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.     

Embryonic death in pregnant rats owing to intercurrent infection with Sendai virus and Pasteurella pneumotropica

During an outbreak of Sendai virus infection in a colony of rats used for embryo production, severe lung lesions due to secondary colonization of the rat lungs with Pasteurella pneumotropica were noted. The effect on pregnancy was to cause embryonic death and resorption, leaving a deciduoma with trophoblastic giant cells as the only embryonic remnants. Up to 30% of fetuses in each pregnant animal were affected at the time of severe maternal lung lesions. Heavy growths of Pasteurella pneumotropica were obtained from the lungs of all affected dams. The formation of deciduomas as a result of embryonic death was due to the indirect effect of damage to the lungs during pregnancy rather than the direct pathogenic effect on the developing embryo of the microbial organisms isolated.     

Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats.

To investigate the pathogenicities of P. pneumotropica (Pp) and V-factor dependent Pasteurellaceae (VFDP) in immunodeficient rats, experimental infections of F344-rnu rats were performed using 3 strains (ATCC 35149, CNP 160 and RPZ) of Pp and 4 strains (V6, V7, V8 and V9) of VFDP. Four animals per experimental group were inoculated twice on day 0 and post-inoculation day (PID) 14 with bacterial suspension intranasally. Two animals from each group were sacrificed on PID 60 and 120, and examined. In the animals inoculated with strains of Pp, sneezing was observed in some animals inoculated with strains ATCC 35149 and CNP 160 until PID 31. No clinical signs were observed in other animals. The strains were mainly isolated from the nasal cavity and trachea on PID 60, and the nasal cavity, trachea and lung on PID 120. Inflammation and necrosis of nasal cavity mucosa were observed in all animals inoculated with strains ATCC 35149 and CNP 160 in a histopathologic examination. No histopathological changes were observed in any other animal. In the animals inoculated with strains of VFDP, neither clinical disorder nor histopathological change was observed. The strains were mainly isolated from the trachea on PID 60, and from the trachea and lungs on PID 120. From these results, the pathogenicity of Pp in immunodeficient rats appears to differ by strain, and VFDP appears to be non-pathogenic in immunodeficient rats.     

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.     

Secondary infection of rat lungs with Pasteurella pneumotropica after Kilham rat virus infection

Lung congestion was observed after an outbreak of Kilham rat virus infection (KRV) in a rat colony, previously free of all rat viruses. A high proportion of congested lungs contained Pasteurella pneumotropica suggesting that KRV might have caused primary damage to the alveoli (hitherto not recorded) which allowed the secondary bacterial colonization. Experimental infection of rats with KRV caused acute damage to the lung alveoli. Since KRV infection is very common in animal facilities it could therefore be a significant agent in the development of respiratory disease.     

Selective media for Pasteurella pneumotropica

Bacteria from the subcutaneous abscesses which appeared in a laboratory colony of DS mouse since October of 1977 were identified as Pasteurella pneumotropica by various biological examinations. The abscess formation was limited to multiparous female mice over 100 day-age, but virgin females were free from the disease. The MIC of various antibacterial substances showed that potassium tellurite, kanamycin and bacitracin were effective to isolate the organism selectively from various infection sites harboring many other species of bacteria. A novel NKBT medium was prepared by adding these antibacterial substances to the heart infusion agar (HIA) supplemented by 10% Fildes digested blood. A fluid culture medium, TGN broth was prepared for multiplication of the organism by adding 10% Fildes digested blood and potassium tellurite to GN broth. To isolate the organism from the pharyngo-larynx a direct application of mucus wiped off the infection site onto the culture medium was sufficient, but pre-multiplication in the TGN broth was required for isolation of the organism from gut contents before inoculation onto the NKBT medium. The pre-cultivation in the TGN broth vastly improved the recovery of the organism especially from feces. Thereby we could easily detect the latent infection of this bacterium without sacrificing animals.     

An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice.

Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice.     

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.     

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.     

Effect of oestrogen on Pasteurella pneumotropica in rat vagina

The effects of ovarian hormones on the vaginal population of Pasteurella pneumotropica in rats were investigated. Specified-pathogen-free adult female Wistar-Imamichi rats with a 4 day oestrous cycle were inoculated with P. pneumotropica in the vagina. Cyclic changes in the vaginal population of P. pneumotropica were not observed in ovariectomized rats and the bacterial population was at a similar level to that at normal dioestrus. Administration of oestrogen to ovariectomized rats caused an increase in the numbers of P. pneumotropica and total bacteria in the vagina nearly equal to that at oestrus in intact rats. The numbers of these organisms in the vagina of ovariectomized rats treated with progesterone did not change and were similar to those of control ovariectomized rats treated with sesame oil. Vaginal smears of ovariectomized rats treated with oestrogen were characterized by abundant cornified non-nucleated epithelial cells with many adherent Gram-negative coccobacilli and were similar to smears from intact rats at oestrus. These findings suggest that the proliferation of P. pneumotropica at oestrus in rat vagina may be primarily due to the environment provided by the degeneration of vaginal epithelial cells promoted by oestrogen secretion from the ovaries.     

嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性研究

目的观察不同来源的嗜肺巴氏杆菌在实验大鼠和小鼠中的传染性.方法取源于野鼠、实验大鼠和小鼠的嗜肺巴氏杆菌3株,对30只受试大鼠和小鼠进行交叉人工感染,并于感染后不同时期取咽拭子分离培养,对感染前后菌株,应用RAPD-PCR、SDS-PAGE和Western blot进行基因型、蛋白和抗原成份比较,以及生物学特性的比较.结果受试实验动物对3株嗜肺巴氏杆菌均易感,被接种的动物能稳定携带嗜肺巴氏杆菌直到试验结束,重新分离的嗜肺巴氏杆菌在生物学特性、蛋白成份、抗原性和基因型方面无明显改变.结论同一株嗜肺巴氏杆菌能在实验大鼠和小鼠中相互传染.     

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.     

嗜肺巴氏杆菌L型的诱导及其流行病学意义初探

目的 探讨L型嗜肺巴氏杆菌在诊断学及流行病学上的意义。方法 青霉素液体法诱导,并对嗜肺巴氏杆菌L型的生物学特性进行系统观察。结果 嗜肺巴氏杆菌L型具有典型的“煎蛋状”（L型）和“丝状”（F 型）菌落,扫描电镜观察,L型菌体呈球状、杆状、长丝状;革兰染色呈阴性、缠绕的长丝体,并具有圆球体及巨型体,细胞壁染色显示细胞壁缺失;对紫外线、新洁尔灭抵抗力较原菌增强5~10倍;自然干燥环境中,原菌2 d死亡,而L型可存活12 d;能透过0.45μm滤膜;回复的最初几代,其菌体形态较原菌大数倍。结论 L型嗜肺巴氏杆菌在形态学方面有较大变化,对理化因素及外界环境抵抗力增强,有可能透过胎盘屏障垂直传播。本研究提示L型嗜肺巴氏杆菌在该菌诊断学及流行病学上有重要意义。     

Evaluation of PCR as a means of identification of Pasteurella pneumotropica.

A polymerase chain reaction with new primers (new PCR) designed from Pasteurella pneumotropica 16S rDNA as an identification system for this organism was compared with the PCR reported by Wang et al. (Wang's PCR) by using 15 bacterial reference species and 70 clinical isolates with the conventional identification system. For the 15 reference strains, both PCRs were identical. For the 70 clinical isolates, the new PCR and Wang's PCR showed consistency with the conventional system in 62.9% (44/70) and 51.4% (36/70), respectively. Twenty-six isolates were inconsistent with the conventional system and the new PCR with respect to morphology and serology. These findings suggested that the new PCR was more sensitive than Wang's PCR, and the new PCR in combination with morphology and serology is useful for P. pneumotropica identification.     

Identification procedure for Pasteurella pneumotropica in microbiologic monitoring of laboratory animals.

Discrepancies have been recognized in the identification of Pasteurella pneumotropica between testing laboratories. To determine the causes of the differences and to propose a reliable identification procedure for P. pneumotropica, a working group was organized and 69 isolates identified or suspected as P. pneumotropica were collected from 8 laboratories in Japan. These isolates were examined by colony morphology, Gram-staining, the slide agglutination test using two antisera (ATCC35149 and MaR), two commercially available biochemical test kits (ID test, API20NE) and two primer sets of PCR tests (Wang PCR, CIEA PCR). The 69 isolates and two reference strains were divided into 10 groups by test results. No single procedure for P. pneumotropica identification was found. Among tested isolates, large differences were not observed by colony morphology and Gram-straining except for colony colors that depended on their biotypes. Sixty-eight out of 69 isolates were positive by the slide agglutination test using two antisera except for one isolate that tested with one antiserum. The ID test identified 61 out of 69 isolates as P. pneumotropica and there was no large difference from the results of CIEA PCR. From these results, we recommend the combination of colony observation, Gram-straining, the slide agglutination tests with two antisera and biochemical test using the ID test for practical and reliable identification of this organism.