Fts insertional mutant of Salmonella typhimurium.

A temperature-sensitive filamentation (fts) Salmonella typhimurium mutant was isolated after transposon mutagenesis with mini-Tn 10dTc. The mutant was unable to form colonies after 20 h incubation at 37 degrees C on LB agar. Colonies appeared, however, after longer incubation at the restrictive temperature. Filamentation affected only part of the bacterial population. Rapid mapping using Mu dP22 hybrid phages revealed that the mutation, ftsD220, lies within minutes 68.5 and 73.6 on the genetic map. Further analysis revealed that the ftsD220 mapped at min 73 and that it is linked to cysG (6%) and to aroB (39%). Complementation tests suggested that the ftsD220 mutation is not homologous to a Escherichia coli ftsH mutation.

Local and systemic immunity against Streptococcus pneumoniae: humoral responses against a non-capsulated temperature-sensitive mutant.

Temperature-sensitive mutants of Streptococcus pneumoniae were isolated after chemical mutagenesis. Intranasal immunization with temperature-sensitive mutant J/3 induced higher levels of circulating antibody than those obtained after immunization with the heat-killed parental wild type. Moreover, local immunization with mutant J/3 induced high levels of anti-S. pneumoniae IgG and IgA in the lower respiratory tract, whereas only moderate IgG (and no IgA) antibodies were detected in lung lavage fluids from mice immunized intranasally with the heat-killed strain.

Intramammary 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 x 10(6) 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.

Intramammary immunization with live-attenuated Staphylococcus aureus protects mice from experimental mastitis.

Female mice were immunized by the intramammary route with live-attenuated Staphylococcus aureus according to different schedules and challenged with virulent S. aureus. Immunization in late pregnancy or early lactation induced a significant decrease (P <0.05) in the number of S. aureus CFU recovered from glands after the challenge and a significant increase (P <0.05) in the levels of milk and serum specific IgG and IgA antibodies. Mice immunized before pregnancy were not protected from S. aureus challenge. Immunization did not increase the number of somatic cells in milk when compared with control mice. Protection from S. aureus intramammary infection may be achieved if mice are locally immunized during late pregnancy or early lactation.

Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes.

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.

Interaction between granulocytes and antibodies in the enhancement of lung defenses against Staphylococcus aureus after intranasal immunization of mice with live-attenuated bacteria.

Immunization with live-attenuated Staphylococcus aureus induced measurable levels of specific IgG and IgA in the lungs, but the pulmonary clearance of S. aureus in immunized mice did not differ from that of control mice. Aerosol exposure of mice to Pseudomonas aeruginosa induced a significant recruitment of polymorphonuclear leukocytes (PMNL) to the lungs in both immunized and control mice, whereas S. aureus challenge did not. However, challenge with a mixture of P. aeruginosa-S. aureus or exposure to an aerosol of Escherichia coli lipopolysaccharide (LPS) before S. aureus challenge induced PMNL migration and a significant enhancement of pulmonary clearance of S. aureus in immunized mice. The presence of both antibodies and PMNL was required for enhancement of S. aureus pulmonary clearance.

Differential persistence, immunogenicity and protective capacity of temperature-sensitive mutants of Salmonella enteritidis after oral or intragastric administration to mice.

The persistence of Salmonella enteritidis temperature-sensitive (ts) mutants of different phenotypes in Peyer's patches (PP) and the spleen, and their immunogenicity after intragastric (i.g.) and peroral (p.o.) administration to mice was investigated. After p.o. administration the ts mutant C/2/2 colonized PP, but was not recovered from the spleen. After i.g. administration the ts mutant E/1/3 colonized both the spleen and PP for at least 2 weeks. Mutant C/2/2 persisted in PP up to 8 days but was not found in the spleen. Mutant H/2/26, although it poorly colonized the PP, was recovered from the spleen up to day 15 after i.g. administration. Immunization with E/1/3 by either the i.g. or the p.o. routes protected mice from challenge with 100 LD50 of the virulent wild-type (wt) strain. Immunization with either C/2/2 or H/2/26 did not confer protection. The three ts mutants induced the production of local IgA after i.g. administration regardless of their protective capacity.

Orally administered attenuated Salmonella enteritidis reduces chicken cecal carriage of virulent Salmonella challenge organisms.

