Four Serogroups of Legionnaires' Disease Bacteria Defined by Direct Immunofluorescence ROGER M. McKINNEY, Ph.D.; LeROY THACKER, B.S.; PATRICIA P. HARRIS, M.S.; KAREN R. LEWALLEN, B.S.; G. ANN HEBERT, B.S.; PAUL H. EDELSTEIN, M.D.; and BERENICE M. THOMASON, B.S.; Atlanta, Georgia; and Los Angeles, California
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. D I R E C T F L U O R E S C E N T antibody (FA) staining is a valuable tool for detecting the Legionnaires' disease ( L D ) bacterium in human lung tissue and respiratory secretions. It is also useful for screening environmental specimens to detect reservoirs of L D bacteria. Cherry and associates (1) raised antisera in rabbits against six of the L D bacterium isolates. When fluorescein isothiocyanate (FITC)-labeled globulins from these rabbit antisera were tested against the 18 available strains of L D bacteria, a 1:80 working dilution of a conjugate prepared from the Knoxville 1 antiserum satisfactorily stained all strains. However, an L D bacterium isolate (Togus 1) obtained in May of 1978 from lung tissue of a victim of Legionnaires' disease in Togus, Maine, did not stain by direct F A staining with the Knoxville 1 conjugate. A conjugate prepared from rabbit antiserum against the Togus 1 isolate stained Togus 1 cells brightly (4 + ) but did not stain either the Knoxville 1 strain or the other 17 available strains of L D bacteria (2). Subsequently, two other isolates designated as Atlanta 1 and Atlanta 2 were obtained, which stained with the Togus 1 conjugate but not with the Knoxville 1 conjugate. This laboratory has now accumulated a total of 35 strains of L D bacteria that have been isolated by various investigators either from diagnostic specimens from the human respiratory system or from environmental samples. Two of these isolates (Bloomington 2 and Los Angeles 1) do not stain with either the Knoxville 1 or the Togus 1 conjugate. In our study, additional antisera and conjugates were prepared against Bloomington 2 and Los Angeles 1. Each of the 35 isolates of L D bacteria currently available was examined separately using the Knoxville 1, Togus 1, Bloomington 2, and Los Angeles 1 conjugates for direct F A staining. A serologic grouping of the L D bacteria based on direct F A results is presented and relationships among the representative strains of each serogroup are examined by direct F A staining and immunoelectrophoresis. • F r o m t h e Center for Disease Control, Public Health Service, U.S. D e p a r t m e n t of Health, Education, and Welfare, Atlanta, Georgia; and the Veterans Administration, Wadsworth Medical Center, and Department of Medicine, University of California School of Medicine, Los Angeles, California.
Materials and Methods Thirty-five strains of L D bacteria were used (Table 1). Pontiac 1 (3), Bloomington 1 and 2 (4), and Memphis 1 (5) were isolated from environmental specimens. Bloomington 1 is the cooling tower water isolate (BL 434), and Bloomington 2 is the creek water isolate (BL 433) (4). T h e other isolates were obtained by various investigators either directly or indirectly (through guinea pigs) from human lung tissues or respiratory fluids. O L D A was isolated in 1947, reported on in 1952 (6), and recently identified as the L D bacterium (7). T h e Los Angeles 1 isolate was obtained from human lung tissue by Paul Edelstein and was documented as being serologically different from the Philadelphia 1 strain by both direct and indirect F A staining (8). The growth characteristics of the 35 isolates were determined by Robert Weaver (9), the gas-liquid chromatography profiles of their cellular fatty acids were obtained by C. Wayne Moss (10), and D N A homology for 17 of the isolates was determined by Don Brenner (11) using methods that are detailed in previous reports on the L D bacterium. All of their data are consistent with the characteristics of the reference strain, Philadelphia 1. T h e D N A homology studies have not yet been completed on Birmingham 1, Miami Beach 1, Allentown £, Rochester 1, Detroit 1 and 2, Los Angeles 1 and 2, Atlanta 2, Indianapolis 1, New York 1 and 2, Memphis 1, Buffalo 1, Orlando 1, Baltimore 1, Oak Lawn 1, and Houston 1. Antisera were raised in rabbits against the Knoxville 1, Togus 1, Bloomington 2, and Los Angeles 1 strains of L D bacteria using a schedule and procedure described previously (2). Immunoglobulin G was isolated from antisera by affinity chromatography on a column of Protein A-Sepharose CL-4B (Pharmacia, Inc., Piscataway, New Jersey) as described by Goding (12). T h e F I T C conjugates of IgG were prepared as described previously (13). Conjugates were adjusted to contain 10 mg of I g G / m L , and had fluorescein-to-protein ratios of approximately 25 jLtg/mg. Smears of L D bacterium cells were prepared and stained as described by Cherry and associates (1). Slides were examined with a Leitz Dialux® microscope (E. Leitz, Rockleigh, New Jersey) using vertical illumination with a 50-watt tungsten-halogen lamp, 2-KP 490 exciter filters, T K 510 beam splitter, and K 510 barrier filter. A 10 X ocular was used with a 100 X objective and 1.25 X prism, giving a total magnification of 1250 X. Conjugates were prepared in serial twofold dilutions, and endpoint titers were reported as the highest dilution of the conjugate that produced 4 + , or very bright staining of the cells. Weaker fluorescence was graded as 3 + or 2 + on a diminishing scale. T h e 4 + end-point dilution titer was determined with each conjugate for homologous strain cells as well as for cells of each strain within the homologous serogroup. T h e conjugates for the representative strains of each of the four serogroups were further tested for cross-reactions by F A staining at a 1:8 dilution (1.25 mg of I g G / m L ) against all strains of the heterologous serogroups. T h e Bloomington 2 conjugate was further tested for cross-staining at higher dilutions against strains Togus 1, Atlanta 1, and Atlanta 2. Solubilized antigen extracts of representative strains of each of the four groups (Knoxville 1, Togus 1, Bloomington 2, and Los Angeles 1) were prepared by extracting the washed cells
Annals of Internal Medicine 90:621-624, 1979
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
621
with hot 0.85% sodium chloride as described previously (2). The extracts were dialyzed against distilled water and lyophilized. Solutions containing 10 mg of solubilized antigen extract per millilitre were prepared in distilled water. Apparatus and procedures used for immunoelectrophoresis were those described previously (2). As a control, immunoelectrophoresis was also performed with the four antisera using a 50 X concentration of the soluble media components that had not been exposed to LD bacterium cells. Results Staining titers obtained for the L D bacteria conjugates are shown in Table 1. Bright staining was obtained in all cases with homologous cells. The Knoxville 1 conjugate stained 30 of the isolates to 4 + brightness but did not stain Togus 1, Bloomington 2, Los Angeles 1, Atlanta 1, and Atlanta 2 isolates. The Togus 1 conjugate stained only Togus 1, Atlanta 1, and Atlanta 2. The Bloomington 2 conjugate stained only Bloomington 2 cells at 4 + brightness but stained the Togus 1, Atlanta 1, and Atlanta 2 cells at 3 + brightness. This staining was completely eliminated only when a 1:512 or higher dilution of the conjugate was used. The Los Angeles 1 conjugate stained only Los Angeles 1 cells. The immunoelectrophoretic patterns obtained are
shown in Figure 1. With Knoxville 1 antiserum (Figure la), Los Angeles 1 antiserum (Figure lc), and Bloomington 2 antiserum (Figure Id) the strong precipitin arcs observed near the wells appear only against the homologous strain antigens. With Togus 1 antiserum (Figure lb) strong precipitin lines are observed near the well with the homologous antigen and with the Bloomington 2 antigen. The immunoelectrophoretic pattern of Los Angeles 1 antigen is unique in that strong anodic (rather than cathodic) lines are formed against homologous antiserum (Figure lc). Common weak precipitin lines appear near the troughs with each antigen against all four antisera. N o precipitin lines were observed in tests using a 50 X concentration of the soluble media components against the four group strains. Discussion The only significant cross-staining that occurred among the four representative strains was between Togus 1 cells and the Bloomington 2 conjugate. This cross-reaction does not appear to be reciprocal, because the Togus 1 conjugate does not stain Bloomington 2 cells. Complete specificity of Bloomington 2 conjugate for Bloomington 2
Table 1. Direc:t Fluorescent Antibody Staining Characteristics of 35 Strains of Legionnaires' Disease Bacterium Number
