Origins of US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.

The US Government Principles for the use of animals in research are a landmark statement of ethical values and guidance for the biomedical research community. However, when The Principles were introduced, a context was not provided for their source or foundation. The US Government Principles were formulated with input from the Council of Europe, World Health Organization, and US Interagency Research Animal Committee. The Principles continue to provide an ethical foundation for the biomedical research community.

The Evaluation of the Containment Efficacy of Semi-Rigid Isolators for Housing Cages of Laboratory Animals Infected With BSL-3 Agents.

Research animals models infected with Biosafety Level-3 (BSL-3) agents need to be housed in specialized biocontainment caging. Most of these specialized cages have input and exhaust that is high efficiency particulate air filtered and sealed to prevent escape of the BSL-3 agent. An alternative to the use of the above BSL-3 biocontainment caging is the use of a flexible film or modified semi-rigid plastic film isolator that has its own high efficiency particulate air-filtered input and exhaust and is sealed with respect to the animal room environment, thus preventing BSL-3 agent escape. Standard caging can be housed within such an isolator. Computational fluid dynamics was used to evaluate the integrity of modified semi-rigid isolators for containment of aerosolized BSL-3 agents. Three isolators were located inside an animal BSL-3 room to provide an extra tier of protection and to permit different infectious studies within the same room while reducing or eliminating the risk of cross-contamination. The isolators were sized to house caging for rabbits and smaller non-human primates such as marmosets, African greens, and macaques. Multiple case studies of failure scenarios were investigated, including isolator breaches through the plastic membrane seam separation and rips, and exhaust fan failure. Breaching the level of containment provided by the isolators required the improbable simultaneous event of a plastic membrane rip in addition to the rare malfunction of the back-up exhaust fans. Each isolator was equipped with 2 blower motors connected in parallel to a common exhaust plenum and a battery backup. Even with this rare double (simultaneous) event, the animal BSL-3 room air exhaust system was able to contain the few droplets released in the simulated computational fluid dynamics breach. The modified semi-rigid isolators with negative airflow proved safe and effective for aerosol studies using BSL-3 agents, even in the unlikely event of a breach in containment.

Immunogenicity and Efficacy of Live-Attenuated Salmonella Typhimurium Vaccine Candidate CVD 1926 in a Rhesus Macaque Model of Gastroenteritis.

Salmonella Typhimurium is a common cause of foodborne gastroenteritis and a less frequent but important cause of invasive disease, especially in developing countries. In our previous work, we showed that a live-attenuated S. Typhimurium vaccine (CVD 1921) was safe and immunogenic in rhesus macaques, although shed for an unacceptably long period (10 days) postimmunization. Consequently, we engineered a new strain, CVD 1926, which was shown to be safe and immunogenic in mice, as well as less reactogenic in mice and human cell-derived organoids than CVD 1921. In this study, we assessed the reactogenicity and efficacy of CVD 1926 in rhesus macaques. Animals were given two doses of either CVD 1926 or saline perorally. The vaccine was well-tolerated, with shedding in stool limited to a mean of 5 days. All CVD 1926-immunized animals had both a serological and a T cell response to vaccination. At 4 weeks postimmunization, animals were challenged with wild-type S. Typhimurium I77. Unvaccinated (saline) animals had severe diarrhea, with two animals succumbing to infection. Animals receiving CVD 1926 were largely protected, with only one animal having moderate diarrhea. Vaccine efficacy in this gastroenteritis model was 80%. S. Typhimurium vaccine strain CVD 1926 was safe and effective in rhesus macaques and shed for a shorter period than other previously tested live-attenuated vaccine strains. This strain could be combined with other live-attenuated Salmonella vaccine strains to create a pan-Salmonella vaccine.

Unusual Myoid Differentiation in a Canine Benign Mixed Mammary Tumour.

