Ectoparasites E-Book

• Herausgeber: Wiley-VCH Verlag GmbH & Co. KGaA
• Kategorie: Wissenschaft und neue Technologien
• Serie: Drug Discovery in Infectious Diseases
• Sprache: Englisch

Seitenzahl: 719

Veröffentlichungsjahr: 2018

Beschreibung

This first book specifically dedicated to ectoparasite drug discovery is unique in providing insights from the veterinary as well as the medical perspective, covering research from both industry and academia while paving the way for new synergies between the two research communities.
Edited by a team combining 80 years of experience in academic research and industrial antiparasitic drug discovery, this volume of Drug Discovery in Infectious Diseases summarizes current knowledge in this rapidly expanding field. Comprehensive yet concise, this ready reference blends solid background information on ectoparasite biology with the very latest methods in ectoparasite drug discovery. Three major parts cover current ectoparasite control strategies and the threat of drug resistance, screening and drug evaluation, and the new isoxazoline class of ectoparasiticides. The future potential of mechanism-based approaches for repellents and parasiticides is thoroughly discussed, as are strategies for vaccines against ectoparasites, making the book ideal for parasitologists in academia as well as researchers working in the pharmaceutical industry.

Leseprobe

Table of Contents

Cover

List of  Contributors

Foreword

Preface

Part One: Strategies & Resistance

Chapter 1: Comparison of Anti‐ectoparasite and Anti‐endoparasite Therapies and Control Strategies

Introduction

Approaches for Ectoparasite and Endoparasite Control

Challenges for Ecto‐ and Endoparasite Control

Perspectives on Current and Future Strategies for Ecto‐ and Endoparasite Control

References

Chapter 2: Vaccination Against Ticks

Tick Evolution and Life Cycle

Commercially Available Vaccines

Rational Tick Vaccine Development

Future Developments

References

Chapter 3: Blocking Transmission of Vector‐borne Diseases

Introduction

Vector‐borne Transmitted Pathogens

Drug Profile for Blocking Pathogen Transmission

Conclusion

Acknowledgments

References

Chapter 4: The Threat and Reality of Drug Resistance in the Cattle Tick

Rhipicephalus (Boophilus) microplus

The Cattle Tick

Rhipicephalus (Boophilus) microplus

History of Acaricidal Compound Classes: Their Introduction on the Market and Emergence of Resistance

Mechanisms of Resistance in Cattle Ticks

Resistance Management

References

Chapter 5: Monitoring Drug Sensitivity in Cattle Ticks

Introduction

How to Identify Acaricide Resistance

Bioassays

Biochemical Tools

Molecular Tools

Use of Resistance Bioassays in the Discovery of New Acaricides

Resistance Management

References

Chapter 6: New Developments in the Control of Human Lice

Introduction

Pediculosis and Medical Importance of Head and Body Lice

Pediculicides and Resistance

Development of the

In Vitro

Rearing System: Maintenance of Insecticide‐susceptible and ‐resistant Strains and Determination of Resistance

Sequencing of the Human Louse Genomes and Transcriptomes

Head Louse Resistance to Pyrethrins, Pyrethroids, and Malathion

Optimization of the Noninvasive Induction Assay to Identify Detoxification Genes Involved in Insecticide Tolerance as a Proactive Resistance Monitoring Approach

New Pediculicides, Infestation Deterrents/Repellents, and Metabolic Synergists with Novel Modes of Action

Sustainable Resistance Management

References

Part Two: Screens & Models

Chapter 7: Molecular Targets to Impair Blood Meal Processing in Ticks

Introduction

Processes Associated with Blood Feeding and Digestion

Conclusions

Acknowledgments

References

Chapter 8: Whole‐organism Screens for Ectoparasites

Purpose, Advantages, and Limitations of Whole‐organism Screens

General Workflow for Ectoparasiticide Discovery Screens

Basic Principles in Ectoparasite Screen Design

Considerations in Assay Implementation and Screen Execution

Specific Whole‐organism Ectoparasite Assays

Conclusion

References

Chapter 9:

In vitro

Feeding Methods for Hematophagous Arthropods and Their Application in Drug Discovery

Introduction

AFS‐Components: The Membrane

AFS Components: The Blood Meal

AFS Components: Temperature Control Systems

Artificial Feeding Methods and Applications

Artificial Feeding Systems in Ectoparasiticide Drug Discovery

Conclusions

References

Chapter 10: Testing in Laboratory Animal Models for Ectoparasiticide Discovery and Development

Introduction

Developing Laboratory Animal Models of Ectoparasite Infestation

Available Ectoparasite Models

Tick Models

Flea Models

Other Ectoparasite Models

Conclusions

Acknowledgments

References

Chapter 11: Testing in Target Hosts for Ectoparasiticide Discovery and Development

Role of Animal Models in Ectoparasiticide Discovery and Development

Alignment of Animal Model Assays with Desired Product Characteristics

Specific Animal Model Ectoparasite Assays

Specific Examples of Animal Model Uses in the Development of Ectoparasiticide Products

Extension to Human Conditions

Conclusion

References

Part Three: Isoxazolines

Chapter 12: Isoxazolines: A Novel Chemotype Highly Effective on Ectoparasites

Arthropod Ectoparasites: Burden to the Agricultural and Veterinary Sectors

Ligand‐gated Chloride Channels as Suitable Targets for Ectoparasiticides

Mode of Action

Isoxazolines: Novel Ectoparasiticides Acting on GABACls and GluCls

Structure and Active Sites of Chloride Channels

Isoxazoline Mode of Action and Binding Site

Selectivity and Safety Profile

Isoxazoline Derivatives: Continuous Exploration of the Novel Chemotype

Conclusions

Acknowledgments

References

Chapter 13: The Discovery of Afoxolaner: A New Ectoparasiticide for Dogs

Introduction

Development of Chemical–Biological Structure–Activity Relationships

Mode of Action

Summary

References

Chapter 14: Development of Afoxolaner as a New Ectoparasiticide for Dogs

Introduction

Study Compliance with Regulatory Requirements

Background

Establishing Proof of Concept in Dogs

Dose Level and Formulation Selection

ADME Properties and Pharmacokinetics

Toxicology and Safety of Afoxolaner

Pivotal Dose Determination, Confirmation and Field Trials

NexGard Spectra

Future Direction

Conclusions

Acknowledgments

Disclaimer

References

Chapter 15: Discovery, Development, and Commercialization of Sarolaner (Simparica

®

), A Novel Oral Isoxazoline Ectoparasiticide for Dogs

Introduction

Discovery of Sarolaner

Laboratory Studies

Flea and Tick Field Studies

Mite Efficacy

Prevention of Tick‐borne Disease Transmission

Comparative Flea and Tick Efficacy

Commercialization of Sarolaner

Acknowledgments

References

Chapter 16: Isoxazolines: Preeminent Ectoparasiticides of the Early Twenty‐first Century

Introduction

Patent Literature

Structural Variations

Conclusion

Disclaimer

References

Index

End User License Agreement

List of Tables

Chapter 3

Table 3.1 Human vector‐transmitted pathogens.

