Fourie

Integrated control of ticks and fleas on dogs
with particular reference

to the prevention of vector-borne diseases
Thesis Fourie NIEUW.indd 1 2-9-2015 15:06:40

Copyright © Josephus J. Fourie, 2015
ISBN/EAN: 978-90-393-6407-9
NUR-code: 886

Integrated control of ticks and fleas on dogs
with particular reference
to the prevention of vector-borne diseases
Geïntegreerde bestrijding van teken en vlooien op honden

met nadruk op de preventie van vector-gebonden ziekten
(met een samenvatting in the Nederlands)
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit Utrecht

op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan,
ingevolge het besluit van het college voor promoties
in het openbaar te verdedigen
op 2 oktober 2015 des middags te 2.30 uur
door
JOSEPHUS J. FOURIE
geboren op 15 Januari 1981 te Cradock, Zuid Afrika

Promotoren:
Prof. dr. H. P. Haagsman
Prof. dr. F. Jongejan

CONTENTS
Chapter 1. Introduction and literature review 9

1.1. Introduction 11
1.2. Ticks 12
1.3. Tick-borne pathogens 16
1.4. Fleas 18
1.5. Flea-borne pathogens 18
1.6. Tick control 20
1.7. Flea control 21
1.8. Mode of action of different classes of acaricides and insecticides 22
1.9. Efficacy and speed of kill 26
1.10. Transmission blocking concept 28
2.0 Aims and Outline of this Thesis 31
References 33
Chapter 2. Control of ticks and fleas on dogs 39

2.1. Fourie JJ, Fourie LJ, Horak IG, Snyman MG. The efficacy
of a topically applied combination of cyphenothrin and
pyriproxifen against the southern African yellow dog tick,
Haemaphysalis elliptica and the cat flea Ctenocephalides
felis. J South Afr Vet Assoc. 2010, 81 (1) : 33-36. 39
2.2. Fourie JJ, Beugnet F, Ollagnier C, Pollmeier MG. Study of
the sustained speed of kill of the combination of fipronil/
amitraz/(S)-methoprene and the combination of imida-
cloprid/permethrin against Dermacentor reticulatus. Para-
site. 2011, 18 (4) : 319-23. 45
2.3. Horak IG, Fourie JJ, Stanneck D. Efficacy of slow-release
collar formulations of imidacloprid/flumethrin and delta-
methrin and of spot-on formulations of fipronil/(s) - metho-
prene, dinotefuran/pyriproxyfen/permethrin and (s) - metho-
prene/amitraz/fipronil against Rhipicephalus sanguineus and
Ctenocephalides felis felis on dogs. Parasit Vectors. 2012,
22 : 5:79. 53

Chapter 3. Prevention of Babesia canis transmission by ticks 69
3.1. Jongejan F, Fourie JJ, Chester ST, Manavella C, Mallouk Y,
Pollmeier MG, Baggott D. The prevention of transmission
of Babesia canis by Dermacentor reticulatus to dogs using a
novel combination of fipronil, amitraz and (S)-methoprene.
Vet Parasitol. 2011, 179 (4) : 343-350. 79
3.2. Fourie JJ, Stanneck D, Jongejan F. Prevention of transmis-
sion of Babesia canis by Dermacentor reticulatus ticks to
dogs treated with an imidacloprid/flumethrin collar. Vet
Parasitol. 2013, 192 (1-3) : 273-278. 87
3.3. Beugnet F, Halos L, Larsen D, Labuschagné M, Erasmus H,
Fourie JJ. The ability of an oral formulation of afoxolaner
to block the transmission of Babesia canis by Dermacentor
reticulatus ticks to dogs. Parasit Vectors. 2014, 7 (1) : 283. 89
Chapter 4. Prevention and speed of Ehrlichia canis

transmission by ticks 97
4.1. Fourie JJ, Ollagnier C, Beugnet F, Luus HG, Jongejan F.
Prevention of transmission of Ehrlichia canis by Rhipicep-
halus sanguineus ticks to dogs treated with a combination of
fipronil, amitraz and (S)-methoprene (CERTIFECT©). Vet
Parasitol. 2012, 193 : 223-228. 97
4.2. Fourie JJ, Luus HG, Stanneck D, Jongejan F. The efficacy
of Advantix© to prevent transmission of Ehrlichia canis to
dogs by Rhipicephalus sanguineus ticks. Parasite. 2013, 20 :
36. 105
4.3. Fourie JJ, Stanneck D, Luus HG, Beugnet F, Wijnveld M,
Jongejan F. Transmission of Ehrlichia canis by Rhipicep-
halus sanguineus ticks feeding on dogs and on artificial
membranes. Vet Parasitol. 2013, 197 (3-4) : 595-603. 115
Chapter 5. Prevention of Dipylidium caninum transmission

by fleas 127
5.1. Fourie JJ, Crafford D, Horak IG, Stanneck D. Prophylactic
treatment of flea-infested dogs with an imidacloprid/flume-
thrin collar to preempt infection with Dipylidium caninum.
Parasitol Res. 2013, 112 : S33-S46. 127

Chapter 6. General discussion 143
6.1. Introduction 145
6.2. Pros and cons of spot-on, collars and oral formulations of
acaricides 154
6.3. Speed of pathogen transmission 157
6.4. Transmission blocking models 158
6.5. Concluding remarks and future directions 162
References 164
Summary 169
Afrikaanse opsomming 172
Nederlandse samenvatting 175
Acknowledgements 178
Curriculum Vitae 181
List of Publications 182

8

Chapter 1. Introduction and literature review

10

1.1. Introduction
According to Saunders Comprehensive Veterinary Dictionary, third edition (2007)

page 413, a companion animal is defined as: “dogs, cats, pleasure horses, birds,
mice, guinea pigs and more exotic species kept by humans for company, amuse-
ment, psychological support, extrovert display and all of the other functions that
humans need to share with animals of other species; companions who will not take
emotional or psychological advantage of the person and will, for the most part, stay
faithful.”
Of these, dogs are among the most common companion animals of man.
Molecular evidence points to an origin of dogs from the wolf, Canis lupus, more
than 14,000 years ago during the hunter-gatherer nomadic period. Less-fearful
wolves were thought to be attracted to human settlements to scavenge kills. These
less-fearful individuals then may have found utility as early guard dogs, warn-
ing settlements through barking of incursions by humans or animals. Over time,
these animals became differentiated from the larger autonomous wolf population
through natural selection and genetic drift. Selection of pups as pets from these
populations based on decreased flight behaviour and increased sociality, two trade-
marks of tameness, laid the foundation for domestication. Once humans were able
to practice selective breeding based on desired traits like obedient behaviour, the
wolf in effect became a dog, ‘man’s best friend’. The large phenotypic variation
that is observed in modern dogs has resulted in more than 400 registered breeds
today (Driscoll et al, 2009).
Because dogs are such loved companion animals, their health and well-
being are of great importance to their human companions. Controlling parasites on
dogs is therefore important not only because of the detrimental impact on the an-
imal’s health, but also the zoonotic risk that some of these parasites pose to their
human companions. Dogs serve as hosts for a considerable number of parasites,
which include ectoparasites (i.e. fleas, ticks, mites and lice) and endoparasites (e.g.
protozoa and helminths). Of these, tick and flea infestations and their associated
pathogens are the most prevalent and constitute a major health concern for dogs.
11

1.2. Ticks
Ticks are obligate, blood-feeding ectoparasites in the phylum Arthropoda. Within

the order Parasitiformes, suborder Ixodida, superfamily Ixodoidea, all ticks are in-
cluded in three families: Ixodidae, Argasidae and Nuttalliellidae. In these families,
the Nuttalliellidae comprises a single species, Nuttalliella namaqua. The remaining
two families consist of the hard ticks (Ixodidae) and the soft ticks (Argasidae)
(Guglielmone et al, 2010). Ixodid ticks are differentiated from Argasid ticks by
the presence of a sclerotized scutum. Fourteen genera are recognized in the family
Ixodidae, and species within the genera Ixodes, Rhipicephalus, Dermacentor, Am-
blyomma, Hyalomma and Haemaphysalis most frequently infest dogs.
The life cycle of ixodid ticks consists of four stages, eggs, six legged larvae,
eight legged nymphs and eight legged adult ticks. Depending on the species, ixodid
ticks have one- two-or three-host life cycles. All ixodid tick species, which feed on
dogs, are characterized by a three-host life cycle (Fig.1). Metastriate ticks which
include all genera except Ixodes mate exclusively on the host, whereas in pros-
triate ticks, of which Ixodes is the only, gametogenesis already begins during
the nymphal to adult moult, resulting in unfed adults sexually active soon after
moulting. In some Ixodes species mating takes place off the host, before the ticks
have fed, while in others mating takes place on the host. Engorgement of female
metastriate and prostriate ticks on the blood of their hosts can be immense, occasion-
ally in excess of 100 times their own unfed body weight. After engorgement, female
ticks detach and drop from their hosts and commence oviposition, which results
in several thousand eggs. The males of most species remain attached for varying
periods of time after the females have dropped.Ticks follow two basic strategies to
access suitable hosts. A passive (ambush) strategy involves a tick remaining quies-
cent in its habitat and depends upon contact with any host that invades this habitat.
These ticks are found questing on the vegetation. Active (hunter) host acquisition
involves a tick leaving the environment where it was sheltering to actively seek a
host. Once a host has been found, tactile stimuli come into play, contributing, along
with short-range odourants and body heat, to the selection of a feeding site and
the commencement of blood-sucking. The mouthparts are composed of a pair
of hollow tubes known as the cheliceral sheaths surrounding a pair of appendages
called chelicerae. The distal extremities of the chelicerae are armed with denticles
12

Fig. 1
Fig. 2
Reproduced with kind permission of Prof. F. Beugnet from the “Pet Owner Educational
Atlas” published by Merial S.A.S., Lyon, France.
13

which are used in a saw-like motion to cut the host’s skin. The hypostome lies ven-
tral to the chelicerae and its ventral surface is armed with variable rows of retrograde
denticles. The hypostome is used to penetrate the laceration created by the chelicerae
and the denticles for attachment to the host. Attachment is secured by means of a
milky white fluid secreted by the salivary glands of the tick, which hardens into
a latex-like cone surrounding the hypostome. This is serves as the initial core of
a cement cone that will eventually secure the attachment site. The secretion of this
substance is continued for up to 72 hours adding cortical layers to the cement, and
may even overflow onto the surface of the host’s skin in some tick species, thus fur-
ther consolidating the attachment site. After the establishment of the cement cone
the salivary glands enlarge and protein synthesis is accelerated. The process of
penetration and probing for blood injures capillaries and small blood vessels causing
an extensive haemorrhagic pool at the tick feeding site. The host’s response to
this type of damage to the dermis is the formation of a haemostatic plug, activa-
tion of the coagulation cascade, vasoconstriction, inflammatory responses leading to
wound healing, all of which have a negative impact on tick feeding (Sonenshine,
1991).
14

Table 1: Some of the most important ixodid tick species infesting dogs in various
zoogeographical regions.
Genus Species Zoogeographical Region

Ixodes I. ricinus (sheep, deer tick) Palearctic
I. hexagonus (hedgehog tick) Palearctic
I. scapularis (black-legged tick) Nearctic
I. pacificus (Western black-legged tick) Nearctic
I. persulcatus (Taiga tick) Palearctic
I. holocyclus (Australian paralysis tick) Australasia
Dermacentor D. variabilis (American dog tick) Nearctic

D. andersoni (Rocky mountain wood tick) Nearctic
D. reticulatus (Ornate dog tick) Palearctic
Rhipicephalus R. sanguineus (Brown dog tick) Worldwide

R. turanicus Afrotropical and Palearctic
Haemaphysalis H. elliptica Afrotropical

Australasia, Oriental,
H. bispinosa
Palearctic
Australasia, Oriental,
H. longicornis
Palearctic
Amblyomma A. americanum (lone star tick) Nearctic

A. maculatum (Gulf coast tick) Nearctic
Hyalomma H. truncatum Afrotropical
15

1.3. Tick-borne pathogens
Two ixodid tick species that commonly infest dogs, Rhipicephalus sanguineus
and Dermacentor reticulatus have been used extensively in the studies that consti-
tute part of this thesis and are therefore described in greater detail.
The brown dog tick, Rhipicephalus sanguineus (Latreille, 1806), has

a worldwide distribution and is found approximately between 50°N and 30°S
(Walker et al, 1999). This tick is a near strict parasite of dogs and is well adapted
to living within human dwellings as well as in gardens and kennels (Dantas-Tor-
res, 2010). It is this adaptability and close association with dogs that facilitated its
worldwide distribution. Depending on the climate, R. sanguineus may be prevalent
throughout the year in tropical and subtropical areas or more active during late
spring to early autumn in temperate regions (Dantas-Torres, 2010).
R. sanguineus is a vector of a number of tick-borne pathogens affecting

dogs, most important of which is Ehrlichia canis, the causative agent of canine
monocytic ehrlichiosis (CME). CME is widely distributed and is found wherever
the brown dog tick occurs. R. sanguineus is also a vector of protozoan tick borne
pathogens, Babesia vogeli and Babesia gibsoni, causative agents of babesiosis in
dogs. Furthermore, it can transmit Anaplasma platys, the cause of thrombocytic an-
aplasmosis, Hepatozoon canis, the cause of hepatozoonosis and Mycoplasma hae-
mocanis, causing canine haemoplasmosis (Harrus and Waner, 2013; Baneth, 2013;
Sykes, 2013).
Although R. sanguineus is in general very host specific, human parasitism

does occur and appears to be more frequent in environmental conditions leading
to high tick densities (Dantas-Torres, 2010). This poses a threat to human health
since R. sanguineus is a competent vector of several Rickettsia species to humans.
Most importantly are Rickettsia conorii, the causative agent of Mediterranean spot-
ted fever and Rickettsia rickettsii, the agent causing Rocky Mountain spotted fever
(RMSF). Mediterranean spotted fever is encountered throughout the Mediterranean
region, sub-Saharan Africa, India and around the Black Sea (Fournier, 2013).
RMSF is present in the south-western region of the USA, where it is usually trans-
mitted to humans by the American dog tick (Dermacentor variabilis) (Drexler
16

et al, 2014). However, transmission of RMSF by R. sanguineus has emerged as a
significant health threat to humans in eastern Arizona (Demma et al, 2005).
The ornate dog tick, Dermacentor reticulatus (Fabricius, 1794), is a Palearctic

species which is characterized by a highly focal distribution pattern (Gray et al,
2009; Estrada-Peña et al, 2013). It occurs in isolated pockets from south-western
England in the west through Central Asia reaching the Yenisei river basin in
Siberia in the east (Dautel et al, 2006; Kubelová et al, 2011; Cochez et al, 2012;
Rubel et al, 2014; Karbowiak, 2014). D. reticulatus is an important vector of a
number of tick-borne protozoan pathogens, in particular Babesia canis, (Fig.2) the
cause of canine babesiosis, and Babesia caballi and Theileria equi, the causative
agents of equine piroplasmosis (Jongejan and Uilenberg, 2004). Moreover, the tick
has proven vectorial competence for Anaplasma marginale, the causative organism
of bovine anaplasmosis (Zivkovic et al, 2007) and A. marginale-infected D.retic-
ulatus have recently been collected in the field in France (Bonnet et al, 2013).
Although D. reticulatus infrequently bites humans, it is associated with a

broad range of human pathogens. For instance, Babesia microti has been identified
in questing adult D. reticulatus, which suggests a role in the transmission of human
babesiosis (Wójcik-Fatla et al, 2012). D. reticulatus has also been incriminated
as a vector of tick-borne encephalitis virus (Wójcik-Fatla et al, 2011). Moreover,
Spotted Fever Group Rickettsiae causing Siberian tick typhus (Rickettsia sibirica)
and human rickettsiosis (Rickettsia slovaca and Rickettsia raoultii) are found in
Dermacentor spp. ticks (Parola et al, 2009). Interestingly, R. raoultii has recently
been isolated and maintained in cell cultures cultivated initiated from eggs origi-
nating from a colony of Dutch D. reticulatus ticks (Alberdi et al, 2012). Last
but not least, Anaplasma phagocytophilum, causing tick-borne fever in ruminants as
well as granulocytic anaplasmosis in dogs, cats, horses and humans, has recently
been detected in questing D. reticulatus collected from the vegetation in Lithuania
(Paulauskas et al, 2012). This finding may suggest a possible role as a vector for
A. phagocytophilum.
17

