Journal articles: 'Leptospira recombinant protein'

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Cullen, Paul A., David A. Haake, Dieter M. Bulach, Richard L. Zuerner, and Ben Adler. "LipL21 Is a Novel Surface-Exposed Lipoprotein of Pathogenic Leptospira Species." Infection and Immunity 71, no. 5 (2003): 2414–21. http://dx.doi.org/10.1128/iai.71.5.2414-2421.2003.

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ABSTRACT Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

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S., Senthil Kumar, M.Parthiban, and Agastian P. "Immunoprotection studies using Leptospiral recombinant proteins from L. interrogans Icterohaemorrhagiae in hamster models : A synergistic approach." Biolife 4, no. 4 (2022): 678–86. https://doi.org/10.5281/zenodo.7350368.

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ABSTRACT Major immunodominant proteins of Leptospira interrogans Icterohaemorrahagiae were cloned, expressed in prokaryotic system was purified and confirmed by western blot. The protective efficacy of purified recombinant proteins were assayed synergistically with adjuvant in hamster models. Three combination were assessed for homologous protection (i.e, rLipL41, rOmpL1, rHapI and rSphH; rLipL41, rOmpL1, rLigA and rLigB; rLigA, rLigB, rHapI and rSphH) each protein at 25 µg and 37.5 µg concentration. Among the combinations tested combination of immunogloblulins (rLigA and rLigB) with hemolysins (rHapI and rSphH) confered 100% protection in both the concentration tested, that is evident through the elevated elicited antibody levels.  The combination of outer membrane proteins and the immunoglobulins stands next which is comparable with the former combination.  The blend of recombinant outermembrane proteins and hemolysins did effect protection which is lesser than the coalescence of immunoglobulins and hemolysins.  Membrane fractions of E. coli harboring pET15b used as internal control showed neither homologous protection nor enhanced antibody levels used to compare the experimental proteins over hamster models.  Placebo controls receiving PBS showed similar results as that of internal controls. Key words : Leptospiral  recombinant protein, recombinant proteins, outer membrane proteins, hemolysins, immunoglobulin like proteins, immune response, challenge studies, , homology modeling REFERENCES: Alves, V.A., Gayotto, L.C., Brito, T.De., Santos, R.T., Wakamatsu, A., Vianna, M.R. and Sakata, E.E. (1992).  Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage. Exp. Toxicol. Pathol. 44(7): 425-434. Artiushin, S., Timoney, J.F., Nally, J. and Verma. A. (2004).  Host inducibile immunogenic Sphingomyelinases like protein, Lk73.5, of Leptospira interrogans.  Infect. Immun. (72): 742-749. Bartlett, J.M.S. and Stirling, D. (2004). DNA Extraction form Fungi, Yeast and Bacteria. PCR Protocols Second Edition, Methods in Molecular Biology, Humana Press, (226): 53-54. Bernheimer , A.W., and Bey, R.F. 1986. Copurification of Leptospira interrogans serovar pomona hemolysin and Sphingomyelinase C. Infect. Immun. 54(1): 262-264. Bolin CA, Cassells, J.A., Zuerner, R.L. and Trueba, G. (1991). Effect of vaccination with a monovalent Leptospira interrogans  serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle. Am. J. Vet. Res. (52): 1639-1643. Branger, C., Sonrier, C., Chatrenet, B., Klonjkowski, B. and Ruvoen-Clouet, N. (2001). Identification of the hemolysis-associated protein I as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination.  Infect. Immun. (69): 6831-6838. Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F. and Aubert, A. (2005). Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein I.  Infect. Immun. (73): 4062-4069. Carvalho, E., Barbosa, A.S,  Gomez, R.M., Oliveira, M.L.S., Romero, E.C.,  Goncales, A.P., Morais, Z.M., Vasconcellos, S.A. and Ho, P.L. (2010).  Evaluation of the Expression and Protective Potential of Leptospiral Sphingomyelinases. Curr. Microbiol. (60): 134-142. Choy, H.A., Kelley, M.M., Chen, T.L., Moller,A.K., Matsunaga, J. and Haake, D.A. (2007).  Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect. Immun. (75): 2441-2450. Coutinho, M.L., Choy, H.A., Kelley, M.M., Matsunaga, J., Babbitt, J.T., Lewis, M.S., Aleixo, J.G. and Haake, D.A. (2011).  A LigA three domain region protects hamsters from lethal infection by Leptospira interrogans.  PLoS Negl. Trop. Dis. 5(12): e1422. Porika Raju and Estari Mamidala (2015). Anti-diabetic activity of compound isolated from Physalis angulata fruit extracts in alloxan induced diabetic rats. The Ame J Sci & Med Res, 2015,1(1); Pages 1 -6. doi:10.17812/ajsmr2015.11.1. Croda, J., Ramos, J.G.R., Matsunaga, J., Queiroz, A., Homma, A., Riley, L.W., Haake, D.A., Reis, M.G. and Ko, A.I. (2007).  Leptospira immunoglobulin like proteins as a serodiagnostic marker for acute Leptospirosis. J. Clin. Microbiol. 45(5): 1528-1534. Croda, J., Figueira, C.P., Wunder Jr., A.E., Santos, C.S., Reis, M.G., Ko, A.I. and Picardeau, M. (2008).  Targeted Mutagenesis in pathogenic Leptospira species: Disruption of the LigB gene does not affect virulence in animal models of leptospirosis.  Infect. Immun. 76(12): 5826-5833. Faine, S. (1994).  Leptospira and leptospirosis, 1st ed. CRC press, Boca Raton, Fla. Faucher, J.F, Hoen, B. and Estavoyer, J.M. (2004). The management of Leptospirosis. Expert Opin. Pharmacother. 5(4): 819-827.. Goldstein, S.F. and Charon, N.W. (1990).  Multiple exposure photographic analysis of motile spirochetes. Proc Natl Acad Sci, (87): 4895-4899. Haake, D.A., Mazel, M.K., McCoy, A.M., Milward, F., Chao, G., Matsunga, J. and Wager, E.A., (1999). Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immune protection. Infect Immun. (67): 6572-6582. Isogai, E., Isogai, H., Kurebayashi, Y. and Ito, N. (1986). Biological activities of Leptospiral lipopolysaccharide. Zentralbl. Bakteriol. Mikrobiol. Hyg. A. (261): 53-64. Koizumi, N. and Watanabe, H. (2004).  Leptopsira immunoglobulin-like protein elicit protective immunity.  Vaccine (22): 1545-1552. Lee, S.H., Sangduk, K., Seung, C.P. and Min, J.K. (2002).  Cytotoxic Activities of Leptospira interrogans  Hemolysin SphH as a Pore-Forming Protein on Mammalian Cells. Infect. Immun. (70): 315-322. Levett, P.N. (2001).  Leptospirosis. Clin. Microbiol. Rev. (14): 296-326. Lourdault, K., Wang, L.C., Vieira, A., Matsunaga, J., Melo, R., Lewis, M.S., Haake, D.A. and Solecki, M.G. (2014).  Oral immunization with Escherichia coli expressing a lapidated form of LigA protects hamsters against challenge with Leptospira interrogans serovar copenhageni.  Infect. Immun. 82(2): 893-902. Lucas, D.S., Cullen, P.A., Lo, M., Srikram, A. and Sermswan, R.W. (2011).  Recombinant LipL32 and LigA from Leptospira are unable to stimulate protective immunity against leptospirosis in the hamster model.  Vaccine. (29): 3413-3418. Matsunaga, J., Barocchi, M.A., Croda, J., Young, T.A., Sanchez, Y., Siqueira, I., Bolin, C.A., Reis, M.G., Riley, L.W., Haake, D.A. and Ko, A.I. 2003.  Pathogenic Leptospira species express surface exposed proteins belonging to the bacterial immunoglobulin superfamily.  Mol. Microbiol. (49): 929-945. Matsunaga, J., Werneid, K., Zuerner, R.L., Frank, A. and Haake, D.A. (2006).  LipL46 is a novel surface exposed lipoprotein expressed during Leptospiral dissemination in the mammalian host. Microbiology. (152): 3777-3786. Merino, S., Rubires, X., Knoche, S. and Tomas, J.M. (1995).  Emerging pathogens: Aeromonas spp. Int. J. Food. Microbiol. (28): 157-168. Natarajaseenivasan, K., Shanmughapriya, S., Velineni, S., Artiushin, S.C. and Timoney, J.F. (2011). Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans  Serovar Autumnalis Strain N2. Genomics Proteomics Bioinform. 9(4-5): 151. Palaniappan, R.U., Chang, Y.F., Jusuf, S.S.D., Artiushin, S., Timoney, J.F., McDonough, S.P., Barr, S.C., Divers, T.J., Simpson, K.W., McDonough, P.L. and Mohammed, H.O. (2002). Cloning and Molecular Characterization of an Immunogenic LigA Protein of Leptospira interrogans. Infect. Immun. 70(11): 5924-5930. Palaniappan, R.U., Chang, Y.F., Hassan, F., McDonough, S.P., Pough, M., Barr, S.C., Simpson, K.W., Mohammed, H.O., Shin, S., McDonough, P.L., Zuerner, R.L., Qu, J. and Roe, B. 2004.  Expression of Leptospiral immunoglobulin like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Palaniappan, R.U., McDonough, S.P., Divers, T.J., Chen, C.S., Pan, M.J., Matsumoto, M. and Chang, Y.F. (2006).  Immunoprotection of recombinant Leptospiral immunoglobulin like protein A against Leptospira interrogans serovar Pomona infection. Infect. Immun. (74(3)): 1745-1750. Parthiban, M., Senthil Kumar, S., Balachandran, C., Kumanan, K., Aarthi, K.S. and Nireesha, G. 2015.  Comparison of Immunoprotection of Leptospira recombinant proteins with conventional vaccine in experimental animals. Ind. J. Exp. Biol. (53): 779-785. Patti, J.M., Allen, B.L., McGavin, M.J. and Hook, M. (1994).  MSCRAMM-mediated adherence of microorganisms to host tissues. Ann. Rev. Microbiol. (48): 585-617. Picardeau, M.D., Bulach, D.M. and Bouchier., C. (2008).  Genome sequence of the saprophyte Leptospira biflexa provides insightsinto the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS ONE 3: 1607 Pinnie, M. and Haake, D.A. (2009).  A comprehensive approach to identification of surface-exposed, outer  membrane spanning proteins of Leptospira interrogans. PLoS One (4): e6071. Seixas, F.K., da Silva, E.F., Hartwig, D.D., Cerqueira, G.M., Amaral, M. (2007).  Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamster from challenge.  Vaccine. 26: 88-95. Silva, E.F., Medeiros, M.A., McBride, A.J., Matsunaga, J., Esteves, G.S., Ramos, J.G., Santos, C.S., Croda, J., Homma, A. and Dellgostin, O.A. (2007).  The terminal portion of Leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis.  Vaccine. 25(33): 6277-6286. Trowbridge, A.A., Green, J.B., Bonnet, J.D., Shohet, S.B., Ponnappa, B.D. and Mccombs, W.B. 1981. Hemolytic anemia associated with leptospirosis morphologic and lipid studies. Am. J. Clin. Pathol. (76): 493-498. Zuerner, R.L., Alt, D.P., Palmer, M.V., Thacker, T.C. and Olsen, S.C. (2011).  A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin. Vaccine. Immunol. (18): 684-691.

