Background and Purpose: The major goal was to determine whether variations in the method of CO₂ euthanasia would induce significant immunologic differences. Methods: Young adult C57BL/6 mice (n = 40) were euthanized, using four regimens: 70% CO₂/30% O₂; 70% CO₂/30%O₂ → 100% CO₂; 100% CO₂-naïve chamber; and 100% CO₂ pre-charged chamber. Time to recumbency and euthanasia and body, liver, lung, spleen, and thymus masses were determined. Blood and spleen were further evaluated for leukocyte, lymphocyte, and thrombocyte counts, erythrocyte characteristics, distribution of lymphocyte subpopulations, spontaneous and mitogen-induced blastogenesis, complement activity, and cytokine production. Results: Time to euthanasia was five- to eightfold longer in mice exposed to 70% CO₂/30% O₂ than that for any other group. There were slight increases in mean erythrocyte volume (MCV) and mean erythrocyte hemoglobin (MCH) for all groups, compared with those for the 100% CO₂ pre-charged group. Circulating cytotoxic T (CD8⁺) lymphocyte percentages and numbers, and spontaneous blastogenesis of leukocytes in blood and spleen, also were affected by euthanasia method. Conclusions: The method of CO₂ euthanasia can result in significant differences in immunologic/hematologic variables. Thus, consistency in euthanasia procedures may be important in accurate interpretation of research data.

Two novel murine monoclonal antibodies (mAbs) were produced to the Thy-1 molecule of Mongolian gerbils (Meriones unguiculatus). These mAbs, HUSM-M. g.27 of IgG1 isotype and HUSM-M. g.40 of IgG2a isotype, immunohistochemically reacted with the thymus, nervous system, and glomerular mesangium in partially different manners, suggesting that they recognize distinct epitopes, although they reacted with Thy-1 antigen, with apparent molecular weight of about 25 kDa, on gerbil thymocytes. Mild and severe forms of mesangioproliferative nephritis after glomerular deposition of the antibody was observed in gerbils administered mAbs HUSM-M. g.27 and HUSM-M. g.40, respectively, intraperitoneally, with or without guinea-pig serum as supplementary complement. Distinct pathogenicity and requirement of guinea pig serum for pathologic sequels are discussed as they relate to the rat model of anti-Thy-1-induced glomerulonephritis.

Purpose: The lacrimal gland is often selected for microscopic examination in toxicologic studies. However, this gland is difficult to find within the orbit in marmosets at necropsy. Therefore, we examined the extraorbital lacrimal glands in marmosets. Methods: The formalin-fixed craniums of four marmosets were used in a topographic study to confirm location of the lacrimal gland, and the results were applied to a routine toxicologic study in marmosets. Results: The extraorbital lacrimal gland was located on the temporal surface of the zygomaticofrontal process and was covered with the temporalis muscle. The gland was easily detached from the surrounding tissue, and its histologic features were the same as those of the intraorbital lacrimal gland. Conclusions: The extraorbital lacrimal glands have been reported in some New World monkeys, but to the authors' knowledge, this is the first report in marmosets. Identification and characterization of this gland will be useful for toxicologic studies in marmosets.

To determine whether the multimammate mouse (Mastomys coucha) could be used to evaluate rodent parvovirus-based vectors, neonates were subcutaneously inoculated with minute virus of mice (prototype strain, MVMp) or rat parvovirus H-1. The course of infection with both viruses was similar. Seroconversion occurred within two weeks after virus inoculation, as detected by use of hemagglutination-inhibition assays, and antibody titers remained high for the entire observation period of 12 months. Viral DNA and infective virions were detected in several organs of inoculated animals prior to seroconversion, as measured by use of Southern blotting and plaque assays, respectively. Infective particles subsequently became undetectable, whereas viral DNA imprints persisted in distinct organs for at least nine months. Clinical signs of parvovirus infection appeared around six weeks after virus inoculation, and consisted of hemorrhages, stunted growth, and transient hair color changes. Sudden death occurred in a significant fraction of animals infected with MVMp, but not H-1 virus, at the time of weaning. Alto- gether, MVMp, which is innocuous to its natural host, the mouse, and H-1 virus, which is poorly pathogenic to the rat, appear to be pathogenic for Mastomys coucha .

