Stability of Doxycycline in Feed and Water and Minimal Effective Doses in Tetracycline-Inducible Systems.

Despite the extensive use of doxycycline in tetracycline-inducible rodent models, little is known regarding its stability in feed or water or the most effective route or dose. We assessed the concentrations of doxycycline in reverse-osmosis-purified (RO; pH 6.0) and acidified RO (pH 2.6) water in untinted or green-tinted bottles. Doxycycline remained stable in all groups for 7 d and in acidified water in untinted bottles for 14 d. Fungal growth occurred in nonacidified water in tinted and untinted bottles by 12 and 14 d, respectively, and in tinted bottles containing acidified water on day 14, but not in untinted bottles with acidified water. Doxycycline concentrations were also assessed before and at various points after the pelleting of feed from 2 vendors. Each batch was divided for storage at 4 °C, at room temperature, or within ventilated mouse isolator cages and then sampled monthly for 6 mo. Drying caused the greatest decline in doxycycline concentration, whereas γ-irradiation plus shipping and storage condition had minimal effect. Two mouse lines with tetracycline-inducible promoters received 25, 150, or 467 µg/mL or 2 mg/mL doxycycline in water and 200 or 625 ppm in feed before analysis of GFP expression. GFP was expressed in Rosa-rtTA2 mice at 150 µg/mL, whereas Cags-rtTA3 mice required 25 µg/mL. These studies indicate that 1) doxycycline-compounded feed can be handled in the same manner as standard rodent feed, 2) tinted water bottles are not necessary for maintaining drug concentrations, and 3) concentrations lower than those used typically may be effective in lines with tetracycline-inducible promoters.

Corynebacterium bovis: epizootiologic features and environmental contamination in an enzootically infected rodent room.

Corynebacterium bovis is a common pathogen in athymic nude mouse colonies. Control and eradication of the organism are challenging because depopulation and restricted colony access are often not options within vivaria. We evaluated potential sources and dissemination routes of C. bovis in an enzootically infected colony. Immunocompetent mice and personnel were evaluated for their potential to carry C. bovis, and husbandry and sanitation methods were evaluated for their efficacy in preventing cross-contamination. C. bovis was detected in furred immunocompetent mice previously exposed to infected athymic nude mice and in the nasopharynx of humans. Microisolation cages were not effective in maintaining athymic nude mice C. bovis-free when they were housed in a room known to contain immunodeficient mice with C. bovis infections. A tunnel washer that provided a ≥180 °F final rinse provided effective elimination of C. bovis from cage components. Passive and active air sampling techniques showed airborne dispersal of C. bovis despite the use of individually ventilated caging systems and stringent operational standards. Bacterial growth was not observed in settle plates placed inside autoclaved individually ventilated microisolation cages on various ventilated racks for 24-h periods. C. bovis aerosolization was shown to be a means of spread of the bacterium during cage-change procedures inside a class II type A2 biosafety cabinet. Our findings indicate that C. bovis can be a pervasive environmental contaminant in infected rodent holding rooms and successful eradication strategies must include environmental decontamination and attention to air quality.

Strategies to prevent, treat, and provoke Corynebacterium-associated hyperkeratosis in athymic nude mice.

Athymic nude mice infected with Corynebacterium bovis typically exhibit transient hyperkeratotic dermatitis. Our vivarium experienced an increased incidence of disease characterized by persistent skin lesions and increased mortality, leading to this study. For detection of infection, skin and buccal swab methods showed comparable sensitivities in nude mice. Various prevention, treatment, and eradication strategies were evaluated through clinical assessment, microbiology, and histopathology. In experimentally naïve athymic nude mice, a 2-wk course of prophylactic amoxicillin-containing diet (1200 ppm amoxicillin; effective dose, 200 mg/kg) was ineffective at preventing infection or disease. There was also no significant difference in disease duration or severity in athymic nude mice that received amoxicillin diet or penicillin-streptomycin topical spray (penicillin, 2500 U/mL; streptomycin, 2500 µg/mL). Prolonged treatment with 4 or 8 wk of amoxicillin diet cleared only a small number of athymic nude mice that had subclinical C. bovis infections. Antibiotic sensitivity of C. bovis isolates demonstrated a small colony isolate with less susceptibility to all antibiotics compared with a large colony isolate. Resistance did not appear to develop after prolonged treatment with amoxicillin. Provocation testing by administration of cyclophosphamide (50 mg/kg i.p. every 48 to 72 h for 90 d) to subclinically infected athymic nude mice resulted in prolonged clinical disease that waxed and waned without progression to severe disease. Our findings suggest that antibiotic prophylaxis and treatment of clinical disease in experimentally naïve mice is unrewarding, eradication of bacterial infection is difficult, and severe disease associated with C. bovis is likely multifactorial.

Treatment and eradication of murine fur mites: I. Toxicologic evaluation of ivermectin-compounded feed.

