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Variability among 29 clinical and environmental strains of Legionella dumoffii was investigated by multilocus enzyme analysis by use of starch gel electrophoresis. Based on results of analysis at 20 enzyme loci, the strains were separated into five closely related electrophoretic types (ETs), which were clearly distinguished from 53 strains representing 53 ETs of L. pneumophila. DNA hybridization results (hydroxyapatite method, 60 and 75 degrees C) for representative strains confirmed that all L. dumoffii ETs were a single genetic species. Although multilocus enzyme analysis indicated that L. dumoffii was genetically a quite uniform species, sufficient variability existed to warrant electromorph fingerprinting for epidemiologic studies.

Diethylaminoethyl (DEAE)-cellulose was used as a support material for preparing bacterial cell columns. Pretreatment of the bacterial cells with formalin was essential in obtaining satisfactory adherence of the cells to DEAE-cellulose. Cross-reacting antibodies were removed from antibody preparations against strains of Streptococcus mutans serotypes a and d by adsorption on appropriate bacterial cell columns. S. mutans serotype d was further divided into two subtypes on the basis of immunofluorescent staining with conjugates of immunospecifically adsorbed immunoglobulin G. The DEAE-cellulose-bacterial cell columns were regenerated after use by desorbing the cross-reacting antibodies with low-pH buffer and were used repeatedly over and 18-month period with no detectable loss in effectiveness.

In the period August 10-29, 1986, 29 confirmed cases of Legionnaires' disease occurred in Sheboygan, Wisconsin; two cases were fatal. No common source of indoor exposure was identified. Water specimens were obtained from all known cooling tower units in Sheboygan; Legionella pneumophila serogroup 1 was isolated at 1 x 10(6) colony-forming units per liter from a specimen obtained August 27 at plant A. This isolate was identical to the only clinical isolate by monoclonal antibody and isoenzyme subgrouping. Of 29 persons with Legionnaires' disease, 21 lived or worked within one mile (1.6 km) of plant A; seven of the remaining eight visited within one to two miles (1.6 to 3.2 km) of plant A from three to seven days before onset of illness. Attack rates were highest for persons living within 0.5 mile (0.8 km) of plant A. These findings associate a cooling tower with community-acquired Legionnaires' disease and suggest that dissemination of Legionella may occur over longer than previously recognized distances.

We developed an enzyme-linked immunosorbent assay (ELISA) system to measure human immunoglobulin G (IgG) and IgM response to the major outer membrane proteins of Brucella melitensis. The ELISA was more sensitive in detecting antibody than a standard microagglutination (MA) test with B. abortus antigen. Of 101 sera from persons with suspected brucellosis, 79 (78.2%) gave ELISA IgM titers greater than or equal to the B. abortus MA titer without 2-mercaptoethanol (2ME), which measures both IgM and IgG. Of the 101 sera, 97% gave ELISA IgG titers greater than or equal to the MA with 2ME titer. A total of 58 sera, drawn from 11 human patients from 1 to 29 weeks after onset of brucellosis, gave higher geometric mean titers for the ELISA IgG test than for the MA with 2ME test. These 58 sera also gave ELISA IgM geometric mean titers that were greater than or within one doubling dilution of the geometric mean titers of MA without 2ME. In addition to detecting antibody response to B. abortus, B. melitensis, and B. suis, the ELISA was sensitive to antibody response to human and canine infections with B. canis. The B. canis antibody response is not detected by the MA test with B. abortus antigen. The ELISA, with a standard preparation of major outer membrane proteins of B. melitensis as antigen, appears to be useful in measuring antibody response in humans to infections by all species of Brucella known to infect humans.

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.

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.