Background and Purpose: Mycoplasma pulmonis is a natural pathogen of the respiratory and genital tracts of rats. Differential susceptibility and severity of the respiratory form of the disease, known as murine respiratory mycoplasmosis (MRM), exist between rat strains. We now report that specific rat strains vary in susceptibility to genital tract infection and pregnancy outcome. Methods: Specific-pathogen-free (SPF) female F344, LEW, Wistar (WIS) and Sprague Dawley (SD) rats were intravaginally inoculated with 3 x 10⁷ colony-forming units (CFU) of M. pulmonis strain X1048 or sterile diluent, and allowed to breed at 10 days after inoculation. Pregnant dams and pups were necropsied within 24 hours of parturition. At necropsy, culture for M. pulmonis was performed on dam and pups, and adverse effects on pregnancy outcome were assessed by determination of the incidence of infertility, fetal resorption, stillbirths, changes in litter size, and pup birth weight. Blood from dams was collected prior to inoculation and at time of necropsy for measurement of IgM and IgG antibodies to M. pulmonis. Results: At time of necropsy, WIS (50%) and SD (60%) dams had a higher frequency of M. pulmonis culture positivity in the genital tract than did LEW (22.2%) and F344 (17.6%) dams. Dams that were still infected with M. pulmonis at time of necropsy had various complications. The SD rats had the greatest degree of adverse effects on pregnancy outcome, which were: infertility, decreased litter size (P ≤ 0.01), decreased pup birth weight (P ≤ 0.01), increased frequency of resorptions, stillbirths (P ≤ 0.05), and the highest rate of pup pulmonary infection (23.1%) (P ≤ 0.001). Despite a 50% colonization rate, WIS dams were the least adversely affected. The WIS pups born from M. pulmonis infected dams had slight decrease in birth weight, and only 6% had pulmonary infections. The LEW infected dams developed infertility and lower numbers of liveborn pups without evidence of vertical transmission. The F344 infected dams had lower numbers of liveborn pups that were smaller than their control counterparts, and none had pulmonary infections. None of the animals had detectable IgM and IgG antibodies to M. pulmonis before inoculation. At time of necropsy, all animals inoculated with M. pulmonis developed significantly (P ≤ 0.001) higher amounts of M. pulmonis IgG and IgM antibodies, with SD rats developing the highest amounts (P ≤ 0.005). Conclusions: Both F344 and LEW rats are more resistant to vaginal inoculation with M. pulmonis than are WIS and SD rats. However, only SD dams suffered severe adverse effects on pregnancy outcome. The SD dams also had the greatest IgM and IgG antibody response to M. pulmonis. Our studies clearly indicate differences among rat strains in their susceptibility to vaginal inoculation with M. pulmonis and in secondary complications associated with infection. This system may be a useful model for determining host-specific factors that influence the outcome of natural mycoplasmal infections of the genital tract.

Background and Purpose: Potential drugs for human acute renal failure are often tested in an animal model of renal ischemia/reperfusion injury. Analgesics are often not given after surgery because of concerns that they would alter renal function. Therefore, we tested whether postoperative analgesia would alter animal health or affect the degree of renal injury. Methods: Mice were subjected to either 32 or 37 minutes of renal ischemia, given two or six doses of buprenorphine or vehicle at 12-hour intervals, and followed for 72 hours. In some animals, we measured body temperature and physical activity by use of telemetry. Results: Animals treated with buprenorphine recovered more rapidly from surgery based on postoperative activity, and had a small but not significant tendency for faster restoration of normal body temperature. Animals treated with buprenorphine had less weight loss after 37 minutes of ischemia. Buprenorphine given after surgery did not influence the degree of renal injury after ischemia/reperfusion. Conclusions: Buprenorphine should be given after renal ischemia-reperfusion surgery because administration of the proper analgesic improved animal health without interfering with the renal ischemia/reperfusion model. Analgesic treatment at the time of the operation and 12 hours after was sufficient. Buprenorphine may reduce the post-surgical stress response, and thus potentially improve the specificity of testing for drugs that reduce or treat renal injury.