Isolate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Knoxville 1 Philadelphia 1 Philadelphia 2 Philadelphia 3 Philadelphia 4 Birmingham 1 Burlington 1 Miami Beach 1 Allentown 1 New York 1 Memphis 1 Buffalo 1 Pontiac 1 Rochester 1 Los Angeles 2 Orlando 1 Flint 1 Flint 2 Albuquerque Indianapolis 1 Berkeley Houston Detroit 2 New York 2 Baltimore 1 Detroit 1 Bloomington 1 Olda Oaklawn 1 Bellingham 1 Togus 1 Atlanta 1 Atlanta 2 Bloomington 2 Los Angeles 1
Conjugate Knoxville 1
Togus 1
Bloomington 2
Los Angeles 1
1024* 2048 2048 4096 4096 4096 4096 4096 4096 2048 4096 4096 2048 2048 2048 2048 2048 512 512 512 512 512 128 64 32 16 16 16 16 16
—t — — — — — — — — — — — — — — — — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 64(3+)§ 64(3+)§ 128(3+)§ 2048 —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — —
— —
— — — — — — 64 512 512 — —
* Reciprocal of the highest dilution of the conjuigate to give a 4 + or very bright staining. t Negative st;aining with 1:8 dilution of the conjuf ?ate. § No staining was obtained at a conjugate dilutioni of 1:512. 622
April 1979 • Annals of Internal Medicine • Volume 90 • Number 4
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
— — 64
Figure 1 . Immunoelectrophoresis of solubilized antigen extracts of the four type strains of Legionnaires' disease bacteria cells against rabbit antisera to the four strains. The antigens are in the wells and are designated by K, Knoxville 1 ; T, Togus 1 ; L, Los Angeles 1 ; and B, Bloomington 2. The antiserum is in the trough in each case. a. Knoxville 1. b. Togus 1. c. Los Angeles 1. d . Bloomington 2.
cells is obtained when a working dilution of 1:512 is used. Considerable specificity of the group antisera is shown for their homologous antigens by immunoelectrophoresis except in the case of Togus 1 antiserum, which reacts to a rather pronounced degree with Bloomington 2 antigens. Since this reaction is the reverse of that obtained with direct F A staining where Bloomington 2 conjugate crossreacts with Togus 1 cells, we must conclude that different antigens are involved in the two systems. Direct F A staining is the most widely accepted diagnostic procedure for identifying the L D bacterium directly in tissue specimens. The clear-cut separation of the 35 strains on the basis of direct F A staining with the four serogroup conjugates is remarkable. We recommend that strains 1-30 in Table 1 be designated as serogroup 1, strains 31-33 as serogroup 2, strain 34 as serogroup 3, and strain 35 as serogroup 4. As can be readily seen in Table 1, most of the currently available strains of L D bacteria are classified in group 1. This fact may reflect the ability of this particular serogroup to adapt to diverse demographic conditions. It is also possible that some cases of infection with L D bacteria belonging to other serogroups have not been identified in the past because only the group 1 conjugate was available. Almost certainly, additional serogroups will be rec-
ognized in the future. Lung tissue specimens that exhibit the pathological features typical of Legionnaires' disease but are negative by direct F A staining with conjugates for the four serogroups should be subjected to conventional isolation procedures for the L D bacterium. On the basis of these results the most practical means of using direct F A staining to screen diagnostic specimens is to use a polyvalent conjugate prepared against strains representative of the four serologic groups. Positive specimens can then be further classified with groupspecific conjugates. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare. • Requests for reprints should be addressed to Roger M. McKinney, Ph.D., Bldg 5, Rm 112, Center for Disease Control; Atlanta, GA 30333 Received 15 November
1978; accepted 14 December 1978.
References 1. C H E R R Y WB, P I T T M A N B, H A R R I S PP, H E B E R T GA, T H O M A S O N BM,
THACKER L, W E A V E R RE: Detection of Legionnaires' disease bacteria by direct immunofluorescent staining. J Clin Microbiol 8:329-338, 1978 2. M C K I N N E Y RM, THOMASON BM, H A R R I S PP, T H A C K E R L, L E W A L LEN KR, WILKINSON HW, H E B E R T GA, Moss CW: Recognition of a
new serogroup of the Legionnaires disease bacterium. J Clin Microbiol 9:103-107, 1979 3. G L I C K TH, G R E G G MB, BERMAN B, M A L L I S O N G, R H O D E S WW J R . , McKinney
etal.
