Parasiticide Screening: Volume 1: 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 One 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: 9780128138915

Book preview

Parasiticide Screening

In Vitro and In Vivo Tests With Relevant Parasite Rearing and Host Infection/Infestation Methods, Volume 1

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. Defining in vitro parasiticide screening and test methods

Abstract

References

Chapter 2. Defining in vivo parasiticide screening and test methods

Abstract

References

Chapter 3. Arthropoda

Chapter 3a. Arthropoda, Diptera, Nematocera

Culicidae

Simuliidae

Ceratopogonidae

Psychodidae

Chapter 3b. Arthropoda, Diptera, Brachycera

Tabanidae

Chapter 3c. Arthropoda, Diptera, Cyclorrhapha

Muscidae

Sarcophagidae: flesh flies

Calliphoridae: blowflies

Oestridae—bot flies

Chapter 3d. Arthropoda, Phthiraptera, Anoplura

Biology and life cycle

Rearing method(s)

In vitro method(s)

In vivo method(s)

Chapter 3e. Arthropoda, Phthiraptera, Mallophaga

Biology and life cycles

Rearing method(s)

In vitro method(s)

In vivo method(s)

Chapter 3f. Arthropoda, Insecta, Siphonaptera

Pulicidae

Pulicidae

Hectopsyllidae

Chapter 3g. Arthropoda, Insecta, Hemiptera

Cimex lectularius Linnaeus, 1758—bed bug

Reduviidae spp. Latreille, 1807—assassin bugs and kissing- or cone-nose bugs

Chapter 3h. Arthropoda, Pentastomida

Lingulatula serrata Fröhlich, 1789—tongueworm

Chapter 4. Arachnida

Chapter 4a. Arachnida, Metastigmata, Argasidae

Otobius megnini—Dugés, 1884—spinose ear tick

Argas persicus—Oken, 1818—fowl or poultry tick

Ornithodoros moubata–Murray, 1877; Ornithodoros turicata—Dugès, 1876

Chapter 4b. Arachnida, Metastigmata, Ixodidae (except Ixodes holocyclus)

Biology and life cycles

Rearing method(s)

In vitro method(s)

In vivo method(s)

Infesting rats with ticks

Counting ticks on rats

Chapter 4c. Arachnida, Metastigmata, Ixodidae, Ixodes holocyclus

Ixodes holocyclus—Neumann, 1899—the Australian paralysis tick

Chapter 4d. Arachnida, Mesostigmata, Dermanyssidae, Macronyssidae, Varroidae

Dermanyssidae

Macronyssidae

Varroidae

Chapter 4e. Arachnida, Astigmata

Sarcoptidae

Notoedres cati (Hering, 1838) Railliet, 1893—feline scabies mite

Psoroptidae

Psoroptes cuniculi (Delafond, 1859)—rabbit ear mite or ear canker mite

Chorioptes bovis (Hering, 1845)—chorioptic mange mite

Otodectes cynotis (Hering, 1838) Canestrini, 1894—dog ear mite

Chapter 4f. Arachnida, Cryptostigmata

Demodex spp

Cheyletiellidae

Psorergatidae

Myobiidae

Trombiculidae

Straelensia cynotis (Le Net et al., 1999)—chigger mite

Chapter 5. Protozoa

Chapter 5a. Protozoa, Sarcomastigophora, Hemoflagellates

Protozoans

Protozoa

Sarcomastigophora

Chapter 5b. Protozoa, Sarcomastigophora, Mucosoflagellates

Trichomonadidae: trichomonads

Giardia Künstler, 1882

Chapter 5c. Protozoa, Apicomplexa: as Eimeria and Cystoispora are genera Eimeria, and Cystoisospora

Eimeria Schneider, 1875

Sarcocystidae Poche, 1913

Chapter 5d. Protozoa, Apicomplexa: Toxoplasma, Neospora, Hammondia, Besnoitia, and Hepatozoon

Toxoplasma gondii Nicolle and Manceaux, 1908

Sarcocystidae

Sarcocystidae

Sarcocystidae

Hepatozoidae

Chapter 5e. Protozoa, Apicomplexa: Cryptosporidium, and Sarcocystis

Cryptosporidium Tyzzer, 1907

Sarcocystidae

Chapter 5f. Protozoa, Apicomplexa, Hemosporidia

Babesia Henry, 1913

Theileria Bettencourt, França, and Borges, 1907

Leucocytozoon Sambon, 1908

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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ISBN: 978-0-12-813890-8

