Parasiticide Screening: Volume 2: In Vitro and In Vivo Tests with Relevant Parasite Rearing and Host Infection/Infestation Methods

By Alan A. Marchiondo, Larry R. Cruthers and Josephus J. Fourie

Parasiticide Discovery: In Vitro and In Vivo Tests with Relevant Parasite Rearing and Host Infection/Infestation Methods, Volume Two presents valuable screening methods that have led to the discovery of the majority of parasiticides commercialized in the animal health industry. As much of the knowledge of parasiticide discovery methods is being lost in the animal health industry as seasoned parasitologists retire, this book serves to preserve valuable methods that have led to the discovery of the majority of parasiticides commercialized in animal health, also giving insights into the in vitro and in vivo methods used to identify the parasiticide activity of compounds.

• Addresses current issues of resistance, along with combination uses for resistant parasites
• Presents useful, authoritative information (chemical, pharmaceutical, clinical, etc.) for the pyrantel family of compounds
• Includes a discussion on screening methods in combination therapies
• Provides cutting-edge material for an evolving area of scientific discussion
• Includes in vitro and in vivo screens and parasite maintenance and culture methods

• Language: English
• Publisher: Academic Press
• Release date: Jun 8, 2019
• ISBN: 9780128165782

Alan A. Marchiondo

• American Association of Veterinary Parasitologists (AAVP) President 2012-2013 • AAVP Vice-President 2010-2011 • AAVP President-Elect & Program Chair, 2011-2012 • AAVP Secretary/Treasurer 2004-2010 • AAVP Program Administrator and Executive Secretary, 2014-Present • Editorial Board Member - Veterinary Parasitology, 1988-Present • 30-year career in the animal health industry • 50+ Publication in Peer reviewed Scientific Journals • Edmonds. M.D., Vatta, A.F., Marchiondo, A.A., Vanimisetti, H.B., Edmonds, J.D. 2018. Concurrent treatment with a macrocyclic lactone and benzimidazoles provides season long performance advantages in grazing cattle harboring macrocyclic lactone resistant nematodes. Vet Parasitol 252:157-162. • Ballweber, L.R., Beugnet, F., Marchiondo, A.A., Payne, P.A. 2014. American Association of Veterinary Parasitologists’ review of veterinary fecal flotation methods and factors influencing their accuracy and use – Is there really one best technique? Vet Parasitol 204:73-80. • Riviere, J.E., Brooks, J.D., Collard, W.T., Deng, J., de Rose, G., Mahabir, S.P., Merritt, D.A., Marchiondo, A.A. 2014. Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture-adjusted quantitative structure permeability relationship. J Vet Pharm Therap 37 (5):435-444. • Marchiondo, A.A., Holdsworth, P.A., Fourie, L.J., Rugg, D., Hellman, K., Synder, D.E., Dryden, M.W. 2013. World Association for the Advancement of Veterinary Parasitology: W.A.A.V.P. second edition: Guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestations on dogs and cats. Vet Parasitol 194:84-97. • Campbell, W.C., Conder G.A, Marchiondo A.A. 2009. Future of the animal health industry at a time of food crisis. Vet Parasitol 163 (3): 188-195. • Marchiondo, A.A., Holdsworth, P.A., Green, P., Blagburn, B.L., Jacobs, D.E. 2007. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestations on dogs and cats. Vet Parasitol 145: 332-344. • Marchiondo, A.A., Meola S.M., Palma K.G., Slusser J.H., Meola R.W. 1999. Chorion formation and ultrastructure of the egg of the cat flea (Siphonaptera, Pulicidae). J Med Entomol 36 (2): 149-157. • Co-Editor of Veterinary Parasitology Special Issue: “Antiparasitic Drug Use and Resistance in Large and Small Ruminants and Equines: Current Status in the United States with Global Perspectives”, 2014, Vet. Parasitology, Vol 204, Issues 1-2, pages 1-73. • Editor of PYRANTEL PARASITICIDE THERAPY IN HUMANS AND DOMESTIC ANIMALS, Academic Press, Elsevier, July 1, 2016, ISBN: 978-0-12-801449-3, 143 pages. • Patent Declaration: Synergistic activity of Fipronil and (S)-methoprene. Declaration, U.S. Patent 45313-2339, Issued August 1, 2000. Insecticidal combination to control mammal fleas, in particular on cats and dogs.

Book preview

Parasiticide Screening

In Vitro and In Vivo Tests With Relevant Parasite Rearing and Host Infection/Infestation Methods, Volume 2

Edited by

Alan A. Marchiondo

Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States

Larry R. Cruthers

LCruthers Consulting, Chesapeake, VA, United States

Josephus J. Fourie

Clinvet International, Bloemfontein, South Africa

Table of Contents

Cover image

Title page

Copyright

Dedication

List of Contributors

About the Authors/Editors

Preface

Acknowledgments

Introduction

Chapter 1. Platyhelminthes

Chapter 1a. Platyhelminthes, Trematoda

Fasciolidae

Fasciola gigantica Cobbold, 1855—giant liver fluke

Paramphistomatidae

Calicophoron microbothrium (Fischoeder, 1901)—conical fluke

Troglotrematidae

Heterophyidae

Dicrocoelilidae

Diplostomatidae

Schistosomatidae

Chapter 1b. Platyhelminthes, Cestoda

Diphyllobothriidae

Mesocestoididae

Chapter 2. Nematoda

Chapter 2a. Nematoda, Strongyloidea

Strongylidae; Strongylinae (large strongyles or palisade worms)

Stephanuridae

Syngamidae

Chapter 2b. Nematoda, Trichostrongyloidea

Haemonchus Cobb, 1898

Trichostrongylus Looss, 1905

Ostertagia (Teladorsagia) Ransom, 1907

Cooperia Ransom, 1907

Hyostrongylus Hall, 1921

Nematodirus Ransom, 1907

Dictyocaulus Railliet and Henry, 1907

Chapter 2c. Nematoda, Ancylostomatoidea

Ancylostomidae

Chapter 2d. Nematoda, Metastrongyloidea

Metastrongylidae

Protostrongylidae

Filaroidae

Crenosomatidae

Angiostrongylidae

Chapter 2e. Nematoda, Oxyurida

Oxyuridae

Rodent pinworms

Chapter 2f. Nematoda, Ascaridida

Ascaris suum Goeze, 1782—pig ascarid or swine roundworm

Parascaris spp. [Parascaris equorum (Goeze, 1782) Yorke and Maplestone, 1926; Parascaris univalens van Benedin, 1883]—equine roundworm

