Abstract
BackgroundLeishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies.Methodology/Principal findingsSeventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1–28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67–81 and 122–135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%).Conclusions/SignificanceOur work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.
Highlights
Visceral leishmaniasis (VL) is an infection caused by various species of Leishmania, an intracellular protozoan parasite
Dogs are the main source of infection in the urban area and, in Brazil, the main strategies of the Visceral Leishmaniasis Control Program are directed to control the canine reservoir
We developed a synthetic bi-epitope peptide as an antigen for immunodiagnostic ELISA to detect canine visceral leishmaniasis (CVL)
Summary
Visceral leishmaniasis (VL) is an infection caused by various species of Leishmania, an intracellular protozoan parasite. A2 is a stress response protein from L. donovani and it is expressed in amastigote and in promastigote cultures It corresponds to the specific virulence factor (LdVFA2) and has been shown to be required for L. donovani amastigote survival in visceral organs of mice [6, 7, 8]. Anti- LdVFA2 antibodies have been detected in sera samples from human patients with active visceral leishmaniasis, confirming that LdVFA2 proteins are expressed during infection [10, 15]. These findings suggest that studies of LdVFA2 proteins antigenic properties might have great potential for the development of vaccines, therapeutics and diagnostics for leishmaniasis. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies
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