Disaster Planning and Research Continuity in Responsible Animal Research.

Disaster preparedness for research facilities can be a daunting task. The purpose of this review is to introduce basic preparedness concepts and terminology so that facilities may begin to develop customized plans for their specific needs. Regulatory requirements are reviewed and an overview of the Incident Command System, National Preparedness System Planning Frameworks, and fundamental terms is provided. Important concepts for successful planning are then explored. Good planning involves fostering a culture of preparedness, resilience, and understanding the interactions and partnerships with other groups that are essential for core functions and incident response. Methods to gain institutional support and set up an advisory committee are examined in detail. Next, the steps to develop and carry out a plan are outlined. Risk assessments using an all hazards approach and tools such as risk indices and risk matrices are explained, and tips to design and test plans, train personnel, and evaluate improvement are discussed. Finally, special challenges unique to animal research facilities are considered along with ways to address them. Examples and information are drawn from a wide variety of organizations both to underscore themes common to all preparedness plans and to introduce new concepts that may be adapted for use in research institutions.

Fecal corticosterone levels in RCAN1 mutant mice.

Regulator of calcineurin 1 (RCAN1) is related to the expression of human neurologic disorders such as Down syndrome, Alzheimer disease, and chromosome 21q deletion syndrome. We showed here that RCAN1-knockout mice exhibit reduced innate anxiety as indicated by the elevated-plus maze. To examine whether glucocorticoids contribute to this phenotype, we measured fecal corticosterone in male wildtype and RCAN1-knockout mice and in male and female transgenic mice with neuronal overexpression of RCAN1 (Tg-RCAN1(TG)). We found no difference in fecal corticosterone levels of RCAN1-knockout mice and their wildtype littermates. As expected, we found differences between sexes in fecal corticosterone levels. In addition, we found higher levels of excreted corticosterone in Tg-RCAN1(TG) female mice as compared with female wildtype mice. Our data indicate normal diurnal corticosterone production in RCAN1 mutant mice and do not suggest a causal role in either the cognitive or anxiety phenotypes exhibited by RCAN1-knockout mice.

Pregnancy following homologous prepubertal ovarian transplantation in the dog.

In several canine models of hereditary human disease the homozygote dogs die prior to puberty, or have substantially reduced fertility. To create a clinically healthy animal that can be bred, but can also transmit the gene of interest, a model of homologous ovarian transplantation in prepubertal dogs was developed. Six dog leukocyte antigen (DLA) identical littermates underwent transplantation of ovarian cortical strips (n = 2) or the entire ovary (n = 4). Immunosuppression was maintained with cyclosporine and MMF in the immediate post-operative period and cyclosporine alone thereafter. All 6 dogs entered puberty and normal semiannual estrus cycles as demonstrated by both physical changes and increasing serum progesterone. Four dogs were bred to a proven stud male, and one became pregnant. Three viable fetuses with observable heart-beats were detected on ultrasound examination. Although the dog eventually aborted the litter, this work represents the first pregnancy achieved following a prepubertal ovarian transplant in the dog.

Investigation of appropriate sanitization frequency for rodent caging accessories: evidence supporting less-frequent cleaning.

The Guide for the Care and Use of Laboratory Animals states that sanitization of caging accessories (for example, filter tops and wire-bar lids) should be done every 2 wk. In this study we tested the hypothesis that organic contamination measured by the presence of ATP associated with organic material (measured with luciferase test swabs) and the number of bacterial colony-forming units (as determined by use of replicate organism detection and counting plates) on caging accessories did not differ significantly at 2 wk versus several months of use. The study evaluated 4 groups: mouse and rat ventilated and static wire-bar cages with or without filter tops (n = 10 per group). The cages were evaluated at several time points from 2 wk to 6 mo. For every cage type, ATP levels did not differ significantly between 14 and 90 d and, in most cases, between 14 and 180 d. In addition the number of bacterial colonies did not differ significantly between 14 and 120 d (and, in some cases, between 14 and 180 d). This study provides data relevant to establishing a validated frequency for sanitization of rodent caging accessories while controlling, and potentially decreasing, costs associated with sanitization.

Cross-fostering in combination with ivermectin therapy: a method to eradicate murine fur mites.