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Barbosa, Angela S., Denize Monaris, Ludmila B. Silva, et al. "Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP." Infection and Immunity 78, no. 7 (2010): 3207–16. http://dx.doi.org/10.1128/iai.00279-10.

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ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

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Cavenague, Maria F., Aline F. Teixeira, Luis G. V. Fernandes, and Ana L. T. O. Nascimento. "LIC12254 Is a Leptospiral Protein That Interacts with Integrins via the RGD Motif." Tropical Medicine and Infectious Disease 8, no. 5 (2023): 249. http://dx.doi.org/10.3390/tropicalmed8050249.

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Pathogenic leptospires can bind to receptors on mammalian cells such as cadherins and integrins. Leptospira effectively adheres to cells, overcomes host barriers and spreads into the bloodstream, reaching internal target organs such as the lungs, liver and kidneys. Several microorganisms produce proteins that act as ligands of integrins through the RGD motif. Here, we characterized a leptospiral RGD-containing protein encoded by the gene lic12254. In silico analysis of pathogenic, intermediate and saprophytic species showed that LIC12254 is highly conserved among pathogenic species, and is unique in presenting the RGD motif. The LIC12254-coding sequence is greatly expressed in the virulent Leptospira interrogans L1-130 strain compared with the culture-attenuated L. interrogans M20 strain. We also showed that the recombinant protein rLIC12254 binds to αVβ8 and α8 human integrins most likely via the RGD motif. These interactions are dose-dependent and saturable, a typical property of receptor–ligand interactions. The binding of the recombinant protein lacking this motif—rLIC12254 ΔRAA—to αVβ8 was almost totally abolished, while that with the α8 human integrin was decreased by 65%. Taken together, these results suggest that this putative outer membrane protein interacts with integrins via the RGD domain and may play a key role in leptospirosis pathogenesis.

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S., Senthil Kumar, M.Parthiban, and Agastian P. "Invivo and Invitro studies on carboxy terminal domains of leptospiral immunoglobulins of L. interrogans Icterohaemorrhagiae." Biolife 4, no. 3 (2022): 521–29. https://doi.org/10.5281/zenodo.7332898.

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ABSTRACT Carboxy terminal domains of leptospiral immunoglobulins of rLigA and rLigB were assessed its hemolytic, cytotoxicity effects invitro and its protective efficacy was studied in hamster models. Experimental recombinant proteins of Leptospira interrogans Icterohaemorrhagiae were individually cloned, expressed in prokaryotic system.  Purified expressed proteins confirmed by western blot, assessed for its protective efficacy using hamster models.  100% and 93% protection on homologues challenge was observed with rLigA and rLigB with adjuvant respectively which is evident with increased levels of antibody levels of rLigA and rLigB with 98% and 95% specificity.  Cytotoxicity was maximum with 14% in Daudi cells with with rLigB and 7% and rLigA.  17% and 4% hemolytic activity with rLigA, and rLigB protein and 11% and 10% in pH-7.0 respectively. E. coli (harboring pET15b) membrane fraction used as placebo neither exerted cytotoxicity nor hemolysis was used to compare the leptospiral proteins.  Leptospiral immunoglobulins showed least cytotoxicity and hemolytic activity with increased homologues protection in experimental animals Keywords: Leptospira recombinant protein, Leptospiral immunoglobulins, immune response, challenge studies, cytotoxic and hemolytic activity. REFERENCES Alves, V.A., Gayotto, L.C., Brito, T.De., Santos, R.T., Wakamatsu, A., Vianna, M.R., and Sakata. E.E (1992). Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage. Exp. Toxicol.  Pathol. (44): 425-434. Andre-Fontaine, G., Branger, C., Gray, A.W and Klaasen, H.L. (2003).  Comparison of the efficiacy of three commercial bacterins in preventing canine leptospirosis.  Vet. Rec. (153): 165-169. Bartlett, J.M.S. and Stirling, D. (2004).  DNA Extraction form Fungi, Yeast and Bacteria.  PCR Protocols Second Edition, Methods in Molecular Biology, Humana Press, (226): 53-54. Bernheimer, A.W. and Bey, R.F. (1986). Copurification of Leptospira interrogans serovar pomona hemolysin and sphingomyelinase C. Infect. Immun. (54): 262-264. Bulach, D.M., Kalambaheti, T., Dela pena, M.A. and Adler, B. (2000). Functional analysis of genus is the rfb locus of Leptospira borgpetersenii serovar hardjo subtype hardjobovis. Infect. Immun.  (68): 3793-3798. Choy, A.H., Kelley, M.M., Chen, T.L., Moller, A.K., Matsunga, J. and Haake., D.A. (2007).  Physiological osmotic induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extra cellular Matrix Proteins and Fibrinogen. Infect. Immun. (75):  2441-2450. Croda, J., Ramos, J.G., Matsunga, J., Queiroz, A., Homma, A., Riley, L.W., Haake, D.A., Reis, M.G. and Ko, A.I. (2007).  Leptospira Immunoglobulin-like proteins as a serodiagnositc marker for acute leptospirosis.  J. Clin. Microbiol. (45): 1528-1534. Croda, J., Figueira C.P., Wunder Jr.,, A.E., Santos, C.S., Reis, M.G. and Ko, A.I. (2008).  Targeted Mutagenesis in Pathogenic Leptospira species: Disruption of the LigB gene does not affect virulence in animal models of Leptospirosis. Infect. Immun.  (76(12)) : 5826-5833. Del Real, G., Segers, R.P.A.M., Van der Zejst, B.A.M. and Gaastra, W. (1989). Cloning of a hemolysin gene from Leptospira interrogans serovar hardjo. Infect. Immun. (57): 2588-2590. Goldstein, S.F. and Charon, N.W. (1990). Multiple exposure photographic analysis of motile spirochetes. Pro. Nat. Acad.  Sci. (87): 4895-4899. Haake, D.A., Mazel, M.K., McCoy, A.M., Milward, F., Chao, G., Matsunga, J. and Wager, E.A. (1999). Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immune protection. Infect. Immun. (67): 6572-6582. Hartwig, D.D., Oliveira, T.L., Seixas, F.K., Forster, K.M., Rizzi, C., Hartleben, C.P., McBride A.J.A. and Dellagostin, O.A. (2010). High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris.  Microb. cell Fact. (9): 98. Isogai, E., Isogai, H., Kurebayashi,Y. and Ito, N. (1986). Biological activities of Leptospiral lipopolysaccharide. Zentralbl Bakteriol Mikrobiol Hyg A. (261): 53- 64. Ito, T. and Yanagawa, R. (1987). Leptospiral attachment to extracellular matrix of mouse fibroblast (L929) cells. Vet. Microbiol. (15): 89-96. Lee, S.H., Sangduk, K., Seung, C.P. and Min, J.K. (2002). Cytotoxic Activities of Leptospira interrogans Hemolysin SphH as a Pore-Forming Protein on Mammalian Cells. Infect. Immun. (70): 315-322. Levett, P. N. (2001). Leptospirosis. Clin. Microbiol. Rev. (14): 296-326. Matsunga, J., barocchi, M.A., Croda, J., Young, T.A., Sanches, Y., Siqueira, I., Bolin, C.A., Reis, M.G., Riley, L.W., Haake, D.A. and Ko, A.I. (2003). Pathogenic Leptospira species express surface exposed proteins belonging to the bacterial immunoglobulin superfamily. Mol. Microbiol. (49): 929-945. Matsunga, J., Sanchez, Y., Xu, X. and Haake, D.A (2005).  Osmolarity, a key environmental signal controlling expression of leptospiral proteins LigA and LigB and the extracellular release of LigA.  Infect. Immun. (73): 70-78. Naiman, B.M., Blumerman, S., Alt, D., Bolin, C.A., Brown, R., Zuerner, R. and Baldwin, C.L. (2002). Evaluation of type 1 immune response in  aive and vaccinated animals following challenge with Leptospira borgpetersenii serovar hardjo: involvement of WC1and CD4 T cells. Infect. Immun. (70): 6147-6157. Natarajaseenivasan, K., Shanmughapriya, S., Velineni, S., Artiushin, S.C. and Timoney, J.F. (2011). Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2. Genomics Proteomics Bioinform. (9(4-5)): 151-157. Palaniappan, R.U., Chang, Y.F., Hassan, F., Jusuf, S.S., Artiushin, Timoney, F.,  McDonough, S.P.,  Barr, S.C.,  Drivers T.J.,  Simpson, K.W., McDounough, P. and Mohammed, H.O.  (2002).  Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Palaniappan, R.U., Chang, Y.F., Hassan, F., McDouough S.P., Pough, M., Barr, S.C., Simpson, K.W., Mohammed, H.O., Shin, S., McDounough, P., Zuerner, R.L., Qu, J. and Roe, B. (2004).  Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA. J. Med. Microbiol. (53): 975-984. Sateesh Pujari and Estari Mamidala (2015). Anti-diabetic activity of Physagulin-F isolated from Physalis angulata fruits. The Ame J Sci & Med Res, 2015,1(1):53-60. doi:10.17812/ajsmr2015113. Palaniappan, R.U., McDouough S.P., Drivers T.J., Chen, C.S., Pan, M.J.,  Matsumoto, M and Chang Y.F. (2006).  Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection. Infect. Immun. (74): 1745-1750. Patti, J.M., Allen, B.L., McGavin, M.J. and Hook, M. (1994). MSCRAMM-mediated adherence of microorganisms to host tissues.  Ann. Rev. Microbiol. (48): 585-617. Pinkney, M., Beachey, E. and Kehoe, M. (1989). The thio-activated toxin streptolysin O does not require a thiol group for cytolytic activity. Infect. Immun. (57): 2553-2558. Raghavan, U., Palaniappan, M., Chang, Y.F., Jusuf, S.S.D., Artiushin, S., Timoney, J.F., McDonough, S.P., Barr, S.C., Divers T.J., Simpson, K.W., McDonough, P.L. and Mohammed H.O. (2002). Cloning and Molecular Characterization of an Immunogenic LigA Protein of Leptospira interrogans. Infect. Immun. (70(11)): 5924-5930. Raghavan, U., Palaniappan, M., Mc Donough, S.P., Divers, T.J., Chen, C.S., Pan, M.J.,  Matsumoto, M. and Chang, Y.F. (2006).  Immunoprotection of Recombinant Leptospiral Immunoglobulin like Protein a against Leptospira interrogans serovar Pomona infection.  Infect. Immun. (74): 1745-1750. Reed, L.J. and Muench, H. (1938).  A simple method of estimating fifty percent end points Am. J. . Hyg. (27): 493. Senthilkumar, T.M.A., Subathra, M. and Ramadass, P. (2008). Latex agglutination test for the detection of canine leptospiral antibodies using recombinant OmpL1 antigen. Veterinarski Arhiv (78): 393-399. Zuerner, R,L, Alt, D.P, Palmer, M.V, Thacker, T.C. and Olsen, S.C. (2011). A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin.  Vac. Immunol. (18): 684.

