PCR Testing of Media Placed in Soiled Bedding as a Method for Mouse Colony Health Surveillance.

Rodent colony health surveillance has traditionally been accomplished by testing sentinel animals that have been exposed to soiled bedding from colony animals. Collecting samples from exhaust plenums on ventilated caging systems, followed by PCR analysis, has emerged as another promising method for health surveillance. However, environmental testing at the rack level is not effective for all ventilated rack designs. In this study, we tested whether media placed in soiled bedding is effective in detecting 3 adventitious agents: mouse norovirus (MNV), Helicobacter spp., and fur mites. Soiled bedding was collected from pathogen-positive colony mice and distributed to traditional sentinel mouse cages and mouse-free experimental cages every 1 to 2 wk for static and ventilated cages, respectively. Experimental cages contained 10 flocked swabs ('passive swabs') and 1 piece of filter media. After 90 d, fresh feces, pelage swabs, and blood were collected from the sentinel cages, and the passive swabs and filter media were collected from the experimental cages. Concurrently, 10 additional flocked swabs ('active swabs') were stirred through the cumulated soiled bedding of each experimental cage. Sentinel mice were positive for MNV and Helicobacter spp., but negative for fur mites by pelage swab PCR. All samples from experimental cages were positive for Helicobacter spp. and fur mites in both caging types. For MNV, passive swabs were most effective at detection (100%), followed by active swabs (80% to 100%) and filter media (60% to 80%). These findings suggest that testing media in pooled soiled bedding samples is more effective than traditional sentinel methods for colony health surveillance and is a viable option when sampling at the rack level is ineffective.

Pleiotropic phenotype of a genomic knock-in of an RGS-insensitive G184S Gnai2 allele.

Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Galpha(i2) subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Galpha(i2)(G184S) knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Galpha(i2)(+/G184S) mice show some but not all of these abnormalities. Thus, loss of RGS actions at Galpha(i2) produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.

Moxidectin toxicity in senescence-accelerated prone and resistant mice.

Moxidectin has been used safely as an antiparasitic in many animal species, including for the eradication of the mouse fur mite, Mycoptes musculinus. Although no side effects of moxidectin have previously been reported to occur in mice, 2 strains of the senescence-accelerated mouse (SAMP8 and SAMR1) sustained considerable mortality after routine prophylactic treatment. To investigate the mechanism underlying this effect, moxidectin toxicosis in these mice was evaluated in a controlled study. Moxidectin was applied topically (0.015 mg), and drug concentrations in both brain and serum were analyzed by using HPLC coupled with mass spectrometry. The moxidectin concentration in brain of SAMP8 mice was 18 times that in controls, and that in brain of SAMR1 mice was 14 times higher than in controls, whereas serum moxidectin concentrations did not differ significantly among the 3 strains. Because deficiency of the blood-brain barrier protein P-glycoprotein leads to sensitivity to this class of drugs in other SAM mice, Pgp immunohistochemistry of brain sections from a subset of mice was performed to determine whether this commercially available analysis could predict sensitivity to this class of drug. The staining analysis showed no difference among the strains of mice, indicating that this test does not correlate with sensitivity. In addition, no gross or histologic evidence of organ toxicity was found in brain, liver, lung, or kidney. This report shows that topically applied moxidectin at a standard dose accumulates in the CNS causing toxicosis in both SAMP8 and SAMR1 mice.

Influence of Pain and Analgesia on Cancer Research Studies.

Mice and rats are valuable and commonly used as models for the study of cancer. The models and methods of experimentation have the potential to cause pain to some degree, and all charged with ensuring animal welfare must determine how to manage it. A commonly posed question, especially from investigators and IACUC, is whether the provision of analgesic agents will render the model invalid. Left untreated, pain is a stressor and has negative consequences, most notably immune system perturbations. In addition, analgesic agents in the opioid and NSAID drug classes exhibit immunomodulatory activity and influence processes such as cell proliferation, apoptosis, and angiogenesis that are important in cancer formation. Therefore, both pain and the agents used to alleviate it have the potential to act as confounding factors in a study. This review article presents data from both human medicine and work with animal models in an attempt to help inform discussions about the withholding of analgesic agents from animals used in cancer studies.

