Letters in Applied Microbio1og.y 1991, 12, 171-176
ADONIS 026682549100047D
Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization P. FACHD , A N I E LTER A P& J.P. GUILLOUCentre National d’Etudes Veterinaires et Alimentaires, Laboratoire Central de Recherches Vttkrinaires, 94703 Maisons-Alfbrt, France Received 18 January 1991 and accepted 21 January 1991 Paper number: A1 TI03
F A C H ,P., T K A P D. , & G U I L L O UJ.P. , 1991. Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization. Letters in Applied Microbiology 12, 171-176. Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (3539%) and owing to the decreasing melting temperature (T,,,) due to overbiotinylation, hybridization and wash temperatures were optimized at 33°C and at 42°C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira bijkxa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.
Leptospirosis is a bacterial infection found in man, domestic and wild animals. This anthropozoonosis is widespread all over the world but the conventional diagnosis based on bacteriological, immunological and histological methods suffers from significant drawbacks. In animals, the presence of serum antibodies bears little relationship to current infection or renal shedding. It is, therefore, preferable to base diagnosis on the detection of leptospires themselves; however, culturing, the more reliable procedure, is time-consuming, delicate, exposed to contamination and needs very fresh material. DNA hybridization techniques with genomic probes are widely used for rapid, specific and sensitive diagnosis of many infectious diseases. The use of such probes for leptospirosis diagnosis has been put forward by different authors (Millar et al. 1987; Terpstra et al. 1986, 1987). A genomic DNA probe specific to serovar hardjo-boois has been perfected by LeFebvre (1987). In this study
a total genomic DNA probe, pathogenic species-specific,is proposed.
Materials and Methods BACTERIAL STRAINS A N D C U L T U R E S
Fourteen serovars of Leptospira interrogans belonging to 1 1 different serogroups and three serovars of Leptospira bijlexa used in this study are listed in Table 1. These strains were obtained from the World Health Organisation Collaborating Center, Leptospirosis Reference Laboratory, Institut Pasteur, Paris, France. Each strain was grown in EMJH medium (Difco Laboratoires, Detroit, MI, USA) as modified by Johnson & Harris (1967) before DNA extraction. The culture was processed in 250 ml flasks containing 90 ml of EMJH medium enriched with 1% rabbit serum (Ellis
P . Fach et al.
172
Table 1. Leptospira strains used
Serogroup Leptospira interrogans grippotyphosa canicola autumnalis bataviae cynopteri hebdomadis icterohaemorrhagiae papyrogenes australis sejroe
Serovar grippotyphosa canicola autumnalis bataoiae cynopteri hebdomadis icterohaemorrhagiae panama pyrogenes australis bratislava muenchen hardjo sejroe
i
Strain Moskva V Chiffon Akiyami A. Swart. 3522 C.
Hebdomadis Verdun CZ 214 K. Salinem Ballico Jez-Bratislava Muenchen C90 Hardjoprajitno M84
Leptospira bij7exa patoc buenos-aires andamana
1986). Each flask was inoculated with 10 ml or 20 ml of bacterial suspension and incubated at 30°C. D N A EXTRACTION A N D PURIFICATION
DNA was purified as described by Marshall et al. (1981), with a few modifications. Exponentially growing leptospiral cultures were collected and centrifuged at 15OOOg for 30 min at 4°C in an SS34 Sorvall rotor. The pellets were collected and washed twice in TEB (10 mmol/l Tris-HC1 pH 8.5, 10 mmol/l sodium EDTA). Bacterial walls were lysed for 30 min at 35°C by lysozyme (Sigma, St Louis, USA) to a final concentration of 1 mg/ml in TEB; SDS to a final concentration of 1% (w/v) was added and the protein digestion was performed by 50 pg/ml of proteinase K (Sigma) at 37°C overnight. After three phenolchloroform (v/v) extractions, nucleic acids were recovered in the aqueous phase, then precipitated with ammonium acetate and ethanol at -20°C. RNA was removed by RNAase to a final concentration of 50 pg/ml, during 30 min at 37°C. Leptospiral DNA purity and concentration were determined by spectrophotometry at 260nm. DNA solutions whose A2,0/A280 ratio ranged between 1.8 and 2 were stored at - 20°C. In vitro L A B E L L I N G