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Lan Anh, Le Thi, Minh Thi Hang, Nguyen Thi Thu Hien, et al. "Cloning, expression and purification of Leptospira LigB antigen in Escherichia coli." Vietnam Journal of Biotechnology 17, no. 3 (2020): 569–75. http://dx.doi.org/10.15625/1811-4989/17/3/14364.

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Leptospira is one of the most common zoonotic diseases in the tropics and subtropics. Humans are infected by exposure to Leptospira contained water or food sources. Leptospirosis usually breaks out after the flood and causes several consequences for people and economy. Leptospirosis disease, if not rapidly detected and treated promptly, it causes serious consequences such as acute hepatitis-kidneys, meningitis and bleeding, heart and nerve complications, and severe illness can lead to death. Therefore, quick and accurate detection of Leptospira pathogen plays a very important role in Leptospirosis disease treatment. Among antigens of Leptospira, a conserved domain of LigB antigen (Leptospiral immunoglobulin-like protein) was reported that is present in the most of pathogenic serovars of Leptospira, but not in the non-pathogenic Leptospira biflexa, thus this conserved domain was used for production of Leptospirosis detection kits as well as vaccine for Leptospirosis. In order to create a kit for Leptospirosis diagonostic, especially detect anti-Leptospira antibodies in Leptospira infected serum and plasma samples, about 1kb gene fragment encoding for conserved domain of LigB (about 36 kb in molecular weight) was used as the material for producing of LigB protein by DNA recombinant technology. In this study, we present the results for cloning, expressing a conserved domain of LigB antigen in E. coli cells and purifying protein by affinity chromatography collumn. The result indicates that recombinant LigB protein was successfully expressed in E. coli Rosetta 1 and purified by Hitrap chealating collumn. The LigB protein concentration after purification reached 60 mg/L medium with 98% purity. This purified protein will be used as the materials for creating Leptospirosis kit.

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Zhu, Weinan, Felipe J. Passalia, Camila Hamond, et al. "MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection." PLOS Pathogens 19, no. 7 (2023): e1011313. http://dx.doi.org/10.1371/journal.ppat.1011313.

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Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch’s molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen’s ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

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Chagas-Junior, Adenizar D., Alan J. A. McBride, Daniel A. Athanazio, et al. "An imprint method for detecting leptospires in the hamster model of vaccine-mediated immunity for leptospirosis." Journal of Medical Microbiology 58, no. 12 (2009): 1632–37. http://dx.doi.org/10.1099/jmm.0.014050-0.

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In determining the efficacy of new vaccine candidates for leptospirosis, the primary end point is death and an important secondary end point is sterilizing immunity. However, evaluation of this end point is often hampered by the time-consuming demands and complexity of methods such as culture isolation (CI). In this study, we evaluated the use of an imprint (or touch preparation) method (IM) in detecting the presence of leptospires in tissues of hamsters infected with Leptospira interrogans serovar Copenhageni. In a dissemination study, compared to CI, the IM led to equal or improved detection of leptospires in kidney, liver, lung and blood samples collected post-infection and overall concordance was good (κ=0.61). Furthermore, in an evaluation of hamsters immunized with a recombinant leptospiral protein-based vaccine candidate and subsequently challenged, the agreement between the CI and IM was very good (κ=0.84). These findings indicate that the IM is a rapid method for the direct observation of Leptospira spp. that can be readily applied to evaluating infection in experimental animals and determining sterilizing immunity when screening potential vaccine candidates.

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Kochi, Leandro Toshio, Luis Guilherme Virgílio Fernandes, and Ana Lucia Tabet Oller Nascimento. "Heterologous Expression of the Pathogen-Specific LIC11711 Gene in the Saprophyte L. biflexa Increases Bacterial Binding to Laminin and Plasminogen." Pathogens 9, no. 8 (2020): 599. http://dx.doi.org/10.3390/pathogens9080599.

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Leptospirosis is a febrile disease and the etiological agents are pathogenic bacteria of the genus Leptospira. The leptospiral virulence mechanisms are not fully understood and the application of genetic tools is still limited, despite advances in molecular biology techniques. The leptospiral recombinant protein LIC11711 has shown interaction with several host components, indicating a potential function in virulence. This study describes a system for heterologous expression of the L. interrogans gene lic11711 using the saprophyte L. biflexa serovar Patoc as a surrogate, aiming to investigate its possible activity in bacterial virulence. Heterologous expression of LIC11711 was performed using the pMaOri vector under regulation of the lipL32 promoter. The protein was found mainly on the leptospiral outer surface, confirming its location. The lipL32 promoter enhanced the expression of LIC11711 in L. biflexa compared to the pathogenic strain, indicating that this strategy may be used to overexpress low-copy proteins. The presence of LIC11711 enhanced the capacity of L. biflexa to adhere to laminin (Lam) and plasminogen (Plg)/plasmin (Pla) in vitro, suggesting the involvement of this protein in bacterial pathogenesis. We show for the first time that the expression of LIC11711 protein of L. interrogans confers a virulence-associated phenotype on L. biflexa, pointing out possible mechanisms used by pathogenic leptospires.

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Kumari, Anita, Mallela Martha Premlatha, Veerapandian Raja, et al. "Protective immunity of recombinant LipL21 and I-LipL21 against Leptospira interrogans serovar Autumnalis N2 infection." Journal of Infection in Developing Countries 12, no. 01 (2018): 022–30. http://dx.doi.org/10.3855/jidc.9545.

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Introduction: Leptospirosis is a zoonotic disease caused by the spirochete of genus Leptospira with widespread distribution in tropical, subtropical and temperate zones. Leptospirosis is often confused with other febrile illnesses including jaundice, dengue, and malaria. Generally, the disease is often underdiagnosed or misdiagnosed. Though leptospirosis is curable with antibiotic treatment, the laboratory diagnosis of the disease is specialized and open to interpretation with multiple kits available to detect the different serological markers of Leptospira. Moreover, when leptospirosis is misdiagnosed, the disease can lead to multi-organ failure and may have fatal effects. There is a need for strategies to develop vaccines and prevent leptospirosis. In the present study, the immunogenic potential of leptospiral recombinant protein LipL21 (rLipL21) and its truncated form I-LipL21 (rI-LipL21) was evaluated. Methodology: The recombinant proteins were established in cyclophosphamide treated BALB/c mice model infected with L. interrogans serovar Autumnalis strain N2. Results: The vaccination study showed 66% and 83% survivability among mice immunized with rLipL21 and rI-LipL21 respectively and post-challenge with leptospiral strain N2 compared to control groups that showed 100% lethality. Additionally, a significant increase in antibody levels and cytokine levels (TNF-a, IFN-γ and IL-10) was observed evidencing a marked stimulation of both humoral and cell-mediated immune response in mice immunized with rLipL21/rI-LipL21 compared to whole cell leptospiral lysate (WCL). Conclusions: This study evidenced protective immunization against leptospirosis with rLipL21 and rI-LipL21 recombinant proteins and are potential candidates for the development of leptospiral vaccine.

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Takahashi, Maria Beatriz, Aline Florencio Teixeira, and Ana Lucia Tabet Oller Nascimento. "Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans." BioTechniques 74, no. 3 (2023): 137–42. http://dx.doi.org/10.2144/btn-2022-0076.

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The production of leptospiral recombinant proteins in the soluble form and in high yield from Escherichia coli is still a challenge. This work presents the cloning, expression and purification of the outer membrane protein of Leptospira interrogans, LipL21, which is considered an interesting target for vaccine and diagnostics development. The expression profile and yield of LipL21 was compared after cloning in the vectors pAE, pET28a and pET-SUMO, and it was observed that LipL21 was expressed in a low amount with pAE vector. By using the pET-28a vector, protein expression was increased, but the majority of the product was obtained as inclusion bodies. As a highlight, using a pET-SUMO vector was shown to overcome the problems of low expression and solubility of the lipoprotein LipL21.

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Atzingen, Marina V., Amane P. Gonçales, Zenaide M. de Morais, et al. "Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans." Journal of Medical Microbiology 59, no. 9 (2010): 1005–15. http://dx.doi.org/10.1099/jmm.0.021485-0.