Background and purpose: Light amplification by stimulated emission of radiation (laser) systems operating in the so-called “eye safe” region are gaining widespread use in industry, medicine, and military applications. This research effort was geared to study the effects of laser tissue interaction on human skin by using in vivo porcine skin as an animal model. The goals of the study were to determine the median effective dose (ED₅₀) for 1540-nm laser exposures, to evaluate the Yorkshire pig and the Yucatan mini-pig as animal models for laser exposure, and to characterize laser-induced skin lesions histologically. Methods: A 1540-nm wavelength laser was used to expose multiple sites on the flanks of 10 pigs, using 0.8-ms pulses, ranging from 7 to 96 joules (J)/cm² . Single pulses were delivered to the flank of Yorkshire and Yucatan pigs in a grid pattern. Exposure sites were evaluated immediately after exposure and at 1 hour and 24 hours for presence of gross lesions. Representative biopsy specimens were collected from lesion sites for histologic evaluation at the 24-hour endpoint. Results: The ED₅₀ for the two breeds differed in the amount of energy required to induce dermal lesions. Grossly, lesions in each breed were well demarcated and pale gray to brightly erythematous. Microscopically, lesions had epidermal layer damage as cellular swelling and nuclear pyknosis, loss of cellular detail, and coagulation necrosis at the dermal layer. Conclusions: Findings suggest the presence of a different mechanism of laser-tissue damage in these two breeds. Photo-thermal mechanism appears to induce the skin lesions in the Yorkshire pig, whereas photo-thermal and photochemical mechanisms appear to be involved in lesion formation in the Yucatan mini-pig. All data obtained in this study will become part of database used by the American National Standards Institute (ANSI) to recommend laser safety standards for the occupational health and safety programs (OHSP), which will be used by industry and the military to base and update their current OHSP.

Purpose: A study was conducted to assess the cardiopulmonary and anesthetic effects of sevoflurane anesthesia on Garnett's Greater Bush Baby (Otolemur garnettii). Methods: Anesthesia was induced in ten animals with 8% sevoflurane and was maintained by use of 2.5% sevoflurane for 30 minutes. Induction and recovery times were recorded. Heart and respiratory rates (RR), end-tidal carbon dioxide concentration (ET CO₂), arterial blood pressures, relative arterial blood oxygen saturation (SpO₂ ), arterial partial pressure of oxygen (P_aO₂), arterial partial pressure of carbon dioxide (P_aCO₂), and pH were monitored. Preand poststudy CBC and serum biochemical values were compared. Results: Anesthesia induction was rapid (75 ± 8.7 seconds [mean ± SEM]) and smooth. Heart rate significantly increased initially, then decreased significantly over the remaining 30 minutes. There were no significant changes in RR, SpO₂, ETCO₂, or arterial blood pressure. The P_aO₂ values significantly increased in the 10- to 30-minute samples. The P_aCO₂ values remained steady in the 10- to 30-minute samples. A significant decrease was seen in white blood count, calcium, and total protein (TP) values, compared with values in pre-anesthesia samples. Recovery from anesthesia was smooth and rapid, with extubation at 24 ± 5.8 seconds. Conclusions: At the concentrations used in this study, sevoflurane appears to be a safe and effective agent for induction and maintenance of anesthesia in O. garnettii.

Objective: An advantage of animal models in cardiopulmonary resuscitation (CPR) research is the possibility to control confounding variables that may be impossible to standardize in clinical trials. A neglected effect of the anesthesia protocol in porcine CPR studies may be its impact on hemodynamic variables before induction of cardiac arrest. Accordingly, the purpose of the study reported here was to evaluate published CPR reports with regard to their anesthesia protocol. Methods: Of 100 articles that reported on laboratory models simulating cardiac arrest between 1987 and 1997 in peer-reviewed journals, 25 met inclusion criteria and were analyzed for values of coronary perfusion pressure, mean arterial pressure, heart rate, temperature, and cardiac index before induction of cardiac arrest. Subsequently, mean values for all animals in a given report were calculated and corrected for group size; statistical analysis was not performed since this was a survey only. Results: Different anesthesia protocols resulted in a widely distributed pattern of hemodynamic variables prior to induction of cardiac arrest. Ranges compared with reference values were: heart rate, 100 to 122 beats/min versus 105 ± 11 beats/min; mean arterial pressure, 68 to 130 mm Hg versus 102 ± 9 mm Hg; coronary perfusion pressure, 55 to 114 mm Hg (no reference value); cardiac index, 69 to 152 ml/kg/min versus 147 ± 22 ml/kg/min; body temperature, 37 to 38.5°C versus 38.5 ± 0.7°C. Conclusion: The anesthesia protocol may have an impact on hemodynamic variables before induction of cardiac arrest in CPR studies.

