title: Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic Leishmania infantum antigens with potential serodiagnosis applications.
abstract: 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 cross-reactivity 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 programs.
authors.affiliation: Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Lourena E
authors.initials: LE
authors.lastname: Costa
authors.forename: Mayara I S
authors.initials: MI
authors.lastname: Lima
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Chávez-Fumagalli
authors.forename: Daniel
authors.initials: D
authors.lastname: Menezes-Souza
authors.forename: Vivian T
authors.initials: VT
authors.lastname: Martins
authors.forename: Mariana C
authors.initials: MC
authors.lastname: Duarte
authors.forename: Paula S
authors.initials: PS
authors.lastname: Lage
authors.forename: Eliane G P
authors.initials: EG
authors.lastname: Lopes
authors.forename: Daniela P
authors.initials: DP
authors.lastname: Lage
authors.forename: Tatiana G
authors.initials: TG
authors.lastname: Ribeiro
authors.forename: Pedro H R
authors.initials: PH
authors.lastname: Andrade
authors.forename: Danielle F
authors.initials: DF
authors.lastname: de Magalhães-Soares
authors.forename: Manuel
authors.initials: M
authors.lastname: Soto
authors.forename: Carlos A P
authors.initials: CA
authors.lastname: Tavares
authors.forename: Luiz R
authors.initials: LR
authors.lastname: Goulart
authors.forename: Eduardo A F
authors.initials: EA
authors.lastname: Coelho
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000302:isolation & purification; D001938:Brazil; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000302:isolation & purification; D005260:Female; D018314:Leishmania infantum / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D008297:Male; D019151:Peptide Library; D010455:Peptides / Q000302:isolation & purification; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides