Bacteria were concentrated 500-fold from 20-liter water samples collected from 67 different lakes and rivers in the United States. The data suggest that Legionella pneumophila is part of the natural aquatic environment and that the bacterium is capable of surviving extreme ranges of environmental conditions. The data further demonstrate the effectiveness of the direct fluorescent-antibody technique for detecting L. pneumophila in natural aquatic systems. Smears of the concentrated samples were screened microscopically for serogroups of L. pneumophila by the direct fluorescent-antibody technique. Virtually all of the 793 samples were found to be positive by this method. The 318 samples containing the largest numbers of positive bacteria which were morphologically consistent with L. pneumophila were injected into guinea pigs for attempted isolations. Isolates were obtained from habitats with a wide range ofphysical, chemical, and biological parameters. Samples collected monthly from a thermally altered lake and injected into guinea pigs demonstrated a seasonality of infection, with the highest frequency of infection occurring during the summer months.

Continuous centrifugation of large volumes of water from natural southeastern lakes allowed quantitative detection of Legionella pneumophila by direct immunofluorescent staining. Positive samples were injected intraperitoneally into guinea pigs, and the L. pneumophila were isolated and identifiea by their morphological, cultural, physiological, and serological characteristics.

We describe a new species of Legionella represented by 10 strains isolated from industrial cooling towers. Legionella oakridgensis differed genetically from the other seven species of Legionella in DNA hybridization studies and differed serologically in direct fluorescent-antibody tests. The new species, unlike all other species except L. jordanis, did not require added L-cysteine for growth in serial transfer on charcoal-yeast extract agar. L. oakridgensis, as well as three other species tested, required L-cysteine for primary isolation from animal tissues. L. oakridgensis was the only species of Legionella that failed to produce alkaline phosphatase at pH 8.5. In all other respects, it resembled other species of Legionella, including having a high content of branched-chain cellular fatty acids and being pathogenic for guinea pigs. These bacteria have not yet been associated with human disease, but they are potential causes of legionellosis. Colonization of air conditioning equipment by legionellae is recognized in the United States and abroad as a potential major public health problem because strong circumstantial evidence indicates that cooling towers and evaporative condensers have frequently been the source of legionellae involved in outbreaks of both mild and severe respiratory disease (2, 11, 12, 16). The maintainence of equipment free of these organisms is a desirable objective but one that has not yet been achieved by practical means. In this study we describe 10 strains isolated from thermally altered water of large industrial cooling towers in two different geographic locations (R. Tyndall, S. B. Gough, C. B. Fliermans, E. Dominque, and C. Duncan, submitted for publication). These cultures were characterized by morphological, cultural, biochemical, genetic, serological, and pathogenicity tests. They represent a new species, for which the name Legionella oakridgensis sp. nov. is proposed. The type strain of L. oakridgensis is Oak Ridge 10 (OR-10; ATCC 33761). MATERIALS AND METHODS Cultures. Tyndall and co-workers in Oak Ridge, Tenn., isolated the strains of L. oakridgensis designatt Publication 2103, Environmental Sciences Division, Oak Ridge National Laboratory.

Legionellajordanis 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. The recognized species of Legionella are L. pneumophila (4), L. bozemanii (3), L. micdadei (16, 27), L. dumoffii (3, 17), L. gormanii (20), and L. longbeachae (18). In this report, we describe a new species for which the name

Methods are described for detection of Legionella pneumophila in cooling tower water or other water sources by direct fluorescent-antibody staining. A procedure for isolation of Legionella bacteria from water samples by guinea pig inoculation is described. Two different serogroups of L. pneumophila were isolated repeatedly from one of the cooling towers.