The aim of the study reported here was to provide some basic and general information on the suitability of an experimental sheep model for conducting in vivo orthopedic studies. The authors have classified the fundamental aspects that should be carefully evaluated when using sheep as an experimental model in orthopedic research: factors strictly related to bone anatomy and formation; and factors strictly affecting bone physiology, such as gastrointestinal mineral and vitamin absorption, and reproductive cycle. Future investigations should address all of the aspects highlighted, since there is no animal with the same anatomic, biochemical, physiologic, and biological characteristics as those of human beings. Moreover, useful data for treating orthopedic patients are based not only on good planning and study design, but also on perfect knowledge of the animal used and the differences between the model and the human being. The authors hope that this report will contribute to extrapolation of reliable data for use of sheep in the orthopedics field.

Studies of immune deficiencies have a history as long as that of immunology. However, reports of two key spontaneous recessive mutations in mice (nude in 1966–1968 and scid in 1983) laid the foundations for widespread application of immune-deficient rodents to a broad range of research topics. More recently, technologies modifying the mouse genome by transgenesis, gene ablation and crossbreeding for lines with multiple immune deficits have provided a large number of new types of immunologically impaired mice. The primary goals of this overview are to help non-immunologists understand key differences between some of the immunodeficient strains, develop an appreciation for the value of information derived from immunodeficient mouse-based research and to encourage expanded, creative use of these specialized research animals. Secondary goals are to promote greater awareness of unexpected outcomes that can arise when working with genetically immune-deficient mice, the need for vigilance in maintaining these research animals, and the care required in interpretation of the data that immune-deficient modeling provides. Two illustrations on developing appropriate immune deficient animal models for a new research application conclude the review.

The deer mouse (Peromyscus maniculatus) is a natural reservoir for several human pathogens, but little is known about the mechanisms by which such pathogens are maintained in nature. As a first step toward developing a colony of deer mice that were permissive for infection with Sin Nombre (SN) hantavirus, we collected 68 wild P. maniculatus rufinus from central New Mexico. Mice from this cohort were used to establish 26 breeding pairs, of which 85% were fertile. In subsequent generations, fertility decreased slightly to 73% (N = 59) in laboratory-reared F1 and F2 pairs. Wild-caught females delivered 7.2 litters on average (range, 1 to18), whereas laboratory-reared pairs delivered 5.5 (range, 1 to 13). The average time between pairing and first litter was 106 days for wild-caught animals, whereas that for laboratory-reared pairs was 71 days. None of the pairs displayed a seasonal breeding preference. Cannibalistic behavior increased from 5% in founders to 26% in laboratory-reared pairs. Mean litter size for wild-caught females was 4.3, whereas that for laboratory-reared dams was 4. Founding animals have been maintained in captivity for longer than 2 years, with only 2 deaths (4.8%). Our colony is competent for infection with SN virus. Thus, it should be useful for testing of models for maintenance of SN virus in wild rodents, and other aspects of the virus-host relationship.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains, although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.

Background and Purpose: The aim of the study reported here was to investigate variations in the serum concentration of α2 -macroglobulin (α2M) from healthy rats and rats inoculated with Staphylococcus aureus or subjected to surgery. Methods: Concentration of α2M was measured by use of an enzyme-linked immunosorbent assay. Results: Serum α 2M in healthy rats at intervals of 3 h, 1 day, and 1 week ranged from 17.5 to 38.0 (mean ± SD, 27.2 ± 6.6) μg/ml, 15.8 to 48.2 (27.9 ±8.7) μg/ml, and 17.2 to 39.6 (23.9 ±5.9) μg/ml, respectively. Concentrations of α2M did not undergo significant variations within individuals or between rats. Serum α2M concentration increased at one day and peaked two days after inoculation with S. aureus or being subjected to surgery. Peak concentration was eight to 33 times pre-inoculation values after inoculation with S. aureus, four to 25 times pre-surgical values when rats were subjected to castration, and seven to 28 times pre-surgical values when rats were subjected to oophorohysterectomy, respectively. Conclusions: Physiologic variation in the concentration of α2M in rats was not found. Induction of α2M in rats inoculated with S. aureus or subjected to surgery was documented.

