Comparison of Rodent Infectious Agent Detection by Exhaust Dust Testing and Traditional Sentinel Testing Using Quantitative Polymerase Chain Reaction.

Improved diagnostic capabilities and a desire to reduce or refine the use of animals as soiled bedding sentinels (SBS) have driven interest in developing the use of PCR-based testing methods, such as exhaust dust testing (EDT), for routine rodent health surveillance. We compared the absolute and quantitative PCR results from EDT filters with SBS mice by routine screening via a panel of 19 infectious agents including agents known to be excluded or present in the colony. In this study, EDT and SBS were compared at days 0, 90, and 180 in 3 facilities (n = 12 rooms) with animals housed on IVC racks (n = 19 double-sided and 23 single-sided racks). All racks were negative for excluded agents (n = 15 agents) during the study. The bacterial agent Helicobacter spp. was consistently detected on EDT filters while less consistently detected via SBS. EDT filters detected Corynebacterium bovis better than SBS in areas where the agent was present. EDT filters and SBS mice tested for murine norovirus (MNV) demonstrated agreement for positive tests by both PCR and serology. For rodent chaphamaparvovirus-1 (RCHPV-1) we compared EDT to urine and feces from SBS. Six cages of SBS were positive for RCHPV-1 by fecal PCR with 5 out of 6 testing positive on urine, while only 3 out of 6 EDT filters tested positive. Since real-time fluorogenic PCR was used for testing, relative PCR copy numbers for each positive finding were evaluated to estimate organism load at the rack level. Copy numbers allowed for further characterization of agent presence within a colony. Furthermore, we compared copy numbers with cage census for MNV and Helicobacter spp., which was positively correlated for EDT testing but not for SBS. Overall, our results demonstrate that EDT's ability to detect many commonly excluded agents is comparable to or better than SBS.

Pharmacokinetic Evaluation of a Topical Extended-release Analgesic in Mice.

Mice often undergo painful procedures and surgeries as part of biomedical research protocols. Buprenorphine, a partial µ-opioid receptor agonist and κ receptor antagonist, is commonly used to alleviate the pain associated with such procedures. Due to its pharmacokinetic profile, buprenorphine requires frequent dosing, resulting in handling stress that can impact animal welfare and study data. A long-acting transdermal buprenorphine formulation (LA-bup) was recently approved for use in cats to provide up to 4 d of postoperative analgesia. In this study, we characterized the pharmacokinetics of a single topical dosing of LA-bup in male and female CD-1 mice administered a 0.36-mg or 18-µL topical dose at select time points. Plasma buprenorphine concentrations were evaluated at 0.25, 0.5, 1, 1.5, 2, 4, 8, 24, 48, and 72 h (n = 3 mice/time point) and remained above the purported therapeutic threshold (1 ng/mL) from 1 to 24 h postadministration. Repeated daily dosing at 24 and 48 h demonstrated plasma levels above 1 ng/mL for up to 72 h with minimal accumulation or changes in maximal concentrations over time. Inadvertent transfer of the topical drug to nondosed mice in the same cage was evaluated by measuring plasma buprenorphine concentrations in nondosed mice cohoused with a single-dosed mouse. Male mice did not demonstrate transfer of drug via grooming or interactions, yet 2 out of 26 nondosed female mice had detectable buprenorphine plasma levels indicating a relatively low incidence of cross-ingestion in cohoused female mice. This study demonstrates that LA-bup is a promising analgesic in mice that could be used for tailored analgesia strategies, depending on the surgical model or duration of analgesic therapy.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Experimental Verification of Inferred Regulatory Interactions of EARLY FLOWERING 3 ( GmELF3-1 ) in Glycine max.

Understanding the roles of evening complex (EC) genes in the circadian clock of plants can inform how diurnal transcriptional loops in the clock gene network function to regulate key physiological and developmental events, including flowering transition. Gene regulatory interactions among soybean's circadian clock and flowering genes were inferred using time-series RNA-seq data and the network inference algorithmic package CausNet. In this study, we seek to clarify the inferred regulatory interactions of the EC gene GmELF3-1. A gene expression analysis using soybean protoplasts as a transient model indicated regulatory roles of GmELF3-1 in expression of selected flowering genes.