Legionella pneumophila urinary antigen subtyping using monoclonal antibodies as a tool for epidemiological investigations

Legionnaires’ disease is diagnosed predominantly by urinary antigen detection, and patient isolates are rarely available. The lipopolysaccharide (LPS) epitope pattern of isolates detected by monoclonal antibodies is an accepted marker for the phenotyping of L. pneumophila serogroup 1 strains into monoclonal subgroups. L. pneumophila LPS is the dominant antigen in patients’ urinary specimens. By using commercially available microtiter wells coated with rabbit anti-Legionella serogroup 1 IgG as the catching antibody, LPS components in urine specimens were bound and detected separately by corresponding monoclonal antibodies of the Dresden Panel. The subtyping of LPS on urinary antigen molecules by using enzyme-linked immunosorbent assay (ELISA) allows deducing of first evidences for the identity/non-identity of environmental isolates and the legionellosis pathogen. Most importantly in our study, urinary antigen typing possesses high probability to distinguish (or does not distinguish) if the pathogen belongs to the MAb 3/1-negative L. pneumophila strains, which are widespread contaminants of water systems, but represent the minority of patient isolates.     

Taxonomic investigation of Legionella pneumophila using monoclonal antibodies.

A panel of 19 monoclonal antibodies was used to produce patterns of immunofluorescent staining of 468 isolates of Legionella pneumophila. Twelve monoclonal antibodies were selected that divided L. pneumophila into 17 phenons which, in the majority of cases, conform to serogroup divisions. These phenons are more easily defined than the present serogroups, and isolates can be placed in them with little ambiguity. The standardized set of monoclonal antibodies was also used to define the subgroups of serogroup 1.     

Production of monoclonal antibodies to Legionella pneumophila serogroups 1 and 6

To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, we have produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common antigen. The second antibody, LP-I-81, was specific for serogroup 1. This antibody was able to agglutinate bacterial cells belonging to the serogroup 1 reference strains. Philadelphia and Knoxville. Microagglutination assays of environmental and clinical isolates revealed a subgroup of serogroup 1 environmental isolates which were not agglutinated by LP-I-81. This subset of isolates was segregated to certain buildings in the medical complex. Immunodiffusion studies showed identity between the LP-I-81 antigen and the serogroup-specific antigen of serogroup 1 organisms. This antigen could be absorbed out of the serogroup 1 organism extract with LP-I-81-coated Staphylococcus aureus, leaving the serogroup-common antigens. Three hybridomas were produced to serogroup 6. All three produced antibodies which were serogroup 6 specific and agglutinated serogroup 6 bacteria.     

Detection of Legionella pneumophila antigens in patients' sera using monoclonal antibodies

Three monoclonal antibodies (McAb) were produced against soluble antigens of Legionella pneumophila serogroup 1 which was cultured on BCYE agar. The McAbs were all of the IgM isotype. The McAbs were used in the McAb-based ELISA for detection of circulating L. pneumophila antigens in 186 sera collected from patients with symptoms and signs suggestive of atypical pneumonia. The normal reference optical (OD) density value of each of the McAbs was determined using 44 sera collected from healthy blood donors. The antigen positivity rates for the McAbs 1C7.2B, 2B2.10F and 2B2.11E were 11.3%, 7.7% and 22.2% respectively. Antigen positivity of the McAb 2B2.10F was significantly higher in the younger age group (p < 0.05). There is no significant association between the antigen positivity with age and sex for all the McAbs. There was no cross-reaction demonstrated between the McAbs with other bacterial antigens.     

Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping

Endemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection.     

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.     

Confirmation of Legionella pneumophila cultures with a fluorescein-labeled monoclonal antibody.

We compared a fluorescein-labeled monoclonal antibody directed against an outer membrane protein of Legionella pneumophila (Genetic Systems Corp. [GSC], Seattle, Wash.) with a similarly labeled polyclonal reagent (L. pneumophila serogroups 1 to 6, poly; BioDx, Inc., Denville, N.J.) for the confirmation of L. pneumophila isolates grown in culture. Duplicate suspensions of 52 organisms, including 21 L. pneumophila and 8 non-L. pneumophila species of legionella, were placed on individual glass slides, fixed, and stained with both reagents, and the results were compared. Both antisera correctly identified all L. pneumophila serogroups 1 to 6, but only the GSC reagent produced definitive staining of the L. pneumophila isolates of serogroups 7, 8, and 9. Additionally, the GSC reagent produced more uniform staining patterns around the legionella bacilli and displayed little background fluorescence when compared with the BioDx reagent.     

Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed.     

Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroup 1.

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to characterize Legionella pneumophila serogroup 1. Cells from a single colony could be subtyped by AP-PCR within a few hours. The discrimination between strains of L. pneumophila serogroup 1 by AP-PCR was equivalent to that by monoclonal antibody analysis and ribotyping. Four strains representing the monoclonal antibody pattern most frequently associated with outbreaks all yielded unique amplicon patterns by AP-PCR.     

Immunologic characterization and specificity of three monoclonal antibodies against the 58-kilodalton protein of Legionella pneumophila.