Toxocara canis (Werner, 1782) Stiles, 1905—roundworm

Toxocara cati (Schrank, 1788) Brumpt, 1927—roundworm

Toxascaris leonina (von Linstow, 1902) Leiper, 1907—roundworm

Ascaridia galli (Schrank, 1788) Freeborn, 1923—chicken ascarid

Heterakis gallinarum (Schrank, 1788) Madsen, 1949—cecal worms

Chapter 2g. Nematoda, Spirurida

Physalopteroidea

Thelaziidae

Spiruroidea

Habronematoidea

Chapter 2h. Nematoda, Filarioidea

Setaria labiatopapillosa (Rudolphi, 1819) Baylis, 1939; Setaria cervi Rudolphi, 1819; Setaria equina (Abildgaard, 1789) Viborg, 1795

Litomosoides sigmodontis (=Litomosoides carinii) (Travassos, 1919)

Onchocerca spp

Wuchereria bancrofti (Cobbold, 1877) Seurat, 1921—Bancroft’s filarial worm

Brugia pahangi (Buckley and Edeson, 1956) Buckley, 1960; Brugia malayi (Brug, 1927) Buckley, 1958—Malayan filarial worm

Parafilaria bovicola Tubangui, 1934; Parafilaria multipapillosa (Condamine and Drouilly, 1878) Yorke and Maplestone, 1926

Dirofilaria immitis (Leidy, 1856) Railliet and Henry, 1911—canine heartworm

Acanthocheilonema (Dipetalonema) reconditum (Grassi, 1889); Acanthocheilonema (Dipetalonema) viteae (Krepkogorskaja, 1933)

Chapter 2i. Nematoda, Trichinelloidea

Trichinella spiralis (Owen, 1835) Railliet, 1895—trichina worm or garbage worm

Trichuris globulosa (von Linstow, 1901) Ransom, 1911—cattle whipworm; Trichuris discolor (von Linstow, 1906) Ransom, 1911—cattle whipworm; Trichuris ovis (Abildgaard, 1795) Smith, 1908—sheep and goat whipworm

Trichuris muris Seurat, 1920—mouse whipworm

Trichuris suis (Schrank, 1788)—swine whipworm

Trichuris vulpis (Froelich, 1789)—canine whipworm

Capillaria species

Chapter 2j. Nematoda, Rhabditida

Rhabdidae

Strongyloididae

Chapter 3. Acanthocephala

Abstract

Acanthocephala

References

Chapter 4. Statistical considerations of parasiticide screening tests and confirmation trials

Abstract

General experimental design considerations

General data summary and analysis considerations

Variables

Data summaries

Statistical analysis

Statistical models

Transformations

Parametric versus nonparametric statistics

Multiple testing

Fixed and random effects

Repeated measurements

More on sample size and power

Choice of estimator for the mean (arithmetic mean vs geometric mean)

In vitro method(s)

In vivo method(s)

References

Appendices

Appendix I. World Association For The Advancement Of Veterinary Parasitology Parasiticide Guidelines

Appendix II. Manual For Maintenance Of Multi-Host Ixodid Ticks In The Laboratory

Glossary: abbreviations and symbols

Index

Copyright

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Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

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A catalog record for this book is available from the Library of Congress

ISBN: 978-0-12-816577-5

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Dedication

This book is dedicated to Dr. William C. Campbell in recognition of his Nobel Prize in Physiology or Medicine, 2015, for the discovery of ivermectin, and to scientists worldwide who are involved in the discovery of parasiticides.

List of Contributors

Douglas D. Colwell, PhD, FRES, Assoc. EVPC,     Agriculture and Agri-Food Canada, Lethbridge, AB, Canada

Dionne Crafford, PhD,     Clinglobal, La Mivoie, Mauritius

Larry R. Cruthers, MS, PhD,     LCruthers Consulting, Chesapeake, VA, United States

Michael T. Dzimianski, DVM,     University of Georgia, Athens, GA, United States

Ivan G. Horak, DVSc, Professor,     Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa

Dawie J. Kok, DSc,     Independent Scientific Consulting, Bloemfontein, South Africa

Sean P. Mahabir, PhD,     Zoetis Inc., Kalamazoo, MI, United States

Alan A. Marchiondo, MS, PhD,     Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States

Craig R. Reinemeyer, DVM, PhD, Dipl. ACVM, EVPC,     East Tennessee Clinical Research, Inc., Rockwood, TN, United States

Daniel E. Snyder, DVM, PhD, Dipl. ACVM,     Daniel E. Snyder DVM, PhD Consulting LLC, Indianapolis, IN, United States

Hima Bindu Vanimisetti, PhD,     Zoetis Inc., Kalamazoo, MI, United States

Thomas A. Yazwinski, PhD, Professor,     Animal Science, University of Arkansas, Fayetteville, AR, United States

Dante S. Zarlenga, PhD,     USDA, ARS Animal Parasitic Disease Lab, Beltsville, MD, United States

About the Authors/Editors

1. Author’s full name: Alan Anton Marchiondo, MS, PhD

2. Position and affiliation: Retired Research Fellow, Zoetis (formally Pfizer Animal Health), Kalamazoo, MI, United States; currently Managing Director, Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States

3. Important biographical notes regarding author (i.e., awards, previous books published):

a. American Association of Veterinary Parasitologists (AAVP) President, 2012–13

b. AAVP Vice President, 2010–11

c. AAVP President-Elect and Program Chair, 2011–12

d. AAVP Secretary/Treasurer, 2004–10

e. AAVP Program Administrator and Executive Secretary, 2014–Present

f. Editorial Board Member—Veterinary Parasitology, 1988–Present

g. 30 years career in the animal health industry

h. 50+ publications in peer-reviewed scientific journals

i. Edmonds MD, Vatta AF, Marchiondo AA, Vanimisetti HB, Edmonds JD. Concurrent treatment with a macrocyclic lactone and benzimidazoles provides season long performance advantages in grazing cattle harboring macrocyclic lactone resistant nematodes. Vet Parasitol 2018;252:157–62.

ii. Ballweber LR, Beugnet F, Marchiondo AA, Payne PA. American Association of Veterinary Parasitologists’ review of veterinary fecal flotation methods and factors influencing their accuracy and use – is there really one best technique? Vet Parasitol 2014;204:73–80.

iii. Riviere JE, Brooks JD, Collard WT, Deng J, de Rose G, Mahabir SP, et al. Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture-adjusted quantitative structure permeability relationship. J Vet Pharm Ther 2014;37(5):435–44.

iv. Marchiondo AA, Holdsworth PA, Fourie LJ, Rugg D, Hellman K, Synder DE, et al. World Association for the Advancement of Veterinary Parasitology: W.A.A.V.P. second edition: guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestations on dogs and cats. Vet Parasitol 2013;194:84–97.

v. Campbell WC, Conder GA, Marchiondo AA. Future of the animal health industry at a time of food crisis. Vet Parasitol 2009;163(3):188–95.

vi. Marchiondo AA, Holdsworth PA, Green P, Blagburn BL, Jacobs DE. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestations on dogs and cats. Vet Parasitol 2007;145:332–44.

vii. Marchiondo AA, Meola SM, Palma KG, Slusser JH, Meola RW. Chorion formation and ultrastructure of the egg of the cat flea (Siphonaptera, Pulicidae). J Med Entomol 1999;36(2):149–57.

viii. Coeditor of Veterinary Parasitology Special Issue. Antiparasitic drug use and resistance in large and small ruminants and equines: current status in the United States with Global Perspectives. Vet Parasitol 2014;204(1–2):1–73.

ix. Editor of Pyrantel parasiticide therapy in humans and domestic animals. Academic Press, Elsevier; 2016. p. 143. ISBN: 978-0-12-801449-3.

x. Patent Declaration. Insecticidal combination to control mammal fleas, in particular on cats and dogs. In: Synergistic activity of Fipronil and (S)-methoprene. Declaration, United States patent 45313-2339. Issued August 1, 2000.

