Modern genetic engineering technologies enable us to manipulate the mouse genome in increasingly complex ways to model human biology and disease. As a result, the number of mouse strains carrying transgenes or induced mutations has increased markedly. Thorough understanding of strain and gene nomenclature is essential to ensure that investigators know what kind of mouse they have, and what to expect in terms of phenotype. Genetically engineered mice alter gene function by over-expressing, eliminating, or modifying a gene product. The resulting phenotype is often unexpected and not completely understood, necessitating special care and potentially complex breeding and husbandry strategies. Animal care technicians responsible for routine maintenance of the colony, facility managers, veterinarians, and research personnel working with mice should be well informed about the nature of the mutation, distinguishing characteristics, and necessary precautions in handling the mice. Personnel working with mice also must be aware of the multitude of factors intrinsic to the mouse and present in the environment that can influence reproductive performance. Finally, diligent adherence to the maintenance of genetic quality in conjunction with cryopreservation of germplasm is the best insurance against loss of a colony.

Over the past decade, breakthrough technologies in transgenic animal technology and functional genomics have played a central role in the explosive growth of rodent modeling and in scientific innovation. Various noninvasive alternatives to routine surgical biopsy have been described for genotypic and phenotypic analyses of laboratory animals. A number of options are available to refine or replace potentially painful and invasive procedures ranging from tissue biopsies (including tail biopsies and toe docking) to several blood sampling techniques. Unfortunately, adoption of many non- or minimally invasive alternatives has proven difficult on a number of fronts ranging from historical reservations to procedural expectations and actual experimental productivity. Similarly, a variety of phenotyping considerations have addressed throughput efficiencies and the health and well being of research animals. From an animal welfare perspective, marked increases in laboratory animal populations have accompanied rapid advancements spanning the life sciences. As described for rodent modeling, but with applications across many laboratory animal species, diverse procedural refinements are available that will readily aid in the analysis of whole animal models. Ultimately, non-invasive technologies and complementary refinements have bearing on the quality and reproducibility of data that are reported, as well as of critical importance to the well being and ethical management of animals at all developmental stages: from fetal existence, to the neonatal period, and on through adulthood.

Established methods for analyzing behavioral traits in mutant lines of mice allow researchers to understand the outcomes of genetic manipulations in the nervous system. A rigorous six-tiered behavioral phenotyping strategy is described. Recommendations are offered for the design of mouse behavioral testing suites in animal housing facilities.

Gene therapy is the purposeful delivery of genetic material to somatic cells for the purpose of treating disease or biomedical investigation. Either viral or non-viral vector methods can be used. The risk of collateral exposure of laboratory animal care personnel to gene therapy vectors is dependent on a number of factors. These factors are intrinsic to the gene therapy vector (the vehicle for genetic conveyance), product encoded by the genetic construct delivered, method of delivery, and immune status of the recipient. The component risks of gene therapy experiments can be analyzed to surmise the overall relative risk of the experiment. Knowledge of the components that contribute potential hazardous risk to a study can assist animal care staff in identifying area(s) where prudent practices should be focused. Gene therapy experiments involving viral vectors are generally performed at either biosafety level 2 or 3. The objective of this review is to report on various components of gene therapy experiments, focusing on characteristics of viral and non-viral vectors, to assist the laboratory animal science community in determining prudent biosafety practices.

Oversight of recombinant DNA research by the National Institutes of Health (NIH) is predicated on ethical and scientific responsibilities that are akin, in many ways, to those that pertain to the oversight of animal research. The NIH system of oversight, which originated more than 25 years ago, is managed by the NIH Office of Biotechnology Activities (OBA), which uses various tools to fulfill its oversight responsibilities. These tools include the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) and the Recombinant DNA Advisory Committee. The OBA also undertakes special initiatives to promote the analysis and dissemination of information key to our understanding of recombinant DNA, and in particular, human gene transfer research. These initiatives include a new query-capable database, an analytical board of scientific and medical experts, and conferences and symposia on timely scientific, safety, and policy issues. Veterinary scientists can play an important role in the oversight of recombinant DNA research and in enhancing our understanding of the many safety and scientific dimensions of the field. These roles include developing appropriate animal models, reporting key safety data, enhancing institutional biosafety review, and promoting compliance with the NIH Guidelines.

