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Antigenic diversity of ruminant Chlamydia psittaci strains demonstrated by the indirect microimmunofluorescence test with monoclonal antibodies
veterinary microbiology Veterinary Microbiology 43 ( 1995) 2 19-226
Antigenic diversity of ruminant Chlamydia psittaci strains demonstrated by the indirect microimmunofluorescence test with monoclonal antibodies Jesus Salinas a,b, Arrnel Souriau b, Francisco Cue110 a, Annie Rodolakis b,* ’ Dept Patologia Animal Facultad Veterinaria Universidad de Murcia, Campus de Espinardo Murcia, Spain ’ Pathologic Infectieuse et Immunologic INRA Nouzilly 37380, France
Received 5 February 1993; accepted 14 July 1994
Abstract Monoclonal antibodies (mAbs) were produced to find strain markers essential to the epidemiological study of chlamydial abortion of ruminants. Their specificity was tested on 53 C. psittuci strains including 35 ruminant strains isolated mainly from abortion, belonging to serotype 1 and which are invasive in our mouse model (Rodolakis et al., 1989), and 14 ruminant strains mostly intestinal, belonging to serotype 2 and non-invasive for mouse. One strain specific mAb was obtained reacting only with the non-invasive strain iB 1. Six sub serotype 2 mAbs were found. They reacted only with some non-invasive strains. They allowed the distinction of 9 patterns of response among the 14 noninvasive strains. No serotype 2 specific mAbs reacting with all non-invasive serotype 2 strains were selected. In return all the invasive strains reacted with all the 18 serotype 1 specific mAbs produced. No cross-reactivities between invasive and non-invasive strains were observed, whereas common epitopes were demonstrated between invasive strains and avian or feline strains. Keyvords:
Chlamydia psittaci; Antigenic diversity; Microimmunofluorescence test
1. Introduction Chlamydia are obligatory intracellular other bacteria by a unique developmental
gram negative bacteria that are differentiated from cycle. They share a common group antigen. They
* Corresponding author. tel (33) 47 42 78 62, FAX (33) 47 42 77 79 0378-1135/95/$09.50 0 1995 Elsevier Science I3.V. All rights reserved .SSDIO378-1135(94)00100-6
220
J. Salinas et al. /Veterinary
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43 (1995) 219-226
infect primates including man, domestic and wild mammals, birds and amphibians. Chlumydia psittuci is an important pathogen in domestic ruminants. In many countries it induces abortion in cattle, goats and sheep. It also contributes to respiratory disease, enteritis, conjunctivitis, arthritis and reproductive disorders, but latent infections such as intestinal colonization without clinical sign are quite frequent. The part played by different strains in the epidemiology of chlamydia-induced abortions is still unclear for lack of strain markers to monitor their distribution and transmission in nature. Diversity among ruminant isolates has been demonstrated by examination of growth characteristics and inclusion morphology in cell cultures (Spears and Storz, 1979a, b), analysis of mouse virulence (Buzoni-Gate1 and Rodolakis, 1983; Anderson, 1986; Rodolakis et al., 1989), protein patterns (Buzoni-Gate1 et al., 1989; McClenaghan et al., 1991; Griffiths et al., 1992; Layachi et al., 1993), serological tests (Schachter et al., 1974, 1975; Eb and Orfila, 1982; Perez-Martinez and Storz, 1985; Delong and Magee, 1986; Andersen and Van Deusen, 1988; Souriau et al., 1993) by restriction endonuclease analysis of total DNA (McClenaghan et al., 1984; Timms et al., 1988; Fukushi and Hirai, 1989; Fukushi and Hirai, 1992; Rodolakis and Souriau, 1992) or of amplified gene (Denamur et al., 1991; Kaltenboeck et al., 1992). More recent DNA-DNA homology studies on a small number of strains (Fukushi and Hirai, 1992) shown sufficient divergence between the DNA of ruminant strains to suggest the creation of a new species Chlamydia pecorum. Focusing our interest on ruminant strains we have shown that these strains could be split into 2 groups of strains according to their virulence for mice (Rodolakis et al., 1989) : one group - the mouse invasive strains - induces splenomegaly and splenic infection after the subcutaneous inoculation of mice in the footpad. Mouse invasive strains are mainly isolated from symptomatic infections and belong to serotype 1 (Souriau et al., 1994) ; the second group - the mouse non-invasive strains - mostly intestinal strains, neither induces a change in spleen weight nor a splenic infection. Mouse non-invasive strains belong to serotype 2. This mouse model for evaluation of virulence of strains is not adequate for epidemiological studies because it is too laborious. In return monoclonal antibodies or specific DNA probes could provide strain markers which could be easily used to test many isolates. For this reason, to identify such strain markers we produced monoclonal antibodies (mAbs) against a mouse invasive strain, abortion strain C. psittuci AB7 and a mouse noninvasive strain, intestinal strain C. psittuci iB 1 and used them for typing ruminant strains in the micro-immunofluorescence (MIF) test.
