USO DE CÉLULAS DERIVADAS DE PROTOESCÓLICES BOVINOS DE ECHINOCOCCUS GRANULOSUS COMO FUENTE ANTIGÉNICA PARA EL DIAGNÓSTICO DE ECHINOCOCCOSIS QUÍSTICA

ANDREA MAGLIOCO; JORGE GENTILE; CLAUDIA HERNANDEZ; JENSEN O; MARÍA LAURA GERTISER; GABRIELA A CANZIANI; ALICIA G FUCHS

Congreso; II Congreso Internacional de Zoonosis IX Congreso Argentino de Zoonosis; 2018

Introduction: Cystic echinococcosis (CE) is an infectious disease produced by Echinococcus granulosusworldwide distributed. Several approaches have been performed to improve the serological diagnosis in CE asparasite?s antigen purification and recombinant proteins, however serological diagnosis have not improved. Acell line from bovine´s protoscolices was developed, defined by the presence of CO1 and antigen B. They havetwo growth stages in culture: in the first seven days cells form clusters and after ten days they aggregated andattached on the self- membranes floating in the medium.The aim of this work was to study in comparative design whether sera from CE patients regarding the differentculture stages recognized cell proteins.Materials and Methods: Protocols and procedures were approved by the Interamerican Open UniversityEthical Committee. Human sera were obtained from CE patients treated in the ?Ramón Santamarina?Municipal Hospital, Tandil, Buenos Aires (years 2012- 2016) or from Chubut (2017) and samples from healthydonors were obtained from the same area (controls). Sera were heat-inactivated at 56 °C for 30 minutes.Antigen preparation and western blotting Cell extracts were obtained after short- (7 days) or long- term cellcultures (20 days). The cell pellet was incubated in ice cold lysis buffer pH 8; containing 0.1 % Bmercaptoethanol;1/ 100 protease inhibitor cocktail. After three freeze-thaw cycles and centrifugation,proteins from supernatants were quantified. Proteins were run under reducing conditions on a 15 % SDS- PAGEmini-gel. After proteins transfer to nitrocellulose membrane each serum (1: 125, 2 h) was processed intoindividual strip. Reaction was revealed with alkaline phosphatase- goat anti- human IgG (1: 10000, 1 h) andBCIP/ NBT. Positive controls was performed with homogenate of crude cyst material, Echinococcus granulosusG1 (Chubut). Bands were analysed with GelAnalyzer software. Bands recognized by controls were excludedfrom the analysis. Statistics: differences among short- and long-term culture band recognition were analysedby Chi-square test.Results: Several protein bands spanning 12 - 94 kDa, were recognized. Differences in recognition betweenprotein source, cell culture stage, were found in 37- 46 kDa (short-term culture: 17 positive over 24 patientsvs long-term culture 8/ 24 patients, p< 0.05) and inverse relation was found in the recognition of proteinsbetween 47-55 kDa (short-term culture: 7/ 24 patients vs long-term culture: 14/ 24 patients, p< 0.05). Patientsera was positive for crude cyst material, positive control. .Discussion and conclusions: The use of cells as controlled antigen source would be advantageous over the useof ex vivo parasite because constitutes a laboratory protein source which presents different growth modulablestages. The clinical mean of the sera recognition differences must be investigated