Evaluation of 4 Presurgical Skin Preparation Methods in Mice.

Mice routinely undergo surgical procedures for use in research; however, studies of skin preparation methods to achieve antisepsis are rare. The present study evaluated 4 skin preparation treatments: depilatory agent followed by povidone-iodine and alcohol scrub; depilatory agent followed by povidone-iodine and saline scrub; electric clippers followed by povidone-iodine and alcohol scrub; and electric clippers followed by povidone-iodine and saline scrub. Swabs for bacterial culture were obtained immediately after hair removal and after scrubbing to measure the reduction in bacterial load. Full-thickness incisions were assigned ASEPSIS wound scores and examined histologically on days 0, 1, and 7 after surgery. Neither bacterial load growth nor ASEPSIS wound scores differed among any of the treatments. Histopathology revealed statistically significant but biologically irrelevant differences. Overall all treatment methods achieved acceptable bacterial load reduction and surgical site healing.

Pharmacokinetic Profiles of Nalbuphine after Intraperitoneal and Subcutaneous Administration to C57BL/6 Mice.

Mice undergo a variety of procedures that necessitate the use of analgesic agents. Opioids are often essential to successful pain management plans, but most are controlled substances, and their use requires appropriate federal and state registrations. Nalbuphine is a potentially effective opioid analgesic for mice that is not currently classified as a controlled substance. This compound has received little attention as an analgesic for mice, and standard dosage regimens have not been developed. Here we compared the pharmacokinetic profiles of 10 mg/kg nalbuphine in male C57BL/6 mice subcutaneous or intraperitoneal administration. Blood was collected from 3 mice per treatment at 5, 10, 20, and 30 min and 1, 2, 3, 6, 12, and 24 h after administration. Plasma concentrations were measured, and standard pharmacokinetic parameters were calculated. Profile characteristics for each route of administration were similar, with significant differences in plasma concentration at 5 and 30 min and 1 and 3 h. Nalbuphine was absorbed more quickly when administered subcutaneously (Tmax, 5 min) than intraperitoneally (Tmax, 10 min), whereas the drug's half-life was similar between the intraperitoneal (0.94 h) and subcutaneous (1.12 h) routes. The AUC0-tldc and AUC0-inf were higher but the apparent clearance and apparent volume of distribution were lower after subcutaneous administration compared with intraperitoneal dosing. Plasma concentrations were below the level of detection by 12 h. These results suggest that nalbuphine is absorbed in and eliminated quickly from mice, making it a possible candidate for acute pain management.

Booklice (Liposcelis spp.), Grain Mites (Acarus siro), and Flour Beetles (Tribolium spp.): 'Other Pests' Occasionally Found in Laboratory Animal Facilities.

Pests that infest stored food products are an important problem worldwide. In addition to causing loss and consumer rejection of products, these pests can elicit allergic reactions and perhaps spread disease-causing microorganisms. Booklice (Liposcelis spp.), grain mites (Acarus siro), and flour beetles (Tribolium spp.) are common stored-product pests that have previously been identified in our laboratory animal facility. These pests traditionally are described as harmless to our animals, but their presence can be cause for concern in some cases. Here we discuss the biology of these species and their potential effects on human and animal health. Occupational health risks are covered, and common monitoring and control methods are summarized.

The effect of light level, CO2 flow rate, and anesthesia on the stress response of mice during CO2 euthanasia.

