|Year : 2002 | Volume
| Issue : 1 | Page : 33--36
Differentiation of pathogenic and saprophytic leptospires by polymerase chain reaction
R Noubade1, GV Krishnamurthy2, S Murag3, MD Venkatesha3, G Krishnappa2,
1 Department of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly - 243 122, India
2 Department of Veterinary Microbiology, Veterinary College, Bangalore - 560 024, India
3 Institute of Animal Health and Veterinary Biologicals, Bangalore - 560 024, India
Department of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly - 243 122
PURPOSE: To study the diferences between pathogenic and saprophytic leptospires. METHOD: A total of 275 samples were collected from different sources out of which 107 were subjected to PCR and bacteriological culturing. Two sets of primers were used for detection of leptospiral DNA and differentiation of pathogenic and saprophytic leptospires. Differentiation was also carried out by conventional methods. RESULTS: Twenty seven samples were found positive by PCR ut of which 26 were pathogenic and one was saprophytic. Culturing in EMJH medium yielded four isolates, of which isolates from sera were found to be pathogenic and isolate from water was found to be saprophytic. CONCLUSION: From the present study, it was concluded that PCR is simple, specific and rapid method for detection as well as differentiation of leptospires when compared to conventional methods.
|How to cite this article:|
Noubade R, Krishnamurthy G V, Murag S, Venkatesha M D, Krishnappa G. Differentiation of pathogenic and saprophytic leptospires by polymerase chain reaction.Indian J Med Microbiol 2002;20:33-36
|How to cite this URL:|
Noubade R, Krishnamurthy G V, Murag S, Venkatesha M D, Krishnappa G. Differentiation of pathogenic and saprophytic leptospires by polymerase chain reaction. Indian J Med Microbiol [serial online] 2002 [cited 2020 Jul 5 ];20:33-36
Available from: http://www.ijmm.org/text.asp?2002/20/1/33/8336
Leptospirosis, an acute febrile septicemic disease, is a worldwide zooanthroponosis with wide geographical, having a broad spectrum of host range and is caused by the spirochete Leptospira interrogans. Many species of domestic and wild animals excrete the causative agent in their urine, contaminating soil and fresh water. The transmission of the disease occurs by direct contact with infected urine or more often through contact with water and soil contaminated with infected urine. The genus Leptospira is conventionally divided into two species: the pathogenic Leptospira interrogans and the free living saprophytic Leptospira biflexa which are indigenous to fresh surface water. Since both of them are morphologically indistinguishable they have to be differentiated to prevent false positive results especially when samples have to be collected from water and soil.
Conventionally, the differentiation between pathogenic and saprophytic leptospires is carried out by tests like pathogenicity to animals, growth response to 8- azaguanine nad low temperature (13°C), conversion to spherical forms by 1M NaCl, lipase activity etc., which are time consuming and laborious. Hence, alternative simple, rapid and reliable methods are the need of the hour and polymerase chain reaction is one such method. Recently several PCR based methods have been reported for detection of leptospires in clinical samples.,,,,,,,
Material and Methods
A total of 275 different samples included blood (n=130), collected during the first week of illness and before any antimicrobial therapy, urine (n=47), collected after first week of the onset of clinical signs in equal quantity of sterile phosphate buffer saline (PBS) of pH 7.2 either by catheterisation with aseptic precautions or the midstream portion during natural voiding, milk (n=4) from mastitis cases suspected due to leptospirosis, kidney (n=38) from either slaughtered animals or carcasses, water (n=47) of pH 6.0-8.0 collected in sterile PBS from taps, sewage, paddy fields and various ponds which were frequently visited by domestic animals and human beings, soil (n=9) collected from wet, shaded areas with pH 6.0-8.0, in the morning hours. These samples were subjected for differential centrifugation of initial speed of 2000 rpm for 10 minutes followed by 15000 rpm for 15 minutes. A loopful of serum, urine, milk and kidney samples was inoculated into Ellinghausen-McCullough-Johnson-Harris (EMJH) semisolid medium containing 5-Fluorouracil (200µg/mL) and of water and soil samples was inoculated after filtering through 0.22 µm membrane filter. The remaining sample was stored at 4°C for PCR assay. All the cultures were examined routinely by dark field microscopy for 12 weeks before they were regarded as negative. The strains isolated were identified by serology (Regional Medical Research Centre, PortBlair) and Random Amplified Polymorphic DNA (RAPD) fingerprinting.