Table 3.2 Companion animal vector‐transmitted pathogens.

Table 3.3 Farm animal vector‐transmitted pathogens.

Chapter 8

Table 8.1 Ectoparasiticide standards in the mosquito larval assay.

Table 8.2 Activity of ectoparasiticide standards in the flea contact assay.

Table 8.3 Activity of ectoparasiticide standards in the flea ingestion assay.

Table 8.4 Activity of ectoparasiticide standards in the tick contact assay.

Table 8.5 Activity of ectoparasiticide standards in the stable fly contact assay.

Chapter 10

Table 10.1 Principal features of laboratory models used for drug screening and characterization.

Table 10.2 Efficacious doses for known ectoparasiticides in the corresponding laboratory models in comparison with dosages used in the marketed product.

Chapter 12

Table 12.1 Ectoparasiticides acting on ligand‐gated chloride channels.

Table 12.2 Isoxazoline‐derived parasiticides.

Table 12.3 Ectoparasiticide activity (IC50 (nM)) in binding assays on

M. domestica

head membrane.

a)

Chapter 13

Table 13.1 Membrane feeding and oral gavage results for monoamide naphthalene isoxazolines on fleas.

Table 13.2 Membrane feeding and oral gavage results for diamide naphthalene isoxazolines on fleas.

Table 13.3 Membrane feeding and oral gavage results for diamide naphthalene isoxazolines on fleas.

Table 13.4 Tick control by oral gavage.

Chapter 14

Table 14.1  Weight bands and active ingredient amounts in the four sizes of NexGard chewable tablets for dogs.

Table 14.2 Mean ± standard deviation of the effective concentration for 90% efficacy (EC

90

) determined using a Sigmoid

E

max

model for percent efficacy against

R. sanguineus, D. variabilis

, and

A. americanum

following oral administration of afoxolaner in a chewable formulation to dogs.

Table 14.3 Mean percent efficacy against eight tick species on day 30 or 31 following treatment with NexGard (48‐ or 72‐h counts).

Chapter 15

Table 15.1 Comparative

in vitro

whole parasite efficacy for afoxalaner, fluralaner, and sarolaner fed in blood to

C. felis

and

O. turicata

(

n

 = 3).

Table 15.2 Summary of dose confirmation laboratory flea efficacy studies [9].

Table 15.3 Summary of dose confirmation laboratory tick efficacy studies [10, 11].

Table 15.4 Flea efficacy: comparative speed of kill [29–31].

List of Illustrations

Chapter 1

Figure 1.1 Consolidation of animal health companies 1990–2016.

Chapter 2

Figure 2.1 Schematic representation of the systems that are being targeted to develop vaccines against tick infestation.

Figure 2.2 Simplified schematic of the blood coagulation processes that are modulated by tick proteins. Compounds in green fields and printed in italics are targets of tick serine protease inhibitors. Compounds in blue fields are substrates of tick proteases. Compounds in yellow are activated by tick proteins. ADP, adenosine diphosphate; VWF, von Willebrand factor.

Chapter 3

Figure 3.1 Generic sketch of transmission of diseases by ectoparasites (vectors). Blocking of transmission can, in principle, occur at every stage, but most drugs aim to interfere during the “Attachment” phase and/or “Feeding and Transmission” phase.

Chapter 4

Figure 4.1 Chronological order of introduction of acaricides for use against cattle ticks (green date markers) and the first documentation of resistance against the respective class (red date markers).

Chapter 5

Figure 5.1 The cattle tick

R

.

(B.) microplus

has attachment sites where it may not be easily detected. In the present case, a heifer was captured on pasture and laid down for determination of the tick burden. Using such a technique before and after acaricide treatment to identify resistance is very resource intensive and time consuming.

Figure 5.2 Schematic depiction of the most commonly used bioassays for

in vitro

identification of cattle tick resistance to acaricides. (a). AIT: Adult engorged female ticks are immersed in defined dilutions of acaricide. The ticks are dried, placed in Petri dishes and kept at 27 to 28 °C and 80–95% relative humidity (RH). After 2 weeks, eggs are weighed, transferred to tubes, and incubated at the before‐mentioned climatic conditions for ~3 additional weeks to evaluate the larval hatching. In a simplified protocol, oviposition is evaluated approximately 1 week after incubation, without further evaluation of the larval hatching. (b). LPT: Tick larvae are incubated at 27 to 28 °C and 85–95% RH for 24 h in filter paper pouches that were impregnated with acaricides. The pouches are opened to visually determine the viability of the larvae. (c). LIT: The tick larvae are immersed for ~10 min in defined concentrations of acaricide. This is followed by a transfer into filter paper pouches (no impregnation with acaricide) in which the larvae are incubated for 24 h at 27 to 28 °C and 80–90% RH. The viability of the larvae is determined directly in the pouch by removing the clips, analogous to the LPT. (d). LTT: Tick eggs are placed into 96‐well flat bottom plates which were pre‐coated with defined concentrations of acaricides. The plates are sealed and incubated at 27 to 29 °C and 70–80% RH. Two weeks after egg hatching the larval mortality is determined in each well.

Figure 5.3 Position of the so far identified mutations in the voltage‐gated sodium channel of

R

.

(B.) microplus,

which lead to resistance to synthetic pyrethroids. Three mutations lead to changes in the S4‐S5 linker of domain II: T170C (red) [20], C190A (green) [21], and G215T (blue) [22]; and one in the S6‐region of domain III: T2134A (violet) [23].

Chapter 6

Figure 6.1 Assembly of the

in vitro

rearing system for lice. ()

Figure 6.2 Comparative sodium current traces from the house fly VSSC variants with and without head louse mutations expressed in

Xenopus

oocytes before and after exposure to increasing concentrations of permethrin. ()

Figure 6.3 Relative transcript levels (panel a and b) and mortality responses (panel c and d) of body louse females to a lethal contact amount of ivermectin (5% IVM) following injection of dsRNA targeting either louse

CYP9AG2

or

ABCC4.

