. perfringens in situ, we analyzed frozen duodenal tissues using both 16S microbiome profiling and RNAseq. Microbiome analysis of the duodenal tissues of 29 marmosets from the MIT colony confirmed an increased abundance of Clostridium in stricture cases. Comparison of the duodenal gene expression from stricture and non-stricture marmosets found enrichment of genes associated with intestinal absorption, and lipid metabolism, localization, and transport in stricture cases. Using machine learning, we identified increased abundance of C. perfringens, as a potential causative agent of GI disease and intestinal strictures in marmosets.National Institutes of Health/[T32 OD010978]/NIH/Estados UnidosNational Institutes of Health/[P30-ES002109]/NIH/Estados UnidosUniversidad de Costa Rica/[803-C1-163]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Evaluating rectal swab collection method for gut microbiome analysis in the common marmoset (Callithrix jacchus)

The common marmoset (Callithrix jacchus) is increasingly used as an animal model for biomedical research; however, gastrointestinal diseases causing significant morbidity are endemic in many captive marmoset colonies. Establishing gut microbiome patterns in a marmoset colony may aid in clinical decision-making and model reproducibility. A standardized method of sample collection and storage is essential for proper interpretation of microbiome data. While microbiome studies commonly utilize fecal samples, the goal of this study was to determine whether the microbiome profile from a rectal swab performed on a sedated animal was comparable to the microbiome profile from a fecal sample. During routine physical exams, paired fecal and rectal swab samples were collected from each of 23 marmosets. DNA was extracted from all fecal and rectal swab samples and 16S ribosomal RNA gene sequences were amplified and analyzed. Initial comparison of the relative abundance of bacterial phyla between paired samples had a r2 value of 0.70 with S of 0.08 with no significant differences in α and β diversity metrics between fecal and rectal samples. Initial analysis however, revealed 5 discordant fecal-rectal pairs which corresponded only with the 5 rectal swabs that were classified as free of visible fecal matter during collection. Exclusion of these 5 pairs resulted in an optimized fit of the data as evidenced by a r2 value of 0.91 with S of 0.05. These results demonstrate that rectal swabs are a reliable method for profiling the fecal microbiome in the marmoset since the bacterial composition from a rectal swab with visible fecal contents correlated well with the bacterial composition from a fecal sample from the same marmoset. This study highlights the importance of standardized sample collection methods and exclusion of inappropriate samples.National Institutes of Health (U.S.) (Grant T32 OD010978)National Institute of Environmental Health Sciences (Award P30-ES002109

Evaluating rectal swab collection method for gut microbiome analysis in the common marmoset (Callithrix jacchus)

The common marmoset (Callithrix jacchus) is increasingly used as an animal model for biomedical research; however, gastrointestinal diseases causing significant morbidity are endemic in many captive marmoset colonies. Establishing gut microbiome patterns in a marmoset colony may aid in clinical decision-making and model reproducibility. A standardized method of sample collection and storage is essential for proper interpretation of microbiome data. While microbiome studies commonly utilize fecal samples, the goal of this study was to determine whether the microbiome profile from a rectal swab performed on a sedated animal was comparable to the microbiome profile from a fecal sample. During routine physical exams, paired fecal and rectal swab samples were collected from each of 23 marmosets. DNA was extracted from all fecal and rectal swab samples and 16S ribosomal RNA gene sequences were amplified and analyzed. Initial comparison of the relative abundance of bacterial phyla between paired samples had a r2 value of 0.70 with S of 0.08 with no significant differences in α and β diversity metrics between fecal and rectal samples. Initial analysis however, revealed 5 discordant fecal-rectal pairs which corresponded only with the 5 rectal swabs that were classified as free of visible fecal matter during collection. Exclusion of these 5 pairs resulted in an optimized fit of the data as evidenced by a r2 value of 0.91 with S of 0.05. These results demonstrate that rectal swabs are a reliable method for profiling the fecal microbiome in the marmoset since the bacterial composition from a rectal swab with visible fecal contents correlated well with the bacterial composition from a fecal sample from the same marmoset. This study highlights the importance of standardized sample collection methods and exclusion of inappropriate samples

Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis

Background: Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10 −/− mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties. Experimental procedures: Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. Results: Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. Conclusions: This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.National Institutes of Health (U.S.) (Grant T32‐OD010978)National Institutes of Health (U.S.) (Grant P30‐ES002109)National Institutes of Health (U.S.) (Grant R01‐OD01141

Lung Lobe Torsion in an Adult Male Common Marmoset (Callithrix jacchus)

