2 research outputs found
Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic leishmania infantum antigens with potential serodiagnosis applications
Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs,
and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite
technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential
subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in
the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative
selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected
dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection
against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified
IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage
enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n 31) compared to those
from vaccinated dogs (n 21), experimentally infected dogs with cross-reactive parasites (n 23), and healthy controls (n
17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no crossreactivity
with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in
Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of
these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can
be applied in CVL-monitoring programsThis work was supported by grants from the Pró-Reitoria de Pesquisa
of UFMG (supported 03/2013), the Instituto Nacional de Ciência e
Tecnologia em Nano-Biofarmacêutica (INCT Nano-Biofar), Rede
Nanobiotec/Brasil-UFU (CAPES), PRONEX-FAPEMIG (APQ-01019-
09), FAPEMIG (APQ-00496-11 and APQ-00819-12), and CNPq (APQ-
472090/2011-9 and APQ-482976/2012-8). E.A.F.C. and L.R.G. are recipients
of grants from CNPq. M.A.C.-F. is the recipient of a grant from
FAPEMIG/CAPE
Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach
In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to
62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition,
problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The
present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis
of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no
homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of
these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent
assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and
specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruziinfected
animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to
differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined
in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected
dogs were used. The study’s findings suggest that these three peptides can constitute a potential tool for more sensitive and specific
serodiagnosis of L. infantum infection in dogsThis work was supported by grants from the Pró-Reitoria de Pesquisa
from UFMG (Edital 07/2012), Instituto Nacional de Ciência e Tecnologia
em Nano-biofarmacêutica (INCT-NANOBIOFAR, Fundação de Amparo
à Pesquisa do Estado de Minas Gerais (FAPEMIG) (CBB-APQ-02364-08,
CBB-APQ-00356-10, CBB-APQ-00496-11, and CBB-APQ-00819-12),
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) (APQ-472090/2011-9), and the Instituto Nacional de Ciência e
Tecnologia em Vacinas (INCT-V). E.A.F.C. and A.P.F. are CNPq grant
recipients. M.A.C.-F. is a FAPEMIG/CAPES grant recipient. This study
was supported in Spain, in part, by grants from the Ministerio de Ciencia
e Innovación (FIS/PI1100095)