Bone ingrowth has been studied extensively in rats by use of bone chambers. However, it is not known whether results in small animals, with respect to bone ingrowth processes, are similar in large animals, in which more realistic models are often used. Since the metabolic rate in small animals is, in general, higher than that in larger species, we hypothesized that bone ingrowth in chambers develops more rapidly in small animals. Therefore, identical bone chambers were placed in the tibias of rats and goats. After 6 and 12 weeks, histologic and histomorphometric examinations were carried out to measure bone and tissue ingrowth distances. Bone ingrowth was higher in both species at 12, compared with 6 weeks (P < 0.01). Tissue ingrowth in general (including soft tissue) was less in rats than in goats at both time periods (P < 0.001). However, bone ingrowth did not differ between species. Thus, when differences in size of an osseous defect are corrected for, there seems to be only little influence of differences in body size.

A pig model of wound healing was developed by excision of 2-cm-diameter full thickness skin in young Yorkshire pigs. The results indicated that wound re-epithelialization in this animal model took an average of 20 days. Analysis of cellular change was assessed by use of DNA quantification and determination of apoptotic cells in tissue sections. The results indicate that RNA and DNA contents paralleled each other throughout the healing process, and observed changes in the pattern of RNA and DNA content of the scar tissues were consistent with cell loss due to apoptosis in this model. Expression of mRNA for relevant genes was assessed by use of semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, using porcine specific primer sets and RNA isolated from normal skin and specimens obtained at various times after wounding. The mRNA values for tumor necrosis factor-α (TNF-α), connective tissue growth factor (CTGF), insulin-like growth factor II (IGF-II), and decorin were significantly high at specific times after wounding, but mRNA values for the transcription factors (c-fos and c-jun) were significantly decreased. Quantitative bacteriologic results indicated that the total bacterial count in this animal model reached 10⁹ colony-forming units (CFU)/g, with the highest value at post-wounding day 7, and Pseudomonas aeruginosa and Staphylocococci aureus were the most common bacteria detected in this model. Further definition of this model should identify unique points in the healing process, and such information could lead to development of therapeutic interventions to improve skin wound healing.

Background and Purpose: The limited availability and cost of many drugs prohibits routine use of the previously developed intestinal and vascular access port (IVAP) canine model by our group. A lower animal species model such as the rabbit is suitable for implanting intestinal and vascular access ports for investigating regiospecific intestinal absorption and hepatic elimination while requiring significantly lower doses of drugs. In addition, expression of certain cytochrome P450 enzymes and apical secretory and absorptive transporters in rabbit intestine is similar to that in humans making the rabbit a suitable model. Methods : Individual 5-F Silastic catheters were placed in the proximal or distal portion of the small intestine or the colon of subject animals, while a 5-F Heparin Coated Polyurethane (HCP) catheter was implanted in the portal vein of each subject. The catheters were tunneled out of the abdomen and attached to separate subcutaneous access ports along the spine. The animals were allowed a two-week minimum recovery period prior to being used in pharmacokinetic studies. Results and Discussion: After some initial difficulties, rabbits with IVAP implants proved to be an efficient and dependable model for investigating intestinal and hepatic extraction of drugs. Fluoroscopic visualization of intestinal and portal venous catheters indicated that surgically implanted catheters did not interfere with gastrointestinal motility or blood flow into the liver, respectively. Acute pH studies in the proximal portion of the small intestine were consistent with normal GI motility patterns.

Two from a group of approximately 50 C. B-17 scid-bg mice were examined because of lethargy, dehydration, and rough coat. Three months prior to development of clinical signs of disease, mice of this study had been surgically implanted with fetal bovine liver, thymus, and lymph node. At necropsy, marked splenomegaly and mild hepatomegaly were observed in both animals. Large areas of necrosis and inflammation, with associated intracytoplasmic granular basophilic inclusions, were observed in histologic sections of multiple organs. Aerobic and anaerobic culturing of the liver yielded negative results. Six months after the initial case, four more reconstituted scid-bg mice from a different fetal donor had identical clinical, gross, and histologic signs of disease. To determine whether the basophilic inclusions represented an infective agent, 4-month-old immune-naïve C. B-17 scid-bg mice were inoculated intraperitoneally with a liver and spleen homogenate from an affected mouse. Two weeks after inoculation, mice developed clinical signs of disease and lesions identical to those seen in the signal mice. On ultrastructural examination of the liver, pleomorphic bacteria were found in large cytoplasmic vacuoles of hepatocytes. Bacterial DNA was amplified from the liver, using primers that amplify a segment of the16S rRNA gene from many bacterial species. Sequencing of the polymerase chain reaction (PCR) product revealed gene sequence identical to that of Coxiella burnetii, the agent of Q-fever. These results highlight the need to consider infective agents of the donor species when working with xenografted animals.

Zebrafish (Brachydanio rerio) have become an important model system for studying vertebrate embryonic development and gene function through manipulation of genotype and characterization of resultant phenotypes. An established research zebrafish colony without substantial disease problems for more than 7 years of operation began experiencing appreciable mortalities in November of 1997. Young fish (fry), from five to 24 days after hatching, spontaneously developed elongate strands of organic material protruding from the mouth, operculum, and anal pore, leading workers in the laboratory to describe the infected fish as “bearded.” Unlike typical freshwater fish fungal infections, the skin surface did not have evidence of fungal colonization. The disease was associated with progressive lethargy, reduced feeding, and subsequent mortality. From 10 to 100% of the fry in a given tank were affected. Initial examination indicated that the biofilm around the head of affected fry consisted of bundles of septate fungal hyphae, large numbers of mixed bacterial populations, and protozoans. Environmental samples of air and water in the laboratory were obtained to ascertain the source of the infective agent and to isolate and identify the fungus. A fungus identified as Lecythophora mutabilis was isolated repeatedly from infected fish and water samples from infected fish tanks, and from the main laboratory water supply tanks, but not from laboratory air. Some biofilm beards on fish were found to consist of relatively pure bacterial populations, and beards on occasional fish examined in the later part of the study consisted of hyphae and spores of the oomycete genus Aphanomyces. Lecythophora mutabilis did not invade tissues; however, elimination of the epizootic correlated with reduction in the number of L. mutabilis conidia in the water following modification of the laboratory water system by use of new filtration and sterilization systems. We conclude that the dense hyphal strands of L. mutabilis composing the predominant biofilm type, along with mixed bacteria and protozoa, contributed to the die-off in young fry by occluding the oral cavity and/or gills, leading to starvation and/or asphyxiation.

Variation in susceptibility to viral infection is well documented across mouse strains. Specific combinations of viral strains and murine hosts may favor viral infection and disease, and could potentially allow the unexpected development of chronic, persistent, or latent infections. In some genetically modified strains of mice, immune function and perhaps other physiologic or metabolic systems may be substantially or marginally impaired. In the case study reported here, we document the apparent persistent transmission of mouse hepatitis virus (MHV) over a twoyear period by MHV-seropositive transgenic mice. Transmission occurred via direct contact with seropositive mice and exposure to contaminated bedding. However, MHV was not detected at diagnostic laboratories by use of viral isolation or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tissues from MHV-seropositive animals. Our observation, together with the constantly expanding varieties of immune-impaired or poorly characterized murine hosts and the burgeoning dissemination of these animals throughout the biomedical research community, indicate that unexpected pathophysiologic presentations of common murine viral diseases may present new challenges to the biomedical research community in the future.