Effects of chronic Helicobacter pylori strain PMSS1 infection on whole brain and gastric iron homeostasis in male INS-GAS mice.

Iron deficiency, the most common micronutrient deficiency in humans, is associated with long-term deficits in cognition and memory if left untreated. Infection with the gastric pathogen Helicobacter pylori has been linked to iron deficiency anemia (IDA). The H. pylori virulence factor cytotoxin-associated gene A (cagA) is proposed to be especially pertinent in iron deficiency. Male INS-GAS/FVB mice were infected with the CagA+ strain pre-murine Sydney strain 1 (PMSS1) for 12-13 or 27-29 weeks to investigate the role of chronic H. pylori infection in iron deficiency and neurological sequelae. Mice at both timepoints demonstrated significantly elevated gastric histopathology scores and inflammatory cytokines compared to sham-dosed controls. However, only mice at 27-29 weeks post infection had changes in hematological parameters, with significantly decreased erythrocyte count, hematocrit, serum hemoglobin, and increased serum total iron binding capacity. Gastric transcription of iron-regulatory genes Hamp and Bmp4 were significantly downregulated at both timepoints. In the brain, iron-dependent myelingergic and synaptic markers were significantly downregulated at 27-29 weeks. These results indicated that long-term infection of the CagA + PMSS1 strain of H. pylori in this study caused anemia, altered gastric iron homeostasis, and neurological changes similar to those reported in other rodent H. pylori CagA- strain infection models.

Factors Affecting Hematologic and Serum Biochemical Parameters in Healthy Common Marmosets (Callithrix jacchus).

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.

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.

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.

Identification of Rodent Husbandry Refinement Opportunities through Benchmarking and Collaboration.

Expanding the use of methods that refine, reduce, and replace (3Rs) the use of animals in research is fundamental for both ethical and scientific reasons. The mission of the 3Rs Translational and Predictive Sciences Leadership Group (3Rs TPS LG) of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) is to promote sharing and integration of science and technology to advance the 3Rs in the discovery and development of new medicines, vaccines, medical devices, and health care products for humans and animals. The 3Rs TPS LG is dedicated to identifying opportunities for member companies to share practices, enhance learning, promote discussions, and advance the 3Rs across the industry. One such opportunity was a benchmarking survey, conducted by the Contract Research Organization (CRO) Outreach Working Group, designed to share practices in rodent husbandry for drug safety research and to identify potential opportunities for refinement. IQ member companies and CROs in Asia, North America, and Europe were surveyed. Areas identified for potential alignment included provision of corncob bedding and wire-grid flooring, management of the nest at cage change, approaches to social housing for male mice, evidence-based enrichment strategies, and evaluating the effects of the timing of studies in relation to the animals' circadian rhythm and light-cycle, with consideration for how such extrinsic factors influence animal welfare and scientific outcomes. This manuscript presents the results of the benchmarking survey, including general trends in mouse and rat husbandry practices in toxicology studies, considerations for social housing, enrichment selection, and potential effects of bedding substrate, emphasizing opportunities for collaboration that can help to identify refinements to rodent husbandry practices.

Pharmacokinetics of Single-Dose Intramuscular and Subcutaneous Injections of Buprenorphine in Common Marmosets (Callithrix jacchus).