In 1981, an outbreak of herpetic disease developed in a colony of DeBrazza's monkeys (Cercopithecus neglectus). In seven of eight infected animals, clinical signs of infection included vesicular and ulcerative lesions on the lips, tongue, and/or palate. Histologic examination of lesions revealed intranuclear inclusion bodies, and electron microscopy revealed nucleocapsids and virions with typical herpesvirus morphology. Although a virus was isolated that appeared similar to monkey B virus, techniques available at the time did not allow precise identification of the virus. Analysis of serum from one surviving monkey collected 12 years after the outbreak revealed a pattern of reactivity characteristic of B virus-positive serum on the basis of results of ELISA and western immunoblot analysis. Polymerase chain reaction analysis of archived paraffin-embedded tissue specimens and molecular analysis of the one viral isolate obtained from a DeBrazza's monkey indicated that the virus responsible for the outbreak was a new genotype of B virus. Testing of sera from lion-tailed macaques (Macaca silenus) housed in an adjacent cage at the same zoo indicated that these animals harbored this virus and, thus, were the likely source of the virus that infected the DeBrazza's monkeys. This study documents usefulness of archiving samples from disease outbreaks for later analysis. In addition, this incident underscores the importance of considering herpes B virus infection when outbreaks of disease having characteristics of herpetic infections develop in nonhuman primates kept at institutions that also house macaques.

Background and Purpose: Wild-caught New World monkeys (NWM) from Central or South America are often infected with Trypanosoma species, including T. cruzi. In humans, T. cruzi causes Chagas' disease. Even in closed monkey colonies, T. cruzi can be propagated by blood-to-blood exposure, sexual activity, and transplacental transmission. Animal handlers and laboratory staff who deal with blood and tissues from infected NWM are at risk for acquiring Chagas' disease via accidental exposure. Methods: We screened 162 blood samples from wild-caught Saimiri sp. monkeys for Trypanosoma species infections by use of blood smear examination, ELISA, and polymerase chain reaction (PCR) analysis. Blood samples from 19 employees with recent history of monkey-associated injuries also were tested. Results: Six percent (10/162) of the monkey samples were T. cruzi positive on the basis of blood smear examination results, 10.4% (17/162) were positive by ELISA results, and 26.5% (43/162) were positive by PCR results. Other organisms identified by PCR analysis included T. rangeli in two animals, Plasmodium spp. in two animals (P. malariae confirmed by PCR results) and microfilariae in one animal (morphologically, Mansonella perstans). Evidence of trypanosome infection was not found in the 19 employee samples on the basis of results of any of the three aforementioned tests. Conclusions: Close attention must be paid to worker safety where wild-caught NWM are used. The PCR analysis has a clear advantage over conventional techniques (ELISA, blood smear) for screening NWM for trypanosome infections during quarantine and after employee injury.

Two established zebrafish colonies experienced increased mortality and decreased reproductive performance. Initial examination of several fish from one facility revealed hyperemic gills, petechia around the opercula, abdominal distention, and emaciation. Affected fish had congested liver with inflammation and multifocal hepatic necrosis. Large numbers of acid-fast-positive, rod-shaped bacteria were evident in multiple tissues and the blood. Mycobacterium fortuitum was subsequently isolated from several fish. Zebrafish from the second facility had skin erosions and ulceration along the flank just caudal to the pectoral fins. Large numbers of acid-fast-positive, rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas in go- nads, liver, and peritoneum from affected fish. Mycobacterium abscessus and M. chelonae were isolated and identified biochemically. Definitive diagnosis in these outbreaks was obtained by culture on selective media. Because Mycobacterium spp. grow extremely slowly and positive confirmation may require 45 to 60 days, Mycobacterium species-specific polymerase chain reaction analysis was used to provide a rapid screening assay for Mycobacterium spp. as well as for verification of culture results. To our knowledge, this is the first documentation of mycobacterial infection in laboratory-maintained zebrafish and provides guidelines for diagnosis, management, and prevention of atypical mycobacteriosis in laboratory zebrafish colonies.

During routine physical examination, a five-year-old male rhesus macaque (Macaca mulatta) was observed to have gaps in the right iris. Ophthalmic examination revealed inferior and superior iridodialysis with an anterior cortical cataract. The optic nerve head and fundus were normal. Uninvolved areas of the iris and anterior-chamber angle were normal on the basis of results of gonioscopy. Tonometry revealed normal intraocular pressure. The cause of the iridodialysis in this monkey's eye was not known. The animal had been housed individually since arrival due to requirements of the research protocol. Although the concomitant cataract supports a traumatic cause, there was no history of cranial or other ocular injuries. Trauma from fighting through the cage walls, self-trauma or falling inside the cage while under sedation cannot be ruled out. Multiple hematologic evaluations disclosed no abnormalities. This animal did not manifest behavioral abnormalities or any indication of pain. Therefore, treatment was not initiated. Intraocular pressure continues to be monitored at least semiannually.