Euthanasia protocols are designed to mitigate the stress experienced by animals, and an environment that induces minimal stress helps achieve that goal. A protocol that is efficient and practical in a typical animal research facility is also important. Light intensity, isoflurane, and CO2 flow rate were studied for their impact on the stress response of mice during CO2 euthanasia. Behavior was observed and scored during euthanasia and serum corticosterone was measured immediately after death. Unsurprisingly, animals euthanized with a high-flow rate of CO2 became unconscious in the least amount of time, while animals euthanized with a low-flow rate required the most time to reach unconsciousness. There was a significant increase in anxious behaviors in animals in the isoflurane group (F1,12 = 6.67, P = 0.024), the high-flow rate CO2 group (F1,12 = 10.24, P = 0.007), and bright chamber group (F1,12 = 7.27, P = 0.019). Serum corticosterone was highest in the isoflurane group (124.72 ± 83.98 ng/ml), however there was no significant difference in corticosterone levels observed for the other study variables of light and flow-rate. A darkened chamber and low CO2 flow rates help to decrease stress experienced during CO2 euthanasia, while the use of isoflurane was observed to increase the stress response during euthanasia.

Use of low-dose chlorpromazine in conjunction with environmental enrichment to eliminate self-injurious behavior in a rhesus macaque (Macaca mulatta).

A 7-year-old, captive-bred, female rhesus macaque was placed in a quarantine facility upon arrival at our institution. At release from quarantine, she was observed pawing at and chewing on her left cheek. Physical examination revealed ulcerative lesions on the buccal surface of the left cheek. Initial differential diagnoses included Cercopithecine herpesvirus 1 (B virus)-induced lesions and bacterial infection. Dental abnormalities and cheek pouch foreign body were ruled out during the physical exam. Treatment with 30 mg/kg cefazolin intramuscularly every 12 h was initiated. Twelve days later, the animal presented with a 2 x 2-cm, full-thickness erosion involving the opposite (right) cheek. Treatment with buprenorphine (0.1 mg/kg intramuscularly every 24 h) was initiated. Cultures for B virus were negative, and only nonpathogenic bacteria were isolated from swabs of the lesions. Hematology and serum chemistry profiles were normal. A wedge biopsy of the lesion revealed no definitive etiology. Further observation revealed that the lesions likely resulted from self-injurious behavior (SIB). Treatment with low-dose chlorpromazine (1 mg/kg intramuscularly once daily for 25 days, and then 0.5 mg/kg intramuscularly once daily for 25 days) was initiated. Bodyweight and condition were maintained during therapy, and serial hematology and serum chemistry profiles were normal. The animal was moved into a different room, and a toy "necklace" was created. The SIB was eliminated, and lesions healed within 35 days. Presently, 20 months after presentation, this animal remains in good health.

Modeling the effect of a prosthetic limb on 4-km pursuit performance.

The individual pursuit is a 4-km cycling time trial performed on a velodrome. Parathletes with transtibial amputation (TTA) have lost physiological systems, but this may be offset by the reduced aerodynamic drag of the prosthesis. This research was performed to understand the effect of a unilateral TTA on Olympic 4-km pursuit performance. A forward-integration model of pursuit performance explored the interplay between power loss and aerodynamic gains in parathletes with TTA. The model is calibrated to a 4-km pursuit time of 4:10.5 (baseline), then adjusted to account for a TTA. Conditions simulated were based on typical pedal asymmetry in TTA (AMP), if foot stiffness were decreased (FLEX), if pedaling asymmetries were minimized (ASYM), if the prosthesis were aerodynamically optimized (AERO), if the prosthesis had a cosmetic cover (CC), and if all variables were optimized (OPT). A random Monte Carlo analysis was performed to understand model precision. Four-kilometer pursuit performances predicted by the model were 4:10.5, 4:20.4, 4:27.7, 4:09.2, 4:19.4, 4:27.9, and 4:08.2 for the baseline, AMP, FLEX, ASYM, AERO, CC, and OPT models, respectively. Model precision was ±3.7 s. While the modeled time decreased for ASYM and OPT modeled conditions, the time reduction fell within model precision and therefore was not significant. Practical application of these results suggests that parathletes with a TTA could improve performance by minimizing pedaling asymmetry and/or optimizing aerodynamic design, but, at best, they will have performance similar to that of intact cyclists. In conclusion, parathletes with TTA do not have a net advantage in the individual pursuit.

Behavioral, clinical and pathological effects of multiple daily intraperitoneal injections on female mice.

