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.     

Microbial contamination of embryos and semen during long term banking in liquid nitrogen

We report on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen. There were 32 bacterial and 1 fungal species identified from randomly drawn liquid nitrogen, frozen semen, and embryos samples stored in 8 commercial and 8 research facility liquid nitrogen (LN) tanks. The identified bacteria represented commensal or environmental microorganisms and some, such as Escherichia coli, were potential or opportunistic pathogens for humans and animals. Stenotrophomonas maltophilia was the most common contaminant identified from the samples and was further shown to significantly suppress fertilization and embryonic development in vitro. Analysis of the strains by pulsed field gel electrophoresis revealed restriction patterns with no relatedness indicating that there was no apparent cross-contamination of S. maltophilia strains between the germplasm and liquid nitrogen samples. In addition, no transmission of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) from infected semen and embryos straws to clean germplasm stored in the same LN tanks or LN was detected.     

Rederivation of inbred strains of mice by means of embryo transfer

Embryo transfers were performed to rederive six inbred strains of mice, A/He, BALB/cByJ, BALB/c Lac, B10.BR/SgSnJ, C57BL/6J and DBA/2J. The aim was to determine whether it is possible to eliminate pathogens like mouse hepatitis virus (MHV) and Pasteurella pneumotropica (P.p.). The embryos were collected, handled and transferred into the oviduct of day one pseudopregnant SPF surrogate mothers under aseptic conditions. In 40.5% of the transfers, embryos developed to term. With respect to surrogate mothers delivering viable litters, 47.9% of the transferred embryos were born alive. Out of these 93.5% were reared. Virological and bacteriological examination of embryo donors verified the presence of P.p. and of antibodies against MHV in all strains. In some embryo donors P.p. could be isolated even from the uterine mucosa. However, neither in the surrogate mothers nor in the offspring could P.p. and antibodies against MHV be detected. Further bacteriological examination revealed that the offspring carried only the microbial flora received from the surrogate mother. The results indicate that embryo transfer is an appropriate tool to rederive mouse strains. In contrast to hysterectomy rederivation, embryo transfer has the advantage of avoiding postimplantational vertical transmissions of infections.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

Implementation of a PCR assay of Pasteurella pneumotropica to accurately screen for contaminated laboratory mice

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.     

Viral contamination of embryos cryopreserved in liquid nitrogen

Despite the worldwide application of embryo-freezing technology as the means of preserving germplasm of mammalian species, there is no information available on the possible transmission of infectious agents to cryopreserved embryos via contaminated liquid nitrogen (LN). Recently, it has been reported that new methods of cryopreservation which employ ultrarapid freezing or vitrification require direct contact between the freezing medium containing oocytes or embryos and liquid phase nitrogen (LPN). As models for human and animal viral pathogens three bovine viruses, bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV), and bovine immunodeficiency virus (BIV), were employed to study the potential for their transmission by experimentally contaminated LN to embryos frozen and stored in open freezing containers. Bovine embryos in a mixture of 20% ethylene glycol, 20% ME(2)SO, and 0.6% sucrose were vitrified in either unsealed standard 0.25 ml or modified open pulled straws or in plastic cryovials and then plunged into contaminated LPN. After 3-5 weeks of storage in LN, embryos were thawed and sequentially washed and only those with intact ZP were pooled together and tested in batches of three for viral contamination. From this pool of 83 batches, 13 of 61 (21.3%) batches exposed to BVDV and BHV-1 tested positive for viral association while all 22 batches exposed to BIV in unsealed containers tested negative. All control embryos vitrified in sealed cryovials and straws were free from viral contamination.     

Ten years-long survey on pathogen status of mouse and rat breeding colonies

Eleven pathogens including P. aeruginosa, Salmonella spp., E. coli O115a, c: K(B), P. pneumotropica, B. bronchiseptica, C. kutscheri, Tyzzer's organism, M. pulmonis, Sendai virus, MHV and Syphacia spp. were surveyed in 217 mouse and rat breeding colonies during 1972-1981. In conventional animals, P. pneumotropica and/or Syphacia spp. were detected in nearly 90% of 89 mouse and 64 rat colonies. Sendai virus, M. pulmonis, P. aeruginosa and MHV were positive in 51.7 to 23.6% of the colonies, and Tyzzer's organism, B. bronchiseptica and probably SDA virus were also detected in more than 10% of the rat colonies. Salmonella spp., E. coli O115a, c: K(B) and C. kutscheri were found in a few colonies. In SPF animals, P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. pneumotropica was also positive in 3 rat colonies. Infection rates of P. pneumotropica, M. pulmonis, Sendai virus and Syphacia spp. were usually higher than 40% of animals sampled from colonies contaminated with them. Accidental contaminations of SPF colonies were usually caused by P. pneumotropica and Syphacia spp.     