This report describes an unusual mesenchymal differentiation in a canine benign mixed mammary tumour. A 13-year-old crossbreed female dog was submitted to surgery to remove an inguinal mammary nodule. The tumour was composed of mammary epithelium and mesenchymal populations, not only of cartilage and bone but also of myoid cells. PTAH demonstrated cross striation of striated muscle, and immunohistochemistry highlighted striated muscle expressing desmin and calponin, and smooth muscle expressing desmin, SMA, and calponin. The tumour was diagnosed as a benign mixed tumour with leio- and rhabdomyoid differentiation. There was no tumour recurrence after one year of clinical follow-up. In conclusion, the well-differentiated features of leiomyocytes and rhabdomyocytes and the growth pattern define the benign origin of the mesenchymal component, which has been confirmed by a benign outcome; therefore, the knowledge of this kind of differentiation is helpful to avoid misdiagnoses.

Fit for Purpose Assessment: A New Direction for IACUCs.

The organization and function of the institutional animal care and use committee (IACUC) is the key component of government regulation and oversight of necessary scientific research using live animals and of AAALAC - International accreditation of animal care and use programs in the United States. The regulations, roles, and responsibilities of IACUCs have evolved since their inception 35 years ago from a limited focus on animal welfare and specific animal procedures to embracing scientific quality, data reproducibility and translation, and animal welfare as inextricably interdependent and critical components of generation of new scientific knowledge and medical treatments. A current challenge for IACUCs is in evaluating whether benefits to be derived (eg, new knowledge or treatments) justify any unavoidable pain, stress, or injury associated with proposed research protocols, because the former are long-term and at best speculative outcomes, whereas the latter are immediate and tangible for the study animals. Scientific consensus is that research most likely to generate significant new knowledge and medical treatments is that conducted to high scientific, technical, and quality standards and reported with full transparency to facilitate reproducibility. As an alternative to current benefits evaluations included in risk benefit and harm benefit constructs, the authors propose that IACUCs assess the proposed research for scientific quality and alignment of study elements with the study purpose (e.g., Fit for Purpose [FfP]), including justifications for study design components, selection of primary endpoints and technologies, rationale for data and statistical analyses, and research communication plans. Fit for Purpose endpoints are objective, immediate, and impactful as are the potential risks for study animals, and at the same time they are the best predictors for achievement of longer-term benefits. We propose that IACUCs and any revision of The ILAR Guide consider FfP concepts in place of traditional benefits assessment to accelerate the generation of new knowledge and treatments benefiting medical and veterinary patients and the environment through better science and animal welfare rather than to continue to rely on speculative future outcomes.

Pathogenic, but Not Nonpathogenic, Rickettsia spp. Evade Inflammasome-Dependent IL-1 Responses To Establish an Intracytosolic Replication Niche.

Rickettsia species (spp.) are strict obligate intracellular bacteria, some of which are pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis is evolving, the mechanisms by which pathogenic Rickettsia spp. evade host innate immune detection remain elusive. Here, we show that disease severity in wild-type (WT) C57BL/6J mice infected with Rickettsia typhi (the etiologic agent of murine typhus) and Rickettsia rickettsii (the etiologic agent of Rocky Mountain spotted fever), but not with the nonpathogenic species Rickettsia montanensis, correlated with levels of bacterial burden as detected in the spleens of mice, as well as the serum concentrations of proinflammatory cytokine interleukin-1α (IL-1α) and, to a lesser extent, IL-1ß. Antibody-mediated neutralization of IL-1α confirmed a key role in controlling mortality rates and bacterial burdens of rickettsia-infected WT mice. As macrophages are a primary source of both IL-1α and IL-1ß cytokines, we determined the mechanism of the antirickettsial activities using bone marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not nonpathogenic R. montanensis, eluded pro-IL-1α induction and benefited predominantly from the reduced IL-1α secretion, via a caspase-11-gasdermin D (Gsdmd)-dependent pathway, to facilitate intracytosolic replication. Adoptive transfer experiments identified that IL-1α secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike nonpathogenic, rickettsiae preferentially target the caspase-11-Gsdmd-IL-1α signaling axis in macrophages, thus supporting their replication within the host. IMPORTANCE Currently, no vaccines are available to prevent rickettsioses, while vector-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the caspase-11-Gsdmd-IL-1α signaling axis in limiting the replication of pathogenic R. rickettsia and R. typhi species in murine macrophages and wild-type (WT) C57BL/6J mice. Adoptive transfer studies further identified IL-1α-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not nonpathogenic, Rickettsia spp. benefit from a reduction in the caspase-11-Gsdmd-mediated release of IL-1α to support host colonization.