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Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6×His were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

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Longhi, Mariana T., Tatiane R. Oliveira, Eliete C. Romero, et al. "A newly identified protein of Leptospira interrogans mediates binding to laminin." Journal of Medical Microbiology 58, no. 10 (2009): 1275–82. http://dx.doi.org/10.1099/jmm.0.011916-0.

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Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host–pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

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Palaniappan, Raghavan U. M., Sean P. McDonough, Thomas J. Divers, et al. "Immunoprotection of Recombinant Leptospiral Immunoglobulin-Like Protein A against Leptospira interrogans Serovar Pomona Infection." Infection and Immunity 74, no. 3 (2006): 1745–50. http://dx.doi.org/10.1128/iai.74.3.1745-1750.2006.

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ABSTRACTWe previously reported the cloning and characterization of leptospiral immunoglobulin-like proteins LigA and LigB ofLeptospira interrogans. LigA and LigB are conserved at the amino-terminal region but are variable at the carboxyl-terminal region. Here, we evaluate the potential of recombinant LigA (rLigA) as a vaccine candidate against infection byL. interrogansserovar Pomona in a hamster model. rLigA was truncated into conserved (rLigAcon) and variable (rLigAvar) regions and expressed inEscherichia colias a fusion protein with glutathione-S-transferase (rLigA). Golden Syrian hamsters were immunized at 3 and 6 weeks of age with rLigA (rLigAcon and rLigAvar) with aluminum hydroxide as an adjuvant. Hamsters given recombinant glutathione-S-transferase (rGST)-adjuvant and phosphate-buffered saline-adjuvant served as nonvaccinated controls. Three weeks after the last vaccination, all animals were challenged intraperitoneally with 108L. interrogansserovar Pomona bacteria (NVSL 1427-35-093002). All hamsters immunized with recombinant LigA survived after challenge and had no significant histopathological changes. In contrast, nonimmunized and rGST-immunized hamsters were subjected to lethal doses, and the hamsters that survived showed severe tubulointerstitial nephritis. All vaccinated animals showed a rise in antibody titers against rLigA. Results from this study indicate that rLigA is a potential vaccine candidate againstL. interrogansserovar Pomona infection.

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Sripattanakul, Sineenat, Teerasak Prapong, Attapon Kamlangdee, et al. "Leptospira borgpetersenii Leucine-Rich Repeat Proteins and Derived Peptides in an Indirect ELISA Development for theDiagnosis of Canine Leptospiral Infections." Tropical Medicine and Infectious Disease 7, no. 10 (2022): 311. http://dx.doi.org/10.3390/tropicalmed7100311.

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Domestic and stray dogs can be frequently infected by Leptospira, and thus may represent a source for transmission of this zoonotic disease in Thailand. Here, we have used peptides derived from a recombinant leucine-rich repeat (LRR) protein of Leptospira, rKU_Sej_LRR_2012M, for the development of an indirect enzyme-linked immunosorbent assay (ELISA) aimed at detecting antibodies against Leptospira interrogans, L. borgpetersenii and L. biflexa, the three major seroprevalences in Thai dogs. The rKU_Sej_LRR_2012M protein is recognized by hyperimmune sera against several leptospiral serovars. The epitope peptides of the rKU_Sej_LRR_2012M showed binding affinities with lower IC50 values than peptides of known antigenic protein LipL32. Four peptides, 2012-3T, 2012-4B, 2012-5B and pool 2012-B, were specifically recognized by rabbit hyperimmune sera against nine serovars from three Leptospira spp. The indirect peptide-based ELISAs with these four peptides were evaluated with the LipL32 ELISA by using a receiver–operator curve (ROC) analysis. All peptides had an area under the curve of ROC (AUC) greater than 0.8, and the sum of sensitivity and specificity for each peptide was greater than 1.5. The degree of agreement of 2012-3T and pool 2012-B and 2012-4B and 2012-5B peptides were in moderate-to-good levels with kappa values of 0.41–0.60 and 0.61–0.80, when compared with LipL32, respectively. This finding would suggest an excellent capability of the 2012-4B and 2012-5B peptide-based ELISAs assay for the diagnosis of canine leptospiral infections.

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Fernandes, Luis G. V., Kátia E. S. Avelar, Eliete C. Romero, Marcos B. Heinemann, Karin Kirchgatter, and Ana L. T. O. Nascimento. "A New Recombinant Multiepitope Chimeric Protein of Leptospira interrogans Is a Promising Marker for the Serodiagnosis of Leptospirosis." Tropical Medicine and Infectious Disease 7, no. 11 (2022): 362. http://dx.doi.org/10.3390/tropicalmed7110362.

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The zoonotic disease leptospirosis is caused by pathogenic species of the genus Leptospira and was recently included in the list of Neglected Diseases by the World Health Organization. Leptospirosis burden is estimated to have over a million human cases and cause 60 thousand deaths annually, in addition to its economic impact and veterinary concern. The microscopic agglutination test (MAT), recommended by the World Health Organization, exhibits reduced sensitivity at the beginning of the disease, in addition to being technically difficult. New recombinant antigens are being pursued for rapid and specific serodiagnostic tests, especially in the initial phase of the disease, and chimeric multiepitope proteins are a strategy with a great potential to be implemented in serology. Based on previous subproteomic results, we designed a synthetic construct comprising 10 conserved leptospiral surface antigens, and the recombinant protein was purified and evaluated regarding its diagnostic potential. The protein termed rChi2 was recognized by antibodies in serum from patients both at the onset (MAT−) and in the convalescent (MAT+) phase in 75 and 82% of responders, respectively. In addition, rChi2 immunization in hamsters elicited a strong humoral response, and anti-rChi2 antibodies recognized several immobilized intact Leptospira species, validating its potential as an early, broad, and cross-reactive diagnostic test.

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Verma, Ashutosh, Jens Hellwage, Sergey Artiushin, et al. "LfhA, a Novel Factor H-Binding Protein of Leptospira interrogans." Infection and Immunity 74, no. 5 (2006): 2659–66. http://dx.doi.org/10.1128/iai.74.5.2659-2666.2006.

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ABSTRACTThe early phase of leptospiral infection is characterized by the presence of live organisms in the blood. PathogenicLeptospira interrogansis resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenicL. interrogansproduces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed byL. interrogansduring mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

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Artiushin, S., J. F. Timoney, J. Nally, and A. Verma. "Host-Inducible Immunogenic Sphingomyelinase-Like Protein, Lk73.5, of Leptospira interrogans." Infection and Immunity 72, no. 2 (2004): 742–49. http://dx.doi.org/10.1128/iai.72.2.742-749.2004.

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ABSTRACT Leptospira interrogans causes a variety of clinical syndromes in animals and humans. Although much information has accumulated on the importance of leptospiral lipopolysaccharide in protective antibody responses, relatively little is known about proteins that participate in immune responses. Identification of those proteins induced only in the host is particularly difficult. Using a novel double-antibody screen designed to identify clones in a gene library of L. interrogans serovar Pomona expressing host-inducible proteins, we have characterized a gene (lk75.3) encoding a sphingomyelinase-like preprotein of 648 amino acids with cytotoxic activity for equine pulmonary endothelial cells and weak hemolytic activity for equine and rabbit erythrocytes. lk73.5 was found as a single gene copy in all serovars of L. interrogans but not in other Leptospira spp. except L. inadai. The open reading frame (ORF) for Lk73.5 is followed by another partially homologous sequence containing an ORF (sph-like 2) for a 28.7-kDa peptide. Lk73.5 and Sph-like 2 share 95.1 and 97.7% amino acid identity with putative sphingomyelinases Sph2 and Sph1 (N terminus) from L. interrogans serovar Lai (S.-X. Ren, G. Fu, X.-G. Jiangk, R. Zeng, Y.-G. Miao, H. Xu, Y.-X. Zhang, H. Xiong, G. Lu, L.-F. Lu, H.-Q. Jiang, J. Jia, Y.-F. Tu, J.-X. Jiang, W.-Y. Gu, Y.-Q. Zhang, Z. Cai, H.-H. Sheng, H.-F. Yin, Y. Zhang, G.-F. Zhu, M. Wank, H.-L. Huangk, Z. Qian, S.-Y. Wang, Wei Ma, Z.-J. Yao, Y. Shen, B.-Q. Qiang, Q.-C. Xia, X.-K. Guo, A. Danchinq, I. S. Girons, R. L. Somerville, Y.-M. Wen, M.-H. Shik, Z. Chen, J.-G. Xuk, and G.-P. Zhao, Nature 422:88-893, 2003). Substantial homologies to sphingomyelinases from other leptospiras and other bacteria are also present. Lk73.5 was not detected in leptospiras cultured at 30 or 37°C. The recombinant protein reacted strongly with sera from recently infected mares but not with sera from horses vaccinated with commercial pentavalent bacterin. The host-inducible immunogenic Lk73.5 should have value in distinguishing vaccine from infection immune response.

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Prapong, Siriwan, Yada Tansiri, Tepyuda Sritrakul, et al. "Leptospira borgpetersenii Leucine-Rich Repeat Proteins Provide Strong Protective Efficacy as Novel Leptospiral Vaccine Candidates." Tropical Medicine and Infectious Disease 8, no. 1 (2022): 6. http://dx.doi.org/10.3390/tropicalmed8010006.

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Leucine-rich repeat (LRR) proteins are advocated for being assessed in vaccine development. Leptospiral LRR proteins were identified recently in silico from the genome of Leptospira borgpetersenii serogroup Sejroe, the seroprevalence of leptospiral infections of cattle in Thailand. Two LRR recombinant proteins, rKU_Sej_LRR_2012M (2012) and rhKU_Sej_LRR_2271 (2271), containing predicted immunogenic epitopes, were investigated for their cross-protective efficacies in an acute leptospirosis model with heterologous Leptospira serovar Pomona, though, strains from serogroup Sejroe are host-adapted to bovine, leading to chronic disease. Since serovar Pomona is frequently reported as seropositive in cattle, buffaloes, pigs, and dogs in Thailand and causes acute and severe leptospirosis in cattle by incidental infection, the serogroup Sejroe LRR proteins were evaluated for their cross-protective immunity. The protective efficacies were 37.5%, 50.0%, and 75.0% based on the survival rate for the control, 2012, and 2271 groups, respectively. Sera from 2012-immunized hamsters showed weak bactericidal action compared to sera from 2271-immunized hamsters (p < 0.05). Therefore, bacterial tissue clearances, inflammatory responses, and humoral and cell-mediated immune (HMI and CMI) responses were evaluated only in 2271-immunized hamsters challenged with virulent L. interrogans serovar Pomona. The 2271 protein induced prompt humoral immune responses (p < 0.05) and leptospiral tissue clearance, reducing tissue inflammation in immunized hamsters. In addition, protein 2271 and its immunogenic peptides stimulated splenocyte lymphoproliferation and stimulated both HMI and CMI responses by activating Th1 and Th2 cytokine gene expression in vaccinated hamsters. Our data suggest that the immunogenic potential renders rhKU_Sej_LRR_2271 protein a promising candidate for the development of a novel cross-protective vaccine against animal leptospirosis.

