Protease production by Burkholderia pseudomallei and virulence in mice.

The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains. Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm. Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6-6 x 10(5) CFU, and the LD50 was calculated after 30 days by the method of Reed and Muench. The lethal activity was studied for five strains of B. pseudomallei and the type strains of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia cepacia. The three type strains appeared to be low protease producers (A405 = 0.11, 0.09 and 0.00, respectively) and avirulent. The two more virulent B. pseudomallei strains exhibited significantly different LD50, 3.5 x 10(2) (IPP 6068 VIR) versus 2.1 x 10(5) CFU/mouse (40/97), and protease activities (A405 = 0.046 and 0.79, respectively). Moreover, the avirulent parent of IPP 6068 (AG), was a better protease producer than the 6068 VIR strain, A405 = 0.26 versus 0.046. These results suggest that there is no correlation between virulence and level of exoproteolytic activity, when B. pseudomallei is injected to mice via the intraperitoneal route.

[Biological safety in the laboratory. Biological risk, standardization and practice]. / Biosécurité au laboratoire. Risque biologique, normalisation et pratique.

Working with pathogens or genetically engineered micro-organisms is a potential hazard for scientists, health care workers, employees of pharmaceutical industry, and also for the environment. Carelessness, poor technique in the handling of infectious materials, needle sting or infectious aerosol exposure are the cause of laboratory acquired infection. Biosafety, corollary of biocontamination, is based on the combination of good microbiological techniques, facility design of the laboratory and safety equipment. So, four biosafety levels are appropriate for the operations performed and the hazard posed by the infectious agents.

Relationship between free amoeba and Legionella: studies in vitro and in vivo.

In 1980, Robowtham demonstrated that Legionella multiplies in free amoeba cytoplasm and hypothesized that the amoeba could act as a reservoir of virulent bacteria. In this paper we report various aspects of the relationship between amoeba and Legionella. A liquid medium co-culture method was applied to Acanthamoeba sp. and Legionella pneumophila serogroup 1. Within 4 days, Legionella growth increased by 2 log s CFU/ml. Using a direct immunofluorescence assay and electron microscopy, Legionella was shown to grow abundantly inside phagosomes, and bacteria and/or antigen were present on the cytoplasmic membrane of the amoeba. These aspects are very similar to those observed with Legionella-infected alveolar macrophages. The morphology and structure of Legionella cells were modified after 20 days of co-culture: - viable bacteria showed large fatty cytoplasmic inclusions, - gas liquid chromatography analysis demonstrated a decrease in the i16:0 fatty acid ratio. Cystic forms of amoeba were abundant but none contained viable Legionella. In an in-vivo study using a guinea-pig aerosol infection model, we compared the virulence of Legionella in co-culture with Legionella grown on charcoal dialysed yeast extract (CDYE) agar medium. The Legionella obtained by co-culture had an LD 50 (50% lethal dose) similar to that obtained for those grown on CDYE, showing that bacterial virulence is preserved in the cellular model.

Immunotoxicity testing of mycotoxins T-2 and patulin on Balb/c mice.

The effects of patulin and T-2 toxin were investigated on immunological responses of Balb/c mice. In vitro patulin had a stimulatory effect on splenocytes at lower concentration (1 nM to 10 nM) and strongly inhibited lymphocyte proliferation at higher concentrations (ID50 from 0.02 to 0.24 microM depending on mitogens). In the same experiments T-2 toxin was 100-fold more potent (ID50 from 0.7 to 2 nM). In vivo studies on immunity were performed in mice receiving Bordetella pertussis antigens and keyhole limpet haemocyanin. Patulin significantly reduced delayed type hypersensitivity to B. pertussis antigen and did not reduce anti-KLH antibody production. T-2 toxin had no effect on delayed type hypersensitivity and reduced anti-KLH antibody production. Splenocytes were harvested in mice with or without antigen stimulation to assess mitogenic responses. Patulin generally increased splenocyte proliferation, therefore T-2 toxin effect depended on the immunological status of mice and on the dose injected. At the lower doses (0.8 mg/kg), T-2 toxin enhanced responses to mitogen, but at the greater dose (1.6 mg/kg) T-2 toxin enhanced responses to mitogen of antigen stimulated mice and decreased responses of unstimulated mice.

Comparative efficacy of five antibiotics on experimental airborne legionellosis in guinea-pigs.

