title: Effect of the preservative and temperature conditions on the stability of Leishmania infantum promastigotes antigens applied in a flow cytometry diagnostic method for canine visceral leishmaniasis.
abstract: The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.
authors.affiliation: Laboratório de Pesquisas Clínicas, Departamento de Análises Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Ouro Preto, MG CEP 35400-000, Brazil.
authors.forename: Henrique
authors.initials: H
authors.lastname: Gama Ker
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Dian de Oliveira Aguiar-Soares
authors.forename: Bruno
authors.initials: B
authors.lastname: Mendes Roatt
authors.forename: Nádia
authors.initials: N
authors.lastname: das Dores Moreira
authors.forename: Wendel
authors.initials: W
authors.lastname: Coura-Vital
authors.forename: Cláudia
authors.initials: C
authors.lastname: Martins Carneiro
authors.forename: Andréa
authors.initials: A
authors.lastname: Teixeira-Carvalho
authors.forename: Olindo Assis
authors.initials: OA
authors.lastname: Martins-Filho
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Cordeiro Giunchetti
authors.forename: Denise
authors.initials: D
authors.lastname: da Silveira-Lemos
authors.forename: Alexandre
authors.initials: A
authors.lastname: Barbosa Reis
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D004283:Dog Diseases / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D004285:Dogs; D005434:Flow Cytometry / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect; D005557:Formaldehyde / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D018314:Leishmania infantum / Q000276:immunology / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000662:veterinary*; D008018:Life Cycle Stages; D011310:Preservatives, Pharmaceutical / Q000737:chemistry; D055550:Protein Stability; D013696:Temperature
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D011310:Preservatives, Pharmaceutical; D005557:Formaldehyde
keywords: Canine visceral leishmaniasis; Diagnosis; ELISA; FC-AFPA-IgG; Flow cytometry; IFAT