Veterinary Microbiology, 31 ( 1992 ) 263-271 Elsevier Science Publishers B,V., Amsterdam
263
Restriction endonuclease analysis of D N A from ruminant Chlamydia psittaci and its relation to mouse virulence Annie Rodolakis and Armel Souriau INRA, DOpartement de Pathologie Animale, Laboratoire de Pathologie Infectieuse et Immunologie, 37380 Nouzilly France (Accepted 24 September 1991 )
ABSTRACT Rodolakis, A. and Souriau, A., 1992. Restriction endonuclease analysis of DNA from ruminant Chlamydia psittaci and its relation to mouse virulence. Vet. Microbiol., 31 : 263-271. DNA from 20 pathogenic or non-pathogenic ruminant strains of Chlamydia psittaci was compared by restriction endonuclease analysis. The strains could be easily differentiated according to their invasiveness for mouse, whatever their pathological origin. DNA patterns of invasive strains were similar, whereas those of non-invasive strains were distributed in two groups.
INTRODUCTION
Chlamydiae are Gram-negative obligate intracellular bacteria. They are characterized by a unique developmental cycle which serves as the basis for their current taxonomic classification. The genus Chlamydia is subdivided into three species, namely: C. trachomatis, C. pneumoniae and C. psittaci (Grayston et al. 1989). C. trachomatis and C. pneumoniae infect mainly humans. For this reason, they have been studied extensively. The classification of C. trachomatis in 15 distinct serovars (Grayston et al. 1975 ) proved to be very useful and a correlation between serovars and virulence has been described (Ito et al. 1990). In contrast the typing of C. psittaci which infects a variety of mammals and birds and exhibits broad pathogenic potential (Storz 1971 ), is less well understood. Mammalian strains of C. psittaci may be separated into nine serovars (Perez-Martinez and Storz 1985). Strains of serovar 1 are principally associated with intestinal infection or abortion, while strains of serovar 2 are isolated from pneumonitis, polyarthritis and conjunctivitis in ruminants (Eb and Orfila 1982; Perez-Martinez and Storz 1985; Schachter et al. 1974; Schachter et al., 1975; Spears and Storz 1979a, b. ) This classification does not distinguish abortion strains from intestinal strains, a 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
264
A. RODOLAKIS AND A. SOURIAU
distinction that is essential for a better understanding of the epidemiology of ovine chlamydial abortion. We have developed a murine model of virulence to distinguish these two categories of strains (Rodolakis et al. 1989). Mouse invasive strains, most often isolated from clinical diseases (abortion, pneumonitis, conjunctivitis, and arthritis), may be recovered from the spleen of mice following subcutaneous inoculation, whereas this does not occur with non-invasive strains, mostly of intestinal origin, treated similarily. But the usefulness of this method remains limited because it is very laborious. Restriction endonuclease analysis (REA) of DNA has proved to be a valuable means of typing in numerous bacterial species particularly in C. trachomatis (Peterson and de la Maza, 1988), C. pneumoniae (Campbell et al. 1987) and in some C. psittaci according to host origin (Fukushi and Hirai 1989; Herring et al., 1987; McClenaghan et al. 1984; Timms et al., 1988). The purpose of this study was to compare the REA of ruminant strains of C. psittaci isolated either from diseased tissues or from feces of healthy animals. MATERIALS AND METHODS
Chlamydial strains Besides our two laboratory reference strains AB7 (from abortion, a mouse invasive strain ) and iB 1 (from feces, a mouse non-invasive strain ), 18 strains of C. psinaci (Table 1) and 1 strain of C. trachomatis ( C. trachomatis L~ pF2) were used. Their geographic origin was described in a previous paper (Rodolakis et al., 1989 ), All the strains were propagated in Mycoplasma-free McCoy cells in plastic flasks ( 150 cm2), 4 and 12 flasks were used for mouse invasive and non-invasive strains respectively. Cell monolayers were infected with about 107 plaque forming units (pfu) (Banks et al. 1970) of yolk sacpropagated chlamydiae and incubated at 37 °C. Seven days after inoculation the m e d i u m which contained chlamydiae was gently collected and replace by fresh medium. At 2 weeks, cells were detached from the plastic by vigourous shaking with glass beads and harvested with the medium. After each harvest, chlamydiae were immediately pelleted at 15 000 g for 2 h at 4 ° C, suspended in 1.5 ml of phosphate glutamine-sucrose buffer, pH 7.6 (Madeley 1977) and stored in phosphate glutamine-sucrose buffer at - 70 ° C.