Improved Tolerability of a Salmonella enterica Serovar Typhimurium Live-Attenuated Vaccine Strain Achieved by Balancing Inflammatory Potential with Immunogenicity.

A notable proportion of Salmonella-associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S Typhimurium vaccine candidate CVD 1921 (I77 ΔguaBA ΔclpP) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA We determined that S Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S Typhimurium ΔpipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (109 CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD50 (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model.

Suspected Lonely Mouse Syndrome as a Cage Effect in a Drug Safety Study.

Studies have demonstrated that buprenorphine, a front line drug for veterinary analgesia, may alleviate symptoms of chronic pain. A cage side observation protocol was used to record behavioral signs in a mouse clinical trial of extended release buprenorphine. A retrospective review of the observations for signs of pain and stress revealed that mice given a fivefold overdose of buprenorphine (16.25 mg/kg) showed lethargy and facial signs associated with stress. However, similar signs were observed in the drug-free control mice as early as Day 3 of single-cage housing. This appears to be the first report of cage effects in a clinical trial for a veterinary drug.

Virulence of invasive Salmonella Typhimurium ST313 in animal models of infection.

Salmonella Typhimurium sequence type (ST) 313 produces septicemia in infants in sub-Saharan Africa. Although there are known genetic and phenotypic differences between ST313 strains and gastroenteritis-associated ST19 strains, conflicting data about the in vivo virulence of ST313 strains have been reported. To resolve these differences, we tested clinical Salmonella Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD50) was determined for three Salmonella Typhimurium ST19 and ST313 strains in mice. For dissemination studies, bacterial burden in organs was determined at various time-points post-challenge. Indian rhesus macaques were infected with one ST19 and one ST313 strain. Animals were monitored for clinical signs and bacterial burden and pathology were determined. The LD50 values for ST19 and ST313 infected mice were not significantly different. However, ST313-infected BALB/c mice had significantly higher bacterial numbers in blood at 24 h than ST19-infected mice. ST19-infected rhesus macaques exhibited moderate-to-severe diarrhea while ST313-infected monkeys showed no-to-mild diarrhea. ST19-infected monkeys had higher bacterial burden and increased inflammation in tissues. Our data suggest that Salmonella Typhimurium ST313 invasiveness may be investigated using mice. The non-human primate results are consistent with clinical data, suggesting that ST313 strains do not cause diarrhea.

Mycobacterium kansasii Isolated from Tuberculinpositive Rhesus Macaques (Macaca mulatta) in the Absence of Disease.

Mycobacterial infections are of primary health concern in NHP colonies in biomedical research. NHP are constantly monitored and screened for Mycobacterium spp. We report 6 Chinese-origin rhesus macaques infected with Mycobacterium kansasii that exhibited positive tuberculin skin tests in the absence of disease. Two of these macaques were being used for research purposes; the remaining 4 macaques were residing at the contract quarantine company. Histopathology and acid-fast staining of fixed tissues from all macaques showed that all were free of disease. Thoracic radiographs were negative for any signs of disease or infection. Samples from bronchial lavage and tissues including lung, spleen, hilar and mesenteric lymph nodes tested negative by PCR assay for Mycobacterium spp. One of the research macaques tested culture-positive for M. kansasii and a poorly characterized M. avium complex organism. One macaque from the contract quarantine facility tested culture positive for M. kansasii. Genomic testing and target gene RNA expression analysis of the 2 M. kansasii isolates were performed to evaluate possible kinship and affected genes that might contribute to susceptibility to mycobacterial infection. Genotyping of the 2 isolates revealed 2 genetically distinct strains (strains 1 and 4). The presence of positive tuberculin skin tests in the absence of disease raises serious concerns regarding diagnostic methods used for infected NHP.

Complete Genome Sequences of Mycobacterium kansasii Strains Isolated from Rhesus Macaques.

Mycobacterium kansasii is a nontuberculous mycobacterium. It causes opportunistic infections with pulmonary and extrapulmonary manifestations. We report here the complete genome sequences of two M. kansasii strains isolated from rhesus macaques. We performed genome comparisons with human and environmental isolates of M. kansasii to assess the genomic diversity of this species.

Spatial learning impairment in prepubertal guinea pigs prenatally exposed to the organophosphorus pesticide chlorpyrifos: Toxicological implications.