1. Author’s full name: Larry R. Cruthers, MS, PhD

2. Position and affiliation: Retired VP of Research and Chief Operating Officer, Professional Laboratory and Research Services, Inc., Corapeake, NC, United States; Currently owner of LCruthers Consulting, Chesapeake, VA, United States

3. Important biographical notes regarding author (i.e., awards, previous books published):

a. Secretary/Treasurer of the New Jersey Society for Parasitology, 1976–79

b. President of the New Jersey Society for Parasitology, 1979–80

c. 49 years career in veterinary parasitology and/or the animal health industry

d. 44 publications/abstracts in peer-reviewed journals/scientific meetings

i. Elsemore DA, Geng J, Flynn L, Cruthers LR, Lucio-Forster A, Bowman DD. Enzyme-linked immunosorbent assay for coproantigen detection of Trichuris vulpis in dogs. J Vet Investig 2014;26(3):404–11.

ii. Snyder DE, Wiseman S, Cruthers LR, Slone RL. Ivermectin and milbemycin oxime in experimental adult heartworm (Dirofilaria immitis) infection of dogs. J Vet Internal Med 2011;25:61–4.

iii. Snyder DE, Cruthers LR, Slone RL. Preliminary study on the acaricidal efficacy of spinosad administered orally to dogs infested with the brown dog tick, Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Vet Parasitol 2009;166:131–5.

iv. McTier TL, Siedek EM, Clemence RG, Wren JA, Bowman DD, Hellmann K, et al. Efficacy of selamectin against experimentally induced and naturally acquired ascarid (Toxocara canis and Toxascaris leonina) infections in dogs. Vet Parasitol 2000;91(3–4):333–45.

v. Cruthers L, Guerrero J, Robertson-Plough C. Evaluation of the speed of kill of fleas and ticks with fipronil or imidacloprid. In: World Veterinary Congress. Lyon, France; September 1999.

vi. Cruthers LR, Hotchkin HD, Sarra L, Perry D. Efficacy of Tiamulin against and experimental Eimeria acervulina and Eimeria tenella infection of broilers. Avian Dis 1980;24:241–6.

vii. Veterinary Master File VMF 005–804 "Induced Whipworm (Trichuris vulpis) Infection in Canine".

viii. Chapter entitled. Pharmaco-therapeutics of gastrointestinal protozoa. In: Scientific foundations of veterinary science. William Heinemann Medical Books Ltd; 1980.

ix. Awarded nine US patents involving antiparasitics.

1. Author’s full name: Josephus J. Fourie, MSc, PhD

2. Position and affiliation: Vice President, Scientific Operations, Clinvet International, Bloemfontein, South Africa

3. Important biographical notes regarding author (i.e., awards, previous books published):

a. 16 years of experience in veterinary contract research

b. Acted as study Director/Investigator on 300+ preclinical and clinical trials

c. 50+ publications in peer-reviewed scientific journals

i. Halos L, Fourie JJ, Fankhauser B, Beugnet F. Knock-down and speed of kill of a combination of fipronil and permethrin for the prevention of Ctenocephalides felis flea infestation in dogs. Parasites Vectors 2016;9:57.

ii. Varloud M, Fourie JJ, Blagburn BL, Deflandre A. Expellency, anti-feeding and speed of kill of a dinotefuran-permethrin-pyriproxyfen spot-on (Vectra 3D) in dogs weekly challenged with adult fleas (Ctenocephalides felis) for 1 month-comparison to a spinosad tablet (Comfortis). Parasitol Res 2015;114(7):2649–57.

iii. Fourie JJ, Joubert A, Labuschagnè M, Beugnet F. New method using quantitative PCR to follow the tick blood meal and to assess the anti-feeding effect of topical acaricide against Rhipicephalus sanguineus on dogs. Comp Immunol Microbiol Infect Dis 2013;37:181–7.

iv. Fourie JJ, Liebenberg J, Nyangiwe N, Austin C, Horak I, Bhushan C. The effects of a pour-on formulation of fluazuron 2.5% and flumethrin 1% on populations of Rhipicephalus decoloratus and Rhipicephalus microplus both on and off bovine (Bonsmara breed) hosts. Parasitol Res 2013;112:67–79.

v. Fourie JJ, Stanneck D, Luus HG, Beugnet F, Wijnveld M, Jongejan F. Transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks feeding on dogs and on artificial membranes. Vet Parasitol 2013;197(3–4):595–603.

vi. Kužner J, Turk S, Fourie JJ, Grace S, Marchiondo AA, Rugg D. Efficacy of a novel fipronil spot-on for the treatment and control of induced infestations of adult cat fleas (Ctenocephalides felis) and castor bean ticks (Ixodes ricinus) on cats. Parasitol Res 2013;112:365–72.

vii. Jongejan F, Fourie JJ, Chester ST, Manavella C, Mallouk Y, Pollmeier MG, et al. The prevention of the transmission of Babesia canis by Dermacentor reticulatus to dogs using a novel combination of fipronil, amitraz and (S)-methoprene. Vet Parasitol 2011;179(4):343–50.

viii. Coauthored book chapter entitled. Biology, ecology and vector role of fleas. Guide to vector borne diseases of pets. Lyon: Merial; 2013.

ix. Coauthored book chapter entitled. The bed bug resurgence in Africa. In: Doggett SL, Miller DM, Lee C-Y, editors. Advances in the biology and management of modern bed bugs. Penang, Malaysia: Wiley Blackwell; 2018.

Preface

The discovery of new and novel parasiticides for veterinary medicine in the animal health and agricultural industries has been driven by (1) the need to increase the spectrum and effectiveness of parasiticide activity and (2) the ongoing emergence and development of parasiticide resistance. Since the introduction of the first commercial parasiticides, parasitologists and entomologists, in animal health and agricultural companies, have devised various screening methods using parasites to identify the parasiticide activities of compounds that would treat and control targeted parasites of animals and crops. The majority of new chemical entities in the animal health and agrochemical industries originate from the chemical evolution approach, which is the empirical screening of more or less randomly selected chemicals in relevant in vitro and in vivo tests in the hope of finding useful biological properties in a novel chemical. This book focuses on those whole organism empirical screening methods used to identify parasiticide activities and does not cover mechanistic, in silico, and receptor-based/genomic screening methods. The inspiration for this book was to capture historically empirical parasiticide screening methods in a single source reference form; lest this information is lost over time.