A colony of mutant mice with sickle cell anemia was infested with the fur mites Myocoptes musculinus and Myobia musculi. Pups of sickle-cell phenotype obtained by cesarean section prior to natural birth were of such poor vigor that none survived the combined insults of delivery by hysterectomy and cross-fostering. Consequently, surgical rederivation, the most reliable means of mite eradication, was not an option. Because furless mice are not susceptible to mite infestation and because neonates putatively remain free of mites until 4 to 5 days after birth, pups born by natural delivery were cross-fostered within 0 to 36 h to outbred lactating females treated once with ivermectin (2 mg/kg topically) at the time of transfer and housed in filter-top cages. Cross-fostering in conjunction with topical ivermectin administered to weaned mice one or more times at approximate 9-day intervals beginning on the day of weaning was successful in reliably eradicating mites. In addition, the 58% postnatal survivability of pups cross-fostered to dams given ivermectin was equivalent to that of natural-born pups that were reared by their untreated biological mothers.

A single dose of topical moxidectin as an effective treatment for murine acariasis due to Myocoptes musculinus.

Murine fur mites are reported to exist in over one-third of research institutions and can be problematic to eliminate. Current treatment strategies can be labor-intensive, toxic, and may confound research studies. The ideal method would be technically simple, safe, effective, and relatively inexpensive. When we found that mice from a noncommercial vendor were infested with Myocoptes musculinus, the animals were treated topically with Cydectin pour-on (containing moxidectin 0.5%) at 0.5 [corrected] mg/kg. After one treatment, mites were eradicated from all infested mice. No toxic effects or clinical signs of illness were observed in the mice. To the authors' knowledge, this is the first report of topical moxidectin as a treatment for murine acariasis.

Multiple vaginal exposures to low doses of R5 simian-human immunodeficiency virus: strategy to study HIV preclinical interventions in nonhuman primates.

A nonhuman-primate model of human immunodeficiency virus type 1 (HIV-1) infection that more closely emulates human heterosexual transmission by use of multiple exposures to low doses of virus is critical to better evaluate intervention strategies that include microbicides or vaccines. In this report, we describe such a system that uses female pig-tailed macaques exposed vaginally to a CCR5-using simian-human immunodeficiency virus (SHIV(SF162P3)) at weekly intervals. Results of dose-titration experiments indicated that 3 once-weekly exposures to 10 tissue culture infectious doses of SHIV(SF162P3) resulted in consistent transmission of virus and establishment of systemic infection. The efficacy of cellulose acetate phthalate (CAP) as a vaginal microbicide was evaluated by applying it to the vaginal vault of macaques (n = 4) 15 min before each weekly exposure to SHIV(SF162P3). One conclusion that can be drawn from the data derived from multiple exposures to virus is that CAP prevented infection in 12 of 13 possible chances for infection, over the course of 39 total exposures. Our findings provide a basis to refine monkey models for transmission of HIV-1, which may be relevant to preclinical evaluation for therapeutic interventions.

Urolithiasis associated with experimental lymphocytic choriomeningitis virus inoculation in Lewis rats.

A high frequency of struvite urolithiasis, hydronephrosis, and other urinary tract lesions developed in a group of Lewis rats inoculated intracranially with lymphocytic choriomeningitis virus (LCMV). Initially, clinically ill rats were referred to necropsy: 30 rats over 3 years. These rats had high frequency of urolithiasis (8/30, 27%), hydronephrosis (12/30, 40%), cystitis (9/30, 30%), transitional cell carcinoma (4/30, 13%), and pyelonephritis (19/30, 63%). Lesions were more common in LCMV-inoculated rats. After this trend was noted, all rats on this protocol were necropsied as part of a cohort study (n = 144). Although the apparent frequency of disease was lower due to increased sampling, there still was a high number of urolithiasis (9/144, 6%) and hydronephrosis (40/144, 28%) cases. All cases of urolithiasis developed in rats inoculated with LCMV (9/44, 20%), as did most cases of hydronephrosis (31/44, 70%). Although sham-injected and uninoculated control rats also had high frequency of hydronephrosis (6/57 [11%] and 3/43 [7%], respectively), LCMV-inoculated rats had a significantly higher frequency of disease than did sham inoculated (P < 0.0001) and uninoculated (P < 0.0001) controls. These results suggest that Lewis rats may be predisposed to developing lesions of the urinary tract, and that intracranial inoculation of rats with LCMV augments this tendency, leading to formation of struvite calculi and associated urinary tract disease.