Chickens were immunized orally with 10(9)cfu of the temperature-sensitive (T(s)) mutant E/1/3 of Salmonella enteritidis at 1, 2, 3 and 7 days of age. The animals were challenged with wild-type strains of Salmonella of different serotypes 7 or 14 days following immunization. Chickens receiving multiple oral doses of the vaccine strain showed no signs of disease. Immunized animals shed the vaccine strain for at least 2 weeks after the last inoculation; on the other hand, colonization by the attenuated mutant of internal organs such as spleen and liver was limited. Early exposure of the immunized animals to the virulent bacteria resulted in a reduced cecal colonization by the pathogen. Visceral invasion by the wild-type strain of S. enteritidis or S. gallinarum was drastically diminished in birds challenged 14 days after immunization. Significant differences in the number of these Salmonella were found in the cecal contents, spleen and liver of immunized birds compared with the control animals. In addition, cecal colonization by the virulent strain was reduced in birds challenged with S. typhimurium. These results demonstrate that immunization of newly hatched chickens with live attenuated T(s) mutant E/1/3 of S. enteritidis is safe and reduces Salmonella shedding.

SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium are synthesized at late stages of infection in mice.

Salmonella pathogenicity island (SPI)-1 is essential for invasion of non-phagocytic cells, whereas SPI-2 is required for intracellular survival and proliferation in phagocytes. Some SPI-1 effectors, however, are induced upon invasion of both phagocytic and non-phagocytic cells, suggesting that they may also be required post-invasion. In the present work, the presence was analysed of SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium in vitro and in vivo during murine salmonellosis. Tagged (3xFLAG) strains of S. enterica serovar Typhimurium were inoculated intraperitoneally or intragastrically to BALB/c mice and recovered from the spleen and mesenteric lymph nodes of moribund mice. Tagged proteins were detected by SDS-PAGE and immunoblotting with anti-FLAG antibodies. In vitro experiments showed that SPI-1 effector proteins SipA, SopA, SopB, SopD and SopE2 were secreted under SPI-1 conditions. Interestingly, it was found that S. enterica serovar Typhimurium continued to synthesize SipA, SopB, SopD and SopE2 in colonized organs for several days, regardless of the route of inoculation. Together, these results indicate that SPI-1 effector proteins may participate in the late stages of Salmonella infection in mice.

Vaccination of chickens with a temperature-sensitive mutant of Salmonella enteritidis.

One-day old chickens were inoculated with temperature-sensitive mutant E/1/3 of S. enteritidis. Two routes of inoculation were used: oral and intraperitoneal (ip). One group of chickens were given two oral inoculations (oral-oral). A second group received two ip inoculations (ip-ip). A third group received the first dose orally and the second ip (oral-ip) and the fourth group was given the first dose ip and the second dose orally (ip-oral). The vaccine strain was safe even when inoculated at high doses, and induced strong protection against virulent S. enteritidis strain after oral challenge. Results show that vaccination with mutant E/1/3 reduced the number of animals shedding the pathogen after challenge. Furthermore, animals immunized oral-oral and oral-ip showed a significant reduction in cecal and spleen colonization by virulent Salmonella.

Vi I typing phage for generalized transduction of Salmonella typhi.

Salmonella typhi Vi typing phages were used to transduce temperature-sensitive (Ts) mutants of Salmonella typhi. Antibiotic resistance and Ts+ markers were transduced at high frequency (> 10(-4) per virulent phage). Several markers were cotransduced by phage Vi I, suggesting that it may be useful for mapping studies of the S. typhi genome.

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.

Replication rate of Pseudomonas aeruginosa in the murine lung.

Using a method recently developed at our laboratory, we determined the initial rate of Pseudomonas aeruginosa replication in the lung under different experimental conditions. Mice were exposed to aerosols containing mixtures of a temperature-sensitive (ts) mutant of P. aeruginosa and its parental wild type (wt). The changes in the ratio of ts:wt were determined by quantitatively culturing homogenates of lungs from animals sacrificed over different time periods. The doubling time (DT) was calculated as the reciprocal of the slope of the linear portion of the curve generated by plotting n = (log [r0/rt])/log 2 against time where r is the ratio of ts:wt at a given time. The DTs measured in both outbred ICR mice and F1 hybrids (DBA/2J X B10.D2/nSnJ) were 32 and 30 min, respectively. These DTs were higher than that determined in the peritoneal cavities of ICR mice (20 min). The DT in the lungs of ICR mice rendered granulocytopenic by treatment with cyclophosphamide was 16 min. Experiments performed with inocula of different sizes showed that DTs tended to be higher in animals aerosolized with low doses of the ts-wt mixture.

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.

Piroxicam treatment protects mice from lethal pulmonary challenge with Pseudomonas aeruginosa.