Lice were also injected with either dsRNA of the odd‐paired gene,

opa,

(

GeneBank

accession # S78339) for P450 silencing or with dsRNA of the

E. coli

plasmid, pQE30, for ABC transporter silencing as sham injected controls. Asterisks (*) in panels a and b indicate that

CYP9AG2 and ABCC4

dsRNA significantly suppress the levels of

CYP9AG2 and ABCC4

transcripts, respectively (Student’s

t

‐test,

P

 < 0.05). In panel c, the bioassay was started 48 h after

CYP9AG2

dsRNA injection. In panel d, the bioassay was started 12 h after

ABCC4

dsRNA injection. Asterisks (*) in panels c and d indicate that the mortality responses of lice injected with dsRNAs were significantly different from their respective controls (buffer or water only injected, maximum log‐likelihood ratio test,

P

 < 0.05). ()

Chapter 7

Figure 7.1 Physiological processes associated with blood meal processing in ticks. A schematic overview of tick tissues and related processes of blood meal uptake, digestion, heme and iron metabolism, detoxification and inter‐tissue transport that may serve as rational targets for “anti‐tick” intervention. The red and blue arrows indicate blood meal uptake and reverse water secretion, respectively.

Figure 7.2 The current model of uptake and digestion of major host blood proteins by

Ixodes ricinus

digestive cells. Red arrows – expressional and secretory pathway of hydrolases involved in protein digestion. Black arrows – endocytic pathways of hemoglobin and serum albumin. RME – receptor‐mediated endocytosis of hemoglobin; FPE – fluid‐phase endocytosis of albumin (and other serum proteins). HbR – hemoglobin receptor (putative, yet not identified); CP – coated pits, E‐l – large endosomal vesicles containing hemoglobin; E‐s – small endosomal vesicles containing dissolved serum albumin [40]; Hs – hemosomes containing condensed heme [42]; Golgi – Golgi apparatus; ER – endoplasmic reticulum; PL – primary lysosomes.

Ir

AE –

I. ricinus

legumain/AE;

Ir

CD –

I. ricinus

cathepsin D;

Ir

CB –

I. ricinus

cathepsin B;

Ir

CC –

I. ricinus

cathepsin C;

Ir

SCP –

I. ricinus

serine carboxypeptidase;

Ir

LAP –

I. ricinus

leucine aminopeptidase. For details, see the text.

Figure 7.3 A model of putative tick iron and heme metabolic pathways. Iron and heme pathways in ticks are independent as ticks are not capable of heme degradation given the absence of heme oxygenase. Iron is most likely acquired from the host serum transferrin digested in the acidic environment of small endosomal vesicles (E‐s) of midgut digestive cells. Upon reduction, ferrous iron is transported from the lysosome by the divalent metal transporter 1 (Dmt1). Once in the cytoplasm, Fe

2+

ions are scavenged by intracellular ferritin 1 (Fer1), whose translation is strictly regulated by the cytoplasmic Aconitase/Iron responsive protein1 (Aco/IRP1), which senses the iron cellular level. Iron destined to be delivered to the peripheral tissues is transported from digestive cells via DMT1 to the hemolymph and bound to the iron secreted ferritin 2 (Fer2) that functions as an iron transporter. The excessive iron in peripheral tissues is scavenged and stored in Fer1. Heme released from the digested host hemoglobin in the large endosomal vesicles (E‐l) is transferred to the cytoplasm via the heme responsive gene 1 (HRG‐1) transporter. Ticks detoxify the majority of acquired heme by an ABC transporter (ABC)‐mediated transport to hemosomes. Glutathione‐S‐transferase(s) (GST) serve as an intracellular scavenger of free heme. A small portion of acquired heme required for proteosynthesis of endogenous hemoproteins is exported from the digestive cells to the hemocoel by FLVCR transporter. In hemolymph, heme is bound by the abundant carrier protein(s), heme‐lipo‐glycoprotein (HeLp), which serves in all developmental stages both as a scavenger of excessive heme and transporter into peripheral tissues. In the post‐repletion period of fully engorged females, most of the heme is bound to vitellogenins (Vg) and transported to the ovaries to supply heme metabolic demands of developing embryos and larvae. After entry into the developing oocytes via vitellogenin receptor (VgR), Vg is proteolytically processed to vitellins (Vns) by aspartic proteases BYC and THAP, and cathepsin L‐like activity of VTDCE. For details, see the text.

Chapter 8

Figure 8.1 Ectoparasiticide discovery flow diagrams: (a) Part 1; (b) Part 2.

Figure 8.2 Typical 96‐well plate setup for mosquito larval assay.

Figure 8.3 (a–c) Apparatus for flea contact assay.

Figure 8.4 (a) Flea eggs, larvae, fras in Petri dish. (b) Flea larvae in Petri dish.

Figure 8.5 (a) Ticks in shipping container. (b) Ticks on white paper.

Figure 8.6 (a) Dv larvae nymph, adult. (b) Vial on hotdog roller. (c) Amitraz effect.

Figure 8.7 A

Cheyletiella

spp. mite.

Figure 8.8 (a) Adult flies in Bugdorm

®

cage. (b) Flies in the Petri dish assay.

Figure 8.9 (a) Fly capture system. (b) Setup to anesthetize the flies.

Chapter 9

Figure 9.1 The “Greyhound” artificial feeding system. ()

Figure 9.2 Rutledge‐type feeder. The Rutledge feeding system consists of a jacketed hollow cone, the base of which can be covered by an artificial membrane or animal skin (a). Blood is introduced into the cone through a tube (b) that extends from its vertex. Using a circulating water bath, heated water is pumped through the cylindrical water jacket that surrounds the cone and tube (c), thereby warming the blood meal. ()

Figure 9.3 Example of an

in vitro

flea feeding system, showing three Rutledge‐type feeders (a) attached to laboratory support stands (b) and connected in series to tubing attached to a circulating water bath. The continuous flow of warm water heats the blood meal inside the Rutledge feeders to approx. 37°C. Flea cages (c) are brought into contact with the Parafilm membrane covering the blood meal using a laboratory jack (d). Each flea cage has a feeding screen which directly touches the Parafilm membrane and a bottom screen which allows flea eggs and feces to fall through in a dish containing culture media (e), from where the eggs can be harvested. ()

Figure 9.4 An

in vitro

feeding unit modified after Ref. [53]. It consists of glass tubing and mosquito netting (N) glued to a silicone membrane (M) which closes the unit on one side. Additional attachment stimuli such as hair or hair extracts can be placed on the membrane. A movable rubber ring (R) around the unit keeps the blood meal below the membrane when the feeding unit is placed in a blood container such as a glass beaker or the well of tissue culture plate. A perforated plastic stopper wrapped with muslin (S) is inserted in the unit to confine the ticks during feeding. ()

Chapter 10

Picture 10.1 Non‐murine rodents like gerbils can be used successfully for evaluating ectoparasiticides. To our experience, they are easy to handle and are very good hosts for ticks and cat fleas.

Figure 10.1 Tick rat model, diagrammed based on description by Gutierrez

et al

., 2006 [18].

Chapter 11

Figure 11.1 Prototypical workflow and decision tree for ectoparasiticide development.

