RESUMO
Human toxocariasis is one of the neglected helminthiases and it is caused by the zoonotic roundworm species Toxocara canis and Toxocara cati. Diagnosis of human toxocariasis is based on the combination of clinical, parasitological, and epidemiological criteria, as well as serology tests that detect anti-Toxocara antibodies. Notwithstanding, due to the absence of pathognomonic symptoms and signs of the disease, serology is the key evidence to support a conclusive diagnosis. TES-ELISA is the most widely used serological test for diagnosis. However, cross-reaction of TES antigens with antibodies produced to other helminth antigens is a major drawback for its application in countries with high parasitic prevalence. T. canis recombinant antigens have been described as an alternative to native TES for diagnosis. Nevertheless, the selection of antigenic proteins is a complex process that requires validation. In this paper, we developed an eGFP carrier-based system to express and purify blocks of recombinant polypeptides of T. canis antigenic proteins. Intense cross-reaction polypeptides were detected by Immunoblot and avoided to finally produce a chimeric prototype protein. Additionally, a control chimeric protein that harbors the complete tested proteins was produced. Purified chimeric antigens were tested in ELISA and Immunoblot assays with 310 sera samples of negative and positive control individuals. Our results showed that chimeric rCHITC0 and rCHITC1 antigens (with sensitivities of 62% 58%, 38% and 16% in IB-rCHITC0, ELISA-rCHITC0, ELISA-rCHITC1 and IB-rCHITC1 respectively for OLMS) can perform better in terms of specificity (being 91%, 89%, 87% and 76% for ELISA-rCHITC1, IB-rCHITC1, ELISA-rCHITC0 and IB-rCHITC0 respectively for OLMS) than T. canis TES-ELISA (with 61% specificity), giving a higher signal with serum samples of infected individuals as well the possibility to discriminate false positive cases with other parasitic infections. Our data suggest that T. canis chimeric proteins, represent candidate antigens for phase II studies.
RESUMO
RESUMEN El cultivo comercial de hortensias para flor de exportación ocupa un renglón importante en el sector económico del oriente antioqueño, por ser fuente de empleo y de desarrollo en la zona. La hortensia (Hydrangea macrophylla) es afectada por numerosos organismos fitopatógenos, entre ellos, nematodos del género Aphelenchoides, los que ocasionan, en el follaje, lesiones necróticas angulares, malformación de flor, enanismo y un daño indirecto en la tasa fotosintética, demeritando los parámetros de calidad para exportación. El objetivo de este estudio fue identificar, molecularmente, las especies del nematodo Aphelenchoides asociadas al cultivo de hortensias de color, en los municipios de Medellín (Santa Elena), La Ceja y Rionegro, siendo este el primer reporte para Colombia, de las especies de este género. Para la ejecución del estudio, se realizaron 10 muestreos en cultivos comerciales, distribuidos entre los tres municipios mencionados. Los nematodos extraídos, se sometieron a pruebas basadas en el análisis de ADN, haciendo uso del marcador ribosomal 18S. Los análisis filogenéticos practicados mostraron la presencia de la especie Aphelenchoides ritzemabosi en cultivos de hortensias, del corregimiento de Santa Elena y, de A. fragarie, en los municipios de La Ceja y Rionegro.
ABSTRACT Hydrangea macrophylla commercial crops have an important economic significance for the eastern region of the department of Antioquia, Colombia, due to its impact on employment and local development. This flowering plant can be attacked by several phytopathogens among them nematodes of the genus Aphelenchoides; which can produce necrotic lesions on the plant leaves as well decreased photosynthetic rate, dwarfism, and flower malformation. Additionally, this pathogen is considered a regulated pest and limits the international commercialization of the flower. The aim of this study was to molecularly identify the Aphelenchoides species associated with infected H. macrophylla plants in crops the municipalities of Medellín, La Ceja, and Rionegro. This is the first report of the species of this phytopathogen for Colombia. The sampling activities were performed in ten commercial crops locate at the three municipalities. The isolated nematodes were subject to DNA-based tests were the 18S rDNA gene was amplified, sequenced and analyzed using phylogenetic methods. The obtained results showed the infection of Aphelenchoides ritzemabosi within the Santa Elena (Medellin) area in crops and A. fragarie within the municipalities of La Ceja, and Rionegro.