2. Materials and methods
2.1. Chlamydia strains Three C. trachomatis and 53 C. psittuci strains (Table 1) including 38 strains isolated from sheep, 8 strains from goats, one strain from cattle, 2 thermosensitive mutants of ovine abortion strain AB7 of C. psittaci, 2 strains from cats and 2 strains from birds were used in this study.
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43 (1995) 219-226
Table I Strains used in this study Strains Ovine abortion: ABl, AB2, AB3, AB4. AB5, AB6, ABS, AB9, ABII. AB15 AB7 ABIO
Ql8 A22, S26/3, HI54 PS22 A03, AOS, A01 Caprine abortion: AC 1,AC2, AC3 Cb4 Bovine abortion: AVI Thermosensitive mutants of AB7 strain: 1B. IH Ovine epididymitis: VBl Ovine pneumonitis: 109-75 Ovine Arthritis: 1H77 LW679 Ovine conjunctivitis: OCI 824 Feces of healthy sheep: iBI. iB2, iB3, iB4, iB5, iB6 w73 R69 Mo907 Feces of healthy goats: ic1 iC2, iC3. iC4 Avian strains: L2A PG.5 Feline strains: FEPN Cat-2 Chlamydia rrachomatis serotypes: Ll L2 D
Geographic _ . AB12, AB13, AB14,
origin
Serotype”
Franceb
1
France’ Franceb Franced Scotland USA’ Spaina
1 2 1 1 1 1
Franceb Spain8
1 1
Franceb
1
France”
1
Franceb
1
France”
1
Scotland’ USA
1 2
Franceb Scotland’
1 2
Franceb Ulster’ Ulster’ USAk
2 2 2
Franceb Franceb
1 2
Franceb Spaina
_
USA’ Spaina
_
Greece” France” Greece”
Ll L2 D
“Souriau et al., 1993. ‘Isolated at the Pathologie Infectieuse et Immunologie from different areas in France. ‘Isolated by Faye, Ecole Nationale Veterinaire Alfort, France. dIsolated by P. Giroud, Institut Pasteur Paris, France. “Isolated at the Moredum Institute Edimburg, Scotland. ‘Isolated by J. Schachter, G.P. Hooper Foundation University of California, San Francisco, eIsolated an the Facultad de Veterlnaria de Murcia from different areas in Spain. “Isolated by P. Russo, CNEVA Sophia Antipolis, France. ‘Isolated by Nortom and Storz ( 1967). ‘Isolated by M.S. McNulty, Veterinary Research Laboratory Stormont Belfast, Ulster. *From Storz ( 1963). ‘Live vaccine, strain Baker from Solvay Sante Animale Tours, France. mIsolated at the Institute Pasteur Athens, Greece. “Isolated by F. Catalan, Institut Foumier Paris, France.
I
USA.
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2.2. Monoclonal
.I. Salinas et al. / Veterinary Microbiology 43 (1995) 219-226
antibodies
Female Balb/c mice (3 to 4 weeks of age) were immunized intraperitoneally by two injections of live chlamydia ( lo6 plaque forming units (PFU) of ovine abortion strain AB7 of C. psittaci and 5 X lo6 PFU for intestinal ovine C. psittaci strain iB1) at an interval of 30 days. Three days after the last injection, spleen cells and myeloma cells (P-X-63 Ag 8613) were fused by using 50% polyethylene glycol 1500 (Boerhing, Germany) by the methods of Kohler and Milstein ( 1975). Antibody production of hybridoma cells surviving in RPMI- 1640 (Gibco, Scotland) supplemented with 10% horse serum (Gibco, Scotland), hypoxanthine ( 100 FM), aminopterine (0.4 PM) and thymidine ( 16 PM) (Sigma, USA) was tested in AB7 strain antigen-specific MIF tests described by Wang and Grayston ( 1970). Antibody producing hybridomas were cloned by the limiting dilution technique. Ascitic mAbs were raised in Freund’s incomplete adjuvant primed Balb/c mice. 2.3. MIF A modified procedure of the MIF test of Wang and Grayston ( 1970) was used. Briefly small amounts of each yolk sac propagated chlamydia strains, and normal yolk sac as control, were spotted on a 24-well multitest slide (CML Nemours, France) in groups of 14 small dots per well. The slides were dried for 30 min at 37°C and fixed in acetone at room temperature for 20 min, dried again and stored at - 20°C until required. The thawed slides were incubated for 30 min at 37°C with 25 ~1 of dilutions of ascitic fluid (from 1: 10’ to 1: 105) before being washed once in deionized water and twice for 10 min in 0.15M saline solution. Rabbit anti-mouse immunoglobulin G and M fluorescein isothiocyanate conjugated (Pasteur Diagnostic, France) with 0.01% Evans blue, at a predetermined working dilution ( 1 / 100) was added to each well. The slides were incubated 30 min at 37°C in the dark and washed before being examined with a Leitz Aristoplan Fluorescent microscope. The highest dilution of mAbs which showed specific fluorescence was considered as the end point.