1.4. Fleas
Fleas are wingless insects within the phylum Arthropoda and are characterized
by laterally compressed bodies. Only four species of fleas, namely Ctenocepha-
lides felis (cat flea), Ctenocephalides canis (dog flea), Pulex simulans and Echid-
nophaga gallinacea (poultry stick-tight flea) within the order Siphonaptera, family
Pulicidae, occur in large enough numbers on dogs to be considered as important
nuisance pests (Blagburn and Dryden, 2009). The flea life cycle consists of eggs,
larvae, pupa and finally the adult stage (Krämer and Mencke, 2001) (Fig.3.). Flea
larvae are free-living and feed on incompletely digested blood excreted daily as
flea frass by female fleas. Since flea larvae are extremely susceptible to heat and
desiccation, they require a protected environment with moderate temperatures and
high humidity for development (Dryden and Rust, 1994). After the development of
1st and 2nd instar larvae, the 3rd instar larvae spin a cocoon in which pupation and
further metamorphosis takes place and from which an adult flea emerges. Upon
emergence, the adult flea almost immediately seeks a host by means of visual and
thermal factors, while an increase in CO2 concentration stimulates an escalation of
flea activity (Krämer and Mencke, 2001). Blood feeding is initiated within seconds
on the host, and mating occurs within the first eight to 24 hours and egg production
by females within 24 to 36 hours. Female fleas are capable of producing approx-
imately 40 to 50 eggs per day, while consuming large volumes of host blood
daily (up to 15 times their own body weight). The entire life cycle can be completed
in 12 to 14 days (Blagburn and Dryden, 2009).
1.5. Flea-borne pathogens
One flea species in particular, C. felis, has been used in studies that constitute part of
this thesis and it will therefore be described in greater detail.
The cat flea, C. felis (Bouche, 1835) has a worldwide distribution and is found
on all continents except Antarctica. Within this species there are four recognized
sub-species. These are, Ctenocephalides felis damarensis and C. felis strongylus oc-
curing primarily in East Africa, C. felis orientis occuring in India and Australia and
18

Fig. 3
Fig. 4
Reproduced with kind permission of Prof. F. Beugnet from the “Pet Owner Educational
Atlas” published by Merial S.A.S., Lyon, France.
19

C. felis felis, which has a worldwide distribution (Blagburn et al, 2009). C. felis is
responsible for a common dermatological disease of dogs, namely flea allergic
dermatitis (FAD). FAD occurs in dogs, which develop a hypersensitive state in
response to antigenic material secreted by the salivary glands of fleas (Krämer et al
2001).
Ctenocephalides felis also serves as a vector of various bacterial pathogens

as well as being the intermediate host for filarid and cestode parasites (Blagburn et
al, 2009). Dipylidium caninum is one of these cestodes, and is of particular impor-
tance as it infects dogs worldwide. Because the cat flea has a low host specificity it
frequently bites humans causing intense pruritus, especially in sensitised children.
In addition to the irritation associated with flea bites, C. felis can also transmit D.
caninum to humans, causing dipylidiosis (Fig.4.). Moreover, fleas have played a
major role in the epidemiology of historically well known diseases like murine
typhus (Rickettsia typhi) and plague (Yersina pestis) (Krämer et al 2001). More re-
cently, C. felis has also been incriminated as vector of emerging diseases such as
flea-borne spotted fever caused by Rickettsia felis (Parola et al, 2005; Beugnet and
Marié, 2009).
1.6. Tick control
An effective tick control strategy involves the control of ticks on the host as well as
in the environment. The control of ticks in the environment is more difficult com-
pared to that of fleas, mainly because ticks use alternative hosts other than dogs to
complete their life cycle (the exception is R. sanguineus). Consequently, effective
tick control in the environment is dependent on limiting the availability of these
alternative hosts. This can be done by destroying the refuge areas of these animals
thus creating an unattractive environment for their maintenance (Blagburn et al
2009). Since R. sanguineus utilises dogs as hosts for all its parasitic life stages, en-
vironmental tick control can be very effective when acaricidal products are applied
in the kennel environment.
A broad range of active substances are available for use in a host-targeted tick
control strategy. Moreover, they can be administered as a topical spot-on, impreg-
nated in a collar or provided in an oral chew. It is also important to consider the
life style of the pet when choosing an acaricidal product. For instance, frequent
20

swimming or bathing can negatively influence the effectiveness of any topical
product that mainly resides in the superficial skin layers of the animal. Another
important factor to consider is the speed of action of the acaricidal product
and whether it has any repellency properties. Topically applied substances such as
amitraz, permethrin, flumethrin and fipronil have dominated the market for a long
time. Recently, however, orally active substances like afoxolaner and fluralaner have
entered the market for tick (and flea) control on dogs (Kunkle et al, 2014;
Hunter et al, 2014; Rohdich et al, 2014).
1.7. Flea control
The effective control of flea infestations on dogs involves a combination of vari-

ous strategies. These not only include the control of flea infestations on the host,
but also the control of flea life stages in the environment using insecticides as well
as mechanical means (Blagburn et al, 2009). In this regard, insect growth reg-
ulators (IGR) acting as juvenile hormone analogues work especially well when
administered to dogs. These IGRs are very persistent in the environment and
contaminate surfaces with which the dog was in contact. Bedding areas are par-
ticularly affected when IGRs are administered to dogs, thus effectively controlling
the further development of flea eggs and larvae in these areas. Other strategies for
controlling fleas in the environment include the regular vacuuming of homes and
frequent washing of pet bedding (Blagburn et al 2009). Host targeted control of flea
infestation involves the use of topical or oral adulticides registered for use on dogs.
Effective topically active substances include imidacloprid, permethrin, fipronil and
dinotefuran and orally active substances include spinosad, afoxolaner and fluralaner.
If the dog is not excessively bathed or wetted many of the topical products remain
effective and this will be indicated as such in the specifications of the product. Oral
products are not affected by wetting or bathing and therefore represent a good
alternative. Other factors to consider are the speed of action, duration of effec-
tiveness and spectrum of activity.
21

1.8. Mode of action of different classes of acaricides and insecticides
A broad range of substances active against ticks and fleas has been used in the studies
on which this thesis is based. They belong to different chemical groups: pyrethroids,
phenylpyrazoles, formamidines, neonicotinoids, isoxazolines and juvenile hormone
analogues. Each group of chemicals has unique characteristics and can be used
either as acaricides or as insecticides and their mode of action targets both ticks
and fleas. Other chemical groups contain substances which also are acaricidal
and/or insecticidal, such as organophosphates (diazinon, dichlorvos e.o.), semicar-
bazone (metaflumizone), oxadiazines (indoxacarb) and several macrocyclic lactones
(spinosad, selamectin).
Pyrethroids
Synthetic pyrethroids such as cyphenothrin, permethrin, flumethrin and deltamethrin
are second generation photostable pyrethroids with a relatively long residual activity
(Beugnet and Franc, 2012). The mode of action of these pyrethroids is mediated
through the closure of the sodium channels leaving the nerve cell membrane in
a permanent state of depolarization, resulting in a sudden “knock down” effect.
Pyrethroids are also volatile, which explains the repellency effect against flying
insects and even against ticks. Cyphenothrin, permethrin and deltamethrin display
both acaricidal as well as insecticidal activity, whereas the activity of flumethrin
is mainly acaricidal.
Phenylpyrazoles
Fipronil is the most widely used phenylpyrazole and studies on it have also been
included in this thesis. Fipronil binds to GABA-gated chloride channels and gluta-
mate-gated chloride channels of arthropods, which prevents the opening of chloride
ion channels, leading to neuronal hyperactivity. Fipronil is a relatively slow-acting
acaricide as well as insecticide and due to its lipophilicity, the molecule remains
active on animals that are shampooed, bathed or exposed to rain (Beugnet and
Franc, 2012).
Formamidines
Amitraz is a formamidine with acaricidal properties without acting directly on nerve
conduction. Instead it induces an alteration in the behaviour of ticks through interfer-
22

ing with the octopaminergic system by binding to octopamine receptors. This results
in the stimulation of monoamine oxidases (adenylate cyclase activity) and the G
protein, thus inducing cAMP and cGMP synthesis, which have various intracellu-
lar consequences. At sublethal doses, these alterations lead to detachment of ticks
from the host or the prevention of attachment and feeding and reduced prolificity.
Interestingly, when amitraz is combined with other acaricides/insecticides signif-
icant synergistic effects have been discovered (Prullage et al, 2011).
Neonicotinoids
Neonicotinoids included in the studies contributing to this thesis are imidacloprid
and dinotefuran. These molecules cause spastic paralysis in insects by acting like
agonists on postsynaptic nicotinic acetylcholine receptors, mainly in motorneurons,
inducing depolarization of nerve membranes. The spectrum of activity of imidaclo-
prid and dinotefuran is limited to insects. However, when these molecules are used
in combination with pyrethroids, the spectrum of activity is increased resulting in
combination products targeting both fleas and ticks (Beugnet and Franc, 2012).
Juvenile hormone analogues

Pyriproxyfen and (S)-methoprene are insect growth regulators (IGRs) of the juve-
nile hormone analogs group. They act by contact or ingestion and end up in the
eggs of fleas either indirectly via adult fleas that had come into contact with these
IGRs or directly through the cuticle of the eggs. IGRs impair the hatching of flea
eggs and as neotenin analogs prevent the 3rd larval instar from moulting into pupae.
Pyriproxyfen and (S)-methoprene can be used for the control of flea populations
in the environment or as part of a host-target strategy in which case these IGRs
are combined with other adulticidally active substances (Beugnet and Franc, 2012).
Isoxazolines
Isoxazolines are a new class of acaricides/insecticides, which after oral administra-
tion act systemically. Afoxolaner, an isoxazoline studied in this thesis, is character-
ized by a high affinity to plasmatic proteins, which are ingested by haematophagous
arthropods during their blood meal. This molecule acts as a ligand to a specific
receptor on both GABA and glutamate receptors on the chloride ion channel in the
neuronal synaps and induces hyper excitation before death of both fleas and ticks
(Shoop et al, 2014).
23

Table 3: Overview of the active ingredients included in the studies and marketed for tick and
flea control on dogs
Route of Main spectrum

Trade name Active substances Chemical group Mode of action
application of activity
Prevents closure of Na+

channels leading to
Insecticidal and
Cyphenothrin Pyrethroids continued Na+ influx and
acaricidal
hyper excitation of nerve
Sergeant’s Topical cells.
Gold® spot-on
Affects fecundity, egg
IGR: Juvenile
hatching, metamorphosis
Pyriproxyfen hormone ana- Insecticidal
of 3rd stage flea larvae to
logues
pupae.
Binding to GABA and

glutamate receptors
Insecticidal and
Fipronil Phenylpyrazoles inhibits opening of Cl⁻ ion
acaricidal
channels leading to
neuronal hyperactivity.
Binds to octopamine re-

Topical ceptors (agonist of octopa-
Certifect®
spot-on Amitraz Formamidines Acaricidal mine) leading to activation
of adenylate cyclase and G
protein.

IGR: Juvenile
hatching, metamorphosis
(S)-methoprene hormone ana- Insecticidal
of 3rd instar flea larvae to
logues
pupae.
Nicotinic acetylcholine
receptor agonist leading to
Imidacloprid Neonicotinoids Insecticidal
membrane depolarization
and spastic paralysis.
Topical
Advantix®
spot-on Prevents closure of Na+
Insecticidal and channels leading to contin-
Permethrin Pyrethroids
acaricidal ued Na+ influx and hyper
excitation of nerve cells.
24

Route of Main spectrum
Trade name Active substances Chemical group Mode of action
application of activity

channels leading to contin-
Flumethrin Pyrethroids Acaricidal
ued Na+ influx and hyper
Nicotinic acetylcholine
receptor agonist leading to
Seresto® Collar Imidacloprid Neonicotinoids Insecticidal
membrane depolarization
and spastic paralysis.

IGR: Juvenile hor- hatching, metamorphosis
(S)-methoprene Insecticidal
mone analogues of 3rd stage flea larvae to
pupae.
Binding on both GABA
and glutamate receptors,
Insecticidal and
Nexgard® Oral chew Afoxolaner Isoxazolines stimulation of Cl⁻ ion
acaricidal
channels, hyper excitation
of nerve cells.
Scalibor® Collar Deltamethrin Pyrethroids
Binding to GABA and
glutamate receptors inhib-
Insecticidal and
Fipronil Phenylpyrazoles its opening of Cl⁻ ion
acaricidal
channels leading to neuro-
Frontline Topical nal hyperactivity.
Combo® spot-on
Decrease in fecundity, egg
IGR: Juvenile hor- hatching, and affects meta-
(S)-methoprene Insecticidal
mone analogues morphosis of 3rd stage
flea larvae to pupae.