• Serogroups Defined by Immunofluorescence
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
623
KASSANOFF I: Pontiac fever. An epidemic of unknown etiology in a health department. 1. Clinical and epidemiologic aspects. Am J Epidemiol 107:149-160, 1978 4. M O R R I S G K , P A T T O N CM, F E E L E Y JC, JOHNSON SE, G O R M A N G, M A R T I N WT, SKALIY P, M A L L I S O N G F , POLITI BD, M A C K E L DC:
Isolation of the Legionnaires' disease bacterium from environmental samples. Ann Intern Med 90:664-666, 1979 5. CENTER FOR DISEASE CONTROL: Isolation of organisms resembling Legionnaires' disease bacterium—Tennessee. Morbid Mortal Weekly Rep 27:368-369, 1978 6. JACKSON EB, CROCKER TT, S M A D E L JE: Studies on two rickettsia-like
agents probably isolated from guinea pigs. Bacteriol Proc 119, 1952 7. M C D A D E JE, BRENNER DJ, BOZEMAN FM: Legionnaires' disease bacterium isolated in 1947. Ann Intern Med 90:659-661, 1979 8. E D E L S T E I N PH, M E Y E R R D , F I N E G O L D SM: Isolation of a new sero-
624
type of Legionnaires' disease bacterium. Lancet 2:1172-1174, 1978 9. WEAVER RE: Cultural and staining characteristics, in Legionnaires': The Disease, the Bacterium and Methodology, edited by JONES GJ, H E B E R T GA. Atlanta, Center for Disease Control, 1978, pp. 17-21 10. Moss CW, W E A V E R RE, D E E S SB, CHERRY WB: Cellular fatty acid composition of isolates from Legionnaires' disease. / Clin Microbiol 6:140-143, 1977 11. B R E N N E R DJ, S T E I G E R W A L T A G , W E A V E R RE, M C D A D E JE, F E E L E Y
JC, M A N D E L M: Classification of the Legionnaires' bacterium: an interim report. Curr Microbiol 1:71-76, 1978 12. G O D I N G JW: Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods 13:215-226, 1976 13. H E B E R T G A , P I T T M A N B, M C K I N N E Y RM, C H E R R Y WB: The prepa-
ration and physicochemical characterization of fluorescent antibody reagents. Atlanta, Center for Disease Control, 1972
Annals of Internal Medicine 90:624-627, 1979
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
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. D I R E C T F L U O R E S C E N T antibody (FA) staining is a valuable tool for detecting the Legionnaires' disease ( L D ) bacterium in human lung tissue and respiratory secretions. It is also useful for screening environmental specimens to detect reservoirs of L D bacteria. Cherry and associates (1) raised antisera in rabbits against six of the L D bacterium isolates. When fluorescein isothiocyanate (FITC)-labeled globulins from these rabbit antisera were tested against the 18 available strains of L D bacteria, a 1:80 working dilution of a conjugate prepared from the Knoxville 1 antiserum satisfactorily stained all strains. However, an L D bacterium isolate (Togus 1) obtained in May of 1978 from lung tissue of a victim of Legionnaires' disease in Togus, Maine, did not stain by direct F A staining with the Knoxville 1 conjugate. A conjugate prepared from rabbit antiserum against the Togus 1 isolate stained Togus 1 cells brightly (4 + ) but did not stain either the Knoxville 1 strain or the other 17 available strains of L D bacteria (2). Subsequently, two other isolates designated as Atlanta 1 and Atlanta 2 were obtained, which stained with the Togus 1 conjugate but not with the Knoxville 1 conjugate. This laboratory has now accumulated a total of 35 strains of L D bacteria that have been isolated by various investigators either from diagnostic specimens from the human respiratory system or from environmental samples. Two of these isolates (Bloomington 2 and Los Angeles 1) do not stain with either the Knoxville 1 or the Togus 1 conjugate. In our study, additional antisera and conjugates were prepared against Bloomington 2 and Los Angeles 1. Each of the 35 isolates of L D bacteria currently available was examined separately using the Knoxville 1, Togus 1, Bloomington 2, and Los Angeles 1 conjugates for direct F A staining. A serologic grouping of the L D bacteria based on direct F A results is presented and relationships among the representative strains of each serogroup are examined by direct F A staining and immunoelectrophoresis. • F r o m t h e Center for Disease Control, Public Health Service, U.S. D e p a r t m e n t of Health, Education, and Welfare, Atlanta, Georgia; and the Veterans Administration, Wadsworth Medical Center, and Department of Medicine, University of California School of Medicine, Los Angeles, California.