Cryopreservation of Musca domestica (Diptera: Muscidae) embryos

Prior studies on cryopreserving embryos of several non-drosophilid flies established that two Drosophila melanogaster embryo cryopreservation protocols were not directly suitable for use with these species. This paper describes our work on developing a protocol for cryopreservation of embryos of the housefly, Musca domestica. Significant progress was made when permeabilization of the vitelline membrane was optimized, a vitrification solution containing ethylene glycol, polyethylene glycol, and trehalose was formulated, and when cooling and recovery of the cryopreservation protocol included a step which passed the embryos through liquid nitrogen vapor. More than 70% of housefly embryos withstand treatments of dechorionation, permeabilization, loading with cryoprotectant, and dehydration in vitrification solution, but the cooling, warming, and poststorage rearing steps still cause a considerable reduction in survival. About 53% of the vitrified M. domestica embryos hatched into larvae. Relative to the percentage of the control adult emergence, about 13% of the embryos stored in liquid nitrogen developed into fertile adults. Hatching of the F(1) progeny of adults having been cryopreserved as embryos was similar to control levels.     

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.     

A trial designed to obtain a specific pathogen free Syrian hamster colony by administration of chemicals]

Conventional Syrian hamsters, contaminated with Giardia spp., Spironucleus muris, Trichomonas spp., Pasteurella pneumotropica and Pseudomonas aeruginosa were treated with chemicals in order to obtain specific pathogen free animals. Hamsters kept in the laminar flow rack were treated orally with metronidazole several times to obtain a flagellate-free colony. After all flagellates had been eradicated, one pair of animals were kept in an isolator and mating was allowed to occur. When their offspring reached the age of seven weeks, they were intramuscularly injected daily with netilmicin sulfate for 10 consecutive days. Following these treatments, all of the hamsters were free of Pasteurella and Pseudomonas. Further breeding of these animals was continued in isolators. To confirm the absence of selected pathogens, they were placed in a barrier room for further breeding as specific pathogen free animals.     

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.     

Production of transgenic mice following deoxyribonucleic acid microinjection and embryo freezing

Experiments with mouse embryos were designed to assess the feasibility of freezing embryos after DNA microinjection. One-cell pronuclear stage mouse embryos were microinjected with cloned deoxyribonucleic acid (DNA) and cultured in vitro to the late eight-cell stage. Microinjected and matched control embryos were frozen and stored in liquid nitrogen. Following thawing, embryos were cultured for 8 h and transferred to recipient females. In a separate set of experiments, embryos were transferred to recipients immediately following DNA microinjection. Control (uninjected) embryos developed to the late eight-cell stage significantly better than surviving microinjected embryos. Of the embryos thawed, 76% of the microinjected and 60% of the control embryos survived to be transferred to recipients. Progeny were obtained with similar survival rates from both groups following embryo transfer with transgenic mice identified among the progeny from microinjected embryos. Mouse embryos can be microinjected with DNA, cultured in vitro, frozen, thawed, transferred to recipients and transgenic progeny can be obtained.     

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.     

Pasteurella pneumotropica and the prevalence of the AHP (Actinobacillus, Haemophilus, Pasteurella)-group in laboratory animals

134 bacterial isolates originally identified as Pasteurella pneumotropica were cultured from healthy, ill or dead mice, rats, hamsters, guinea pigs, rabbits and cats originating from 7 conventional laboratory animal facilities. They occurred seldom in pure culture and were found in a variety of organs. Thorough identification (41 criteria) revealed that only 83 isolates (62%) were P. pneumotropica and could be subdivided into 3 biotypes. 3 isolates were P. aerogenes, 1 was P. ureae, 11 (8%) were qualified as Actinobacillus spp. and 13 (10%) as Haemophilus spp. Close relationship of the 3 genera--the 'AHP-group' --made the differentiation difficult. 23 atypical cultures were discarded at the beginning of the study as not belonging to the 'AHP-group'. Two-thirds of isolates were associated with inflammation or suppuration; Haemophilus spp. seemed to be more pathogenic than Pasteurella and Actinobacillus species.     

Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen in dry shippers

Cryopreservation and storage of germplasm is an important factor in the prevention of disease transmission by embryo transfer and artificial insemination. Here we report the results of an investigation on transmission of selected bacterial and viral pathogens by the vapour phase of liquid nitrogen (VPLN) to embryos and semen in dewars designed for short-term storage and transportation of biological specimens. In this study transmission of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, BVDV, and BHV-1 was examined from: (1) contaminated dry shippers to germplasm; (2) between contaminated and non-contaminated cryopreserved germplasm; and (3) between stock culture of pathogenic agents and germplasm. Contaminated and non-contaminated samples of embryos and semen were stored in proximity in the vapour phase LN in open containers for 7 days prior to testing for the presence of microbes. The results showed that there was no cross-contamination from either the contaminated dewars to germplasm or between contaminated and non-contaminated samples of embryos and semen during 7 days storage under LN vapours. The outcome of our investigation indicates that VPLN is a safe means for short-term storage of embryos and semen in dry shipper dewars commonly used for transportation of bovine germplasm.     

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。     

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.     

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.     

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。     

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.