Vol. 29, No. 4

0095-1137/91/040707-05$02.00/0 Copyright © 1991, American Society for Microbiology

Serotyping of Chlamydia psittaci Isolates Using Serovar-Specific Monoclonal Antibodies with the Microimmunofluorescence Test ARTHUR A. ANDERSEN

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010 Received 9 October 1990/Accepted 6 January 1991

A panel of 10 serovar-specific monoclonal antibodies that could distinguish 10 distinct serovars of Chiamydia psittaci was prepared. The panel included one monoclonal antibody to each of the 10 serovars. Monoclonal antibodies were selected for their specificity in the indirect microimmunofluorescence test. Each of the monoclonal antibodies had a titer of 1:1,280 or higher to the homologous strain, with only two showing any cross-reactivity at a dilution of 1:10. Chlamydial antigen derived from organisms growing in tissue culture of one well of a 96-well multiwell dish was usually sufficient for the serotyping of an isolate. Infected yolk sac preparations were also suitable for serotyping. The panel of monoclonal antibodies was used to serotype 55 mammalian and avian strains. All except five of the strains were successfully serotyped; these five strains are presumed to represent at least two additional serovars. The use of a panel of monoclonal antibodies in the indirect microimmunofluorescence test provides a rapid and reliable method for serotyping new isolates. Monoclonal antibodies to new serovars can easily be added to the panel.

The genus Chlamydia is divided into three species: C. trachomatis, C. pneumoniae, and C. psittaci (15, 20). C. trachomatis is primarily a pathogen of humans and has been divided into 15 serovars. C. pneumoniae, also a pathogen of humans, has one known serovar. C. psittaci encompasses a diverse group of strains that have a broad host range and pathogenic potential; it has been isolated from most species of birds and animals. These strains have been grouped by a number of methods (5, 8, 9, 31, 33, 34, 36). Most of these studies have involved a narrow range of strains and have had limited success. The more extensive studies involving larger numbers of strains have used biotyping (36), conventional sera (8, 9, 31), monoclonal antibodies (MAbs) (1, 2, 14, 39, 41), and genetic diversity (12, 13, 40). The MAb studies in laboratories other than our own have used primarily groupreactive MAbs (14, 39, 41). These studies have demonstrated that extensive diversity exists within the group and that a number of serologically distinct strains exist. Using serovarspecific MAbs, I have identified four serovars within the avian strains (2). The development of a rapid and precise method of serotyping C. psittaci isolates would benefit C. psittaci research. It would provide a tool for epidemiological studies to determine the distribution of a given serovar in both a geographic area and a host range. It would provide needed information about whether a given serovar is limited to specific clinical syndromes and/or to specific anatomical sites in the host. Serotyping will help answer questions about latency of infection and about the immune process by determining whether an infection is a recurrent infection or an exposure to a new serovar. Most important, serotyping will make it easier to compare results from different laboratories. For a serotyping system to be successful for C. psittaci, it must be easy to perform, be reproducible, require a minimum of sample and materials, and be expandable to include new serovars. The use of the indirect microimmunofluorescence (IMIF) test with serovar-specific MAbs meets these requirements and has proven to be usable with C. trachomatis isolates (22, 43). Earlier, I and others reported on the production of serovar-specific MAbs to C. psittaci and

demonstrated that they could be used for serotyping isolates in the indirect fluorescent antibody technique (1-3). These MAbs have proven to be highly specific with little crossreactivity. The goal of this research was to develop a panel of MAbs that could be used with the IMIF test to rapidly serotype new C. psittaci isolates. MATERIALS AND METHODS

Chlamydiae. The C. psittaci strains used and their origins are given in Table 1. The strains have been maintained in our laboratory by passage in embryonated eggs. The feline and polyarthritis strains were used as yolk sac harvests because of difficulty in obtaining adequate growth in Vero cells. The remaining strains were adapted for growth in Vero cells by centrifuging the inoculum onto 24-h-old confluent Vero cell monolayers (3). Yolk sac harvest or tissue culture harvest was used as the source of chlamydial elementary bodies for serotyping of the strains. IMIF. The IMIF test was performed essentially as described by Wang and Grayston (42). Briefly, the chlamydial antigen, either in yolk sac harvest or mixed with a normal yolk sac preparation, was placed in 0.5-,ul dots on a standard microscope slide and allowed to air day for 30 min. The slides were then fixed with acetone for 10 min. Two microliters of the MAbs at the proper dilution was added to each dot. The slides were incubated for 30 min in a humidified chamber at 37°C. The slides were then carefully washed four times with phosphate-buffered saline (pH 7.2) and four times with distilled water and allowed to air day. The slides were then stained with fluorescein-conjugated anti-mouse immunoglobulin G (heavy- and light-chain-specific) serum (Organon Technika, Malvern, Pa.) at a dilution of 1:30 containing 0.5% Evans blue. The staining was done by adding 4 pl of fluorescein-conjugated anti-mouse serum to each dot and incubating and washing the slides as described above. The antigen dots were placed on the slide in a pattern of four rows containing seven dots each. This permitted the use of four dilutions for each of seven MAbs on one microscope slide. The first column of four dots was routinely assigned a 707