The effect of treatment with the nonsteroidal anti-inflammatory agent piroxicam on leukocyte migration to the lungs was investigated after aerosol administration of sublethal doses of Pseudomonas aeruginosa to mice. Piroxicam decreased, in a dose-related fashion, the polymorphonuclear leukocyte recruitment to, and the degree of perivascular and peribronchial infiltration in, the lungs. Piroxicam treatment also protected the animals in a dose-dependent manner from challenge with lethal doses of P. aeruginosa. The effect of piroxicam was not related to direct action of the drug on the microorganisms. Piroxicam treatment maintained the animal's pulmonary defenses against infection while diminishing inflammatory responses against P. aeruginosa, an occurrence decreasing the potential for tissue damage due to phagocytes migrating from circulation.

Specific pulmonary defences against Pseudomonas aeruginosa after local immunization with temperature-sensitive mutants.

The specificity of the enhancement in lung defences after local immunization of mice with three temperature-sensitive (ts) mutants of Pseudomonas aeruginosa was investigated. The three selected mutants display altered growth characteristics when transferred from 29 degrees C to mammalian body temperature. Mice immunized with the live ts mutants by aerosol exposure or multiple intranasal inoculations were challenged with aerosols containing wild-type (wt) P. aeruginosa. Aerosol immunization with ts mutant A/10/25 significantly enhanced the lung clearance of the wt but did not enhance the clearance of either Klebsiella pneumoniae or Staphylococcus aureus. Aerosol immunization with ts mutants D/1/8 or E/9/9 enhanced the lung defences against the parental wt (of identical immunotype 1) but not against immunotype 4; similarly, intranasal immunization enhanced the lung defences against the parental wt but not against immunotypes 4 or 5. We conclude that local immunization with ts mutants of P. aeruginosa enhances lung defences against the wt in a genus- and immunotype-specific fashion. It is suggested that local immunity may play a central role in immunoprophylaxis against P. aeruginosa lung infection.

A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium.

A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet. The mutant D220 grows well at 28 degreesC but has a lower growth rate and forms filaments at 37 degreesC. Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced. The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein. The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype. The insertion dam-231 : : Tn10d Tet resulted in a dam "leaky" phenotype since methylated and unmethylated adenines in GATC sequences were present. In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S. typhimurium strain. The wild-type dam gene of S. typhimurium exhibited 82% identity with the Escherichia coli dam gene.

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization.

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10(-6). Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10(8) colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.

Protective capacity of a temperature-sensitive mutant of Salmonella enteritidis after oral and intragastric inoculation in a murine model.

Temperature-sensitive (ts) mutant E/1/3 of Salmonella enteritidis was selected to evaluate its capacity to induce protective responses after peroral (p.o.) or intragastric (i.g.) inoculation to mice. This ts mutant of coasting phenotype was detected in Peyer's patches until day 4, and in spleen by days 3 and 4 after the mice were inoculated by the p.o. route with 10(10) colony forming units. Peroral immunization induced significant protection from oral challenge with 240 LD50 of the wild-type (wt) strain. Higher protection was achieved when the animals were boosted intraperitoneally after p.o. immunization. Intragastric inoculation with the same dose of the ts mutant increased both the level of protection, and colonization and persistence of the micro-organism in Peyer's patches and spleen. Immunization with a single i.g. inoculation induced 70% protection from p.o. challenge of the animals with the wt S. enteritidis. Two i.g. immunizations with E/1/3 raised the level of protection to 90%. Specific IgG, IgM and IgA antibodies, measured in plasma using a micro-ELISA method, were detected after i.g. immunization with ts mutant E/1/3. In addition, specific antibody-secreting cells were detected by means of an ELISPOT assay in spleen and mesenteric nodes of mice immunized with the ts mutant.

Impaired lung defenses against Staphylococcus aureus in mice with hereditary deficiency of the fifth component of complement.

Mice of the C5-deficient DBA/2J and B10.D2/oSnJ inbred strains were aerosolized with Staphylococcus aureus, and the pulmonary clearance of bacteria was determined 4 h later. Both C5-deficient strains had a significantly decreased lung clearance of S. aureus compared with their genetically closest C5-sufficient relatives, DBA/1J and B10.D2/nSnJ strains, respectively. Serum hemolytic activity and pulmonary clearance of S. aureus were also investigated in F1 and F2 progenies (DBA/1J X DBA/2J and DBA/2J X B10.D2/oSnJ). Serum hemolytic activity was present in all F1 (DBA/1J X DBA/2J) mice, and their pulmonary clearance of S. aureus was no different from that of the C5-sufficient parents (DBA/1J). The absence of serum hemolytic activity (absence of C5) in all mice from F1 and F2 (DBA/2J X B10.D2/oSnJ) and 20% of the F2 (DBA/1J X DBA/2J) was related to a decreased lung clearance of S. aureus. These results are consistent with an autosomal recessive pattern of heredity for the murine abnormality in pulmonary clearance of S. aureus.