Purification of chlamydial elementary bodies Chlamydiae were purified on Renografin as described by Caldwell et al. (1981 ), using 150 m M Tris-potassium chloride buffer (pH 7.5 ). The purified elementary bodies were stored at - 70 ° C.
DNA extraction Genomic DNA was extracted from purified elementary bodies by a modification of the method of Wenman and Lovett ( 1982 ). The purified elemen-
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTACI
265
TABLE 1 Characteristics o f r u m i n a n t C. psittaci Strains a n d associated disease
Ovine Abortion AB7 AB10 AB13 H5742 $26/32 Bovine A b o r t i o n AV 1 Caprine Abortion AC1 Thermosensitive Mutant 1B 3 1H 3 O v i n e Arthritis LW6794 Ovine Conjunctivitis OC1 8242 Ovine Pneumonitis 109-75 s Intestinal ( f r o m faeces o f h e a l t h y sheep or goat) iB1 iB3 iB5 iB6 M O 9074 iB3-ip 6 iCl
Invasiveness for m o u s e 1
REA p a t t e r n type
+ + + +
1 3 1 1 1
+
1
+
1
+ +
1 1
-
3
+ -
1 3
+
1
+ + +
2 3 3 3 1 1 1
~Established by splenic colonization following footpad inoculation: + = i n v a s i v e , - = non-invasive. 2From I.D. Aitken, M o r e d u n R e s e a r c h Institute, E d i n b u r g h Scotland. 3 T h e r m o s e n s i b l e m u t a n t o b t a i n e d f r o m AB7 strain (Rodolakis 1983 ) 4From J. Storz School o f Veterinary Medicine, L o u i s i a n a State University, Baton Rouge, L o u i s i a n a U S A (Spears a n d Storz 1979a) 5From P. R u s s o CNEVA, Lab. Pathol. Petits R u m i n a n t s Nice France 6From M c C o y cells persistently infected with iB3 (Rodolakis et al. 1989 )
tary bodies were homogenized in 1 ml of 10 m M Tris, 1 m M EDTA pH 8 buffer (TE) containing 0.584M sucrose and 0.8/lg/ml of proteinase K. They were then incubated at 55°C for 15 min. Then, 200/~1 of 0.6% (w./v.) Nlauroylsarcosine was added. This mixture was incubated at 55 ° C for 15 min and then at 37 °C overnight. The resulting viscous solution was extracted once with phenol, twice with phenol-chloroform and once with chloroform. Finally the solution was dialysed for 72 h at 4 ° C against TE before precipitation
266
A. R O D O L A K I S A N D A. S O U R I A U
1 2 1 2 2 1 1 2
18-5
10"6 9 7-4
5-6
4
I
I
A
•
I
B
I
|
C
I
I
D
Fig. 1. Restriction endonuclease analysis of DNA extracts from iB1 ( 1) and AB7 (2) by ApaI (A), HaeIII(B), EcoRI (C), HincII (D). The molecular weight of Raoull (Appligbne Illkirch France) are indicated in kilobases on the left of the gel. of the D N A with 2 vols of ethanol at - 2 0 °C overnight. The precipitated D N A was dissolved in TE to a concentration of 100-500/~g/ml. Restriction endonuclease treatment Samples of chlamydial D N A ( 1-2/~g) were digested with 10 U of either ApaI, B a m H I (Boerhinger Mannheim, G e r m a n y ) , EcoRI, HaeIII, HhaI, HincII, HindIII, Kpn, MspI, PstI, SalI, or Xhol (Appligene, Illkirch, France) for 3-5 h, under the conditions r e c o m m e n d e d by the manufacturers. Agarose
267
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTA(7
electrophoresis was carried out in horizontal gels of 1% agarose in 80 mM Tris- phosphate, 2 mM EDTA pH 8 (TPE), for 24 h. Gels were stained with ethidium bromide (0.5/~g/ml) after electrophoresis and photographed for comparison of digest profiles. Raoul I (Appligene, Illkirch, France) was used as size marker. RESULTS
Twelve different restriction enzymes including ApaI, BamHI, EcoRI, HaeIII, HhaI, HincII, HindIII, Kpn, MspI, PstI, SalI, and XhoI were used on
18-5 10-6 9 7-4
1
2 3