Permethrin Pyrethroids
Topical Nicotinic acetylcholine re-

Vectra 3D® Dinotefuran Neonicotinoids Insecticidal ceptor agonist, stimulation
spot-on
of postsynaptic neurons.
IGR: Juvenile hor- hatching, metamorphosis
Pyriproxyfen Insecticidal
mone analogues of 3rd stage flea larvae to
pupae.
25

1.9. Efficacy and speed of kill
In order to protect dogs against the detrimental effects of tick and flea infestations,
effective acaricidal and insecticidal products are required. The efficacy can, howev-
er, not be entirely defined by a single value, but rather as a combination of different
facets of activity ultimately defining the profile of a specific product (Marchiondo
et al, 2013). Aspects of activity that are important contributors to the profile of a
product are therapeutic or immediate effectiveness, persistent or residual efficacy,
speed of kill, repellency, tick expellency and anti-feeding effect. The therapeutic
or immediate efficacy of a compound is the ability to kill existing tick or flea infes-
tations on a dog, while persistent or residual efficacy is the ability to prevent the es-
tablishment of new infestations. Speed of kill refers to the rate at which the product
induces mortality over time. The ability of a product to cause an irritant effect on
ticks and fleas, thereby causing them to leave their host immediately after contact
or to entirely avoid contact with a treated host has recently been defined as repellen-
cy (Halos et al, 2012). Tick expellency refers to the ability of a product to disrupt the
mechanism of tick attachment thereby leading to the detachment of already attached
ticks or the prevention of attachment of new ticks (Halos et al, 2012). The ability
of a product to prevent ticks or fleas taking a blood meal is the anti-feeding effect.
To determine whether a product has acaricidal or insecticidal properties various
in vitro assays can be employed. However, determination of the different aspects
of activity ultimately contributing to product efficacy on an animal, can only be
evaluated in vivo on the target species.
In an attempt to ensure scientifically acceptable standardised trial designs

for the evaluation of product effectiveness against ticks and fleas a number of re-
gional guidance documents with specific study design requirements and criteria for
evaluation of effectiveness have been published. The European Medicines Agency
(EMEA) has compiled a guidance document, entitled “Guideline for the testing and
evaluation of the efficacy of antiparasitic substances for the treatment and preven-
tion of tick and flea infestations in dogs and cats” (EMEA/CVMP/005/2000-Rev2)
with a revision published in 2011 (EMEA/CVMP/055/00-Rev 2). Similarly, the En-
vironmental Protection Agency (EPA) in the USA has published a guidance docu-
ment for the evaluation of externally applied parasiticides which are not internally
absorbed by the treated pet. This document, entitled, “(OPPTS 810.3300) Product
26

Performance Test Guidelines, Treatments to Control Pests of Humans and Pets
(EPA 712-C-98-411)” was published in March 1998 by the Office of Prevention
Pesticides and Toxic Substances.
In addition to the regional guidance documents for the EU and the USA, there
is also a scientific guidance document published by the 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”.(Marchiondo et al, 2013). In the W.A.A.V.P.
guidelines, general principles for good scientific design for efficacy studies
against ticks and fleas on dogs are given. For instance, laboratory study designs
assessing the effectiveness of compounds against ticks and fleas must incorporate
the use of a negative control group. The negative control group must remain un-
treated and allows for the comparison of parasite counts at specific pre-determined
time points between the treated and the untreated control group. Dogs must be
assigned to study groups based on pre-allocation parasite counts, thus allowing for
ranking according to parasite establishment, followed by random allocation to study
groups. This ensures that the study groups are well balanced with regard to host
suitability. Once post-treatment parasite counts are generated, the percentage effica-
cy can then be calculated using the formula developed by Abbott (1925)
Efficacy (%) = 100 x (C – T) / C, whereby:
C = Mean live parasite count on the control group; T = Mean live parasite count
on the treated group.
Although the above formula is suitable for topically applied compounds,

evaluating the efficacy of systemic compounds requires additional categorization of
whether a live tick is attached or not. The rationale behind this is that ticks must in-
gest a systemic compound via a blood meal, therefore the use of only live attached
ticks to evaluate the efficacy is more appropriate.
Survival status Attachment status Interpretation

1
Live Free or attached Acaricidal effect not demonstrated
Dead Free or attached Acaricidal effect demonstrated
1
The use of only live attached ticks for assessment of systemic efficacy may be acceptable if justified.
27

For the calculation of efficacy, arithmetic and geometric means may be used. Geo-
metric means are generally considered to be a more accurate estimate in view
of the general tendency that parasite counts may be skewed. Alternative views do,
however, exist and arithmetic means are considered by the EMEA guidelines to be
the only appropriate basis for calculating product efficacy.
In addition to infesting dogs with ticks and/or fleas at pre-determined time

points, the infestation load for each challenge must be standardised. It is generally
acceptable to infest dogs with ~50 ticks and ~100 fleas. Except when using a
species where often only the females attach and ingest a blood meal (e.g. Ixodes
spp.), there must be an equal gender distribution of ticks (50% male/50% female).
It is also important to evaluate tick and flea retention before the allocation of dogs
to groups, as well as throughout the assessment period on control animals, since this
indicates host suitability and parasite viability. In general, retention of at least 25%
of fleas and 20% of ticks is regarded as acceptable. Opinions on suitable parasite
retention do, however, differ and the EMEA guidelines require at least 50% retention
for fleas and 25% for ticks.
Other important considerations are the acclimatisation of dogs to the envi-

ronmental conditions and husbandry practices of the particular laboratory facility,
and acclimatisation should last at least seven days. This improves animal welfare
and limits inter-animal variation, which may impact on the study outcome. It is also
important that all animal husbandry practices comply with local standards and that
research protocols are approved by an Institutional Animal Care and Use Committee
(IACUC).
1.10. Transmission blocking concept
The concept “transmission blocking” implies the ability of an anti-parasitic product

to forestall infection with a specific vector-borne pathogen. The blocking ability of
an antiparasitic product is dependent on (a) the time it takes for a product to reach
its full adulticidal effect (100% efficacy) and (b) the time interval before the vector
transmits the pathogen. For example the duration of attachment that is required for
ticks to transmit protozoan, bacterial or viral pathogens differs considerably. For in-
stance, the protozoan parasite, Babesia microti, is transmitted by Ixodes scapularis
between 36 and 48 hours after tick attachment (Piesman and Spielman, 1980),
28

Fig 5: Risk balance between adulticidal effect and Babesia transmission time
Fig 6: Risk balance between adulticidal effect and Ehrlichia canis transmis-
sion time
29

whereas the rickettsia E. canis can be transmitted within a few hours after attach-
ment of R. sanguineus (Fourie et al, 2013). Viral pathogens in general are mostly
transmitted within minutes after tick attachment has taken place.
A “grace” period is therefore present for several acaricidal/insecticidal

products in which to reach their full adulticidal effect in order to prevent trans-
mission by the targeted vector. The balance between adulticidal efficacy and patho-
gen transmission time is illustrated in Figures 5 and 6 for Babesia and Ehrlichia.
Relatively little research has been done on dogs to evaluate the transmission
blocking capability of acaricidal and insecticidal compounds under laboratory
conditions. However, several papers have been published on the pathogen blocking
ability of acaricidal/insecticidal compounds under field conditions. For instance,
research done in South Africa showed that the application of amitraz-impregnated
collars prevented infections with Babesia rossi transmitted by Haemaphysalis ellip-
tica. In this study eight of 30 control dogs (26.6%) became infected over a six-month
period and none of the 20 treated dogs (Last et al, 2007). Similarly fipronil spot-on
was shown to be effective in preventing infection with E. canis transmitted by R.
sanguineus in a West African field trial (Davoust et al, 2003). Field trials, however,
depend on naturally occurring challenge pressure, which often results in unpredict-
able numbers of untreated control animals actually contracting the target tick-borne
disease. In contrast, controlled laboratory studies presented in this thesis allow for
a much more standardised evaluation of the transmission blocking ability of a range
of acaricidal and insecticidal compounds.
Finally, in addition to vector-borne diseases, the transmission blocking con-

cept can also be applied to other infections where an intermediate host that itself
is a parasite, is involved. An example presented in this thesis is Dipylidium caninum
and its intermediate flea hosts belonging to the genus Ctenocephalides. As is the
case for vector-borne infectious diseases, a grace period is also present based on the
maturation time of the metacestode in its the flea intermediate host. Consequently
if the insecticidal activity of a product is rapid enough to kill infected fleas on
dogs before metacestode maturation occurs, infection will be forestalled.
30

2.0 Aims and Outline of this Thesis
Because dogs are such loved companion animals, their health and wellbeing is of
great importance to their human companions. Moreover, controlling ticks and fleas
on dogs is also important in respect of the zoonotic risk that some of these parasites
pose to their human companions. Numerous products are available to veterinarians
and dog owners for controlling ticks and fleas. By comparing the efficacy of these
products, important information can be collected that will help the end-user to de-
cide on the most appropriate product.
It was hypothesized that the combination of different active substances and

alternative routes of administration using improved delivery systems, could enhance
the speed of kill as well as duration of efficacy of various compounds.
Moreover, if the speed of kill was rapid enough, these products could
potentially prevent infections with tick-borne and flea-borne pathogens.
Therefore the aim of the studies described in this thesis was to evaluate and
compare the efficacy of various classes of compounds, with different modes of ac-
tion, used for the control of tick and flea infestation on dogs. The efficacy of these
compounds was evaluated either separately, or administered in combination, using
different application modes, such as topical application, impregnated collars or oral
formulations. Moreover, the ability of several compounds to prevent transmission of
selected tick- and flea-borne pathogens was also assessed.
In Chapter 2 the efficacy of various compounds against Haemaphysalis

elliptica, Dermacentor reticulatus and Rhipicephalus sanguineus as well as Cteno-
cephalides felis was evaluated on dogs in controlled laboratory studies.
In order to protect a dog from the detrimental effects of tick infestation phar-
maceutical products must not only be able to control ticks, but the onset of action
must be fast enough to prevent transmission of harmful tick-borne pathogens.
In Chapter 3 the ability of different compounds to prevent the transmission

of the tick-borne pathogen Babesia canis by Dermacentor reticulatus is evaluated in
controlled laboratory studies.
31

In Chapter 4 the ability of different compounds to prevent the transmission
of the tick-borne pathogen Ehrlichia canis by Rhipicephalus sanguineus is evaluat-
ed in controlled laboratory studies. Moreover, the speed of transmission of E. canis
by infected R. sanguineus was evaluated and compared in vivo on dogs and in vitro
on artificial feeding membranes.
Finally, in addition to the immediate irritation that flea infestations cause

to dogs, fleas are also intermediate hosts of the metacestode stages of the cestode
Dipylidium caninum. In Chapter 5 the prophylactic effectiveness of a collar formu-
lation against infected fleas is evaluated and consequently also its ability to prevent
D. caninum infection in dogs.
32

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37

38

Chapter 2. Control of ticks and fleas on dogs
2.1. Fourie JJ, Fourie LJ, Horak IG, Snyman MG.

The efficacy of a topically applied combination of cyphenothrin and
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39

40

Article — Artikel
The efficacy of a topically applied combination of cyphenothrin and

pyriproxyfen against the southern African yellow dog tick, Haemaphysalis
elliptica, and the cat flea, Ctenocephalides felis, on dogs
a* a,b b c
J J Fourie , L J Fourie , I G Horak and M G Snyman
South Africa 11 and is likely to occur

ABSTRACT wherever there are adequate rodent and
The objective of this study was to determine the therapeutic and residual efficacy of a dog populations to sustain it17. It is a
topically applied combination of cyphenothrin (40 %) and pyriproxyfen (2 %) against the 3-host tick, of which the larvae and
tick Haemaphysalis elliptica and the flea Ctenocephalides felis on dogs. Twelve dogs were nymphs use murid rodents as their hosts
infested with 50 ticks 2 days before they were treated and with approximately 100 fleas of choice15. The adults infest domestic
6 days before treatment and again 2 days before treatment and with 50 ticks and approxi- dogs and cats and the larger wild felids
mately 100 fleas at weekly intervals thereafter. They were ranked according to their flea
and some canids6,10. Adult ticks are most
counts and sex 5 days before treatment and randomly allocated to an untreated
abundant on dogs in the Eastern Cape
control group of 6 dogs and a treated group of 6 dogs. Ticks and fleas were collected from
Province from June to February9, during
the dogs 48 h after treatment and 48 h after each infestation and live and dead ticks and live
fleas were counted. The counts of ticks and fleas were transformed to geometric means, and
the period May to September in the West-
efficacy was calculated by comparing these means. The product had a therapeutic efficacy ern Cape Province10, during the period
of 83.1 % against H. elliptica and 97.5 % against C. felis 2 days after treatment. The residual October to February in Free State Prov-
period of protection during which efficacy was ≥ 90% was 5 weeks for both H. elliptica and ince12 and from January to April in north-
C. felis. eastern KwaZulu-Natal Province, South
Keywords: Ctenocephalides felis, cyphenothrin, dogs, efficacy study, Haemaphysalis elliptica, Africa7.
pyriproxyfen, topical application. The most commonly encountered flea
on dogs in South Africa is the cat flea,
Fourie J J, Fourie L J, Horak I G, Snyman M G The efficacy of a topically applied combination
Ctenocephalides felis19. The numbers of
of cyphenothrin and pyriproxyfen against the southern African yellow dog tick
Haemaphysalis elliptica and the cat flea Ctenocephalides felis on dogs. Journal of the South
Ctenocephalides spp., amongst which C. felis
African Veterinary Association (2010) 81(1): 33–36 (En.). ClinVet International, PO Box 11186, was predominant, increased on kennelled
Universitas, 9321 South Africa. dogs, on which no insecticide was used,
from very low numbers in winter to reach
a peak during late summer, decreasing
again during autumn5. Effective control
INTRODUCTION tick, Haemaphysalis elliptica, the kennel of C. felis will reduce irritation in healthy
Ticks and fleas are without doubt the tick, Rhipicephalus sanguineus, and the dogs, while absolute control is necessary
most important ectoparasites of domestic glossy brown tick Rhipicephalus simus. In when dogs display flea-bite allergies.
dogs. Ticks are important because they the western Free State Province and the Control of both C. felis and D. caninum is
can give rise to ‘tick worry’, transmit southwestern Western Cape Province necessary to reduce the likelihood of
diseases to dogs, and may also bite and R. simus is replaced by Rhipicephalus infection in dogs and humans with this
transmit diseases to the owners of the gertrudae10,12. The 2 most important of tapeworm.
dogs8,20. Fleas are of veterinary impor- the last 4 tick species are H. elliptica, The present paper describes an efficacy
tance because they cause intense irrita- the only proven vector of Babesia canis study in kennelled dogs artificially in-
tion in healthy dogs, flea-bite allergies in rossi14, the cause of virulent babesiosis in fested with adult H. elliptica and C. felis
sensitised dogs and are the intermediate domestic dogs, and R. sanguineus the and to which a combination of the insecti-
hosts of the larval stage of the dog tape- vector of Ehrlichia canis, the cause of cides cyphenothrin (40 %) and pyriproxy-
worm Dipylidium caninum. This tape- ehrlichiosis in dogs4. Both ticks are also fen (2 %) (Sergeant’s Gold®: Sergeant’s Pet
worm may also infect humans, and capable of transmitting Rickettsia conorii, Care Products, Omaha, USA) was applied
especially children17,18. the cause of tick-bite fever or tick typhus in a spot-on formulation.
At least 22 ixodid tick species have been in humans3,13,16.
collected from dogs in South Africa7,9,10,12. Haemaphysalis elliptica is an old South MATERIALS AND METHODS
The most commonly encountered of African taxon and has recently been The study was a parallel group design,
these are the southern African yellow dog re-established as such, as opposed to blinded, randomised, controlled efficacy
a
ClinVet International, PO Box 11186, Universitas, 9321
Haemaphysalis leachi, a tick that occurs study. Although it was not a good clinical
South Africa. north of South Africa and with which it practice investigation, it was carried out
b
Department of Zoology and Entomology, University of the had long been confused2. In our experience in line with the VICH GL9 guideline on
Free State, PO Box 339, Bloemfontein, 9300 South
Africa. H. leachi does not occur in South Africa good clinical practice1. The study was
c
Vetrem Developments, PO Box 908-477, Montana, 0151 and where it is mentioned in the pre-2007 conducted on 2 groups of dogs, each
South Africa.
South African literature it should be read consisting of 6 animals. Each group
*Author for correspondence.
E-mail: josephus@clinvet.com as H. elliptica. comprised 3 male and 3 female dogs, all
Received: March 2009. Accepted: February 2010. Haemaphysalis elliptica is widespread in more than 6 months old, and weighing
0038-2809 Jl S.Afr.vet.Ass. (2010) 81(1): 33–36 33
41

