Use of absorbed antisera for demonstration of antigenic variation among strains of Legionella pneumophila serogroup 1.

Antigenic typing of strains of Legionella pneumophila serogroup 1 (Lp1) has been shown to be useful in epidemiological studies of outbreaks of legionellosis. Selective absorption of rabbit antibodies produced against five strains of Lp1 resulted in the recognition of 17 somatic types among the 176 strains tested. A comparison was made of our results and those obtained by McKinney et al. (Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 255:91-95, 1983) and Joly et al. (J. Clin. Microbiol. 18:1040-1046, 1983), who used monoclonal antibodies to subgroup Lp1 strains. The results indicate that antigens are present in Lp1 strains that were undetected by either system. The data presented in this study may be helpful in selecting for the production of additional monoclonal or absorbed antibodies for diagnostic purposes or epidemiological studies.

Recognition of a second serogroup of Legionella longbeachae.

A strain of Legionella longbeachae (Tucker 1) that was isolated from the postmortem lung tissue of a pneumonia patient was serologically distinct from four other strains of L. longbeachae. The recognition of a second serogroup of L. longbeachae represents the first reported instance of serogroup diversity within a species of Legionella other than L. pneumophila. The disease caused by the Tucker 1 strain does not seem to be readily distinguishable from that of pneumonia caused by other legionellae.

Flagella on Legionnaires' disease bacteria: ultastructural observations.

Scanning and transmission electron microscopic studies were done on five strains of flagellated Legionella pneumophila cultured for 1 to 3 days on charcoal yeast extract agar or yeast extract broth. Ultrastructurally, each strain consisted of pleomorphic, flagellated bacilli, many of which showed the typical pinching type of division as seen with other gram-negative bacteria. Most of the flagellated organisms, regardless of the strain, had a single, relatively straight or undulant polar flagellum, about 25 nm in diameter. In some instances, retraction of the bacterial cell membranes made the flagellar basal bodies (bulbs) visible when observed by transmission electron microscopy. The bulbous nature of these bodies appears to be different from the basal structures described for Escherichia coli and other gram-negative bacteria. Long "streamers," previously observed with the light microscope, appear to be fimbriae (or pili) that have a diameter approximatley half that of the flagella and are somewhat coiled and bent at irregular intervals.

Pathogenesis of acute Legionnaires' disease pneumonia.

Immunofluorescent study of the lungs in cases of fatal suspected acute Legionnaires' disease enabled confirmation of the presence of Legionella pneumophila. In addition, probable pathogenetic mechanisms that had not been as clearly visualized by light microscopy became apparent: the retrograde involvement of the larger bronchioles and proximal airways, invasion of the interstitium, extension to pleura, and lymphatic and hematogenous spread. Organisms were demonstrated to occur in the liver and spleen of one patient. The development of technics for the earliest possible diagnostic verification of Legionnaires' disease, with specimens obtained as untraumatically as practical from selected sites and screened by specific immunofluorescent microscopic examination, should contribute to patient survival.

Fatal pneumonia caused by Legionella pneumophila, serogroup 3: demonstration of the bacilli in extrathoracic organs.

An immunosuppressed patient with malignant lymphoma died of acute pneumonia caused by Legionella pneumophila. Bacilli of serogroup 3 were detected in areas of pnemonia, in a mediastinal lymph node, and in the liver and spleen by direct immunofluorescence done on tissue obtained at autopsy. That the extrathoracic fluorescent material represented intact bacteria rather than antigenic fragments or antigen-antibody complexes was confirmed by finding intracellular bacilli in the liver by electron microscopy. To our knowledge, this case represents the first example of fatal disease attributed to serogroup 3 L. pneumophila and the first case in which L. pneumophila has been demonstrated in extrathoracic organs.

"Pittsburgh pneumonia agent": a bacterium phenotypically similar to Legionella pneumophila and identical to the TATLOCK bacterium.

The "Pittsburgh pneumonia agent," isolated by Pasculle and co-workers from human lung tissue, has been cultured on artificial media and characterized. The "Pittsburgh" bacterium and the TATLOCK and HEBA bacteria have identical cultural, biochemical, and antigenic characteristics. They also have the same cellular fatty-acid composition, and DNA relatedness indicates that they belong to the same species.