Materials and Methods Thirty-five strains of L D bacteria were used (Table 1). Pontiac 1 (3), Bloomington 1 and 2 (4), and Memphis 1 (5) were isolated from environmental specimens. Bloomington 1 is the cooling tower water isolate (BL 434), and Bloomington 2 is the creek water isolate (BL 433) (4). T h e other isolates were obtained by various investigators either directly or indirectly (through guinea pigs) from human lung tissues or respiratory fluids. O L D A was isolated in 1947, reported on in 1952 (6), and recently identified as the L D bacterium (7). T h e Los Angeles 1 isolate was obtained from human lung tissue by Paul Edelstein and was documented as being serologically different from the Philadelphia 1 strain by both direct and indirect F A staining (8). The growth characteristics of the 35 isolates were determined by Robert Weaver (9), the gas-liquid chromatography profiles of their cellular fatty acids were obtained by C. Wayne Moss (10), and D N A homology for 17 of the isolates was determined by Don Brenner (11) using methods that are detailed in previous reports on the L D bacterium. All of their data are consistent with the characteristics of the reference strain, Philadelphia 1. T h e D N A homology studies have not yet been completed on Birmingham 1, Miami Beach 1, Allentown £, Rochester 1, Detroit 1 and 2, Los Angeles 1 and 2, Atlanta 2, Indianapolis 1, New York 1 and 2, Memphis 1, Buffalo 1, Orlando 1, Baltimore 1, Oak Lawn 1, and Houston 1. Antisera were raised in rabbits against the Knoxville 1, Togus 1, Bloomington 2, and Los Angeles 1 strains of L D bacteria using a schedule and procedure described previously (2). Immunoglobulin G was isolated from antisera by affinity chromatography on a column of Protein A-Sepharose CL-4B (Pharmacia, Inc., Piscataway, New Jersey) as described by Goding (12). T h e F I T C conjugates of IgG were prepared as described previously (13). Conjugates were adjusted to contain 10 mg of I g G / m L , and had fluorescein-to-protein ratios of approximately 25 jLtg/mg. Smears of L D bacterium cells were prepared and stained as described by Cherry and associates (1). Slides were examined with a Leitz Dialux® microscope (E. Leitz, Rockleigh, New Jersey) using vertical illumination with a 50-watt tungsten-halogen lamp, 2-KP 490 exciter filters, T K 510 beam splitter, and K 510 barrier filter. A 10 X ocular was used with a 100 X objective and 1.25 X prism, giving a total magnification of 1250 X. Conjugates were prepared in serial twofold dilutions, and endpoint titers were reported as the highest dilution of the conjugate that produced 4 + , or very bright staining of the cells. Weaker fluorescence was graded as 3 + or 2 + on a diminishing scale. T h e 4 + end-point dilution titer was determined with each conjugate for homologous strain cells as well as for cells of each strain within the homologous serogroup. T h e conjugates for the representative strains of each of the four serogroups were further tested for cross-reactions by F A staining at a 1:8 dilution (1.25 mg of I g G / m L ) against all strains of the heterologous serogroups. T h e Bloomington 2 conjugate was further tested for cross-staining at higher dilutions against strains Togus 1, Atlanta 1, and Atlanta 2. Solubilized antigen extracts of representative strains of each of the four groups (Knoxville 1, Togus 1, Bloomington 2, and Los Angeles 1) were prepared by extracting the washed cells
Annals of Internal Medicine 90:621-624, 1979
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
621