4
5
6
7
8
9 10
Fig. 2. Similarity of DNA fragment patterns of invasive strains of C. psittaci. Xhol digests of genomic DNA extracted from C. psittaci invasive strains AB7 ( 1 ), OC1 (2), H574 (3), AB13 (4), $26 / 3 ( 6 ), Mo907 ( 7 ), iB3ip ( 8 ), non-invasive strain iB l ( 5 ) and C. trachomatis L2 ( 9 ), Raoul I (10). Arrows indicated minor differences between strains.
268
A. RODOLAKIS AND A. S O U R I A U
the twenty strains of C. psittaci isolated from ruminants. Restriction pattern that divided the strains in two distinct groups correlating with mouse invasive and non-invasive strains were observed with all enzymes used. For example, Fig. 1 shows fragment differences of the two reference strains iB 1 and AB7 digested with ApaI, EcoRI, HaelII, and HinclI. The most notable feature of the D N A fragment patterns of mouse invasive strains was their high degree o f similarity whatever their pathological or geographical origin. Within the limits of resolution of the method, restriction patterns of the mouse invasive strains were identical with ten o f the enzymes
18.5 10.6 9 7.4 5.6 4 2-9 2-3
1
2
3
4
5
Fig. 3. EcoRl digests ofgenomic DNA extracted from non-invasive C. psittaci strains iB1 ( 1), iB5 (2), 824 (3), AB 10 (4). Lane 5: Raoul I. Arrows indicated minor differences between iB5 and 824 or ABI0.
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTACI
269
used. When the DNA was digested with XhoI (Fig. 2) or HaeIII (data not shown) minor reproducible differences could be detected. Some differences could be observed between mouse non-invasive strains suggesting that they were less homogenous than mouse invasive strains. After digestion with EcoRI (Fig. 3 ), BamHI or XhoI (data not shown) the DNA fragment pattern of iB 1 could be easily distinguished from those of other mouse non-invasive strains. Moreover while mouse non-invasive strains shared a number of common bands, some variations in a few bands enabled the discrimination of strains as shown for strains iB5, AB 10 and 824 in Fig. 3. With the nine other enzymes similar but not indentical patterns were observed for iB 1 and other mouse non-invasive strains. DISCUSSION
In this report, we have shown that REA of chlamydia DNA allows differentiation among the strains isolated from ruminants. They can be grouped by DNA patterns based on their virulence for mouse, but not on their tropism for particular tissue or cells. Strains AB 10 and 824 isolated from ovine abortion or ovine conjunctivitis respectively, had similar DNA patterns while exhibiting major differences with the other abortion strains and the conjunctivitis strain OC 1. McClenaghan et al. (1984) using REA distinguished abortion strains from the polyarthritis strain P867, but this strain has been demonstrated as non-invasive in our mouse model (Anderson 1986). The only polyathritis strain that we tested was a mouse non-invasive strain. Timms et al. ( 1988 ) and Fukushi and Hirai (1989) were able to differentiate one ovine abortion strain from ruminant strains of other pathological origin, but we have no information on their invasiveness for mouse. From our results the distinction between strains seems to be between those which are mouse invasive and those which are not, rather than reflecting their association with disease. The REA patterns clearly showed that all the mouse invasive strains are closely related. Over 95% of the bands were the same. McClenaghan et al. (1984) have reported