Poliomyelitis in MuLV-infected ICR-SCID mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus

The arterivirus lactate dehydrogenase-elevating virus (LDV) causes life-long viremia in mice. Although LDV infection generally does not cause disease, infected mice that are homozygous for the Fv1(n) allele are prone to develop poliomyelitis when immunosuppressed, a condition known as age-dependent poliomyelitis. The development of age-dependent poliomyelitis requires coinfection with endogenous murine leukemia virus. Even though LDV is a common contaminant of transplantable tumors, clinical signs of poliomyelitis after inadvertent exposure to LDV have not been described in recent literature. In addition, LDV-induced poliomyelitis has not been reported in SCID or ICR mice. Here we describe the occurrence of poliomyelitis in ICR-SCID mice resulting from injection of LDV-contaminated basement membrane matrix. After exposure to LDV, a subset of mice presented with clinical signs including paresis, which was associated with atrophy of the hindlimb musculature, and tachypnea; in addition, some mice died suddenly with or without premonitory signs. Mice presenting within the first 6 mo after infection had regions of spongiosis, neuronal necrosis and astrocytosis of the ventral spinal cord, and less commonly, brainstem. Axonal degeneration of ventral roots prevailed in more chronically infected mice. LDV was identified by RT-PCR in 12 of 15 mice with typical neuropathology; positive antiLDV immunolabeling was identified in all PCR-positive animals (n = 7) tested. Three of 8 mice with neuropathology but no clinical signs were LDV negative by RT-PCR. RT-PCR yielded murine leukemia virus in spinal cords of all mice tested, regardless of clinical presentation or neuropathology.     

Microbiological survey of Korean mouse facilities from 2014 to 2019

We surveyed mouse microbiological contamination rates by testing rates for common contaminants using serological, culture, and parasitological methods. A total of 21,292 experimentally housed mice from 206 animal facilities, including hospitals, universities, companies, and research institutes, were tested over a 6-year period from 2014 to 2019. The most commonly found contaminants were various species of nonpathogenic protozoa (47.2%). The most common pathogenic bacteria were Staphylococcus aureus (21.2%), Pasteurella pneumotropica (12.5%), and Pseudomonas aeruginosa (5.8%). Mouse hepatitis virus (6.1%) was detected, but no other viral or bacterial pathogens were found. These results establish that the main pathogens that currently contaminate mouse facilities in Korea are opportunistic pathogens and that contamination with important pathogens, such as those in Categories B or C, has decreased.     

Elimination of Pasteurella pneumotropica from a contaminated mouse colony by oral administration of Enrofloxacin.

Enrofloxacin, a fluoroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment. Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.     

Evaluation of a forced-air-ventilated micro-isolation system for protection of mice against Pasteurella pneumotropica.

Studies to date have established that the physical environment inside cages can be controlled adequately by setting the intra-cage ventilation at 60 air changes per hour in a forced-air-ventilated micro-isolation system (FVMIS). In this study, the capability of FVMIS to prevent inter-cage transmission of microorganisms was evaluated using Pasteurella pneumotropica as a reference microorganism. One FVMIS rack and a conventional rack were used, and cages with mice positive for P. pneumotropica and those with P. pneumotropica-free mice were housed on both racks. The mice were examined for P. pneumotropica contamination every 4 weeks after initiating the experiment for 12 weeks using a polymerase chain reaction method. Some P. pneumotropica-free mice housed in open air cages in the conventional rack became positive for P. pneumotropica (four of 28 animals after 4 weeks; eight of 28 animals after 12 weeks), but all P. pneumotropica-free mice housed in the FVMIS cages remained negative for the bacterium throughout the experiment. The results demonstrate that FVMIS can prevent inter-cage transmission of P. pneumotropica when proper cage handling practice is under taken.     

Upgrading mouse health and welfare: direct benefits of a large-scale rederivation programme

We report the outcome of a 30-month programme to rederive 310 specific pathogen-free mouse strains to populate a new individually ventilated cage barrier facility at the Mary Lyon Centre (MLC), Medical Research Council (MRC) Harwell. The mice were rederived in a self-contained quarantine suite and embryo-recipient females were health-screened to assess microbiological status, before moving their offspring into the new facility. The MLC currently houses approximately 49,000 mice in about 9750 cages and we have 30 months of follow-up health screen data. Embryo rederivation and hysterectomy have high safety margins; however, the precaution of performing the programme in isolators facilitated the containment and decontamination of two mouse hepatitis virus (MHV) infection outbreaks. Rederivation of the colony has eliminated endemic MHV, mouse adenovirus type 2 (MAV-2), Theiler's murine encephalomyelitis virus, pinworms, intestinal protozoa, Pasteurella pneumotropica, Helicobacter spp. and mites. The improvements in microbiological status have had notable benefits for mouse health and welfare and the science at MRC Harwell. Previously important clinical entities such as sudden death associated with lactation ileus in C3H/HeH mice, early weight loss associated with inflammatory bowel disease in B6-TgN(HDexon1)61Gpb and B6TgN-(HD82Gln)81Dbo (Huntington) mice and early weight loss in male mice mutagenized with N-ethyl-N-nitrosourea have been markedly reduced or eliminated.     