A 6-y-old, intact, pair-housed male common marmoset (Callithrix jacchus) presented with acute onset dyspnea and tachypnea immediately after sedation with alfaxalone; a history of gradual weight loss initiated the examination under sedation. Thoracic radiographs revealed significant right-lung consolidation, with a vesicular gas pattern in the right caudodorsal lung field, pleural effusion, and dorsal displacement of the heart. The marmoset was euthanized due to his unstable condition and poor prognosis. At necropsy, the cranial and middle lobes of the right lung were homogenously dark red-brown, enlarged, edematous, and twisted around the longitudinal axis at the hilus. The left lung lobes were pale pink and slightly edematous. In light of the clinical and gross necropsy findings, acute torsion of the right cranial and middle lung lobes was diagnosed. Predisposing conditions for lung lobe torsion include trauma, neoplasia, pulmonary disease, previous thoracic surgery, and diaphragmatic hernia, but none of these applied to this case. Initial therapy for lung lobe torsion is to stabilize the patient and treat for underlying conditions, with prompt surgical resection as the treatment of choice. To our knowledge, this report is the first description of lung lobe torsion in an experimentally unmanipulated New World NHP.National Institutes of Health (U.S.) (Grant T32-OD010978)National Institutes of Health (U.S.) (Grant P30 ES02109

Host-directed combinatorial RNAi improves inhibition of diverse strains of influenza A virus in human respiratory epithelial cells

<div><p>Influenza A virus infections are important causes of morbidity and mortality worldwide, and currently available prevention and treatment methods are suboptimal. In recent years, genome-wide investigations have revealed numerous host factors that are required for influenza to successfully complete its life cycle. However, only a select, small number of influenza strains were evaluated using this platform, and there was considerable variation in the genes identified across different investigations. In an effort to develop a universally efficacious therapeutic strategy with limited potential for the emergence of resistance, this study was performed to investigate the effect of combinatorial RNA interference (RNAi) on inhibiting the replication of diverse influenza A virus subtypes and strains. Candidate genes were selected for targeting based on the results of multiple previous independent genome-wide studies. The effect of single and combinatorial RNAi on the replication of 12 diverse influenza A viruses, including three strains isolated from birds and one strain isolated from seals, was then evaluated in primary normal human bronchial epithelial cells. After excluding overly toxic siRNA, two siRNA combinations were identified that reduced mean viral replication by greater than 79 percent in all mammalian strains, and greater than 68 percent in all avian strains. Host-directed combinatorial RNAi effectively prevents growth of a broad range of influenza virus strains <i>in vitro</i>, and is a potential therapeutic candidate for further development and future <i>in vivo</i> studies.</p></div

Analysis of gut microbiome profiles in common marmosets (Callithrix jacchus) in health and intestinal disease

Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets. To understand the role of the microbiome in GI diseases, we characterized the gut microbiome of 91 healthy marmosets (303 samples) and 59 marmosets diagnosed with inflammatory bowel disease (IBD) (200 samples). Healthy marmosets exhibited “humanized,” Bacteroidetes-dominant microbiomes. After up to 2 years of standardized diet, housing and husbandry, marmoset microbiomes could be classified into four distinct marmoset sources based on Prevotella and Bacteroides levels. Using a random forest (RF) model, marmosets were classified by source with an accuracy of 93% with 100% sensitivity and 95% specificity using abundance data from 4 Prevotellaceae amplicon sequence variants (ASVs), as well as single ASVs from Coprobacter, Parabacteroides, Paraprevotella, Phascolarctobacterium, Oribacterium and Fusobacterium. A single dysbiotic IBD state was not found across all marmoset sources, but IBD was associated with lower alpha diversity and a lower Bacteroides:Prevotella copri ratio within each source. IBD was highest in a Prevotella-dominant cohort, and consistent with Prevotella-linked diseases, pro-inflammatory genes in the jejunum were upregulated. RF analysis of serum biomarkers identified serum calcium, hemoglobin and red blood cell (RBC) counts as potential biomarkers for marmoset IBD. This study characterizes the microbiome of healthy captive common marmosets and demonstrates that source-specific microbiomes can be retained despite standardized diets and husbandry practices. Marmosets with IBD had decreased alpha diversity and a shift in the ratio of Bacteroides:Prevotella copri compared to healthy marmosets.Universidad de Costa Rica/[803-C1-163]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Description of influenza virus strains used.

<p>Description of influenza virus strains used.</p

siRNA knockdown of mRNA in NHBE and the impact on influenza A/WSN/1933 (WSN) replication.

<p>(a) Knockdown efficiency of siRNA in NHBE cells; the components within Combo1 are shown as a representative example. Bars represent mean (+ SEM) knockdown relative to non-targeting siRNA 24 hours post transfection. Knockdown data for additional combinations of targets is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197246#pone.0197246.s001" target="_blank">S1 Table</a>. (b) Inhibition of viral replication when genes were targeted singly, or in combination. For each bar, the targeted genes are shown (X) below. The Allstars negative control (N) and siNP positive control (P) are shown. Results are displayed as normalized to viral growth in cells transfected with non-targeting siRNA. (* p < 0.05, ** p < 0.01). NHBE cells were transfected with siRNA 24 hours after plating. Cells were infected with WSN virus (MOI = 0.2) 24 hours following siRNA transfection and incubated for 48 hours prior to evaluating viral titer by plaque assay.</p