Table 1: Experimental design of an efficacy study against Haemaphysalis elliptica and Ctenocephalides felis on dogs.
Acclimatisation to Tick infestations Flea infestations Spot-on treatment Tick and flea counts
pen environment (3 m /dog)
Day: –7 to –1 Day: –2, +7, +14, +21, +28, Day: –6, –2, +7, +14, +21, Day: 0 Day: –5, +2, +9, +16,
+35 and +42 +28, +35 and +42 +23, +30, +37 and +44
Tick and flea counts were carried out approximately 48 hours after treatment or artificial infestation.
Day-5 flea counts were used for group allocation purposes.
between 10.6 kg and 16.4 kg, with Table 2: Parameters used to assess the condition of live or dead ticks.
hair-lengths varying between 18.7 mm
and 60.0 mm. Each dog was fitted with Category General findings Attachment status
an electronic transponder with unique
1 Live Free
alphanumeric numbers and harboured 2 Live Attached; unengorged*
no ticks or fleas at the commencement of 3 Live Attached; engorged**
the study. The dogs had not been dipped 4 Dead Free
in an acaricide/insecticide during the 5 Dead Attached; unengorged*
8 weeks prior to the day of treatment, or 6 Dead Attached; engorged**
treated with an acaricide/insecticide
*No filling of the alloscutum evident.
spot-on/spray during the preceding **Obvious or conspicuous filling of the alloscutum evident.
12 weeks. They were individually housed
in pens with concrete floors and a sleep Group 1 served as negative controls and where Gmc = geometric mean number of
bench in an indoors animal unit. No received no treatment, whereas the dogs live ticks (categories 1–3, Table 2) on dogs
contact between dogs was possible. The in Group 2 were treated with the product in the control group on Days 9, 16, 23, 30,
animals were fed a commercially avail- at a label recommended dosage of 37 and 44 after treatment of the dogs in
able animal feed once daily according to 3 m /dog. the treated group, and Gmr = geometric
the manufacturer’s recommendation. Ticks were collected from the dogs 48 h mean number of live ticks (categories 1–3,
Potable water was available ad libitum. after treatment and 48 h after each infesta- and 6) on dogs in the treated group on
The accommodation was in compliance tion by direct observation following part- Days 9, 16, 23, 30, 37 and 44 after treat-
with the ‘National Code for Animal Use in ing of the hair and palpation. All ticks ment. Even though category 6 ticks
Research, Education, Diagnosis and were removed and the condition of each (Table 2) were dead on the day of exami-
Testing of Drugs and Related Substances tick was recorded using the parameters nation, they had managed to engorge
in South Africa’ (Director General of Agri- listed in Table 2. Fleas were collected from before dying and were therefore included
culture, Pretoria, South Africa). the dogs 48 h after treatment and again with the live ticks.
A laboratory-bred strain of H. elliptica, of after each infestation by combing the hair Efficacy against fleas was calculated as
which the larvae and nymphs were fed coat with a fine-toothed flea comb. follows:
on rabbits, was used for artificial infesta- Combing was performed by making
Efficacy (%) = 100 × (mc – mt) / mc,
tion of the dogs. The adult ticks used for several strokes with the comb in each area
these infestations were unfed, at least of the animal, each time following the lie where mc = geometric mean number of
1 week old and of a balanced sex ratio. of the hair. Movement, from one part of live fleas on the control group of dogs
Each dog was artificially infested with ap- the animal’s body surface to the next was (Group 1), and mt = geometric mean
proximately 50 ticks on the days indicated via strokes overlapping each other, so that number of live fleas on the treated group
in the experimental design summarised no site was left uncombed. (Group 2).
in Table 1. A laboratory-bred strain of C. The primary criterion for the assessment The groups were compared using an
felis (routinely fed on cats) was used for all of efficacy was based on the number of ANOVA with a treatment effect after
infestations. Fleas were unfed and of live ticks and fleas collected from dogs in logarithmic transformation of the tick and
mixed sex. Each dog was infested with ap- the control group compared with the flea counts. The level of significance of the
proximately 100 fleas on the days indi- treated group on those days on which tests was set at 5 %. The residual period
cated in Table 1. After treatment had been counts were conducted. of protection against ticks and fleas was
administered to the dogs, subsequent Percentage therapeutic efficacy against prescribed as the number of weeks
batches of fleas were not placed on or near ticks was calculated as follows: post-treatment during which efficacy was
the site at which the medication had been ≥90 %.
Therapeutic efficacy (%) = 100 × (Gmc –
applied. Gmt) / Gmc,
The study followed a randomised block RESULTS
design and the Day 5 flea counts of each where Gmc = geometric mean number of Mean tick and flea counts and the effi-
dog were used for ranking and group live ticks (categories 1–3, Table 2) on dogs cacy of the product against H. elliptica and
allocation purposes. The 12 dogs were in the control group on Day 2 after treat- C. felis on the various days of assessment
ranked, within sex, in descending order of ment of the dogs in the treated group, and are summarised in Tables 3 and 4.
individual flea counts. Within each sex Gmt = geometric mean number of live Therapeutic efficacy against H. elliptica
animals were subsequently blocked into ticks (categories 1–3) on dogs in the based on geometric means was 83.1 %,
2 blocks of 3 dogs each, and within each treated group on Day 2 after treatment. and the residual period of protection
block dogs were randomly allocated to Percentage residual efficacy was calcu- against ticks during which efficacy was
Groups 1 and 2. The groups were coded to lated as follows: ≥90 % was 5 weeks. Therapeutic efficacy
blind the persons performing the post- Residual efficacy (%) against ticks = against C. felis was 97.5 %, and the resid-
treatment assessments. The dogs in 100 × (Gmc – Gmr) / Gmc, ual period of protection was 5 weeks.
34 0038-2809 Tydskr.S.Afr.vet.Ver. (2010) 81(1): 33–36
42

Table 3: Arithmetic and geometric mean tick counts and the efficacy of a combination of cyphenothrin (40 %) and pyriproxyfen (2 %) against
Haemaphysalis elliptica on dogs.
Days post-treatment Group 1: untreated control dogs Group 2: treated dogs Geometric mean efficacy (%)
Arithmetic mean Geometric mean Arithmetic mean Geometric mean
+2 21.7 19.7 5.8 3.3* 83.1

+9 25.5 24.5 0.3 0.2* 99.2
+16 27.8 27.0 0.0 0.0* 100.0
+23 29.3 28.3 1.3 1.0* 96.5
+30 28.2 27.8 1.5 0.7* 97.5
+37 27.5 27.1 1.5 0.9* 96.7
+44 32.8 32.0 11.7 4.4* 86.3
*Values for Group 2 differ significantly from those of the untreated control
Group 1 (P = 0.05).
Table 4: Arithmetic and geometric mean flea counts and the efficacy of a combination of cyphenothrin (40 %) and pyriproxyfen (2 %) against
Ctenocephalides felis on dogs.
Days post treatment Group 1: untreated control dogs Group 2: treated dogs Geometric mean efficacy (%)
Arithmetic mean Geometric mean Arithmetic mean Geometric mean
+2 52.5 48.0 2.2 1.21 97.5

+9 56.3 52.2 0.0 0.01 100.0
+16 49.3 44.6 0.2 0.11 99.7
+23 70.0 66.5 0.0 0.01 100.0
+30 71.0 67.2 3.7 1.81 97.4
+37 66.7 64.9 11.3 6.21 90.4
+44 38.7 36.9 31.7 16.0 56.8
1
Values for Group 2 differ significantly from those of the untreated control Group 1 (P = 0.05).
DISCUSSION ing and thereafter to keep numbers Huxsoll D L 1975 Transmission of Ehrlichia
It must be born in mind that a therapeutic down. This would be during November canis to dogs by ticks (Rhipicephalus
sanguineus). American Journal of Veterinary
efficacy of 83.1 % for the product investi- or December in northern Gauteng Prov- Research 36: 937–940
gated was obtained against H. elliptica ince5 and probably also at other localities 5. Horak I G 1982 Parasites of domestic and
that had already been attached for 2 days. in South Africa with a similar climate. wild animals in South Africa. XIV. The
On the other hand, its residual efficacy, seasonal prevalence of Rhipicephalus sangui-
which was ≥90 % for 5 weeks after treat- ACKNOWLEDGEMENTS neus and Ctenocephalides spp. on kennelled
dogs in Pretoria North. Onderstepoort Jour-
ment, was measured against ticks that We express our sincere thanks to the nal of Veterinary Research, 49: 63–68
were placed on dogs after the animals had technical staff of Clinvet Laboratories for 6. Horak I G, Braack L E O, Fourie L J, Walker J
been treated. Efficacy thus appears to be their assistance in maintaining the tick B 2000 Parasites of domestic and wild
greater against unattached ticks than and flea colonies and with the infestation animals in South Africa. XXXVIII. Ixodid
against attached ticks. This pattern of effi- ticks collected from 23 wild carnivore
and collection of ticks and fleas from the
species. Onderstepoort Journal of Veterinary
cacy makes it a suitable parasiticide for dogs. Dr H. Luus of Clinvet Laboratories Research 67: 239–250
use at the commencement of the tick was responsible for the statistical process- 7. Horak I G, Emslie F R, Spickett A M 2001
season to prevent a rapid seasonal in- ing of the data. Parasites of domestic and wild animals in
crease in the numbers of H. elliptica. Initial South Africa. XL. Ticks on dogs belonging
treatment should be administered during to people in rural communities and carni-
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taxon of the Haemaphysalis (Rhipistoma) wild animals in South Africa. XXII. Ixodid
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D 1995 Prevalence of rickettsia-like organ- cats in the Western Cape Province. Onderste-
its appropriateness for use during the isms in ticks collected around Zimbabwe. poort Journal of Veterinary Research 70: 187–
commencement of the flea season when Journal of Medical Entomology 32: 787–792 195
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44

2.2. Fourie JJ, Beugnet F, Ollagnier C, Pollmeier MG.

Study of the sustained speed of kill of the combination of fipronil/ami-
traz/(S)-methoprene and the combination of imidacloprid/permethrin
against Dermacentor reticulatus. Parasite. 2011, 18 (4) : 319-23.
45

46

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
STUDY OF THE SUSTAINED SPEED OF KILL OF THE COMBINATION OF

FIPRONIL/AMITRAZ/(S)-METHOPRENE AND THE COMBINATION OF IMIDACLOPRID/PERMETHRIN
AGAINST DERMACENTOR RETICULATUS, THE EUROPEAN DOG TICK
FOURIE J.J.*, BEUGNET F.**, OLLAGNIER C.*** & POLLMEIER M.G.**
Summary: Résumé : ÉTUDE DE LA RÉMANENCE ET DE LA RAPIDITÉ D’ACTION DE

DEUX COMBINAISONS ANTIPARASITAIRES EXTERNES, FIPRONIL/AMITRAZ/(S)-
The sustained speed of kill against Dermacentor reticulatus of two MÉTHOPRÈNE ET IMIDACLOPRIDE/PERMÉTHRINE, SUR DERMACENTOR
topical combinations, one containing fipronil/amitraz/(S)-methoprene RETICULATUS, LA TIQUE EUROPÉENNE DU CHIEN
and the other, imidacloprid/permethrin, was evaluated in dogs. La rémanence et la rapidité d’action de deux antiparasitaires
Two treated groups and one untreated control group of eight adult externes destinés au chien, l’un contenant fipronil/amitraz/(S)-
Beagle dogs each were randomly formed based on pre-infestation méthoprène et l’autre imidaclopride/perméthrine, ont été évaluées.
rates and bodyweight. Each treatment was administered topically Deux groupes traités et un groupe témoin de chacun huit chiens
once on Day 0, according to the recommended label dose and de race Beagle ont été constitués après une infestation préalable
instructions for use. All dogs were infested with 50 adult unfed par des tiques. Chaque traitement topique en pipette est appliqué
D. reticulatus starting on Day 1, then weekly, for a total of five au jour 0, selon les recommandations et instructions d’utilisation
weeks. While most studies determine tick efficacy at 48 hours (h), des laboratoires fabriquant. Tous les chiens sont infectés par 50
in this study, all remaining ticks were counted and categorized 24 h D. reticulatus adultes au jour 1, puis chaque semaine pendant cinq
following each infestation. The numbers of ticks (living or dead) that semaines. Alors que la plupart des études regardent l’efficacité
remained attached on treated dogs were compared to those on the anti-tiques à 48 heures (h), dans cette étude, les tiques restant
control animals. The percent reduction of attached ticks (disruption sur les chiens ont été comptées et classées selon leur état (fixées
of attachment) at 24 h on dogs treated with fipronil/amitraz/(S)- ou non fixées, vivantes ou mortes, gorgées ou non gorgées) après
methoprene remained above 92 % for four weeks. The reduction of 24 h de contact. Le nombre de tiques (vivantes ou mortes) qui
attached ticks at 24 h on dogs treated with imidacloprid/permethrin restaient attachées sur les chiens traités a été comparé avec celui
did not reach 80 % during the entire study. The number of ticks dénombré sur les chiens témoins non traités. Le pourcentage de
attached at 24 h was significantly (p < 0.05) lower in the fipronil/ réduction d’attachement (ou de perturbation d’attachement) à
amitraz/(S)-methoprene group than in the imidacloprid/permethrin 24 h chez les chiens traités par la combinaison fipronil/amitraz/(S)-
group in assessments on Days 2, 15, 22, 29 and 36. When méthoprène est resté supérieur à 92 % pendant quatre semaines.
assessing efficacy based upon live ticks on treated versus control La réduction d’attachement des tiques à 24 h chez les chiens traités
dogs, fipronil/amitraz/(S)-methoprene 24 h efficacy was above par l’association imidaclopride/perméthrine n’a pas dépassé 80 %
95 % for four weeks, decreasing to 77.8 % at Day 36. The 24 h durant cet essai. Le nombre de tiques fixées sur les chiens à 24 h
efficacy of imidacloprid/permethrin ranged from 56.2 % to 86.7 % était significativement plus faible (p < 0.05) dans le groupe traité
through Day 29, never achieving 90 % throughout the study. The fipronil/amitraz/(S)-méthoprène que dans le groupe imidaclopride/
24-hour efficacy of fipronil/amitraz/(S)-methoprene was significantly perméthrine aux jours 2, 15, 22, 29 et 36. Si l’on étudie l’efficacité
(p < 0.05) higher than imidacloprid/permethrin at all time points, acaricide par comparaison au nombre de tiques vivantes sur les
including Day 36. chiens témoins, cette efficacité à 24 h est restée supérieure à 95 %
pendant quatre semaines, puis a diminué à 77,8 % au jour 36 dans
KEY WORDS: Dermacentor reticulates, fipronil/amitraz/(S)-methoprene,
le groupe fipronil/amitraz/(S)-méthoprène. L’efficacité acaricide à
imidacloprid/permethrin, ADVANTIX®, CERTIFECT®.
24 h dans le groupe traité imidaclopride/perméthrine a été comprise
entre 56,2 et 86,7 % jusqu’au jour 29, ne dépassant jamais 90 %.
L’efficacité acaricide à 24 h du groupe traité par l’association
fipronil/amitraz/(S)-méthoprène a été significativement plus forte
que dans le groupe imidaclopride/perméthrine à tous les points de
comptage, jusqu’au jour 36.
MOTS-CLÉS : Dermacentor reticulatus, fipronil/amitraz/(S)-méthoprène,

imidaclopride/perméthrine, ADVANTIX®, CERTIFECT®.
* ClinVet, P.O. Box 11186, Universitas, Bloemfontein, 9321, Republic

of South Africa.
** Merial S.A.S., 29, avenue Tony Garnier, 69007 Lyon, France.
*** Merial S.A.S., PIPA, CRSV, 1, allée des Cyprès, 01150 Saint-Vulbas,
T icks and fleas are the primary ectoparasites
infesting dogs (Pfister, 2011). Dermacentor tick
species are present worldwide and can be vec-
tors of disease for humans and animals (Berrada et
al., 2009; Nicholson et al., 2010). In Western Europe,
France.
Correspondence: Frédéric Beugnet. Dermacentor reticulatus is a common tick species
E-mail: frederic.beugnet@merial.com affecting dogs and is the primary vector of Babesia
Parasite, 2011, 18, 319-323