Exposure of the developing brain to chlorpyrifos (CPF), an organophosphorus (OP) pesticide used extensively in agriculture worldwide, has been associated with increased prevalence of cognitive deficits in children, particularly boys. The present study was designed to test the hypothesis that cognitive deficits induced by prenatal exposure to sub-acute doses of CPF can be reproduced in precocial small species. To address this hypothesis, pregnant guinea pigs were injected daily with CPF (25mg/kg,s.c.) or vehicle (peanut oil) for 10days starting on presumed gestation day (GD) 53-55. Offspring were born around GD 65, weaned on postnatal day (PND) 20, and subjected to behavioral tests starting around PND 30. On the day of birth, butyrylcholinesterase (BuChE), an OP bioscavenger used as a biomarker of OP exposures, and acetylcholinesterase (AChE), a major molecular target of OP compounds, were significantly inhibited in the blood of CPF-exposed offspring. In their brains, BuChE, but not AChE, was significantly inhibited. Prenatal CPF exposure had no significant effect on locomotor activity or on locomotor habituation, a form of non-associative memory assessed in open fields. Spatial navigation in the Morris water maze (MWM) was found to be sexually dimorphic among guinea pigs, with males outperforming females. Prenatal CPF exposure impaired spatial learning more significantly among male than female guinea pigs and, consequently, reduced the sexual dimorphism of the task. The results presented here, which strongly support the test hypothesis, reveal that the guinea pig is a valuable animal model for preclinical assessment of the developmental neurotoxicity of OP pesticides. These findings are far reaching as they lay the groundwork for future studies aimed at identifying therapeutic interventions to treat and/or prevent the neurotoxic effects of CPF in the developing brain.

Clinical Trypanosoma cruzi Disease after Cardiac Transplantation in a Cynomolgus Macaque (Macaca fascicularis).

A cynomolgus macaque received a heterotopic cardiac allograft as part of a transplant study, with monoclonal antibodies targeted to specific immune costimulation molecules (CD154, CD28) but no traditional immunosuppressive therapy after surgery. Clinical anemia was detected on postoperative day (POD) 35 and had worsened (Hgb, 2.3 g/dL; Hct = 7.3%) by POD 47, despite type-matched whole-blood transfusions. After a total of 4 blood transfusions, hematologic parameters were improved (Hgb, 5.9 g/dL; Hct, 18.7%). On POD 50, a peripheral blood smear revealed trypomastigotes, and qualitative RT-PCR of whole blood identified the organism as Trypanosoma cruzi. Although clinically stable initially, the macaque soon developed sufficient weight loss to necessitate euthanasia on POD 64. The final diagnosis was clinical anemia due to T. cruzi infection. This study represents the first reported case of Chagas disease after heart transplant in a NHP.

Safety studies of post-surgical buprenorphine therapy for mice.

The use of appropriate analgesia in laboratory mice may be suboptimal because of concerns about adverse events (AE). Target Animal Safety trials were conducted to determine the safety of an extended-release suspension of buprenorphine. Drug or control suspensions were injected subcutaneously in surgically-treated BALB/c mice anesthetized with ketamine-xylazine to mimic post-operative conditions in which the compound might commonly be administered. Single and repeat five-fold (5×) excesses of the 3.25 mg/kg intended dose were used to provoke potential AE. Trials included prospective measurements of weight changes, blood chemistry, hematology, and histopathology. Clinical and histopathology findings were similar in drug-treated and control mice in a four-day trial using a single 16.25 mg/kg, 5× overdose of the drug. In a 12-day trial, which used a total buprenorphine dose of 48.75 mg/kg, clinical and histopathology values were also similar in control and drug-treated female mice. In the male arm of the repeat-overdose trial, two of eight mice died on the morning of day 12, three days following the third 16.25 mg/kg overdose administration. Histopathology did not reveal a cause of death. In a 14-month trial using a single 3.25 mg/kg dose of the drug, no significant findings identified potential AE. These findings indicate a high tolerance to an extended-release buprenorphine suspension administered post-operatively in mice with appropriate husbandry.

A rabbit model of non-typhoidal Salmonella bacteremia.

Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.