the homogeneity of 8 ovine abortion strains by both EcoRI and HindlII digests, two of their strains (H574 and $26/3) were included in our study. In contrast the mouse non-invasive strains form a less homogeneous group. Their heterogeneity was confirmed by REA patterns of the maj or outer membrane protein (MOMP) gene ( Denamur et al. in press ), the analysis of enzyme electrophoretic polymorphism and the polymorphism of ribosomal DNA (unpublished data), while with the 3 methods mouse invasive strains belong to a unique group. The REA appears to be useful technique for comparison of strains: the differences between mouse invasive and non-invasive strains were very clear and the results suggest that specific probes better suited for routine studies
270
A. RODOLAK1S AND A. SOURIAU
could be found in order to distinguish the strains isolated from ruminants according to their virulence. This method could be a sound alternative to the mouse model. ACKNOWLEDGMENT
This work was supported by a grant of"R6gion Centre".
REFERENCES Anderson, I.E., 1986. Comparison of the virulence in mice of some ovine isolates of Chlamydia psittaci. Vet. Microbiol., 12:213-220. Banks, J,, Eddie, B., Schachter, J. and Meyer, K.F., 1970. Plaque formation by chlamydia in L cells. Infect. lmmun. 1: 259-262. Caldwell, H.D., Kromhout, J. and Schachter, J., 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun., 31:11611176.
Campbell, L.A., Kuo, C-C. and Grayston, J.T., 1987. Characterization of the new Chlamydia agent TWAR, as a unique organism by restriction endonuclease analysis and D N A - D N A hybridization. J. Clin. Microbiol., 25:1911-1916. Denamur, E., Sayada, C., Souriau, A., Orfila, J., Rodolakis, A., Elion, J., 1991. Restriction pattern of the Major Outer Membrane Protein (MOMP) gene provide evidence for a homogeneous invasive group among Chlamydia psittaci ruminant isolates. J. Gen. Microbiol., in press. Eb, F., and Orfila, J., 1982. Serotyping of Chlamydia psittaci by microimmunofluorescence test: isolates of ovine origin. Infect. Immun., 37:1289-1291. Fukushi, H., and Hirai, K., 1989. Genetic diversity of Avian and mammalian Chlamydia psittaci strains and relation to host origin. J. Bacteriol., 171: 2850-2855. Grayston, J.T., and Wang, S.P. 1975. New knowledge of chlamydia and the diseases they cause. J. Infect. Dis., 132: 87-105. Grayston, J.T., Kuo, C-C., Campbell, L.A. and Wang, S.P., 1989. Chlamydia pneumoniae sp. nov. for Chlamydia sp. Strain TWAR. Int. J. Syst. Bacteriol., 39: 88-90. Herring, A.J., Anderson, I.E., McClenaghan, M., Inglis, N.F., Williams, H., Matheson, B.A., West, C.P., Rodger, M. and Brettle, R.P., 1987. Restriction endonuclease analysis of DNA from two isolates of Chlamydia psittaci obtained from human abortions. Br. Med. J., 295: 1239. Ito, J.I., Lyons, J.M. and Airo-Brown, L.P., 1990. Variation in virulence among oculogenital serovars of Chlamydia trachomatis in experimental genital tract infection. Infect. Immun., 58: 2021-2023. Madeley, C.R., 1977. Guide pour le pr61~vement et le transport des 6chantillons dans les maladies/t virus, Rickettsiae et Chlamydiae. Organisation Mondiale de la Sant6, Genbve. 49 pp. McClenaghan, M., Herring, A.J. and Aitken, I.D., 1984. Comparison of Chlamydiapsittaci isolates by DNA restriction endonuclease analysis. Infect. Immun., 45: 384-389. Perez-Martinez, J.A. and Storz, J., 1985. Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence. Infect. Immun., 50:905-910.