Chapter 12

Figure 12.1 Schematic representation of the transmembrane domain of a ligand‐gated chloride channel. (a) Four transmembrane helices (M1–M4) that make up one subunit are depicted with their connecting loops. (b) Arrangement of the transmembrane subunits to form a pentameric ion channel with M2 Cl

−

helices (orange) lining the pore, M1 and M3 linking the adjacent subunits, and M4 facing the outside of the pore. Helices M3

′

and M4

′

of the foremost subunit have been omitted for clarity. The composition of helices is illustrated for only three of the five subunits.

Figure 12.2 Homology model of GABACl in a proposed closed form. Sequences of

Erwinia chrysanthemi

GABACl and dlr‐GABACl of

Ctenocephalides felis

were aligned using the BLAST algorithm, and final modeling was performed with the MOE software package [41]. The five individual subunits are shaded in different colors. The Cys loop of one subunit is highlighted in light green (light‐green arrows), the resistance‐inducing mutation in dark green (dark‐green arrow). (a) Protein shown parallel to membrane; horizontal gray bars indicate membrane boundaries. (b) View along the channel pore axis from the extracellular side, with M2 helices visible as the inner pore lining. (c) View along the channel pore axis from the intracellular side, with resistance‐inducing residue (red dot) highlighted with a red arrow.

Figure 12.3 GluCl crystal structure of

C. elegans

represented in an activated, open‐channel state. These images are based on an original X‐ray crystallographic structure (PDB ID: 3RHW) [49] and were produced with the MOE software package [41]. Fab molecules are omitted for clarity. The five individual subunits are shaded in different colors. The surface of one ivermectin‐binding pocket is highlighted in yellow. The Cys loop of one subunit is highlighted in light green (light‐green arrows). (a) Protein parallel to membrane, horizontal gray bars indicate membrane boundaries. (b) View along the channel pore axis from the extracellular side, with M2 helices visible as the inner pore lining. (c) View along channel pore axis from the intracellular side.

Figure 12.4 Schematic representation of CysLGCCs in a sectional view with only three units of the pentameric transmembrane region depicted for clarity. Positioning of isoxazolines is putative.

Chapter 13

Figure 13.1 Isoxazoline insecticide afoxolaner.

Scheme 13.1 Lead compounds in the discovery of afoxolaner.

Scheme 13.2 Synthesis of

4

. (a) nBuLi, DMF, THF; (b) NH

2

OH, EtOH; (c) NCS, Et

3

N, DMF; (d) CO, PdCl

2

dppf, NH

2

CH

2

CF

3

, toluene.

Scheme 13.3 Synthesis of

14

. (a) NCS, Et

3

N, DMF; (b) PdCl

2

dppf, CO, NH

2

CHR

3

CO

2

Me, Et

3

N, toluene; (c) LiOH, THF/H

2

O (1 : 1) then HCl; (d) (COCl)

2

, DMF, CH

2

Cl

2

, Et

3

N, NH

2

R

2.

Scheme 13.4 Synthesis of DD‐16 and DD‐17. (a) CO, PdCl

2

dppf, MeOH, Et

3

N, toluene; (b) LiOH, THF/H

2

O then HCl; (c) oxalyl chloride; (d) (

R

)‐1‐(methylthio)propan‐2‐amine, Et

3

N, CH

2

Cl

2;

(e) HPLC separation of diastereomers by chiral OJ‐RH column with MeOH/CH

3

CN as eluent

Scheme 13.5 Synthesis of

22

. (a) Pd(PPh

3

)

4

, 4 N KOH, THF/DME, 80 °C; (b) CuCN, NMP 150 °C 15 h; (c) NH

2

OH, EtOH, rt, 3 h; (d) NaOCl, THF, 0 °C to rt, 30 min; (e) triazole, K

2

CO3, CH

3

CN, 80 °C, 18 h.

Figure 13.2 CPD I (

22

) inhibits GABA‐gated currents in American cockroach,

P. americana,

neurons. Dissociated neurons were clamped at a holding potential of −60 mV and repeatedly stimulated with pulses of 100 μM GABA (inset, solid trace). Perfusion of CPD I (

22

) inhibited the GABA response (inset, dashed trace) in a dose‐dependent manner with an IC

50

 = 10.8 nM. Following prolonged saline rinse, a partial recovery of the GABA response (inset, dotted trace) was observed. ()

Figure 13.3 Inhibitory effect of afoxolaner, on GABA‐gated Cl

−

currents recorded from

Xenopus

oocytes expressing either

wt

RDL or

A302S

RDL(resistant) receptors. Oocytes were recorded using TEVC (two electrode voltage clamp method) with a holding potential of ‐60 mV. ()

Figure 13.4 Contact toxicity of afoxolaner (square), fipronil (triangle), and dieldrin (circle) against wild‐type (Canton‐S, closed symbol) and cyclodiene‐resistant (Rdl, open symbol) strains of

Drosophila

. Mortality measurements were taken 72 h after flies were transferred to treated glass vials. The resistance ratio (RR) was calculated as Rdl LD

50

/Canton‐S LD

50

for each compound.

Chapter 14

Figure 14.1 Percent efficacy of afoxolaner against

Rhipicephalus sanguineus

following a single oral treatment to dogs at 3 dose levels: 1.5, 2.5, and 3.5 mg/kg.

Figure 14.2 Percent efficacy against

Dermacentor variabilis

versus afoxolaner plasma concentration following oral administration of afoxolaner in a chewable formulation to Beagle dogs.

Figure 14.3 Proposed pathway for the formation of the hydroxylated metabolite of afoxolaner.

Figure 14.4 Percent efficacy of afoxolaner against

C. felis

following a single oral treatment of NexGard to dogs.

Figure 14.5 Efficacy of NexGard against existing flea (

C. felis

) infestations on dogs.

Figure 14.6 Efficacy results from NexGard field trials.

Chapter 15

Figure 15.1 Structure of sarolaner (1‐(5′‐((5S)‐5‐(3,5‐dichloro‐4‐fluorophenyl)‐5‐(trifluoromethyl)‐4,5‐dihydroisoxazol‐3‐yl)‐3′‐H‐spiro(azetidine‐3,1′‐(2) benzofuran)‐1‐yl)‐2‐(methylsulfonyl) ethanone): (1) phenyl head group, (2) isoxazoline core, (3) spiroazetidinebenzofuran moiety, and (4) methylsulfonylethanone tail.

Chapter 16

Scheme 16.1 Diamide starting point resulting in divergent compounds.

Figure 16.1 Other commercial isoxazolines for use against fleas and ticks.

Figure 16.2 Potential first commercial isoxazoline for crop protection.

Scheme 16.2 Two general pathways for the synthesis of parasiticidal isoxazolines.

Scheme 16.3 Fractional crystallization and recycling processes in the synthesis of Lotilaner.

Scheme 16.4 An alternate route to access enantiomerically enriched antiparasitic isoxazolines.