ANDERSEN TABLE 1. C. psittaci strains and designated serotypes Serovar



80022 79068 80032 78001 Vsla



VS2 80026 80033 SE45 6BC M-2 NADC-3 NC-2 DD34 SCP IP-1 CP3 a 80066 WWD CT-2

CT-4 CS-i IT-2 SG125 FalTex 78015 TT3 VT

Duck Abortion (type I)

SCT NJla VS21 VS22 CC NC-1 TT Borg GDa B577a

OSP EBA IA Id Polyarthritis (type II)


Feline pneumonitis


Wolfsen cattle Muskrat Guinea pig Untyped

Cello 9277 WCa M56a GPICa MN 87-30985 BP-1 BP-4 z


Parrot Cockatiel Parrot Cockatiel Parakeet Lovebird Parrot Cockatiel Snowy egret Parakeet

Quail Parakeet Turkey Parrot Pigeon Pigeon Pigeon Pigeon White wing dove Turkey Turkey Sparrow Turkey Sea gull Turkey Budgerigar Turkey Turkey Turkey Turkey Turkey Turkey Canine Turkey Turkey Human

Duck Sheep Sheep Bovine

Sheep Sheep Bovine

Sheep Cat Cat Cat Bovine Muskrat Guinea pig Human?

Pigeon Bovine Bovine Bovine


Reference or source


1980 1979 1980 1978 1985 1985 1980 1980 1950 1943 1988 1985 1990 1949 1972 1967 1957 1980 1959 1956 1958 1958 1973 1957 1975 1978 1975 1965 1973 1954 1986 1986 1989 1990 1952 1944

Texas Texas Texas Texas

Georgia Minnesota Texas Texas Louisiana California Arizona Iowa North Carolina

44 44 44 44 3 3 44 44 32 ATCCb

Reggiardoc Andersen Fickend

7 South Carolina Iowa California South Carolina Texas California California California Iowa

Pagee Pagee 28 44 16 27 27 27


Texas Texas Texas

44 44 44 26



South Carolina New Jersey Minnesota Minnesota Texas North Carolina Texas Louisiana

30 24



1962 1985 1959 1972 1987 1940 1968 1944

Utah Oregon California Iowa Idaho Iowa Iowa New Jersey California Arkansas California Canada Massachusetts

17 37 3 38

1990 1963 1961 1964 1934 1987 1961 1961


Colorado Wisconsin Wisconsin Minnesota

NVSLf NVSL Grimesg Fickend

19 23

Pagee Andersenh 18 29

4 6

Parsley' 25 35 21 10 NVSL 11 11 Werdin and Johnson'

a Used to produce the monoclonal antibody. b ATCC, American Type Culture Collection, Rockville, Md. Isolated by C. Reggiardo, Veterinary Diagnostic Lab., Tucson, Ariz. d Isolated by M. Ficken, College of Veterinary Medicine, Raleigh, N.C. Isolated by L. Page, Ames, Iowa (present address, Escondido, Calif.). f NVSL, Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa. g Isolated by J. Grimes, College of Veterinary Medicine, College Station, Tex. h Isolated from material supplied by B. Leamaster, Washington State University, Pullman. Isolated by M. Parsley, Arkansas Veterinary Diagnostic Lab, Little Rock. J Isolated by R. Werdin and D. Johnson, College of Veterinary Medicine, St. Paul, Minn.