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTACI
271
Peterson, E.M. and de la Maza, L.M., 1988. Restriction endonuclease analysis of DNA from Chlamydia trachomatis biovars. J. Clin. Microbiol., 26: 625-629. Rodolakis, A., 1983. In vitro and in vivo properties of chemically induced temperature sensitive mutants of Chlamydia psittaci var. ovis. Screening in a murine model. Infect. Immun., 42: 525-530. Rodolakis, A., Bernard, F., and Lantier, F., 1989a. Mouse models for evaluation of virulence of Chlarnydia psittaci isolated from ruminants. Res. Vet. Sci., 46: 34-39. Rodolakis, A., Bernard, F., Souriau, A., Layachi K. and Buzoni-Gatel, D., 1989b. Relationship between virulence of Chlamydia psittaci strains and establishment of persistent infection of McCoy cells. Vet. Microbiol., 19: 65-73. Schachter, J., Banks, J., Sugg, N., Sung, N. Storz J. and Meyer, K.F., 1974. Serotyping of Chlamydia. I. Isolates ofovine origin. Infect. Immun., 9: 92-94. Schachter, J., Banks, J., Sugg, N., Sung, M., Storz J. and Meyer, K.F., 1975. Serotyping of Chlamydia: Isolates of bovine origin. Infect. Immun., 11: 904-907. Spears, P. and Storz, J., 1979a. Biotyping of Chlamydia psittaci based on inclusion morphology and response to diethylaminoethyldextran and cycloheximide. Infect. Immun. 24: 224-232. Spears, P. and Storz, J., 1979b. Chlamydia psittaci: growth characteristics and enumeration of serotype 1 and 2 in cultured cells. J. Infect. Dis., 140: 959-967. Storz, J., 1971. Chlamydia and Chlamydia-induced disease. C.C. Thomas Publisher, Springfield, Ill, 358 pp. Timms P., Eaves, F.W. Girjes, A.A. and Lavin, M.F., 1988. Comparison of Chlamydia psittaci isolates by restriction endonuclease and DNA probe analyses. Infect. Immun., 56: 287-290. Wenhman W.M. and Lowett, M.A., 1982. Expression in E. coli of Chlamydia trachomatis antigen recognized during human infection. Nature (London). 269: 68-70.
263
Restriction endonuclease analysis of D N A from ruminant Chlamydia psittaci and its relation to mouse virulence Annie Rodolakis and Armel Souriau INRA, DOpartement de Pathologie Animale, Laboratoire de Pathologie Infectieuse et Immunologie, 37380 Nouzilly France (Accepted 24 September 1991 )
ABSTRACT Rodolakis, A. and Souriau, A., 1992. Restriction endonuclease analysis of DNA from ruminant Chlamydia psittaci and its relation to mouse virulence. Vet. Microbiol., 31 : 263-271. DNA from 20 pathogenic or non-pathogenic ruminant strains of Chlamydia psittaci was compared by restriction endonuclease analysis. The strains could be easily differentiated according to their invasiveness for mouse, whatever their pathological origin. DNA patterns of invasive strains were similar, whereas those of non-invasive strains were distributed in two groups.
