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Year : 2006  |  Volume : 24  |  Issue : 4  |  Page : 346-348

Evaluation of recombinant Leptospira interrogans serovar canicola outer membrane proteins as diagnostic antigen

Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh - 22, India

Correspondence Address:
S K Srivastava
Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh - 22
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.29417

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How to cite this article:
Srivastava S K, Chaudhuri P, Thangapandian E, Mariya R, Amutha R. Evaluation of recombinant Leptospira interrogans serovar canicola outer membrane proteins as diagnostic antigen. Indian J Med Microbiol 2006;24:346-8

How to cite this URL:
Srivastava S K, Chaudhuri P, Thangapandian E, Mariya R, Amutha R. Evaluation of recombinant Leptospira interrogans serovar canicola outer membrane proteins as diagnostic antigen. Indian J Med Microbiol [serial online] 2006 [cited 2020 Oct 28];24:346-8. Available from:

Leptospirosis of animals and man is an important re-emerging infectious disease worldwide. In domestic animals it is an important cause of abortion, stillbirth, infertility, decreased milk production and death.[1],[2] In dairy cattle, it is responsible for great economic losses as a consequence of agalactia, abortion, stillbirth, birth of weak calves and reduced fertility.[3],[4] The standard serological test, such as microscopic agglutination test (MAT) is time-consuming and requires technical skill. The demonstration of sero-conversion by using this test requires paired acute and convalescent serum samples limiting it to a few reference laboratories.[5] The other available serological methods uses whole cell antigen preparations[6],[7] for diagnosis as well as for screening of the cases of leptospirosis. The whole-cell antigen preparations possess broadly reactive immunodominant epitopes[8] present in both, pathogenic and non-pathogenic leptospires as well as a diverse group of non-leptospiral species.[9] Recently, recombinant antigens based sero-diagnostic strategies have been developed to conduct seroprevalence studies in human leptospirosis.[10] The important protein antigens residing in the outer membrane of leptospires reported in recent years are LipL32, LipL41 and OmpL1; they are expressed in-vivo during infection.[11],[12] LipL32 and LipL41 are important and surface-exposed leptospiral outer membrane proteins expressed only in pathogenic Leptospira spp.[13] In this study, recombinant LipL32 and LipL41 proteins were tested an antigens in ELISA against serum bovine samples.

 ~ Materials and Methods Top

Growth and maintenance of bacterial strains

The leptospiral strains were maintained in semi-solid EMJH medium. LB broth and LB agar medium were used for E. coli cultures.

Preparation of antisera

The hyper-immune serum against serovar Canicola cells was raised in New Zealand white rabbits of about one kilogram body weight according to the method described by Faine.[14]

For preparation of antiserum against the recombinant proteins, 0.25 mg of the protein in 0.5 ml PBS was mixed with Freund's incomplete adjuvant (FIA, DIFCO Laboratories, USA) and inoculated subcutaneously at two to four sites on the back into each of the two rabbits. The first booster 0.25 mg protein in FIA was given after 14 days of first inoculation followed by two more inoculations of the protein (0.25 mg each) at seven days intervals. Blood was collected three weeks after the first inoculation and titrated using MAT.

Production of rLipL32 and rLipL41 antigens

The primers were designed with restriction sites from the previously reported gene sequences of L. kirschneri serovar Grippotyphosa.[13] The genes encoding LipL32 and LipL41 were amplified from the genomic DNA of Leptospira interrogans serovar Canicola and cloned into pDrive (Qiagen) cloning vector and sequenced. The nucleotide sequence was submitted to GenBank under the accession number AY642287. The genes was sub-cloned into the pPROEXHTb (LipL41) and pPROEXHTc (LipL32) expression vector (Life Technologies) and transformed into  Escherichia More Details coli DH5a cells. The recombinant clones were screened using 1mM IPTG (Isopropyl-a-D-thiogalactopyranoside). The polyhistidine (6X-His) tagged fusion proteins were purified under denaturing conditions by Nickel chelating affinity chromatography. The recombinant proteins were dialyzed and the concentration was determined by Lowry method (Bangalore Genei, India)

Bovine serum samples

A total of 515 bovine serum samples obtained from various parts of the country were screened for the presence of anti-leptospiral antibodies. These animals had the history of reproductive problems like abortion, stillbirth, repeat breeding, weak calves and mastitis.

Microscopic agglutination test

The microscopic agglutination test (MAT) was carried out according to Faine[14] using reference strains of eight different leptospiral serovars viz, Leptospira interrogans serovars Hardjo (Hardjoprajitno), Pomona, Pyrogenes, Icterohaemorrhagiae, L. borgpetersenii serovars Tarassovi, Javanica, Sejroe, Ballum. Reciprocal agglutination titres of greater than or equal to 100 were considered positive.

 ~ Enzyme-linked immunosorbent assay (ELISA) Top

ELISA was done as per standard protocols,[15] with initial standardization done with 10 test serum samples and an equal number of known negative sera. Serum was used at 1:100 dilution. Individual serum samples were tested in duplicate on three different plates. The cut-off O.D. value was calculated as double of the mean value of negative control plus twice the S.D.

The relative sensitivity and specificity of the ELISA were evaluated in comparison to MAT as described below.

Sensitivity = a / (a+b) x 100, where 'a' is the number of sera positive by ELISA and MAT, 'b' the number of sera positive by MAT but negative by ELISA.

Specificity = d / (c+d) x 100, where'd' is the number of sera negative by ELISA and MAT, 'c' the number of sera negative by MAT but positive by ELISA.