The target audience of this book is not only the basic researcher in parasiticides and the field researcher involved in parasite control but also advanced undergraduates, graduate students, university and industrial research workers, and teachers in biology, the health sciences, veterinary, and human medicine. Animal health programs can contribute to screening models and expertise to human parasite discovery efforts.¹

In a comprehensive review of this kind, it is probably inevitable that some errors and omissions will have crept in, in spite of careful checking. The literature on the subject was huge and we acknowledge that some references have been overlooked or omitted. We apologize up front for any omissions, but we were bound by editorial limitations.

The authors and contributors cannot verify that all parasiticide testing and rearing methods involving mammalian species reported herein have been reviewed, approved, and conducted under an IACUC protocol or other regional, national, and international regulatory laws and policies applicable to animal welfare in research. Therefore the authors and contributors do not regard or treat as acceptable or endorse from an animal welfare standpoint any parasiticide testing or rearing method reported in this book. The use of these parasiticide testing or laboratory rearing methods is to be conducted under current animal welfare and regulatory guidelines. Most methods and tests reported herein are as they appeared in print, appropriately cited with limited minor editing, so as not to lose the original meaning. For the most part, the taxonomic classification scheme and scientific nomenclature follow that in Bowman et al.²

Due to the length of this book, it has been divided into two volumes. Volume 1 consists of five chapters on parasiticide screening tests beginning with an introductory section that sets the stage for the content that follows. Due to space constraints, the screening and rearing methods reported have been culled, abbreviated, and/or combined where appropriate. References to specific scientific papers have been kept to a minimum with some details and subject reviews to be sought in original citations. Since the chapters have been written by different authors, the format may vary slightly, but the content is consistent throughout. The first and second chapters define in vitro and in vivo parasiticide screening, respectively. Both chapters include details and guidance on protocol design and execution. Chapter 3, Arthropoda, focuses on the in vitro and in vivo parasiticide discovery screening tests and host evaluation methods for arthropod parasite species with information on parasite biology and rearing methods. This theme is continued in Chapter 4, Arachnida, on arachnid parasites and Chapter 5, Protozoa, on protozoan parasites.

Volume 2 of the book begins with Chapter 1, Platyhelminthes, on the Platyhelminthes, followed by Chapter 2, Nematoda, on the Nematoda, and a short Chapter 3, Acanthocephala, on acanthocephalan parasites. The final chapter, Chapter 4, Statistical considerations of parasiticide screening tests and confirmation trials, provides the statistical considerations of parasiticide screening methods for parasite and host species. Biometrics, the application of the mathematical statistics to biology, is essential both in the planning of the bioassay or applied studies and the interpretation of the results of parasiticide screens and test methods.

A useful Appendix I, World Association for the Advancement of Veterinary Parasitology Parasiticide Guidelines, containing the WAAVP parasiticide testing guidelines has been printed in toto with permission from the publisher, so the reader has direct access in this text to these valuable documents to dovetail with the testing and rearing methods. Appendix II contains a useful manual printed in toto with permission entitled Manual for Maintenance of Multi-Host Ixodid Ticks in the Laboratory by M.L. Levin and L.B.M. Schumacher. Finally, a glossary of abbreviations is provided.

Respectfully,

Alan A. Marchiondo, Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States

Larry R. Cruthers, LCruthers Consulting, Chesapeake, VA, United States

Josephus J. Fourie, Vice President: Scientific Operations, Clinvet International, Bloemfontein, South Africa

Date: April 1, 2019

References

1. Geary TG, Woods DJ, Williams T, Nwaka S. Target identification and mechanism-based screening for anthelmintics: application of veterinary antiparasitic research programs to search for new antiparasitic drugs for human indications. In: Selzer PM, ed. Antiparasitic and antibacterial drug discovery: from molecular targets to drug candidates. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2009;3–15.

2. Bowman DD, Lynn RC, Eberhard ML, Alcaraz A. Georgi’s parasitology for veterinarian’s 8th ed. St. Louis, MO: Saunders; 2003;422.

Acknowledgments

The idea for the book was conceived on March 10, 2004 by Drs. Alan A. Marchiondo and Larry R. Cruthers. Dr. Josephus J. Fourie eagerly agreed to coauthor the book.

The book could not have been completed without the contributor’s scientific expertise and their cooperation in meeting timelines. All contributing authors are acknowledged in the contributor section.

We are indebted and express our appreciation to Linda Versteeg, Acquisition Editor, Elsevier, Inc. for her encouragement and enthusiasm for this book and to Tracy I. Tufaga, Editorial Project Manager, Academic Press/Elsevier, San Diego, CA, United States for support and assistance in the publication of the book. The Elsevier staff were most valuable to us in obtaining permission to cite tables and figures from previous publications and in providing editing assistance.

We thank the following individuals for their assistance: Carmen Neethling and Dr. Dionne Crafford, Clinvet International, Bloemfontein, South Africa for research and literature searches; Lisa A. Marchiondo, PhD, Department of Organizational Studies, Anderson School of Management, University of New Mexico, Albuquerque, NM, United States as a reviewer and technical resource; Dr. Tom J. Kennedy for his Strongylus vulgaris artificial infection methods; Dr. Tom Nolan for the Strongyloides stercoralis rearing and infection methods; Dr. Tom McTier for his review of the Siphonaptera section; and Dr. Dan Ostlind for his review of the Hemiptera section and for providing numerous screening references.

We also thank Jason C. Liddell, Tijeras, NM, United States for the book cover illustration of the ancient ouroboros symbol redesigned as a morphologically similar female hookworm (teeth resemble Ancylostoma caninum), consuming its own tail, reflecting an image of circular reasoning that knowledge of veterinary parasitology feeds even more knowledge of veterinary parasitology.

Finally, a special thanks to Dr. William C. Campbell, 2015 Nobel Laureate, for reviewing the Preface and Introduction of the book and granting permission to dedicate the book in his honor.

The images, diagrams, and articles printed in toto have been reproduced with kind permission from the following: Elsevier permission to reprint in toto the WAAVP testing guidelines, as well as the Entomological Society of America and the Springer Science for various figures and permission to reprint in toto the Manual for Maintenance of Multi-Host Ixodid Ticks in the Laboratory by M.L. Levin and L.B.M. Schumacher.

Introduction

Alan A. MarchiondoAdobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States

Larry R. CruthersLCruthers Consulting, Chesapeake, VA, United States

Josephus J. FourieClinvet International, Bloemfontein, South Africa

Abstract

This book, provided in two parts (Volume 1 and Volume 2), provides the biology, life cycles, laboratory rearing methods, and in vitro and in vivo parasiticide screening methods of major parasite species of veterinary importance. The history of screening for parasiticides now firmly establishes the paradigm of data-driven compound activities identified through extensive experimentation and empirical testing in relevant in vitro and in vivo screens that vary by target parasite and host species. The in vitro and in vivo parasiticide screening methods provided herein serve as a resource for animal health companies and parasitology researchers in the discovery of new and novel parasiticides.

