Persistent Corynebacterium bovis Infectious Hyperkeratotic Dermatitis in Immunocompetent Epidermal-Mutant dep/dep Mice.

During a previously reported program-wide Corynebacterium bovis outbreak, both immunocompetent depilated (dep/dep) mutant mice and transgenic mice that express the papillomavirus E6 oncoprotein became persistently infected with C. bovis. An orthokeratotic, hyperkeratotic, acanthotic dermatitis developed in the C. bovis-infected dep/dep mice, which remained C. bovis PCR-positive for >45 days prior to euthanasia as part of the program-wide C. bovis eradication effort. Since both affected strains of mice have altered skin homeostasis, immune status or the presence of hair may not alone be sufficient to explain strain susceptibility to C. bovis-related cutaneous disease. In order to avoid invalidation of preclinical studies due to C. bovis infection, it may be necessary to isolate immunodeficient mouse strains, implement facililty-wide surveillance for C. bovis, and sterilize equipment with vaporized hydrogen peroxide.

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.

Enumeration of Mycobacterium leprae using real-time PCR.

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology.

The purpose of this study was to characterize a spontaneous disease condition causing hyperkeratosis in nude mice and to explore the etiologic role of a particular species of coryneform bacteria in this disease, colloquially known as "scaly skin disease." The study was divided into two parts. In the first phase, a series of inoculation experiments was conducted with a field isolate of the coryneform species used to study the clinical and histopathologic development of the disease syndrome. Athymic nude mice (4 to 5 weeks old) were inoculated on the skin of the back with a suspension of a pure culture of the coryneform bacterium that had been isolated from a field case. The culture was applied with a sterile cotton swab in concentrations varying from 6.1 x 10(4)/ml to 5.0 x 10(7)/ml. All inoculated mice became persistently infected throughout the 33 days of the experiment. Clinically evident hyperkeratosis in inoculated animals developed more frequently in mice housed in a microisolator cage than in a semi-rigid isolator and more frequently in mice inoculated with higher numbers of organisms. In all animals in which hyperkeratosis developed, it was first noted on day 7 after inoculation. The second series of experiments was designed to determine the success of various housing methods in excluding the infection, mechanisms of transmission, susceptibility of other stocks and strains of mice to the organism, and whether the other strains might serve as a source of the organism. Results of the study in various strains indicated that both immunocompetent and immunodeficient mice, whether glabrous or hirsute, could be infected with the organism, but only glabrous animals developed hyperkeratosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Elimination of Pseudomonas aeruginosa from an experimental nude mouse colony].

In order to eliminate Pseudomonas (P.) aeruginosa from a contaminated nude mouse colony, the following procedures were carried out: improvement of environmental sanitation using an effective disinfectant against the organism; supply of tap water acidified with hydrochloric acid at pH2.5-3.0; elimination of mice positive in isolation of P. aeruginosa from the nude mouse colony. The experimental results indicated that P. aeruginosa were successfully eliminated from the colony though a combination of the above three procedures.

Susceptibility of BL6 nude (congenitally athymic) mice to Japanese encephalitis virus by the peripheral route.

Studies on the susceptibility of adult BL6 nude (congenitally athymic, nu/nu) mice, their euthymic littermates (+/nu) and Swiss mice to Japanese encephalitis (JE) virus inoculated subcutaneously were carried out. The mice were observed over a period of 60 days p.i. for sickness and/or death, which was noticed only in nu/nu mice. However, the onset and the duration of sickness varied and no definite pattern was observed. Thirty four of 53 nu/nu, 28 of 42 +/nu and 30 of 52 Swiss mice bled during the first 5 days p.i. exhibited viraemia. Interestingly, only nu/nu mice had secondary viraemia during the period of sickness. The cause of sickness and death in nu/nu mice was confirmed by recovering the virus from the blood, the brain and other organs. Antibodies were detected in the sera of +/nu and Swiss mice from the 10th day p.i. onwards but not in nu/nu mice. These findings indicate the important role of functional T cells both in induction of active immunity and protection against JE virus infection in mice.

Experimental mycoses in congenitally immunodeficient rodents.

Congenitally immunodeficient rodents are playing a major role in clarifying host defense mechanisms and elucidating the virulence factors of a variety of mycotic agents. These murine models are also being used to study the histopathology and chemotherapy of a variety of fungal infections. Recent development of immunodeficient transgenic mice will make these murine models even more valuable for studies on the diagnosis, pathogenesis, immunity, and chemotherapy of fungal infections.

Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines.

Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.

Lymphocytic choriomeningitis outbreak associated with nude mice in a research institute.

OBJECTIVE: After an employee at a cancer research institute was diagnosed with lymphocytic choriomeningitis, an investigation was performed to determine the extent of lymphocytic choriomeningitis virus (LCMV) infections among the institute's employees and to identify risk factors for infection. DESIGN: Retrospective cohort study. SETTING: A US cancer research institute. PARTICIPANTS: Eighty-two of 90 institute employees. MAIN OUTCOME MEASURES: Serum LCMV antibodies. RESULTS: Seven workers (9%) with definite LCMV infection (LCMV IgG antibody titer greater than or equal to 16) and one worker (1%) with probable infection (IgG titer = 8) were identified (10% overall seroprevalence). All infected employees handled animals or animal tissues and were more likely than other animal handlers to have worked with nude mice (Mus musculus) (P less than .02). Among the 31 employees who worked with nude mice at the institute, infected workers were more likely to clean the cages of nude mice (P much less than .001), change their bedding (P less than .01), and change their water (P less than .001). The institute had been injecting nude mice with LCMV-infected tumor cell lines and had recently increased the nude mouse population and the duration of experiments. These changes would have increased the LCMV burden at the facility and were temporally associated with the cluster of LCMV infections in employees. CONCLUSIONS: This LCMV outbreak, the first reported since 1974, is the first associated with nude mice. It illustrates the ongoing hazard LCMV poses in research laboratories. Since the symptoms of LCMV infection can be nonspecific, clinicians should consider this diagnosis in ill patients who report laboratory rodent exposure.

Intracellular fate of Mycobacterium leprae in cultured murine macrophages in 24-well trays.

Mycobacterium leprae is an obligate intracellular parasite which grows within mononuclear phagocytes. In clinical cases, M. leprae reaches enormous numbers in the macrophage-rich granulomas of leprosy. Peritoneal macrophages from CBA mice were cultured in Waymouth medium containing fresh horse serum in Costar 3424 trays (24 wells, 16 mm in diameter) each containing 9 x 12 mm cover slips. This medium was supplemented with 0.5 micrograms/ml of cycloheximide. These cells were infected with M. leprae Thai-53 strain obtained by nude mice inoculation. Significant multiplication of the acid-fast bacilli in the macrophages was observed three weeks after inoculation. This experiment showed M. leprae mainly multiplied in cells and not by rephagocytization of M. leprae derived from destroyed cells.

A survey of Pneumocystis carinii infection in research mouse colonies in Japan.

To determine the frequency of Pneumocystis carinii infection in mouse colonies maintained for biomedical research in medical colleges or medical faculties in universities in Japan, 409 nu/nu mice were sent to 43 animal facilities from a P. carinii-free colony. The animals were housed for 6 months in groups of 3 to 10 animals per room, and examined for the presence of parasites and infection. Colonies in 10 (24.4%) of 41 facilities were positive for the infection. Of 383 animals in 69 rooms, the organism was detected in 66 (17.2%) animals in 13 (18.8%) rooms. The difference in the proportion of rooms where mice were positive for P. carinii is clearly seen among these three groups; SPF mouse rooms (4 of 38 rooms, 10.5%), SPF mouse rooms with breeding units (5 of 25 rooms, 20.0%) and conventional mouse rooms (4 of 6 rooms, 66.7%). The survey indicates that strict housing arrangements and husbandry techniques are necessary to keep SPF mice free from P. carinii infection.

Interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin 2 infection.

Athymic nude mice recover from an infection with recombinant vaccinia virus (VV) encoding murine interleukin 2 (IL-2), but treatment with a mAb to IL-2 accentuated infection. Administration of a mAb against interferon gamma (IFN-gamma) to mice infected with the IL-2-encoding virus completely prevented the IL-2-induced mechanisms of recovery. Both asialo-GM1+ (NK) and asialo-GM1- (non-NK) cells were participants in the IFN-gamma-mediated recovery of nude mice from infection with the IL-2-encoding VV recombinant. Depletion of asialo-GM1+ cells exacerbated infection, though not as much as anti-IFN-gamma mAb. In vitro, both asialo-GM1+ and asialo-GM1- nude mouse splenocytes produced IFN-gamma in response to IL-2.