3. Results Among the 39 mAbs obtained a representative panel of 18 mAbs selected by MIF was used to test all the strains in our collection. Their specificities were the following: 1 mAb was genus specific, 7 mAbs were subspecies specific, 3 mAbs were serotype 1 specific (all the 18 obtained serotype 1 specific mAbs gave the same patterns of response in MIF) and 7 mAbs were subserotype 2 specific. The 49 ruminant strains of C. psittaci were split into 2 groups by the serotype 1 specific mAbs: all the 35 serotype 1 strains reacted with them whereas none of the serotype 2 did (Table 2). No differences were observed between the strains of serotype 1. With the 7 anti-iB1 mAbs, 9 different reaction patterns in MIF test could be observed (Table 3) : Only 1 strain, the homologous iB 1 strain reacted with the 7 mAbs with high titers. One strain, the iB2 strain reacted with 4 mAbs, and only 1 other, the R69 strain reacted with 3 mAbs. Four strains showed reaction with 2 mAbs and three strains reacted
J. Salinas et al. /Veterinary Table 2 MIF titers of anti-AB7 monoclonal mAbs
M. Weight’
Microbiology
antibodies against several chlamydial
Hotyped
Chlamydial
IgGl
Other strains
SZb
L2A
PG5
FEPN
Cat-2
Ll ,L2,D
5’
5
4
4
5
4
5
-
_
3
_
_
-
_ _
3 3
-
_
4 3
_ -
4 4
3 3
_ _
_
4 4 3
_ _ _
3 3 4
4 4
-
4 3 _ _ _
_ _ _
IgG2a
5
_a
IgG2a IgG2a
4 5
_ -
IgG2a IgG2a
4 4
FClA2 LA2GlO AD2C9
IgG2a IgG2a IgG2a
4 4 4
IgG2a IgG2a
4 4
CB3C8 KD3E4
-
“35 strains of serotype 1. b14 strains of serotype 2. ‘Molecular weight in kDa (Soutiau et al., 1994). %ouriau et al.. 1994. ‘Log 10 of the reciprocals of the highest dilution producing ‘No reaction in immunoblotting. “No reaction to l/ 10 dilution.
Table 3 MIF titers of anti-i8 1 monoclonal mAbs
SD5B5 PC6G8 PD3 PD4 TDlG7&TB3B9 OB6E7
M. Weight”
c 52 43 :::
c. trachomatis
Sl”
IC4C7 80-85-90 JC3A5 Subspecies-specific BA6H6 30 DA4E8 8&85-90 80-85-90 30 .._
strains
strains
Ruminant strains
Genus-specific AD5A8
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43 (1995) 219-226
3 4
a positive reaction.
antibodies against serotype 2 strains Isotypeb
1gM IgM IgGl IgM 1gM IgG2a
Strains iB1
iB2
R69
iB3 LW679
W73
824
iB5 iB6
ABlO
iB4 iC2 iC3 iC4
3d 4 4 4 4 4
3 3 4 -
3 3 3 -
3 3 -
3 _ 3 _ _
3 3 _
3 _
-e 3 _
_ _ _ _
“Molecular weight in kDa (as described by Souriau et al., 1994). %ouriau et al., 1994. ‘No reaction in immunoblotting. dLog 10 of the reciprocals of the highest dilution producing a positive reaction, ‘No reaction to l/ 10 dilution.
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only with one mAb. The 3 caprine strains iC2, iC3, iC4, and the intestinal ovine strain iB4 did not react with any mAbs. The 2 avian and the 2 feline strains gave 4 different reaction patterns (Table 2): PG 5 reacted only with genre specific mAbs whereas strain L2A reacted with all subspecies specific mAbs obtained. One of the feline strains, FEPN, reacted with 4 of the subspecies specific mAbs whereas the other Cat-2 gave a weak reaction with 3 serotype specific mAbs and the seven subspecies specific mAbs.