Rodent vendor apparent source of mouse parvovirus in sentinel mice.

Mouse parvovirus (MPV) has been increasingly prevalent in laboratory animal facilities, and the source of infection often can be difficult to determine. After 4 years of sporadic MPV detected in our sentinel mice and continual failure to identify index cases in colony mice, we developed a regimen to house newly arrived vendor mice in large sterile cages with a high stocking density. Some of these mice were retained in isolation after the remaining mice were deployed as sentinels. After detecting MPV seropositive sentinel mice 4 weeks after introduction to the mouse colonies in one facility, the remaining naïve mice that had been previously housed with those sentinels also tested positive for MPV, despite never having been exposed to colony mice. These results suggest that commercially bred mice intended for use as sentinels may, in fact, arrive at animal facilities already infected with MPV. Depending upon numerous factors, including the health surveillance methods used, it is possible that a low prevalence of MPV may exist undetected at rodent vendors.

Chronic HIV-2 infection protects against total CD4+ cell depletion and rapid disease progression induced by SHIV89.6p challenge.

OBJECTIVE: To better understand HIV-1 sexual transmission risk, we have studied the susceptibility of HIV-2-exposed, uninfected (EU) female pig-tailed macaques to intravaginal (IVAG) re-challenge with the homologous HIV-2 strain, followed by heterologous SHIV89.6p. METHODS: Nine female macaques, previously protected by a post-exposure prophylaxis (PEP) regimen, along with one mock-treated EU animal, were re-exposed to HIV-2 by the IVAG route approximately 1.5 years later. A single follow-up challenge was performed approximately 1 year later with SHIV89.6p to assess susceptibility of chronic HIV-2-infected animals to further re-infection and pathogenic effects with a heterologous virus, somewhat mimicking HIV-1. RESULTS: Eight of ten macaques (80%) became infected systemically with HIV-2, and plasma or cervicovaginal vRNA levels did not appreciably differ from prior historic non-PEP control macaques. Interestingly, all eight HIV-2-infected females were susceptible to SHIV89.6p infection by either intravenous (n = 4) or IVAG exposure (n = 4) after one inoculation. Plasma vRNA levels in these groups were controlled by week 8 and there were no decrease in CD4+ T cells > 50%. The remaining two HIV-2 EU macaques, inoculated intrarectally with SHIV89.6p, were unable to control virus replication and succumbed to disease by week 25 or week 61. CONCLUSIONS: Our findings demonstrate that successful PEP regimens to prevent an initial infection do not have any lasting protective effects. The observed lack of cross-protection against SHIV89.6p transmission among chronic HIV-2-infected macaques provides modeling support for limited epidemiologic data indicating that human HIV-2 infection does not protect against HIV-1 infection, but may serve to alter overt clinical outcome.

Long-term results of dietary fenbendazole to eradicate Syphacia muris from rat colonies.

The pinworm Syphacia muris was eradicated from rats after treatment with fenbendazole-medicated chow (150 ppm) and without environmental decontamination for > 54 months. However, this regimen was successful only when the treatment was delivered and efficacy monitoring was done by personnel of the institutional animal resources program. The same pinworm elimination program failed 7 to 24 months after the cessation of treatment in a satellite colony in which animal care, including provision of medicated diet and sample collection for efficacy monitoring, was provided by research personnel. A failure to uniformly deliver adequate therapeutic doses or reinoculation of rats with pinworm eggs from the contaminated environment could not be excluded as causes of the failure. However, there were risk factors, and animal care practices unique to the satellite colony that may have facilitated the re-emergence of pinworms. These risk factors included hand-washing of cages, storage of contact bedding in areas that were not vermin-proof, and animal care provided by personnel having contact with rodents of pet-store origin.

L-arginine improves endothelial vasoreactivity and reduces thrombogenicity after thrombolysis in experimental deep venous thrombosis.