The efficacy of preventive and curative treatment of experimental airborne legionellosis in guinea-pigs was studied for erythromycin, rifampicin, minocycline, doxycycline and pefloxacin. The aerosol was produced from a strain of Legionella pneumophila serogroup 1 and dispersed in a chamber maintained below atmospheric pressure. Guinea-pigs were exposed for 30 min to 1 or 10 LD50 (10(3) or 10(4) viable inhaled organisms). Treatment was preventive or curative. Antibiotic concentrations in lungs and serum were evaluated during treatment. The action of the antibiotics on guinea-pigs infected by 1 LD50 resulted in 100% survival. For the 10 LD50 infection, pefloxacin, minocycline and erythromycin were more active (with 87.5% survivors) than doxycycline (75%) and rifampicin (62.5%). Preventive treatment with pefloxacin or doxycycline gave 100% protection, compared with 87.5% for rifampicin, 50% for erythromycin and 37.5% for minocycline. In this study, treatment of experimental legionellosis was improved, compared with previous publications, by early administration of antibiotics and increased dosage and duration.

Efficacy of spiramycin on experimental airborne legionellosis in guinea pigs.

The effect of preventive and curative spiramycin therapy was studied in guinea pigs infected by aerosol with the experimental model previously tested. The infectious aerosol was obtained from a virulent strain of Legionella pneumophila (Philadelphia ATCC 33 152). Male guinea pigs (Dunkin-Hartley) weighing 250-300 g were exposed for 30 min to an aerosol of 1 or 10 LD50 (10(3) or 10(4) viable inhaled organisms). Spiramycin was administered intraperitoneally (150 mg/kg/day) 18 h after infection for five days for curative therapy; for preventive therapy it was administered on the day before and on the day of aerosol administration (10 LD50). The animals were observed during seven days for weight and temperature and 28 days for survival; bacterial (lungs, spleen) and serological tests were performed. Spiramycin levels (lungs, serum) were evaluated during treatment by a microbiological method. The survival rate in the treated guinea pigs after inhalation of 1 LD50 was 100%. For the 10 LD50 aerosol, curative and preventive therapy gave a survival rate of 87.5%; these results are significant when compared with results of non-treated animals, P less than 0.05. Spiramycin merits further study in experimental and human legionellosis.

[Doxycycline treatment of experimental legionellosis in guinea pigs infected by aerosol]. / Traitement par la doxycycline de la légionellose expérimentale du cobaye infecté par aérosol.

The efficacy of curative and preventive treatment by doxycyclin was studied with the same experimental model as during the study of erythromycin [14]. The infectious aerosol was done with a strain of Legionella pneumophila serogroup 1, Philadelphia (ATCC 33152). Male albinos Dunkin-Hartley guinea-pigs were exposed for 30 min to an aerosol dose of 1, 10 or 100 LD 50 (10(3), 10(4) or 10(5) viable organisms). After preliminary assays to find out the efficient posology, doxycyclin was administered intraperitoneally (IP) 18 h after the infection at a dosage of 60 mg/kg/day for 5 days in the animals infected with 1 LD 50, and 75 mg/kg/day in the animals with 10 and 100 LD 50. The preventive treatment (75 mg/kg/day) administered one day before and the day of the infection was tested with animals infected with 10 LD 50. The guinea-pigs were observed 8 days (weight, rectal temperature) and watched over 3 weeks after the end of treatment; serological and bacteriological tests (cardiac blood, lungs, spleen) were performed in every animal; doxycyclin assays (serum, lungs) during treatment were performed by high performance liquid chromatography (HPLC). The treatment of guinea-pigs infected by 1, 10 and 100 LD 50 gave a survival rate of 100%, 75% and 50% respectively. On the other hand the animals which received preventive treatment were all survivors. Seroconversions with antibody titres from 64 to 512 were observed for all the survivors. Bacteriological tests were all negative. The mean doxycyclin concentrations in serum (microgram/ml) and lung (microgram/g) were 1.66 and 11.42 after 24 h of treatment and 5.71 and 18.93 after 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of culture medium on morphology and virulence of Legionella pneumophila serogroup 1.

In a preliminary study, the preparation of a modified charcoal yeast extract by predialysis of yeast extract (CDYE) allowed us to obtain short non filamentous forms of Legionella pneumophila ser 1 (Philadelphia) found to be more virulent in the chick embryo than the long forms grown on conventional media. We confirmed these findings in guinea pigs inoculated by either intraperitoneal injection or aerosol inhalation. LD50s were calculated using the method of Reed and Muench. Survival curves were established using Liddell's method. If for chick embryo the most virulent organisms were those derived from yolk sac culture, organisms grown on CDYE agar were more virulent than those grown on the other media. There was a significant positive correlation between the mean length of the bacilli and the log 10 of the LD50 (r = 0.96; 0.02 less than p less than 0.05). For guinea pigs by either intraperitoneal injection or inhalation we confirmed that the bacteria cultured on CDYE were more virulent than those grown on other solid media. Thus for the guinea pig inoculated intraperitoneally, the LD50s of the CDYE and BCYE cultures were 1.4 X 10(7) and greater than 3 X 10(9) CFU, respectively. The mortality of guinea pigs inoculated by aerosol with CDYE cultures was significantly higher than that of guinea pigs infected with BCYE cultures using suspensions of 10(8) and 10(9) CFU/ml (p less than 0.01) and 10(10) CFU/ml (p less than 0.05).