Fur mite outbreaks remain a persistent problem in laboratory mouse colonies. All currently published treatment methods are labor-intensive, expensive, or unreliable. During a recent outbreak with Myobia musculi and Myocoptes musculinus in a large colony (approximately 30,000 cages), we developed a feed-based treatment regime in which ivermectin was the active ingredient. Rodent feed was compounded with 3 different concentrations of ivermectin (12, 24, and 48 ppm) and γ-irradiated. Postcompounding analysis revealed loss of ivermectin during manufacturing, but the remaining drug was stable for at least 6 mo. In an 8-wk toxicity study in a C57BL/6NTac mouse breeding colony, ad-libitum feeding of the 3 diets yielded estimated doses of 1.3, 2.7, and 5.4 mg/kg. Adult mice lacked adverse clinical effects, except that 1 of the 144 mice in the 48-ppm group developed tremors and ataxia and was euthanized. No significant differences between doses were revealed by CBC, serum chemistry, body weight, or gross necropsy. Plasma drug concentrations plateaued at a dose-dependent level 7 to 10 d after initiation of treatment and decreased to undetectable levels 6 to 9 d after its discontinuation. Fertility of the P0 generation was unaffected. Pup mortality was higher in the 24- and 48-ppm groups, reaching 100% at the higher dose. Animals exposed to ivermectin as neonates had normal weaning weights, but mice receiving 24-ppm feed had lower adult weights. Our results indicate that using feed containing 12 ppm ivermectin (estimated ingested dose, 1.3 mg/kg) was safe in a C57BL/6NTac breeding colony.

Treatment and eradication of murine fur mites: II. Diagnostic considerations.

Fur mites are a persistent problem in contemporary laboratory mouse colonies. We conducted several studies to evaluate fur mite diagnostic methodologies and interpretation of results. Retrospective analysis of test results from sentinel mice exposed to soiled bedding collected from colonies infested with Myobia musculi and Myocoptes musculinus revealed the skin scrape test to be more reliable than pelt examination, provided that both the head and dorsal thoracolumbar regions were sampled. To assess their diagnostic accuracy, 3 commercial laboratories were sent positive control slides containing mites, mite parts, or eggs in sets of slides containing diagnostic skin scrapings in varying ratios. Laboratory B correctly identified the positive control slide. Laboratory A identified 1 of 3 positive control slides, whereas laboratory C failed to identify both positive control slides submitted. To determine the time required for a mouse to shed its entire hair coat, fur of Crl:CD1(ICR), BALB/cAnNCrl, and Crl:CFW(SW) albino mice was dyed black and the presence of dyed fur evaluated monthly for 8 mo. Limited dyed hair was still present at 8 mo; therefore, finding eggs or egg casings many months after treatment cessation does not necessarily imply treatment failure. To evaluate the effectiveness of soiled bedding sentinels for detection of fur mites in a mite-infested colony, we exposed naïve mice to varying amounts (100%, 50%, 25%, 2.5%, and 0%) of soiled bedding in clean bedding. As little as 2.5% soiled bedding resulted in detection of a positive sentinel within a 2-mo period.

Treatment and eradication of murine fur mites: III. Treatment of a large mouse colony with ivermectin-compounded feed.

We determined the efficacy of ivermectin-compounded feed against fur mites in mice and describe its use to eradicate mites in vivaria holding approximately 30,000 cages. C57BL/6NCrl mice infested with Myobia musculi and Myocoptes musculinus were treated with ivermectin-compounded feed (approximate ingested dose, 1.3 mg/kg) for 1, 4, or 8 consecutive weeks. Regardless of treatment duration, all treated mice, as well as contact sentinels, remained free of fur mites for as long as 21 wk after treatment. No adverse effects were observed. Subsequently, facility-wide treatment was implemented in an attempt to eradicate fur mites from 3 vivaria housing approximately 120,000 mice. Medicated feed was provided for 8 wk to ensure that all cages and mice were treated. A single investigative group reported adverse effects in their colony 4 wk after treatment was initiated; mortality was attributed to ivermectin toxicity after an intracranial injection at 1 d of age. Naïve pups were unaffected. No other adverse effects were noted. Approximately 14,500 skin scrape samples were evaluated during the 12-mo posttreatment surveillance period. All samples were negative for mites. To our knowledge, this is the first report of successful eradication of fur mites from a mouse colony of this large size.

Euthanasia by CO2 inhalation affects potassium levels in mice.

We and others frequently have noted serum potassium levels of 8.0 +/- 0.85 mEq/L or greater in laboratory mice; this concentration has even been published as the upper limit of a 'normal' reference range. However, if bone fide, this potassium concentration would be incompatible with life in all species. We investigated conditions frequently encountered in the research setting to distinguish artifactual from true hyperkalemia. Variables evaluated included site of collection, time allowed for clot formation before serum separation, time elapsed between collection and analysis of samples collected in a serum separator tube, precollection method of anesthesia, and euthanasia technique. Serum potassium was measured from 75 C57BL/6NTac 10-wk-old female mice and divided into at least 5 mice per variable. Animals were euthanized by exsanguination immediately after terminal CO2 or ketamine-xylazine (KX) administration. Mice euthanized with CO2 had higher mean serum potassium (7.0 +/- 0.5 mEq/L) and range serum potassium (6.0 to 8.1 mEq/L) than did KX-treated mice. CO2 inhalation resulted in significantly lower blood pH (6.9 +/- 0.1), higher pCO2 (153.3 +/- 38.8 mm Hg), and higher lactate levels (3.9 +/- 0.9 mmol/L) than did KX anesthesia followed by exsanguination. These results suggest that antemortem respiratory acidosis from CO2 administration causes artifactual hyperkalemia in mice. Therefore, blood collection under KX anesthesia is preferable over CO2 inhalation to obtain accurate potassium values from mice.

Animalfree screening of biological materials for contamination by rodent viruses.

Pathogenic bacteria and viruses may be transmitted to laboratory rodents by contaminated biological materials potentially causing disease and confounding research results. Biological materials have historically being screened by using the mouse antibody production (MAP) test. We developed an alternative assay using polymerase chain reaction (PCR/real-time-PCR) technology to detect viral contamination directly in biological material. The assay was found to be of equal or greater sensitivity when compared to the MAP test.