Pharmacological agents are commonly administered to mice through multiple intraperitoneal (i.p.) injections. The i.p. route of administration is usually considered safe, but questions of animal welfare arise when protocols require that multiple injections be given to the same animal. IACUCs must consider the potential risks associated with multiple i.p. injections in order to determine the maximum number of injections an animal can receive within a study protocol, but there are no published studies of such potential risks. The authors investigated the effects of 30 daily i.p. saline injections on the behavior, body condition, weight, fecal corticosterone levels, hematology and pathology of female adult mice. Results indicate that multiple i.p. injections do not cause any ill effects in mice.

Survey of laboratory animal technicians in the United States for Coxiella burnetii antibodies and exploration of risk factors for exposure.

Little is known about the prevalence of zoonotic infections among laboratory animal care technicians (LAT). Q fever, a disease caused by Coxiella burnetii, is a known occupational hazard for persons caring for livestock. We sought to determine the seroprevalence of C. burnetii antibodies among LAT and to identify risk factors associated with C. burnetii seropositivity. A survey was administered and serum samples collected from a convenience sample of 97 LAT. Samples were screened by using a Q fever IgG ELISA. Immunofluorescent antibody assays for phase I and phase II IgG were used to confirm the status of samples that were positive or equivocal by ELISA; positive samples were titered to endpoint. Antibodies against C. burnetii were detected in 6 (6%) of the 97 respondents. In our sample of LAT, seropositivity to C. burnetii was therefore twice as high in LAT as compared with the general population. Age, sex, and working with sheep regularly were not associated with seropositivity. Risk factors associated with seropositivity included breeding cattle within respondent's research facility, any current job contact with waste from beef cattle or goats, and exposure to animal waste during previous jobs or outside of current job duties. Only 15% of responding LAT reported being aware that sheep, goats, and cattle can transmit Q fever. Research facilities that use cattle or goats should evaluate their waste-management practices and educational programs in light of these findings. Additional efforts are needed to increase awareness among LAT regarding Q fever and heightened risk of exposure to infectious materials. Physicians should consider the risk of infection with C. burnetii when treating LAT with potential occupational exposures.

Assessment of sterility in fluid bags maintained for chronic use.

Fluid therapy is an integral component of standard supportive care in veterinary medicine and is often given subcutaneously for ease of administration. Multiple animals may be treated by using the same bag. These bags often are used repeatedly until they are empty or until a time specified by an IACUC or similar advisory group. This practice introduces the risk of contamination. Here we assessed the sterility of multiple-use fluid bags in the laboratory setting for a maximum of 60 d. Bags were manipulated to mimic infrequent and frequent use. Bacterial cultures of fluid and bag wall and assays for endotoxin and ATP activity were negative at all time points through 30 d. Two fluid samples yielded bacterial growth at 60 d, although all other tests were negative. These results suggest that fluid bags used chronically can be maintained in a sterile condition for a maximum of 30 d.

Analgesic effects of meloxicam, morphine sulfate, flunixin meglumine, and xylazine hydrochloride in African-clawed frogs (Xenopus laevis).

We evaluated analgesic use and analgesiometry in aquatic African-clawed frogs (Xenopus laevis). We used the acetic acid test (AAT) to assess the analgesic potential of systemic xylazine hydrochloride, meloxicam, flunixin meglumine, and morphine sulfate after injection into the dorsal lymph sac. Flunixin meglumine provided better analgesia than did the other drugs, most evident at 5 and 9 h after administration. Because the AAT was associated with the development of dermal lesions, we discontinued use of this assay and chose the Hargreaves test as an alternative method of measuring nociception in Xenopus. This assay is commonly performed in rodents, but its efficacy in an aquatic species such as Xenopus was unknown prior to this study. We found that the Hargreaves test was an effective measure of nociception in Xenopus, and we used it to evaluate the effectiveness of the nonopiod agents xylazine hydrochloride, meloxicam, and flunixin meglumine both in the absence of surgery and after surgical oocyte harvest. Similar to findings from the AAT, flunixin meglumine provided better analgesia in the Hargreaves test than did the other agents when analyzed in the absence of surgical intervention. Results were equivocal after oocyte harvest. Although surgical oocyte harvest is a common procedure in Xenopus, and currently there are no published recommendations for analgesia after this invasive surgery. Future studies are needed to clarify the efficacy of nonsteroidal antiinflammatory drugs for that purpose.

