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.

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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.

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Of nine patients with Legionnaires' disease, seven were receiving corticosteroids, and all nine had serious underlying diseases. Direct immunofluorescent examination of respiratory secretions, including sputum and transtracheal aspirates, showed the Legionnaires' disease (LD) bacterium in five of seven patients who seroconverted and in a sixth patient with a single elevated titer to the LD bacterium. All nine patients received erythromycin therapy, and five survived. Two patients showed persistence of their infection after receiving 2 weeks of erythromycin therapy, and two patients developed pulmonary abscesses. These cases of Legionnaires' disease show the occurrence of pulmonary abscesses, the possibility of relapse after giving only 2 weeks of erythromycin therapy, and the utility of direct immunofluorescence for early diagnosis.

Screening enrichments of surface water specimens by means of a polyvalent fluorescent antibody reagent for the salmonellae yielded approximately 60% more positive specimens than was obtained by cultural procedures. It is not known what fraction of the excess of fluorescent antibody-positive over culturally positive specimens represents staining of non-salmonellae or non-arizonae as opposed to the staining of non-cultivatable organisms of these two genera. Cotton gauze and rayon-polypropylene fiber swabs were equally sensitive for collecting salmonellae from the streams examined. Tetrathionate enrichment incubated at 41.5 C appeared to be superior to selenite-cystine for isolation of salmonellae from surface waters. Twenty-eight serotypes of Salmonella and two serotypes of Arizona were identified in the 121 positive specimens. In water rated moderately polluted, 65% of all specimens tested were positive; in minimally polluted waters, 38% were positive; and in unpolluted streams, 44% were positive.

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.

When the data from performance and physicochemical studies of conjugates are combined for analysis, the performance data and specific titers show a direct relationship to the physicochemical data (Table 2). These reagents were prepared from the same lot of antiserum. The specific titers are very misleading without the accompanying data (Table 2). The protein concentrations range from 4 to 10 mg/ml, the F/P ratios from 10 to 30, and CASE shows gamma-globulin to constitute 30 to 100% of the protein. CASE also shows the gamma-globulin F/P ratio to be only 10 to 20. Using these data, we calculated the concentrations of the gamma-globulins and normalized their titers to 10 mg/ml. The value of good fractionation procedures for recovering gamma-globulin and the desirability of obtaining optimal F/P ratios are reflected in the adjusted titers. Physicochemical characterization of conjugates identifies superior and deficient reagents and frequently reveals the cause of inadequate performance. In this way it serves as a quide for improving reagent quality.