OF LEPTOSPIRAL D N A
One pg of DNA extracted from serovar grippotyphosa, strain Moskva V and serovar canic-
ola, strain Chiffon, was labelled in vitro by nick-translation or random priming. Biotin-16 dUTP was incorporated according to the instructions of the manufacturer (GIBCO BRL, Paisley, Scotland) with the following modification: & of the available biotin-16 dUTP was substituted for dTTP to prevent overbiotinylation. Probes were purified by gel filtration on Sephadex G 50 (Pharmacia, Uppsala, Sweden). The concentration of the probe was 2.5 pg/ml. D N A HYBRIDIZATION A N D DETECTION
OF LEPTOSPIRES
Denatured target DNA was spotted onto nylon membranes, air-dried and baked for 1 h at 80°C. For in situ hybridization, cultured leptospires (2 x lo8 micro-organisms/ml) were layered on microscope slides pre-coated with poly L-lysine (0.5 mg/ml) (Sigma), air-dried and fixed in anhydrous methanol for 10 min. Pre-hybridization
Nylon membranes were pre-hybridized at 65°C for 1 h in 5 x Denhart, 0.2% (w/v) SDS mix containing denatured salmon-sperm DNA (500 Pg/mo. Hybridization Membranes Hybridization on nylon filters took place at 3 3 T , overnight, in the presence of
Leptospira D N A probes the denatured labelled DNA probe (125 ndml), denatured salmon-sperm DNA (500 pglml), 5 x Denhart, 5 x SSPE, 0.2% SDS. Slides Slides were incubated for 10 min at 37°C in a 50 mmol/l Tris-HC1 pH 7.5, 5 mmol/l EDTA, 1% SDS buffer containing 100 pg/ml of proteinase K (Sigma), then washed thrice for 1 rnin in a 50 mmol/I Tris-HC1 pH 7.5, 5 mmol/l EDTA buffer containing 2 mdml of glycine. After dehydration for 1 rnin in successive baths of 30%, 6O%, 95% ethanol and air-drying, a hybridization mixture containing 0.412 pg/ml of denatured labelled DNA probe, 2 x SSC, 100 pg/ml of denatured salmon-sperm DNA, 10% dextran sulphate and 50% (v/v) formamide was dropped on glass slides. The DNA on the slides was denatured by steam-heating for 10 rnin at 85°C. Hybridization was performed at 33°C in a moist chamber, overnight. Visualization Membranes Membranes were washed twice in 2 x SSC for 10 min at room temperature in the presence of 0.1% SDS, followed by one wash in 2 x SSC for 10 min at room temperature and then by a 10 min wash in 0.1 SSC at 42°C. Slides Slides were washed for 10 rnin at 33°C with 2 x SSC in the presence of 50% formamide, as described by Maniatis et al. (1982),
173
then washed in 2 x SSC for 10 min at room temperature. Subsequently the filters and slides were washed for 1 rnin in 0.1 mol/l Tris-HCI pH 7.5 containing 0.15 mol/l NaCI, incubated for 1 h with 3% (w/v) BSA at 42"C, for 10 rnin with conjugate (streptavidin-alkaline phosphatase) solution at room temperature, and washed twice for 15 rnin in 0.1 mol/l Tris-HC1 pH 9.5 buffer containing 0.1 mol/l NaCl, 50 mmol/l MgCl,. Finally, they were incubated in the dark, for 30 rnin to 3 h with freshly prepared 0 3 3 mg/ml nitroblue tetrazolium (NBT), 0.16 mg/ml 5bromo-4-chloro-3-indolyl phosphate (BCIP) in 0.1 mol/l Tris-HC1 pH 9.5 and 50 mmol/l MgCI, buffer. After drying, the slides were examined by light microscopy (lo00x ) and the membranes were selected on the basis of a strong hybridization signal.
Results and Discussion The deficiency of conventional bacteriological methods (Turner 1970) still used in leptospirosis diagnosis, such as culturing, dark-field examination, silver and immunofluorescence staining, naturally leads to perfect nucleic probes and DNA hybridization which is a highly specific and sensitive reaction (Meinkoth & Wahl 1984). According to the weak G + C content of leptospires (3539%) and to the decreasing T, of probes due to biotinylation, it was necessary
n
R
Fig. 1. Dot-blot analysis of several serovars of L. interrogans, using (A) serovar australis DNA probe and (B) serovar icterohaemorrhagiae DNA probe. Doubling solutions starting at 50 ng run from columns 1 to 10. (1) icterohaemorrhagiae; ( 2 ) grippotyphosa; ( 3 ) australis.