Whole-cell protein patterns of nonhemolytic group B, type Ib, streptococci isolated from humans, mice, cattle, frogs, and fish.

Whole-cell protein and physiological patterns of nonhemolytic group B, type Ib, streptococci isolated from humans, cattle, frogs, fish, and mice were compared. Isolates from humans, fish, and mice were identical. Only minor differences were seen in the isolates from human, bovine, and frog sources. Nonhemolytic group B streptococci from humans, fish, and mice and, to a lesser extent, from cattle and frogs share several characteristics, including a high similarity in proteins (on the basis of molecular weight); this suggests that they may have a common ancestry.

Respiratory disease and wasting in athymic mice infected with pneumonia virus of mice.

Although pneumonia virus of mice (PVM) is ubiquitous among rodent colonies in the United States, it has not been reported to cause clinically apparent disease in euthymic mice. However, PVM has been reported to cause respiratory disease and death in experimentally infected euthymic and athymic mice. A group of nu/nu mice, housed in quarantine in a Trexler-type isolator, had weight loss and dyspnea. Gross necropsy findings included cachexia and diffuse pulmonary edema or lobar consolidation. Histologically there was diffuse interstitial pneumonia. Electron microscopy revealed filamentous virions budding from plasma membranes, and immunohistochemical staining of lung tissue was positive for PVM antigen. PVM was isolated from affected lung tissue in BHK 21 cells and mouse antibody production tests resulted in seroconversion to PVM. Experimental inoculation of athymic mice with lung homogenate from spontaneously infected mice resulted in clinically apparent respiratory disease and histologic lung changes similar to those in naturally infected mice. Inoculation of athymic mice with infected BHK 21 cell culture fluid resulted in pneumonia which was qualitatively similar to, but less severe than, that observed in mice with spontaneous disease. These findings indicate that naturally occurring PVM infection in athymic mice may cause respiratory disease and wasting.

[An epizootic Staphylococcus infection in a nude mouse colony].

An epizootic of Staphylococcus infection causing abscesses was encountered in a small-scale breeding colony of nude mice of BALB/c background. The incidence of abscess was sporadic and mostly nude (nu/nu) mice aged over 3 months were affected. Staphylococcus aureus was isolated from the face, oral cavity, and feces of almost all nu/nu and heterozygous (nu/+) mice in the colony. After a prolonged time period, up to 10 to 14 months of age, almost all the S. aureus-carrying nu/nu mice produced abscesses and eventually died. Athymicity of the host seemed to be a prerequisite for the abscess formation since nu/+ mice were spared from the lesions. Also, transfer of immunocompetent spleen cells cured the abscesses of the affected nude mice.

Biological characteristics of Staphylococcus aureus isolated from nude mice.

The characteristics of 50 strains of Staphylococcus aureus isolated from BALB/c nude mice (nu/nu, nu/+) with or without subcutaneous abscesses [13] were examined. All the 50 strains belonged to biotype B according to the classification by Hájek and Marsálek. All of them were phage typable, showing a single phage pattern of 52A/79/47/53/77/83A/85. The coagulase type was classified as VII. All of the 50 strains were highly sensitive to penicillin, methylphenylisoxazolyl penicillin, erythromycin, spiramycin, lincomycin, chloramphenicol, tetracycline, kanamycin, gentamicin and cephaloridine, but were resistant to sulfisoxazole. Two S. aureus strains isolated from the nostril and finger of one person working in the mouse colony were identified as the same biotype as the murine strains but different in phage type, coagulase type and drug resistance pattern.

Multiplication of Mycobacterium leprae in the nude mouse, and some applications of nude mice to experimental leprosy.

Two aspects of the immune deficiency of nude mice make these animals particularly useful tools for leprosy research. Nude mice are capable of supporting multiplication of M. leprae to levels approaching 10(10) per g in peripheral body tissues. In addition, nude mice may be inoculated with greater than 10(4) (in fact, with as many as 10(8) organisms per foot pad, without provoking an immune response that prevents multiplication of the organisms. Thus, the nude mouse should be particularly suitable for detecting persisting M. leprae in treated patients, and as a model of the patient for evaluating chemotherapeutic regimens.