Figure 16.3 Lipophilicity and molecular weight of representative compounds.

Figure 16.4 Hydrogen bond acceptors and donors for representative compounds.

Figure 16.5 Calculated solubility and polar surface area for representative compounds.

Figure 16.6 General structures for literature analyses.

Figure 16.7 Patent applications 2005–present.

Figure 16.8 Common substitution patterns on the isoxazoline core.

Figure 16.9 Phenyl group replacements.

Figure 16.10 Phenyl group replacements.

Figure 16.11 Common amide substitutions.

Figure 16.12 Amide‐related functional groups in isoxazoline analogs.

Figure 16.13 Alternative substitutions

para

to the isoxazoline.

Figure 16.14 Thio substitutions in lieu of an amide.

Figure 16.15 Ketone substitutions on the phenyl linker.

Figure 16.16 Substitutions on the isoxazoline linker.

Figure 16.17 Single‐atom variants of isoxazolines.

Figure 16.18 Additional isoxazoline replacement rings.

Figure 16.19 Fused ring isoxazoline replacements.

Figure 16.20 Acrylamide replacements for the isoxazoline ring.

Figure 16.21 “Open ring” isoxazoline replacements from Dow Agrosciences.

Figure 16.22 Vinyl‐extended phenyl isoxazolines from Syngenta.

Guide

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Selzer, P.M. (ed.)

Antiparasitic and Antibacterial Drug Discovery

From Molecular Targets to Drug Candidates

2009

Print ISBN: 978-3-527-32327-2, also available in digital formats

Becker, K. (ed.)

Apicomplexan Parasites

Molecular Approaches toward Targeted Drug Development

2011

Print ISBN: 978-3-527-32731-7, also available in digital formats

Caffrey, C.R. (ed.)

Parasitic Helminths

Targets, Screens, Drugs and Vaccines

2012

Print ISBN: 978-3-527-33059-1, also available in digital formats

Jäger, T., Koch, O., Flohé, L. (eds.)

Trypanosomatid Diseases

Molecular Routes to Drug Discovery

2013

Print ISBN: 978-3-527-33255-7, also available in digital formats

Doerig, C., Späth, G., Wiese, M.

Protein Phosphorylation in Parasites

Novel Targets for Antiparasitic Intervention

2013

Print-ISBN: 978-3-527-33235-9, also available in digital formats

Unden, G., Thines, E., Schüffler, A. (eds)

Host - Pathogen Interaction

Microbial Metabolism, Pathogenicity and Antiinfectives

2016

Print-ISBN: 978-3-527-33745-3, also available in digital formats

Müller, S., Cerdan, R., Radulescu, O. (eds.)

Comprehensive Analysis of Parasite Biology

From Metabolism to Drug Discovery

2016

Print-ISBN: 978-3-527-33904-4, also available in digital formats

Edited by

Charles Q. Meng and Ann E. Sluder

Ectoparasites

Drug Discovery Against Moving Targets

Copyright

Editors

Charles Q. Meng

Boehringer Ingelheim Animal Health

3239 Satellite Boulevard

Duluth, GA 30096

United States

Ann E. Sluder

Vaccine and Immunotherapy Center

Massachusetts General Hospital

149 13th St.

Charlestown, MA 02129

United States

Series Editor

Paul M. Selzer

Boehringer-Ingelheim Animal Health

Binger Straße 173

55216 Ingelheim am Rhein

Germany

Cover

Hungry Ixodes ricinus females gathered on a small tree seedling questing for a host in front of a molecular cartoon of a ligand gated chloride channel (LGCC). The photo was taken by Jan Erhart in March 2012 in an oak wood in South Bohemia, Czech Rep. Courtesy of Jan Erhart and Petr Kopáček, Institute of Parasitology, BC CAS, Czech Rep. The schematic representation of the CysLGCC sectional view was taken from figure 12.4, chapter 12 by Tina Weber & Paul M. Selzer.

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.

© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978-3-527-34168-9

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Cover Design Adam Design, Weinheim, Germany

List of  Contributors

Eric A. Benner

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

John M. Clark

*

University of Massachusetts

Department of Veterinary & Animal Science

Amherst, MA 01003

USA

[email protected]

Jeffrey N. Clark

*

JNC Consulting Services

38 Wild Rose Lane

Pittsboro, NC 27312

USA

[email protected]

Pat N. Confalone

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Daniel Cordova

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Andrew A. DeRosa

Zoetis, Veterinary Medicine Research and Development

333 Portage St. Kalamazoo

MI 49007

USA

Robert F. Dietrich

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Christian Epe

Boehringer Ingelheim Animal Health

3239 Satellite Blvd

Duluth, GA 30096

USA

Brandon R. Gould

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Ondřej Hajdušek

Czech Academy of Sciences

Institute of Parasitology Biology Centre

České Budějovice

Czech Republic

Eric J. Hartline

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Gail S. Jones

DuPont Crop Protection

Stine‐Haskell Research Center

1090 Elkton Rd.

Newark, DE 19714

USA

Ronald Kaminsky

*

paraC Consulting for Parasitology and Drug Discovery

79685 Haeg‐Ehrsberg

Germany

[email protected]

John B. Kinney

DuPont Crop Protection

Stine‐Haskell Research Center

1090 Elkton Rd.

Newark, DE 19714

USA

Petr Kopáček

*

Czech Academy of Sciences

Institute of Parasitology Biology Centre

Czech Academy of Sciences

České Budějovice

Czech Republic

[email protected]

George P. Lahm

*

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

[email protected]

Diane Larsen

Boehringer Ingelheim Animal Health

3239 Satellite Blvd

Duluth, GA 30096

USA

Laura Letendre

*

Boehringer Ingelheim Animal Health

631 Route 1 South

North Brunswick, NJ 08902

USA

laura.letendre@boehringer‐ingelheim.com

Alan Long

*

Boehringer Ingelheim Animal Health

3239 Satellite Blvd

Duluth, GA 30096

USA

[email protected]

Jeffrey K. Long

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Léonore Lovis

Elanco Animal Health

Mattenstrasse 24A

4058 Basel

Switzerland

Michael Mahaffey

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Richard G. McDowell

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Tom L. McTier

Zoetis, Veterinary Medicine Research and Development

333 Portage St. Kalamazoo

MI 49007

USA

Ard M. Nijhof

*

Freie Universität Berlin

Institute for Parasitology and Tropical Veterinary Medicine

Robert‐von‐Ostertag‐Str. 7–13

14163 Berlin

Germany

ArdMenzo.Nijhof@fu‐berlin.de

Sandra Noack

Boehringer Ingelheim Animal Health

Binger Str. 173

55216 Ingelheim

Germany

Thomas F. Pahutski

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Cedric J. Pearce

Mycosynthetix, Inc.