Research note 319
47

FOURIE J.J., BEUGNET F., OLLAGNIER C. & POLLMEIER M.G.
canis canis (Beugnet & Marie, 2009). Effective control MATERIALS AND METHODS
of tick infestations on dogs is based on several stra-
tegies, including avoidance of infested environments,
environmental controls, and regular application of STUDY DESIGN
T
acaricides to the dog. Environmental control measures his study was a randomized, blinded, negative
are often complex and have varying success rates. Tick controlled comparative-efficacy study. All dogs
infested environments are not easily identifiable; there- were managed similarly and with due regard
fore avoidance is not always possible. Thus, regular for their well-being. All were handled in compliance
topical applications of acaricides or combination pro- with local and Merial Institutional Animal Care and
ducts (e.g. insecticide/acaricide) are often used to con- Use Committee approvals and in accordance with
trol external parasite infestations in domestic animals any applicable laws and regulations. Each dog was
(Brianti et al., 2010). Spot-on formulations of insec- individually housed throughout the study.
ticides and/or acaricidal drugs provide a convenient From a pool of 28 healthy dogs, 24 dogs demonstra-
method for external parasite control in dogs and cats. ting the highest tick counts following a prequalifica-
In this study, two topical insecticide/acaricide combi- tion infestation were selected for use in this study.
nation products with labelled activity against ticks were Dogs were allocated to one of eight replicates of
chosen for comparison: the new topical combination three dogs each based on pre-treatment bodyweight,
fipronil/amitraz/(S)-methoprene in a dual chamber then the dogs within each replicate were randomly
pipette (fipronil 10 % w/v and (S)-methoprene 9 % allocated to one of three groups of eight dogs each.
w/v in chamber one and amitraz 20 % w/v in chamber Dogs used in this study hadn’t been treated with any
two; CERTIFECT® Spot-On Dog [registered trademark ectoparasiticides in the preceding three months. Dogs
of Merial. All other marks are the property of their were shampooed seven days prior to treatment with a
respective owners]) and imidacloprid/permethrin in non-insecticidal shampoo (Purl® Shampoo, registered
a single chamber pipette (imidacloprid 10 % w/v + trademark of Kryon laboratories, South Africa).
permethrin 50 % w/v; ADVANTIX® [Europe; Bayer Treatments were applied by weight, in accordance
AG] or K9 ADVANTIX® [United States; Bayer Animal with approved-label directions on Day 0. The dogs
Health]). were weighed to determine appropriate dosing. If
As a single entity, fipronil provides broad spectrum the weight of any dog did not fall exactly on a whole
of activity against insects (including fleas and lice) pound, their weight was rounded up to the next whole
and acari (including ticks and other mites). The new pound. Dogs weighed between 27 and 53 lbs (12.2
combination with amitraz was formulated and deve- to 24 kg).
loped to significantly increase the speed of kill of Dogs in Treatment Group 1 remained untreated.
fipronil on ticks, due to a potentiation effect provided
Dogs in Treatment Group 2 (Table I) were treated with
by amitraz (Pfister et al., 2011; Prullage et al., 2011).
the appropriate size of CERTIFECT® Spot-On for dogs
This new spot-on combination has been registered
(in total volume applied on dogs: fipronil 6.26 % w/v,
in 2011 in both the USA and Europe. Imidacloprid is
amitraz 7.48 % w/v and (S)-methoprene 5.63 % w/v).
an insecticide with no labelled activity against acari;
The total volume was applied on the dog’s midline; half
thus, a combination product including permethrin
midway up the neck, half between the shoulder blades.
was developed to create a product with a broadened
range of efficacy. It is commercialized in the USA and Dogs in Treatment Group 3 (Table I) were treated with
in European countries since a few years. the appropriate size pipette of ADVANTIX® spot-on
for dogs (imidacloprid 10 % w/v + permethrin 50 %
Tick efficacy guidelines for approval in both EU and
w/v). For treatment administration, the total volume
USA typically require efficacy ≥ 90 % at 48-hour
was applied according to package instructions by
counts (Marchiondo et al., 2007). The purpose of the
parting the hair and applying directly on the skin in
study was to assess the ability of each product to
three spots along the midline from the top back of the
disrupt attachment of ticks and to assess the sustained
shoulder blades to the base of the tail.
speed of kill activity at 24-hour counts. Both rapid kill
and disruption of attachment of newly acquired ticks The D. reticulatus ticks used in this study were a labo-
would lower the numbers of engorged ticks seen, and ratory strain, obtained from the ClinVet colony, free
potentially reduce the risk of transmission of many from known tick-borne pathogens and from a strain
tick-borne pathogens. While rapid activity is desirable, with no known resistance to any ectoparasiticide.
it is equally important that rapid activity is sustained Dogs were infested with 50 D. reticulatus (50:50 sex
throughout the treatment interval. This study assessed ratio), for pre-treatment (Day -3) and for each subse-
the 24-hour activity against weekly infestations of quent infestation (Days 1, 7, 14, 21, 28 and 35). Each
D. reticulatus ticks, through Day 36. dog was sedated, and the ticks were gently deposited
Parasite, 2011, 18, 319-323

320 Research note
48

EFFICACY OF TOPICAL COMBINATIONS AT 24 HOURS AGAINST D. RETICULATUS
Treatment Bodyweight range – lbs (kg) Pipette size Pipette volumes – mL

Certifect® Spot-On for dogs 23-44 (10-20) M 2.14
45-88 (20-40) L 4.28
Advantix® spot-on for dogs 21-55 (10-25) Red/55 2.5
Table I. – Dosage of dogs in the treated groups.
Category General findings Attachment status Interpretation for prevention Interpretation for killing effect
of attachment (acaricidal)
1 Live Free Demonstrated Not demonstrated
2 Live Attached; unengorged Not demonstrated Not demonstrated
3 Live Attached; engorged1 Not demonstrated Not demonstrated
4 Dead Free Demonstrated Demonstrated
5 Dead Attached; unengorged Not demonstrated Demonstrated
6 Dead Attached; engorged1 Not demonstrated Not demonstrated
Adapted from Marchiondo et al., 2007.

1
Engorged tick: a tick with a conspicuous enlargement of the alloscutum that has blood in its digestive tract, as shown by squeezing/
crushing of the tick on white paper.
Table II. – Categorization of ticks for counting and interpretation.
on the lateral aspect of the dog’s chest. Any remaining For both analysis, percent reduction from the negative
ticks were counted 24 hours after each infestation. control group (Treatment Group 1) mean was calcu-
All personnel involved with evaluation of efficacy lated for Treatment Groups 2 and 3, if applicable, at
were unaware of treatment status of dogs in the study. every post-treatment time point using the formula
Personnel with access to the treatment assignments [(C - T) / C] × 100, where C is the geometric mean
were identified prior to the first treatment being admi- for the negative control group and T is the geometric
nistered, and blinding was maintained throughout the mean for Treatment Group 2 or 3. Treatment Group 2
study. was compared to each of the other treatment groups
(Treatment Groups 1 and 3) using Analysis of Variance
SPECIFICATION OF STUDY VARIABLES on log count. All testing was two-sided at the signifi-
cance level α = 0.05.
The ticks were categorized according to Table II. Cate-
gorization of the ticks allow calculation of the percent
efficacy (killing effect), as well as the calculation of
the attachment rate, in comparison with the untreated RESULTS
control group.
DISRUPTION OF ATTACHMENT
DATA
T
ANALYSIS
he disruption of attachment is based on the
To measure disruption of attachment rates, total counts counts of all attached ticks, live or dead, that
of ticks in categories 2, 3, 5, and 6 (Table II), i.e. all are present at assessment, which in this study
“attached” categories (live or dead), were transformed occurred at the 24-hour count (Table III, Fig. 1) (Prullage
to the natural logarithm of (count + 1) for calculation of et al., 2011). The percentage of attachment at 24 hours
geometric means by treatment group at each time point. was significantly higher (p < 0.05) in the control group
To measure the killing effect (% acaricidal efficacy), than in the treated groups at all time points. The rate
the total counts of adult ticks in categories 1 through of attachment was significantly lower (p < 0.05) in the
3 and 6 were transformed to the natural logarithm CERTIFECT®-treated group than the ADVANTIX®-treated
of (count + 1) for calculation of geometric means by group throughout the entire study. In the CERTIFECT®-
treatment group at each time point. The ticks in the treated group, 92.3 % to 98.5 % of ticks were prevented
three “Live” categories, as well as in the “Dead, atta- from attachment compared to the untreated group until
ched, engorged” category, were interpreted as treat- Day 29. In the ADVANTIX®-treated group 54.7 % to
ment failures. Their counts were combined and the 78.4 % of ticks were prevented from attachment com-
total for each dog was used in the subsequent analysis. pared to the untreated group until Day 29 (Table III).
Parasite, 2011, 18, 319-323

Research note 321
49

FOURIE J.J., BEUGNET F., OLLAGNIER C. & POLLMEIER M.G.
Categories 2, 3, 5 and 6 Categories 1, 2, 3 and 6

Control Certifect® Advantix® Control Certifect® Advantix®
Day Cnt.1 Cnt. %2 Cnt. % Cnt. Cnt. % Cnt. %
2 23.46 1.28 94.6 8.26 64.8* 23.03 0.19 99.2 6.50 71.8*
8 24.75 1.21 95.1 5.34 78.4 23.42 0.00 100.0 3.11 86.7*
15 30.60 0.45 98.5 9.96 67.5* 30.49 0.22 99.3 8.93 70.7*
22 33.43 2.58 92.3 11.30 66.2* 33.18 1.59 95.2 8.90 73.2*
29 33.56 2.02 94.0 15.19 54.7* 33.43 1.65 95.1 14.63 56.2*
36 29.37 6.30 78.6 20.92 29.3* 29.46 6.55 77.8 21.56 27.3*
1
Cnt. = geometric mean tick count.
2 % = percent disruption of attachment at 24 hours for categories 2, 3, 5 and 6 or percent killing efficacy at 24-hour counts for categories
1, 2, 3 and 6.
* Significant difference between the two treatment groups at p < 0.05.
Table III. – Geometric means of Dermacentor reticulatus ticks counts by category at 24-hour post-infestation and percent of disruption of
attachment and percent killing efficacy at 24-hour counts.
100
90
80
Disruption of attachment (%)
70
Certifect®
60
Advantix®
50
40 * Significant difference
between the two groups at p < 0.05
30
20
10
0
2* 8* 15* 22* 29* 36*
Day
Fig 1. – Disruption of attachment of Dermacentor reticulatus at 24-hour post-infestation.
100
90
80
Acaricidal efficacy (%)
70
Certifect®
60
Advantix®
50
* Significant difference
40
between the two groups at p < 0.05
30
20
10
0
2* 8 15* 22* 29* 36*
Day
Fig 2. – Comparative acaricidal efficacy at 24-hour tick counts.
Parasite, 2011, 18, 319-323

322 Research note
50

EFFICACY OF TOPICAL COMBINATIONS AT 24 HOURS AGAINST D. RETICULATUS
SPEED OF KILL of companion animals in Europe. Veterinary Parasitology,

