Methods in Molecular Biology 2137 Martin Cancela Gabriela Maggioli Editors Fasciola hepatica Methods and Protocols M ETHODS IN M OLECULAR B IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed. Fasciola hepatica Methods and Protocols Edited by Martin Cancela Laboratório de Biologia Molecular de Cestódeos, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil Gabriela Maggioli Unidad de Biología Parasitaria, Instituto de Higiene, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay Editors Martin Cancela Gabriela Maggioli Laborato´rio de Biologia Molecular Unidad de Biologı´a Parasitaria, Instituto de Higiene de Cesto´deos Facultad de Ciencias Centro de Biotecnologia Universidad de la Repu´blica Universidade Federal Montevideo, Uruguay do Rio Grande do Sul (UFRGS) Porto Alegre, Rio Grande do Sul, Brazil ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0474-8 ISBN 978-1-0716-0475-5 (eBook) https://doi.org/10.1007/978-1-0716-0475-5 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Preface In the last 20 years, advances in molecular biology techniques have emerged, expanding our knowledge in many aspects of Fasciola hepatica way of life. This book comprises 17 chapters, which describe basic and advanced protocols to study F. hepatica parasite biology. We start this book describing protocols to obtain and maintain different developmental stages of F. hepatica at the laboratory for different applications such as molecular/cellular biology studies and vaccination trials (Chapter 1). Besides, Chapter 2 refers to protocols to evaluate host tissue changes induced during F. hepatica acute and chronic infection by analyzing peritoneal cells and hepatic lesions in sheep. Chapters 3–7 describe a diverse set of protocols to obtain and characterize tegumental and secreted proteins (Chapter 3) and extracellular vesicles (Chapter 4) by using proteomic approaches and also to perform functional studies by producing recombinant protein in E. coli (Chapter 5). A very detailed protocol to perform gene knockdown by using the RNAi technique in F. hepatica (Chapter 6) including protocol to obtain in-house double strand RNA by in vitro transcription is presented in this book. In Chapter 7, we introduce two advanced protocols to study spatial and temporal gene expression patterns using in situ and in toto hybridization techniques in different F. hepatica developmental stages. The second part of this book (Chapters 8–15) is dedicated to introduce the readers to a complex repertoire of molecular biology techniques to study immunological changes induced by F. hepatica antigens and experimental infection. In Chapter 8, we start with a revision in molecular aspect of F. hepatica infection related to the evasion of host immune response. Excretion–secretion products (ESP) of F. hepatica are well-known to be involved in the evasion mechanisms. Chapters 9–12 describe different protocols to evaluate immu- nological effects of ESP on macrophages (Chapter 9), eosinophil apoptosis (Chapter 10), macrophages alternative activation, (Chapter 11), and toll-like receptor interactions (Chapter 12). Chapter 13 describes protocols to obtain peritoneal and splenic dendritic cells of mice experimentally infected by F. hepatica and the effect of adult worm extracts on DC maturation. Finally, we provide protocols to perform in silico analysis to find immunogenic epitopes in F. hepatica protein antigens (i.e., leucine aminopeptidase and cathepsins) and the design of a chimeric protein to be used in vaccination trials (Chapter 14). A vaccination protocol in a ruminant model to evaluate the immunoprotective effect of F. hepatica antigens (i.e., LAP) is described (Chapter 15). Also, we include some protocols to detect F. hepatica albendazole resistance (Chapter 16) and for the screening of small molecules as enzyme inhibitors (Chapter 17) to identify novel antiparasitic compounds. We selected different topics in areas such as biochemistry, immunology, molecular biology, microscopy, and vaccinology in order to extend the interest in this book to research on community working with Fasciola hepatica and related trematodes. This book is the effort of many researchers who share their time and expertise, and without them it would be impossible to culminate this work. We would like to express our warm thanks to their contributions and patience. Porto Alegre, Rio Grande do Sul, Brazil Martin Cancela Montevideo, Uruguay Gabriela Maggioli v Contents Preface . ................................................................... v Contributors................................................................. ix 1 Maintenance of Life Cycle Stages of Fasciola hepatica in the Laboratory . .................................................... 1 Valeria Gayo, Martin Cancela, and Daniel Acosta 2 Microscopical Techniques to Analyze the Hepatic and Peritoneal Changes Caused by Fasciola hepatica Infection ............................. 15 M. T. Ruiz-Campillo, V. Molina-Herna´ndez, J. Pe´rez, I. L. Pacheco, R. Pe´rez, A. Escamilla, F. J. Martı´nez-Moreno, A. Martı´nez-Moreno, and R. Zafra 3 Isolation of Secreted and Tegumental Surface Proteins from Fasciola hepatica ................................................... 27 Eduardo de la Torre-Escudero and Mark W. Robinson 4 Isolation and Analysis of Fasciola hepatica Extracellular Vesicles............... 37 Alicia Galiano, Maria Teresa Minguez, Christian M. Sa´nchez, and Antonio Marcilla 5 Cloning and Heterologous Expression of Protein-Coding Sequences in Escherichia coli ....................................................... 51 Martin Cancela and Gabriela Maggioli 6 Gene Silencing in the Liver Fluke Fasciola hepatica: RNA Interference ........ 67 Gabriel Rinaldi, Nicola´s Dell’Oca, Estela Castillo, and Jose´ F. Tort 7 Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization........................................... 93 Estela Castillo and Uriel Koziol 8 Evasion of Host Immunity During Fasciola hepatica Infection. ............... 107 Robin J. Flynn and Mayowa Musah-Eroje 9 Immunomodulatory Effect of Fasciola hepatica Excretory–Secretory Products on Macrophages. ............................................. 117 Lorena Guasconi, Marianela C. Serradell, Diana T. Masih, and Laura S. Chiapello 10 Study of Eosinophil Apoptosis Induced by Fasciola hepatica Excretory–Secretory Products ............................................ 133 Marianela C. Serradell, Lorena Guasconi, Laura Cervi, Laura S. Chiapello, and Diana T. Masih 11 Purification of Native Fasciola hepatica Fatty Acid-Binding Protein and Induction of Alternative Activation of Human Macrophages ............. 149 Olgary Figueroa-Santiago 12 Possible Role for Toll-Like Receptors in Interaction of Fasciola hepatica Excretory–Secretory Products with Human Monocyte Cell Line . .................................................... 171 Olgary Figueroa-Santiago vii viii Contents 13 Evaluation of the Immune