Keywords: Discovery, parasiticide, screening, methods, in vitro, in vivo, whole organism

The control of arthropod, helminth, and protozoan parasites remains a primary focus of animal care in veterinary medicine in both companion and production animals. The introduction of new parasiticides that prevent, treat, and control infections and infestations of parasites has provided remarkable productivity benefits to livestock producers and major health benefits to pets. Notwithstanding these achievements, only a few parasites of veterinary importance have been eradicated regionally,¹ therefore the search for new treatments must continue. The prominent trend in chemotherapy and management of parasitic diseases has been driven by the continual search for new drugs with greater efficacy, a broader spectrum of activity, greater ease and safety of use, and relative cheapness. Such drugs could provide marked advances in the field of parasite control, while addressing the ongoing emergence and development of parasiticide resistance.²,³ The effects of parasitism on animals, along with the associated impact on pet and livestock owners, have become well embraced by the animal health industry, which is traditionally linked with the business of developing and marketing parasiticides. Threats (anthelmintic/insecticide resistance) and gaps (lack of broad-spectrum activity, emerging diseases, unmet needs in new species, reformulations, and combinations) in the treatment and control of animal parasites are likely to be considered opportunities for exploitation and differentiation in the discovery, development, and marketing of new animal parasiticides by the animal health industry.

For the purpose of this text an animal parasiticide (Latin parasītus, Greek parásītos, one who eats at another’s table, 1530–1540, + Latin caedo, cide to kill) is defined as a chemotherapy (chemical agent) used in veterinary medicine to treat, control, expel, destroy, or kill an external or internal parasite infesting or infecting a domestic (dog, cat, and horse) or commercial/livestock (cattle, sheep, goat, pig, and chicken) animal. Endoparasiticides that include anthelmintics and some insecticides, such as dichlorvos, famphur, trichlorfon, are used to treat, control, or prevent internal parasite infections and infestations, respectively. Antiprotozoal drugs are a class of pharmaceuticals that treat infections caused by parasitic protozoa. Failure to treat and control parasitic protozoa, trematodes, tapeworms, roundworms, etc. in livestock and companion animals can lead to the loss of condition and productivity. Ectoparasiticides are insecticides/acaricides that control external parasites of animals. These parasites include flies, bugs, fleas, ticks, lice, and mites. Endectocides are anthelmintics/insecticides/acaricides that have endoparasiticide properties but also have ectoparasiticide application in some host species, for example, avermectins and milbemycins.

Searching for treatments of parasitic diseases has been ongoing for literally thousands of years, ever since diseases have been known. Many older drugs used for the treatment of infections of helminth parasites were plant/herbal products widely utilized since ancient times as evidenced by Galen’s dogmatic herbalism from about 200 AD through the Middles Ages, the value of which had been inadequately assessed by clinical observations. Experience with this multitude of remedies supports the old adage that where there are many cures, there are no cures and grew out of favor during the Renaissance when Paracelsus introduced chemistry of a sort by attempting to cure specific diseases with laboratory-produced mineral drugs.⁴,⁵ Ancient antiparasitic lore has been surveyed by Hoeppli,⁶ and the history of antiparasitic therapy has been reviewed up to the middle of the present century by Hawking.⁷ Nobel Laureate William C. Campbell has provided a general, but compelling, evolution of antiparasitic chemotherapy within each category of protozoan, helminth, and arthropod parasites.⁸

Treatment practices of disease founded on incorrect reasoning without scientifically proven facts have often prevailed for years or centuries being often more harmful than beneficial. One night with Venus, a lifetime with Mercury was a common late 1800s dictum among physicians for the standard treatment for syphilis with mercury,⁹ although doctors doubted the efficacy of mercury aborning the terrible side effects, with many patients dying of mercury poisoning. In 1909 the laboratory screening by Paul Ehrlich (1854–1915) led to discovery of the arsenic-based compound arsphenamine (Salvarsan) that was the first effective medical treatment of syphilis. Paul Ehrlich is credited with the concept of chemotherapy, that is, the treatment of a parasitic disease with a chemical pharmaceutical of known constitution, thus postulating his concept and term magic bullet.¹⁰ Ehrlich’s approach to chemotherapy discovery was to start with a known organic arsenic compound and then synthesize hundreds of related organoarsenic compounds for in vivo screening against Treponema pallidum–infected rabbits. Relying on experience and observation alone, empirical evidence of his in vivo screening proved that the 606th compound screened (Compound 606, Salvarsan) was the best candidate based on biological activity and safety. Thus it seemed that a magic bullet (magische Kugel), Ehrlich’s term for an ideal therapeutic agent that selectively killed only the targeted organism without damaging the host, had been found and his hypothesis proven. This is perhaps the best example of faith in a hypothesis triumphing over seemingly insuperable difficulties in the history of the study of disease.¹¹ An interesting historical note is that the chemist who synthesized the screening compounds for Dr. Ehrlich, Sahachiro Hata, studied at the Kitasato Institute in Tokyo, which was the same institution where Dr. Satoshi Ōmura isolated Streptomyces avermitilis in 1974.¹² A research team at the Merck Institute of Therapeutic Research, the United States, led by Dr. William C. Campbell, studied Dr. Ōmura’s Streptomyces cultures for effectiveness in treating parasitic infections in domestic and farm animals, leading to the isolation and discovery of the extremely potent endectocide, avermectin. Avermectin was refined into ivermectin, and this drug revolutionized parasitic control in animals and in control of human filarial parasites. Dr. Ōmura and Dr. Campbell were jointly awarded the 2015 Nobel Prize for physiology or medicine.¹³

In the search for chemotherapeutic agents, chance has been cited as an important factor in discovery. Wise historic quotes include Chance favours only those who know how to court her by Charles Nicolle (1866–1936); In the field of observation, chance favours only the prepared mind by Louis Pasteur (1822–95); the 1605 quote Men are rather beholden … generally to chance, or anything else, than to logic, for the invention of arts and sciences by Francis Bacon (1561–1626); and Discovery should come as an adventure rather than as the result of a logical process of thought by Theobald Smith (1859–1934).¹⁴ The word serendipity (a happy accident that, through sagacity, is transformed into opportunity) was first coined in 1754 by Horace Walpole (1717–97), the fourth Earl of Orford, who derived the new word from a set of fables about princes of Serendip (now Sri Lanka), wherein the princes excelled at making clever deductions on the basis of flimsy and fragmented evidence. Chance is fundamentally unrelated to rationality, whereas serendipity presupposes a smart mind based on two factors: (1) a discovery that is not merely aided by chance but arises from chance; and (2) an intellectual effort or intuitive leap that derives the discovery from chance events.¹⁵ As originally coined, serendipity meant the ability to apply sagacity to chance observation and thereby find something other than what one was looking for. Thus discoveries made in this fashion are considered examples of serendipity, hence serendipitous. Sir William Paton wrote There is an extra dimension to the possible outcomes of an experiment. What happens quite commonly is not an answer to the question put, but something quite unexpected.¹⁶