2009, 163, 298-305.
The killing efficacy is shown on Table III and Fig. 2. BRIANTI E., PENNISI M.G., BRUCATO G., RISITANO A.L., GAGLIO
The fipronil/amitraz/(S)-methoprene efficacy at 24- G., LOMBARDO G., MALARA D., FOGLIAZZA A. & GIANNETTO
hour counts was above 95 % through the four weeks S. Efficacy of the fipronil 10 % + (S)-methoprene 9 %
following treatment, decreasing to 77.8 % following combination against Rhipicephalus sanguineus in natu-
the Day 35 challenge. The imidacloprid/permethrin rally infested dogs: speed of kill, persistant efficacy on
24 hours efficacy did not reach 90 % efficacy at any immature and adult stages and effect of water. Veterinary
assessment throughout the study, with killing efficacy Parasitology, 2010, 170, 96-103.
through the four weeks ranging from 56.2 % to 86.7 % DOYLE V., BEUGNET F. & CARITHERS D. Comparative efficacy
at 24-hour counts. The difference between the two of the combination fipronil-(S)-methoprene and the com-
treatment groups was significant at all time points bination permethrin-imidacloprid against Dermacentor
reticulatus, the European dog tick, applied topically to
including Day 36.
dogs. Veterinary Therapeutics, 2005, 6 (4), 303-310.
DRYDEN M.W., PAYNE P.A., SMITH V. & HOSTETLER J. Evaluation
of an imidacloprid (8.8 % w/w)-permethrin (44 % w/w)
DISCUSSION topical spot-on and a fipronil (9.8 % w/w)-(S)-methoprene
(8.8 %w/w) topical spot on to repel, prevent attachment,
T he tick counts in this study were conducted at and kill adult Rhipicephalus sanguineus and Dermacentor
variabilis ticks on dogs. Veterinary Therapeutics, 2006,
24 hours following each challenge, rather than
7, 187-198.
48 hours assessments. Previous studies have
DRYDEN M.W., PAYNE P.A., MCBRIDE A., MAILEN S., SMITH V.
demonstrated that the efficacy of the imidacloprid/
& CARITHERS D. Efficcay of fipronil (9.8 % w/w) + (S)-
permethrin combination at 48 hours is typically above methoprene (8.8 % w/w) and imidacloprid (8.8 % w/w)
90 % for three to four weeks or more, dependant upon + permethrin (44 % w/w) against Dermacentor variabilis
the tick species studied (Doyle et al., 2005; Dryden et (American dog tick) on dogs. Veterinary Therapeutics,
al., 2008; Tielemans et al., 2010). Another study per- 2008, 9 (1), 15-25.
formed against two tick species reported a rapid, but MARCHIONDO A.A., HOLDSWORTH P.A., GREEN P., BLAGBURN B.L.
short-term activity of imidacloprid/permethrin causing & JACOBS D.E. World Association for the Advancement of
a significant number of one of the tick species to Veterinary Parasitology (WAVVP) guidelines for evaluating
remain unattached and repelled following infestation the efficacy of parasiticides for the treatment, prevention
(Dryden et al., 2006). The quicker a product acts, the and control of flea and tick infestation on dogs and cats.
lower the probability that a tick will initiate its blood Veterinary Parasitology, 2007, 145, 332-344.
feeding and transmit pathogens. Short-term rapid NICHOLSON W.L., ALLEN K.E., MCQUISTON J.H., BREITSCHWERDT
activity is important, but pets can be exposed to ticks E.B. & LITTLE S.E. The increasing recognition of rickettsial
pathogens in dogs and people. Trends in Parasitology,
throughout the treatment interval, not just in the days
2010, 26, 205-212.
immediately following treatment. Therefore, the dura-
PFISTER K. Fipronil, amitraz and (S)-methoprene – a novel
tion of that rapid activity is key. By 24 hours after each
ectoparasiticide combination for dogs. Veterinary Special
infestation the fipronil/amitraz/(S)-methoprene combi- Edition, 2011, 179 (4), 291-353.
nation showed disruption of attachment > 90 % for a
PRULLAGE J., HAIR J., EVERETT W., YOON S.Y., CRAMER L., FRANKE
full month and efficacy > 95 % against D. reticulatus
S., CORNELISON M. & HUNTER J. The prevention of attach-
for a full month, demonstrating both a rapid and most ment and the detachment effects of a novel combination
importantly a consistent performance throughout the of fipronil, amitraz and (S)-methoprene for Rhipicephalus
treatment period. Such consistency would be impor- sanguineus and Dermacentor variabilis on dogs. Veteri-
tant for pet owners, who do not want to see engorged nary Special Edition, 2011, 179 (4), 302-310.
ticks on their dogs. Moreover, it is expected that the PRULLAGE J.B., CAWTHORNE W.G., LE HIR DE FALLOIS L.P. &
disruption of attachment and efficacy provided by 24 TIMMONS P.R. Synergy between fipronil and amitraz in a
hours would reduce the risk of transmission of many Rhipicephalus sanguineus tick residual contact test. Expe-
pathogens transmitted by ticks. rimental & Applied Acarology, 2011, 54, 173-176.
TIELEMANS E., MANAVELLA C., POLLMEIER M., CHESTER T., MURPHY
M. & GALE B. Comparative acaricidal efficacy of the topi-
cally applied combinations fipronil/(S)-methoprene, per-
REFERENCES methrin/imidacloprid and metaflumizone/amitraz against
Dermacentor reticulatus, the Eurpean dog tick (Ornate
BERRADA Z.L. & TELFORD III S.R. Burden of tick-borne infec- dog tick, Fabricius, 1794) in dogs. Parasite, 2010, 17,
tions on American companion animals. Topics in Compa- 343-348.
nion Animal Medicine, 2009, 24, 175-181. Received on July 1st, 2011
BEUGNET F. & MARIE JL. Emerging arthropod borne diseases Accepted on July 13th, 2011
Parasite, 2011, 18, 319-323

Research note 323
51

52

2.3. Horak IG, Fourie JJ, Stanneck D.

Efficacy of slow-release collar formulations of imidacloprid/flumethrin
and deltamethrin and of spot-on formulations of fipronil/(s) -methopre-
ne, dinotefuran/pyriproxyfen/permethrin and (s) -methoprene/amitraz/
fipronil against Rhipicephalus sanguineus and Ctenocephalides felis felis
on dogs.
Parasit Vectors. 2012, 22 : 5:79.
53

54

Horak et al. Parasites & Vectors 2012, 5:79
http://www.parasitesandvectors.com/content/5/1/79
RESEARCH Open Access
Efficacy of slow-release collar formulations of

imidacloprid/flumethrin and deltamethrin and of
spot-on formulations of fipronil/(s) - methoprene,
dinotefuran/pyriproxyfen/permethrin and
(s) –methoprene/amitraz/fipronil against
Rhipicephalus sanguineus and Ctenocephalides felis
felis on dogs
Ivan G Horak1,2*, Josephus J Fourie3 and Dorothee Stanneck4
Abstract
Background: Two studies evaluating the efficacy of an imidacloprid/flumethrin collar (SerestoW, Bayer Animal Health,
IVP), a deltamethrin collar (ScaliborW, MSD, CP1), a fipronil/(s)-methoprene spot-on (Frontline ComboW, Merial, CP2), a
dinotefuran/pyriproxyfen/permethrin spot-on (Vectra 3DW, Ceva, CP3) and an amitraz/fipronil/(s)-methoprene spot-on
(CertifectW, Merial, CP4/CP5) against repeated infestations with Rhipicephalus sanguineus and Ctenocephalides felis felis
on dogs were conducted over periods of 226 days and 71 days respectively.
Methods: The first study comprised 4 groups of treated dogs and one untreated control group, and the second 3
groups of treated dogs and one control group. Each group consisted of 8 dogs. All dogs were infested with ticks and
fleas at regular intervals. Ticks were counted 6 h, 18 h or 48 h after infestations and fleas 24 h after infestations.
Efficacies of the treatments were calculated by comparison with the untreated control groups using standard
descriptive statistics.
Results: The protective 48 h tick efficacy was 97.8% to 100% for the IVP (226 days), 69.3% to 97.4% for CP1 (170 days),
99.6% to 43.4% for CP2 (35 days) and 98% to 61.4% for CP3 (35 days).
The protective 18 h tick efficacy was 98% to 99.6% for the IVP (71 days), 100% to 86.5% for CP4 (29 days), 100% to
72.8% for CP4 after re-treatment (35 days) and 98.8% to 54.3% for CP5 (35 days).
The protective 6 h tick efficacy was 85.6% at Day 7 and 90.1% to 97.1% from Day 14 onwards for the IVP (70 days),
92.3% to 70.7% for CP4 (35 days), 97.5% to 65.2% for CP4 after re-treatment (35 days) and 95.1% to 51.8% for CP5
(35 days).
The protective 24 h flea efficacy was 99.5/90.9% to 100% for the IVP (71/226 days), 66.7% to 83% for CP1 (170 days),
100% to 88.5% for CP2 (35 days), 100% to 73.3% for CP3 (35 days), 100% to 98.7% for CP4 (35 days), 100% to 87.5% for
CP4 after re-treatment (35 days) and 100% to 79.5% for CP5 (35 days).
* Correspondence: ivan.horak@up.ac.za
1
Department of Zoology and Entomology, University of the Free State,
Bloemfontein 9301, South Africa
2
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science,
University of Pretoria, Onderstepoort 0110, South Africa
Full list of author information is available at the end of the article
© 2012 Horak et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
55

Horak et al. Parasites & Vectors 2012, 5:79 Page 2 of 14
Conclusions: These data suggest that the long-term efficacy provided by a medicated collar that is effective, is a
means to overcome the fluctuating efficacy of spot-on treatments resulting from a lack of pet owner re-treatment
compliance, and consequently protect animals successfully against ectoparasites and probably vector-borne
diseases.
Keywords: Fleas, Ticks, Efficacy, Imidacloprid, Flumethrin, Collar, Deltamethrin, Fipronil, Methoprene, Amitraz,
Dinotefuran, Spot-on
Background infestations of R. sanguineus and C. felis felis. For the

Ticks and fleas are parasites of dogs practically world- sake of clarity the methods applicable to both studies
wide. Although certain tick species are predominant on have been presented as an entity, and thereafter the
dogs in some regions, the brown dog tick, Rhipicephalus experimental designs and results of each study are
sanguineus is present on these animals in several regions presented separately.
and predominant in others [1-3]. On the other hand the
cat flea, Ctenocephalides felis felis is widespread through- Methods: general
out most regions, and infests both dogs and cats [4,5]. The studies were conducted in South Africa and were
The effective control of these ectoparasites not only alle- parallel group-designed, randomised, unicentre, con-
viates the immediate distress caused to their hosts, such trolled efficacy studies and were performed on groups of
as itching, skin lesions and blood loss, but may also pre- eight dogs each. The dogs enrolled in the studies were
vent the direct effects of infestation such as tick-induced sub-adult and adult male and female mixed-breeds
paralysis and flea allergy dermatitis [6]. Furthermore, a weighing between 9.26 and 25.30 kg. They were main-
reduction in parasite numbers will inevitably have an tained and handled with due regard for their welfare,
effect on the prevalence of the diseases that they transmit. and were acclimatized to the kennel environment seven
It is also possible that chemicals that have a repellent, or to ten days prior to the commencement of the studies.
a particularly rapid killing effect, could eliminate ticks The dogs were individually housed in pens in animal
and fleas before they can transmit vector-borne organ- units that conformed to the South African National
isms with which they may be infected. For instance R. Standards (SANS 10386:2008 The care and use of
sanguineus is a vector of Babesia canis, Babesia vogeli, animals for scientific purposes). Water was available
Babesia gibsoni, Hepatozoon canis and Ehrlichia canis, ad libitum and an adequate amount of a commercial
the causative organisms of tick-borne diseases that dog food towards their maintenance was provided
affect dogs in different regions of the world [7]. On the daily.
other hand C. felis felis is the intermediate host of A laboratory-bred strain of R. sanguineus, originating
the larval stage of the tapeworm Dipylidium caninum from France and subsequently maintained for at least
of dogs and the vector of the bacterium Rickettsia 3 years on rabbits in South Africa, was used for infest-
typhi, the organism responsible for murine typhus in ation of the dogs. Adult ticks used for infestation were
humans [8]. unfed, at least one week old, and of a balanced sex ratio
Several chemicals, or combinations of chemicals, with (50% female: 50% male). Each dog was infested with 50
acaricidal or insecticidal properties and which are appro- ticks on the days indicated in the respective experimental
priate and safe for treatment of domestic dogs and cats, designs.
have been formulated for application either orally, paren- A laboratory-bred strain of C. felis felis, originating
tally, topically or as medicated collars [9,10]. Depending from Hanover University, Germany, and maintained on
on the active ingredients of the chemicals or combina- cats in South Africa for at least 2 years prior to the stud-
tions of chemicals, they are effective against fleas or ies, was used for all infestations. Each dog was infested
ticks, or both [11,12], and may in addition also be suit- with approximately 100 unfed fleas of mixed sex on the
able for the treatment of lice and mites [13,14]. Some of days indicated in the respective experimental designs. At
these chemicals also have a persistent effect lasting for the time of infestation neither the ticks nor the fleas
several weeks after their initial application [12], while were placed near the collars, or on or near the site, or
others are effective for several months [15]. sites, where the spot-on treatments were or had been
The aims of the present investigation were to evaluate administered.
the efficacies of various remedies against ticks and fleas The time at which each animal was treated or at which
on dogs. To this end two studies were performed during it was infested with ticks was recorded. This was done to
which the efficacies of five chemicals or combinations ensure that counting and removal of ticks were as close
of chemicals were assessed against laboratory-induced as possible to the specified target times (6 h ± 30 min, or
56

18 h ± 1 h, or 48 h ± 2 h post infestation or treatment). in dogs and cats; EMEA/CVMP/005/2000- Rev.2: per-

During the in situ counts, ticks were counted but not cent efficacy for the in situ tick counts was calculated as
removed. Ticks were found by direct observation follow- follows:
ing parting of the hair coat and palpation over the whole
skin surface of the dog. The same procedure was fol- Efficacy ð%Þ ¼ 100xðmc –mt Þ=mc ; where
lowed for the removal tick counts, but in addition the
dogs were combed after the ticks had been removed to mc = arithmetic mean number of live ticks (categories
ensure that all ticks were found and counted. Ticks that 1-3) on dogs in the untreated control group at a specific
were removed were classified according to the para- time-point.
meters listed in Table 1. mt = arithmetic mean number of live ticks (categories
The time at which each animal was treated or at 1-3) on dogs in the treated groups at a specific time-
which it was infested with fleas was recorded. This point.
was done to ensure that counting was as close as Percent efficacy for the removal tick counts was calcu-
possible to the specified target times (48 ± 2 h post- lated as follows:
treatment or 24 ± 2 h post-infestation). During count-
ing a fine-toothed flea comb was used to recover Efficacy ð%Þ ¼ 100xðmc –mt Þ=mc ; where
fleas from the animal’s hair. Combing was performed
by several strokes of the comb over each part of the mc = arithmetic mean number of live ticks (categories
dog’s skin surface, each time in the same direction 1-3) on dogs in the untreated control group at a specific
and following the lie of the hair coat. Movement, time-point.
from one part of the animal’s body to the next, was mt = arithmetic mean number of live and dead ticks
via strokes overlapping each other, so that no part of (categories 1-3 and 6) on dogs in the treated groups at a
the skin surface was missed. After the completion of specific time-point.
combing, the whole procedure was repeated so that Percent efficacy against fleas was calculated as follows:
all areas were combed at least twice. If necessary a
third combing was performed until no live fleas were Efficacy ð%Þ ¼ 100xðmc –mt Þ=mc ; where
found.
The pre-treatment flea counts of each dog were used mc = arithmetic mean number of live fleas on dogs in
for ranking and group allocation purposes. The dogs the untreated control group
were ranked within gender, in descending order of indi- mt = arithmetic mean number of live fleas on dogs in
vidual pre-treatment flea counts. Animal IDs were used the treated groups.
to break ties. Within each gender, animals were then Descriptive statistics (mean, minimum, maximum,
blocked and within each block dogs were randomly allo- standard deviation, CV%, arithmetic mean and median)
cated to the various treatment groups. on tick and flea counts on the various assessment days
Efficacy against ticks and fleas was calculated for each were also performed. SAS Version 8 (Release 8.02 TS
treatment group at each assessment day according to the Level 02 M0) was used for all the statistical analyses.
formulas below. All efficacy calculations were performed
on the arithmetic means of the tick or flea counts. Methods: study 1
In accordance with the Guidelines for the testing and This study was designed to ascertain the immediate
evaluation of the efficacy of antiparasitic substances for and the long-term efficacy of a collar containing imi-
the treatment and prevention of tick and flea infestation dacloprid 10% and flumethrin 4.5% (w/w) (SerestoW,
Bayer Animal Health) and a collar containing delta-
methrin 4% (w/w) (ScaliborW, MSD) against R. sangui-
Table 1 Classification of ticks on their state of attachment
neus and C. felis felis on groups of experimentally
and engorgement and whether they are alive or dead
infested dogs, compared to an untreated control
Category Condition Attachment status
group of dogs infested with the same ectoparasites. In
1 Alive Unattached addition a group of dogs treated with a spot-on for-
2 Alive Attached; unengorged* mulation of fipronil 10% (w/v) and (s)-methoprene 9%
3 Alive Attached; engorged** (w/v) (Frontline ComboW, Merial), and another group
4 Dead Unattached treated with a spot-on formulation of dinotefuran
5 Dead Attached; unengorged*
4.95% (w/w), pyriproxyfen 0.44% (w/w) and permeth-
rin 36.08% (w/w) (Vectra 3DW, Ceva) were included
6 Dead Attached; engorged**
* no filling of the alloscutum evident.
in the study 160 days after its commencement. Each
** obvious or conspicuous filling of the alloscutum. of the treated groups and the control group consisted
57