4. Discussion The 49 C. psitfaci strains isolated from ruminants were separated into 2 groups by the 18 monoclonal antibodies according to their mouse invasiveness and their serotype but regardless of their pathological origin. We did not find common epitopes between the 2 groups whereas there were common epitopes between invasive-serotype 1 strains and some avian or feline C. psittuci strains. Others, Kikuta et al. ( 1991) and Andersen ( 1991) found no cross-reaction between avian strain and ruminant abortion strain B577, but using DNADNA hybridization Cox et al. ( 1988) and Fukushi and Hirai ( 1989) found 80% homology between the avian strain 6BC and a ruminant abortion strain (B577 for Fukushi and Hirai and a local strain for Cox et al.) whereas there was only 1% homology between strain B577 and 3 other ruminant strains, the serotype of one of which (IPA = ATCC VR629) was known as serotype 2. Monoclonal antibodies raised against invasive-serotype 1 strain AB7 underlined the antigenic homogeneity of the serotype 1 and those obtained with non-invasive-serotype 2 strain iB 1 displayed the antigenic heterogeneity of the serotype 2. All the invasive strains presented exactly the same reaction patterns including the 3 Scottish strains A22, S26/3 and H574 which exhibited in vivo immunogenic variations (Rodolakis and Bernard, 1984). Our previous study on restriction fragments length polymorphism (RFLP) of total DNA (Rodolakis and Souriau, 1992) or of major outer membrane protein (MOMP) gene (Denamur et al., 1991) demonstrated the genetic homogeneity of invasive-serotype 1 strains whatever their geographical or pathological origin and the heterogeneity of non-invasiveserotype 2. The group of non-invasive-serotype 2 strains was so heterogeneous that we did not fmd a mAb which reacted with all the strains since 4 of them (one ovine and the 3 caprine strains) failed to react with any of the anti-iB1 mAbs, but SD5B5 was common to 9 strains. We are currently seeking the existence of a common antigen to non-invasiveserotype 2 strains and will be producing mAbs against ABlO, iB4 and iC4 strains which were the less recognized by anti-iB 1 mAbs. Nevertheless these results and those of RFLP of ompl suggest that it would be possible to identify each non-invasive strains by the combination of mAbs and DNA probes.
References Andersen, A.A., 199 1. Serotyping of Chlamydia psitfaci isolates using serovar-specific with the microimmunofiuorescence test. J. Clin. Microbial., 29: 707-711.
monoclonal
antibodies
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Andersen, A.A. and Van Deusen, R.A., 1988. Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates. Infect. Immun.. 56: 2075-2079. Anderson, LE., 1986. Comparison of the virulence in mice of some ovine isolates of Chlamydia psirtaci. Vet, Microbial., 12: 213-220. Buzoni-Gatel, D. and Rodolakis, A., 1983. A mouse model to compare virulence of abortive and intestinal ovine strains of Chlamydia psiffaci. Influence of the route of inoculation. Ann. Microbial. (Inst. Pasteur), 134A: 91-99. Buzoni-Gatel, D., Layachi, K., Dubray, G. and Rodolakis, A.. 1989. Comparison of protein patterns between invasive and non-invasive ovine strains of Chlamydia psiffaci. Res. Vet. Sci., 46: 4042. COX. R.L., Kuo, C.C., Grayston, J.T. and Campbell, L.A., 1988. Deoxyribonucleic acid relatedness of Chiamydia sp. strain TWAR to Chlamydia trachomatis and Chlamydia psittaci. Int. J. Syst. Bacterial., 38: 265-268. Drlong, W.J. and Magee, W.E.. 1986. Distinguishing between ovine abortion and ovine arthritis Chlamydia psitfaci isolates with specific monoclonal antibodies. Am. J. Vet. Res., 47: 1520-1523. Denamur, E.. Sayada, C., Souriau, A., Otlila, J., Rodolakis, A. and Elion, J., 1991. Restriction pattern of the outermembrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydiapsitfaci. J. Gen. Microbial., 137: 2525-2530. Eb, F. and Orfila, J., 1982. Serotyping of Chlamydia psitcaci by the micro-immunofluorescence test: isolates of ovine origin. Infect. Immun., 37: 1288-1291. Fukushi. H. and Hirai. K., 1989. Genetic diversity of avian and mammalian Chlamydia psittaci relation to host origin. J. Bacterial., 171: 2850-2855. Fukushi, H. and Hirai, K., 1992. Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants. Int. J. Syst. Bacterial., 42: 306-308. Griffiths, P.C., Philips, H.L.. Dawson, M. and Clarkson, M.J., 1992. Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydiapsiftaci of ovine origin. Vet. Microbial.. 30: 165-167. Kikuta, A.. Furukawa, N., Yoshida, T.. Fukushi, H., Yamaguchi, T. and Hirai, K., 1991. Antigenic analysis of avian Chlamydia psiftoci using monoclonal antibodies to the major outer membrane protein. J. Vet. Med. Sci., 53: 385-389. Kaltenboeck, B.. Kousoulas, K.G. and Storz, J., 1992. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate omp A DNA of Chlamydia spp. J. Clin. Microbial.. 