Perhaps the most striking examples of empirical discoveries are found in chemotherapy.¹⁷ The majority of new chemical entities introduced, particularly in parasitology, has arisen, and continue to arise, from chance observation, controlled experimentation, clinical experience, and the empirical search for substances active against parasites. The basis of most biological experimentation is the controlled test. The parasiticide discovery has historically been empirical, based on trial and error or on particular clinical observation. The role of chance does not produce drugs, but it is the interpretation of chance observation that merely provides the opportunity. Where chance plays a pivotal role in drug discovery, the event may be considered serendipitous to a greater or lesser degree. Empirical discoveries have occurred by finding impurities in other substances which were being tested (e.g., sulfanilamide in the dye of prontosil), an unexpected pharmacological effect (e.g., piperazine introduced for the treatment of gout dewormed the patient; sildenafil citrate given for the treatment of heart disease was developed to treat sexual dysfunction), or happy accidents in screening with the wrong animal model so characteristic of serendipity (e.g., the in vitro nematode activity of thiazothienol was inactive in worm-infected rats and mice but was metabolized to thiazothilite and excreted in the feces of chickens, leading to the discovery of tetramisole and levamisole).¹⁸

Scientists have a strong bias for attributing creative insight to empirical evidence.¹⁹ The use of initial in vitro screens is carried out on a large number of chemical substances to detect biological activity and determine which of them warrant further testing. Such basic screening activity is often pooh-poohed as dog work of unimaginative drones. However, no discovery of new parasiticides has occurred without the foundation that these data provide to the parasitologist and medicinal chemist. Data-driven decisions ensure that the best candidates are advanced into the development pipeline.²⁰

In parasiticide discovery, it is often good policy to start with a modest preliminary experiment to get an indication as to whether a full-scale experiment is warranted. Whole parasite (whole organism) in vitro screening, followed by in vivo confirmation and spectrum of activity validation, has been most valuable in the discovery of new anthelmintics and has led to the discovery of the majority of currently available anthelmintics.²¹,²²

Parasiticides do not originate from de novo design but, rather, are the products of evolution by exploiting existing related chemicals or classes of compounds (i.e., the systematic process of changing established structures with established biological properties with the intention of improving the profile of the biological properties).²³ The chemical evolution approach is driven by empirical screening of a more or less random selection of chemicals in relevant in vitro and in vivo tests in the hope of finding useful biological properties in a novel chemical; that is, lead identification compounds.⁷ The chances of success in the identification of a lead compound depend upon the number and structural variety of the compounds screened and the reliability and relevance of the screening tests.⁷ Identified leads are then optimized via structure–activity relationship information on the chemicals, the extension of this information to a group of related compounds, and the subsequent arrival at a drug candidate with optimal properties.

The processes involved in lead identification and optimization are not to be taken lightly and involve more failures than successes. A typical antiparasiticide lead seeking paradigm might begin with a battery of in vitro whole-organism parasite screens coupled with small animal model in vivo parasite screens to identify a hit compound that meets the profile and spectrum of parasiticide activity. Bona fide and optimized hit compounds subjected to toxicological testing are then tested in proof of concept in vivo efficacy testing in the target host against one or more target parasite species. If the in vivo efficacy and safety testing shows promise, then compounds are advanced to a new project phase that may lead to development of a new parasiticide.

Screening paradigms might focus on a specific target parasite, for example, fleas, ticks, helminths, that the veterinary market and/or research companies have identified as a potential unmet need or commercial opportunity. Before the discovery and commercialization of ivermectin, both the cattle nematode and canine heartworm markets consisted of older endoparasiticides that were relatively toxic, difficult, or inconvenient to administer, and relatively short acting in efficacy. But the development of ivermectin formulations specifically for these two disease areas revolutionized these markets. In the same way the flea and tick companion animal market was dominated by organophosphates, carbamates, pyrethroids, etc. which only provided 1–2 weeks of control and had potential adverse reactions on treated pets. However, the advent of topical fipronil with consistently proven once a month efficacy against both fleas and ticks revolutionized the companion animal ectoparasite control market. So, a screening paradigm for these disease areas might concentrate on in vitro tests of target parasites to identify and determine the intrinsic activity of a hit compound, that is, a compound which has the desired activity in an applicable screen. However, this strategy depends on competitive intelligence via patent, published literature, and scientific meetings; novel target identification; mechanism-based screens, chemical libraries; and a commitment to the synthesis of lead compounds for screening. It also runs the risk of missing parasiticide activities against other species of parasites. Hence, a more general compound screening paradigm might include a broad number of in vitro screens of different parasite species to identify parasiticide activities with hit compounds being confirmed in secondary parasite screens for validation and progression to lead candidate compounds for potential treatment of a particular parasite disease. These examples of compound screening strategies are a bit oversimplified, and researchers devote extensive time and resources to develop optimal compound screening processes in ever-changing parasite target goals and timelines.

Typically in chemotherapy, rational experimentation after the initial empirical discovery opens up the field of research that usually leads to a series of similar compounds; some of which may have important improvements. An example includes the avermectins and milbemycins that contain the common macrocyclic lactone ring but are fermentation products of different organisms. Structurally, avermectins have sugar groups at C13 of the macrocyclic ring, whereas the milbemycins are protonated at C13. Another example is the number of isoxazolines (fluralaner, sarolaner, and lotilaner) identified following the initial discovery of afoxolaner.

An excellent example of optimization of structure–activity relationships for broad-spectrum ectoparasiticidal potency, pharmacokinetics, and safety in the host species in arriving at an optimal drug candidate is provided in the discovery of sarolaner, a novel isoxazoline administered orally for systemic control of fleas and ticks on dogs and cats.²⁴ Wherein the addition of a 4-substituted fluorine to a 3,5-dichlorophenyl head unit provided superior tick potency, the single S-enantiomer provided all of the activity residing in this chiral conformation; the spiroazetidinebenzofuran moiety, a unique structure, previously undescribed in the parasitology literature, providing rigidity, potency, and novelty to the molecule; and the final piece of optimization, yielded the methylsulfonylethanone tail, increasing the polar surface area of the molecule and maximizing the pharmacokinetic exposure to target the rapid kill of fleas and ticks.²⁴

Once in vitro hit compounds are identified, the development of in vivo models in rodents to facilitate lead optimization and spectrum profiling provided the advantage of testing compounds in a living host with a known parasite infection or infestation, while requiring less compound than that in a larger target host (i.e., dog or calf), often times shorter study duration, increased capacity to screen more compounds, and lower screening costs. The discovery of the tetrahydropyrimidines provides an early example of parasiticide lead identification and optimization using novel in vivo rodent screens of one up to three species of parasites, followed by confirmation tests against target parasites within host species.²⁵,²⁶ Initially, researchers at Pfizer established a screening program to find new anthelmintic agents. They employed infections of the gastrointestinal nematodes Nematospiroides dubius (Heligmosomoides polygyrus bakeri) in mice and Nippostrongylus muris in rats.²⁷ But as the screening program progressed, the need for broader anthelmintic spectrum increased evolving to a triple parasite infection model in mice consisting of two nematode suborders (Strongylata, Ascaridata) and one cestode order (Cyclophyllidea) that covered three intestinal niches within the host (the duodenum, ileum, and cecum).²⁵ Close and competitive collaboration between medicinal chemists and parasitologists yielded a highly successful outcome. The identification of the laboratory anthelmintic spectrums of activity of pyrantel, morantel, and oxantel rapidly led to the development and commercialization of products for the treatment and control of nematode infections in animals and humans.