The reference strains [Table:1] representing different serogroups of both pathogenic and saprophytic leptospires were procured from Regional Medical Research Centre, Port Blair, Andaman and Nicobar Islands, the national reference laboratory for leptospirosis. They were maintained in EMJH semisolid medium and passaged at eight weeks interval.
Polymerase chain reaction (PCR)
Two sets of oligonucleotide primers A and B and G1 and G2, as described eralier, were used for PCR with the following sequences.
A 5' - GGC GGC GCG TCT TAA ACA TG - 3'
B 3' - TTC CCC CCA TTG AGC AAG ATT - 5'
G1 5' - CTG AAT CGC TGT ATA AAA GT - 3'
G2 3' - GAA GGC TGG TAA ACA AAA GG - 5'
The reaction was carried out in a total volume of 50µL consisting of a reaction buffer of 100 mM of Tris HCl (pH 9.0), 500 mM KCl, 15 mM MgCl2 and 0.1% gelatin, Deoxyribonucleotide triphosphates (dATP, dGTP, dCTP and dTTP) to a final concentration of 2.5 mM and 100 pmol of each primer. For each reaction, 0.5U of Taq. Polymerase (Bangalore Genie, Bangalore) was added.
Fifty µL of the samples was suspended in 500µL of sterile PBS in an Eppendorf tube and centrifuged at 15000 rpm for 30 minutes. The resulting pellet was resuspended in 20µL distilled water and boiled for 10 minutes in water bath, which was immediately transferred to ice box. The PCR reaction was carried out in MJ Research thermal cycler. For primers A and B, the program of amplification included 30 cycles. The first cycle consisted of denaturation of DNA at 94°C for 3.0 min, annealing of primers at 63°C for 1.5 min. and extension at 72°C for 1.0 min. The next 29 cycles consisted of denaturation at 940C for 1.0 min, annealing at 63°C for 1.5 min, and extension at 72°C for 2.0 min. and an additional 10 minutes was included in the last cycle to complete extension of primers.
The program of amplification for the primers G1 and G2 included 32 cycles. Each cycle consisted of denaturation of 94°C for 1.5 min, annealing at 55°C for 1 min, and extension at 72°C for 2.0 min.
The reference strains maintained in the laboratory served as the positive controls and the master mix without any sample served as the negative control. The amplified products were analyzed by electrophoresis through 1.5% agarose gel using 10µL of the product, and identified by visualizing the bands of expected size on ethidium bromide stained gel under UV transilluminator.
Differentiation of pathogenic and saprophytic leptospires by conventional methods
i) Growth at low temperature (13°C)
The reference strains belonging to both pathogenic and saprophytic leptospira along with the isolates were incubated in a BOD incubator for 21 days.
ii) Growth response to 8-azaguanine
The reference strains belonging to both pathogenic and saprophytic leptospira and the isolates were grown in the medium containing 225 µg/mL of 8-azaguanine and incubated at room temperature.
Specificity of PCR amplification
The oligonucleotide primers A and B amplified all the reference strians, including saprophytes producing an amplicon of 331 bp whereas the oligonucleotide primers G1 and G2 amplified only the pathogenic reference strains but not saprophytic ones, producing an amplicon of 285 bp. Further, both the primers were found to be specific to leptospiral DNA as no amplification was detected when DNAs from other microorganisms were tested.
Detection and differentiation of leptospires by PCR
Of the 107 samples tested by PCR with primers A and B, 27 were found positive producing an amplicon of 331 bp [Figure:1]. Among these 26 comprised of sera and urine and the other was water sample. All the 26 positive samples when further tested with the oligonucleotide primers G1 and G2 produced an amplicon of size 285 bp [Figure:2] indicating that they may be pathogenic whereas the one water sample was negative with oligonucleotide primers G1 and G2 indicating that it may be a saprophytic leptospira.
Differentiation of pathogenic and saprophytic leptospires by conventional methods.
