Costa Lourena E, Lima Mayara I S, Chávez-Fumagalli Miguel A, Menezes-Souza Daniel, Martins Vivian T, Duarte Mariana C, Lage Paula S, Lopes Eliane G P, Lage Daniela P, Ribeiro Tatiana G, Andrade Pedro H R, de Magalhães-Soares Danielle F, Soto Manuel, Tavares Carlos A P, Goulart Luiz R, Coelho Eduardo A F
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.
Clin Vaccine Immunol. 2014 Jan;21(1):96-106. doi: 10.1128/CVI.00583-13. Epub 2013 Nov 20.
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.
内脏利什曼病（VL）是一种人畜共患病，在巴西流行，犬类是主要的家养寄生虫宿主，在犬内脏利什曼病（CVL）流行地区，感染犬的比例在10%至62%之间。尽管技术不断进步，但CVL血清学诊断仍存在一些问题。本研究描述了一种通过噬菌体展示技术从阴性和阳性血清的多克隆抗体中进行序列消减筛选的方法，该方法鉴定出了对CVL抗体具有高敏感性和特异性的潜在噬菌体融合肽。进行了阴性筛选，将噬菌体克隆与来自健康犬和感染克氏锥虫犬的纯化IgG结合，以消除交叉反应性噬菌体。将剩余的未结合上清噬菌体用于针对感染婴儿利什曼原虫犬血清IgG的阳性筛选。选择与CVL感染血清样本的纯化IgG结合的噬菌体克隆。鉴定出18个克隆，并通过噬菌体酶联免疫吸附测定（噬菌体-ELISA）测试它们与感染犬（n = 31）、接种疫苗犬（n = 21）、实验性感染交叉反应性寄生虫犬（n = 23）和健康对照犬（n = 17）血清样本的反应性。8个克隆的敏感性、特异性、阳性和阴性预测值均为100%，并且它们与感染克氏锥虫或犬埃立希体的犬或接种巴西两种不同商业CVL疫苗的犬无交叉反应。我们的研究以100%的准确率鉴定出了8个婴儿利什曼原虫抗原模拟表位用于CVL血清学诊断。通过噬菌体-ELISA使用这些模拟表位被证明是一种出色的检测方法，具有可重复性、简单、快速且廉价的特点，可应用于CVL监测项目。