Another rodent discovery screening example applicable to screening compounds against nematode infections in ruminants was developed by Conder et al.²⁸ using an immunosuppressed Mongolian gerbil (Meriones unguiculatus) model infected with Haemonchus contortus and Trichostrongylus colubriformis. The uniqueness of this model was that various different classes of anthelmintics (i.e., benzimidazoles, febantel, ivermectin, levamisole, and milbemycin) were efficacious in the jird against these two species of ruminant parasites harboring different locations in the gastrointestinal tract at doses comparable to those effective in the treatment of sheep and cattle. Subsequent to the dual infection in the jird model, other ruminant helminth parasites such as Ostertagia ostertagi and O. circumcincta have been used to profile the anthelmintic activity of the cyclodepsipeptide PF1022A.²⁹ A triple infection model consisting of H. contortus, T. colubriformis, and T. sigmodontis was used to profile the spectrum and efficacy of paraherquamide.³⁰

Sarolaner emerged as the most potent of more than 3000 compounds tested using an in vitro and in vivo whole-organism parasite screening system.²⁴ Preliminary efficacy screening was conducted in vitro against the cat flea, Ctenocephalides felis felis, with active hits subsequently screened against the soft tick, Ornithodoros turicata. Once initial efficacy had been profiled in vitro, the safety of selected compounds was assessed in a mouse symptomatology model, and those compounds with an acceptable rodent safety profile were progressed to target animal toleration, pharmacokinetics, and efficacy studies in the dog. The novel ectoparasiticide class of the isoxazolines has been thoroughly reviewed along with the discovery and development of afoxolaner and sarolaner.³¹

The history of screening for parasiticides now firmly establishes the paradigm of data-driven compound activities identified through extensive experimentation and empirical testing in relevant in vitro and in vivo screens that vary by target parasite and host species. The ancient ouroboros symbol of a serpent consuming its own tail reflects an image of circular reasoning that past knowledge of veterinary parasitology screening feeds even more knowledge of future veterinary parasitology screening. This circular reasoning also applies to veterinary parasitology knowledge.

No current body of work captures the plethora of in vitro and in vivo parasiticide screening methods. Therefore the rearing and screening methods provided in this text will be valuable for research and teaching. This book, provided in two parts (Volume 1 and Volume 2), provides the biology, life cycles, laboratory rearing methods, and in vitro and in vivo parasiticide screening methods of major parasite species of veterinary importance.

References

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29. Conder GA, Johnson SS, Nowakowski DS, et al. Anthelmintic profile of the cyclodepsipeptide PF1022A in in vitro and in vivo models. J Antibiot (Tokyo). 1995;48:820–823.

30. Ostlind DA, Cifelli S, Mickle WG, et al. Evaluation of broad-spectrum anthelmintics activity in a novel assay against Haemonchus contortus, Trichostrongylus colubriformis, and T. sigmodontis in the gerbil Meriones unguiculatus. J Helminthol. 2006;80:393–396.

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Chapter 1

Platyhelminthes

Chapter 1a

Platyhelminthes, Trematoda

Ivan G. Horak, DVSc, Professor¹, Alan A. Marchiondo, MS, PhD² and Douglas D. Colwell, PhD, FRES, Assoc. EVPC³,    ¹Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa,    ²Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States,    ³Agriculture and Agri-Food Canada, Lethbridge, AB, Canada

Abstract

This large chapter covers the biology, life cycles, laboratory rearing, and cultivation methods, in vitro parasiticide methods, and in vivo parasiticide methods of veterinary and medically important species of the Trematoda, Digenea. Selected fluke species of the families Fasciolidae, Paramphistomatidae, Troglotrematidae, Heterophyidae, Dicrocoelidae, Diplostomatidae, and Schistosomidae are included.

Keywords: Platyhelminthes, Trematoda, Digenea, Fasciolidae, Fasciola hepatica, liver fluke of sheep and cattle, Fasciola gigantica, giant liver fluke, Calicophoron microbothrium, conical fluke, Troglotrematidae, Nanophyetus salmincola, salmon poisoning fluke, Paragonimus kellicotti, North American lung fluke, Heterophyidae, Heterophyes heterophyes, minute intestinal fluke, Metagonimus yokogawai, Opisthorchis felineus, Opisthorchis tenuicollis, cat liver fluke, Dicrocoelium dendriticum, lancet liver fluke, Platynosomum concinnum, Platynosomum fastosum, feline liver fluke, lizard poisoning, Eurytrema procyonis, Diplostomatidae, Alaria canis (americana), Alaria marcianae, Schistosomidae, Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Schistosoma bovis, Schistosoma mattheei, Schistosoma douthitti, Heterobilharzia americana, dog schistosome, biology, life cycle, rearing, cultivation, in vitro, in vivo, parasiticides

Platyhelminthes

Trematoda

Digenea

Fasciolidae

Fasciola hepatica Linnaeus, 1758—liver fluke

Biology and life cycle

Fasciola hepatica occurs worldwide where sheep and cattle are found but is dependent on climates and soil characteristics that favor the intermediate host, lymnaeid snails.¹ Fasciolosis caused by F. hepatica is a zoonotic disease with 7071 human cases reported from 51 countries in all continents over a 25-year period.² The fascinating history of F. hepatica has been described in detail by Schmidt and Roberts³ and extensively reviewed by Dalton⁴ and Beesley et al.⁵

Adult flukes live in the bile ducts of the liver, particularly in ruminants and occasionally humans. The adult flukes are leaf shaped and measure ~30 mm long×~13 mm wide and taper from relatively broad shoulders anteriorly to a pointed or narrowly rounded tip posteriorly. The flukes are dorsoventrally flat and have undulating margins. A more substantial cone-shaped projection is present anteriorly with an oral sucker surrounding a mouth at its tip. The ventral sucker is situated at the base of the cone, and the genital pore lies between the two suckers. Living flukes are light to dark pink in color and when filled with blood the branched and rebranched caeca can clearly be seen through the integument. Blood may be regurgitated by flukes placed in water or normal saline. The anterior and ventral suckers are used for attachment to the epithelium of the bile ducts to assist with migratory movements. Most of the body surface is covered with closely packed minute spines that point posteriorly. The number of spines and their length increases as the flukes grow, and at 3-week PI the spines metamorphose from being single pointed to multipointed. The spines assist the flukes to maintain their position in the bile ducts and at the same time erode the epithelium and pierce the finer blood vessels as an aid to feeding.