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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

Ronnie L. Byford, PhD,     Center for Animal Health & Food Safety, New Mexico State University, Las Cruces, NM, United States

Leszek J. Choromanski, DVM, PhD,     Retired Research Scientist, Zoetis, Inc., Lenexa, KS, United States

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

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

Richard G. Endris, PhD,     Endris Consulting, Inc., Bridgewater, NJ, United States

Josephus J. Fourie, PhD,     Clinvet International, Bloemfontein, South Africa

Maxime Madder, PhD, Professor

Clinglobal, La Mivoie, Mauritius

Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa

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

Marco Pombi, PhD,     Dipartimento di Sanità Pubblica e Malattie Infettive, Sapienza University of Rome, Rome, Italy

Ian S. Ridley, BRurSc,     Invetus Pty Ltd., Armidale, NSW, Australia

Theo P.M. Schetters, PhD, Professor

Clinglobal, La Mivoie, Mauritius

Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa

Philip J. Scholl, PhD,     Retired Entomologist, Oxford, FL, United States

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

Maurice C. Webster, BVSc, MACVSc,     Invetus Pty Ltd., Armidale, NSW, Australia

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+ publication 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, Merritt DA, Marchiondo AA. 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. 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. 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+ publication 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(2013):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, 1530–40, and 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, and 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 the 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 that were widely utilized since ancient times as evidenced by Galen’s dogmatic herbalism from about AD 200 through the Middle 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 for Therapeutic Research, 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, have 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. 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

The 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, and 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. that 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 A close and competitive collaboration between medicinal chemists and parasitologists yielded a highly successful outcome. 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 comprising H. contortus, Trichostrongylus 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 ouroborus 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 (Volumes 1 and 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

1. Bowman DD. Successful and currently ongoing parasite eradication programs. Vet Parasitol. 2006;139(4):293–307.

2. Geary TG, Conder GA, Bishop B. The changing landscape of antiparasitic drug discovery for veterinary medicine. Trends Parasitol. 2004;20(10):449–455.

3. Woods DJ, Knauer CS. Veterinary antiparasitic agents in the 21st century: a review from industry. Int J Parasitol. 2010;40:1177–1181.

4. Ackerknecht EH. Historical aspects of medicinal drug control. In: Blake JB, ed. Safeguarding the public. Baltimore, MD: John Hopkins Press; 1970;51–58.

5. Marchiondo AA. Introduction. In: Marchiondo AA, ed. Pyrantel parasiticide therapy in humans and domestic animals. San Diego, CA: Academic Press; 2016;143.

6. Hoeppli R. Parasites and parasitic infections in early medicine and science Singapore: University of Malaya Press; 1959;526.

7. Hawking F. History of chemotherapy. In: Schnitzer RJ, Hawking F, eds. Experimental chemotherapy. New York: Academic Press; 1963;1–24.

8. Campbell WC. Historical introduction. In: Campbell WC, Rew RS, eds. Chemotherapy of parasitic diseases. New York: Plenum Press; 1989;3–21.

9. Sides H. In the Kingdom of Ice New York: Doubleday; 2014;178.

10. Thorburn AL. Paul Ehrlich: pioneer of chemotherapy and cure by arsenic (1854–1915). Br J Vet Dis. 1983;59(6):404–405.

11. Beveridge WIB. Chapter 4: The art of scientific investigation. Hypothesis New York: Norton WW & Company; 1957;44.

12. Burg RW, Stapley EO. Chapter 2: Isolation and characterization of the producing organism. In: Campbell WC, ed. Ivermectin and abamectin. New York: Springer-Verlag; 1989;24–32.

13. Nobelprize.org. The Nobel Prize in physiology or medicine 2015. Nobel Media AB. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/2015/>; 2014 [Web 08.02.16].

14. Beveridge WIB. Chapter 3: The art of scientific investigation. Chance. 81 New York: Norton WW & Company; 1957;27–40.

15. Campbell WC. Serendipity in research involving laboratory animals. ILAR J. 2005;46(4):329–331.

16. Paton W. Man and mouse Animals in medical research Oxford, UK: Oxford University Press; 1984;22.

17. Beveridge WIB. Chapter 3: The art of scientific investigation. Chance New York: Norton WW Company; 1957;31.

18. Campbell WC. Serendipity and new drugs for infectious disease. ILAR J. 2005;46(4):352–356.

19. Gould SJ. Ever since Darwin—reflections in natural history New York: Norton WW & Co., Inc.; 1977;31.

20. Fannkhauser R, Cozzie LR, Nare B, Powell K, Sludr AE, Hammerland LG. Chapter 11: Use of rodent models in the discovery of novel anthelmintics. In: Caffrey CR, ed. Parasitic helminths: targets, screens, drugs and vaccines. Boschstr 12, 69469 Weinham, Germany: Wiley VCH Verlag & Co. KGaA; 2012;181–199.

21. Campbell WC. Ivermectin, an antiparasitic agent. Med Res Rev. 1993;13:61–79.

22. Geary TG, Sangster NC, Thompson DP. Frontiers in anthelmintic pharmacology. Vet Parasitol. 1999;84(3–4):275–295.

23. Freter KR. Drug discovery—today and tomorrow: the role of medicinal chemistry. Pharm Res. 1988;5(7):397–400.

24. McTier TL, Chubb N, Curtis MP, et al. Discovery of sarolaner: a novel, orally administrated, broad-spectrum, isoxazoline ectoparasiticide for dogs. Vet Parasitol. 2016;222:3–11.

25. Austin WC, Courtney W, Danilewicz JC, et al. Pyrantel tartrate, a new anthelmintic effective against infections of domestic animals. Nature. 1966;212:1273–1274.

26. Howes Jr HL, Lynch JE. Anthelmintic studies with pyrantel I Therapeutic and prophylactic efficacy against the enteral stages of various helminths in mice and dogs. J Parasitol. 1967;53(5):1085–1091.

27. McFarland JW. In: New York: John Wiley & Sons; 1983;87–108. Bindra JS, Lednicer D, eds. Chronicles of drug discovery. vol. 2.

28. Conder GA, Johnson SS, Guimond PM, Cox DL, Lee BL. Concurrent infections with the ruminant nematodes Haemonchus contortus and Trichostrongylus colubriformis in jirds, Meriones unguiculatus, and use of this model for anthelmintic studies. J Parasitol. 1991;77:621–623.

29. Conder GA, Johnson SS, Nowakowski DS, et al. Anthelmintic profile of the cyclodepsipeptide PF1022A in in vitro and in vivo models. Antibiotics. 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

Defining in vitro parasiticide screening and test methods

Alan A. Marchiondo, MS, PhD¹, Larry R. Cruthers, MS, PhD² and Josephus J. Fourie, PhD³,    ¹Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States,    ²LCruthers Consulting, Chesapeake, VA, United States,    ³Clinvet International, Bloemfontein, South Africa

Abstract

This chapter defines in vitro testing as it relates to the whole organism laboratory rearing and screening methods used to test parasiticides against single cell and multicellular parasites of veterinary importance. Advantages and disadvantages of in vitro testing methods are discussed. Guidelines and study design in the development of in vitro protocols are provided. The end points of in vitro testing are discussed as related to repellency, morbidity, and mortality expressed as the percentage of lethal dose concentration or efficacy of parasiticides tested in vitro. In vitro cultivation of parasite stages is a hallmark of parasiticide screening and testing.

Keywords

In vitro; definition; advantages; disadvantages; regulated guidelines; repellency; morbidity; mortality; lethal dose concentration; in vitro cultivation; parasites; ex vivo

In vitro comes from the Latin root vitreus or vitrum which means glassy, glass, transparent (origin 1890–95). A term which is cognate to this root is in vitro which means in glass.¹ In vitro, often not italicized in English because it has become part of the standard English usage, refers to techniques, experiments, or studies conducted by a given procedure in a controlled environment outside of a living organism or outside their normal biological context.²,³ Colloquially called test tube experiments, these studies in biology, medicine, and its subdisciplines are traditionally done in test tubes, flasks, Petri dishes, etc.

Rearing a parasite adult or immature stage(s) in the laboratory without or on/in a mammalian host to yield large numbers of organisms for testing purposes, including in vivo tests, and to cultivate (maintain/propagate) the parasite in the laboratory will be considered an in vitro test for the purpose of this text. On animal bioassays, where the animal is treated PO or parenterally and the target organism/stage is allowed to imbibe/feed on blood, flesh, or host secretions or infest/infect the treated host in order to determine the efficacy of the parasiticide will be considered an in vivo test.