Tuberculosis is a significant threat to non-human primates and their caretakers. The diagnosis of tuberculosis in living non-human primates is currently based on the tuberculin skin test, which is cumbersome and sometimes inaccurate. Development of an accurate serodiagnostic test requires identification of the key antigens of Mycobacterium tuberculosis involved in antibody production. When sequential serum samples obtained from 17 cynomolgus, rhesus, and African green monkeys up to seven months since experimental infection with M. tuberculosis Erdman were screened for antibody against purified proteins of M. tuberculosis, three highly seroreactive antigens were identified. One protein, ESAT-6, reacted with sera from all infected animals. Two additional proteins, α-crystallin and MTSA-10, were recognized by sera from approximately 90% of infected animals. Time course analysis of antibody production indicated that the earliest response was usually to ESAT-6 alone or to ESAT-6 and other antigen(s). These results provide experimental evidence of the potential value of ESAT-6 as an antigen for use in serodiagnosis of tuberculosis in non-human primates.

Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM in immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer and probe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virus and PVM, respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, and the Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.

The aim of the study reported here was to assess the prognostic value of gastric tonometry and its implications in the initial phases of hemorrhagic shock. Hemorrhagic shock was induced by use of femoral arterial bleeding in 12 hybrid swine under general anesthesia. Approximately 30% of the circulating blood volume was removed, until mean arterial pressure of 45 mmHg was reached. The shock conditions were observed over a limited period (90 min) by comparing traditional hemodynamic parameters with gastric tonometric measurements and tissue oxygenation. After a shock period of 90 min without pharmacologic treatment, blood was collected in acid-citrate dextrosetreated bags and was reinfused via the right femoral vein. At the end of the experiment, seven animals had good hemodynamic recovery on reinfusion (group A), whereas values in five animals deceased in the same phase (group B). Hemodynamic and gastric tonometric results were compared between survivors and nonsurvivors. Intravascular volume restoration and reduction of systemic vascular resistance (SVR) enabled the animals of group A to maintain standard ventricular kinetics and recover in terms of splanchnic regional flow. In addition, increase in intramucosal gastric pH (pHi), decrease in the pH-gap (pHa – Hi), and progressive restoration in gastric wall tissue oxygenation (PtO₂) also were observed. These results suggest that useful diagnostic and therapeutic indications can be obtained by acquisition of simple hemodynamic measurements at the beginning of the shock period. On the basis of results of statistical analysis, only mean arterial pressure and SVR were good indicators of shock development, whereas pHi was not a significant factor in this experimental model.

Melatonin (MLT), the circadian neurohormone secreted by the pineal gland in mammals during darkness, eicosapentanoic acid (EPA), and conjugated linoleic acid (CLA) have established regulatory roles in cancer growth. Investigations in our laboratory have indicated that these agents inhibit fatty acid (FA) transport by tumors and several sub-types of white adipose tissue via inhibitory G protein-coupled receptor mechanisms. Skeletal muscle constitutes over 45% of human body mass and plays an important role in cancer cachexia and obesity-related diseases. Since fatty acid oxidation is a major source of energy for this tissue, we tested the hypothesis that physiologic MLT levels, EPA, or CLA injected intravenously, inhibit FA uptake in rat skeletal muscle in vivo. We used a surgical technique for catheterizing the femoral vein in rats that allows rapid blood collection from the entire hind limb, while ensuring continuous blood flow to the tissue. Blood acid/gas tensions and hematocrit were monitored and remained constant during the course of each experiment. The MLT, EPA, and CLA inhibited FA uptake by the tissue and lowered cAMP values. Glucose uptake and glycerol production in the hind limb were not affected. These investigations suggest a novel role for MLT, omega-3 FAs, and CLA in the regulation of FA transport and fat metabolism in skeletal muscle.

Although cardioviruses related to Theiler's murine encephalomyelitis virus (TMEV) appear to be common in mice and rats, few TMEV isolates have been obtained from rat colonies. In 1991, a cardiovirus isolate designated NGS910 was obtained from sentinel rats exposed to cage bedding previously used by adult rats that were TMEV seropositive, but had never manifested clinical signs of disease. To determine to which group and subgroup of cardiovirus this virus belongs, the sequence of the viral genome was determined. The NGS910 genome consisted of 8,021 nucleotides and the 5'-nontranslated region had a predicted secondary structure that is similar to members of the TMEV group of cardioviruses. The Leader-P3D open reading frame (L ORF) of NGS910 had strong homology with L ORFs of other TMEVs (72% identity), but lower homology with EMCV cardioviruses (55 to 56%). Phylogenetic analyses on the basis of aligned nucleotide sequences of the L ORF (6,924 b) and the internal L* ORF (471 b) supported this classification of NGS910 as a TMEV strain. However, within the TMEV group, NGS910 was sufficiently divergent from other isolates that it could not be regarded as simply a mutant strain of a known TMEV. As genetic distances between NGS910 and other TMEVs were greater than those between Mengo virus of EMCV and other EMCVs, we propose to designate the NGS910 isolate as a rat Theiler-like virus.