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Dezhbord, Mehrangiz, Majid Esmaelizad, Pejvak Khaki, Fariba Fotohi, and Athena Zarehparvar Moghaddam. "Molecular identification of the ompL1 gene within Leptospira interrogans standard serovars." Journal of Infection in Developing Countries 8, no. 06 (2014): 688–93. http://dx.doi.org/10.3855/jidc.3174.

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Introduction: Leptospirosis, caused by infection with pathogenic Leptospira species, is one of the most prevalent zoonotic diseases in the world. Current leptospiral vaccines are mainly multivalent dead whole-cell mixtures made of several local dominant serovars. Therefore, design and construction of an efficient recombinant vaccine for leptospirosis control is very important. OmpL1 is an immunogenic porin protein that could be of special significance in vaccination and serodiagnosis for leptospirosis. Methodology: Three strains belonging to pathogenic L. interrogans were analyzed. The specific primers for proliferation of the ompL1 gene were designed. The amplified gene was cloned. In order to investigate the ompL1 nucleotide sequence and homological analysis of this gene, ompL1 genes cloned from standard vaccinal Leptospira serovars prevalent in Iran were sequenced and cloned. Results: PCR amplification of the ompL1 gene using the designed primers resulted in a 963 bp ompL1 gene product. The PCR based on the ompL1 gene detected all pathogenic reference serovars of Leptospira spp. tested. Based on alignment and phylogenetic analysis, although the ompL1 nucleotide sequence was slightly different within three vaccinal serovars (100%-85% identity), amino acid alignment of the OmpL1 proteins revealed that there would be inconsiderable difference among them. Conclusion: The ompL1 gene of the three isolates was well conserved, differing only by a total of 6 bp and the proteins by 2 amino acids. The cloned gene could be further used for expression and recombinant OmpL1 as an efficient and conserved antigen, and may be a useful vaccine candidate against leptospirosis in our region.

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Hoke, David E., Suhelen Egan, Paul A. Cullen, and Ben Adler. "LipL32 Is an Extracellular Matrix-Interacting Protein of Leptospira spp. and Pseudoalteromonas tunicata." Infection and Immunity 76, no. 5 (2008): 2063–69. http://dx.doi.org/10.1128/iai.01643-07.

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ABSTRACT LipL32 is the major outer membrane protein in pathogenic Leptospira. It is highly conserved throughout pathogenic species and is expressed in vivo during human infection. While these data suggest a role in pathogenesis, a function for LipL32 has not been defined. Outer membrane proteins of gram-negative bacteria are the first line of molecular interaction with the host, and many have been shown to bind host extracellular matrix (ECM). A search for leptospiral ECM-interacting proteins identified the major outer membrane protein, LipL32. To verify this finding, recombinant LipL32 was expressed in Escherichia coli and was found to bind Matrigel ECM and individual components of ECM, including laminin, collagen I, and collagen V. Likewise, an orthologous protein found in the genome of Pseudoalteromonas tunicata strain D2 was expressed and found to be functionally similar and immunologically cross-reactive. Lastly, binding activity was mapped to the C-terminal 72 amino acids. These studies show that LipL32 and an orthologous protein in P. tunicata are immunologically cross-reactive and function as ECM-interacting proteins via a conserved C-terminal region.

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Kamalzadeh, K., M. Esmaelizad, J. Ghalani, P. Khaki, and M. Tebianian. "High expression of LipL21 protein of Iranian Leptospira interrogans in E. coli, applicable for diagnostic ELISA." BULGARIAN JOURNAL OF VETERINARY MEDICINE 26, no. 3 (2023): 342–50. http://dx.doi.org/10.15547/bjvm.2021-0052.

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Leptospirosis is an emerging infectious zoonotic disease caused by pathogenic Leptospira. The disease is more prevalent among farmers in hot and humid areas of Iran. Lack of clear clinical signs have impeded the diagnosis of leptospirosis. In this study, we attempted to produce a recombinant LipL21 protein of Leptospira based on a dominant pattern of Iranian isolates and to evaluate it in ELISA test. One hundred and sixty-two complete sequences of LipL21 available in GenBank until January 1, 2019 were compared. One dominant LipL21 protein pattern was selected. The codon optimised sequence was cloned into the pET32a+ expression vector. Trx-LipL21 fusion protein was induced, purified and confirmed by 10% SDS-PAGE followed Coomassie blue staining and immune blotting. For evaluation of effectiveness of rLip21 in ELISA test, 200 µg rLip21 with Montanide ISA70 adjuvant was injected subcutaneously in rabbits three times. Results showed that rLipL21 protein was highly expressed in 2YT media in presence of 0.1 mM IPTG after 16 hours incubation at 37 oC. Recombinant protein was purified 36 mg per liter using affinity batch formation method by Ni-NTA resin. ELISA with micro plate coated with 250 ng rLipL21 protein demonstrated prominently differences between test and control groups (P<0.01). The rLipL21 protein produced large amounts of antibodies in the rabbit. The protein was also able to detect high levels of antibody in animals immunised with Leptospira vaccine. The rLipL21 might be a good candidate for diagnosis and evaluation of antibody levels against Leptospira.

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Telfer, Janice C., Lauren Le Page, Dipika Nandi, et al. "WC1 is a hybrid gammadelta TCR coreceptor and pattern recognition receptor for pathogenic bacteria." Journal of Immunology 198, no. 1_Supplement (2017): 226.19. http://dx.doi.org/10.4049/jimmunol.198.supp.226.19.

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Abstract WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. Present in variable, and sometimes high, numbers in most animals with adaptive immune systems, in cattle there are 13 distinct genes (WC1-1 to WC1-13). The structure of a multigenic WC1 gene array, with each WC1 protein containing multiple SRCR domains in its extracellular domain, has been conserved over millions of years of evolutionary time, suggesting that they act as a panel of pattern recognition receptors for conserved pathogen ligands. All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation-dependent manner and some are required for the γδ T cell response to Leptospira. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is comprised of amino acids in three discontinuous regions. In addition, recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. WC1 SRCR domains also bind to Borrelia burgdorferi and Mycobacterium spp. WC1 in other artiodactyls such as goat, sheep and swine are closely related, but show some divergence that may have been driven by natural selection by species-specific pathogens. Our data suggest that WC1 gene arrays play a multi-faceted role in the γδ T cell response to bacteria in multiple species, including acting as hybrid pattern recognition receptors and TCR coreceptors, and may function as anti-microbials.

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Hsu, Shen-Hsing, Li-Fang Chou, Chung-Hung Hong та ін. "Crosstalk between E-Cadherin/β-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis". International Journal of Molecular Sciences 22, № 23 (2021): 13132. http://dx.doi.org/10.3390/ijms222313132.

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Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor β-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/β-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

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Padilha, Bárbara Couto Roloff, Henrique Queiroz Simão, Thais Farias Collares, et al. "ErpY-like protein, a promising antigen to leptospirosis control: characterization of antigenic and immunogenic potential." Acta Scientiarum. Biological Sciences 44 (August 22, 2022): e61160. http://dx.doi.org/10.4025/actascibiolsci.v44i1.61160.

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ErpY-like protein (LIC11966) is an antigen from Leptospira spp., which is possibly involved in the infection process and, consequently, can be a promising solution for the development of new diagnostic tests and vaccines. Here, the presence of the erpY-like gene was evaluated in several Leptospira serovars by polymerase chain reaction (PCR), and the ErpY-like recombinant protein was produced and characterized in terms of antigenicity and immunogenicity in vivo. The erpY-like gene was detected by PCR in all Leptospira pathogenic serovars tested (n = 8) and was absent in the saprophytic ones. The rErpY-like protein was recognized by antibodies present in the sera of humans and animals (swine and canine) naturally infected, suggesting ErpY-like expression during natural infection. The rErpY-like protein used to immunize mice with Freund’s adjuvant stimulated a mixed Th1/Th2 response, an important protective immunity against leptospirosis

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Humphryes, P. C., M. E. Weeks, M. AbuOun, G. Thomson, A. Núñez, and N. G. Coldham. "Vaccination with Leptospiral Outer Membrane Lipoprotein LipL32 Reduces Kidney Invasion of Leptospira interrogans Serovar Canicola in Hamsters." Clinical and Vaccine Immunology 21, no. 4 (2014): 546–51. http://dx.doi.org/10.1128/cvi.00719-13.

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ABSTRACTTheLeptospira interrogansvaccines currently available are serovar specific and require regular booster immunizations to maintain protection of the host. In addition, a hamster challenge batch potency test is necessary to evaluate these vaccines prior to market release, requiring the use of a large number of animals, which is ethically and financially undesirable. Our previous work showed that the N terminus of the outer membrane protein LipL32 was altered inLeptospira interrogansserovar Canicola vaccines that fail the hamster challenge test, suggesting that it may be involved in the protective immune response. The aim of this study was to determine if vaccination with LipL32 protein alone could provide a protective response against challenge withL. interrogansserovar Canicola to hamsters. Recombinant LipL32, purified from anEscherichia coliexpression system, was assessed for protective immunity in five groups of hamsters (n= 5) following a challenge with the virulentL. interrogansserovar Canicola strain Kito as a challenge strain. However, no significant survival against theL. interrogansserovar Canicola challenge was observed compared to that of unvaccinated negative controls. Subsequent histological analysis revealed reduced amounts ofL. interrogansin the kidneys from the hamsters vaccinated with recombinant LipL32 protein prior to challenge; however, no significant survival against theL. interrogansserovar Canicola challenge was observed compared to that of unvaccinated negative controls. This finding corresponded to a noticeably reduced severity of renal lesions. This study provides evidence that LipL32 is involved in the protective response againstL. interrogansserovar Canicola in hamsters and is the first reported link to LipL32-induced protection against kidney invasion.

