Development of leptospiral outer membrane protein-based dot-ELISA assays and immunoprofiling for diagnosis of acute canine leptospirosis
The present study was carried out to evaluate outer membrane protein (OMP)-based relatively simple dot-ELISA assays for the diagnosis of acute canine leptospirosis and to identify the differential expression of leptospiral OMPs for identifying the markers of acute infection, if any. Among 91 canine serum samples screened, 63 samples were found to be positive by MAT. All the samples were tested by OMP-based IgM and IgG dot ELISAs. The sensitivities of IgM and IgG dot ELISA in comparison to MAT (titer: ≥1:400) were found to be 100%, whereas the specificities were found to be 89.71% and 45.31%, respectively. IgM and IgG immunoblotting of OMPs was standardized and tested with the MAT-positive samples. IgM immunoblotting of leptospiral OMP revealed proteins of 18, 21, and 32 kDa as major reactors. With IgG, proteins of 32, 41, and 66 kDa were recognized as major reactors and an 86-kDa protein was identified as a minor reactor. The 32-kDa protein was the predominant reactor in both IgM and IgG immunoblots. Thus, a combination of 18-, 21-, and 32-kDa proteins could be a good choice for the detection of IgM antibodies indicative of acute infection while that of 32-, 41-, and 66-kDa proteins could be employed for seroepidemiological studies.
___
- Vijayachari P. Leptospirosis, Laboratory Manual. New Delhi,
India: World Health Organization Country Office for India;
2007.
- Koteeswaran A. Seroprevalence of leptospirosis in man and
animals in Tamil Nadu. Indian J Med Microbiol 2006; 24: 329-
331.
- Balakrishnan G, Govindarajan R, Meenambigai TV, Jayakumar
V, Manohar MB. Seroprevalence of animal leptospirosis in
certain parts of Tamil Nadu. Indian Vet J 2008; 85: 227-228.
- Abhinay G, Joseph S, Ambily R. Seroprevalence of canine
leptospirosis. Indian Vet J 2012; 89: 72-73.
- Ambily R, Mini M, Jospeh S, Abhinay G, Krishna SV.
Outer
membrane protein based
latex agglutination test for diagnosis
of canine leptospirosis in Kerala, India. International Journal of
Advanced Research 2014; 2: 684-687.
- Levett PN. Leptospira and leptonema. In: Murray PR, Baron
EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Manual
of Clinical Microbiology. Washington, DC, USA: American
Society for Microbiology; 2003. pp. 929-936.
- Levett PN. Leptospirosis. Clin Microbiol Rev 2001; 14: 296-326.
- Singh A, Kumar M, Srivastava SK. Diagnostic utility of a
dominant outer membrane protein of
Leptospira borgpetersenii
serovar Hardjo
.
Indian J Comp Microbiol Immunol Infect Dis
2004;
25: 90-93.
- Srivastava SK, 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: 346-348.
- Flannery B, Costa D, Carvalho FP, Guerreiro H, Matsunaga
J, Silva ED, Ferreira AGP, Riley LW, Reis MG, Haake DA
et al. Evaluation of recombinant
Leptospira
antigen-based
enzyme-linked immunosorbent assays for the serodiagnosis of
leptospirosis. J Clin Microbiol 2001; 39: 3303-3310.
- Abhinay G, Joseph S, Ambily R. IgM Immunoprofiling
of leptospiral outer membrane proteins in acute canine
leptospirosis. Indian Vet J 2012; 89: 9-10.
- Office International-des-Epizooties. Manual of Standards for
Diagnostic Tests and Vaccines. 4th ed. Paris, France: OIE; 2008.
- Nicholson VM, Prescott JF. Outer membrane proteins of three
pathogenic
Leptospira
spp.
Vet Microbiol 1993; 36: 123-138.
- Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein
measurement with Folin-phenol reagent. J Biol Chem 1951;
193: 265-271.
- Laemmli UK. Cleavage of structural proteins during the
assembly of the head of bacteriophage T
4
. Nature 1970; 227:
680-685.
- Towbin H, Stachelin T, Gerdon J. Electrophoretic transfer of
protein from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. P Natl Acad Sci USA 1979;
76: 4350-4354.
- Sharma R, Tuteja U, Shukla RKJ, Batra HV. Application of
rapid dot-ELISA for antibody detection of leptospirosis. J Med
Microbiol 2007; 56: 873-874.
Alves FSF, Lefebvre RB, Probert W. Identification of outer
envelope proteins conserved among
Leptospira
serovars. Rev
Med Vet 1999; 150: 877-884.
- Cullen PA, Cordwell SJ, Bulach DM, Haake DA, Adler B.
Global analysis of outer membrane proteins from
Leptospira
interrogans
serovar Lai. Infect Immun 2002; 70: 2311-2318.
- Joseph S. Efficacy of recombinant LipL21 protein of
Leptospira
interrogans
serovar Canicola for the diagnosis of leptospirosis
in animals. PhD, Indian Veterinary Research Institute, Bareilly,
Uttar Pradesh, India, 2007.
- Sankar S, Harshan HM, Somarajan SR, Srivastava SK. Evaluation
of a recombinant LigB protein of
Leptospira interrogans
serovar
Canicola in an enzyme-linked immunosorbent assay for the
serodiagnosis of bovine leptospirosis. Res Vet Sci 2010; 88:
375-378.
- Joseph S, Thomas N, Thangapandian E, Singh VP, Verma
R., Srivastava SK. Evaluation and comparison of native and
recombinant LipL21 protein-based ELISAs for diagnosis of
bovine leptospirosis. J Vet Sci 2012; 13: 99-101.
- Vijayachari P, Sugunan AP, Umapathi T, Sehgal SC. Evaluation
of dark ground microscopy as a rapid diagnostic procedure in
leptospirosis. Indian J Med Res 2001; 114: 54-58.
- Pappas MG, Ballou WR, Gray MR, Takafuji ET, Miller RN,
Hockmeyer WT. Rapid serodiagnosis of leptospirosis using the
IgM-specific dot-ELISA: Comparison with the microscopic
agglutination test. Am J Trop Med Hyg 1985; 34: 346-354.
- Tansuphasiri U, Deepradit S, Phulsuksombati D, Tangkanakul
W. A test strip IgM dot-ELISA assay using leptospiral antigen
of endemic strains for serodiagnosis of acute leptospirosis. J
Med Assoc Thai 2005; 88: 391-398.
- Guerreiro H, Croda J, Flannery B, Mazel M, Matsunaga J,
Reis MG, Levett PN, Ko AI, Haake DA. Leptospiral proteins
recognized during the humoral immune response to
leptospirosis in human. Infect Immun 2001; 69: 4958-4968.
- Natarajaseenivasan K, Vijayachari P, Sugunan AP, Sharmaan
S, Sehgal SC. Leptospiral proteins expressed during acute and
convalescent phases of human leptospirosis. Indian J Med Res
2004; 151-159.