505 Meadowland Drive

Suite 103

Hillsborough, NC 27278

USA

Jan Perner

Czech Academy of Sciences

Institute of Parasitology Biology Centre

Czech Academy of Sciences

České Budějovice

Czech Republic

Daniel F. Rhoades

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Heinz Sager

*

Elanco Animal Health

Mattenstrasse 24A

4058 Basel

Switzerland

[email protected]

Theo P. M. Schetters

*

ProtActivity R&D

Cuijk

The Netherlands

and

Université de Montpellier

Laboratoire de Biologie Cellulaire et Moléculaire

Montpellier

France

[email protected]

Sandra Schorderet‐Weber

*

Sablons 30

2000 Neuchâtel

Switzerland

[email protected]

Mark E. Schroeder

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Paul M. Selzer

*

Boehringer Ingelheim Animal Health

Binger Str. 173

55216 Ingelheim

Germany

paul.selzer@boehringer‐ingelheim.com

Rafael Shapiro

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Wesley L. Shoop

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Radek Šíma

Czech Academy of Sciences

Institute of Parasitology Biology Centre

Czech Academy of Sciences

České Budějovice

Czech Republic

Ben K. Smith

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Daniel Sojka

Czech Academy of Sciences

Institute of Parasitology Biology Centre

Czech Academy of Sciences

České Budějovice

Czech Republic

Mark Soll

Boehringer Ingelheim Animal Health

3239 Satellite Blvd

Duluth, GA 30096

USA

Katharine R. Tyson

AgBiome

Research Triangle Park, NC 27709

USA

Molly E. Waddell

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Ty Wagerle

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Tina Weber

Merck KGaA

Frankfurter Str. 250

64293 Darmstadt

Germany

Debra J. Woods

*

Zoetis, Veterinary Medicine Research and Development

333 Portage St. Kalamazoo

MI 49007

USA

[email protected]

Ming Xu

Stine‐Haskell Research Center

DuPont Crop Protection

1090 Elkton Rd.

Newark, DE 19714

USA

Note

*

Corresponding author.

Foreword

The attempts of humans to control the influence of ectoparasites on the health of themselves and their associated animals have been documented throughout recorded time. Within the past 100 years, we have witnessed major gains for ectoparasite control with the use of synthetic insecticides; but through time, we have found that these gains are episodic, primarily because of environmental issues and selection of drug resistance in arthropod populations. Therefore, the constant discovery of novel and safe drugs for ectoparasite control is a modern need. Volume 8 of the series Drug Discovery in Infectious Diseases provides a valuable snapshot of the timeline in the battle to control ectoparasites. The contributing authors have provided current perspectives on control of ectoparasites and transmission of agents of disease, strategies for discovery and development of drugs, and the development and potential uses of isoxazolines.

Ectoparasites have impacts on human and animal health by both direct and indirect mechanisms, and the reduction of these different impacts can be achieved by approaches that are not dependent on pesticides. The control program for the New World screwworm using the area‐wide release of sterile males has been highly effective in controlling the direct impact of obligatory myiasis in North and Central America. Area‐wide programs to control the indirect effects of ectoparasites, such as using vaccines for protection against agents of vector‐borne diseases like yellow fever, and controlling onchocerciasis by targeting the microfilarial populations of humans also have been effective. However, the success of these programs is based on very specific parameters that lead to narrow applications, which leaves the need for broader spectrum control methods as a top priority.

The need for drug discovery for use in the control of ectoparasites of humans and animals will continue to be a major factor in the preservation of human and animal health. The One Health approach considers the facts that these entities cannot be separated and will only become more important due to global changes in the environment, as well as human population growth and movement. The majority of vector‐borne human diseases have zoonotic cycles which can be affected by the effective use of ectoparasite control. Even for anthroponoses such as malaria and visceral leishmaniasis, zoonotic blood sources maintain many species of potential vectors of pathogens that are drivers of major causes of death in humans. Ectoparasites do truly represent a moving target for control efforts relative to population density and susceptibility. The timely and rational use of extant and novel drugs against these moving targets and upon a changing global stage can provide leverage for humans in our race against ectoparasites, as long as the discovery and development of new and effective drugs can maintain the pace.

April 2018

Lane Foil

Professor of Entomology

Louisiana State University

Preface

Infestation by ectoparasites has plagued humans, figuratively and literally, since ancient times; for example, lice are listed among the Biblical plagues visited upon Egypt (Exodus 8:17, KJV) and fleas transmitting bubonic plague have had devastating impacts on numerous civilizations over the centuries. Strategies for battling ectoparasites have an equally deep history, as evidenced by mummified lice found in ancient Egyptian combs and by perforated necklace beads that doubled as personal flea traps in medieval Europe. Although human ectoparasite infestations are less prevalent in modern developed countries due to dramatically improved living and hygiene conditions, infestation on domesticated animals remains a major challenge, causing nuisance in companion animals and livestock as well as lowering livestock productivity. Ectoparasites can move between animals and from animals to humans, potentially transmitting various diseases in the process. Ectoparasite control strategies must therefore contend with the ability of the target to move, often quite quickly, as anyone who has ever wanted to kill a flea can attest. This eighth volume in the Drug Discovery for Infectious Diseases series reviews strategies and models for discovery and development of ectoparasiticidal treatments for use in both human and animal health. The challenges presented by moving targets are a common theme throughout, ranging from the market requirement for a rapid speed of kill to the design of effective containment strategies in whole‐organism drug screening assays.

The first section of the volume, Strategies & Resistance, presents various perspectives on what is needed to achieve effective therapeutic control of ectoparasite infestations. The section begins with a comparison by Woods et al. of therapeutic strategies against moving target ectoparasites with those against the less‐mobile endoparasites. Weber et al. review strategies for preventing disease transmission by ectoparasite vectors, for which speed of kill is an important consideration. Schetters reviews promising progress toward development of vaccines against ticks. The emergence of drug resistance threatens the utility of ectoparasiticides, especially for cattle tick and human head lice. Sager et al. and Lovis et al. discuss the threat, reality, and monitoring of drug resistance in cattle tick, particularly relevant for Southern Hemisphere markets such as Brazil and Australia. Clark reviews new developments in the control of human lice.