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Pires, Henrique M., Igor R. M. Silva, Aline F. Teixeira, and Ana L. T. O. Nascimento. "Revisiting Host-Binding Properties of LigA and LigB Recombinant Domains." Microorganisms 13, no. 6 (2025): 1293. https://doi.org/10.3390/microorganisms13061293.

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Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Despite the increasing number of studies, the mechanisms of leptospiral pathogenesis remain poorly comprehended. In this study, we report various interactions of the LigA7’-13’ and LigB1’-7’ domains with host components. The LigA7’-13’ and LigB1’-7’ were cloned into the pET28a vector, and the recombinant proteins were expressed in E. coli C43 (DE3) and E. coli BL21 (DE3), respectively. Both recombinant protein domains were expressed in soluble form and purified using nickel-chelating chromatography. The rLigA7’-13’ and rLigB1’-7’ domains exhibited binding to several types of integrins, with most interactions occurring in a dose-dependent and saturable manner, consistent with the characteristics of typical receptor-ligand interactions. The recombinant domain LigA7’-13’ demonstrated affinity for the glycosaminoglycans (GAGs) chondroitin-4-sulfate, chondroitin sulfate, heparin, chondroitin sulfate B, and heparan sulfate, while no binding was detected for LigB1’-7’ with these molecules. Both rLigA7’-13’ and rLigB1’-7’ interacted with components of the terminal complement pathway and were capable of recruiting C9 from normal human serum (NHS). These interactions may inhibit the formation of polyC9, ultimately preventing the assembly of the membrane attack complex (MAC). Collectively, our data expand the repertoire of host components that interact with rLigA7’-13’ and rLigB1’-7’, opening new avenues for understanding leptospiral immune evasion and broadening the roles of these domains in bacterial virulence.

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Fernandes, Luis G. V., Monica L. Vieira, Karin Kirchgatter, et al. "OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp." Infection and Immunity 80, no. 10 (2012): 3679–92. http://dx.doi.org/10.1128/iai.00474-12.

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ABSTRACTLeptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genusLeptospira. OmpL1 is an outer membrane protein ofLeptospiraspp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed inEscherichia coliBL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, withKD(equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with aKDof 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein ofLeptospiraspp. that may play a role in bacterial pathogenesis when expressed during infection.

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Llanos Salinas, Samantha Paulina, Luz Olivia Castillo Sánchez, Giselle Castañeda Miranda, et al. "GspD, The Type II Secretion System Secretin of Leptospira, Protects Hamsters against Lethal Infection with a Virulent L. interrogans Isolate." Vaccines 8, no. 4 (2020): 759. http://dx.doi.org/10.3390/vaccines8040759.

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The wide variety of pathogenic Leptospira serovars and the weak protection offered by the available vaccines encourage the search for protective immunogens against leptospirosis. We found that the secretin GspD of the type II secretion system (T2S) of Leptospira interrogans serovar Canicola was highly conserved amongst pathogenic serovars and was expressed in vivo during infection, as shown by immunohistochemistry. Convalescent sera of hamsters, dogs, and cows showed the presence of IgG antibodies, recognizing a recombinant version of this protein expressed in Escherichia coli (rGspDLC) in Western blot assays. In a pilot vaccination study, a group of eight hamsters was immunized on days zero and 14 with 50 µg of rGspDLC mixed with Freund’s incomplete adjuvant (FIA). On day 28 of the study, 1,000 LD50 (Lethal Dose 50%) of a virulent strain of Leptospira interrogans serovar Canicola (LOCaS46) were inoculated by an intraoral submucosal route (IOSM). Seventy-five percent protection against disease (p = 0.017573, Fisher’s exact test) and 50% protection against infection were observed in this group of vaccinated hamsters. In contrast, 85% of non-vaccinated hamsters died six to nine days after the challenge. These results suggest the potential usefulness of the T2S secretin GspD of Leptospira as a protective recombinant vaccine against leptospirosis.

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Seixas, Fabiana Kömmling, Claudia Hartleben Fernandes, Daiane Drawanz Hartwig, Fabricio Rochedo Conceição, José Antônio Guimarães Aleixo, and Odir Antônio Dellagostin. "Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis." Canadian Journal of Microbiology 53, no. 4 (2007): 472–79. http://dx.doi.org/10.1139/w06-138.

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Leptospirosis, caused by bacteria of the genus Leptospira , is a direct zoonosis with wide geographical distribution. The implications in terms of public health and the economical losses caused by leptospirosis justify the use of a vaccine against Leptospira in human or animal populations at risk. In this study, we used the external membrane protein LipL32 as a model antigen, as it is highly immunogenic. The LipL32 coding sequence was cloned into several expression vectors: (i) pTarget, to create a DNA vaccine; (ii) pUS973, pUS974, and pUS977 for expression in BCG (rBCG); and (iii) pAE, to express the recombinant protein in Escherichia coli , for a subunit vaccine. Mice were immunized with the various constructs, and the immune response was evaluated. The highest humoral immune response was elicited by the subunit vaccine (rLipL32). However, with rBCG, the titer was still rising at the end of the experiment. The serum of vaccinated animals was able to recognize LipL32 on the membrane of the Leptospira, detected by indirect immunofluorescence. A monoclonal antibody anti-LipL32 was shown to inhibit the growth of Leptospira in vitro, indicating potential protection induced by the LipL32 antigen.

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Sumarningsih, Sumarningsih, Gita Sekarmila, Andi Mulyadi, Ahpas Ahpas, and Simson Tarigan. "Development of In-House ELISA using recombinant LipL32 for Detection of Human Leptospirosis in Indonesia." Jurnal Sain Veteriner 42, no. 1 (2024): 32. http://dx.doi.org/10.22146/jsv.90085.

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Early laboratory confirmation is important for the accurate diagnosis and treatment of patient infected by leptospirosis. However, Microscopic agglutination test (MAT) as the gold standard for detection of human leptospirosis has many limitation and only available in reference laboratories. Therefore, many studies suggested LipL32 protein as a good candidate for development of leptospirosis detection kit because it is highly conserved and produced only in pathogenic Leptospira species. In this study, we aim to investigate the performance of our in-house ELISA using recombinant LipL32 to detect leptospirosis in Indonesia. Fourteen human sera were used in this study and the infection status were determine using MAT. The result showed that nine of eleven MAT positive sera were successfully recognized by LipL32 ELISA. The antibody binding to LipL32 was also confirm by immunoblot. There was one of three MAT negative sera has high OD above 0.5 in ELISA, but it showed negative reaction in immunoblot result. Overall, this study demonstrated that recombinant LipL32 protein can recognized antibody from human leptospirosis and can be used as a universal antigen to detect infection by any serovars of pathogenic leptospira.

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Techawiwattanaboon, Teerasit, Christophe Barnier-Quer, Tanapat Palaga, et al. "A Comparison of Intramuscular and Subcutaneous Administration of LigA Subunit Vaccine Adjuvanted with Neutral Liposomal Formulation Containing Monophosphoryl Lipid A and QS21." Vaccines 8, no. 3 (2020): 494. http://dx.doi.org/10.3390/vaccines8030494.

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Leptospirosis vaccines with higher potency and reduced adverse effects are needed for human use. The carboxyl terminal domain of leptospiral immunoglobulin like protein A (LigAc) is currently the most promising candidate antigen for leptospirosis subunit vaccine. However, LigAc-based vaccines were unable to confer sterilizing immunity against Leptospira infection in animal models. Several factors including antigen properties, adjuvant, delivery system, and administration route need optimization to maximize vaccine efficacy. Our previous report demonstrated protective effects of the recombinant LigAc (rLigAc) formulated with liposome-based adjuvant, called LMQ (neutral liposome combined with monophosphoryl lipid A and Quillaja saponaria fraction 21) in hamsters. This study aimed to evaluate the impact of two commonly used administration routes, intramuscular (IM) and subcutaneous (SC), on immunogenicity and protective efficacy of rLigAc-LMQ administrated three times at 2-week interval. Two IM vaccinations triggered significantly higher levels of total anti-rLigAc IgG than two SC injections. However, comparable IgG titers and IgG2/IgG1 ratio was observed for both routes after the third immunization. The route of vaccine administration did not influence the survival rate (60%) and renal colonization against lethal Leptospira challenge. Importantly, the kidneys of IM group showed no pathological lesions while the SC group showed mild damage. In conclusion, IM vaccination with rLigAc-LMQ not only elicited faster antibody production but also protected from kidney damage following leptospiral infection better than SC immunization. However, both tested routes did not influence protective efficacy in terms of survival rate and the level of renal colonization.

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Barbosa, Liana Nunes, Alejandro LIanes, Swetha Madesh, et al. "Enhancement of clinical signs in C3H/HeJ mice vaccinated with a highly immunogenic Leptospira methyl-accepting chemotaxis protein following challenge." PLOS Neglected Tropical Diseases 18, no. 9 (2024): e0012155. http://dx.doi.org/10.1371/journal.pntd.0012155.

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Leptospirosis is the most widespread zoonosis and a life-threatening disease in humans and animals. Licensed killed whole-cell vaccines are available for animals; however, they do not offer heterologous protection, do not induce long-term protection, or prevent renal colonization. In this study, we characterized an immunogenic Leptospira methyl-accepting chemotaxis protein (MCP) identified through a reverse vaccinology approach, predicted its structure, and tested the protective efficacy of a recombinant MCP fragment in the C3H/HeJ mice model. The predicted structure of the full-length MCP revealed an architecture typical for topology class I MCPs. A single dose of MCP vaccine elicited a significant IgG antibody response in immunized mice compared to controls (P < 0.0001), especially the IgG1 and IgG2a subclasses. The vaccination with MCP, despite eliciting a robust immune response, did not protect mice from disease and renal colonization. However, survival curves significantly differed between groups, and the MCP-vaccinated group developed clinical signs faster than the control group. There were differences in gross and histopathological changes between the MCP-vaccinated and control groups. The factors leading to enhanced disease process in vaccinated animals need further investigation. We speculate that anti-MCP antibodies may block the MCP signaling cascade and may limit chemotaxis, preventing Leptospira from reaching its destination, but facilitating its maintenance and replication in the blood stream. Such a phenomenon may exist in endemic areas where humans are highly exposed to Leptospira antigens, and the presence of antibodies might lead to disease enhancement. The role of this protein in Leptospira pathogenesis should be further evaluated to comprehend the lack of protection and potential exacerbation of the disease process. The absence of immune correlates of protection from Leptospira infection is still a major limitation of this field and efforts to gather this knowledge are needed.

