M. Cristina Cerquetti

Intranasal immunization with temperature-sensitive mutants protects granulocytopenic mice from lethal pulmonary challenge withPseudomonas aeruginosa

Intranasal (i.n.) immunization with two temperature-sensitive (ts) mutants ofPseudomonas aeruginosa protected, in a dose-related manner, granulocytopenic (GCP) mice challenged with a lethal dose of the wild-type (wt) organism. The number of ts mutants in oronasopharyngeal lavage fluids and stools decreased steadily in both normal and GCP mice after i.n. immunization. Intranasal immunization with 107 colony-forming units (CFU) of either mutant induced significant protection, whereas intraperitoneal (i.p.) immunization with similar doses induced lower protection. Protection induced by i.n. immunization was accompanied by increased levels of anti-P. aeruginosa IgA in lung lavage fluids. The results of this study demonstrate the usefulness of ts mutants ofP. aeruginosa for local immunization to protect GCP hosts from fatalP. aeruginosa pneumonia.

Research articleIntramammary immunization with live-attenuated Staphylococcus aureus: microbiological and immunological studies in a mouse mastitis model

Mammary infection was induced in lactating mice by intramammary injection of Staphylococcus aureus. Histopathological analysis revealed infiltration and lesions of varying magnitude that were still apparent 21 days after the challenge. Concomitantly, viable S. aureus was recovered from infected mammary glands. Mice were immunized by the intramammary route with 5 × 106 colony forming units of a temperature-sensitive mutant of S. aureus and subsequently received a boosting injection seven days later. On day 14 mice were challenged by the intramammary route with the wild-type strain. Intramammary immunization induced a significant increase in milk IgA (P < 0.05), serum IgG (P < 0.05) and serum IgA (P < 0.05) on the day of the challenge, when compared with non-immunized mice. Immunization decreased significantly (P < 0.01) the number of S. aureus colony forming units recovered 96 h after intramammary challenge. In conclusion, the feasibility of immunizing locally with temperature-sensitive S. aureus to induce immunity in the mouse mammary gland was demonstrated. The mouse model of mastitis is proposed as a useful system for screening temperature-sensitive S. aureus strains to be utilized in the development of a vaccine.

Evaluation of different temperature-sensitive mutant phenotypes ofSalmonella enteritidis as vaccine potentials

Temperature-sensitive (ts) mutants ofSalmonella enteritidis were isolated after mutagenesis with UV light and enrichment with antibiotic. Mutants were characterized according to their growth profile at the permissive (28°C) and the nonpermissive (37°C) temperatures, persistence of surface antigens, reversion frequencies, and potentials for inducing humoral immunity and protection against challenge with the parental wild-type (wt) in mice. We obtained 32 strains ofS. enteritidis able to grow well at 28°C, but capable of only limited or no replication at 37°C. The ts mutants were positive for factor 9 in an agglutination assay and were susceptible to infection with phage P22. Three mutants of different phenotypes were selected for protection studies. A single intraperitoneal (i.p.) immunization with any of the mutants studied induced significant protection from i.p. challenge with 100 LD50 of the wt strain.

Pulmonary Clearance of Staphylococcus Aureus and Plasma Angiotensin-Converting Enzyme Activity in Hydrocarbon Pneumonitis

Summary: Pulmonary clearance of Staphylococcus aureus and plasma angiotensin-converting enzyme (ACE) activity were investigated in an experimental mouse model of kerosene aspiration. Twenty-four hours after acute kerosene aspiration, mice were exposed for 30 min to an aerosol containing the pathogen, and the uncleared bacteria ratio (UBR) determined 4 h after nebulization. The results showed a significant increase (P = 0.004) in UBR in animals with severe pneumonitis (0.44 ± 0.05) when compared with controls (0.24 ± 0.03). This impairment in lung clearance correlated with the increase in lung weight and the decrease in plasma ACE levels. Mice with kerosene pneumonitis had zones of lung injury, and areas with no gross signs of tissue damage. Lung clearance of S. aureus was significantly impaired in damaged areas whereas it was no different from controls in the non-affected areas. It is suggested that the measurement of plasma ACE activity may be an adjunct in the assessment of the extent of lung injury in hydrocarbon aspiration in children.

Genetically stable temperature-sensitive mutants of Salmonella typhi induce protection in mice

Temperature-sensitive (ts) mutants of Salmonella typhi were isolated following treatment with nitrosoguanidine, and characterized with respect to cut-off temperature, ts phenotype and reversion frequency. Linkage of the ts mutations to selectable chromosomal markers was established by generalized transduction with bacteriophage phage Vi I, and the appropriate antibiotic resistances were transduced into the ts mutants. Multiple mutant S. typhi were then constructed by combining three independent ts mutations in one strain, utilizing linkage of three of the mutations to erythromycin-, streptomycin- and methylglyoxal-resistance. Several recombinants are genetically stable, with calculated reversion rates of less than 10(-22), and induce both protection from intraperitoneal challenge with the virulent parental wild-type S. typhi in mice and the formation of antibodies to the somatic O-9 and O-12, the flagellar H and the capsular Vi antigens.

Immunization with temperature-sensitive mutants of Salmonella typhi induces protection in mice

Temperature-sensitive (TS) mutants of Salmonella typhi were isolated following mutagenesis with nitrosoguanidine and two cycles of enrichment with penicillin and D-cycloserine. Several of the TS mutants were characterized with respect to growth profiles at permissive (29 degrees C) and non-permissive (36 degrees C) temperatures, reversion rates, and the potential for inducing protection against challenge in an animal model. All three TS mutants tested were immunogenic in mice; antibodies measured by enzyme-linked immunosorbent assay were produced following intraperitoneal (i.p.) immunization with three different doses of each of the mutants; i.p. immunization with the same mutants also induced highly significant protection (100%) from i.p. wild-type challenge; and oral immunization with one of the mutants significantly reduced shedding of the wild-type following oral challenge.

Oral immunization of mice with temperature-sensitive Pseudomonas aeruginosa enhances pulmonary clearance of the wild-type

DBA/2J mice were immunized daily for 3 days per os with 10(8)-10(9) colony forming units (c.f.u.) of two different temperature-sensitive (TS) mutants of Pseudomonas aeruginosa. At varying times after the final immunization the animals were exposed to aerosols of the parental immunotype 1, and the ability of the immunized and control mice to clear their lungs of the wild-type (WT) challenge was measured 4 h later. The number of c.f.u. remaining in the lungs of mice immunized with one mutant, D/1/8, was significantly less (p less than 0.01) than the number remaining in the lungs of control mice and mice immunized with a second TS mutant, E/9/9.

Defective Mononuclear Phagocytic Function in Mice Homozygous for the Cribriform Degeneration Autosomic Recessive Mutation

Decreased lung clearance of Staphylococcus aureus has been reported in mice homozygous for the cribriform degeneration (cri) autosomal recessive mutation. In the present study, the phagocytic capacities of alveolar and peritoneal macrophages were quantitated by applying kinetics of the first order reaction criteria. The characteristics of the pulmonary and peritoneal mononuclear cell populations from mutant and control mice were indistinguishable. The kinetic assays revealed decreased phagocytosis work in both alveolar and peritoneal macrophages from cri/cri mice. The results lend support to this mutation as a possible model system to study the early stages of lung disease physiopathology in cystic fibrosis.