INTRODUCTION
Chlamydiae are Gram-negative obligate intracellular bacteria. They are characterized by a unique developmental cycle which serves as the basis for their current taxonomic classification. The genus Chlamydia is subdivided into three species, namely: C. trachomatis, C. pneumoniae and C. psittaci (Grayston et al. 1989). C. trachomatis and C. pneumoniae infect mainly humans. For this reason, they have been studied extensively. The classification of C. trachomatis in 15 distinct serovars (Grayston et al. 1975 ) proved to be very useful and a correlation between serovars and virulence has been described (Ito et al. 1990). In contrast the typing of C. psittaci which infects a variety of mammals and birds and exhibits broad pathogenic potential (Storz 1971 ), is less well understood. Mammalian strains of C. psittaci may be separated into nine serovars (Perez-Martinez and Storz 1985). Strains of serovar 1 are principally associated with intestinal infection or abortion, while strains of serovar 2 are isolated from pneumonitis, polyarthritis and conjunctivitis in ruminants (Eb and Orfila 1982; Perez-Martinez and Storz 1985; Schachter et al. 1974; Schachter et al., 1975; Spears and Storz 1979a, b. ) This classification does not distinguish abortion strains from intestinal strains, a 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
264
A. RODOLAKIS AND A. SOURIAU
distinction that is essential for a better understanding of the epidemiology of ovine chlamydial abortion. We have developed a murine model of virulence to distinguish these two categories of strains (Rodolakis et al. 1989). Mouse invasive strains, most often isolated from clinical diseases (abortion, pneumonitis, conjunctivitis, and arthritis), may be recovered from the spleen of mice following subcutaneous inoculation, whereas this does not occur with non-invasive strains, mostly of intestinal origin, treated similarily. But the usefulness of this method remains limited because it is very laborious. Restriction endonuclease analysis (REA) of DNA has proved to be a valuable means of typing in numerous bacterial species particularly in C. trachomatis (Peterson and de la Maza, 1988), C. pneumoniae (Campbell et al. 1987) and in some C. psittaci according to host origin (Fukushi and Hirai 1989; Herring et al., 1987; McClenaghan et al. 1984; Timms et al., 1988). The purpose of this study was to compare the REA of ruminant strains of C. psittaci isolated either from diseased tissues or from feces of healthy animals. MATERIALS AND METHODS
Chlamydial strains Besides our two laboratory reference strains AB7 (from abortion, a mouse invasive strain ) and iB 1 (from feces, a mouse non-invasive strain ), 18 strains of C. psinaci (Table 1) and 1 strain of C. trachomatis ( C. trachomatis L~ pF2) were used. Their geographic origin was described in a previous paper (Rodolakis et al., 1989 ), All the strains were propagated in Mycoplasma-free McCoy cells in plastic flasks ( 150 cm2), 4 and 12 flasks were used for mouse invasive and non-invasive strains respectively. Cell monolayers were infected with about 107 plaque forming units (pfu) (Banks et al. 1970) of yolk sacpropagated chlamydiae and incubated at 37 °C. Seven days after inoculation the m e d i u m which contained chlamydiae was gently collected and replace by fresh medium. At 2 weeks, cells were detached from the plastic by vigourous shaking with glass beads and harvested with the medium. After each harvest, chlamydiae were immediately pelleted at 15 000 g for 2 h at 4 ° C, suspended in 1.5 ml of phosphate glutamine-sucrose buffer, pH 7.6 (Madeley 1977) and stored in phosphate glutamine-sucrose buffer at - 70 ° C.
Purification of chlamydial elementary bodies Chlamydiae were purified on Renografin as described by Caldwell et al. (1981 ), using 150 m M Tris-potassium chloride buffer (pH 7.5 ). The purified elementary bodies were stored at - 70 ° C.