VOL. 29, 1991


VS1/E8 CP3/2A7

NJ1/B1O GR9/B6

B577/F3 IPA/2G7 M56/E6 WC/E4 FP/F1O GPIC/C5 NJ1/D3


TABLE 2. Titers of MAbs in the IMIF test with homologous strains and representative strains of other serovars Titer' of MAb to: FP WC M56 IPA B577 GD VS1 NJ1 CP3 20,480 10 5,120 10,240 1,280 20,480 10 10,240 10,240 10,240 10,240 10,240 10,240 10,240 10,240 10,240 10,240 10,240 10,240 10,240




10,240 10,240

a The reciprocal of the highest dilution giving a clearly distinguishable positive reaction. -, No detectable cross-reaction at a dilution of 1:10.

group-reactive MAb to monitor the antigen preparation for adequate quantities of chlamydial elementary bodies. Each of the remaining six columns was used either as a negative control or for testing four dilutions of the appropriate MAbs. The chlamydial antigen used in the IMIF test was from either yolk sac or tissue culture harvest. The tissue culture harvest material was prepared from infected Vero cell monolayers. The tissue culture media, along with the disrupted tissue culture monolayer, were thoroughly mixed with a vortex mixer. The tissue culture harvest was mixed with a normal 20% yolk sac preparation at a ratio of 3:1. Yolk sac harvests of chlamydiae were used as a 10% yolk sac preparation. Samples were further diluted in phosphate-buffered saline (pH 7.2) if they contained too much yolk sac material for proper testing. MAbs to 10 distinct serovars of C. psittaci were produced by the procedure reported earlier (3). The clones were selected for their ability to produce high levels of fluorescence with the homologous strain and no fluorescence with heterologous serovars. One serovar-specific MAb to each of the known 10 serovars was selected for use in the panel of MAbs. The 10 MAbs were tested for serovar specificity by the IMIF test. They were screened at dilutions of 1:10, 1:20, 1:40, and 1:80 with the homologous strains and with representatives of the other known serovars. Positive clones were retested with a twofold dilution to determine an endpoint. Serotyping. Fifty-five strains of C. psittaci were serotyped by the IMIF test as described above. Chlamydial antigen prepared as described above was dotted in the same pattern on two microscope slides. The first column of four wells was used for a group-reactive MAb. The remaining columns were used to test the sample with serovar-specific MAbs at dilutions of 1:100, 1:400, 1:1,600, and 1:6,400. A strain was considered to be a particular serotype if reactions were observed at 1:1,600 to 1:6,400 with one MAb and no reactions at 1:100 with the remaining serovar-specific MAbs. Isolates that reacted with the group-reactive MAb and not with any of the 10 serovar-specific MAbs were presumed to be members of a currently undefined serotype. RESULTS After MAbs from a number of fusions were tested for cross-reactions to different C. psittaci strains, a panel of 10 MAbs was selected to represent 10 serovars (Table 2). These MAbs were tested by the IMIF test for titers to each of 10 strains that were used to produce the MAbs (Table 2). The

results show that the MAbs are highly specific to the homologous strain, having titers of 1:1,280 or higher. Only two MAbs showed cross-reactions at 1:10. The MAb for the pigeon serovar (CP3/2A7) gave a weak reaction at 1:10 with the duck isolate (GD), and the MAb to the polyarthritis serovar (IPA/2G7) gave a reaction at 1:10 with the muskrat isolate (M56). The 10 MAbs were used at dilutions of 1:100, 1:400, 1:1,600, and 1:6,400 to serotype 55 avian and mammalian strains (Table 1). Fifty of the strains reacted to only one serovar-specific MAb in the panel. These reactions were at the 1:1,600 or 1:6,400 dilution, easily serotyping the strain. Five strains failed to react with any of the serotype-specific MAbs, even though chlamydia was detected with the groupreactive MAb used as a control. These are presumed to be members of new serovars. DISCUSSION The use of serovar-specific MAbs in the IMIF test provides a rapid and highly specific method to serotype isolates of C. psittaci. Serotyping could be performed with small quantities of crude antigen grown in either tissue culture or eggs. The MAbs, when used at dilutions of 1:100, 1:400, 1:1,600, and 1:6,400, provided clear differentiation of the strains into different serovars. Each strain reacted with only one serovar-specific MAb at the 1:1,600 or higher dilution. No cross-reactions were observed even at the 1:100 dilution of the MAbs. The group-reactive MAb serves as a reliable control which demonstrates that adequate antigen is present for serotyping when isolates which fail to react with the serovar-specific MAbs are found. An isolate that fails to react can be assumed to be a member of an additional serovar; a serovar-specific MAb to it can readily be produced and added to the testing format. The panel of 10 MAbs used in the IMIF test serologically differentiated 50 of the 55 strains into 10 serological groups. The strains were grouped as four avian isolates (psittacine, pigeon, turkey, and duck) and six mammalian isolates (abortion [type I], polyarthritis [type II], feline pneumonitis, guinea pig inclusion [GPIC], muskrat [M56], and Wolfsen cattle [WC]). The results are in close agreement with other studies on the classification of C. psittaci. Examination of serological data from these laboratories indicates that our serotyping corresponds with that in previous reports (5, 8, 9, 11-14, 31, 33, 34, 36, 44). For example, the abortion and polyarthritis serovars correlate with biotypes and immunotypes 1 and 2



