The antigenic profiles of six strains of Chlamydia pneumoniae were analyzed by the ... infections in humans, especially atypical pneumonia (12). In.
Vol. 29, No. 7
JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1312-1316
0095-1137/91/071312-05$02.00/0 Copyright X 1991, American Society for Microbiology
Antigenic Variation among Strains of Chlamydia pneumoniae CAROLYN M.
BLACK,'* JAMES E. JOHNSON,'
CAROL E. AND BJ0RN P. BERDAL2
FARSHY,1 TERESA M. BROWN,'
Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Institute of Medical Biology, University of Troms0, Troms0, Norway2 Received 1 October 1990/Accepted 8 April 1991 The antigenic profiles of six strains of Chlamydia pneumoniae were analyzed by the microimmunofluorescence test (MIF) and immunoblotting with human serum and murine monoclonal antibody. MIF-derived antibody titers in serum samples from culture-positive patients were four- to eightfold higher against autologous isolate antigen than they were against the prototype antigen strain TW-183. Sera of patients with respiratory illness that were culture negative and complement fixation positive for Chlamydia spp. produced higher titers by MIF against a strain of C. pneumoniae isolated in the area than they did against TW-183. For two of five cases, the criteria for establishing the diagnosis of acute infection were met only with use of the antigen from the local strain; TW-183 was inadequate for this purpose. Immunoblot profiles revealed antigenic differences between strains that varied with the human serologic response; i.e., unique antigens were recognized by the sera of some individuals and not by the sera of others. Using the reactivity of a genus-specific monoclonal antibody against a major outer membrane protein, we found that strain CWL-011, isolated in Atlanta, Ga., may possess a major outer membrane protein with a molecular mass between those of C. trachomatis L2 and other C. pneumoniae strains. These data provide evidence of several new and unique serotypes of C. pneumoniae and suggest that the serologic diagnosis of C. pneumoniae infection may require the use of antigens from more than one strain of this species.
Chlamydia pneumoniae is a recently recognized species consisting of the strains commonly referred to as TWAR (11). These strains are associated with acute respiratory infections in humans, especially atypical pneumonia (12). In adult populations, the prevalence of antibody titers indicative of exposure to the organism has been reported to be in the range from 50 to 78% (12, 14). Culture-confirmed diagnosis of infection followed by propagation of clinical isolates has proven difficult to date, and the methods for this procedure have not yet been standardized. Diagnosis is based on the detection of fourfold rises in antibody titer in the microimmunofluorescence test (MIF) or an elevated immunoglobulin G (IgG) or IgM titer when only a single serum sample is available (12). Conventionally, the prototype strain TW-183 (Washington Research Foundation, Seattle, Wash.) is used as the only C. pneumoniae antigen in serologic tests, with additional use of Chlamydia trachomatis or Chlamydia psittaci antigen pools, depending on the laboratory. Grayston and colleagues (12) at the University of Washington have recently reported that all the isolates of C. pneumoniae in their collection appear to be members of a single strain, designated TWAR, on the basis of multiple parameters of protein and nucleic acid measurements. Several new isolates of C. pneumoniae from cases of community-acquired pneumonia have been characterized at the Centers for Disease Control, Atlanta, Ga. (6, 7). We report here an antigenic analysis of six C. pneumoniae strains and present evidence that these isolates represent serotypes unique from that of the prototype strain TW-183. MATERIALS AND METHODS Strains. C. pneumoniae CWL-011, CWL-029 (on deposit as ATCC VR1310), CWL-050, and CM-1 were isolated at the *
Corresponding author.
Centers for Disease Control from patients at a local hospital with radiologic evidence of pneumonia (1). The CWL strains were cultured from oropharyngeal swabs. Strain CM-1 was cultured from sputum. C. pneumoniae FML-10 was one of five strains isolated from nasal swabs from patients in Norway with respiratory infection associated with an outbreak of adenovirus (4). C. pneumoniae TW-183 was obtained from the Washington Research Foundation. All C. pneumoniae strains tested were reactive with a Chlamydia genus-specific monoclonal antibody (Kallestad Diagnostics, Austin, Tex.) and a C. pneumoniae-specific monoclonal antibody (Washington Research Foundation) but not with a C. trachomatis species-specific monoclonal antibody (Syva Diagnostics, Palo Alto, Calif.). These strains also reacted positively in a C. pneumoniae-specific polymerase chain reaction test (2). Preliminary studies by electron microscopy of infected HeLa cells demonstrated that elementary bodies of strain CWL-029 lack the loose periplasmic membrane described by Chi et al. (10) for strain TW-183. Elementary bodies of strain TW-183 tested in parallel did exhibit this loose periplasmic membrane; thus, strain CWL-029 may differ from strain TW-183 in its ultrastructural morphology. Isolations were performed by conventional procedures by using the HeLa 229 cell line as the host, centrifuge-assisted infections, and cycloheximide in the medium (13, 14). Antigen for immunoblotting was prepared from elementary bodies purified by centrifugation through a cushion of 30% Renografin-76 (Squibb Diagnostics, New Brunswick, N.J.) (5). Sera and monoclonal antibody. Serum samples for immunoblots were from three patients with culture-confirmed C. pneumoniae infections who presented with community-acquired pneumonia at a hospital in Atlanta, Ga., during a 10-month period in 1987 and 1988 (6, 7). The CWL strains of C. pneumoniae used in this study (described above) were cultured from specimens from these patients and are thus described as autologous antigen in immunoblot studies where appropriate. An additional serum specimen included 1312
VOL. 29, 1991
ANTIGENIC VARIATION IN C. PNEUMONIAE
TABLE 1. Titers of IgG determined by MIF in serum samples from patients with culture-confirmed C. pneumoniae infection by using autologous and heterologous antigen Strain
Patient nsrm serum
CWL-011 CWL-029
CWL-050C
S1 S2 S1 S2
IgG titer determined with the following antigens:
TABLE 2. MIF-derived antibody titers of complement fixationpositive serum samples from patients with acute-phase respiratory infections by using C. pneumoniae TW-183 and Norwegian strain FML-10 as antigens Antibody titer with the following
CWL-011 CWL-029 CWL-050 TW-183 CJAb 512 1,024
32 256
256 512
32 256
32 128
128 1,024
128 2,048
128 1,024
16 512
256
256
128
256
64