of eight dogs. All dogs were infested on multiple Methods: study 2

occasions and examined for live parasites at predeter- This study was designed to evaluate the immediate
mined time intervals after infestation. and persistent “repellent” efficacies (measured at 6 h)
The medicated collars were fastened around the dogs’ and “fast killing” efficacies (measured at 18 h) of a
necks and adjusted until a comfortable fit was achieved. collar containing imidacloprid 10.5% (w/w) and flume-
Excess collar was pulled through the collars’ loops and thrin 4.5% (w/w) (SerestoW, Bayer Animal Health)
any excess length beyond 2 cm was cut off. The fipronil/ against R. sanguineus and C. felis felis on dogs over a
(s)-methoprene spot-on formulation was administered at period of 71 days. In addition it was so designed that
a weight specific dosage of 0.067 ml/kg body weight and the immediate and persistent efficacy of a spot-on for-
applied as a single spot between the shoulder blades. mulation of (s)-methoprene 5.8%/amitraz 7.6%/fipronil
The spot-on formulation of dinotefuran/pyriproxyfen/ 6.4% (w/v) (CertifectW, Merial) administered twice,
permethrin was administered at a dosage level of 35 days apart, could be determined, and also so that
0.14 ml/kg body weight. The calculated dose was applied the efficacy of a single treatment with the latter rem-
at three spots along the dog’s back, namely between the edy on a group of dogs introduced into the study
shoulder blades, in the middle of the back and at the 35 days after its commencement could be determined.
base of the tail. The choice of dosage level, dose regimen The imidacloprid/flumethrin collars were fastened
and of evaluation time-points will be dealt with more ex- around the dogs’ necks as previously described. The (s)-
tensively in the discussion. methoprene/amitraz/fipronil combination was applied
The experimental design of the study is summarized in topically on the skin of the neck between the base of the
Table 2. skull and the shoulder blades as two spots of
Table 2 Experimental design of an ectoparasiticidal efficacy study against Rhipicephalus sanguineus and
Ctenocephalides felis felis on dogs, Study 1
Study Day Activity
-6 24 dogs each infested with 100 fleas

-5 Fleas counted and flea numbers used for ranking and allocation to groups
-1 Allocation to control group of 8 dogs (Group 1) and two treated groups (Groups 2, 3)
0 Dogs infested with 100 fleas and 50 ticks immediately prior to collaring
2 48 h flea and tick counts*
7 50 ticks
8 100 fleas
9 24 h flea, 48 h tick counts
14 to 30 Infestation and counting at weekly intervals the same as for Days 7 to 9 above
56 50 ticks
57 100 fleas
84 to 142 Infestation and counting at 4-weekly intervals the same as for Days 56 to 58 above
160 Two fresh groups of 8 dogs each (Groups 4 and 5) added to the study
161 Infestation with fleas and ticks (Groups 1, 4 and 5), followed by spot-on treatment of dogs in groups 4 and 5.
163 Flea and tick counts 48 h after treatment on dogs in Groups 1, 4 and 5, thereafter dogs in all five groups subject to the same infestation
and counting regimen
168 50 ticks
169 100 fleas
170 End of label claim for Group 3, no further evaluation
175 to 198Infestation and counting at weekly intervals the same as for Days 168 to 170 above
199 End of label claim for Groups 4 and 5, no further evaluation
224 50 ticks
225 100 fleas
* ticks and fleas counted 48 h after application of the collars.
58

approximately equal volume at a dosage rate of Immediate efficacies were similar on the imidacloprid/
0.107 ml/kg body weight. Exact weight dependent flumethrin, deltamethrin, fipronil/(s)-methoprene and
dosages were chosen for the spot-on product to ensure dinotefuran/pyriproxyfen/permethrin treated groups of
appropriate comparability with the collar and this will be dogs (78.3%, 86.5% 89.1% and 79.9%). Thereafter the per-
addressed in greater detail in the discussion. sistent efficacies of the imidacloprid/flumethrin collar
The study was conducted in two phases. Phase 1 was exceeded 97.8% until the termination of the study on
carried out on 24 dogs allocated to three groups and the Day 226. Persistent, preventive acaricidal efficacies
dogs in these groups also participated in Phase 2 of the recorded for this group of dogs were also consistently
study. In Phase 2 eight fresh dogs were added to the higher than those recorded for the other treated groups
study 35 days after its commencement. The experimental of dogs. With the exception of Days 16, 23 and 114 after
design of the study is summarized in Table 3. infestation, when efficacies above 90% were recorded for
the deltamethrin treated group of dogs, persistent effica-
Results cies for this group remained below the 90% level. Persist-
Results: study 1 ent efficacies exceeding 90% lasted for four weeks on the
Acaricidal (48 h) efficacy against R. sanguineus dogs treated with fipronil/(s)-methoprene, and for three
The arithmetic mean tick counts of the untreated control weeks on the dogs treated with dinotefuran/pyriproxy-
group of dogs 48 h after infestation varied between 23.6 fen/permethrin whereafter efficacy decreased to 43.4%
and 38.4, thus ensuring a robust challenge with ticks on for the fipronil/(s)-methoprene treated group and 61.4%
all assessment days. The tick counts of the dogs in each for the dinotefuran/pyriproxyfen/permethrin treated
of the treatment groups are summarized in Table 4. group, five weeks after treatment.
The mean tick counts recorded for the four treated
groups of dogs differed significantly (p < 0.05) from Insecticidal (24 h) efficacy against C. felis felis
those of the untreated control group of dogs (Group 1) The arithmetic mean flea counts recorded for the un-
on all assessment days. The imidacloprid/flumethrin col- treated control group of dogs varied between 69.6 and
lared dogs (Group 2) harboured significantly (p < 0.05) 97.1, thus ensuring a robust flea challenge on all assess-
fewer ticks 48 h after infestation on Days 58, 86 and 170 ment days. The mean flea counts of the dogs 48 h after
than the group of deltamethrin collared dogs (Group 3), treatment and 24 h after each re-infestation are summar-
significantly (p < 0.05) fewer ticks on Day 198 than the ized in Table 5.
dogs treated with fipronil/(s) methoprene (Group 4), and The flea counts recorded for the four treated groups
significantly (p < 0.05) fewer ticks on Days 191 and 198 of dogs differed significantly (p < 0.05) from those of
than the dogs treated with dinotefuran/pyriproxyfen/ the untreated control group (Group 1) on all assess-
permethrin (Group 5). ment days. The imidacloprid/flumethrin collared
The immediate efficacy of the various remedies against group of dogs (Group 2) had significantly (p < 0.05)
ticks 48 h after treatment and persistent efficacy 48 h after fewer fleas 24 h post-infestation than the deltamethrin
each re-infestation are graphically illustrated in Figure 1. collared group of dogs (Group 3) on all assessment
Table 3 Experimental design of an ectoparasiticidal efficacy study against Rhipicephalus sanguineus and
Ctenocephalides felis felis on dogs, Study 2
Study day Activity
-5 to -3 Flea infestation, counting and allocation to 3 groups of 8 dogs (Groups 1, 2, 3)

0 Controls (Group 1) and treated dogs (Groups 2 and 3) infested with fleas and ticks, and treated directly thereafter (Groups 2 and 3)
2 48 h flea + tick counts
7 50 ticks, 100 fleas 6 h tick counts
8 18 h tick counts 24 h flea counts
14 to 29 Infestation and counting at weekly intervals the same as for Days 7 and 8 above
34 Fresh group of 8 dogs (Groups 4) added to study
35 Groups 1, 2 and 3 infested with fleas + ticks, Group 3 dogs re-treated and Group 4 dogs treated for the first time directly thereafter
6 h tick counts (Groups 1, 2, and 3)
36 18 h tick counts and 24 h flea counts (Groups 1, 2, and 3)
42 (all groups) 50 ticks, 100 fleas 6 h tick counts
43 18 h tick counts 24 h flea counts
49 to 71 Infestation and counting at weekly intervals the same as for Days 42 and 43 above
59

Table 4 Study 1: Mean numbers of ticks on treated dogs 48 h after treatment and 48 h after each re-infestation with
Rhipicephalus sanguineus
Day Mean numbers of ticks (48 h after re-infestation)
Untreated controls Imidacloprid/flumethrin Deltamethrin Fipronil/(s)-methoprene Dinotefuran/pyriproxyfen/
(Group 1) (Group 2) (Group 3) (Group 4) permethrin (Group 5)
2* 28.8 6.3 3.9 - -

9 36.0 0.3 4.3 - -
16 33.9 0.8 0.9 - -
23 38.4 0.0 2.8 - -
30 33.4 0.1 3.4 - -
58 26.5 0.1 8.1 - -
86 31.1 0.3 8.3 - -
114 29.1 0.4 2.3 - -
142 33.3 0.0 5.9 - -
163 29.9 - - 3.3* 6.0*
170 31.3 0.0 5.1 0.1 0.6
177 33.1 0.1 - 0.5 1.5
184 32.8 0.1 - 2.4 2.8
191 34.1 0.3 - 2.5 5.5
198 23.6 0.0 - 13.4 9.1
226 29.4 0.4 - - -
* Flea counts 48 h after treatment.
days except Day 9. No significant (p > 0.05) differ- The immediate efficacy of the various remedies against
ences between the arithmetic mean numbers of fleas fleas 48 h after treatment and persistent efficacy 24 h
on the imidacloprid/flumethrin collared, fipronil/(s)- after each re-infestation are graphically illustrated in
methoprene and dinotefuran/pyriproxyfen/permethrin Figure 2.
spot-on treated groups (Groups 4 and 5) were The immediate efficacy of the imidacloprid/flumethrin
observed. collar against C. felis felis 48 h after collaring was 99.8%,
100
90
80
70
Efficacy (%)
60
50
40
Rhipicephalus sanguineus (48 h after each infestation)
30
Imidacloprid/flumethrin
20 Deltamethrin
Fipronil/(s) methoprene
10 Dinotefuran/pyriproxyfen/permethrin
0
2 9 16 23 30 58 86 114 142 163 170 177 184 191 198 226
Study Day
Figure 1 Study 1. Efficacy of imidacloprid/flumeththrin and deltamethrin collars, and fipronil/(s) methoprene and dinotefuran/pyriproxyfen/
permethrin spot-on formulations against Rhipicephalus sanguineus on dogs 48 h after treatment and 48 h after each re-infestation.
60

Table 5 Study 1: Mean numbers of fleas on treated dogs 48 h after treatment and 24 h after each re-infestation with
Ctenocephalides felis felis
Day Mean numbers of fleas (24 h after re-infestation)
Untreated controls Imidacloprid/flumethrin Deltamethrin Fipronil/(s)-methoprene Dinotefuran/pyriproxyfen/
(Group 1) (Group 2) (Group 3) (Group 4) permethrin (Group 5)
2* 70.4 0.1 21.4 - -

9 70.1 0.0 15.3 - -
16 78.8 0.0 21.1 - -
23 80.0 0.0 14.3 - -
30 83.8 0.0 17.9 - -
58 69.6 0.0 13.9 - -
86 72.0 0.0 19.5 - -
114 72.5 0.3 24.1 - -
142 72.3 0.4 16.4 - -
163 72.5 - - 0.0* 0.0*
170 78.0 2.8 13.3 0.0 0.0
177 88.8 6.0 - 0.0 2.0
184 89.1 2.3 - 0.0 1.1
191 91.4 5.0 - 1.4 13.3
198 97.1 8.9 - 11.1 25.5
226 82.6 5.9 -
* Flea counts 48 h after treatment.
and with the exception of Day 177 when an efficacy of 48 h after collaring, was 69.6%, and persistent efficacies,
93.2% was recorded, persistent efficacies, assessed 24 h assessed 24 h after each re-infestation, varied between a
after each re-infestation, varied between 94.5% and 100% low of 66.7% on Day 114 and a high of 83.0% on Day
until Day 191, decreasing thereafter to 90.9% and 92.9% 170. The spot-on treatments with fipronil/(s) metho-
on Days 198 and 226 respectively. The immediate effi- prene and dinotefuran/pyriproxyfen/permethrin resulted
cacy of the deltamethrin collars against fleas, assessed in immediate efficacies of 100%. Persistent efficacies
100
90
80
70
60
Efficacy (%)
50
Ctenocephalides felis felis (24 h after each infestation)
40
30
Deltamethrin
Fipronil/(s) methoprene
20
Dinotefuran/pyriproxyfen/permethrin
10
0
2 9 16 23 30 58 86 114 142 163 170 177 184 191 198 226
Study Day
Figure 2 Study 1. Efficacy of imidacloprid/flumeththrin and deltamethrin collars, and fipronil/(s) methoprene and dinotefuran/pyriproxyfen/
permethrin spot-on formulations against Ctenocephalides felis felis on dogs 48 h after treatment and 24 h after each re-infestation.
61

greater than 95% were recorded for four weeks on the flumethrin collars assessed 18 h post-infestation was ≥ 98%
fipronil/(s) methoprene treated group of dogs and three for the duration of the study, whereas persistent efficacy
weeks on the dinotefuran/pyriproxyfen/permethrin on the dogs treated twice with (s)-methoprene/amitraz/
group of dogs, thereafter efficacy decreased to 88.5% for fipronil exceeded 90% on Days 8, 15 and 22 after the first
the fipronil/(s)-methoprene treated group and 73.7% for treatment and on the same days after the second treat-
the dinotefuran/pyriproxyfen/permethrin treated group, ment. Persistent tick efficacy on the group of dogs treated
five weeks post treatment. with (s)-methoprene/amitraz/fipronil for the first time on
Day 35 exceeded 90% in the first two weeks after treat-
Results: study 2 ment (Days 43 and 50).
“Fast killing” acaricidal (18 h) efficacy against R. sanguineus
The arithmetic mean numbers of ticks counted on dogs “Repellent” or acaricidal (6 h) efficacy against R. sanguineus
in the untreated control group 18 h after infestation var- The arithmetic mean tick counts recorded on the dogs
ied between 20.5 and 36.9, thus ensuring a robust tick in the untreated control group 6 h after infestation var-
challenge on all assessment days. The tick counts of the ied between 27.8 and 36.9, thus ensuring a robust tick
dogs in each of the treatment groups are summarized in challenge on all assessment days. The mean tick counts
Table 6. of the dogs in the different treatment groups are sum-
The mean tick counts recorded 18 h after infestation marized in Table 7.
for all treated groups of dogs (Groups 2, 3 and 4) dif- The tick counts of the dogs in each of the treated
fered significantly (p < 0.05) from those of the untreated groups 6 h after infestation differed significantly (p
control group (Group 1) on all assessment days. The < 0.05) from those of the untreated control group on all
mean tick counts recorded for the imidacloprid/ assessment days. The tick counts of the dogs in the imi-
flumethrin treated group were significantly (p < 0.05) dacloprid/flumethrin treated group were significantly (p
lower on Days 29, 64 and 71 than those of the group < 0.05) lower on Days 28, 56, 67 and 70 than those of
treated twice with (s)-methoprene/amitraz/fipronil, and the dogs treated twice with (s)-methoprene/amitraz/
also significantly lower on Days 57, 64 and 71 than those fipronil, and also significantly (p < 0.05) lower on Days
of the group of dogs treated for the first time on Day 35. 49, 56, 63 and 70 than those of the dogs in the group
The immediate efficacy of the various remedies against treated for the first time on Day 35.
ticks 48 h after treatment and persistent efficacy 18 h The efficacies of the various treatments against ticks
after each re-infestation are graphically illustrated in 6 h after each infestation are graphically illustrated in
Figure 3. Figure 4.
The immediate efficacy of (s)-methoprene/amitraz/ Except for Days 7 and 42, the persistent efficacies
fipronil against ticks was markedly superior to that against R. sanguineus recorded 6 h after infestation
recorded for the imidacloprid/flumethrin collars. However, on the group of dogs fitted with imidacloprid/flume-
persistent fast-killing efficacies for the imidacloprid/ thrin collars were consistently higher than those of
Table 6 Study 2: Mean numbers of ticks on treated dogs 48 h after treatment and 18 h after each re-infestation with
Rhipicephalus sanguineus
Untreated controls Imidacloprid/flumethrin (S)-methoprene/amitraz/fipronil (S)-methoprene/amitraz/fipronil
(Group 1) (Group 2) (Group 3)re-treatment: Day 35 (Group 4)
2* 20.5 4.5 1.6 -