A newly identified bacterium phenotypically resembling, but genetically distinct from, Legionella pneumophila: an isolate in a case of pneumonia.

A bacterium with growth characteristics similar to, but genetically distinct from, either Legionella pneumophila or WIGA (a "rickettsia-like agent") was obtained from a postmortem lung specimen of a patient with fatal atypical pneumonia at the M. D. Anderson Hospital and Tumor Institute in Houston, Texas. This bacterium and WIGA have essentially the same cellular fatty acid composition, which is distinct from that of L. pneumophila. Deoxyribonucleic acid-reletadness studies show that the isolate from Texas is only about 10% related to both L. pneumophila and WIGA and there fore may represent a new species. This new bacterium should be considered in selecting laboratory procedures in the diagnosis of atypical pneumonia.

A Legionella-like bacterium related to WIGA in a fatal case of pneumonia.

An unusual bacterium serologically related to a "rickettsia-like agent," designated previously as WIGA, was seen in lung tissue from a patient who died of pneumonia of unknown cause. A fluorescent antibody conjugate prepared with the WIGA organism, isolated in 1959, was used to stain the lung tissue. Enormous numbers of fluorescent bacteria in the lungs of this patient confirm the pathogenicity of this unusual bacterium.

Effect of various histological fixatives on fluorescent antibody detection of Legionnaires disease bacteria.

Human lung tissue containing the Legionnaires disease bacterium was fixed in seven different histological fixatives, processed, and embedded in paraffin. Deparaffined sections from each were stained by fluorescent antibody and by Dieterle silver impregnation. With the fluorescent antibody stain, the Legionnaires disease bacterium could be detected in tissues prepared with any of the fixatures, but the Dieterle silver impregnation was not satisfactory on Zenker-fixed tissues.

Four serogroups of Legionnaires' disease bacteria defined by direct immunofluorescence.

Thirty-five strains of Legionnaires' disease bacteria were shown to belong in four distinct serologic groups on the basis of findings obtained with direct fluorescent antibody testing. Thirty of the strains were placed in group 1, three in group 2, one in group 3, and one in group 4. Immunoelectrophoretic studies showed both unique and common antigens among the representative strains of the four serogroups.

Pathologic findings in guinea pigs inoculated intraperitoneally with the Legionnaires' disease bacterium.

Tissues from guinea pigs inoculated intraperitoneally with the Legionnaires' disease (LD) bacterium were studied with light, immunofluorescent, and electron microscopy. The principal gross lesion was diffuse peritonitis of varying severity. Microscopically, the peritonitis of covered by a mixed inflammatory infiltrate of macrophages, neutrophils, fibrin, and cellular debris. Foci of inflammation and necrosis were consistently observed in the splenic parenchyma, and similar lesions were often found in the lungs, liver, lymph nodes, pancreas, heart, and other organs. Numerous LD bacteria were seen in the peritoneal exudate; fewer were found in disseminated lesions. In electron micrographs, the highest concentrations were seen in macrophages, with fewer organisms present in neutrophils or extracellular spaces. Although the lung is the primary organ konwn to be affected by Legionnaires' disease in humans, our findings indicate that the LD bacterium is capable of dissemination.

Recognition of a new serogroup of Legionnaires disease bacterium.

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.

Enrichment procedures for isolating salmonellae from raw meat and poultry.

The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and preenrichment in buffered peptone-water followed by enrichment in tetrathionate broth yielded the maximal recovery of salmonellae from raw meat and poultry samples.

Detection of Legionnaires disease bacteria by direct immunofluorescent staining.

Antisera and fluorescein isothiocyanate conjugates prepared for five strains of the Legionnaires bacteria were tested in both homologous and heterologous staining reactions with 10 isolates of the organism from patients in seven geographic areas. The strains were related but not identical as judged by the results of direct immunofluorescence staining. The conjugates were successfully used to detect Legionnaires disease bacteria in Formalin-fixed lung scrapings, in histological sections, and in fresh lung tissue obtained at biopsy or autopsy. In addition, the labeled antibodies are valuable for staining suspected cultures of the bacterium and for searching for the source of these organisms in soil, water, and other environmental niches. The reagents are highly specific for detecting the Legionnaires organism in clinical specimens.