 ~ Results and Discussion Top

The optimum concentration of each of the purified recombinant antigens, which showed maximum difference between the positive and negative sera, was determined to be 100 ng/well. Of the total 515 samples, 331 were negative as tested by MAT and ELISA to these recombinant antigens. 95 serum samples were positive by MAT, recombinant(r) LipL32 ELISA and rLipL41 ELISA; 89 sera were negative by MAT of which 71 were positive by both rLipL32 as well as rLipL41 ELISA and 18 were positive by rLipL32 ELISA. The sensitivity and specificity of rLipL32 ELISA as against MAT was calculated to be 100% and 78.8% respectively, whereas using rLipL41 ELISA it was 100% and 83% [Table - 1].

The relative sensitivity using either of the antigens was 100% suggesting they were as effective as M at0 in detecting the true positive cases. However, the specificity was less than MAT. The observation that ELISA was positive in MAT negative cases could be due to the fact that MAT was done with a limited number of leptospiral serovars. Thus our findings support the prevailing view that the ELISA is more sensitive than MAT.[16],[17]

The seroprevalence of leptospirosis based on MAT in animals of various states is given in [Table - 2]. Maximum positive samples were from Uttar Pradesh followed by Tamil Nadu, Gujarat and Kerala. A high prevalence of the disease was observed in animals suggesting that control measures are to be adapted in future to contain the infection. One of the methods of control is the use of a suitable vaccine in the affected population. Before any such attempt is envisaged, information on the prevalence of various serovars in a given population is essential. The study indicated that commonly occurring serovars in animals were Canicola, Pomona and Pyrogenes. Predominance of these serovars in animals has been reported earlier in the country. Development of a vaccine incorporating these serovars will be of advantage for future vaccination studies.

 ~ Acknowledgements Top

The authors are thankful to the Director, IVRI for providing necessary facilities for conducting the research. Thanks are also due to the Department of Biotechnology, Government of India for the financial support.

 ~ References Top

1.Haake DA, Martinich A, Summers TA, Shang ES, Pruetz JD, McCoy AM, et al . Characterization of leptospiral outer membrane lipoprotein LipL36: Downregulation associated with late log-phase growth and mammalian infection. Infect Immun 1998; 66: 1579-87.  Back to cited text no. 1    
2.Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, et al . Peru-United States Leptospirosis Consortium, Leptospirosis: A zoonotic disease of global importance. Lancet Infect Dis 2003; 3 :757-71.  Back to cited text no. 2    
3.Thiermann AB. Leptospirosis: Current developments and trends. J Am Vet Med Assoc 1984; 184 :722-5.  Back to cited text no. 3  [PUBMED]  
4.Yan KT, Ellis WA, Mackie DP, Taylor MJ, McDowell SW, Montgomery JM. Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar Hardjo in cattle. Vet Microbiol 1999; 69 :173-87.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Levett PN, Morey RE, Galloway RL, Turner DF, Steigerwalt AG, Mayer LW. Detection of pathogenic leptospires by real time quantitative PCR. J Med Microbiol 2005; 54 :45-9.  Back to cited text no. 5    
6.Yersin C, Bovet P, Smits HL, Perolat P. Field evaluation of a one-step dipstick assay for the diagnosis of human leptospirosis in the Seychelles. Trop Med Int Hlth 1999 ;4 :38-45.  Back to cited text no. 6    
7.Smits HL, van der Hoon MA, Goris MG, Gussenhoven GC, Yersin C, Sasaki DM, et al . Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis. J Clin Microbiol 2000; 38 :1272-5.  Back to cited text no. 7    
8.Terpstra WJ, Ligthart GS, Schoone GJ. ELISA for the detection of specific IgM and IgG in human leptospirosis. J Gen Microbiol 1985; 131: 377-85.  Back to cited text no. 8  [PUBMED]  
9.Matsuo K, Isogai E, Araki Y. Utilization of extracellular mannan from Rhodotorula glutinis as an immunoreactive antigen in diagnosis of leptospirosis. J Clin Microbiol 2000; 38 :3750-4.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Flannery B, Costa D, Carvalho FP, Guerreiro H, Matsunaga J, da Silva ED, et al . Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis. J Clin Microbiol 2001; 39 :3303-10.  Back to cited text no. 10    
11.Barnett JK, Barnett D, Bolin CA, Summers TA, Wagar EA, Chebille NF, et al . Expression and distribution of leptospiral outer membrane components during renal infection of hamsters. Infect Immunol 1999; 67 :853-61.   Back to cited text no. 11    
12.Matsunaga J, Barocchi MA, Croda J, Young TA, Sanchez Y, Siqueira I, et al . Pathogenic Leptospira species express surface exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol Microbiol 2003; 49 :929-45.  Back to cited text no. 12    
13.Shang ES, Summers TA, Haake DA. Molecular cloning and sequence analysis of the gene encoding LipL41: A surface-exposed lipoprotein of pathogenic Leptospir a species. Infect Immun 1996; 64 :2322-30.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Faine S. Guidelines for the control of leptospirosis . Vol. 27, WHO offset Publication: 1982.  Back to cited text no. 14    
15.Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immuno Chem 1971; 8 :871-4.  Back to cited text no. 15    
16.Ribotta MJ, Higgins R, Gottschalk M, Lalier R. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs. Can J Vet Res 2000; 64: 32-7.  Back to cited text no. 16    
17.Agunloye CA, Alabi FO, Odemuyiwa SO, Olaleye OD. Leptospirosis in Nigerians: A seroepidemiological survey. Indian Vet J 2001; 78 :371-5.  Back to cited text no. 17    


[Table - 1], [Table - 2]

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