30: 10981104. Kohler, G. and Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256: 495497. Layachi. K., Rodolakis, A., Buzoni-Gatel, D., 1993. Identification by Western blots of virulence specific antigens of C’hlamydiapsitraci isolated from ewes. Ann. Rech. Vet., 24: 55-65. McClenaghan. M.. Herring, A.J. and Aitken. I.D., 1984. Comparison of Chlamydia psittaci isolates by DNA restriction endonuclease analysis. Infect. Immun., 45: 384-389. McClenaghan. M., Inglis, N.F., Herring, A.J., 1991. Comparison of isolates of Chlamydia pshraci of ovine, avian and feline origin by analysis of polypeptide profiles from purified of elementary bodies. Vet. Microbial.. 26: 2699278. Perez-Martinez, J.A. and Storz, J., 1985. Antigenicdiversityof Chlamydiapsirtaciofmammalianorigindetermined by microimmunofluorescence. Infect. Immun., 50: 905-910. Rodolakis, A. and Bernard, F., 1984. Vaccination with temperature-sensitive mutant of Chlamydiapsittaci against enzootic abortion of ewes. Vet. Rec., 114: 193-194. Rodolakis, A., Bernard, F. and Lantier, F., 1989. Mouse models for evaluation of virulence of Chlamydia psittaci isolated from ruminants. Res. Vet. Sci., 46: 34-39. Rodolakis, A. and Souriau, A., 1992. Restriction endonuclease analysis of DNA from ruminant Chlamydiapsirtaci and its relation to mouse virulence. Vet. Microbial. 3 1: 263-271. Schachter, J.. Banks, J., Sugg, N., Sung, M., Storz, J. and Meyer, K.F.. 1974. Serotyping of Chlamydia: 1 Isolates of ovine origin. Infect. Immun., 9: 92-94. Schachter. J., Banks, J., Sugg, N.. Sung, M., Storz, J. and Meyer, K.F., 1975. Serotyping of Chlamydia: 11 Isolates of bovine origin. Infect. Immun., 11: 904-907. Somiau, A., Le Rouzic, E., Bernard, F. and Rodolakis, A., 1993. Differentiation of abortion and intestinal strains of Chlam_vdiapsiftaciisolated from ruminants by the microimmunofluorescence test, Vet. Rec. 132: 217-219.
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Souriau, A., Salinas, J., De Sa, C., Layachi, K. and Rodolakis. A., 1994 Identification of subspecies- and serotype l-specific epitopes on the 80- to 90-kilodalton protein region of Chlamydia psittaci that may be useful for diagnosis of chlamydial induced abortion. Am. J. Vet. Res. 55: 510-514. Spears, P. and Storz, J., 1979a. Biotyping of Chlamydia psittaci based on inclusion morphology and response to diethylaminoethyl-dextran and cycloheximide. Infect. Immun., 24: 224-232. Spears, P. and Storz, J., 1979b. Chlamydia psittaci: growth characteristics and enumeration of serotypes 1 and 2 in cultured cells. J. Infect. Dis., 140: 959-967. Timms, P.F., Eaves, W., Gijes, A.A. and Lavin, M.F., 1988. Comparison of Chlamydia psittuci isolates by restriction endonuclease and DNA probe analysis. Infect. Immun., 56: 287-290. Wang, S.P. and Grayston, J.T., 1970. Immunologic relationship between genital TRIC, lymphogranuloma venereum and related organisms in a new microtiter indirect immunofluorescence test. Am. J. Ophtalmol., 70: 367374.
Antigenic diversity of ruminant Chlamydia psittaci strains demonstrated by the indirect microimmunofluorescence test with monoclonal antibodies Jesus Salinas a,b, Arrnel Souriau b, Francisco Cue110 a, Annie Rodolakis b,* ’ Dept Patologia Animal Facultad Veterinaria Universidad de Murcia, Campus de Espinardo Murcia, Spain ’ Pathologic Infectieuse et Immunologic INRA Nouzilly 37380, France
Received 5 February 1993; accepted 14 July 1994
Abstract Monoclonal antibodies (mAbs) were produced to find strain markers essential to the epidemiological study of chlamydial abortion of ruminants. Their specificity was tested on 53 C. psittuci strains including 35 ruminant strains isolated mainly from abortion, belonging to serotype 1 and which are invasive in our mouse model (Rodolakis et al., 1989), and 14 ruminant strains mostly intestinal, belonging to serotype 2 and non-invasive for mouse. One strain specific mAb was obtained reacting only with the non-invasive strain iB 1. Six sub serotype 2 mAbs were found. They reacted only with some non-invasive strains. They allowed the distinction of 9 patterns of response among the 14 noninvasive strains. No serotype 2 specific mAbs reacting with all non-invasive serotype 2 strains were selected. In return all the invasive strains reacted with all the 18 serotype 1 specific mAbs produced. No cross-reactivities between invasive and non-invasive strains were observed, whereas common epitopes were demonstrated between invasive strains and avian or feline strains. Keyvords:
Chlamydia psittaci; Antigenic diversity; Microimmunofluorescence test
1. Introduction Chlamydia are obligatory intracellular other bacteria by a unique developmental
gram negative bacteria that are differentiated from cycle. They share a common group antigen. They
* Corresponding author. tel (33) 47 42 78 62, FAX (33) 47 42 77 79 0378-1135/95/$09.50 0 1995 Elsevier Science I3.V. All rights reserved .SSDIO378-1135(94)00100-6
220
J. Salinas et al. /Veterinary
Microbiology
43 (1995) 219-226