The second section focuses on laboratory screens and in vivo models for discovery of new treatments against ectoparasites. Compared to human diseases, the molecular targets of parasites, especially ectoparasites, are much less clear, and few can be utilized for screening. The chapter by Kopáček considers the challenges in identifying candidate small‐molecule drug targets in ticks. Currently, discovery of new treatments against ectoparasites relies heavily on phenotypic‐based screening against whole organisms such as fleas and ticks. Chapters by Clark and Pearce and by Nijhof and Tyson discuss the design and implementation of various whole‐organism assays to detect different aspects of the desired treatments, for example, the flea ingestion assay to detect the ability of a compound to work through ingestion rather than through contact. Compared to drug discovery for humans, a major advantage of drug discovery for animal health is that a new investigative drug can be tested in the target host much sooner in the latter. This might seem to make testing in rodent models less critical. However, testing in rodent models remains an important step in drug discovery for animal health, because these models require much less quantity of a compound and save valuable animals of the target species, as discussed in depth by Weber et al. Of course, testing in the target host species is an essential aspect of late‐stage development of a new drug for animal health, and in the concluding chapter of this section Clark reviews protocols for controlled laboratory testing in host species and provides numerous examples of how these testing strategies have been applied in successful ectoparasiticide development programs.

Drugs effective against ectoparasites comprise only a few chemical classes, the pyrethroids, the phenylpyrazoles, and the macrocyclic lactones being the major ones. On average a new class appears about every 20 years. The isoxazolines are the most recent addition to the roster. The last section of this volume is devoted exclusively to this fascinating new class of ectoparasiticides, which has attracted tremendous interest in the animal health and crop protection industries. Weber and Selzer first discuss the new mode of action that underlies the rapid speed of kill by the isoxazolines. Chapters by Lahm et al. and by Letendre et al. detail the complete drug discovery and development process for afoxolaner, the first commercial product launched from this class. The development of sarolaner, reviewed by Woods and McTier, gives another story from a different setting. The final chapter by Long presents a comprehensive overview of the entire isoxazoline chemical class to date.

We thank Dr. Paul M. Selzer, the series editor, and the various representatives of Wiley for the opportunity to shepherd this volume, and for their guidance and support. We also thank the authors who have generously contributed their time and expertise. The combined result of their efforts is a volume designed to be of both interest and utility to those scientists in academia and industry willing to undertake the discovery of drugs aimed at moving targets.

April 2018

Charles Q. Meng

Duluth, Georgia

Ann E. Sluder

Boston, MA

Part OneStrategies & Resistance

1Comparison of Anti‐ectoparasite and Anti‐endoparasite Therapies and Control Strategies

Debra J. Woods*, Tom L. McTier and Andrew A. DeRosa

Abstract

In this chapter, we consider the similarities and differences between management of ecto‐ and endoparasites. We discuss the general approaches of prevention and control of ecto‐ and endoparasites (historic and current chemotherapies, environmental management/host management), while considering the different challenges faced relating to lifecycle, host distribution, genetics, and selection pressure.

Introduction

The Merriam Webster dictionary defines a parasite as an organism living in, with, or on another organism. “Parasitism” refers to the intimate association between the parasite and host, whereby the parasite obtains part or all of its nutrition or needs from the host and results in an overall negative effect on the host. Simply, ectoparasites live on the outside of the animal and endoparasites on the inside. Microparasites (bacteria, viruses, protozoa) establish infections where it is hard to quantify numbers of infectious agents present, so numbers of infected hosts are quantified, rather than numbers of parasites within each host. Microparasites are small and have rapid generation times relative to their hosts. Macroparasites (nematodes, flies, ticks, etc.) are larger and can be counted; so the unit of study is the individual parasite, not the infected host. Macroparasites are also small and have rapid generation times, but there is less of a difference than between microparasites and host. Epiparasites are an interesting class of parasites whereby a parasite parasitizes a parasite in a host–parasite interaction referred to as hyperparasitism (as referred to in the well‐known poem by Jonathan Swift: “a flea has smaller fleas that on him prey, And these have smaller still to bite ’em: And so proceed ad infinitum”). Examples of this are the larvae of the tapeworm, Dipylidium caninum, which infect fleas (Ctenocephalides species) and biting lice (Trichodectes canis). When a dog ingests a parasitized flea/louse when grooming, the tapeworm develops into an adult in the dog’s intestine.

Fleas, ticks, and flies are the most visible and treated ectoparasites, but lice and mites also affect health and wellness. Infestation with ectoparasites causes many pathogenic effects, including tissue damage and blood loss due to feeding; hypersensitivity responses following exposure to ectoparasite antigens; secondary infections; and, most importantly disease transmission. Ectoparasites have evolved to fill many niches, but may be considered in terms of their host association. Many mites and lice live almost completely in permanent association with their host and, as such, have fairly low mobility and are open to risk of desiccation and death without the protection of their host. Other parasites, such as fleas, ticks, and flies, are more mobile and relatively resistant to damaging factors when off the host. As a result, the first category of organisms, mites and lice, often has a commensal relationship with the host as opposed to a parasitic interaction. The latter are able to find new hosts relatively easily, so are less impacted by death of a host and therefore likely to impose greater harm to the host. Most medically important ectoparasites have short generation times, large numbers of offspring, and very high rates of population growth [1].

Roundworms are the major infective internal parasite in both humans and animals, although cestodes (tapeworms) and trematodes (flukes) also have a significant impact on health. Helminth infections cause significant long‐term, chronic debilitating disease and even death. In humans, it is estimated that around 125 000 deaths occur every year, and these are mainly due to infections with the hookworms, Ancylostoma duodenale and Necator americanus, or the roundworm, Ascaris lumbricoides [2]. In companion animals, endoparasite infections are primarily a disease of younger animals, with peak occurrence in dogs less than 6 months old and cats under 18 months old [3], with prevalence ranging from 5% to 70% worldwide [4]. Clinically, symptoms can vary from zero to critical (emaciation, anemia, death) and the zoonotic risks associated with some helminths are an additional concern. The economic impact of helminth infections on livestock, especially ruminant, production is well recognized [5, 6]; in pigs, it has been shown that the presence of endoparasites induces a reduction in body weight [7]. The mechanisms for the impact of helminths on production include direct tissue damage and diminished function of the affected organs; diversion of energy and protein resources of the host from production toward defense and immune mechanisms and reduced feed intake. In companion animals, there are similar adverse effects on health; unfortunately, roundworm infection is common, due to the ubiquity of infective stage larvae in the environment, and concerns are elevated due to zoonotic health risks.