with hot 0.85% sodium chloride as described previously (2). The extracts were dialyzed against distilled water and lyophilized. Solutions containing 10 mg of solubilized antigen extract per millilitre were prepared in distilled water. Apparatus and procedures used for immunoelectrophoresis were those described previously (2). As a control, immunoelectrophoresis was also performed with the four antisera using a 50 X concentration of the soluble media components that had not been exposed to LD bacterium cells. Results Staining titers obtained for the L D bacteria conjugates are shown in Table 1. Bright staining was obtained in all cases with homologous cells. The Knoxville 1 conjugate stained 30 of the isolates to 4 + brightness but did not stain Togus 1, Bloomington 2, Los Angeles 1, Atlanta 1, and Atlanta 2 isolates. The Togus 1 conjugate stained only Togus 1, Atlanta 1, and Atlanta 2. The Bloomington 2 conjugate stained only Bloomington 2 cells at 4 + brightness but stained the Togus 1, Atlanta 1, and Atlanta 2 cells at 3 + brightness. This staining was completely eliminated only when a 1:512 or higher dilution of the conjugate was used. The Los Angeles 1 conjugate stained only Los Angeles 1 cells. The immunoelectrophoretic patterns obtained are
shown in Figure 1. With Knoxville 1 antiserum (Figure la), Los Angeles 1 antiserum (Figure lc), and Bloomington 2 antiserum (Figure Id) the strong precipitin arcs observed near the wells appear only against the homologous strain antigens. With Togus 1 antiserum (Figure lb) strong precipitin lines are observed near the well with the homologous antigen and with the Bloomington 2 antigen. The immunoelectrophoretic pattern of Los Angeles 1 antigen is unique in that strong anodic (rather than cathodic) lines are formed against homologous antiserum (Figure lc). Common weak precipitin lines appear near the troughs with each antigen against all four antisera. N o precipitin lines were observed in tests using a 50 X concentration of the soluble media components against the four group strains. Discussion The only significant cross-staining that occurred among the four representative strains was between Togus 1 cells and the Bloomington 2 conjugate. This cross-reaction does not appear to be reciprocal, because the Togus 1 conjugate does not stain Bloomington 2 cells. Complete specificity of Bloomington 2 conjugate for Bloomington 2
Table 1. Direc:t Fluorescent Antibody Staining Characteristics of 35 Strains of Legionnaires' Disease Bacterium Number
Isolate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Knoxville 1 Philadelphia 1 Philadelphia 2 Philadelphia 3 Philadelphia 4 Birmingham 1 Burlington 1 Miami Beach 1 Allentown 1 New York 1 Memphis 1 Buffalo 1 Pontiac 1 Rochester 1 Los Angeles 2 Orlando 1 Flint 1 Flint 2 Albuquerque Indianapolis 1 Berkeley Houston Detroit 2 New York 2 Baltimore 1 Detroit 1 Bloomington 1 Olda Oaklawn 1 Bellingham 1 Togus 1 Atlanta 1 Atlanta 2 Bloomington 2 Los Angeles 1
Conjugate Knoxville 1
Togus 1
Bloomington 2
Los Angeles 1
1024* 2048 2048 4096 4096 4096 4096 4096 4096 2048 4096 4096 2048 2048 2048 2048 2048 512 512 512 512 512 128 64 32 16 16 16 16 16
—t — — — — — — — — — — — — — — — — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 64(3+)§ 64(3+)§ 128(3+)§ 2048 —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — —
— —
— — — — — — 64 512 512 — —
* Reciprocal of the highest dilution of the conjuigate to give a 4 + or very bright staining. t Negative st;aining with 1:8 dilution of the conjuf ?ate. § No staining was obtained at a conjugate dilutioni of 1:512. 622
April 1979 • Annals of Internal Medicine • Volume 90 • Number 4
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
— — 64
Figure 1 . Immunoelectrophoresis of solubilized antigen extracts of the four type strains of Legionnaires' disease bacteria cells against rabbit antisera to the four strains. The antigens are in the wells and are designated by K, Knoxville 1 ; T, Togus 1 ; L, Los Angeles 1 ; and B, Bloomington 2. The antiserum is in the trough in each case. a. Knoxville 1. b. Togus 1. c. Los Angeles 1. d . Bloomington 2.