Gamma interferon-deficient (IFN-γ KO) mice developed a wasting syndrome and were found to be co-infected with Helicobacter sp., and a new isolate of mouse hepatitis virus (MHV) designated MHV-G. The disease was characterized by pleuritis, peritonitis, hepatitis, pneumonia, and meningitis. Initial experiments used a cecal homogenate inoculum from the clinical cases that contained H. hepaticus and MHV-G to reproduce the development of peritonitis and pleuritis in IFN-γ KO mice. In contrast, immunocompetent mice given the same inoculum developed an acute, self-limiting infection and remained clinically normal. This result confirmed the importance of IFN-γ in preventing chronic infection and limiting viral dissemination. To understand the role of both agents in the development of peritonitis and pleuritis, IFN-γ KO mice were infected with either agent or were co-infected with H. hepaticus and MHV-G. Infection with MHV-G induced a multisystemic infection similar to that described in the original cases, with multifocal hepatic necrosis, acute necrotizing and inflammatory lesions of the gastrointestinal tract, and acute peritonitis and pleuritis with adhesions on the serosal surfaces of the viscera. However, mice given H. hepaticus alone had minimal pathologic changes even though the organism was consistently detected in the cecum or feces. Although co-infection with H. hepaticus and MHV-G induced lesions similar to those associated with MHV-G alone, the pathogenesis of the MHV infection was modified. Helicobacter hepaticus appeared to reduce the severity of MHV-induced lesions during the acute phase of infection, and exacerbated hepatitis and meningitis at the later time point. We conclude that infection of IFN-γ KO mice with MHV-G results in multisystemic infection with peritonitis, pleuritis, and adhesions due to the aberrant immune response in these mice. In addition, co-infection of these mice with H. hepaticus results in alterations in the pathogenesis of MHV-G infection.

Vascular leak syndrome (VLS) is a common and often fatal sequela of multiple bone traumas, and of infectious, toxic, and allergic insults in human patients. Although an animal model for VLS has not been fully established, rats have shown sensitivity to the syndrome that approximates that of the human population. We describe cases of VLS in three-month-old adult and one-month-old Sprague-Dawley rats in an osteogenesis study aimed at optimizing correction of bone hypoplasias and other craniofacial deformities in children, using a mandibular distraction device. In the study reported here, VLS was diagnosed in 46% of the rats that were necropsied after dying or being euthanized early, subsequent to mandibular osteotomy, a procedure that involves minimal bone trauma. The gross and histologic findings, as well as the clinical course of VLS in the rats of the osteogenesis study, were similar to those of documented human cases. Hence, the rat may be a useful animal model to help characterize the physiologic and molecular events that accompany this syndrome.

Ferrets with adrenal gland dysfunction have alopecia as their most common clinical sign of disease. Two cases of alopecia in neutered female ferrets are reported that were associated instead with neoplastic tissue found at the site of an ovarian pedicle. Androstenedione and 17-hydroxyprogesterone, but not estradiol, concentrations were high in both ferrets. Following surgical resection of the abnormal tissue in one ferret, the high hormone values decreased quickly and hair regrowth ensued. In both cases, histologic examination revealed features consistent with classical sex cord-stromal (gonadostromal) tumors: prominent spindle cells, along with polyhedral epithelial cells and cells with vacuolated cytoplasm. Although similiar cell types have been described in the adrenal glands of ferrets with adrenal-associated endocrinopathy, an ovarian origin for the current neoplasms is considered likely on the basis of their anatomic location; accessory adrenal tissue has only been described close to an adrenal gland or in the cranial perirenal fat of ferrets. Immunohistochemical analysis, using an antibody against Mullerian-inhibiting substance, failed to prove definitively the source of the steroidogenic cells.