DNA extraction Genomic DNA was extracted from purified elementary bodies by a modification of the method of Wenman and Lovett ( 1982 ). The purified elemen-
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTACI
265
TABLE 1 Characteristics o f r u m i n a n t C. psittaci Strains a n d associated disease
Ovine Abortion AB7 AB10 AB13 H5742 $26/32 Bovine A b o r t i o n AV 1 Caprine Abortion AC1 Thermosensitive Mutant 1B 3 1H 3 O v i n e Arthritis LW6794 Ovine Conjunctivitis OC1 8242 Ovine Pneumonitis 109-75 s Intestinal ( f r o m faeces o f h e a l t h y sheep or goat) iB1 iB3 iB5 iB6 M O 9074 iB3-ip 6 iCl
Invasiveness for m o u s e 1
REA p a t t e r n type
+ + + +
1 3 1 1 1
+
1
+
1
+ +
1 1
-
3
+ -
1 3
+
1
+ + +
2 3 3 3 1 1 1
~Established by splenic colonization following footpad inoculation: + = i n v a s i v e , - = non-invasive. 2From I.D. Aitken, M o r e d u n R e s e a r c h Institute, E d i n b u r g h Scotland. 3 T h e r m o s e n s i b l e m u t a n t o b t a i n e d f r o m AB7 strain (Rodolakis 1983 ) 4From J. Storz School o f Veterinary Medicine, L o u i s i a n a State University, Baton Rouge, L o u i s i a n a U S A (Spears a n d Storz 1979a) 5From P. R u s s o CNEVA, Lab. Pathol. Petits R u m i n a n t s Nice France 6From M c C o y cells persistently infected with iB3 (Rodolakis et al. 1989 )
tary bodies were homogenized in 1 ml of 10 m M Tris, 1 m M EDTA pH 8 buffer (TE) containing 0.584M sucrose and 0.8/lg/ml of proteinase K. They were then incubated at 55°C for 15 min. Then, 200/~1 of 0.6% (w./v.) Nlauroylsarcosine was added. This mixture was incubated at 55 ° C for 15 min and then at 37 °C overnight. The resulting viscous solution was extracted once with phenol, twice with phenol-chloroform and once with chloroform. Finally the solution was dialysed for 72 h at 4 ° C against TE before precipitation
266
A. R O D O L A K I S A N D A. S O U R I A U
1 2 1 2 2 1 1 2
18-5
10"6 9 7-4
5-6
4
I
I
A
•
I
B
I
|
C
I
I
D
Fig. 1. Restriction endonuclease analysis of DNA extracts from iB1 ( 1) and AB7 (2) by ApaI (A), HaeIII(B), EcoRI (C), HincII (D). The molecular weight of Raoull (Appligbne Illkirch France) are indicated in kilobases on the left of the gel. of the D N A with 2 vols of ethanol at - 2 0 °C overnight. The precipitated D N A was dissolved in TE to a concentration of 100-500/~g/ml. Restriction endonuclease treatment Samples of chlamydial D N A ( 1-2/~g) were digested with 10 U of either ApaI, B a m H I (Boerhinger Mannheim, G e r m a n y ) , EcoRI, HaeIII, HhaI, HincII, HindIII, Kpn, MspI, PstI, SalI, or Xhol (Appligene, Illkirch, France) for 3-5 h, under the conditions r e c o m m e n d e d by the manufacturers. Agarose
267
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTA(7
electrophoresis was carried out in horizontal gels of 1% agarose in 80 mM Tris- phosphate, 2 mM EDTA pH 8 (TPE), for 24 h. Gels were stained with ethidium bromide (0.5/~g/ml) after electrophoresis and photographed for comparison of digest profiles. Raoul I (Appligene, Illkirch, France) was used as size marker. RESULTS
Twelve different restriction enzymes including ApaI, BamHI, EcoRI, HaeIII, HhaI, HincII, HindIII, Kpn, MspI, PstI, SalI, and XhoI were used on
18-5 10-6 9 7-4
1
2 3
4
5
6
7
8
9 10
Fig. 2. Similarity of DNA fragment patterns of invasive strains of C. psittaci. Xhol digests of genomic DNA extracted from C. psittaci invasive strains AB7 ( 1 ), OC1 (2), H574 (3), AB13 (4), $26 / 3 ( 6 ), Mo907 ( 7 ), iB3ip ( 8 ), non-invasive strain iB l ( 5 ) and C. trachomatis L2 ( 9 ), Raoul I (10). Arrows indicated minor differences between strains.