Rabbits were inoculated with whole, formalinized Campylobacter jejuni, C. coli, and C. fetus cells; the C. jejuni and C. coli immunogens were identified by their DNA relatedness at the species level to the type strains of C. jejuni or C. coli. The designation C. coli was not used among the other C. jejuni strains; they were classified as hippurate-positive or hippurate-negative C. jejuni. Immunoglobulin G was isolated from the antisera and labeled with fluorescein isothiocyanate. These conjugates defined 10 serogroups of C. jejuni, 2 serogroups of C. coli, and 2 serogroups of C. fetus. Of the 316 strains of C. jejuni tested, 258 (82%) were groupable; 173 were single-serogroup strains, and 85 were multiple-serogroup strains. Of the 226 strains in C. jejuni serogroups, 223 (98.7%) were hippurate positive; of the 27 strains in C. coli serogroups, 26 (96.3%) were hippurate negative. Five strains were equally reactive in immunofluorescent staining with a conjugate for a C. jejuni serogroup and a conjugate for a C. coli serogroup. A total of 58 strains of C. jejuni were ungroupable: 33 (13%) of the 259 hippurate-positive strains and 25 (44%) of the 57 hippurate-negative strains. All 121 strains of C. fetus tested were groupable as A, B, or A:B. The 14 conjugates used to define serogroups of C. jejuni, C. coli, and C. fetus reacted with the flagella but not the cells of other Campylobacter species and were negative on 256 other bacteria from 21 genera.

A number of bacterial systems were studied with specific direct fluorescent-antibody reagents prepared from rabbit antiserum fractions and having a wide range of fluorescein-to-protein ratios. These systems included Bacteroides, Bordetella, Clostridium, Escherichia, Legionella, Listeria, Salmonella, Shigella, and Streptococcus. For all systems studied, a fluorescein-to-protein ratio of 30 was optimal for conjugates prepared from ammonium sulfate fractions (greater than 75% gamma globulin) and pure immunoglobulin G desorbed from the Sepharose-bound protein A of Staphylococcus aureus. A pepsin digestion procedure is described that yielded the F(ab')2 piece of pure immunoglobulin G; this was labeled and studied at two fluorescein-to-protein ratios.

Isolates of Legionella pneumophila that are serologically different from strains of serogroups 1 through 5 were obtained from lung biopsy tissue or pleural fluid from three renal transplant recipients in Chicago, Ill. These strains were placed in a newly designated L. pneumophila serogroup, serogroup 6, on the basis of fluorescent-antibody staining characteristics. An L. pneumophila strain obtained from Bethesda, Md., one from Houston, Tex., and one from Oxford, England, also belong to this new serogroup. L. pneumophila serogroup 6 appears to be widely distributed geographically.

As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.

Although direct fluorescent-antibody (DFA) testing has been used successfully for a number of years to detect legionellae in clinical specimens, the number of known species and serogroups of Legionella has now increased to such an extent that the performance of DFA testing for all serological variants is impractical. Lung homogenates that were submitted to the Centers for Disease Control, Atlanta, Ga., from patients with suspected legionellosis, from November 1977 through May 1982, were originally screened by DFA testing. In our study 498 of these lung homogenates were screened by indirect fluorescent-antibody (IFA) testing, using a panvalent antiserum pool containing antibodies to 25 serological variants of Legionella spp. Fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin was used as the second antibody of the sandwich system. For positive homogenates, i.e., those containing Legionella organisms, species and serogroup identification was made by IFA staining with polyvalent serum pools and then with monovalent antiserum. Of the 498 homogenates screened, 39 (7.8%) were positive by IFA testing. Four (0.8% of total; 10.3% of positive homogenates) of these had previously been negative by DFA testing, but subsequent testing showed that they contained Legionella organisms for which DFA reagents were not available at the initial screening. All specimens that were positive by DFA testing were also positive by IFA testing. IFA testing with polyvalent antisera is a simple, efficient method which is at least as sensitive as DFA testing and which can be used by clinical laboratories to cope with the increasing number of known serological variants of Legionella spp.

Rabbits were inoculated with whole, formalinized cells from eight passive hemagglutination reference strains of Campylobacter. Fluorescein isothiocyanate-labeled immunoglobulin G from these antisera defined seven new direct fluorescent-antibody serogroups of C. jejuni and one new serogroup of C. coli. This expanded the Campylobacter direct fluorescent antibody system to include 17 serogroups of C. jejuni, 3 serogroups of C. coli, and 2 serogroups of C. fetus. We then compared the passive hemagglutination method (57 serotypes) and the direct fluorescent-antibody method (20 serogroups) for typing strains of C. jejuni and C. coli. The data obtained by testing 101 strains by both methods revealed that the two test systems were measuring completely different sets of antigen complexes. The two serogrouping methods were complementary, and their combined use discriminated among strains more effectively than did either method individually.