Eggs are yellowish brown, oval, 130–145 µm long×70–90 µm wide, and the operculum is indistinct. The miracidium is ~130 µm long; it is broad anteriorly and tapers posteriorly to a narrowly rounded end. It has a pair of eyespots close to the anterior end of the body and a mobile papilliform anterior protrusion. The surface of the miracidium is covered by large ciliated epithelial plates arranged in sequence from 6 anteriorly to 2 posteriorly.

The sporocyst is elongate and sac like in appearance and contains balls of germinal cells, which give rise to rediae. The rediae are sausage shaped with a raised collar-like structure in the anterior one-fourth of their length and a pair of lateral projections posteriorly. They have a mouth and pharynx that leads to an unbranched intestine. Mature rediae may contain germinal cells, daughter rediae, and cercariae and have a birth pore situated posterior to the collar. The cercariae are tadpole like in appearance. The length of their body varies between 250 and 350 µm, and their tail is about twice as long. The anterior and ventral suckers are visible. The metacercariae are circular in shape with a diameter of ~200 µm, and the cyst wall consists of four layers. The external casing of the metacercariae is at first white, but later becomes ivory colored.

Life cycle in the intermediate host

The eggs are passed with the feces of the infected host and must be freed from fecal material and end up in water for miracidia to develop. The miracidia hatch from the eggs after ~12 days on exposure to bright light and seek a snail host. The miracidia have ~24 hr to find and penetrate a suitable freshwater snail of the family Lymnaeidae, particularly Galba truncatula in Europe, Asia, Africa, and South America; Lymnaea viator, Lymnaea neotropica, Lymnaea cubensis, and Pseudosuccinea columella in Central and South America; Lymnaea tomentosa in Australia; and Fossaria modicella or Stagnicola bulimoides in the United States. The miracidia are phototropic and swim toward the water surface where they are more likely to find an amphibious intermediate snail host. Their host-seeking activity is affected by temperature; below 5°C, they are inactive while in temperatures between 15°C and 26°C they are optimal. They live for ~24 h. Using their apical papilla, most miracidia penetrate the epithelium of the snail close to its pulmonary cavity, shedding their epithelial plates and cilia during their migration into the snail’s tissue. After penetration of the snail a miracidium metamorphoses into a mother sporocyst, a process that takes ~12 h. The mother sporocyst grows, and the first generation of ~8 daughter rediae develops internally. The rediae rupture the wall of the sporocyst, and it subsequently dies. This first generation of rediae may give rise to a second generation of rediae, and these and the first generation of rediae give rise to large numbers of cercariae. The cercariae leave the rediae via the birth pore and require a period of maturation in the snail’s tissues before emergence. The cercariae may emerge from the snails as early as 27 days PI. The emergence of the cercariae from the intermediate snail host G. truncatula is triggered by bright light, whereas the emergence from P. columella is triggered by a cold shock, followed by a rise in temperature. The cercariae swim to the water surface, lose their tails and encyst, becoming metacercariae on vegetation at the water’s edge.

Life cycle in the definitive host

The life cycle of F. hepatica in cattle or sheep has been described by several authors.⁶ The metacercariae encysted on vegetation are ingested by animals grazing in snail-infested marshy areas or at the water’s edge. The metacercariae excyst in the small intestine after the exposure to HCl in the abomasum, and trypsin and bile salts in the duodenum. The young flukes exit the metacercariae via a ventral plug in its base and burrow through the wall of the small intestine into the peritoneal cavity within a few hours after excystation. By ~6 days PI the majority of young flukes have penetrated the liver capsule and migrated within the liver parenchyma, leaving tracts of necrotic cells and other debris behind. Some immature flukes may reach the liver via the blood stream and others via the common bile duct, but the usual route is via the peritoneal cavity. The migration in the liver continues for 5–6 weeks, and the first young flukes are found in the main bile ducts ~6 weeks PI, and the first F. hepatica eggs are detected in the feces of sheep at ~8 weeks PI.

However, an alternative migratory route has been suggested by Moazeni and Ahmadi.⁷ According to these authors, there is no microscopic evidence of a young fluke actually in the process of penetrating the wall of a bile duct after completing its migration through the parenchyma of the liver. They suggest that some of the very small juvenile flukes may enter the bile ducts immediately after reaching the liver parenchyma, or that when newly excysted flukes are penetrating the intestinal wall, a number of them may enter the choleduct and from there reach the hepatic bile ducts.

The migration of a large number of immature flukes in the parenchyma of the liver and the arrival of some of them in the bile ducts give rise to acute fasciolosis, particularly in sheep. At necropsy the liver is enlarged, and there are subcapsular hemorrhages, and numerous tracts filled with parenchymatous debris are obvious on the liver surface and in the hepatic tissue. Few, if any flukes are visible in the bile ducts and those that are, are small and immature. Ascites, abdominal hemorrhage, pale membranes, anemia, hypoalbuminemia, and eosinophilia may be present. The adult flukes in the bile ducts feed on the hypertrophic epithelium of the ducts and on the increased blood supply to the hypertrophic tissue and cause chronic fasciolosis. This is characterized by weight loss, pale mucous membranes, anemia, hypoalbuminemia, and sometimes submandibular edema. In addition to the debilitating effects of fasciolosis the livers of infected animals are condemned for human consumption during routine meat inspection at state or municipally regulated abattoirs. The adult flukes produce eggs and can live for as long as 11 years.

Rearing method(s)

Easy but effective methods for maintaining and propagating the snails P. columella, Lymnaea natalensis, Bulinus (Physopsis) tropicus, and Bulinus (Physopsis) globosus are provided. The methods for infecting the first three species with the miracidia of F. hepatica, Fasciola gigantica, and Calicophoron microbothrium, respectively, and the collection of the metacercariae from them are described. Because of the possibility of human infection, the utmost care must be taken when working with infected snails or with the metacercariae. Disposable gloves must be worn at all times, and it is advisable to have a tap delivering extremely hot water as well as a stainless-steel sink and stainless-steel surfaces in the room in which infected snails and the metacercariae are kept. Consequently, all infected snails and the metacercariae that are surplus to immediate requirements can be killed with hot water. The aquaria in which infected snails had been kept are washed out with very hot water, then scrubbed with a nailbrush, and the stainless-steel surface of the sink and other surfaces thoroughly cleaned with extremely hot water.

Establishment of a snail colony

Oblong fish tanks (~30 cm long×25 cm broad and 25 cm deep) with a glass bottom and glass sides inside a metal frame with the corners of the glass sealed with a nontoxic black