Preparation Of Monoclonal Antibodies Against L.pneumophila Serogroup 8, And Cellular Fatty Acid Profiling And Partial 16S RRNA Gene Sequencing Of Environmental Legionella Isolates

Background: Legionella pneumophila was identified as the causative agent of Legionnaires'disease in 1977, which led to the description of a new genus: Legionella, the sole member of the family Legionellaceae. It has already been established that Legionella spp. is responsible for 2–5% of community-acquired pneumonia cases. Fifty two species,3 subespecies and more than 70 serogroups in total have been described so far. L. pneumophila consists of 15 serogroups, of which serogroup 6 is the most common one found in China, followed by serogroups 1 and 8. Legionnaires'disease is a common form of severe pneumonia, however, it is quite difficult to be diagnosed only based on clinical symptoms. Failure to diagnose Legionnaires'disease is largely due to the lack of clinical awareness. In addition, Legionella spp, is fastidious and not easily detected. It is hardly possible to clinically distinguish patients with Legionnaires'disease from patients with other types of pneumonia, and chest X-ray patterns will not help to differentiate this infection from other types of pneumonia. Reliable protocols for isolating, detecting and typing Legionella spp. are in urgent need to address the above issues.PART ONE: preparation of monoclonal antibodies against L.pneumophila serogroup 8Method: L. pneumophila serogroup 8 (ATCC 35906) grown on BCYE agar plates was harvested and formalin killed, then, 3×10⁸ cells were used to immunize female BALB/c mice (six to eight-week-old) via intraperitoneal route every two weeks over a period of 6 to 8 weeks. The primary injection was accompanied with complete Freund adjuvant, while the second injection was given with incomplete Freund adjuvant instead, and the subsequent injections were given with physiological saline. At the end of the immunization regimen, the mice were splenectomized, and the spleen cells were fused with SP2/0 cells. The culture supernatant fluids of growing clones were screened by ELISA with whole L. pneumophila SG 8 cells as antigens. The resulting hybridomas were cloned by limiting dilution. Selected hybridoma cells were injected intraperitoneally into paraffin oil-pretreated BALB/c mice for producing ascitic fluid. By using two purified monoclonal antibodies obtained from ascites fluids, an antibody-sandwich ELISA was established to detect L. pneumophila SG 8. The sensitivity of this assay was measured using sonicated cells, and the specificity was evaluated by using 14 other serogroups of L. pneumophila, 17 non-pneumophila legionellae and 11 other bacteria. Result: Eight hybridomas producing antibodies against L. pneumophila serogroup 8 were obtained in this study and they were identified as IgM, IgG₃ and IgG₁, respectively. An antibody-sandwich ELISA was established, using McAb 6G10 as the capture antibody and McAb 6C7 as the detecting antibody. Detection limit of this assay for sonicated cells was about 2.6×10⁵cfu/ml. No cross-reactivity was detected in 14 other serogroups of L. pneumophila, 17 non-pneumophila legionellae and 11 other bacteria, except for Staphylococcus aureus which gives a quite weak cross reaction. Immnobloting and proteinase K assays implied that McAbs 6G10 and 6C7 targeted lipopolysaccharide antigens.Conclusion: Two hybridomas producing McAb with high specificity and moderate affinity against L. pneumophila serogroup 8 were obtained in this study, which can be used in antibody-sandwich ELISA for detecting this serogroup.PART TWO: cellular fatty acid profiling and partial 16S rRNA gene sequencing of environmental Legionella isolatesMethod: 156 environmental Legionella strains isolated in Guangzhou and Xinhui were identified by cellular fatty acid profiling and partial 16S rRNA gene sequencing. Bacterial cells were harvested after incubating for 72 h, then, the fatty acid methyl esters (FAMEs) were obtained by saponification, methylation, and extraction, as recommended by the MIDI Sherlock (MIDI Inc. USA) standard protocol. The separation of FAMEs was achieved by using a Hewlett-Packard model 6890 gas chromatograph controlled by the Sherlock software. Performance of the test was controlled by repeated runs of an internal standard quantitative FAME mixture (MIDI). The reproducibility of FAME profiling was evaluated by using a control strain L. jamestowniensis (ATCC 35298). A dendrogram based on the fatty acid patterns of the strains was generated by Euclidean distances, utilizing UPGMA (unweighted pair group method with arithmetic average) algorithm provided by BioNumerics software package. Mapped features were also applied to achieve better differentiation. Clusters were delineated in the dendrogram to differentiate individual species. 16S rRNA gene fragments of the tested stains were amplified by PCR and the 5'partial of them were sequenced. The raw data was handled by DNAStar software package, and then input into Bionumerics. A dendrogram based on partial 16S rRNA gene sequences of the strains, was generated using UPGMA. Cellular fatty acids profiling and partial 16S rRNA gene, together with biochemical assay results and other phenotypic characteristics, were used identify all the 156 strains.Result: Triplicate runs of L. pneumophila ATCC 35298 gave a maximum coefficient of variation of 2%, which implies an excellent reproducibity of celluar fatty acid profiling protocol used in this study. At least 73 different fatty acids were detected in the 156 strains tested, and 16 of them were the most common fatty acids detected in the tested strains. In total, 149 out of the 156 strains were identified as Legionella spp. by cellular fatty acids profiling, including 109 L. pneumophila strains, 21 L. feeleii strains, 17 L. longbeachae strains, and 2 L . oakridensis strains. Dendrogram based on FAME profiles of the tested strains revealed 5 distinct clusters, with 4 strains remaining unclustered. Four of the clusters corresponded directly to Legionella species: cluster 1 was composed of the 21 L. feeleii strains; 17 L. longbeachae strains were grouped as cluster 2; 109 isolates identified as L. pneumophila were grouped as cluster 3. Two L. oakridensis strains were split into clusters 4. Strains LG149, LG151 and LG152 were grouped as cluster 5, showing distinct FAME profiles of the known species, and these strains might be undescribed Legionella species or atypical strains of the known Legionella strains. Four other isolates LG 77, LG145, LX05 and LG 95 had fatty acid profiles different from any of those of the validly described species and may represent distinct FAME groups of the known species or undescribed Legionella species. Dendrogram based on partial 16S rRNA genes of the tested stains resembles the FAME dendrogram.Conclusion: The numerical analysis of fatty acid profiles is very helpful in identifying Legionella species, especially suitable for screening a large number of Legionella isolates. Identification results of the environmental Legionellae strains, based on FAME profiling, partial 16S rRNA and phenotypic assays, provide valuable data for identifying the suspected Legionella isolates.