Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA

Abstract

The search for novel antigens suitable for improved vaccines and diagnostic reagents against leptospirosis led to the identification of LigA and LigB. LigA and LigB expression were not detectable at the translation level but were detectable at the transcription level in leptospires grown in vitro. Lig genes were present in pathogenic serovars of Leptospira, but not in non-pathogenic Leptospira biflexa. The conserved and variable regions of LigA and LigB (Con, VarA and VarB) were cloned, expressed and purified as GST-fusion proteins. Purified recombinant LigA and LigB were evaluated for their diagnostic potential in a kinetic ELISA (KELA) using sera from vaccinated and microscopic agglutination test (MAT)-positive dogs. Sera from vaccinated dogs showed reactivity to whole-cell antigens of leptospires but did not show reactivity in the KELA assay with recombinant antigens, suggesting a lack of antibodies to Lig proteins in the vaccinated animals. The diagnostic potential of recombinant Lig antigens in the KELA assay was evaluated by using 67 serum samples with MAT >1600, which showed reactivity of 76, 41 and 35 % to rConA, rVarA and rVarB, respectively. These findings suggest that recombinant antigen to the conserved region of LigA and LigB can differentiate between vaccinated and naturally infected animals.

Leptospira Immunoglobulin-Like Proteins as a Serodiagnostic Marker for Acute Leptospirosis

“…Initial studies found that acute leptospirosis produces robust antibody responses to Lig proteins (28,39), albeit these evaluations were performed with small numbers of patient samples. Palaniappan et al found that levels of antibodies to recombinant Lig fragments as detected by ELISA correlated with MAT titers in dogs (46). However, this study did not specifically determine the performance of the assay in diagnosing acute leptospirosis in these animals.…”

“…Furthermore, they are expressed in virulent strains but not in strains that have been attenuated by culture passaging (39). Lig proteins are expressed during host infection (39) and appear to induce strong antibody responses in patients (28,39) and infected animals (28,46,47). However, previous studies were performed with limited numbers of leptospirosis patients (28,39).…”

“…More recently, a novel family of surface-associated proteins, Lig proteins, were identified (28,39,47) which have Big tandem-repeat domains. These proteins were identified through screening of genomic DNA expression libraries with patient (28,39) and infected animal sera (46) and appear to be preferentially expressed during host infection (39,46). Initial studies found that acute leptospirosis produces robust antibody responses to Lig proteins (28,39), albeit these evaluations were performed with small numbers of patient samples.…”

“…The study's findings indicate that the first six Big domains of LigB proteins form a candidate antigen for such an assay. Previous studies found that the C-terminal Big domain regions of LigA (domains 7 to 12) and LigB (domains 7 to 11) are seroreactive as well (28,39,46). Inclusion of these fragments may potentially increase assay performance.…”

Functional and immunological evaluation of two novel proteins of Leptospira spp.

“…Lsa44 and Lsa45 share serum recognition properties with the adhesins described elsewhere, LipL32 (Flannery et al, 2001;Xu et al, 2008), LigA and LigB proteins (Palaniappan et al, 2004;Srimanote et al, 2008), Lsa27 (Longhi et al, 2009), Lsa63 (Vieira et al, 2010a), Lsa66 (Oliveira et al, 2011), and OmpL37 (Pinne et al, 2010), which have shown positive reactivity with serum samples from patients diagnosed with leptospirosis. Protein recognition by serum (1), or lsPBS containing PBS-(2), Lsa44-(3) or Lsa45-injected mouse serum (4), at a dilution of 1 : 100.…”