PURPOSE: Nitric oxide (NO) is important in regulation of platelet aggregation, endothelial function, and intravascular thrombosis. The purposes of this study were to assess the effect of thrombolysis on endothelial function in a porcine model of deep venous thrombosis (DVT) and to evaluate the effect of NO precursor l-arginine on endothelial function after thrombolytic therapy. METHODS: DVT was created in bilateral iliac veins by deploying a self-expanding stent-graft that incorporated an intraluminal stenosis, from a groin approach. Five pigs underwent sham operation. After 7 days of DVT, animals were randomized to three groups: saline pulse-spray (saline group, n = 5), thrombolytic pulse-spray with tissue plasminogen activator (alteplase, 8 mg; t-PA group, n = 5), and thrombolytic pulse-spray plus intravenous l-arginine (20 mmol/L; arginine group, n = 5). At 2 weeks iliac vein patency was evaluated at venography and intravascular ultrasound scanning. NO level was determined with a chemiluminescent assay of the nitrite and nitrate metabolites (NO(x)). Thrombogenicity was evaluated with radiolabeled platelet and fibrin deposition. Veins were harvested and evaluated with light microscopy and scanning electron microscopy. Endothelial function was evaluated with organ chamber analysis. RESULTS: All iliac veins remained patent at 2 weeks. The luminal areas in the sham, saline, t-PA, and arginine groups were 53 +/- 23 mm(2), 14 +/- 11 mm(2), 34 +/- 19 mm(2), and 42 +/- 21 mm(2), respectively. No difference in endothelial cell structure was observed between the three treatment groups at light microscopy or scanning electron microscopy. Although no difference in fibrin deposition was noted among the three treatment groups, decreased platelet deposition occurred in the arginine group compared with the saline or t-PA groups (P <.05). The arginine group showed greater endothelial-dependent relaxation compared with the t-PA or saline groups (73% +/- 23% vs 49% +/- 18% and 32% +/- 21%; P <.05). Local NO(x) level in the arginine group was correspondingly higher compared with the saline or t-PA groups (1.8 +/- 0.3 micromol/L vs 0.3 +/- 0.05 micromol/L and 0.2 +/- 0.04 micromol/L; P <.05). CONCLUSIONS: NO precursor l-arginine supplementation enhances NO production at sites of venous thrombosis. Moreover, l-arginine preserves endothelial vasoreactivity and reduces platelet deposition after thrombolysis in iliac DVT. These data suggest that l-arginine may preserve endothelial function after thrombolysis and may reduce the likelihood of postthrombotic syndrome.

Confirmed persistent mouse hepatitis virus infection and transmission by mice with a targeted null mutation of tumor necrosis factor to sentinel mice, using short-term exposure.

Mouse hepatitis virus (MHV) infection in immunocompetent mice is typically self limiting, and transmission is short lived. With the recent surge in the development of genetically engineered mutant mice with alterations in immune system components, however, MHV clearance may be disrupted. We report confirmed persistent transmission of MHV from tumor necrosis factor (TNF) knockout mice, B6.129S1-Tnftm1Lj (TNF -/-), to nude and immunocompetent sentinel mice over a period of five months. Infection with MHV was confirmed in nude sentinel mice by use of reverse transcriptase-polymerase chain reaction (RT-PCR) detection of viral RNA in ascending colon and feces. The RT-PCR-analyzed specimens recovered from sentinel animals were sequenced, and 92% homology to the N region of the MHV strain S genome was documented. In addition, immunocompetent mice had evidence of seroconversion to MHV infection and RT-PCR-positive fecal and ascending colon specimens after only 24 h of direct contact with the TNF -/- mice. To the authors' knowledge, this is the first reported experimental evidence that MHV transmission can occur for several months, from persistently infected mice to sentinel mice, over a short-term exposure period.

Postanesthesia death and suspected peracute endotoxic shock due to Pseudomonas putida in a cynomolgous macaque (Macaca fascicularis).

An adult male cynomolgous macaque (Macaca fascicularis) died suddenly after anesthesia for a positron emission tomography scan. Bacteriologic culture of the mucopurulent secretions recovered from the endotracheal tube yielded heavy growth of Pseudomonas putida, a known endotoxin producer. Histologically, the lungs had severe, diffuse perivascular edema and neutrophils marginating to the endothelium. The sudden death and the pathologic findings were consistent with peracute endotoxic shock. Numerous environmental swab specimens of the surgical suite and equipment were submitted for bacteriologic culture, as were swab specimens of endotracheal secretions from a control animal; however, Pseudomonas putida was not isolated from any specimen. The animal in this report may have carried Pseudomonas putida as a commensal in the oropharynx, and the stress of anesthesia may have resulted in increased sensitivity to the endotoxin.