Legionella jordanis sp. nov., as found in two cultures, is described. One isolate was from river water in Indiana and the other isolate was from sewage in DeKalb County, Ga. The former is the type strain of the species, and is designated BL-540 (ATCC 33623). L. jordanis had a partial relationship to L. bozemanii by direct fluorescent-antibody tests but was unrelated to L. pneumophila, L. dumoffii, L. micdadei, L. gormanii, or L. longbeachae. Legionella phenotypic characteristics, including large amounts of branched-chain cellular fatty acids, were shown by the isolates. Studies of DNA relatedness showed that the two cultures of L. jordanis were only slightly related to the six previously described species of Legionella but were more than 90% related to each other. Indirect fluorescent-antibody tests with human sera suggested that unrecognized human infections with L. jordanis may be occurring.

A strain of the Legionnaires disease bacterium (LDB) that was isolated by Joseph E. McDade from a postmortem lung specimen of a patient with fatal atypical pneumonia at the Veterans Administration Hospital in Togus, Maine was serologically different from 16 other strains of LDB that had been isolated previously from patients in other geographic locations. The serological differences of the Togus isolate were shown in results of direct and indirect fluorescent antibody staining and of immunoelectrophoresis with soluble antigen extracts. Seroconversion for the Togus strain of LDB in acute- and convalescent-phase sera from a second patient with atypical pneumonia at the Veterans Administration Hospital in Togus indicated that this patient had been infected with an LDB that was serologically similar or identical to the Togus isolate. The Togus serogroup of LDB should be considered when performing serological tests for Legionnaires disease.

The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources. Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. Principal coordinates and clustering analyses demonstrated that isolates received as L. pneumophila were a heterogeneous array of genotypes that included two previously undescribed species. For 50 ETs of L. pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources. Cluster analysis revealed four major groups or lineages of ETs in L. pneumophila. Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs. Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total. Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET. The genetic structure of L. pneumophila is clonal, and many clones apparently are worldwide in distribution. The fact that L. pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole.

Legionella pneumophila subsp. pneumophila serogroup 6 is second in importance only to L. pneumophila serogroup 1 as a cause of legionellosis. Monoclonal antibody (MAb) reactivity and multilocus enzyme electrophoretic analyses were used to subtype serogroup 6 isolates as a potential aid for epidemiologic and virulence studies. Forty-eight serogroup 6 isolates submitted to the Centers for Disease Control from 1980 to 1985 were examined by these methods. The isolates were divided into two groups based on differential reactivity with two MAbs. Thirty-two of the isolates were of a single electrophoretic type (ET) and were reactive with both MAbs. The remaining 16 isolates were distributed among 10 ETs and were reactive with one or both MAbs. The mean genetic diversity for serogroup 6, as determined from the degree of variability at 20 enzyme loci, was found to be essentially the same as that for L. pneumophila subsp. pneumophila as a whole. The ETs of serogroup 6 isolates were unique but closely related genetically to the ETs of L. pneumophila subsp. pneumophila serogroups 1 to 5, 7, and 8. The range of serogroup 6 subtypes distinguished by MAbs and enzyme electrophoresis suggests that the combination of these two methods can be useful as a typing system.

Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3.