In vitro studies are conducted with whole living organisms, while ex vivo (Latin=out of the living, outside the normal living organism) uses components of an organism that have been isolated from their usual biological surroundings. In science, ex vivo means that something is experimented or measured in or on cells/tissues from an organism in an external environment with minimal alteration of its natural in vivo conditions. Electrophysiology experiments on Ascaris suum muscle strips are an example. Ex vivo conditions allow experimentation on an organism’s cells or tissue under more controlled conditions than is possible in in vivo experiments (in the intact organism), at the expense of altering the natural environment. The term ex vivo means that the samples to be tested have been extracted from the organism. The term in vitro (within or in glass) means the parasites to be tested are obtained from a repository, culture, or colony, such as nematodes from the gastrointestinal tract of sheep and cattle or fleas from natural or artificial rearing. These two terms are not synonymous even though the testing in both cases is within the glass.

One factor during in vitro testing, often overlooked but emphasized by Redi in 1684,⁴ is that a parasite removed from its host is being tested under abnormal conditions and is actually a dying organism. In vitro results should be interpreted carefully and cautiously as changes resulting from molecular, cellular, physiological, biochemical, and environmental conditions are always open to discussion. An excellent example of an in vitro false lead is the activity of triclabendazole against Hymenolepis in vitro, but later found to be inactive in vivo using a rodent model.⁵

Advantages of in vitro tests include (1) species-specific results (using the target parasite and stage without the influences of the hosts humeral and cellular responses), (2) enormous level of simplification of the system, (3) simpler and more convenient procedural methods, (4) faster screening and more rapid result often predictive of the likelihood of advancing to in vivo testing, (5) reduced use of test animals, (6) relatively inexpensive, (7) reduced use of the amount of test compound, (8) adaptable to allowing for miniaturization and automation yielding high-throughput screening methods to cope with increasing demands for new parasiticides, and (9) more detailed physiological, biochemical, and morphological analysis than can be done with the whole organism. For confirmation of mortality/lethality in the target parasite species, however, there is no substitute for in vivo parasite screens.

Disadvantages of in vitro tests are that they fail to replicate the precise microphysiological and microenvironmental conditions of a parasite in/on a host which makes results very challenging to extrapolate to the target host. Because of this, in vitro studies are complex methods with difficult quality control and may lead to results that do not correspond to the circumstances occurring around a living parasite within the definitive host. The results must never be overinterpreted leading to erroneous conclusions about the parasite and its biological systems. Typically, many candidate drugs that are effective in vitro prove to be ineffective in vivo. This can be due to (1) issues with drug delivery and uptake to the parasite and its microenvironment; (2) absorption, distribution, metabolism, and excretion of the drug and its metabolites in the target host as well as the parasite; (3) parasite species and surrogate differences not applicable to the target host; (4) false positives and reproducibility of the assay; (5) false negatives; (6) target host toxicity; (7) lack of biotransformation; and (8) absence of biokinetics.

In vitro laboratory studies include screening studies of the active ingredient, used to establish an innate toxic effect on the test parasite species, dose–response tests, or simple laboratory studies, including surface contact tests, etc. These tests should provide an indication of the intrinsic/inherent parasiticidal activity and/or the range of concentrations (dose–response curve) over which such activity would be expected. Tests are usually conducted using technical material and performed against well-characterized susceptible and/or resistant strains of laboratory-reared parasite stages under standardized and controlled testing conditions. Negative (no compound) and positive controls (reference compounds) should be included to assess validation of the assay and potential cross-resistance with commonly used parasiticides. Efficacy against a specific parasite depends on the delivery form, application/exposure method, and on the dose administered.

Bioassays for in vitro testing of parasiticides against parasites in general should contain a protocol or test method that may specify but is not limited to a study accession number; title; background information, rationale, and/or justification for the study; clearly stated objective(s) with measurable variables; parasite origin, stage, sex, date isolated, and period of time in laboratory cultivation; parasite chemical susceptibility profile; test article chemical characteristics and purity; negative and/or positive controls; handling, disposal, and accountability of test articles; materials specifications and suppliers as appropriate; weight scale verification and validation; inclusion/exclusion criteria (stage, age, sex, weight, etc., and other viability characteristics of the test parasite); parasite randomization to treatments; study design; treatment method, dose, and regimen; evaluation criteria and variable measurements/observations; human accidental exposure; standard operating procedures for the facility and laboratory; biometrics; and data capture forms as appropriate. Good laboratory practice guidelines should be followed to promote the quality, validity, and reliability of nonclinical test data, common understanding of, and harmonization approach.