infect primates including man, domestic and wild mammals, birds and amphibians. Chlumydia psittuci is an important pathogen in domestic ruminants. In many countries it induces abortion in cattle, goats and sheep. It also contributes to respiratory disease, enteritis, conjunctivitis, arthritis and reproductive disorders, but latent infections such as intestinal colonization without clinical sign are quite frequent. The part played by different strains in the epidemiology of chlamydia-induced abortions is still unclear for lack of strain markers to monitor their distribution and transmission in nature. Diversity among ruminant isolates has been demonstrated by examination of growth characteristics and inclusion morphology in cell cultures (Spears and Storz, 1979a, b), analysis of mouse virulence (Buzoni-Gate1 and Rodolakis, 1983; Anderson, 1986; Rodolakis et al., 1989), protein patterns (Buzoni-Gate1 et al., 1989; McClenaghan et al., 1991; Griffiths et al., 1992; Layachi et al., 1993), serological tests (Schachter et al., 1974, 1975; Eb and Orfila, 1982; Perez-Martinez and Storz, 1985; Delong and Magee, 1986; Andersen and Van Deusen, 1988; Souriau et al., 1993) by restriction endonuclease analysis of total DNA (McClenaghan et al., 1984; Timms et al., 1988; Fukushi and Hirai, 1989; Fukushi and Hirai, 1992; Rodolakis and Souriau, 1992) or of amplified gene (Denamur et al., 1991; Kaltenboeck et al., 1992). More recent DNA-DNA homology studies on a small number of strains (Fukushi and Hirai, 1992) shown sufficient divergence between the DNA of ruminant strains to suggest the creation of a new species Chlamydia pecorum. Focusing our interest on ruminant strains we have shown that these strains could be split into 2 groups of strains according to their virulence for mice (Rodolakis et al., 1989) : one group - the mouse invasive strains - induces splenomegaly and splenic infection after the subcutaneous inoculation of mice in the footpad. Mouse invasive strains are mainly isolated from symptomatic infections and belong to serotype 1 (Souriau et al., 1994) ; the second group - the mouse non-invasive strains - mostly intestinal strains, neither induces a change in spleen weight nor a splenic infection. Mouse non-invasive strains belong to serotype 2. This mouse model for evaluation of virulence of strains is not adequate for epidemiological studies because it is too laborious. In return monoclonal antibodies or specific DNA probes could provide strain markers which could be easily used to test many isolates. For this reason, to identify such strain markers we produced monoclonal antibodies (mAbs) against a mouse invasive strain, abortion strain C. psittuci AB7 and a mouse noninvasive strain, intestinal strain C. psittuci iB 1 and used them for typing ruminant strains in the micro-immunofluorescence (MIF) test.
2. Materials and methods
2.1. Chlamydia strains Three C. trachomatis and 53 C. psittuci strains (Table 1) including 38 strains isolated from sheep, 8 strains from goats, one strain from cattle, 2 thermosensitive mutants of ovine abortion strain AB7 of C. psittaci, 2 strains from cats and 2 strains from birds were used in this study.
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Table I Strains used in this study Strains Ovine abortion: ABl, AB2, AB3, AB4. AB5, AB6, ABS, AB9, ABII. AB15 AB7 ABIO
Ql8 A22, S26/3, HI54 PS22 A03, AOS, A01 Caprine abortion: AC 1,AC2, AC3 Cb4 Bovine abortion: AVI Thermosensitive mutants of AB7 strain: 1B. IH Ovine epididymitis: VBl Ovine pneumonitis: 109-75 Ovine Arthritis: 1H77 LW679 Ovine conjunctivitis: OCI 824 Feces of healthy sheep: iBI. iB2, iB3, iB4, iB5, iB6 w73 R69 Mo907 Feces of healthy goats: ic1 iC2, iC3. iC4 Avian strains: L2A PG.5 Feline strains: FEPN Cat-2 Chlamydia rrachomatis serotypes: Ll L2 D
Geographic _ . AB12, AB13, AB14,
origin
Serotype”
Franceb
1
France’ Franceb Franced Scotland USA’ Spaina
1 2 1 1 1 1
Franceb Spain8
1 1
Franceb
1
France”
1
Franceb
1
France”
1
Scotland’ USA
1 2
Franceb Scotland’
1 2
Franceb Ulster’ Ulster’ USAk
2 2 2
Franceb Franceb
1 2
Franceb Spaina
_
USA’ Spaina
_
Greece” France” Greece”
Ll L2 D
“Souriau et al., 1993. ‘Isolated at the Pathologie Infectieuse et Immunologie from different areas in France. ‘Isolated by Faye, Ecole Nationale Veterinaire Alfort, France. dIsolated by P. Giroud, Institut Pasteur Paris, France. “Isolated at the Moredum Institute Edimburg, Scotland. ‘Isolated by J. Schachter, G.P. Hooper Foundation University of California, San Francisco, eIsolated an the Facultad de Veterlnaria de Murcia from different areas in Spain. “Isolated by P. Russo, CNEVA Sophia Antipolis, France. ‘Isolated by Nortom and Storz ( 1967). ‘Isolated by M.S. McNulty, Veterinary Research Laboratory Stormont Belfast, Ulster. *From Storz ( 1963). ‘Live vaccine, strain Baker from Solvay Sante Animale Tours, France. mIsolated at the Institute Pasteur Athens, Greece. “Isolated by F. Catalan, Institut Foumier Paris, France.