Comparison of the effects of ketamine, ketamine-medetomidine, and ketamine-midazolam on physiologic parameters and anesthesia-induced stress in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

This study compared the cardiovascular, respiratory, anesthetic, and glucocorticoid effects of ketamine alone with ketamine-medetomidine and ketamine-midazolam in rhesus and cynomolgus macaques. Macaques were given either intramuscular ketamine (10 mg/kg), intramuscular ketamine-medetomidine (3 mg/kg; 0.15 mg/kg), or oral midazolam (1 mg/kg) followed by intramuscular ketamine (8 mg/kg). The addition of medetomidine, but not midazolam, provided muscle relaxation and abolishment of reflexes that was superior to ketamine alone. In addition, medetomidine did not cause clinically relevant effects on cardiovascular and respiratory parameters when compared with ketamine. These 3 protocols did not have significantly different effects on fecal glucocorticoid metabolites. These results suggest that medetomidine can be a valuable addition to ketamine for healthy patients, whereas oral midazolam at the tested dose does not provide additional benefits.

Isoflurane waste anesthetic gas concentrations associated with the open-drop method.

The open-drop technique is used frequently for anesthetic delivery to small rodents. Operator exposure to waste anesthetic gas (WAG) is a potential occupational hazard if this method is used without WAG scavenging. This study was conducted to determine whether administration of isoflurane by the open-drop technique without exposure controls generates significant WAG concentrations. We placed 0.1, 0.2, or 0.3 ml of liquid isoflurane into screw-top 500 or 1000 ml glass jars. WAG concentration was measured at the opening of the container and 20 and 40 cm from the opening, a distance at which users likely would operate, at 1, 2, or 3 min WAG was measured by using a portable infrared gas analyzer. Mean WAG concentrations at the vessel opening were as high as 662 +/- 168 ppm with a 500 ml jar and 122 +/- 87 ppm with a 1000 ml jar. At operator levels, WAG concentrations were always at or near 0 ppm. For measurements made at the vessel opening, time was the only factor that significantly affected WAG concentration when using the 500 ml jar. Neither time nor liquid volume were significant factors when using 1000 ml jar. At all liquid volumes and time points, the WAG concentration associated with using the 500 ml container was marginally to significantly greater than that for the 1000 ml jar.

Injury related to environmental enrichment in a dog (Canis familiaris): gastric foreign body.

A pregnant 7-y-old Beagle crossbred dog (Canis familiaris) presented with clinical signs of lethargy, dehydration, and occasional vomiting. The dog was managed with fluids, antibiotics, and supportive care for several days in an effort to maintain the pregnancy. The bitch aborted the pups at approximately 50 d of gestation and was euthanized due to her poor reproductive performance and age. Necropsy revealed a compact mass of plastic pieces in the pylorus of the stomach. The gastric foreign body was discovered to be the vinyl covering of a bed that was in the dog's run as part of the environmental enrichment program for this animal. The use of that type of dog bed was discontinued. This case emphasizes that any type of enrichment can cause harm and the risks must be assessed carefully before implementing any enrichment device.

Thrombospondin-2 influences the proportion of cartilage and bone during fracture healing.