Animal models that best reproduce the clinical manifestations of human intoxication with organophosphorus compounds.

The translational capacity of data generated in preclinical toxicological studies is contingent upon several factors, including the appropriateness of the animal model. The primary objectives of this article are: 1) to analyze the natural history of acute and delayed signs and symptoms that develop following an acute exposure of humans to organophosphorus (OP) compounds, with an emphasis on nerve agents; 2) to identify animal models of the clinical manifestations of human exposure to OPs; and 3) to review the mechanisms that contribute to the immediate and delayed OP neurotoxicity. As discussed in this study, clinical manifestations of an acute exposure of humans to OP compounds can be faithfully reproduced in rodents and nonhuman primates. These manifestations include an acute cholinergic crisis in addition to signs of neurotoxicity that develop long after the OP exposure, particularly chronic neurologic deficits consisting of anxiety-related behavior and cognitive deficits, structural brain damage, and increased slow electroencephalographic frequencies. Because guinea pigs and nonhuman primates, like humans, have low levels of circulating carboxylesterases-the enzymes that metabolize and inactivate OP compounds-they stand out as appropriate animal models for studies of OP intoxication. These are critical points for the development of safe and effective therapeutic interventions against OP poisoning because approval of such therapies by the Food and Drug Administration is likely to rely on the Animal Efficacy Rule, which allows exclusive use of animal data as evidence of the effectiveness of a drug against pathologic conditions that cannot be ethically or feasibly tested in humans.

Niemann-Pick Disease Type C: Induced Pluripotent Stem Cell-Derived Neuronal Cells for Modeling Neural Disease and Evaluating Drug Efficacy.

Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-ß-cyclodextrin, methyl-ß-cyclodextrin, and δ-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and δ-tocopherol shows an additive or synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. In addition, we found that hydroxypropyl-ß-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells compared to the NPC1 fibroblasts. Miglustat, suberoylanilide hydroxamic acid, curcumin, lovastatin, pravastatin, and rapamycin did not, however, have significant effects in these cells. The results demonstrate that patient-derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.

Subcutaneous implants for long-acting drug therapy in laboratory animals may generate unintended drug reservoirs.

BACKGROUND: Long-acting therapy in laboratory animals offers advantages over the current practice of 2-3 daily drug injections. Yet little is known about the disintegration of biodegradable drug implants in rodents. OBJECTIVE: Compare bioavailability of buprenorphine with the biodegradation of lipid-encapsulated subcutaneous drug pellets. METHODS: Pharmacokinetic and histopathology studies were conducted in BALB/c female mice implanted with cholesterol-buprenorphine drug pellets. RESULTS: Drug levels are below the level of detection (0.5 ng/mL plasma) within 4-5 days of implant. However, necroscopy revealed that interstitial tissues begin to seal implants within a week. Visual inspection of the implant site revealed no evidence of inflammation or edema associated with the cholesterol-drug residue. Chemical analyses demonstrated that the residues contained 10-13% of the initial opiate dose for at least two weeks post implant. DISCUSSION: The results demonstrate that biodegradable scaffolds can become sequestered in the subcutaneous space. CONCLUSION: Drug implants can retain significant and unintended reservoirs of drugs.

Thermal latency studies in opiate-treated mice.

BACKGROUND: The change in the reaction time of a tail or paw exposed to a thermal stimulus is a measure of nociceptive activity in laboratory animals. Tail-flick and plantar thermal sensitivity (Hargreaves) tests are non-invasive, minimize stress, and can be used to screen animals for phenotype and drug activity. OBJECTIVE: Hargreaves testing has been widely used in rats. We investigated its use to measure the activity of opiate analgesia in mice. METHODS: Mice were used in thermal stimulus studies at 1-5 hours and 1-5 days to test acute and extended release preparations of buprenorphine. RESULTS: Hargreaves testing had limited value at 1-5 hours because mice can have an obtunded response to opiate therapy. Tail-flick studies with restrained mice are not affected by the initial locomotor stimulation. DISCUSSION: The present report describes a simple restraint system for mice. The utility of the system is demonstrated by examining the efficacy of acute and extended release buprenorphine injections in Balb/c and Swiss mice. CONCLUSION: Standardized tail-flick testing provides a sensitive robust method to monitor opiate activity in mice.