Although buprenorphine is the most frequently used opioid analgesic in common marmosets (Callithrix jacchus), there is limited information in the literature supporting current dosing regimens used for this species. The purpose of this study was to determine the pharmacokinetic profiles of single-dose buprenorphine HCl administered intramuscularly (IM) at 0.01 mg/kg in 6 adult marmosets (1.8 to 12.8 y old; 2 males, 4 females) and subcutaneously (SQ) at 0.01 mg/kg in 6 adult marmo- sets (2.3-4.4 y old; 3 males, 3 females) by mass spectrometry. Blood was collected at multiple time points from 0.25 to 24 h from unsedated animals following a hybrid sparse-serial sampling design. The maximal observed plasma concentration of buprenorphine (Cmax ) administered IM (2.57 ± 0.95 ng/mL) was significantly higher than administered SQ (1.47 ± 0.61 ng/mL). However, the time to Cmax (Tmax) was not statistically different between routes (17.4 ± 6 min for IM and 19.8 ± 7.8 min for SQ). The time of the last quantifiable concentration of buprenorphine was 5 ± 1.67 h for IM compared with 6.33 ± 1.51 h for SQ, which was not statistically different. The mean buprenorphine plasma concentration-time curves were used to propose a dosing frequency of 4 to 6 h for buprenorphine at 0.01 mg/kg IM or SQ based on a theoretical therapeutic plasma concentration threshold of 0.1 ng/mL. Based on the mean pharmacokinetic parameters and plasma-concentration time curves, both IM and SQ routes of buprenorphine at this dose provide a rapid increase in the plasma concentration of buprenorphine above the therapeutic threshold, and may be more effective for acute rather than long-lasting analgesia. Further studies are needed to examine repeated dosing regimens and the efficacy of buprenorphine in common marmosets.

Cytotoxic Escherichia coli strains encoding colibactin, cytotoxic necrotizing factor, and cytolethal distending toxin colonize laboratory common marmosets (Callithrix jacchus).

Cyclomodulins are virulence factors that modulate cellular differentiation, apoptosis, and proliferation. These include colibactin (pks), cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt). Pathogenic pks+, cnf+, and cdt+ E. coli strains are associated with inflammatory bowel disease (IBD) and colorectal cancer in humans and animals. Captive marmosets are frequently afflicted with IBD-like disease, and its association with cyclomodulins is unknown. Cyclomodulin-encoding E. coli rectal isolates were characterized using PCR-based assays in healthy and clinically affected marmosets originating from three different captive sources. 139 E. coli isolates were cultured from 122 of 143 marmosets. The pks gene was detected in 56 isolates (40%), cnf in 47 isolates (34%), and cdt in 1 isolate (0.7%). The prevalences of pks+ and cnf+ E. coli isolates were significantly different between the three marmoset colonies. 98% of cyclomodulin-positive E. coli belonged to phylogenetic group B2. Representative isolates demonstrated cyclomodulin cytotoxicity, and serotyping and whole genome sequencing were consistent with pathogenic E. coli strains. However, the presence of pks+, cnf+, or cdt+ E. coli did not correlate with clinical gastrointestinal disease in marmosets. Cyclomodulin-encoding E. coli colonize laboratory common marmosets in a manner dependent on the source, potentially impacting reproducibility in marmoset models.

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 ß 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.

Daily Water Intake by Common Marmosets (Callithrix jacchus) and Recommendations Regarding Fluid Regulation.

The typical daily water intake of common marmosets (Callithrix jacchus) in a research setting has not been well characterized. Because these New World primates are in demand as animal models for neurobehavioral experiments, which can include the potential use of fluid regulation for training, veterinary and research staff need to understand how marmosets keep hydrated under normal circumstances. In the current study, we measured the water consumption of older (age, 5 to 12 y; n = 11) and younger (age, 1 to 2 y; n = 11) marmosets every 3 h during the 12-h light phase in 2 different months (January and July). The overall daily water intake (mean ± 1 SD) was 61.3 ± 20.4 mL/kg (range, 36.3 to 99.0 mL/kg); water intake by an individual marmoset or cohoused pair was fairly consistent from day to day. Water intake did not change across the four 3-h periods measured during the day, and minimal water was consumed overnight when the room lights were off. In addition, daily water intake did not differ between the 2 mo of measurements. Older animals drank significantly more than the younger group, and weight was directly correlated with water intake. Water intake was not affected by body condition score or housing status. The variation in water consumption among marmosets underscores the need for individualization of fluid regulation guidelines.

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.