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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgements
 ~  References

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Year : 2001  |  Volume : 19  |  Issue : 2  |  Page : 26-29

Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda)

Biotechnology section (MDV), Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore - 560 024, India

Correspondence Address:
Biotechnology section (MDV), Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore - 560 024, India

 ~ Abstract 

DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

How to cite this article:
Venkatesha M D, Ramadass P. Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda). Indian J Med Microbiol 2001;19:26-9

How to cite this URL:
Venkatesha M D, Ramadass P. Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda). Indian J Med Microbiol [serial online] 2001 [cited 2020 May 30];19:26-9. Available from:

Leptospirosis is an acute febrile septicaemic disease caused by spirocheates of species  L.interrogans  . The impact of the disease in the public health aspects acquires more significance especially in countries like India because of large livestock, rodent and wild life populations, animal management practices and close association between man and animals providing a congenial environment for spread of disease. The disease was first observed in dogs by Ayyar[1] and then many reports have been published regarding its prevalence and other aspects from different parts in India. Kaveri and Upadhye[2] isolated serovar icterohaemorrhagiae from urine of a dog. Venkataraman et al [3] isolated serovar canicola from dogs. Ratnam[4] reviewed the status of leptospiral infection in human beings, domestic animals and rodents in India.
Serovar identification of leptospiral isolates is tedious and time consuming but is very essential if detailed information on the epidemiology of leptospirosis in particular geographical area is to be obtained. The conventional method of identification of leptospiral isolates is based on serotyping which involves the comparison of surface antigens of isolates with those of reference strains, measuring the reactions of leptospires with antisera, specific for unknown strains, reference group and serovars. The factor analysis method later developed is also cumbersome and difficult to reproduce in different laboratories. In view of these limitations, the search for alternative methods of identification of isolates have been focused on DNA based techniques like bacterial restriction endonuclease analysis. Though this technique was standardized by Marshall et al [5], in developing countries like India, we still depend on serological typing or sending our isolates for typing to leptospiral reference laboratories in developed countries, primarily because of lack of facilities or expertise. Keeping these in view, the present study was undertaken to prepare standard patterns for reference strains of each serogroup and compare with that of isolates for their identification.

 ~ Materials and Methods Top

A total of 19 serovars representing 19 serogroups of L.interrogans, were used and the serovars represented were autumnalis (Akiyami), australis (Ballico), ballum (Mus 127), bataviae (Swart), canicola (Hond Utrecht IV), cynopteri (3522C), grippotyphosa (Moskva V), hebdomadis ( Hebdomadis), icterohaemorrhagiae (RGA), javanica (Veldrat Bat 46 ), Louisiana (LSU 1945 ), mini
( Sari ), panama (CZ 214), pomona (Pomona) , pyrogenes (Salinem ), ranarum (ICF), sarmin (Sarmin), hardjo ( Hardjoprajitno), tarassovi (Peripellicin), and two reference serovars from L. biflexa namely patoc (Patoc-I), and andamana ( CH-11) were procured from KIT , Amsterdam for the present work.
EMJH media was procured from Difco (Detroit,MI, USA) for maintenance of reference cultures and isolation from clinical samples .Clinical samples such as Blood / urine were collected from suspected cases of leptospirosis from patients from the hospitals. Isolation was also tried from rats and water samples. The clinical samples were subjected to initial concentration by centrifugation and used for isolation work, and the isolates were purified by dilution method. Two isolates VRN8 and VRN9, the media contaminants received from leptospira laboratory, Madhavaram, VRA1 and VRA2 two human isolates received from Regional Medical Research Centre (RMRC), Andaman and Nicobar islands were used for typing. R1, the rat isolate received from department of Animal Biotechnology which was earlier typed as serovar javanica by both serotyping and DNA restriction analysis from reference laboratory was also used in this study.
DNA extraction procedure was followed as per Fischer and Lerman[6] from 7-10 days old liquid cultures. Fifty ml liquid cultures were centrifuged and the pellet was washed twice with PBS (pH 7.2) resuspended in TE buffer (pH8.0). The cells were treated with lysozyme at 370C for 15 min, then with sodium lauryl sulphate and protease at 560C over night, and DNA was extracted with Phenol: Chloroform: Iso amyl alcohol (25:24:1), the DNA was precipitated by ethanol and stored at -200C. BRENDA test was conducted using EcoR I and Hae III enzymes (procured form Bangalore Genei and Amersham, respectively) as per Senthil Kumar et al7. In brief 20mg of DNA was digested in 40mL volume in appropriate restriction buffer with 10-15units of appropriate restriction enzyme at 370C overnight. Digested DNA samples were separated by running on 0.8% agarose gel in a horizontal submarine gel electrophoresis along with DNA molecular markers at 5V/cm at room temperature for 4-6 hr, after which the gel was visualised under UV transilluminator (Fotodyne,USA) and photographed using a Polaroid camera with wratten gelatin filter.

