Immunofluorescence characterization of Legionella: narrow specificity of polyclonal immunsera to various serogroups and species.

This study was to determine by direct fluorescent antibody staining with antibodies prepared in rabbits, the degree of cross-reactions between serogroups of Legionella pneumophila (1 to 6) and the other antigenic species of Legionella (L. bozemanii, L. dumoffii, and L. micdadei), in order to increase the accuracy of diagnosis and to allow us to reduce the number of conjugates. The polyclonal antibodies were highly species and serogroup-specific without absorption for characterization of Legionella either in patient specimens or in isolated cultures by direct fluorescent antibody staining. No cross-reaction was observed with non-legionella bacteria isolated from sputum specimens. A battery of conjugates for different serogroups and species is necessary for increasing the accuracy of diagnosis of legionellosis.

Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping.

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome of Legionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls. A polymorphism was observed for seven markers among the L. pneumophila strains tested. To check whether these markers could be used to compare strains of L. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of the L. pneumophila colonies and gave a first hint as to the presence of several strains in a single sample.

Francisella tularensis bacteremia.

Bacteremia caused by Francisella tularensis is rare and has been reported mainly in the United States and infrequently in Europe. We report herein the first case of bacteremic F. tularensis pneumonia in an immunocompetent individual in southern Europe.

Detection of Legionella pneumophila antigen by ELISA in urine or experimentally infected guinea-pigs.

Guinea-pigs were experimentally infected with Legionella pneumophila. L. pneumophila antigen was detected in urine samples by a double antibody sandwich ELISA. Urinary antigen was present from the beginning of the acute phase of the disease. In two out of five cases, this antigen was found with no antibody detectable in sera by indirect immunofluorescence staining.

Characterization, serological specificity, and diagnostic possibilities of monoclonal antibodies against Legionella pneumophila.

Hybridoma-producing monoclonal antibodies against Legionella pneumophila were produced by the fusion of nonsecreting mouse myeloma cells (NS-1) with splenocytes of BALB/c mice immunized by heat-killed L. pneumophila of serogroup I. Of 96 wells, 85 produced clones, of which 28 were positive as measured by an enzyme-linked immunosorbent assay technique. Ten of the hybridoma supernatants, remaining positive after 2 months of culture, were tested against the other Legionella serogroups and the atypical strains. None showed significant cross-reaction. Six of the positive clones were subcloned by limiting dilution, and two subclones were put into ascites in BALB/c mice. The monoclonal antibody obtained from the II-6-18 subclone was of the gamma-3 isotype. In this report, we describe the conditions for the use of this monoclonal antibody as a diagnostic tool for the detection of serogroup I L. pneumophila.

In vitro expression of a 22-kilodalton Yersinia pestis polypeptide immunologically related to the 25-kilodalton plasmid-encoded protein of the three pathogenic Yersinia species.

Antibodies raised against the 25-kilodalton (p25) plasmid-encoded polypeptide of Yersinia enterocolitica recognized the homologous protein in the three Yersinia species grown in vitro. This polypeptide was recovered from whole cells as well as from the fluid supernatant of bacteria grown at 37 degrees C in a Ca2+-deficient medium. Furthermore, a 22-kilodalton (p22) plasmid-encoded polypeptide immunologically related to p25 was found only in Y. pestis during early growth. After 30 h of culture, the Y. pestis p25 and p22 were completely degraded, whereas the intensity of the Y. enterocolitica p25 was decreased, but the protein was still detectable in the fluid supernatant. This proteolytic activity was independent of the presence of the virulence plasmid. Some disulfide bonds are probably involved in the quaternary structure of the p25 of the three pathogenic species and of the Y. pestis p22.

Standardization for diagnostic purposes of a monoclonal antibody against Legionella pneumophila.

A monoclonal antibody against Legionella pneumophila has been produced and characterized. The antibody was of the gamma-3 isotype and recognised the lipopolysaccharides of the bacteria as was confirmed by Western blotting. Preliminary assays were performed for the detection of the antigen in clinical samples in order to set up a rapid laboratory technique for the early diagnosis of legionellosis. By using an immuno-enzymatic technique, less than 30 000 bacteria or corresponding antigens could be detected in a few hours.

Molecular typing of nosocomial isolates of Legionella pneumophila serogroup 3.

In Paris, France, an outbreak of pneumonia due to Legionella pneumophila serogroup 3 was observed in Necker (four cases) and Pitié (six cases) hospitals. Neither the 10 clinical isolates nor 5 tap water isolates from Necker Hospital harbored plasmids. Clinical and environmental serogroup 3 isolates and serogroup 3 reference strain Bloomington 2 were analyzed by chromosomal probe fingerprinting. rRNA, 16S and 23S from Escherichia coli and a randomly cloned 15-kilobase-pair nucleotide sequence from L. pneumophila serogroup 3 were used as probes. All strains tested showed a single pattern after HindIII digestion of DNA and hybridization with the 32P-end-labeled rRNA probe, whereas three patterns were obtained after hybridization with the 32P-labeled 15-kilobase-pair DNA probe. One pattern was given by all clinical and tap water isolates from Necker Hospital, another one was given by all clinical isolates from Pitié Hospital, and a last one was given by reference strain Bloomington 2. Thus, molecular analysis showed that the two hospital outbreaks of legionellosis were unrelated and could link the outbreak in Necker Hospital to contaminated tap water.