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Tamanna, Shakila, Fahmida Rahman, TH Tang, et al. "Seroprevalence of Leptospira infection in selected rural and urban areas of Bangladesh by rLipL32 based ELISA." IMC Journal of Medical Science 11, no. 2 (2017): 50–55. http://dx.doi.org/10.3329/imcjms.v11i2.33095.

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Background and objectives: Leptospirosis is a zoonotic infection with worldwide distribution caused by the Leptospira species and predominant in the tropical and subtropical regions. Information on leptospirosis in Bangladesh is limited. The present study was designed to detect anti-leptospiral antibodies in human serum samples in Bangladeshi population by developing an in-house ELISA using recombinant LipL32 (rLipL32) antigen. The study was conducted from April 2014 to December 2014.Method: Healthy individuals from two rural areas and fever cases from one urban healthcare center were enrolled in the study. Rural health centers were located at Sonargoan and Bajitpur sub-district (Upozilla) of Narayaganj and Kishorganj districts. Sonargoan health center is located 26 km south-east and Bajitpur is located 71 km north-east of Dhaka city. About 1-2 ml of blood was collected with aseptic measure and serum was separated and stored at -200C until used. Anti-leptospiral IgG antibody was determined by recombinant LipL32 (rLipL32) antigen based indirect enzyme linked immunosorbent assay (ELISA). Seropositive cases were further confirmed by commercial Leptospira IgG ELISA.Results: The study included 250 febrile cases and 376 healthy individuals from urban and rural areas, respectively. Out of total 626 study population, anti-LipL32 specific IgG antibody was detected in 70 individuals (11.2%). The rate of positivity of anti-LipL32 antibody among the healthy individuals from rural area was 10.6% while the rate was 12.0% in urban febrile population. The rate of positivity in rural and urban population was not significantly (p>0.05) different. Among the urban population, the rate of seropositivity was 9.1% and 16.4% in 21- 40 yrs and above 40 years age group respectively while the rate was 7.2% and 14.0% in rural population respectively. Out of 70 seropositive cases detected by LipL32 ELISA, 65 (92.9%) were positive by commercial ELISA.Conclusion: The present study has revealed that leptospirosis is prevalent in Bangladesh and should be looked for in febrile and clinically suspected cases. The study has also demonstrated that rLipL32 protein may be used as a candidate antigen for the serodiagnosis of leptospirosis.IMC J Med Sci 2017; 11(2): 50-55

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Bulach, Dieter M., Thareerat Kalambaheti, Alejandro de la Peña-Moctezuma, and Ben Adler. "Functional Analysis of Genes in the rfbLocus of Leptospira borgpetersenii Serovar Hardjo Subtype Hardjobovis." Infection and Immunity 68, no. 7 (2000): 3793–98. http://dx.doi.org/10.1128/iai.68.7.3793-3798.2000.

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ABSTRACT Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiralrfb locus. This approach was used to investigate the function of three genes in the leptospiral rfb locus and demonstrated function for orfH10, which complemented awbpM strain of Pseudomonas aeruginosa, andorfH13, which complemented an rfbW strain ofVibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded byorfH8 is similar to GalE from a number of organisms. ASalmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.

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Bich Thuy, Vo Thi, Nguyen Tuan Hung, and Nghiem Ngoc Minh. "Analyzing 16S rRNA sequences from Vietnamese pathogenic Leptospira strains and in-silico prediction of potential antigenic epitopes on LipL21, LipL32 outer membrane lipoproteins." Vietnam Journal of Biotechnology 16, no. 4 (2020): 745–56. http://dx.doi.org/10.15625/1811-4989/16/4/15348.

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Leptospirosis, a zoonosis caused by Leptospira, is recognized as an emergent infectious disease. In currently, the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. In this experiment, a 550 bp fragment of large ribosomal RNA gene (16S rRNA) was sequenced and constructed phylogenetic tree from a panel of six Vietnamese pathogenic strains of Leptospira spirochetes (e.g., Pomona, Canicola, Mitis, Ictero haemohagiae, Bataviae, and Grippotyphosa). The results showed a close relationship of L.Pomona_VN and L.Hardjo (bootstrap: 99%). L.Canicola_VN and L.Ictero haemohagiae_VN appeared to be weak related to the classic L.Canicola, L. Grippotyphosa, these assemblage have a bootstrap support of 62%. The other strains (L.Mitis_VN and L.Grippotyphosa_VN) were appeared monophyletic, while their sister group (L.Bataviae_VN) relationship found only weak support (bootstrap: 62%). We also selected six genes [e.g. the immunoglobulin like proteins A and B (LigA and LigB genes), outer membrane protein (OmpL1 gene), and lipopolysaccharide (LipL32, LipL41, and LipL21 genes)] and checked gene expression in these Leptospira strains by polymerase chain reaction (PCR) method. There were three genes (e.g., LipL32, LipL21, and LigA genes) expressed in all strains, OmpL1 gene occured in 4 strains (L.Bataviae_VN, L.Canicola_VN, L.Grippotyphosa_VN and L.Mitis_VN), whereas LipL41 and LigB genes did not appear in any Leptospira strains. A multi-antigenic epitope potential of two gene (Lip L21 and Lip L32) was predicted by bioinformatic tools for designing a recombinant vaccine against leptospirosis. There were 3 multi-epitope regions (1 region and 95 antigenic epitope for B and T cells of LipL21 peptide; 2 regions and 124 antigenic epitope for both B and T cells of LipL32 peptide). It should be more of the deeply molecular biology studies to confirm the level agglutinating, antigen cleavage, peptide specificity matrices as well as neutralizing antibodies in the immune responses of DNA vaccine of these genes.

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Mohd Ali, Mohammad Ridhuan, Jia Siang Sum, Nurul Najian Aminuddin Baki, et al. "Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology." International Journal of Biological Macromolecules 168 (January 2021): 289–300. http://dx.doi.org/10.1016/j.ijbiomac.2020.12.062.

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OKUDA, Masaru, Yoshiko SAKAI, Megumi MATSUUCHI, et al. "Enzyme-Linked Immunosorbent Assay for the Detection of Canine Leptospira Antibodies Using Recombinant OmpL1 Protein." Journal of Veterinary Medical Science 67, no. 3 (2005): 249–54. http://dx.doi.org/10.1292/jvms.67.249.

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Phoka, Theerapat, Lenka Fule, Juliana Pipoli Da Fonseca, Thomas Cokelaer, Mathieu Picardeau, and Kanitha Patarakul. "Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp." PLOS ONE 16, no. 12 (2021): e0260981. http://dx.doi.org/10.1371/journal.pone.0260981.

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Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

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Kanagavel, Murugesan, Santhanam Shanmughapriya, Kumarasamy Anbarasu, and Kalimuthusamy Natarajaseenivasan. "B-Cell-Specific Peptides of Leptospira interrogans LigA for Diagnosis of Patients with Acute Leptospirosis." Clinical and Vaccine Immunology 21, no. 3 (2014): 354–59. http://dx.doi.org/10.1128/cvi.00456-13.

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ABSTRACTLeptospirosis is a reemerging infectious disease that is underdiagnosed and under-recognized due to low-sensitivity and cumbersome serological tests. Rapid reliable alternative tests are needed for early diagnosis of the disease. Considering the importance of the pathogenesis-associated leptospiral LigA protein expressedin vivo, we have evaluated its application in the diagnosis of the acute form of leptospirosis. The C-terminal coding sequence ofligA(ligA-C) was cloned into pET15b and expressed inEscherichia coli. Furthermore, the B-cell-specific epitopes were predicted and were synthesized as peptides for evaluation along with recombinant LigA-C. Epitope 1 (VVIENTPGK), with a VaxiJen score of 1.3782, and epitope 2 (TALSVGSSK), with a score of 1.2767, were utilized. A total of 140 serum samples collected from leptospirosis cases during the acute stage of the disease and 138 serum samples collected from normal healthy controls were utilized for evaluation. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the recombinant LigA-C-specific IgM enzyme-linked immunosorbent assay (ELISA) and were found to be 92.1%, 97.7%, 92.8%, and 97.5%, respectively. Epitopes 1 and 2 used in the study showed 5.1 to 5.8% increased sensitivity over recombinant LigA-C in single and combination assays for IgM antibody detection. These findings suggest that these peptides may be potential candidates for the early diagnosis of leptospirosis.

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Asuthkar, Swapna, Sridhar Velineni, Johannes Stadlmann, Friedrich Altmann, and Manjula Sritharan. "Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, from Leptospira interrogans Serovar Lai." Infection and Immunity 75, no. 9 (2007): 4582–91. http://dx.doi.org/10.1128/iai.00324-07.

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ABSTRACTIn an earlier study, based on the ferric enterobactin receptor FepA ofEscherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome ofLeptospira interrogansserovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-bindingprotein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein fromL. interrogansserovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA inL. interrogans. Southern blot analysis confirmed our earlier observation that thehbpAgene was present only in some of the pathogenic serovars and was absent inLeptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenicL. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

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Eshghi, Azad, Kristel Lourdault, Gerald L. Murray, et al. "Leptospira interrogans Catalase Is Required for Resistance to H2O2and for Virulence." Infection and Immunity 80, no. 11 (2012): 3892–99. http://dx.doi.org/10.1128/iai.00466-12.