Approaches for Ectoparasite and Endoparasite Control

Treatment of parasites results in removal of an existing infection, whereas prevention is a process by which infection is deterred. For dog and cat ectoparasite infections, experts generally recommend prophylaxis (year round in some climates) over therapeutic treatment, to effectively manage control of the lifecycle, as well as to reduce the risk of disease transmission from ectoparasite vectors [8, 9]. The benefit from regular preventative treatment is particularly recognized for the control of fleas due to the nature of their lifecycle; an adult flea infestation is only a very small part of the population, which includes immature stages present in the pet’s environment. It is critical to control these stages, either by the use of products that target these early lifecycle stages or by regular use of products that eliminate adult fleas on the animal, which will progressively lead to the reduction of environmental lifecycle stages. CAPC (Companion Animal Parasite Council) goes as far as to recommend “avoiding initial infestation altogether by placing pets on life‐long prevention programs is the best option for pets and their owners” [8]. Transmission of diseases (i.e., Rickettsia rickettsia and Borrelia burgdorferi) by vectors, especially ticks, in dogs and cats is a major concern, and reducing the ability of a vector to attach and/or feed with an effective ectoparasite control program will reduce the risk of disease transmission. Tick‐borne diseases in dogs and cats are becoming increasingly important, with several tick species responsible for the continued spread of multiple diseases. Among the other more important diseases are babesiosis, hepatazoonosis, Ehrlichiosis, anaplasmosis, cytauxzoonosis (cats), and tick paralysis. Although control of internal parasites is the primary concern for horses, ectoparasites can also impact the welfare of horses, either through dermatological effects or nuisance bites, which affect the ability of horses to thrive. The primary ectoparasites of horses are houseflies, stable flies, mosquitoes, and horse and deer flies; ticks, lice, and mites also parasitize horses. The major problem is a limited supply of effective, licensed products for horses [10], combined with the challenges of managing ectoparasite species that are able to live for extensive periods off the animal, requiring frequent treatment. Fly repellents tend to have a very short duration of efficacy, if any, and need frequent reapplication. Taylor’s 2001 review [11] highlighted how few pharmaceutical agents are available for treating horse ectoparasites and this situation has improved little in the intervening years.

For livestock, as for companion animals, ectoparasite control is dependent on the parasite lifecycle – do they spend their whole life on the host, like lice; or only spend time on the animal to feed, as for some species of mites, which then return to protected spaces in the environment? For the former, treating just the animal will suffice; for the latter, the environment must also be treated. In a 1992 review [12], Byford et al. gave an authoritative overview of the commercial and health impact of ectoparasite infestation in the United States, focusing on the horn fly, Haematobia irritans, commercially the most important and widespread pest in cattle in the southern United States. Although a complicated condition, the overall implication was that the damaging effect on production and performance of cattle results from an alteration of the total energy balance following ectoparasite infestation. This is a major problem, considering the widespread resistance of horn flies to pyrethroids, probably accelerated by the use of pyrethroid‐impregnated ear tags [13].

Humans are as susceptible to ectoparasite infestation as animals are, and are often affected by the same pests; for example, close contact with pets can result in infestation with fleas, ticks, lice, and mites and, although more common in animals, humans can also suffer from myiasis, especially in tropical regions. Scabies and head lice [14], as well as being socially embarrassing, can cause significant health problems. Resistance is a major issue, with multiple resistance mechanisms identified in different populations of head lice, including kdr (knockdown resistance) mutations of the sodium channel and oxidative metabolism resistance mechanisms (see chapter 6 by J. M. Clark in this volume). Although head lice are the most prevalent parasites causing pediculosis, body louse prevalence is also increasing, which heightens the public health threat due to risk of transmission of a number of diseases, including typhus (Rickettsia prowazekii), louse‐borne relapsing fever (B. recurrentis), and quintana (trench) fever (Bartonella quintana). Tungiasis occurs in tropical and subtropical regions and is caused by the tiny flea, Tunga penetrans, the chigoe flea or jigger, which embeds itself under the stratum corneum and can lead to dangerous complications from secondary infections.

However, the biggest impact on human health globally is from ectoparasite vectors. Malaria, caused by the protozoan parasite Plasmodia spp., is commonly transmitted by infected female Anopheles spp. mosquitoes and, in 2015, there were approximately 214 million malaria cases and an estimated 438 000 malaria deaths [15]. Ticks are becoming increasingly important as a cause of significant disease in humans, as well as their pets. Examples of disease common to both pets and humans include the bacterial Lyme disease (B. burgdorferi), transmitted by the deer tick, Ixodes scapularis (Ixodes ricinus in the European Union); Rocky Mountain spotted fever (R. rickettsia), transmitted by Dermacentor variabilis; and ehrlichiosis (Ehrlichia chaffeensis), transmitted by the lone star tick, Amblyomma americanum and I. scapularis. The protozoal disease babesiosis is caused by infection with Babesia microti or Babesia equi, transmitted by I. scapularis and Ixodes pacificus. Viral diseases can also be transmitted by ticks, for example, tick‐borne encephalitis (TBE) (caused by the flavivirus, TBE virus), transmitted by Ixodes spp. and there are even toxins, such as the tick paralysis toxin transmitted by Dermacentor spp. in the United States and Ixodes holocyclus in Australia. Ticks and mosquitoes may cause significant disease, but fleas have also had a major effect on human history. The vector for bubonic plague, Xenopsylla cheopis, transmits the bacterium Yersinia pestis when it feeds and this was thought to be the cause of the Black Death, which killed an estimated 50 million people in the fourteenth century [16].

For helminth infections, prevention is managed by disrupting the lifecycle of the parasite, which, in humans, is usually achievable by good sanitation and hygiene; but in animals, this is often less feasible. For livestock, experts recommend combining anthelmintic control with minimizing exposure to reinfection; while in companion animals with exposure to the external environment. Where contamination of the environment with infective larvae is extensive, prevention usually requires a strict treatment regimen, combined with regular egg production monitoring. A unique situation exists with heartworm, where a very high degree (up to 100%) of efficacy is required to control this potentially life‐threatening disease of dogs and cats. Fortunately, regular dosing (1‐month and 6‐month products) with a macrocyclic lactone (ML)‐based anthelmintic prevents development of the larval‐stage heartworms. Heartworm larvae are very sensitive to ML products and until recently efficacy was thought to be 100% for the various products. However, more recent evidence of heartworm resistance to MLs has been detected in some areas of the United States (Mississippi Delta) and is a cause for concern. The American Heartworm Society [17] generates guidelines for canine and feline heartworm prevention, which it updates regularly based on the latest scientific understanding of the disease; the most recent revision was in 2014. For horses, as mentioned earlier, internal parasites are a major concern, especially as few new drugs are being approved for horses. In the face of increasing anthelmintic resistance [18], more sustainable methods for helminth control are being sought.

Ectoparasiticides

There are many mechanisms of action utilized in the management of ectoparasites in animals and humans, most older ectoparasiticides being historically leveraged from the crop protection industry. Numerous agricultural pests and veterinary ectoparasites are insects and acarines; and agrochemicals with activity against crop pests also frequently work against animal health ectoparasites. Add to this the fact that the market for Animal Health ectoparasiticides is significantly smaller than the market for agricultural pesticides, and it makes commercial sense to leverage the learnings and assets for animal health utility. Ivermectin is a major exception, being discovered by a pharmaceutical company animal health group (Merck Sharp & Dohme), and was first used on animals and later for agriculture and human medicine.

A primary driver for the development of these multiple therapies is the development of resistance. Resistance is a shift in susceptibility to a drug [19