 ~ Results Top

[Figure - 1] and [Figure - 2] show comparative restriction fragment patterns of 21 reference serovars digested with EcoR I enzyme. Restriction patterns observed with this enzyme were unique to each serovar tested and differences were observed mainly at high molecular weight region i.e. between 5.6-21.2 kb. In lane 4 of [Figure - 2] the serovar pyrogenes showed an unique thick band of size more than 7.4 kb and not observed with any other serovars. The banding patterns of saprophytic serovars semaranga and andamana (lanes 9 and 10 ,[Figure - 2] were similar at the region around 4.8kb at higher molecular weight region (i.e.7.4kb) were different.
[Figure - 3] and [Figure - 4] show comparative restriction fragment patterns of all leptospiral reference serovars digested with Hae III enzyme. Complete digestion and resolution of DNA fragments were observed with this enzyme. DNA fragment patterns of each serovar were found unique and could easily be differentiated from other serovars especially at higher molecular weight region i.e. above 3.5kb. The restriction patterns of saprophytic serovars semaranga and andamana (lanes 9 and 10, [Figure - 4] were different through out.
[Figure:5] shows comparative restriction fragment patterns of leptospiral isolates digested with Hae III enzyme. Complete digestion of DNA of all isolates were obtained and were compared with that of reference strains for their identification. Lanes 2, 3 and 4 representing VRM13, VRA1 and VRA2 had banding patterns similar to lane 1 containing serovar australis The lanes 5 and 6 containing VRN8 and VRN9 showed similar patterns with serovar andamana ( lane 10, [Figure - 4]. Based on restriction patterns comparison , a total of ten isolates were typed [Table - 1], amongst which three belongs to the Australis serogroup, two belongs to serogroup Grippotyphosa and one belongs to Autumnalis serogroup. The two isolates from media contaminants referred by Leptospira laboratory, Madhavaram, TANUVAS were found belongs to saprophytic group Andamana. The rat isolate R1 was earlier typed as javanica along with the other rat isolate VRR2 were showed similarity with serovar javanica..

 ~ Discussion Top

In recent years examination of genomic DNA by restriction endonuclease analysis has gained much attention in the classification and identification of bacteria and viruses[8] From the results of present study it is observed that the digestion of genomic DNA from various serovars with EcoR I enzyme has produced unique banding patterns for each serovar. The patterns for each serovar was distinct and could be easily differentiated from the other with a number of differences through out the length of the patterns with the exception of saprophytic serovars which have similar pattern at region around 4.8kb. Our findings are in agreement with the earlier reports that, Restriction endonuclease analysis (REA) patterns produced by each serovar was distinct and represent a different spacing of EcoR I cleavage site in the nuclear DNA and this enzyme was found to produce stable and reproducible patterns.[5],[9] Theirman et al [10] reported that restriction enzymes EcoR I and Hha I were found to be enzymes of choice for restriction enzyme analysis as they produced complete digestion and good resolution of high molecular weight fragments. But the restriction patterns produced by Hae III enzyme used in this study was also found to be superior and could be used for differentiation of serovars. The digestion patterns with this enzyme showed complete digestion and best resolution of DNA fragments. The differentiation could be possible especially at higher molecular weight region i.e. above 3.5 kb . The restriction patterns of saprophytic serovars were not similar through out. The results are on par with earlier findings. [7],[11]
This study throws light on the prevalence of these four pathogenic serovars namely australis, autumnalis, grippotyphosa and javanica. We believe that the bacterial restriction endonuclease analysis (BRENDA) technique is a sensitive and objective method for differentiation of leptospiral serovars and identification of isolates.

 ~ Acknowledgements Top

Authors are grateful to Koninklijk Institut voor de Tropen (KIT) NH, Amsterdam, for providing leptospiral cultures for this work. 

 ~ References Top

1.Ayyar KB. A note on out break of leptospiral jaundice among Madras haunds. Ind J Vet Sci Anim Husb 1932; 2 : 160.   Back to cited text no. 1    
2.Kaveri SV. Upadhye AS. Isolation of L.ictero-haemorrhagiae from dogs. Ind J Comp Microbiol Immunol Infect Dis 1981; 2 : 19 -20.  Back to cited text no. 2    
3.Venkataraman K S, Nedunchellian S, Ramakrishna J, Ramadass P, Raghavan N. Isolation of canicola leptospiral serovar from urine of a dog. Ind Vet J 1992; 69 : 866.  Back to cited text no. 3    
4.Ratnam S. Leptospirosis: An Indian perspective Ind J Med Microbiol 1994; 12 :228-239.  Back to cited text no. 4    
5.Marshall RB, Whitton BE and Robinson AJ. Identification of leptospira serovars by restriction endonuclease analysis. J Clin Microbiol 1981; 14: 163-166.  Back to cited text no. 5    
6.Fischer SG. Lerman LS. Length dependent separation of DNA restriction fragments in two dimensional gel electrophoresis. Cell 1979; 16: 191.  Back to cited text no. 6    
7.Senthil Kumar A, Ramadass P, Nachimuthu K. Typing of leptospiral serovars by DNA restriction enzyme analysis. J Anim Sci 1997; 67: 457-459.  Back to cited text no. 7    
8.O Hara MJ, Collins DM, De Lise GW. Restriction endonuclease analysis of Brucella species. Vet Microbiol 1985; 10: 425-429.   Back to cited text no. 8    
9.Bolin CA. Zuerner R. Correlation between DNA restriction fragment length polymorphisms in Leptospira interrogans serovar pomona type Kennewicki and host animal source. J Clin Microbiol 1996; 34: 424-425.  Back to cited text no. 9    
10.Thiermann AB, Handsaker AL, Mosely SL, Kingscote B. New method for classification of leptospiral isolates belonging to serogroup pomona by restriction endonuclease analysis. J Clin Microbiol 1985; 21: 585-587.  Back to cited text no. 10    
11.Tamai T, Sada E, Kobayashi Y. Restriction endonuclease DNA analysis of L.interrogans serovars icterohaemorrhagiae and copenhageni. Microbiol Immunol 1988;32:887-894.  Back to cited text no. 11    
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