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ABSTRACTPathogenicLeptospiraspp. are likely to encounter higher concentrations of reactive oxygen species induced by the host innate immune response. In this study, we characterizedLeptospira interroganscatalase (KatE), the only annotated catalase found within pathogenicLeptospiraspecies, by assessing its role in resistance to H2O2-induced oxidative stress and during infection in hamsters. PathogenicL. interrogansbacteria had a 50-fold-higher survival rate under H2O2-induced oxidative stress than did saprophyticL. biflexabacteria, and this was predominantly catalase dependent. We also characterized KatE, the only annotated catalase found within pathogenicLeptospiraspecies. Catalase assays performed with recombinant KatE confirmed specific catalase activity, while protein fractionation experiments localized KatE to the bacterial periplasmic space. The insertional inactivation ofkatEin pathogenicLeptospirabacteria drastically diminished leptospiral viability in the presence of extracellular H2O2and reduced virulence in an acute-infection model. Combined, these results suggest thatL. interrogansKatE confersin vivoresistance to reactive oxygen species induced by the host innate immune response.

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Porrozzi, Renato, Marcos V. Santos da Costa, Antonio Teva, et al. "Comparative Evaluation of Enzyme-Linked Immunosorbent Assays Based on Crude and Recombinant Leishmanial Antigens for Serodiagnosis of Symptomatic and Asymptomatic Leishmania infantum Visceral Infections in Dogs." Clinical and Vaccine Immunology 14, no. 5 (2007): 544–48. http://dx.doi.org/10.1128/cvi.00420-06.

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ABSTRACT The diagnosis of visceral leishmaniasis remains difficult in rural areas where the disease is endemic, and serologic methods still need assessment, as they are not very sensitive for the detection of asymptomatic infectious dogs. Here we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based methods for the detection of antibodies against recombinant leishmanial antigens (namely, the recombinant K26 [rK26] and rK39 antigens from Leishmania infantum and the rA2 protein from Leishmania donovani) in comparison to ELISAs employing crude soluble antigen (CSA). The assays utilized sera from known negative controls (n = 25) and clinically asymptomatic (n = 50) and symptomatic (n = 50) dogs with confirmed L. infantum infections. Additional studies were also done using sera from animals harboring other infections (n = 14) for the evaluation of cross-reactivity. Our study indicated that rK26 and rK39 used in ELISAs provided very high sensitivities for the detection of symptomatic dogs (94% and 100%, respectively), followed by CSA (88%) and rA2 (70%). Conversely, rA2 was more sensitive for asymptomatic dogs (88%) than rK39 and rK26 (both 66%) and CSA (30%). Some cross-reactivity in sera from dogs with other infections (Leishmania braziliensis and Leptospira interrogans) was identified, but the rA2 protein provided the greatest specificity (98%). Data further indicate that all three recombinant proteins must be used in parallel to detect essentially all infected dogs. Efforts should be made to develop a cheap and reliable serologic test based on epitope selection from these diagnostic markers for the sensitive detection of L. infantum-infected dogs.

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., Sumarningsih, Susanti ., and Simson Tarigan. "Evaluation of LipL32 ELISA for detection of bovine leptospirosis in West Java." Jurnal Ilmu Ternak dan Veteriner 22, no. 2 (2018): 80. http://dx.doi.org/10.14334/jitv.v22i2.1610.

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The current diagnosis of leptospirosis, micro Agglutination Test (MAT) and isolation, is expensive, impractical and technically demanding. This study was aimed at developing an ELISA based on recombinant LipL32 as a practical, inexpensive test for Leptospirosis. The DNA encoding LipL32 was isolated from Leptospira pomona, inserted into pRSET-C plasmid then expressed in E.coli BL21 as a poly-histidine-tagged protein. The amount of LipL32 protein, which was purified from the supernatant of lysed cells by a Ni-NTA column, was 1mg/l culture. This purified LipL32 was used as the coating antigen at 5µg/ml. The accuracy of ELISA was evaluated based on ROC analysis, by comparing the ELISA and MAT results of 517 bovine sera. Result in this study showed that the area under curve (AUC) was 0.853, which categorised the LipL32 ELISA as a “moderately accurate” test and indicates that the ELISA was able to differentiate positive and negative Leptospirosis serum. The result also showed ELISA LipL32 could detect serum positive MAT to Hardjo, Grippotyphosa, Tarrasovi, Rachmati and Bataviae. The optimal cut off for OD ELISA determined based on ROC curve was 0.504, and it showed sensitivity and specificity of ELISA LipL32 relative to MAT were 86.0% and 69.5%, respectively. Overall, the result in this study showed that ELISA LipL32 can be used as a rapid test for identification of anti-Leptospira antibodies in bovine.

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Kamaruzaman, Intan Noor Aina, Gareth James Staton, Stuart Ainsworth, Stuart D. Carter, and Nicholas James Evans. "Characterisation of Putative Outer Membrane Proteins from Leptospira borgpetersenii Serovar Hardjo-Bovis Identifies Novel Adhesins and Diversity in Adhesion across Genomospecies Orthologs." Microorganisms 12, no. 2 (2024): 245. http://dx.doi.org/10.3390/microorganisms12020245.

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Leptospirosis is a zoonotic bacterial disease affecting mammalian species worldwide. Cattle are a major susceptible host; infection with pathogenic Leptospira spp. represents a public health risk and results in reproductive failure and reduced milk yield, causing economic losses. The characterisation of outer membrane proteins (OMPs) from disease-causing bacteria dissects pathogenesis and underpins vaccine development. As most leptospire pathogenesis research has focused on Leptospira interrogans, this study aimed to characterise novel OMPs from another important genomospecies, Leptospira borgpetersenii, which has global distribution and is relevant to bovine and human diseases. Several putative L. borgpetersenii OMPs were recombinantly expressed, refolded and purified, and evaluated for function and immunogenicity. Two of these unique, putative OMPs (rLBL0972 and rLBL2618) bound to immobilised fibronectin, laminin and fibrinogen, which, together with structural and functional data, supports their classification as leptospiral adhesins. A third putative OMP (rLBL0375), did not exhibit saturable adhesion ability but, together with rLBL0972 and the included control, OmpL1, demonstrated significant cattle milk IgG antibody reactivity from infected cows. To dissect leptospire host–pathogen interactions further, we expressed alleles of OmpL1 and a novel multi-specific adhesin, rLBL2618, from a variety of genomospecies and surveyed their adhesion ability, with both proteins exhibiting divergences in extracellular matrix component binding specificity across synthesised orthologs. We also observed functional redundancy across different L. borgspetersenii OMPs which, together with diversity in function across genomospecies orthologs, delineates multiple levels of plasticity in adhesion that is potentially driven by immune selection and host adaptation. These data identify novel leptospiral proteins which should be further evaluated as vaccine and/or diagnostic candidates. Moreover, functional redundancy across leptospire surface proteins together with identified adhesion divergence across genomospecies further dissect the complex host–pathogen interactions of a genus responsible for substantial global disease burden.

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Carvalho, Enéas de. "Leptospira Hemolytic protein analysis by the recombinant expression: detection of native expression, biological activity and vaccination potential." Revista do Instituto de Medicina Tropical de São Paulo 50, no. 3 (2008): 138. http://dx.doi.org/10.1590/s0036-46652008000300012.

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Yan, Weiwei, Syed M. Faisal, Sean P. McDonough, et al. "Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model." Microbes and Infection 11, no. 2 (2009): 230–37. http://dx.doi.org/10.1016/j.micinf.2008.11.008.

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Monaris, D., M. E. Sbrogio-Almeida, C. C. Dib, et al. "Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant." Clinical and Vaccine Immunology 22, no. 8 (2015): 965–73. http://dx.doi.org/10.1128/cvi.00285-15.

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ABSTRACTLeptospirosis is a global zoonotic disease caused by differentLeptospiraspecies, such asLeptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinantLeptospira interrogansouter membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) orSalmonellaflagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigACor LigACcoadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.

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Zhang, Xiang-Yan, Yang Yu, Ping He, et al. "Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21, LipL32 and OmpL1 in Epidemic Leptospires." Acta Biochimica et Biophysica Sinica 37, no. 10 (2005): 649–56. http://dx.doi.org/10.1111/j.1745-7270.2005.00094.x.

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AbstractLeptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China. Immunoblot analysis was further performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the recombinant OMPs. Both immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that these three OMPs were conservatively expressed in pathogenic L. interrogans strains and other pathogenic leptospires. Additionally, the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of leptospirosis was evaluated. The recombinant LipL32 and OmpL1 proteins showed a high degree of ELISA reactivity with sera from patients infected with L. interrogans strain Lai and other pathogenic leptospires. These results may contribute to the identification of candidates for broad-range vaccines and immunodiagnostic antigens in further research.

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Bughio, Nasreen I., Min Lin, and Om P. Surujballi. "Use of Recombinant Flagellin Protein as a Tracer Antigen in a Fluorescence Polarization Assay for Diagnosis of Leptospirosis." Clinical Diagnostic Laboratory Immunology 6, no. 4 (1999): 599–605. http://dx.doi.org/10.1128/cdli.6.4.599-605.1999.

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ABSTRACT The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, ofLeptospira interrogans serovar pomona in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloacetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluorescein isothiocyanate (FITC). Western blotting was performed by using bovine sera with microscopic agglutination test (MAT) titers of antibodies against L. interrogans serovarpomona and L. bergpetersenii serovarshardjo and sejroe to confirm the antigenicity of FlaB. Western blot analysis demonstrated that labeled as well as unlabeled FlaB was recognized by the positive sera tested, indicating the broad serovar cross-reactivity of this protein. It also indicated that labeling with FITC did not affect the antigenicity. By using FITC-labeled FlaB as a tracer antigen, a homogeneous FPA was developed to detect antileptospiral antibodies in bovine sera. A population of 208 MAT-positive and 208 MAT-negative serum samples was tested by FPA. The FPA cutoff was determined by receiver operating characteristic analysis. By FPA, 83.7% of the MAT-positive serum samples were positive and 81.2% of the MAT-negative serum samples were negative. Compared to the results of MAT, the positive predictive value of FPA was 81.7% and the negative predictive value of FPA was 83.3%. The FPA is a simple and rapid technique for the detection of anti-Leptospira antibodies.

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Journal articles on the topic 'Leptospira recombinant protein'

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