Lane 1. Molecular marker of 100 bp lader, Lane 2. Isolate D7, Lane 3. Isolate D14, Lane 4. Isolate H12, Lane 5. Isolate W41, Lane 6. Positive Control (Canicola).
Lane 1. Isolate D7, Lane 2. Isolate D14, Lane 3. Sample D18, Lane 4. Sample H6, Lane 5. Isolate H12, Lane 6. Sample H58, Lane 7. Positive Control (Canicola), Lane 8. Molecular marker of 100 bp ladder.
The pathogenic reference strains failed to grow at low temperature (13°C) and in the medium containing 8-azaguanine even after 3-4 weeks of incubation whereas the saprophytic leptospires showed the growth in 4-7 days. When the four isolates obtained were tested, the isolates from serum samples were found to be belonging to the pathogenic group and the isolate from water to saprophytic group.
The conventional methods for differentiation of pathogenic and saprophytic leptospires in addition to being slow and laborious require the isolation of the organisms, which is difficult due to their fastidious nature, fragility for artificial media and overgrowth of contaminants. Moreover, the isolation requires viable organisms to be present in the sample, which is again influenced by various factors such as method of collection, transportation and storage. PCR has the advantage that it does not require the isolation of the organism and detects DNA from both viable and nonviable organisms. This was evident from our results. Out of 27 samples that were found positive by PCR, isolations were obtained from only four samples, which is in accordance with earlier reports.,
In the present study, the primers A and B amplified the DNA of both pathogenic and saprophytic leptospires with an amplicon of size 331 bp which is in agreement with earlier reports.,, The primers G1 and G2 amplified the DNA of only the pathogenic leptospires with an amplicon of size 285 bp which is agreement with earlier reports. , The earlier studies used the PCR for clinical cases but in the present study PCR has been extended to environmental samples.
PCR was found to be a simple and rapid method for differentiation of pathogenic and saprophytic leptospires when compared to conventional method which were time consuming (3-4 weeks), laborious and required the isolation of the organism from the samples. We conclude that PCR is simple, specific and rapid method for detection as well as differentiation of leptospires when compared to conventional methods.
|1||Bal AE, Gravekamp C, Hartskeerl RA, MezaBrewster JD, Korver H, Terpstra WJ. Detection of leptospires in urine by PCR for early diagnosis of leptospires. J Clin Microbiol 1994; 32:1894-1898.|
|2||Brown PD, Gravekamp C, Carringtop DG, Van De Kamp H, Hartskeerl RA, Edwards CN, Reverard CO, Terpstra WJ, Levett PN. Evaluation of polymerase chain reaction for early diagnosis of leptospirosis. J Med Microbiol 1995; 43:110-114.|
|3||Gravekamp C, Van De Kemp H, Franzen M, Carrington D, Schoone GJ, Van Eys GJJM, Everard COR, Hartskeerl RA, Terpstra WJ. Detection of seven species of pathogenic leptospires by PCR using two sets of primers. J Gen Microbiol 1993;139:1691-1700.|
|4||Merien F, Amouriaux P, Perolat P, Baranton G, Girrons IS. Polymerase chain reaction for detection of leptospira species in clinical samples. J Clin Microbiol 1992;30:2219-2224.|
|5||Merien F, Perolat P, Moncel D, Persan D, Baranton G. Detection of leptospira DNA by polymerase chain reaction in aqueous humor of a patient with unilateral uveitis. J Infect Dis 1993;168:1335-1336.|
|6||Merien F, Baranton G, Perolat P. Comparison of Polymerase chain reaction with microagglutination test and culture for diagnosis of leptospirosis. J Infect Dis 1995;172:281-285.|
|7||Romero EC, Billerbeck AEC, Lando VS, Camargo ED, Souza CC, Yasuda PH. Detection of Leptospira DNA in patients with Aseptic meningitis by PCR. J Clin Microbiol 1998;36:1453-1455.|
|8||Van Eys GJJM, Gravekemp C, Gerritsen MJ, Quint W, Cornelissen MTE, Ter Schegget J, Terpstra WJ. Detection of leptospires in urine by polymerase chain reaction. J Clin Microbiol 1989;27:2258-2262. |