I
USA.
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2.2. Monoclonal
.I. Salinas et al. / Veterinary Microbiology 43 (1995) 219-226
antibodies
Female Balb/c mice (3 to 4 weeks of age) were immunized intraperitoneally by two injections of live chlamydia ( lo6 plaque forming units (PFU) of ovine abortion strain AB7 of C. psittaci and 5 X lo6 PFU for intestinal ovine C. psittaci strain iB1) at an interval of 30 days. Three days after the last injection, spleen cells and myeloma cells (P-X-63 Ag 8613) were fused by using 50% polyethylene glycol 1500 (Boerhing, Germany) by the methods of Kohler and Milstein ( 1975). Antibody production of hybridoma cells surviving in RPMI- 1640 (Gibco, Scotland) supplemented with 10% horse serum (Gibco, Scotland), hypoxanthine ( 100 FM), aminopterine (0.4 PM) and thymidine ( 16 PM) (Sigma, USA) was tested in AB7 strain antigen-specific MIF tests described by Wang and Grayston ( 1970). Antibody producing hybridomas were cloned by the limiting dilution technique. Ascitic mAbs were raised in Freund’s incomplete adjuvant primed Balb/c mice. 2.3. MIF A modified procedure of the MIF test of Wang and Grayston ( 1970) was used. Briefly small amounts of each yolk sac propagated chlamydia strains, and normal yolk sac as control, were spotted on a 24-well multitest slide (CML Nemours, France) in groups of 14 small dots per well. The slides were dried for 30 min at 37°C and fixed in acetone at room temperature for 20 min, dried again and stored at - 20°C until required. The thawed slides were incubated for 30 min at 37°C with 25 ~1 of dilutions of ascitic fluid (from 1: 10’ to 1: 105) before being washed once in deionized water and twice for 10 min in 0.15M saline solution. Rabbit anti-mouse immunoglobulin G and M fluorescein isothiocyanate conjugated (Pasteur Diagnostic, France) with 0.01% Evans blue, at a predetermined working dilution ( 1 / 100) was added to each well. The slides were incubated 30 min at 37°C in the dark and washed before being examined with a Leitz Aristoplan Fluorescent microscope. The highest dilution of mAbs which showed specific fluorescence was considered as the end point.
3. Results Among the 39 mAbs obtained a representative panel of 18 mAbs selected by MIF was used to test all the strains in our collection. Their specificities were the following: 1 mAb was genus specific, 7 mAbs were subspecies specific, 3 mAbs were serotype 1 specific (all the 18 obtained serotype 1 specific mAbs gave the same patterns of response in MIF) and 7 mAbs were subserotype 2 specific. The 49 ruminant strains of C. psittaci were split into 2 groups by the serotype 1 specific mAbs: all the 35 serotype 1 strains reacted with them whereas none of the serotype 2 did (Table 2). No differences were observed between the strains of serotype 1. With the 7 anti-iB1 mAbs, 9 different reaction patterns in MIF test could be observed (Table 3) : Only 1 strain, the homologous iB 1 strain reacted with the 7 mAbs with high titers. One strain, the iB2 strain reacted with 4 mAbs, and only 1 other, the R69 strain reacted with 3 mAbs. Four strains showed reaction with 2 mAbs and three strains reacted
J. Salinas et al. /Veterinary Table 2 MIF titers of anti-AB7 monoclonal mAbs
M. Weight’
Microbiology
antibodies against several chlamydial
Hotyped
Chlamydial
IgGl
Other strains
SZb
L2A
PG5
FEPN
Cat-2
Ll ,L2,D
5’
5
4
4
5
4
5
-
_
3
_
_
-
_ _
3 3
-
_
4 3
_ -
4 4
3 3
_ _
_
4 4 3
_ _ _
3 3 4
4 4
-
4 3 _ _ _
_ _ _
IgG2a
5
_a
IgG2a IgG2a
4 5
_ -
IgG2a IgG2a
4 4
FClA2 LA2GlO AD2C9
IgG2a IgG2a IgG2a
4 4 4
IgG2a IgG2a
4 4
CB3C8 KD3E4
-
“35 strains of serotype 1. b14 strains of serotype 2. ‘Molecular weight in kDa (Soutiau et al., 1994). %ouriau et al.. 1994. ‘Log 10 of the reciprocals of the highest dilution producing ‘No reaction in immunoblotting. “No reaction to l/ 10 dilution.