P . Fach et al.
174 A I
2 3
4 5
6 7 8
9 10
I1 12
13 14
15 16 17 18 19
Fig. 2. Detection, using (A) serovar grippotyphosa DNA probe and (B) serovar canicola DNA probe, of different heterologous leptospires DNA. For each dot approximately 0.5 pg DNA was spotted. (1) grippotyphosa; (2) canicola; (3) autumnalis; (4) batauiae; ( 5 ) cynopteri; (6) hebdomadis; (7) icterohaemorrhogiae; (8) panama; (9) pyrogenes; ( 1 0 F ; (11) australis; (12) bratislaua; (13) muenchen; (14)-; (15) hardjo; ( 1 6 F ; (17) buenos-aires; (18)patoc; (19)andamana.
to alter subsequently the operative conditions in comparison with those described by Brendle et al. (1974), Terpstra et al. (1986) and van Eys et al. (1988), especially in the hybridization and washing temperatures which were optimized at 33°C and 42°C respectively. Under these conditions, we showed there is enough genomic difference between saprophytic and pathogenic strains to obtain a specific total DNA probe for the saprophytic serovar buenos-aires: this probe showed hybridization only with homologous DNA but no cross-hybridization with any different pathogenic serovars DNA or with two other saprophytic serovars DNA (L. patoc and L. andamana) (Fig. 3). The dot blot analysis with a few leptospire strains showed many cross-hybridizations between different pathogenic serovars of L. interrogans. Total genomic DNA probes
Fig. 3. Dot blot analysis using the saprophytic Serovar buenos-aires DNA probe. For each dot approximately 0.5 pg DNA was spotted. (1) grippotyphosa; (2) canicola; (3) autumnalis; (4) batauiae; ( 5 )
cynopteri; (6) hebdomadis; (7) icterohaemorrhagiae; (8) panama; (9) pyrogenes; (lo)-; (11) australis; (12) bratislaua; (13) muenchen; ( 1 4 b ; (15) hardjo; (16)-; (17) buenos-aires; (18) patoc; (19) andamana; (20) negative
diluent control.
prepared from serovars australis, icterohaemorrhagiae, grippotyphosa and canicola exhibited a strong hybridization with other pathogenic serovars DNA used in this study (Figs 1 and 2). Two such probes, prepared from serovars canicola and grippotyphosa, which hybridized with every pathogenic DNA strain were tested against certain saprophytic L. biJlexa strains (L. patoc, L. buenos-aires, L. andamana): no cross-hybridization was observed with those saprophytic strains (Fig. 2). The total genomic DNA probes prepared in this study from a single serovar probably recognize common repetitive sequences along the bacterial genome of leptospires. These seem to be specific to pathogenic species and thereby can certainly be used for a rapid diagnosis of leptospirosis. It should be possible to produce more serovar-specific probes by molecular cloning but from a clinical point of view it is
Leptospira D N A probes
175
combines the specificity of the hybridization reaction with the visualization of the leptospiral morphology, and may be useful for direct observation and identification of pathogenic leptospires in clinical samples, as described by Terpstra et al. (1987) and Millar et al. (1987). These procedures are rapid and very specific. No cross-hybridizations were observed with Borrelia or with various other micro-organisms (Terpstra et al. 1987). However, the low sensitivity of the technique is still an obstacle in using these probes for routine diagnosis: approximately 100 pg can be detected with these probes and according to Terpstra et al. (1986) the detectable amounts of DNA were in the range 15 to 375 pg. Consequently, these probes will be used after previous leptospiral enrichment or in uitro amplification by polymerase chain reaction (PCR). To improve the sensitivity of the procedure, PCR experiments are currently in progress in our laboratory.