Three monoclonal antibodies against the Legionella pneumophila 58-kDa protein were produced. By using immunoblot analysis, the percentages of reactivity against 47 serogroups of Legionella representing 29 species were determined to be 80.9, 87.2, and 95.6 for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. Specificities obtained from testing 63 heterologous organisms representing 22 genera and 46 species were 90.7, 92.2, and 95.3% for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. No single heterologous strain was reactive with all three monoclonal antibodies. These monoclonal antibodies successfully identified all 10 clinical isolates of Legionella examined in a dot blot assay and should be excellent reagents for use in genuswide diagnostic immunoassays.     

Detection of soluble Legionella pneumophila antigens in serum and urine specimens by enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies

Urine and serum specimens from three patients with pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) were tested by enzyme-linked immunosorbent assay (ELISA) for Lp1-soluble antigen. A three-layer direct ELISA with polyclonal antibodies and a four-layer indirect ELISA with both polyclonal and monoclonal antibodies were used. Lp1 antigen was detected in both urine and serum from the three patients. As determined by ELISA, the concentration of antigen was 30- to 100-fold less in serum than in urine collected on the same day. In some instances the indirect ELISA was more sensitive than the direct ELISA, but in others it was less sensitive, depending on the monoclonal antibody used. The subgroup of the infecting Lp1 organism was determined based on antigenic determinants expressed in the urine. This study illustrates the use of serum as well as urine as an antigen reservoir in the laboratory diagnosis of legionellosis by ELISA and the potential for developing more sensitive antigen detection systems by the judicious use of monoclonal antibodies.     

Rapid subtyping of equine herpesvirus 1 with monoclonal antibodies.

Two antigenically similar subtypes of equine herpesvirus 1 (EHV-1) cause disease in horses. A procedure for rapid differentiation of the two EHV-1 subtypes with monoclonal antibodies was developed. Subtype-specific pools of monoclonal antibodies were constructed, characterized, and used in enzyme immunofiltration and indirect immunofluorescence assays to subtype 50 epizootiologically unrelated field isolates of EHV-1. Both assays allowed accurate subtype identification of each EHV-1 isolate with the monoclonal antibody pools. The subtyping procedures were simple and amenable to typing many isolates at one time and permitted unambiguous EHV-1 subtype identification with 3 h after isolation of the virus in tissue culture.     

Induction and protective role of antibodies in Legionella pneumophila infection

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium found in aquatic environments and is the causative agent of Legionnaires' disease, a severe form of pneumonia. We have used Lpn-permissive A/J mice as a model to analyze the B cell response upon intravenous (i.v.) and intranasal (i.n.) infection with Lpn. A strong antibody (Ab) response was observed upon i.v. infection with wild-type (WT) Lpn and an icmT mutant strain, which is unable to replicate within permissive host cells. In contrast to i.v. infection, only WT but not icmT mutant Lpn was able to induce specific Ab responses upon i.n. infection. After primary i.n. infection with WT Lpn, a strict compartmentalization of Lpn-specific Ab isotypes was observed, as IgG was found exclusively systemically, while IgA was detectable only locally in the lung. Regardless of the infection route, isotype switching to IgG and to IgA was strictly dependent on CD4+ T cells, whereas IgM production was completely Th-independent. Finally, we analyzed the protective capacity of the Lpn-specific Ab response. Actively or passively immunized mice or mice that were infected with opsonized Lpn had 50-100-fold reduced bacterial titers compared to naive animals, clearly demonstrating the capacity of Ab to protect against infection with Lpn.     

Legionella micdadei and Legionella dumoffii monoclonal antibodies for laboratory diagnosis of Legionella infections.

Two different monoclonal antibodies directed against Legionella micdadei and L. dumoffii (Genetic Systems Corp., Seattle, Wash.) were evaluated for their specificity and ability to detect L. micdadei and L. dumoffii in human and animal clinical samples and bacterial isolates in an indirect immunofluorescence assay. All three frozen sputum samples and all three Formalin-fixed sputum and liver samples from patients with culture-documented L. micdadei pneumonia were positive when tested with the L. micdadei monoclonal antibody. A Formalin-preserved lung sample from a patient with culture-documented L. dumoffii pneumonia was positive with its homologous monoclonal antibody. No cross-staining reactions were found with either monoclonal antibody on any of 25 human sputum samples tested from patients without Legionella infections. A total of 66 Legionella strains and 56 non-Legionella strains including 22 Pseudomonas strains and 34 other bacterial strains were studied. No cross-staining reactions were found except in Staphylococcus aureus Cowan 1 ATCC 12598. The lower limit of detection in seeded sputum samples was about 7 X 10(4) cells per ml for both monoclonal antibodies. Lung and tracheal lavage specimens from L. micdadei- or L. dumoffii-infected guinea pigs showed specific staining only with their respective monoclonal antibodies. The monoclonal antibodies stained homologous bacteria slightly less intensely than did the polyclonal antisera, but the signal-to-noise ratio was considerably higher for the monoclonal antibodies. No differences in sensitivity of staining of clinical specimens or bacterial isolates were noted between the monoclonal antibodies and the polyclonal reagents for L. micdadei and L. dumoffii (Centers for Disease Control, Atlanta, Ga., and BioDx, Denville, N.J. These monoclonal antibodies ae sensitive and specific, making them good candidates for laboratory diagnostic purposes.     

Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila

A total of 57 isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody subgrouping, seven-gene locus sequence-based typing (SBT) scheme and a newly developed variable element typing (VET) system based on the presence or absence of ten variable genetic elements. These elements were detected while screening a genomic library of strain Corby, as well as being taken from published data for PAI-1 (pathogenicity island) from strain Philadelphia. Specific primers were designed and used in gel-based polymerase chain reaction (PCR) assays. PCR amplification of the mip gene served as a control. The end-point was the presence/absence of a PCR product on an ethidium bromide-strained gel. In the present study, the index of discrimination was somewhat lower than that of the SBT (0.87 versus 0.97). Nevertheless, the results obtained showed as a ‘proof of principle’ that this simple and quick typing assay might be useful for the epidemiological characterisation of L. pneumophila strains.     

Identification of Legionella pneumophila antigens and antibodies by immunoferritin electron microscopy

Material from 18 lungs positive for Legionella pneumophila and 21 strains of legionella grown on bacteriological media or in the yolk sac of fertile hen's eggs were examined by direct negative stain and immunoferritin electronmicroscopy. The 18 lung samples and all preparations of cultivated organisms showed the presence of bacteria with the typical morphology of L. pneumophila. By immunoferritin electronmicroscopy, with specific antisera, it was possible to serogroup the organisms. The immunoferritin method in reverse made it possible to detect and titrate antibody in sera from patients. Direct and immunoferritin electronmicroscopy were as sensitive as immunofluorescent antibody tests for detecting the antigens and antibodies of Legionnaires' disease. Additional advantages of the ultrastructural technique are discussed.     

Comparison of cross-staining reactions by Pseudomonas spp. and fluorescein-labeled polyclonal and monoclonal antibodies directed against Legionella pneumophila.

Commercially prepared polyclonal antisera to Legionella pneumophila are known to cross-react with organisms of the genus Pseudomonas. To determine whether a commercially available monoclonal antibody reagent specific for L. pneumophila would also cross-react with pseudomonads, a two-laboratory study was undertaken to test both monoclonal and polyclonal reagents against 33 isolates of Pseudomonas spp., including 25 Pseudomonas aeruginosa, 4 P. putida, 2 P. maltophilia, 1 P. fluorescens, and 1 P. alcaligenes. Four antisera were tested; polyclonal anti-legionella antisera pools A and B (Centers for Disease Control [CDC], Atlanta, (Ga.), polyclonal 1-6 antisera (BioDx, Inc., Denville, N.J.), and a monoclonal antibody reagent produced by Genetic Systems Corp., Seattle, Wash. All reagents were labeled with fluorescein. Cross-staining reactions were found with the BioDx L. pneumophila antisera and 10 isolates of Pseudomonas. Four of these isolates demonstrated cross-staining with CDC pool A. When tested with individual serotype-specific reagents (CDC), three of four cross-reacted with L. pneumophila serotype 1 antisera; the fourth cross-reacted with serotype 3. No cross-staining reactions were noted with the monoclonal reagent and any of the pseudomonads tested, demonstrating that the Genetic Systems Corp. monoclonal reagent is the most specific of the four reagents tested.     

Prevalence of antibodies to Legionella pneumophila in animal populations.

We examined more than 2,800 human and animal sera for antibodies to four serogroups of Legionella pneumophila by using the microagglutination test. Antibody titers of greater than or equal to 1:64 were considered positive. The occurrence of positive equine sera (31.4%) was significantly higher than the occurrence of positive sera in cattle (5.1%), swine (2.9%), sheep (1.9%), dogs (1.9%), goats (0.5%), wildlife (0%), and humans (0.4%). The highest titer measured in horses was 1:512. The occurrence of positive sera in horses was related directly to age. In horses less than or equal to 1, 2 to 3, 4 to 7, 8 to 12, and greater than or equal to 13 years old, the percentages of positive sera were 0, 10.1, 30.3, 44.9 and 58.1%, respectively. When we compared age-specific serogroup-specific rates in horses from Colorado and Pennsylvania, we found differences. With horses 8 to 12 and greater than or equal to 13 years old, there was a significantly higher (P less than 0.05) occurrence of sera that reacted to serogroups II and III in horses from Pennsylvania. Of 242 positive sera, 43.8% reacted to a single serogroup (serogroup III or I most commonly), and 56.2% reacted to multiple serogroups (serogroups II and III or serogroups I, II, and III most commonly). A high percentage of seropositive horses suggested that horses are commonly infected with L. pneumophila or related organisms, and the age-specific rates of occurrence indicated that infection was related directly to duration of exposure. A definitive demonstration of equine infection will depend on isolation of the agent and repetition of this serological study with antigens obtained from organisms isolated from horses.