Epidemiological evidence of legionellosis transmission through domestic hot water supply systems and possibilities of control.

The use of epidemiological markers for a survey is essential because of the ubiquity of legionellae, particularly L. pneumophila serogroup (SG) 1. The two settings under study were an administrative building associated with a fatal Legionnaires' Disease (LD) case due to L. pneumophila SG 1, and a hospital with 11 cases due to L. pneumophila SG 1 and 3 cases due to L. anisa. Monoclonal antibody serotyping allowed us to establish a link between the outbreaks of LD and the contamination of the hot water supply systems. Two subtypes of L. pneumophila SG 1 and L. anisa were detected in the hospital water system. However the finding of only one subtype of L. pneumophila SG 1 in the patients raised the problem of the difference in virulence of each Legionella strain. By means of aerosol tests on normal and cyclophosphamide-immunosuppressed guinea pigs, we demonstrated the potential pathogenicity of hospital water in the immunosuppressed animals. Two decontamination methods were applied: chlorination and a rise in hot water temperature. Time-limited control measures in the hospital were inadequate, resulting in only temporary eradication, followed by a rapid recolonization of legionellae and the appearance of new nosocomial cases. Decontamination of the administrative building was found to be effective when a constant concentration of 5 ppm free chlorine was obtained at tap outlets, and the water temperature was maintained at 55 C.

[Treatment with erythromycin of experimental legionellosis in guinea pigs infected by aerosol]. / Traitement par l'érythromycine de la légionellose expérimentale du cobaye infecté par aérosol.

The infectious strain L. pneumophila serogroup 1 Philadelphia (ATCC 33152) was cultured on charcoal dialysed yeast extract agar medium (CDYE agar) which produces more virulent strains than those grown on classical agar media. The aerosol was dispersed in a depression chamber by means of a nebuliser and the density was controlled by a density probe. Male albinos Dunkin-Hartley guinea pigs weighing 250-300 g were exposed for 30 minutes to an aerosol dose of 1 LD50 (10(3) viable organisms) and 10 LD50 (10(4) viable organisms). Erythromycin lactobionate (Abbott) was administered subcutaneously 18 hours after the infection, at dosages of 270 mg/kg/day for 4 days in the animals treated with 1 LD50 and for 6 or 7 days in the animals treated with 10 LD50. The guinea pigs were observed for 9 days (weight, rectal temperature; serological and bacteriological tests (cardiac blood, lungs, spleen) and erythromycin assays (serum, lungs) were performed and compared in the treated animals, the non-treated infected control animals and the control animals which only received erythromycin. The percentage survival in the treated guinea pigs after inhalation of 1 LD50 and 10 LD50 (2 tests) were 100%, 75% and 87.5% respectively. Three weeks after treatment, the survivors had antibody titres from 32 to 1,024; the bacteriological cultures and erythromycin assays were negative. In this study, an improvement in the treatment of experimental Legionnaires' disease was observed in comparison with previous experiments. The increased dosage and duration and the early initiation of treatment resulted in survival rates of 75%.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunomodulation in offspring mice after neonatal immunostimulation of mothers or newborn mice.

Mothers of offspring Balb/c mice were stimulated after birth by two substances, a bacterial lysate (LAB) and a chemical, diethyldithiocarbamate (DETC). Anti-sheep red blood cell (SRBC) antibodies were studied after immunization of stimulated mothers or offspring. An increase of anti-SRBC was observed in LAB-stimulated mothers, but these antibodies were decreased in their offspring before weaning. Sometimes, these antibodies were increased in LAB-stimulated newborn mice. DETC stimulation of mothers induced an elevation of antibody response in mothers and newborns. The same results were obtained in previous investigations where the pregnant mother was stimulated with the same agents.

[In vitro activity of an extract of Yersinia enterocolitica 0:3 biotype 4 on lymphoblastic transformation]. / Etude in vitro de l'activité d'un extrait de Yersinia enterocolitica 0:3 biotype 4 sur la transformation lymphoblastique.

Effects of an extract from Y. enterocolitica 0:3 biotype 4 on mouse splenocytes and human lymphocytes cultures were examined. Low doses had a mitogenic action on splenocytes, while high doses inhibited tritiated thymidine incorporation into the cells. With human lymphocytes only inhibitory effect was observed.

Interaction between thymosin, testosterone and estradiol on natural killer cell activity in mice.

Thymosin fraction 5, an immunopotentiating thymic preparation, significantly increases the cytotoxic capacity of NK cells isolated from the spleen. This stimulation is inhibited by testosterone and estradiol.