Hazel W. Wilkinson

Ten New Species of Legionella

Ten new Legionella species were characterized on the basis of biochemical reactions, antigens, cellular fatty acids, isoprenoid quinones, and deoxyribonucleic acid relatedness. Nine of the new species were isolated from the environment, and one, Legionella hackeliae, was isolated from a bronchial biopsy specimen obtained from a patient with pneumonia. The species all exhibited the following biochemical reactions typical of the legionellae: growth on buffered cysteine-yeast extract agar, but not on blood agar; growth requirement for cysteine; gram negative; nitrate negative; urease negative; nonfermentative; catalase positive; production of a brown pigment on tyrosine-containing yeast extract agar; liquefaction of gelatin; and motility. Legionella spiritensis was weakly positive for hydrolysis of hippurate; the other species were hippurate negative. Legionella cherrii, Legionella steigerwaltii, and Legionella parisiensis exhibited bluish white autofluorescence. Legionella rubrilucens and Legionella erythra exhibited red autofluorescence. The other species, L. spiritensis, L. hackeliae, Legionella maceachernii, Legionella jamestowniensis, and Legionella santicrucis did not autofluoresce bluish white or red. All species had cellular fatty acid contents qualitatively similar to those of previously described legionellae and had major amounts of ubiquinones with more than 10 isoprene units in the side chains. Each new species was serologically distinct from previously described Legionella species. As determined by the hydroxyapatite method at 60°C, two strains of L. maceachernii were 100% related, and four strains of L. cherrii were 94 to 99% related. The other new species were represented by single strains. The levels of relatedness of the new species to each other and to previously described legionellae ranged from 1 to 67%. L. maceachernii, L. jamestowniensis, and L. hackeliae were less than 25% related to other species. L. rubrilucens and L. erythra, and two red-autofluorescing species, were about 60% interrelated. L. spiritensis (a non-autofluorescing species) was 34% related to L. rubrilucens. L. santicrucis was 64% related to Legionella sainthelensi. The three bluish white-autofluorescing species, L. parisiensis, L. cherrii, and L. steigerwaltii, were most closely related to other bluish white-autofluorescing species, especially Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and “Legionella anisa” (35 to 67%).

A New Legionella Species, Legionella feeleii Species Nova, Causes Pontiac Fever in an Automobile Plant

From 15 to 21 August 1981, Pontiac fever affected 317 automobile assembly plant workers. Results of serologic tests were negative for Mycoplasma, Chlamydia, respiratory tract viruses, and previously described legionellae. A gram-negative, rod-shaped organism (WO-44C) that did not grow on blood agar, required L-cysteine for growth, and contained large amounts of branched-chain fatty acids was isolated from a water-based coolant. The organism did not react with antisera against other legionellae, and on DNA hybridization the organism was less than 10% related to other Legionella species. Geometric mean titers found by indirect fluorescent antibody testing to WO-44C were significantly higher in ill employees than in controls (p = 0.0001). Attack rates by department decreased linearly with the departments distance from the implicated coolant system. The etiologic agent apparently was a new Legionella species; we propose the name Legionella feeleii species nova (AATC 35072). This is the first outbreak of nonpneumonic legionellosis in which the etiologic agent is not L. pneumophila, serogroup 1.

Legionella quinlivanii sp. nov. isolated from water

SixLegionella-like organisms were isolated from the evaporative air conditioning system of a bus in South Australia. All six isolates were presumptively identified as legionellae by their growth requirement forl-cysteine and their cellular branched-chain fatty acids. They were serologically distinct from other legionellae in the slide agglutination test. DNA hybridization studies showed that the six isolates belong to a new species ofLegionella, Legionella quinlivanii (ATCC 43830).

Isolation of Legionellae from Oxidation Ponds and Fishponds in Israel and Description of Legionella israelensis sp. nov.

Strains of Legionella pneumophila serogroup 4 and Legionella bozemanii and three strains of a new species, for which the name Legionella israelensis is proposed, were isolated from an oxidation pond in Israel. L. israelensis exhibited the following biochemical reactions typical of legionellae: Growth on buffered cysteine-yeast extract agar but not on chocolate agar or on buffered yeast extract agar without cysteine; nitrate negative; urease negative; nonfermentative; catalase positive; and motile. It did not hydrolyze hippurate, autofluoresce, or produce a brown pigment on tyrosine-containing media, and it had weakly positive gelatinase activity. It had predominantly branched-chain cellular fatty acids, similar to those described for other legionellae, and had major amounts of ubiquinones with more than 10 isoprene units in their side chains. It could be distinguished from all previously described Legionella species and serogroups by using antisera in slide agglutination tests. As determined by the hydroxyapatite method at 60

Legionella pneumophila Serogroup 9: A Cause of Human Pneumonia

A new serogroup of Legionella pneumophila was isolated from bronchoscopic washings and an open-lung biopsy specimen of a patient from California with pneumonia. A serologically identical isolate was obtained from tap water of a hospital ward in the Netherlands, and a fatal case of pneumonia in a patient from Virginia was shown retrospectively to have been caused by this new organism. The type strain of what is now serogroup 9 of L. pneumophila is IN-23-G1-C2 (American Type Culture Collection no. 35289). Disease caused by L. pneumophila serogroup 9 is not apparently different from that caused by other L. pneumophila serogroups.

Antigenic relatedness of immunoglobulins toLegionella pneumophila serogroups 1–6 from humans and the nonhuman primateMacaca fascicularis

The purpose of this study was to determine whether cynomolgus monkey antisera toLegionella pneumophila serogroups 1–6 antigens could be used as positive controls in the indirect immunofluorescence assay (IFA) for legionellosis. Immunoelectrophoretic mobilities and Ouchterlony analyses with heavy chain-specific antisera and IFA titers with immunoglobulin class-specific conjugates were used to show antigenic relatedness of immunized monkey immunoglobulins to those produced as a result of infection in humans. Identical immunoelectrophoretic precipitation patterns were obtained for human and monkey sera with antihuman gamma, mu, and alpha heavy-chain-specific antisera. Ouchterlony analyses showed precipitin bands of partial identity between human and monkey IgG, IgM, and IgA classes. IFA titers in the monkey hyperimmune antisera were >16,000 with antihuman conjugate. These data suggest that hyperimmune cynomolgus monkey antisera are suitable alternatives to human sera for IFA-positive controls.

Status of Serologic Tests for Legionella Antigen and Antibody at the Centers for Disease Control

Nine species and 16 serogroups have been defined for the genus Legionella. Direct immunofluorescence assay (DFA) results with 12 conjugates in 3 polyvalent pools showed that L. pneumophila serogroup 1 comprised 63% of the legionellae identified in specimens from patients with legionellosis. Indirect immunofluorescence assay (IFA) titers of paired sera from patients with suspected legionellosis against multiple Legionella antigens showed preferential reactivity for the L. pneumophila antigens. The multiple-antigen IFA was 96% specific when sera from patients with disease due to other etiologic agents were tested. The prevalence of antibody among well individuals is relatively high in some population groups.