Twenty-six hybridoma cell lines that produced monoclonal antibodies to toxic shock syndrome toxin 1 (TSST-1) were generated by immunizing mice with a highly purified preparation of TSST-1 and fusing their splenic lymphocytes with SP2/0-Ag-14 cells. One monoclonal antibody of the immunoglobulin G1 isotype, designated as PEC-1 10-2SCH, was selected for extensive study. The specificity of this antibody was determined by testing spent culture fluid filtrates of TSST-1- and non-TSST-1-producing strains of Staphylococcus aureus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunoelectrotransfer blot techniques. Monoclonal antibody PEC-1-10-2SCH was specific for TSST-1-producing strains of S. aureus, reacting with TSST-1 and two other proteins which appear to be unique to S. aureus strains that produce TSST-1. Monoclonal antibody PEC-1-10-2SCH was used in conjunction with polyclonal rabbit antibodies to TSST-1 in a rapid, one-step, sensitive, specific, and quantitative enzyme-linked immunosorbent assay. This assay was shown to be more sensitive, faster, and simpler to perform than previously described isoelectric focusing, immunodiffusion, and solid-phase radioimmunoassays for TSST-1. Monoclonal antibody PEC-1-10-2SCH was not reactive with Staphylococcus protein A under the conditions of the test.

Autopsy specimens of lung tissues from 15 patients that contracted legionellosis during the 1976 Philadelphia outbreak of Legionnaires disease were examined for the presence of Legionella organisms and soluble antigens by indirect fluorescent-antibody (IFA) testing and by an enzyme-linked immunosorbent assay (ELISA) with both polyclonal and monoclonal antibodies. In all 15 cases, at least one specimen was positive for Legionella pneumophila serogroup 1 (Lp-1) antigens by a polyclonal antibody ELISA system. Of the 15 cases tested for Lp-1, 9 were positive by a polyclonal antibody IFA test. Nine mouse monoclonal antibodies to Lp-1 gave essentially the same reactivity pattern with extracts from lung tissue homogenates as that obtained with a Philadelphia 1 culture extract by using a monoclonal antibody ELISA system. The same monoclonal antibody panel gave similar results when used in the IFA system with tissue homogenates. Monoclonal antibodies can be used as epidemiological marker systems with both IFA and ELISA testing. This study provides evidence that the 1976 common source outbreak in Philadelphia was probably caused by a single Lp-1 strain. ELISA testing of the soluble antigen of Lp-1 from lung tissue homogenate supernatants was more sensitive than IFA testing of the homogenates and should be extremely useful as either a primary test or as an adjunct to fluorescent antibody testing for legionellosis.

Seven cases of nosocomial legionellosis occurred between February and September 1982 in a small community hospital in Upstate New York. All seven were cases of Legionella pneumophila serogroup 1; six were hospital patients and one a hospital employee. None of the cases died. During the peak of the outbreak, the incidence of nosocomial legionellosis was 1.2 cases per 100 patient discharges. An epidemiologic comparison of the six patient cases with 21 matched patient controls suggested that longer hospital stay (chi 1(2) = 24.2, p less than 0.001) and the proximity of patients' rooms to ward showers (chi 1(2) = 4.4, p less than 0.04) were significant risk factors for acquiring legionellosis. An environmental investigation demonstrated that the ward showers and the hospital hot water system were contaminated with L. pneumophila serogroup 1. Monoclonal antibody subtyping performed on isolates obtained during the outbreak investigation confirmed that the hot water system and patient isolates had an identical pattern of reactivity. The outbreak demonstrates that legionellosis can be a significant cause of nosocomial pneumonia in a community hospital and that transmission can occur from contaminated potable hot water sources, potentially via shower aerosols.

The fluorescent treponemal antibody absorption (FTA-ABS) double-staining procedure was reproducible, comparable to the conventional test, and easy to read. We recommend the use of the FTA-ABS double-staining procedure for microscopes with incident illumination, the 100 x/1.30 oil achromatic objective and the 6.3 x ocular to obtain optimal fluorescence, and the KP560 as a barrier filter to exclude rhodamine emission when fluorescein fluorescence is read. With this system, errors related to poor focusing or failure to visualise treponemes on all smears should be eliminated. [ABSTRACT FROM PUBLISHER]

Three branched-chain hydroxy acids not previously reported in other bacteria were found in extracts from saponified whole cells of Pseudomonas maltophilia. On the basis of evidence from mass spectrometry, infrared spectroscopy, and gas chromatographic procedures, they were identified as 2-hydroxy-9-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, and 3-hydroxy-11-methyldodecanoic acid. These acids appeared to be tightly bound to other cellular components since they were not extracted from lyophilized cells with a chloroform-methanol (3:1) mixture.