Fig. 4. In situ hybridization on smear of leptospiral suspension, using a biotin-labelled DNA probe prepared from Leptospira interrogans. Light microscopy ( x 1OOO) shows leptospires (A) serovar canicola and (B) serovar grippotyphosa with their characteristic morphology.
important that these probes are species-specific for pathogens and can be used to determine whether animals have leptospirosis or not. The serovar-status of the leptospire causing the disease is of secondary importance but it may be more useful for epidemiological studies or taxonomic identification. Nevertheless, in North America, a genetic recombinant probe (pTL1) specific for the single serovar hardjo-bouis which causes bovine abortion has been described for the diagnosis of the most important cattle leptospirosis in that country (LeFebvre 1987; van Eys et al. 1988). The genomic DNA probes of serovars canicola and grippotyphosa, as described in this study, can also be used for in situ hybridization: Fig. 4 shows DNA of serovars canicola and grippotyphosa hybridized with a homologous probe labelled with biotin-16 dUTP. In situ hybridization using these biotin-labelled probes
The authors thank C. Karoui, C. Vallet, A.M. Mahe, and S. Martin for their excellent technical assistance. c. Tommasino is gratefully acknowledged for her help in typing the manuscript as well as G. Garin-Bastuji for his advice.
References BRENDLE,J.J., RCCUL, M. & ALEXANDER, A.D. 1974 Deoxyribonucleic acid hybridization among selected leptospiral serotypes. International Journal of Systematic Bacteriology 24, 205-214. ELLIS,W.A. 1986 The diagnosis of leptospirosis in farm animals. In The Present State of Leptospirosis Diagnosis and Control ed. Ellis, W.A. & Little, T.W.A. Dordrecht : Martinus Nijhoff. JOHNSON, R.C. & HARRIS, V.G. 1967 Differentiation of pathogenic and saprophytic leptospires. I. Growth at low temperatures. Journal of Bacterioiogy 94, 27-31. LEFEBVRE, R.B. 1987 DNA probes for detection of the Leptospira interrogans serovar hardjo genotype hardjo-bovis. Journal of Clinical Microbiology 25, 22362238. MANIATIS, T., FRITSCH,E.F. & SAMBROOK, J. 1982 Molecular Cloning. N.Y.: Cold Spring Harbor Laboratory. MARSHALL,R.B.. WILTON,B.E. & ROBINSON, A.J. 1981 Identification of Leptospira serovars by restriction endonuclease analysis. Journal of Medical Microbiology 14, 1 6 f 1 6 6 . MEINKOTH, J. & WAHL, G. 1984 Hybridization of nucleic acids immobilized on solid supports. Analytical Biochemistry 138, 267-284. R.J. & ADLER,B. 1987 DetecMILLAR,B.D., CHAPPEL,
176
P . Fac:h et al.
tion of leptospires in biological fluids using DNA hybridisation. Veterinary Microbiology 15, 71-78. TERPSTRA, W.J., SCHOONE,G.J. & TER SCHEGGET, J. 1986 Detection of leptospiral DNA by nucleic acid hybridisation with 32 P- and biotin-labelled probes. Journal of Medical Microbiology 22,23-28. TERPSTRA, W.J., SCHOONE, G.J., LIGTHART, G.S. & TER SCHEGGET, J. 1987 Detection of Leptospira interrogans in clinical specimens by in situ hybridization using biotin-labelled DNA probes. Journal of General Microbiology 133,911-914.
TURNER, L.H. 1970 Leptospirosis 111. Maintenance, isolation and demonstration of leptospires. Transactions of the Royal Society of Tropical Medicine and Hygiene 64,623-646. VAN EYS, G.J.J.M., ZAAL, J., ScHooNE, G.J. & TERPSTRA, W.J. 1988 DNA hybridization with hardjobovis-specific recombinant probes as a method for type discrimination of Leptospira interrogans serovar hardjo. Journal of General Microbiology 134,567-574.
ADONIS 026682549100047D
Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization P. FACHD , A N I E LTER A P& J.P. GUILLOUCentre National d’Etudes Veterinaires et Alimentaires, Laboratoire Central de Recherches Vttkrinaires, 94703 Maisons-Alfbrt, France Received 18 January 1991 and accepted 21 January 1991 Paper number: A1 TI03
F A C H ,P., T K A P D. , & G U I L L O UJ.P. , 1991. Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization. Letters in Applied Microbiology 12, 171-176. Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (3539%) and owing to the decreasing melting temperature (T,,,) due to overbiotinylation, hybridization and wash temperatures were optimized at 33°C and at 42°C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira bijkxa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.