Comparison of the mouse antibody production (MAP) assay and polymerase chain reaction (PCR) assays for the detection of viral contaminants

Mouse antibody production (MAP) tests have become the standard assay for the detection of murine viral contamination in biologic materials, such as cell lines and transplantable tumors. However, newly developed PCR assays offer the advantage of lower cost, faster turn around times, and eliminate the use of live animals. In this study, the MAP test and a panel of PCR assays were compared for the detection of 11 different viral contaminants of cell lines and transplantable tumors. The PCR assays had either better or comparable results to the MAP test for all agents tested. The results of this study confirm that PCR assays are an effective method for detection of viral contamination and can be used as an alternative to the MAP test.     

Role of Pasteurella pneumotropica and Mycoplasma pulmonis in Murine Pneumonia

Pneumonia caused by Mycoplasma pulmonis and Pasteurella pneumotropica was studied in conventional, specific pathogen-free (SPF), and germ-free mice. When P. pneumotropica was serially passed in conventional mice, M. pulmonis, as well as P. pneumotropica, was recovered from mice with gross lesions. When M. pulmonis was serially passed in conventional mice, both organisms were recovered. SPF mice given a nasal instillation of M. pulmonis alone, P. pneumotropica alone, or a combination of the two developed pneumonia when both organisms were present. These findings suggested that both organisms contribute to typical murine pneumonia. That M. pulmonis might be an L form of P. pneumotropica was suggested because some SPF mice inoculated with either organism yielded both on culture. This possibility was investigated with mole per cent guanine plus cytosine (GC) content and nucleic acid hybridization techniques. The GC content of P. pneumotropica is 42.2 mole per cent and that of M. pulmonis is 28.6 mole per cent. No specific hybrids between deoxyribonucleic acid (DNA) from M. pulmonis and DNA from P. pneumotropica were detected. This and the wide disparity in GC content showed that M. pulmonis is not an L form of P. pneumotropica. In germ-free mice, intranasal instillation with either organism alone produced pneumonia. The lesions produced when each organism was inoculated independently were characterized by areas of consolidation with perivascular and peribronchial lymphocytic infiltration. Qualitatively, the lesions produced when both organisms were inoculated simultaneously more closely resembled those seen in naturally occurring murine pneumonia. Statistical analysis indicated that the quantitative effect of the two organisms was additive. The indirect fluorescent antibody technique was used to locate organisms in lung tissue sections. M. pulmonis localized in the bronchial epithelium and P. pneumotropica localized in the alveolar lesions.     

嗜肺巴斯德杆菌研究进展

嗜肺巴斯德杆菌(Pasteurella pneumotropica)是一种条件致病菌,人兽共患。主要危害啮齿类动物,特别是免疫缺陷或抑制的动物,可引起炎症和脓肿等症状。该菌是实验动物中感染率最高的病原菌之一,多呈隐性感染,给动物实验带来了极大的干扰。本文针对嗜肺巴斯德杆菌的流行病学、检测和鉴定、分子分型和防治等方面进行综述。     

Apoptosis in irradiated murine tumors

Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.     

Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy

Summary To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu (nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients’ tumors, allowing assessment of the effect of therapy when confronted with different breast tumors’ genotype and phenotype.     

Proliferation of Pasteurella pneumotropica at oestrus in the vagina of rats

Using a colony of Wistar-Imamichi rats contaminated with P. pneumotropica, the vaginal microflora was qualitatively and quantitatively investigated by swabbing. P. pneumotropica was the most dominant organism in the majority of rats examined. The population of P. pneumotropica and indigenous bacteria increased significantly higher at oestrus than in other oestrous stages. By the vaginal flushing technique changes in the population of P. pneumotropica and total bacteria, and changes in vaginal cell type and bacterial counts adhering to vaginal epithelial cells were consecutively investigated. The populations of P. pneumotropica and total bacteria were maximal at oestrus. The increase was correlated with an increase in cornified non-nucleated cells, with large numbers of adherent Gram-negative coccobacilli. The findings indicate that the vagina is a suitable site for colonization by P. pneumotropica in adult female rats, and that proliferation of P. pneumotropica may be due to increased affinity of the organism for cornified non-nucleated cells.     

Isolation of members of the Haemophilus-Pasteurella-Actinobacillus group from feral rodents

46 feral rodents, including a common vole (Microtus arvalis), house mice (Mus musculus), muskrats (ondatra zibetica), house rats (Rattus rattus) and brown rats (R. norvegicus) were examined for bacteria of the Haemophilus-Pasteurella-Actinobacillus group. Haemophilus spp. (only M. musculus examined) were not obtained. All animal species were found contaminated by P. pneumotropica and/or Actinobacillus spp. Almost all M. musculus (96%) and most Rattus spp. (76%) were contaminated by P. pneumotropica and/or Actinobacillus spp. These bacteria were obtained most frequently from the upper respiratory tract, to a lesser extent from the lung and rarely from caecal contents. It is concluded that feral rodents might constitute an important source of contamination of laboratory rodents by members of the HPA-group.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.     

嗜肺巴斯德杆菌选择性培养基研制及应用

目的　研制一种对嗜肺巴斯德杆菌表现出强选择作用的选择性培养基,用于该菌的常规检测。方法　药敏试验及抗生素最小抑菌浓度测定。结果　研制了嗜肺巴氏杆菌选择性培养基(PPSM培养基)及嗜肺巴斯德杆菌增菌液(PP肉汤)。嗜肺巴斯德杆菌在PPSM培养基上,37℃48h培养,形成1mm左右,凸起、湿润、灰黑色并有金属光泽的特殊菌落;对表皮葡萄球菌和大肠埃氏菌的抑制率为100%,对变形杆菌的抑制率为76%,并能抑制其迁徙生长;通过PP肉汤增菌培养,PPSM培养基使SPF小鼠粪便中嗜肺巴斯德杆菌检出率从0增至67.2%;用小鼠咽拭子接种该培养基,其初代培养物几乎为纯培养物。结论　该培养基对嗜肺巴氏杆菌具有较强的选择作用,使用该培养基对嗜肺巴氏杆菌进行检测可以简化检测程序、防止漏检、在不处死动物的情况下对嗜肺巴斯德杆菌进行常规监测。     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Pasteurella pneumotropica in murine colonies

An ELISA for the detection of class specific IgG antibodies to Pasteurella pneumotropica was developed for the serological diagnosis of infections in mouse colonies. Heat inactivated whole cell preparations of an isolate of P. pneumotropica biotype Heyl (strain P 166) served as antigen for the ELISA procedure and for immune serum production in germ-free Han:NMRI mice. Cross reactions with the autochthonous flora of Han:NMRI SPF-mice were not observed, but were evident when a P. pneumotropica antiserum was tested against other antigens of the Pasteurella-Actinobacillus group. According to the reclassification of this bacterial group proposed by Mutters et al. (1), strains of the following species were tested: P. anatis, P. canis, P. dagmatis, P. langaa, Pl multocida sub. multocida, P. pneumotropica biotype Jawetz, P. stomatis, Actinobacillus equuli and A. lignieresii. Clear cross reactions could be shown with P. pneumotropica biotype Jawetz and A. equuli and to a lesser extent with P. anatis. Antibody formation profiles after nasal infection of Han:NMRI mice exhibited a primary rise of IgG-type antibody titer between 17 to 21 days post infection. Investigations of different mouse colonies free and infected with P. pneumotropica revealed good correlations between serological and bacteriological findings.     

Decontamination of spices by gamma radiation

Spices such as coriander, cumin, turmeric, chilli collected from a local market were found to be highly contaminated with bacteria and fungi. A dose of 10 kGy was required to reduce the total bacterial count below detectable levels, while a dose of only 5 kGy was required to eliminate the fungal contamination. Coliforms were totally eliminated at a radiation dose of 5 kGy. During a 6 months storage of irradiated and unirradiated spices, the irradiated spices were found to retain good microbiological quality.     

Survival and transmissibility of Pasteurella pneumotropica

Survival has been determined for Pasteurella pneumotropica on various surfaces found in an animal room at 23+/-1 degrees C and 50+/-10% relative humidity. Longest survival (120 min) was found on mouse hair, shortest (< 30 min) on laboratory coat fabric. Transmission experiments were performed using sentinel animals in order to evaluate the efficiency of their use for the detection of P. pneumotropica in quarantined mice. In sentinels exposed to infected mice by close contact, P. pneumotropica was detected by culture 2 weeks post-exposure and seroconversion 3 weeks after contact. Transfer of soiled bedding from Pasteurella-infected mice did not infect sentinels within a period of 12 weeks as tested by cultivation or serum antibodies.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Contamination of a monoclonal antibody with LDH-virus causes interferon induction

Interferon induction occurred unexpectedly during an in vivo study using a mouse monoclonal antibody. The interferon was typed alpha/beta and the titer reached a maximum at 24 hours in contrast with other inducers. Similar results were obtained with a virus pool derived from the antibody and with a LDV reference strain. MAP-testing of the monoclonal antibody revealed contamination with lactate dehydrogenase virus (LDV). The production of IFN seems to be controlled genetically. This experimental error demonstrates the importance of an appropriate quality control of biological materials.