Thrombospondin-2 (TSP2) is a matricellular protein with increased expression during growth and regeneration. TSP2-null mice show accelerated dermal wound healing and enhanced bone formation. We hypothesized that bone regeneration would be enhanced in the absence of TSP2. Closed, semistabilized transverse fractures were created in the tibias of wildtype (WT) and TSP2-null mice. The fractures were examined 5, 10, and 20 days after fracture using microCT, histology, immunohistochemistry, quantitative RT-PCR, and torsional mechanical testing. Ten days after fracture, TSP2-null mice showed 30% more bone by microCT and 40% less cartilage by histology. Twenty days after fracture, TSP2-null mice showed reduced bone volume fraction and BMD. Mice were examined 5 days after fracture during the stage of neovascularization and mesenchymal cell influx to determine a cellular explanation for the phenotype. TSP2-null mice showed increased cell proliferation with no difference in apoptosis in the highly cellular fracture callus. Although mature bone and cartilage is minimal 5 days after fracture, TSP2-null mice had reduced expression of collagen IIa and Sox9 (chondrocyte differentiation markers) but increased expression of osteocalcin and osterix (osteoblast differentiation markers). Importantly, TSP2-null mice had a 2-fold increase in vessel density that corresponded with a reduction in vascular endothelial growth factor (VEGF) and Glut-1 (markers of hypoxia inducible factor [HIF]-regulated transcription). Finally, by expressing TSP2 using adenovirus starting 3 days after fracture, chondrogenesis was restored in TSP2-null mice. We hypothesize that TSP2 expressed by cells in the fracture mesenchyme regulates callus vascularization. The increase in vascularity increases tissue oxemia and decreases HIF; thus, undifferentiated cells in the callus develop into osteoblasts rather than chondrocytes. This leads to an alternative strategy for achieving fracture healing with reduced endochondral ossification and enhanced appositional bone formation. Controlling the ratio of cartilage to bone during fracture healing has important implications for expediting healing or promoting regeneration in nonunions.

Study of two devices used to maintain normothermia in rats and mice during general anesthesia.

Rodents are very susceptible to hypothermia during anesthetic events because of their high body surface-to-mass ratio. This study examined the effectiveness of 2 heating devices, a heatpad and a circulating hot-water blanket, during 60 min of isoflurane general anesthesia in rats and mice (n = 6 per treatment). In addition, 1 control group of animals for each species was anesthetized with no heat source (n = 6). Both devices carried minimal risk of causing thermal burns or hyperthermia. Rats on the circulating water blanket showed a slight decrease (0.11 +/- 0.19 degrees C) from the initial (time 0) body temperature (mean +/- standard error), whereas the heatpad was associated with a significant increase (0.96 +/- 0.10 degrees C). Mice on the circulating water blanket showed a significant decrease (0.46 +/- 0.05 degrees C) in body temperature. The trend in mice on the heatpad was similar to that in rats, with a significant increase (0.94 +/- 0.13 degrees C) from the body temperature at time 0. Although statistically significant, these deviations from baseline body temperature were not considered physiologically relevant. In comparison, body temperatures decreased significantly in rats and mice (4.42 +/- 0.60 and 9.90 +/- 0.35 degrees C, respectively) with no heat source. Both heating devices were safe and effective, but the low cost, ease of maintenance, and portability of the heatpad may make it a more desirable choice in some facilities.

Lanolin as a treatment option for ringtail in transgenic rats.

Ringtail is a condition characterized by dry skin and annular constrictions that sometimes result in loss of portions of the tail. This condition most commonly affects preweanling rats, and low relative humidity is thought to be a principal cause. The use of transgenic rats in our facility has been increasing since 2002, and we recently diagnosed several litters from transgenic Fischer 344 rats (Rattus norvegicus) with ringtail. Treatment was necessary to maintain the health and integrity of the tails to allow genotyping. Lanolin ointment was chosen because it is a nontoxic, inexpensive, effective moisturizer used for treating human skin conditions. We examined 5 litters comprising 37 pups total, ranging in age from 7 to 17 days at the time of presentation. Animals in 3 litters were randomly assigned to a treatment or nontreatment group, and all animals in the remaining 2 litters were treated. Lanolin was applied to the tails of treatment groups once daily for 6 d. Treatment was tolerated well by pups and no animals were rejected by the dams. After treatment, tail condition was scored from 0 to 3, with 0 representing a tail normal in appearance, and 3 representing severe disease. Chi square testing showed marginal statistical significance, with a trend for a higher percentage of treated rats having healthier tails on day 7 compared to untreated pups. The Pearson correlation between treatment and tail condition scores was significant. Results indicate that lanolin was an efficacious treatment option for ringtail.