Leptospirosis is a bacterial infection found in man, domestic and wild animals. This anthropozoonosis is widespread all over the world but the conventional diagnosis based on bacteriological, immunological and histological methods suffers from significant drawbacks. In animals, the presence of serum antibodies bears little relationship to current infection or renal shedding. It is, therefore, preferable to base diagnosis on the detection of leptospires themselves; however, culturing, the more reliable procedure, is time-consuming, delicate, exposed to contamination and needs very fresh material. DNA hybridization techniques with genomic probes are widely used for rapid, specific and sensitive diagnosis of many infectious diseases. The use of such probes for leptospirosis diagnosis has been put forward by different authors (Millar et al. 1987; Terpstra et al. 1986, 1987). A genomic DNA probe specific to serovar hardjo-boois has been perfected by LeFebvre (1987). In this study
a total genomic DNA probe, pathogenic species-specific,is proposed.
Materials and Methods BACTERIAL STRAINS A N D C U L T U R E S
Fourteen serovars of Leptospira interrogans belonging to 1 1 different serogroups and three serovars of Leptospira bijlexa used in this study are listed in Table 1. These strains were obtained from the World Health Organisation Collaborating Center, Leptospirosis Reference Laboratory, Institut Pasteur, Paris, France. Each strain was grown in EMJH medium (Difco Laboratoires, Detroit, MI, USA) as modified by Johnson & Harris (1967) before DNA extraction. The culture was processed in 250 ml flasks containing 90 ml of EMJH medium enriched with 1% rabbit serum (Ellis
P . Fach et al.
172
Table 1. Leptospira strains used
Serogroup Leptospira interrogans grippotyphosa canicola autumnalis bataviae cynopteri hebdomadis icterohaemorrhagiae papyrogenes australis sejroe
Serovar grippotyphosa canicola autumnalis bataoiae cynopteri hebdomadis icterohaemorrhagiae panama pyrogenes australis bratislava muenchen hardjo sejroe
i
Strain Moskva V Chiffon Akiyami A. Swart. 3522 C.
Hebdomadis Verdun CZ 214 K. Salinem Ballico Jez-Bratislava Muenchen C90 Hardjoprajitno M84
Leptospira bij7exa patoc buenos-aires andamana
1986). Each flask was inoculated with 10 ml or 20 ml of bacterial suspension and incubated at 30°C. D N A EXTRACTION A N D PURIFICATION
DNA was purified as described by Marshall et al. (1981), with a few modifications. Exponentially growing leptospiral cultures were collected and centrifuged at 15OOOg for 30 min at 4°C in an SS34 Sorvall rotor. The pellets were collected and washed twice in TEB (10 mmol/l Tris-HC1 pH 8.5, 10 mmol/l sodium EDTA). Bacterial walls were lysed for 30 min at 35°C by lysozyme (Sigma, St Louis, USA) to a final concentration of 1 mg/ml in TEB; SDS to a final concentration of 1% (w/v) was added and the protein digestion was performed by 50 pg/ml of proteinase K (Sigma) at 37°C overnight. After three phenolchloroform (v/v) extractions, nucleic acids were recovered in the aqueous phase, then precipitated with ammonium acetate and ethanol at -20°C. RNA was removed by RNAase to a final concentration of 50 pg/ml, during 30 min at 37°C. Leptospiral DNA purity and concentration were determined by spectrophotometry at 260nm. DNA solutions whose A2,0/A280 ratio ranged between 1.8 and 2 were stored at - 20°C. In vitro L A B E L L I N G
OF LEPTOSPIRAL D N A
One pg of DNA extracted from serovar grippotyphosa, strain Moskva V and serovar canic-