Internationally accepted in vitro standard methods for toxicological evaluations by the OECD (http://www.oecd.org/) have been developed and the ZEBET (http://www.bfr.bund.de/en/zebet_alternative_methods_to_animal_experiments-53868.html) has collected all available methods for the pharmacological and toxicological testing and assessing the potential effects of chemicals on human health and the environment. Numerous organizations, such as the FAO of the United Nations (http://www.fao.org), IRAC (http://www.irac-online.org/), and WHOPES (http://www.who.int/whopes/en/), have developed validated in vitro screening methods for the determination of insecticide resistance. These standard screening methods have served as a basis to researchers, who have used or modified these standard methods to determine the inherent parasiticide activity of various compounds and have adapted the methods to various parasitic organisms. This is exemplified by the numerous methods for the determination of the lethal concentration of parasiticides provided in Chapters 3–5 in Volume 1 and Chapters 1–3 in Volume 2. However, neither standardized nor regulated in vitro guidelines exist for parasiticides, as opposed to the in vivo guidelines by the WAAVP.

Typical end points of in vitro tests include morbidity, mortality, and repellency. Morbidity is the quality or state or rate of being morbid, which is defined as a state or symptom of objective aberration in function (sensation, integration, and response) and behavior. Symptoms of peculiar behavior are lack of feeding, inhibited development and phenotype characteristics, and unusual positioning or motor activity (motility and migration). A micromotility meter and motility index (inhibitor concentration) have been developed and adapted to high-throughput screening paradigms for parasiticide discovery and resistance.⁶–⁸

Mortality (Latin=mortalitas 1300–50) is defined as susceptible to death, the state, or condition of death. Neurotoxicity of parasiticides is exhibited by the two general effects of excitation and inhibition caused by interactions between the target site and parasiticide. These physiological and biochemical interactions can occur in receptor agonism or antagonism, ion channel modulation, and enzyme inhibition, all of which can result in and be expressed as mortality. The concept of selective activity of anthelmintics and other parasiticides is based on biochemical and physiological differences between the parasite and the host.⁹,¹⁰

Mortality as a measure of the number of dead can be expressed as a lethal dose (LD) at a specific concentration of parasiticide, for example, LD50 or LD90, wherein the amount of substance required to kill 50% or 90% of the parasites (population) under test, respectively. The use of death as a target allows for a quantal test (all or nothing) comparisons between chemicals that kill or do not kill. An effective dose is the dose that measures the reasonable expectancy of the desired or therapeutic effect. Mortality can be calculated according to the Busvine formula.¹¹ The dose–effect relationship should be calculated statistically by using an appropriate linear regression method:

where mcorr is the corrected mortality at each concentration tested (in percent), mo is the mean observed mortality in the treated groups (in percent), and mc is the mean observed mortality in the control groups (in percent).

Mortality can also be expressed as percent efficacy using Abbott’s formula,¹² wherein the percent or number of live organisms in the control group minus the percent or mean number of dead or killed organisms in the treated group equals the percent or mean number dead or killed by the treatment divided by the percent or mean number of live organisms in the control group times 100 equals the percent controlled (efficacy), expressed by the equation:

Mortality bioassays of parasiticides utilize contact exposure and systemic activity via feeding on treated blood or a suitable substance. Contact exposure by direct application on the parasite or immersions and exposing the parasite to a treated substrate (filter paper, glass surfaces, test tubes, Petri dish lids, vials, watch glasses, etc.) have led to unique in vitro tests specific for parasite adult species and stages. Feeding bioassays utilize artificial feeding membranes through which parasites imbibe parasiticide-treated blood or simply allowing parasite stages to feed and crawl through treated rearing medium allow for the assessment of systemic active drugs.

In general, a repellent effect will cause the parasite to avoid contact with a treated substrate or animal completely and/or to leave/move away/fall off a host soon after contact with the treated host surface. Classically, this definition has been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from the chemically treated source. This definition needs to be used with care for crawling arthropod-like fleas and ticks that require physical contact for exposure and may cause the parasite to avoid or leave the host without attaching, biting or feeding, or prevention and disruption of attachment.¹³,¹⁴ In vitro assays to evaluate repellency will be covered in more detail by parasite species.

