POTENTIAL APPLICATION OF RECOMBINANT LIGBREP REGION TO THE DIAGNOSIS OF LEPTOSPIROSIS CAUSED BY DIFFERENT SEROVARS

Marina da Silva Rosa (Biomanguinhos); Ana Paula Correa Argondizzo (Biomanguinhos); Rafael Lawson Ferreira (Biomanguinhos); Gabriela dos Santos Esteves (Biomanguinhos); Mitermayer Galvão dos Reis (CPqGM); Albert Iksang Ko (WMCCU); Marco Alberto Medeiros (Biomanguinhos)

Leptospirosis is the most widespread zoonosis in the world and is a major human and veterinary health problem in developing countries. LigA and B are members of a family of high molecular weight proteins that have bacterial immunoglobulin-like (Lig) repeat domains and are present only in pathogenic Leptospira. The protein LigB is associated with the cell surface and induces high titres of antibodies in sera recovered from infected patients. LigB protein sequence homology, among the prevalent pathogenic serovars is >80%.  LigB is composed of 12 domains, with domains 1-7 (LigBRep region) sharing 100% sequence identity with the same domains present in LigA. Lig proteins were shown to be high-performing serodiagnostic markers for leptospirosis in immunoblot and ELISA studies. In this work, the aim was to evaluate the cross reaction between Ligs fragments of Leptospiras for feasibility of a development assay for diagnostic kit with global applicability for human and veterinary. In the present study, the ligBRep encoding regions of L. interrogans serovars Copenhageni, Pomona, Canicola, L. Noguchii and L. Weilii were amplified by PCR and cloned into two E. coli expression vectors (pAE and pET100-D/TOPO). The recombinant proteins were purified and then used in ELISA and Western blotting. These assays were performed using human serum pools generated from patients infected with serovar Copenhageni and from healthy controls. It was shown that the recombinant proteins derived from the three serovars and two species were each recognized by the convalescent and acute phase patient serum pools, the healthy control serum pools were all unreactive. The ELISA data showed the  strongest cross-immunoreactivity of the recombinant proteins and supports previous work demonstrating elevated degree of amino acid sequence homology (63- 98%) among these species and serovars. Our data support further study on the use of recombinant Lig proteins as components of diagnostic tests applied to humans and animals infected with different leptospiral serovars and species.