cells is obtained when a working dilution of 1:512 is used. Considerable specificity of the group antisera is shown for their homologous antigens by immunoelectrophoresis except in the case of Togus 1 antiserum, which reacts to a rather pronounced degree with Bloomington 2 antigens. Since this reaction is the reverse of that obtained with direct F A staining where Bloomington 2 conjugate crossreacts with Togus 1 cells, we must conclude that different antigens are involved in the two systems. Direct F A staining is the most widely accepted diagnostic procedure for identifying the L D bacterium directly in tissue specimens. The clear-cut separation of the 35 strains on the basis of direct F A staining with the four serogroup conjugates is remarkable. We recommend that strains 1-30 in Table 1 be designated as serogroup 1, strains 31-33 as serogroup 2, strain 34 as serogroup 3, and strain 35 as serogroup 4. As can be readily seen in Table 1, most of the currently available strains of L D bacteria are classified in group 1. This fact may reflect the ability of this particular serogroup to adapt to diverse demographic conditions. It is also possible that some cases of infection with L D bacteria belonging to other serogroups have not been identified in the past because only the group 1 conjugate was available. Almost certainly, additional serogroups will be rec-
ognized in the future. Lung tissue specimens that exhibit the pathological features typical of Legionnaires' disease but are negative by direct F A staining with conjugates for the four serogroups should be subjected to conventional isolation procedures for the L D bacterium. On the basis of these results the most practical means of using direct F A staining to screen diagnostic specimens is to use a polyvalent conjugate prepared against strains representative of the four serologic groups. Positive specimens can then be further classified with groupspecific conjugates. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare. • Requests for reprints should be addressed to Roger M. McKinney, Ph.D., Bldg 5, Rm 112, Center for Disease Control; Atlanta, GA 30333 Received 15 November
1978; accepted 14 December 1978.
References 1. C H E R R Y WB, P I T T M A N B, H A R R I S PP, H E B E R T GA, T H O M A S O N BM,
THACKER L, W E A V E R RE: Detection of Legionnaires' disease bacteria by direct immunofluorescent staining. J Clin Microbiol 8:329-338, 1978 2. M C K I N N E Y RM, THOMASON BM, H A R R I S PP, T H A C K E R L, L E W A L LEN KR, WILKINSON HW, H E B E R T GA, Moss CW: Recognition of a
new serogroup of the Legionnaires disease bacterium. J Clin Microbiol 9:103-107, 1979 3. G L I C K TH, G R E G G MB, BERMAN B, M A L L I S O N G, R H O D E S WW J R . , McKinney
etal.
• Serogroups Defined by Immunofluorescence
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
623
KASSANOFF I: Pontiac fever. An epidemic of unknown etiology in a health department. 1. Clinical and epidemiologic aspects. Am J Epidemiol 107:149-160, 1978 4. M O R R I S G K , P A T T O N CM, F E E L E Y JC, JOHNSON SE, G O R M A N G, M A R T I N WT, SKALIY P, M A L L I S O N G F , POLITI BD, M A C K E L DC:
Isolation of the Legionnaires' disease bacterium from environmental samples. Ann Intern Med 90:664-666, 1979 5. CENTER FOR DISEASE CONTROL: Isolation of organisms resembling Legionnaires' disease bacterium—Tennessee. Morbid Mortal Weekly Rep 27:368-369, 1978 6. JACKSON EB, CROCKER TT, S M A D E L JE: Studies on two rickettsia-like
agents probably isolated from guinea pigs. Bacteriol Proc 119, 1952 7. M C D A D E JE, BRENNER DJ, BOZEMAN FM: Legionnaires' disease bacterium isolated in 1947. Ann Intern Med 90:659-661, 1979 8. E D E L S T E I N PH, M E Y E R R D , F I N E G O L D SM: Isolation of a new sero-
624
type of Legionnaires' disease bacterium. Lancet 2:1172-1174, 1978 9. WEAVER RE: Cultural and staining characteristics, in Legionnaires': The Disease, the Bacterium and Methodology, edited by JONES GJ, H E B E R T GA. Atlanta, Center for Disease Control, 1978, pp. 17-21 10. Moss CW, W E A V E R RE, D E E S SB, CHERRY WB: Cellular fatty acid composition of isolates from Legionnaires' disease. / Clin Microbiol 6:140-143, 1977 11. B R E N N E R DJ, S T E I G E R W A L T A G , W E A V E R RE, M C D A D E JE, F E E L E Y
JC, M A N D E L M: Classification of the Legionnaires' bacterium: an interim report. Curr Microbiol 1:71-76, 1978 12. G O D I N G JW: Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods 13:215-226, 1976 13. H E B E R T G A , P I T T M A N B, M C K I N N E Y RM, C H E R R Y WB: The prepa-
ration and physicochemical characterization of fluorescent antibody reagents. Atlanta, Center for Disease Control, 1972
Annals of Internal Medicine 90:624-627, 1979
Downloaded From: http://annals.org/pdfaccess.ashx?url=/data/journals/aim/19545/ by a University of California San Diego User on 06/01/2017