reported earlier (31, 36). The results with feline pneumonitis and GPIC isolates are in agreement with those in earlier studies (13, 31, 36). The divisions of the avian strains are more difficult to compare, as laboratories often use different strains. However, reports have shown differences in virulence among avian strains, and the highly virulent strains correspond with the turkey serovar in this study (44). Others have also reported on differences between the psittacine and pigeon strains (5). On the other hand, our current panel does not include some identified serovars, including serovars from swine, sheep, and cattle (31). The distribution of the hosts from which the strains were obtained clearly indicates that a relationship exists between the serovar of the strain and the species of the host. However, the natural relationship between serovars and hosts cannot be determined from our strains, as our collection includes strains submitted to us because of the unusual nature of the disease outbreak. The distribution of our strains among the serovars also is influenced by the perceived importance of chlamydiae in a given species, the ease of growing a given strain, and the emphasis in our laboratory over the past 25 years on avian species. Five of the strains failed to react with any of the serovarspecific MAbs and are presumed to be members of additional serovars. These were from both avian and mammalian sources and are expected to represent two or more new serovars. We are currently seeking to determine the identities of these strains and will be producing MAbs to those representing new serovars. These MAbs will then be added to our panel of MAbs for serotyping. REFERENCES 1. Andersen, A. A. 1989. The use of serovar-specific monoclonal antibodies for the serotyping of Chlamydia psittaci isolates, p. 374-377. In W. R. Bowie, H. D. Caldwell, R. P. Jones, P.-A. Mardh, G. L. Ridgway, J. Schachter, W. E. Stamm, and M. E. Ward (ed.). Chlamydial infections: proceedings of the Seventh International Symposium on Human Infections. Cambridge University Press, Cambridge. 2. Andersen, A. A. 1991. Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies. J. Clin. Microbiol. 29:244-249. 3. Andersen, A. A., and R. A. Van Deusen. 1988. Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates. Infect. Immun. 56:2075-2079. 4. Baker, J. A. 1944. A virus causing pneumonia in cats and producing elementary bodies. J. Exp. Med. 79:159-172. 5. Banks, J., B. Eddie, M. Sung, N. Sugg, J. Schachter, and K. R. Meyer. 1970. Plaque reduction technique for demonstrating neutralizing antibodies for chlamydia. Infect. Immun. 2:443447. 6. Cello, R. M. 1967. Ocular infections with PLT (Bedsovia) group agents. Am. J. Ophthalmol. 63:1270-1273. 7. Davis, D. J. 1949. Use of phenolized allantoic fluid antigen in complement fixation test for psittacosis. J. Immunol. 62:193200. 8. Eb, F., and J. Orfila. 1982. Serotyping of Chlamydia psittaci by the micro-immunofluorescence test: isolates of ovine origin. Infect. Immun. 37:1289-1291. 9. Eb, F., J. Orfila, A. Milon, and M. F. Geral. 1986. Interet epidemiologique du typage par immunofluorescence de Chlamydia psittaci. Ann. Inst. Pasteur/Microbiol. 137B:77-93. 10. Francis, T., Jr., and T. P. Magill. 1938. An unidentified virus producing acute meningitis and pneumonia in experimental animals. J. Exp. Med. 68:147-160. 11. Frazer, D. E. O., and D. T. Berman. 1965. Type-specific antigens in the psittacosis-lymphogranuloma venereum group of organisms. J. Bacteriol. 89:943-948. 12. Fukushi, H., and K. Hirai. 1988. Immunochemical diversity of the major outer membrane protein of avian and mammalian


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Serotyping of Chlamydia psittaci isolates using serovar-specific monoclonal antibodies with the microimmunofluorescence test.

A panel of 10 serovar-specific monoclonal antibodies that could distinguish 10 distinct serovars of Chlamydia psittaci was prepared. The panel include...
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