In vitro testing methods are of no value unless one has an ample supply of test organisms. Therefore in vitro cultivation of parasites is invaluable, providing information on the biology and development of the parasite as well as new approaches to the treatment and control of parasites. It is important in diagnosis, research, epidemiology, and teaching.¹⁵ In vitro cultivation methods include (1) xenic cultures, that is, cultures of parasites grown in association with an unknown or undefined microbial population, such as the cultivation of Entamoeba histolytica¹⁶; (2) monoxenic cultures, that is, parasites grown in association with a single known additional species, such as Acanthamoeba, cultured from a corneal biopsy with Escherichia coli¹⁷; (3) axenic (Greek a=free from; xenos=a stranger) cultures, that is, pure culture without contaminating bacterial association and without the presence of the host, Leishmania species culture¹⁸ and Brugia pahangi¹⁹ as examples. Parasitic helminths and arthropods are perhaps more challenging to cultivate than many other organisms because of their complex life cycles (free-living to parasitic stages) that require different sets of nutritional and physicochemical conditions.²⁰–²²

Chapters 3–5 of Volume 1 and Chapters 1–3 of Volume 2 of this book focus on in vitro tests developed for parasiticide discovery screening and their related rearing/cultivation methods, as applicable and known to the authors, some of which have led to the discovery of novel parasiticides used in veterinary medicine.

References

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6. Folz SD, Pax RA, Thomas EM, Bennett JL, Lee BL, Conder GA. Detecting in vitro anthelmintic effects with a micromotility meter. Vet Parasitol. 1987;24(2–3):241–250.

7. Smout MJ, Kotze AC, McCarthy JS, Loukas A. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility. PLoS Negl Trop Dis 2010; https://doi.org/10.1371/journal.pntd.0000885.

8. Paveley RA, Mansour NR, Hallyburton I, et al. Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases. PLoS Negl Trop Dis 2012; https://doi.org/10.1371/journal.pntd.0001762.

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11. Busvine JR. Toxicological statistics. A critical review of the techniques for testing insecticides. 2nd ed. Slough, England: Commonwealth Agricultural Bureaux; 1971. p. 263–88.

12. Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol. 1925;18:265–267.

13. Halos L, Banet G, Beugnet F, et al. Defining the concept of tick repellency in veterinary medicine. Parasitology. 2012;139(4):419–423.

14. Prullage JB, Hair JA, Everett WR, et al. The prevention of attachment and the detachment effect of a novel combination of fipronil, amitraz and (S)-methoprene for Rhipicephalus sanguineus and Dermacentor variabilis on dogs. Vet Parasitol. 2011;179(4):311–317.

15. Ahmed NH. Cultivation of parasites. Trop Parasitol. 2014;4(2):80–89.

16. Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev. 2002;15(3):329–341.

17. Garcia LS. Diagnostic medical parasitology 4th ed. Washington, DC: ASM Press; 2001;850–872.

18. Gupta N, Goyal N, Rastogi AK. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol. 2001;17(3):150–153.

19. Wisnewski N, Weinstein PP. Growth and development of Brugia pahangi larvae under various in vitro conditions. J Parasitol. 1993;79(3):390–398.

20. Weinstein PP, Jones MF. Development in vitro of some parasitic nematodes of vertebrates. Ann NY Acad Sci. 1959;77:137–162.

21. Taylor AER, Baker JR. The cultivation of parasites in vitro Hoboken, NJ: Blackwell Scientific; 1968;377.

22. Smyth JD. In vitro cultivation of parasitic helminths Boca Raton, FL: CRC Press; 1990;144.

Chapter 2

Defining in vivo parasiticide screening and test methods

Alan A. Marchiondo, MS, PhD¹, Larry R. Cruthers, MS, PhD² and Josephus J. Fourie, PhD³,    ¹Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States,    ²LCruthers Consulting, Chesapeake, VA, United States,    ³Clinvet International, Bloemfontein, South Africa

Abstract

This chapter defines the meaning of in vivo testing of parasites of veterinary importance in their natural or experimental definitive hosts as related to the methods described in subsequent chapters of this book. The advantages and disadvantages of in vivo testing are also listed and discussed. An extensive section on regulatory guidance of parasiticide preclinical and clinical testing and harmonization is provided along with an outline of the desired elements of protocol development.

Keywords

In vivo;