Transluminal stent graft repair with Wallgraft endoprosthesis in a porcine arteriovenous graft pseudoaneurysm model.

PURPOSE: Pseudoaneurysm is a known complication of arteriovenous grafts in chronic hemodialysis and can result in graft disruption or thrombosis if left untreated. This study evaluated the safety and efficacy of endovascular repair with Wallgraft endoprosthesis (Boston Scientific, Inc, Watertown, Mass) in a porcine arteriovenous graft (AVG) pseudoaneurysm model. MATERIALS AND METHODS: Bilateral groin AVG pseudoaneurysms (n = 18) were created with an oversized Dacron interposition graft within a polytetrafluoroethylene femoral AVG in nine domestic swine and allowed to mature 28 +/- 4 days (standard deviation). Transluminal placement of Wallgraft was performed to exclude the pseudoaneurysm from the AVG circulation. Hemodialysis was performed (400 mL/min x 1 hour, with intravenous heparin 30 units/kg) every 4 days for a total of 6 weeks via 15-gauge needles in the treated AVG pseudoaneurysm site. Arteriography and duplex ultrasound scan were performed to determine AVG patency and pseudoaneurysm flow. Histologic evaluation was performed to determine Wallgraft morphology. In vitro pulsatile flow chamber was used to determine maximal flow volume without peri-Wallgraft endoleak. RESULTS: All AVG pseudoaneurysms were successfully excluded with the Wallgrafts. Twelve AVG (67%) remained patent at the completion of the study. No Wallgraft migration occurred from hemodialysis. Transient peri-Wallgraft endoleak (<2 hours after hemodialysis) was present in 13 of 18 (72%) and four of 12 (33%) AVG pseudoaneurysms by weeks 1 and 6, respectively. With maintenance of an intraluminal pressure of 80, 100, 120, 140, and 160 mm Hg in the pulsatile flow chamber, the maximal flow rates without peri-Wallgraft endoleak were 625 +/- 120, 650 +/- 145, 620 +/- 95, 425 +/- 110, and 262 +/- 86 mL/min. Scanning electron microscopy showed a neointimal layer covered with thrombus on the Wallgraft surface. CONCLUSION: Endoluminal placement of Wallgraft endoprosthesis provides adequate structural support for continuous hemodialysis after AVG pseudoaneurysm exclusion. Transient blood flow in the pseudoaneurysm cavity may occur immediately after the hemodialysis, which may represent the effect of heparin used during hemodialysis. This study suggests Wallgraft is a safe and effective treatment for AVG pseudoaneurysm and permits continuous hemodialysis.

High Mortality in Zebrafish (Danio rerio).

A group of 100 adult zebrafish were housed in a new system at a stocking density of 20 fish per tank. Four weeks after arrival, 15 fish presented with petechial hemorrhages and ulceration on the surfaces of the skin. Samples of the fish were collected for histopathology, fungal culture, and bacterial culture and sensitivity. Water samples were analyzed for pH, ammonia, nitrite, and submitted for bacterial and fungal culture. Histologically, the epidermis had multiple areas of ulceration and mononuclear cell infiltrate. Gram-positive bacteria were observed beneath the surface of the skin and surrounding the outer aspect of the spinal cord. Both Aeromonas hydrophila and A. sobria were isolated from the affected fish, and a diagnosis of motile aeromonad septicemia (MAS) was made. Water from the tanks had a nitrite level of 1-5 ppm, a toxic concentration that indicated poor water quality. Because the housing system had been seeded with Nitrobacter spp. and Nitrosomonas spp. only 2 weeks prior to the arrival of the fish, a lack of colonizing nitrifying bacteria was deemed to be the cause of the high nitrite level, which, along with over-crowding, stressed the fish and increased their susceptibility to MAS. No further cases of septicemia were observed once the nitrite level and stocking density were reduced.