Table 3 MIF titers of anti-i8 1 monoclonal mAbs
SD5B5 PC6G8 PD3 PD4 TDlG7&TB3B9 OB6E7
M. Weight”
c 52 43 :::
c. trachomatis
Sl”
IC4C7 80-85-90 JC3A5 Subspecies-specific BA6H6 30 DA4E8 8&85-90 80-85-90 30 .._
strains
strains
Ruminant strains
Genus-specific AD5A8
223
43 (1995) 219-226
3 4
a positive reaction.
antibodies against serotype 2 strains Isotypeb
1gM IgM IgGl IgM 1gM IgG2a
Strains iB1
iB2
R69
iB3 LW679
W73
824
iB5 iB6
ABlO
iB4 iC2 iC3 iC4
3d 4 4 4 4 4
3 3 4 -
3 3 3 -
3 3 -
3 _ 3 _ _
3 3 _
3 _
-e 3 _
_ _ _ _
“Molecular weight in kDa (as described by Souriau et al., 1994). %ouriau et al., 1994. ‘No reaction in immunoblotting. dLog 10 of the reciprocals of the highest dilution producing a positive reaction, ‘No reaction to l/ 10 dilution.
224
J. Satinas et al. / Veterinary Microbiotogy 43 (1995) 219-226
only with one mAb. The 3 caprine strains iC2, iC3, iC4, and the intestinal ovine strain iB4 did not react with any mAbs. The 2 avian and the 2 feline strains gave 4 different reaction patterns (Table 2): PG 5 reacted only with genre specific mAbs whereas strain L2A reacted with all subspecies specific mAbs obtained. One of the feline strains, FEPN, reacted with 4 of the subspecies specific mAbs whereas the other Cat-2 gave a weak reaction with 3 serotype specific mAbs and the seven subspecies specific mAbs.
4. Discussion The 49 C. psitfaci strains isolated from ruminants were separated into 2 groups by the 18 monoclonal antibodies according to their mouse invasiveness and their serotype but regardless of their pathological origin. We did not find common epitopes between the 2 groups whereas there were common epitopes between invasive-serotype 1 strains and some avian or feline C. psittuci strains. Others, Kikuta et al. ( 1991) and Andersen ( 1991) found no cross-reaction between avian strain and ruminant abortion strain B577, but using DNADNA hybridization Cox et al. ( 1988) and Fukushi and Hirai ( 1989) found 80% homology between the avian strain 6BC and a ruminant abortion strain (B577 for Fukushi and Hirai and a local strain for Cox et al.) whereas there was only 1% homology between strain B577 and 3 other ruminant strains, the serotype of one of which (IPA = ATCC VR629) was known as serotype 2. Monoclonal antibodies raised against invasive-serotype 1 strain AB7 underlined the antigenic homogeneity of the serotype 1 and those obtained with non-invasive-serotype 2 strain iB 1 displayed the antigenic heterogeneity of the serotype 2. All the invasive strains presented exactly the same reaction patterns including the 3 Scottish strains A22, S26/3 and H574 which exhibited in vivo immunogenic variations (Rodolakis and Bernard, 1984). Our previous study on restriction fragments length polymorphism (RFLP) of total DNA (Rodolakis and Souriau, 1992) or of major outer membrane protein (MOMP) gene (Denamur et al., 1991) demonstrated the genetic homogeneity of invasive-serotype 1 strains whatever their geographical or pathological origin and the heterogeneity of non-invasiveserotype 2. The group of non-invasive-serotype 2 strains was so heterogeneous that we did not fmd a mAb which reacted with all the strains since 4 of them (one ovine and the 3 caprine strains) failed to react with any of the anti-iB1 mAbs, but SD5B5 was common to 9 strains. We are currently seeking the existence of a common antigen to non-invasiveserotype 2 strains and will be producing mAbs against ABlO, iB4 and iC4 strains which were the less recognized by anti-iB 1 mAbs. Nevertheless these results and those of RFLP of ompl suggest that it would be possible to identify each non-invasive strains by the combination of mAbs and DNA probes.
References Andersen, A.A., 199 1. Serotyping of Chlamydia psitfaci isolates using serovar-specific with the microimmunofiuorescence test. J. Clin. Microbial., 29: 707-711.
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