ola, strain Chiffon, was labelled in vitro by nick-translation or random priming. Biotin-16 dUTP was incorporated according to the instructions of the manufacturer (GIBCO BRL, Paisley, Scotland) with the following modification: & of the available biotin-16 dUTP was substituted for dTTP to prevent overbiotinylation. Probes were purified by gel filtration on Sephadex G 50 (Pharmacia, Uppsala, Sweden). The concentration of the probe was 2.5 pg/ml. D N A HYBRIDIZATION A N D DETECTION
OF LEPTOSPIRES
Denatured target DNA was spotted onto nylon membranes, air-dried and baked for 1 h at 80°C. For in situ hybridization, cultured leptospires (2 x lo8 micro-organisms/ml) were layered on microscope slides pre-coated with poly L-lysine (0.5 mg/ml) (Sigma), air-dried and fixed in anhydrous methanol for 10 min. Pre-hybridization
Nylon membranes were pre-hybridized at 65°C for 1 h in 5 x Denhart, 0.2% (w/v) SDS mix containing denatured salmon-sperm DNA (500 Pg/mo. Hybridization Membranes Hybridization on nylon filters took place at 3 3 T , overnight, in the presence of
Leptospira D N A probes the denatured labelled DNA probe (125 ndml), denatured salmon-sperm DNA (500 pglml), 5 x Denhart, 5 x SSPE, 0.2% SDS. Slides Slides were incubated for 10 min at 37°C in a 50 mmol/l Tris-HC1 pH 7.5, 5 mmol/l EDTA, 1% SDS buffer containing 100 pg/ml of proteinase K (Sigma), then washed thrice for 1 rnin in a 50 mmol/I Tris-HC1 pH 7.5, 5 mmol/l EDTA buffer containing 2 mdml of glycine. After dehydration for 1 rnin in successive baths of 30%, 6O%, 95% ethanol and air-drying, a hybridization mixture containing 0.412 pg/ml of denatured labelled DNA probe, 2 x SSC, 100 pg/ml of denatured salmon-sperm DNA, 10% dextran sulphate and 50% (v/v) formamide was dropped on glass slides. The DNA on the slides was denatured by steam-heating for 10 rnin at 85°C. Hybridization was performed at 33°C in a moist chamber, overnight. Visualization Membranes Membranes were washed twice in 2 x SSC for 10 min at room temperature in the presence of 0.1% SDS, followed by one wash in 2 x SSC for 10 min at room temperature and then by a 10 min wash in 0.1 SSC at 42°C. Slides Slides were washed for 10 rnin at 33°C with 2 x SSC in the presence of 50% formamide, as described by Maniatis et al. (1982),
173
then washed in 2 x SSC for 10 min at room temperature. Subsequently the filters and slides were washed for 1 rnin in 0.1 mol/l Tris-HCI pH 7.5 containing 0.15 mol/l NaCI, incubated for 1 h with 3% (w/v) BSA at 42"C, for 10 rnin with conjugate (streptavidin-alkaline phosphatase) solution at room temperature, and washed twice for 15 rnin in 0.1 mol/l Tris-HC1 pH 9.5 buffer containing 0.1 mol/l NaCl, 50 mmol/l MgCl,. Finally, they were incubated in the dark, for 30 rnin to 3 h with freshly prepared 0 3 3 mg/ml nitroblue tetrazolium (NBT), 0.16 mg/ml 5bromo-4-chloro-3-indolyl phosphate (BCIP) in 0.1 mol/l Tris-HC1 pH 9.5 and 50 mmol/l MgCI, buffer. After drying, the slides were examined by light microscopy (lo00x ) and the membranes were selected on the basis of a strong hybridization signal.
Results and Discussion The deficiency of conventional bacteriological methods (Turner 1970) still used in leptospirosis diagnosis, such as culturing, dark-field examination, silver and immunofluorescence staining, naturally leads to perfect nucleic probes and DNA hybridization which is a highly specific and sensitive reaction (Meinkoth & Wahl 1984). According to the weak G + C content of leptospires (3539%) and to the decreasing T, of probes due to biotinylation, it was necessary
n
R
Fig. 1. Dot-blot analysis of several serovars of L. interrogans, using (A) serovar australis DNA probe and (B) serovar icterohaemorrhagiae DNA probe. Doubling solutions starting at 50 ng run from columns 1 to 10. (1) icterohaemorrhagiae; ( 2 ) grippotyphosa; ( 3 ) australis.
P . Fach et al.