268
A. RODOLAKIS AND A. S O U R I A U
the twenty strains of C. psittaci isolated from ruminants. Restriction pattern that divided the strains in two distinct groups correlating with mouse invasive and non-invasive strains were observed with all enzymes used. For example, Fig. 1 shows fragment differences of the two reference strains iB 1 and AB7 digested with ApaI, EcoRI, HaelII, and HinclI. The most notable feature of the D N A fragment patterns of mouse invasive strains was their high degree o f similarity whatever their pathological or geographical origin. Within the limits of resolution of the method, restriction patterns of the mouse invasive strains were identical with ten o f the enzymes
18.5 10.6 9 7.4 5.6 4 2-9 2-3
1
2
3
4
5
Fig. 3. EcoRl digests ofgenomic DNA extracted from non-invasive C. psittaci strains iB1 ( 1), iB5 (2), 824 (3), AB 10 (4). Lane 5: Raoul I. Arrows indicated minor differences between iB5 and 824 or ABI0.
ANALYSIS OF DNA FROM RUMINANT CHLAMYDIA PSITTACI
269
used. When the DNA was digested with XhoI (Fig. 2) or HaeIII (data not shown) minor reproducible differences could be detected. Some differences could be observed between mouse non-invasive strains suggesting that they were less homogenous than mouse invasive strains. After digestion with EcoRI (Fig. 3 ), BamHI or XhoI (data not shown) the DNA fragment pattern of iB 1 could be easily distinguished from those of other mouse non-invasive strains. Moreover while mouse non-invasive strains shared a number of common bands, some variations in a few bands enabled the discrimination of strains as shown for strains iB5, AB 10 and 824 in Fig. 3. With the nine other enzymes similar but not indentical patterns were observed for iB 1 and other mouse non-invasive strains. DISCUSSION
In this report, we have shown that REA of chlamydia DNA allows differentiation among the strains isolated from ruminants. They can be grouped by DNA patterns based on their virulence for mouse, but not on their tropism for particular tissue or cells. Strains AB 10 and 824 isolated from ovine abortion or ovine conjunctivitis respectively, had similar DNA patterns while exhibiting major differences with the other abortion strains and the conjunctivitis strain OC 1. McClenaghan et al. (1984) using REA distinguished abortion strains from the polyarthritis strain P867, but this strain has been demonstrated as non-invasive in our mouse model (Anderson 1986). The only polyathritis strain that we tested was a mouse non-invasive strain. Timms et al. ( 1988 ) and Fukushi and Hirai (1989) were able to differentiate one ovine abortion strain from ruminant strains of other pathological origin, but we have no information on their invasiveness for mouse. From our results the distinction between strains seems to be between those which are mouse invasive and those which are not, rather than reflecting their association with disease. The REA patterns clearly showed that all the mouse invasive strains are closely related. Over 95% of the bands were the same. McClenaghan et al. (1984) have reported the homogeneity of 8 ovine abortion strains by both EcoRI and HindlII digests, two of their strains (H574 and $26/3) were included in our study. In contrast the mouse non-invasive strains form a less homogeneous group. Their heterogeneity was confirmed by REA patterns of the maj or outer membrane protein (MOMP) gene ( Denamur et al. in press ), the analysis of enzyme electrophoretic polymorphism and the polymorphism of ribosomal DNA (unpublished data), while with the 3 methods mouse invasive strains belong to a unique group. The REA appears to be useful technique for comparison of strains: the differences between mouse invasive and non-invasive strains were very clear and the results suggest that specific probes better suited for routine studies
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could be found in order to distinguish the strains isolated from ruminants according to their virulence. This method could be a sound alternative to the mouse model. ACKNOWLEDGMENT
This work was supported by a grant of"R6gion Centre".
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