174 A I
2 3
4 5
6 7 8
9 10
I1 12
13 14
15 16 17 18 19
Fig. 2. Detection, using (A) serovar grippotyphosa DNA probe and (B) serovar canicola DNA probe, of different heterologous leptospires DNA. For each dot approximately 0.5 pg DNA was spotted. (1) grippotyphosa; (2) canicola; (3) autumnalis; (4) batauiae; ( 5 ) cynopteri; (6) hebdomadis; (7) icterohaemorrhogiae; (8) panama; (9) pyrogenes; ( 1 0 F ; (11) australis; (12) bratislaua; (13) muenchen; (14)-; (15) hardjo; ( 1 6 F ; (17) buenos-aires; (18)patoc; (19)andamana.
to alter subsequently the operative conditions in comparison with those described by Brendle et al. (1974), Terpstra et al. (1986) and van Eys et al. (1988), especially in the hybridization and washing temperatures which were optimized at 33°C and 42°C respectively. Under these conditions, we showed there is enough genomic difference between saprophytic and pathogenic strains to obtain a specific total DNA probe for the saprophytic serovar buenos-aires: this probe showed hybridization only with homologous DNA but no cross-hybridization with any different pathogenic serovars DNA or with two other saprophytic serovars DNA (L. patoc and L. andamana) (Fig. 3). The dot blot analysis with a few leptospire strains showed many cross-hybridizations between different pathogenic serovars of L. interrogans. Total genomic DNA probes
Fig. 3. Dot blot analysis using the saprophytic Serovar buenos-aires DNA probe. For each dot approximately 0.5 pg DNA was spotted. (1) grippotyphosa; (2) canicola; (3) autumnalis; (4) batauiae; ( 5 )
cynopteri; (6) hebdomadis; (7) icterohaemorrhagiae; (8) panama; (9) pyrogenes; (lo)-; (11) australis; (12) bratislaua; (13) muenchen; ( 1 4 b ; (15) hardjo; (16)-; (17) buenos-aires; (18) patoc; (19) andamana; (20) negative
diluent control.
prepared from serovars australis, icterohaemorrhagiae, grippotyphosa and canicola exhibited a strong hybridization with other pathogenic serovars DNA used in this study (Figs 1 and 2). Two such probes, prepared from serovars canicola and grippotyphosa, which hybridized with every pathogenic DNA strain were tested against certain saprophytic L. biJlexa strains (L. patoc, L. buenos-aires, L. andamana): no cross-hybridization was observed with those saprophytic strains (Fig. 2). The total genomic DNA probes prepared in this study from a single serovar probably recognize common repetitive sequences along the bacterial genome of leptospires. These seem to be specific to pathogenic species and thereby can certainly be used for a rapid diagnosis of leptospirosis. It should be possible to produce more serovar-specific probes by molecular cloning but from a clinical point of view it is
Leptospira D N A probes
175
combines the specificity of the hybridization reaction with the visualization of the leptospiral morphology, and may be useful for direct observation and identification of pathogenic leptospires in clinical samples, as described by Terpstra et al. (1987) and Millar et al. (1987). These procedures are rapid and very specific. No cross-hybridizations were observed with Borrelia or with various other micro-organisms (Terpstra et al. 1987). However, the low sensitivity of the technique is still an obstacle in using these probes for routine diagnosis: approximately 100 pg can be detected with these probes and according to Terpstra et al. (1986) the detectable amounts of DNA were in the range 15 to 375 pg. Consequently, these probes will be used after previous leptospiral enrichment or in uitro amplification by polymerase chain reaction (PCR). To improve the sensitivity of the procedure, PCR experiments are currently in progress in our laboratory.
Fig. 4. In situ hybridization on smear of leptospiral suspension, using a biotin-labelled DNA probe prepared from Leptospira interrogans. Light microscopy ( x 1OOO) shows leptospires (A) serovar canicola and (B) serovar grippotyphosa with their characteristic morphology.
important that these probes are species-specific for pathogens and can be used to determine whether animals have leptospirosis or not. The serovar-status of the leptospire causing the disease is of secondary importance but it may be more useful for epidemiological studies or taxonomic identification. Nevertheless, in North America, a genetic recombinant probe (pTL1) specific for the single serovar hardjo-bouis which causes bovine abortion has been described for the diagnosis of the most important cattle leptospirosis in that country (LeFebvre 1987; van Eys et al. 1988). The genomic DNA probes of serovars canicola and grippotyphosa, as described in this study, can also be used for in situ hybridization: Fig. 4 shows DNA of serovars canicola and grippotyphosa hybridized with a homologous probe labelled with biotin-16 dUTP. In situ hybridization using these biotin-labelled probes
The authors thank C. Karoui, C. Vallet, A.M. Mahe, and S. Martin for their excellent technical assistance. c. Tommasino is gratefully acknowledged for her help in typing the manuscript as well as G. Garin-Bastuji for his advice.
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