8 25.4 0.3 0.0 -
15 30.9 0.1 0.6 -
22 31.5 0.1 1.9 -
29 34.4 0.3 4.6 -
36 34.9 0.4 0.5** -
43 32.1 0.4 0.0 0.4
50 33.6 0.1 1.0 2.3
57 35.8 0.4 2.5 3.9
64 36.9 0.8 5.8 12.0
71 33.1 0.1 9.0 15.1
* Tick counts 48 h after treatment.
** Tick counts 18 h after re-treatment.
62

100
90
80
70
Efficacy (%)
60
50
40
30
20 (S)-methoprene/amitraz/fipronil Days 0 and 35
10 (S)-methoprene/amitraz/fipronil Day 35
0
2 8 15 22 29 36 43 50 57 64 71
Study Day
Figure 3 Study 2. Efficacy of an imidacloprid/flumeththrin collar and a (s) –methoprene/amitraz/fipronil spot-on formulation against
Rhipicephalus sanguineus on dogs 48 h after treatment and 18 h after each re-infestation.
the groups of dogs treated twice or once with (s)- collars (Group 2) and treated twice with the metho-
methoprene/amitraz/fipronil. prene/amitraz/fipronil formulation (Group 3) were
observed. The mean flea counts on Day 71 of dogs in the
Insecticidal (24 h) efficacy against C. felis felis group fitted with imidacloprid/flumethrin collars were
The arithmetic mean flea counts of the untreated control significantly (p < 0.05) lower than those of the dogs trea-
group of dogs varied between 69.3 and 85.4, thus ensur- ted for the first time on Day 35 with (s)-methoprene/
ing a robust challenge with fleas on all assessment days. amitraz/fipronil (Group 4).
The mean flea counts of the dogs in each of the treat- Efficacy values derived from the 24 h flea counts are
ment groups are summarized in Table 8. graphically illustrated in Figure 5.
The flea counts of the dogs in the treated groups dif- Immediate efficacies against fleas recorded for both
fered significantly (p < 0.05) from those of the untreated treatment groups on Day 2 were similar. Persistent
control group of dogs (Group 1) on all assessment days. efficacies assessed 24 h after re-infestation exceeded
No significant differences (p > 0.05) in the mean flea 98% in all treated groups up to Day 57. However, ef-
counts of dogs treated with the imidacloprid/flumethrin ficacy in the group of dogs treated twice with (s)-
Table 7 Study 2: Mean numbers of ticks on treated dogs 6 h after each infestation with Rhipicephalus sanguineus
(Group 1) (Group 2) (Group 3)re-treatment: Day 35 (Group 4)
7 27.8 4.0 2.1 -

14 32.9 2.3 5.8 -
21 32.4 1.8 7.4 -
28 34.1 1.4 10.0 -
35 36.9 1.1 4.0* -
42 30.4 0.9 0.8 1.5
49 34.6 1.9 4.4 9.3
56 35.3 3.5 8.1 14.8
63 36.9 2.5 10.4 12.8
70 35.3 1.4 12.3 17.0
* Tick count 6 h after re-treatment.
63

100
90
80
70
60
Efficacy (%)
50
40
30 Imidacloprid/flumethrin
(S)-methoprene/amitraz/fipronil Day 0 and Day 35

20
(S)-methoprene/amitraz/fipronil Day 35
10
0
7 14 21 28 35 42 49 56 63 70
Study Day
Rhipicephalus sanguineus on dogs 6 h after each infestation.
methoprene/amitraz/fipronil declined from 98.6% on Discussion

Day 64 (4 weeks after re-treatment) to 87.5% a week Discussion of study design
later. Efficacy in the group of dogs treated for the When conducting a study comparing the long-term acar-
first time on Day 35 declined to 91.9% by Day 64 icidal efficacy of collars against that of the medium-term
(4 weeks after treatment) and 79.5% a week later. efficacy of spot-on products, a number of difficulties
Persistent efficacy in the imidacloprid/flumethrin arise. These are related to the nature of the remedies,
treated group exceeded 99% for the duration of the the choice of treatment, the evaluation time-points and
study. the choice of the dose regimen.
Table 8 Study 2: Mean numbers of fleas on treated dogs 48 h after treatment and 24 h after each re-infestation with
Ctenocephalides felis felis
Day Mean numbers of fleas (24 h after re-infestation)
(Group 1) (Group 2) (Group 3)retreatment: Day 35 (Group 4)
2* 75.3 0.3 0.5 -

8 69.3 0.0 0.0 -
15 83.4 0.0 0.1 -
22 80.9 0.0 0.5 -
29 85.4 0.0 1.1 -
36 73.9 0.4 0.0** -
43 75.0 0.0 0.0 0.0
50 74.1 0.0 0.1 0.0
57 75.8 0.1 0.8 0.0
64 72.5 0.0 1.0 5.9
71 85.3 0.4 10.6 17.5
* flea counts 48 h after treatment.
** flea counts 24 h after re-treatment.
64

100
90
80
70
Ctenocephalides felis felis (24 h after each infestation)
60
Efficacy (%)
50
40 Imidacloprid/flumethrin
(S)-methoprene/amitraz/fipronil Days 0 and 35

30
(S)-methoprene/amitraz/fipronil Day 35
20
10
0
2 8 15 22 29 36 43 50 57 64 71
Study Day
Ctenocephalides felis felis on dogs 48 h after treatment and 24 h after each re-infestation.
General arrangement of treatment and comparison 4-week period of efficacy. This was done to assess the
time-points The aim of the study was to give an ex- persistent effectiveness of the spot-on treatments should
perimentally-based assessment of the performance a pet owner decide to extend the period between treat-
over time of a variety of remedies that are available ments by a week because of a reliance on the assumed
for the treatment and control of ticks and fleas on slow decrease in efficacy of these remedies. The latter
dogs. Because of the difficulties listed above, a practice can, however, result in unwanted gaps in
straightforward, direct comparison embracing all treat- efficacy.
ments and starting on Day 0 would not result in an The choice of treatment time-points for Study 2 fol-
ideal outcome. However, as both the imidacloprid/ lowed a similar rationale. The (s)-methoprene/amitraz/
flumethrin and the deltamethrin collars claim long- fipronil spot-on and the imidacloprid/flumethrin collar
term efficacy of 8 months and 6 months respectively, were directly compared from Day 0 and onwards. Then
the decision was taken to start simultaneous treat- on Day 35, subsequent to the claimed period of efficacy
ment with them on Day 0 of Study 1. In addition, of the spot-on formulation, the same group of dogs was
because the imidacloprid/flumethrin collar claims effi- re-treated with the same remedy. This was done to de-
cacy lasting 8 months it would only be fair to com- termine whether regular re-treatment would enhance the
pare its efficacy towards the end of this period of performance of the spot-on product, and also so that the
time with that of freshly applied spot-on treatments effectiveness of multiple treatments with the spot-on for-
that claim efficacy of four weeks. Consequently treatment mulation could be compared with a single application of
with the two spot-on products included in Study 1 was the collar formulation.
only administered on Day 161 (5 months + 11 days into The efficacy claim for the (s)-methoprene/amitraz/
the study). This allowed for a comparison of efficacy be- fipronil spot-on is 4 weeks, however, its efficacy against
tween the imidacloprid/flumethrin collar towards the end ticks and fleas is said to be 5 weeks according to the pro-
of its claimed period of efficacy and those of the two spot- duct’s SPC and package leaflet, namely one treatment
on treatments applied during this time. prevents further infestation for 5 weeks by ticks and for up
The investigative periods for the imidacloprid/ to 5 weeks by fleas. On Day 35 a second (s)-methoprene/
flumethrin and the deltamethrin collars comprised the amitraz/fipronil spot-on treated group of dogs was en-
length of time claimed for their respective efficacies. In rolled. This was done so that the efficacy of this spot-on
contrast the investigative periods for the spot-on pro- treatment could be compared with that of the collar
ducts was increased by one week beyond their claimed that had already been in place for 35 days and also
65

with that of the second treatment with the spot-on time as the collars, or 5 weeks or 5½ months after the
administered to the dogs that had been treated 35 days collars had been applied. Furthermore, while the effica-
previously. If re-treatment enhanced the efficacy of cies of the spot-on formulations generally decreased dur-
the spot-on formulation, a series of regular re- ing the successive weeks following their application,
treatments would be a more appropriate comparison those of the imidacloprid/flumethrin collars persisted at
with the long-term efficacy of the collars. the same high level. The decrease in efficacy of the spot-
on treatments five weeks after their application indicates
Infestation and evaluation time-points The infest- that re-treatment subsequent to their 4-week claims of
ation and evaluation time-points in Study 1 were effectiveness is advisable. A second treatment with the
chosen in accordance with the products’ label claims (s)-methoprene/amitraz/fipronil spot-on formulation did
and the Guidelines for the testing and evaluation of not enhance or prolong its efficacy during the ensuing
the efficacy of antiparasitic substances for the treat- evaluation period.
ment and prevention of tick and flea infestation in Although the immediate acaricidal efficacy of the imi-
dogs and cats; EMEA/CVMP/005/2000- Rev.2 and dacloprid/flumethrin collars against R. sanguineus at the
current regulatory practice regarding efficacy evalu- start of the study (Day 2) was below 90% in the two
ation, namely flea counts after 24 h and tick counts studies reported here, it equalled or exceeded 78%. With
after 48 h. Because the efficacies against ticks of the the exception of these results, the persistent acaricidal
remedies under evaluation in Study 2 were expected preventative efficacy of these collars either significantly
to be similar at the 48 h assessment time-points, the or noticeably exceeded that of the deltamethrin collars at
choice of the tick counting time-point was based on each assessment time-point. On most occasions persist-
data already published on the efficacy of the (s)- ent efficacy also exceeded those of the spot-on formula-
methoprene/amitraz/fipronil spot-on [16], namely tions of fipronil/(s)-methoprene and dinotefuran/
“rapid killing” (18 h post-infestation) or “repellence” pyriproxyfen/permethrin, as well as those measured 18 h
(6 h post-infestation). after infestation, for the (s)-methoprene/amitraz/fipronil
spot-on formulation.
Dosage Exact weight dependent dosages were chosen Unexpectedly, considerable variation in the acaricidal
for the spot-on remedies as this neutralizes the influence effectiveness of the deltamethrin collar between individ-
of different animal sizes and body weights in a relatively ual dogs was observed. This variability contributed to the
limited sample size, thus making an equitable compari- overall lower than anticipated efficacy of this collar com-
son between spot-on remedies possible. In spot-on for- pared to published results on its effectiveness 48 h after
mulations the total amount of active ingredient, or re-infestation [18]. In contrast there appeared to be no
ingredients, are immediately released on the treated ani- marked variability in the efficacy of the imidacloprid/
mal’s skin. This is not so with collar formulations, which flumethrin collars in the present and other studies
rely on the slow-release of active ingredients. Conse- [19,20].
quently a weight range dosage level was chosen for the Repellent effects are a general property of pyrethroids.
collars, which were carefully adjusted to fit each dog’s Their in-contact efficacy comprises a mixture of a very
neck circumference, and any excess length cut off. The rapid lethal effect, a knock down effect and the so-called
release of active ingredients from the collar is, at least in “hot-foot effect”, instead of a vapour-based classical
the case of the imidacloprid/flumethrin collar, a “release repellent effect. Besides the well-described difference of
on demand” and is therefore nearly directly animal- specific ectoparasiticidal potency amongst different pyre-
surface and consequently size related. Thus correctly throids of even the same chemical subclass [e.g. Mendes
applied imidacloprid/flumethrin collars deliver a daily [21] described a 125-fold and 400-fold higher efficacy
active ingredient dosage level that is close to being (EC50 after 5 min contact) of flumethrin on Boophilus
weight dependent [17]. Furthermore, the daily dosage microplus than deltamethrin and cyfluthrin, respectively],
levels calculated from the release-values over 8 months the expression of efficacy can also vary within the same
were found to be comparable for dogs and cats of differ- molecule. There is a dose-time-dependency for lethal
ent sizes [17]. effects in pyrethroids [22] so that the presence of a very
fast acaricidal “repellent” effect is perhaps an indication
Discussion of results for higher doses on the hair coat when compared to the
The efficacy of the imidacloprid/flumethrin collars dose of the same pyrethroid that only results in an acari-
against R. sanguineus and C. felis felis, weeks or even cidal effect 48 h after its application. In the case of the
months after their application, was as high as that deltamethrin collar, for which 24 h efficacy against ticks
recorded initially for three spot-on formulations admi- does not exceed the 90% threshold [18], the hair coat
nistered to separate groups of dogs either at the same concentration of deltamethrin released by the collar is
66

apparently not sufficient to cause a rapid “repellent”-type The studies above were all laboratory based, while
efficacy. In contrast the hair coat concentrations after ap- in the field various challenges to the efficacy of the
plication of the imidacloprid/flumethrin collar obviously medicated collars may occur. The most obvious of
exceed the critical dose required to achieve 18 h and these is that dogs are inevitably going to be washed
even 6 h efficacy. This suggests that the 48 h acaricidal or shampooed, or swim or go out into the rain while
efficacy of the latter collar is backed by a resilient safety being walked by their owners or working in the field.
margin of active ingredient on the animal’s hair coat. Importantly collars do not have to be removed during
The long-term repellent efficacy of the imidacloprid/ any of these events [20], and that their efficacy against
flumethrin collars in Study 2, measured 6 h after infest- re-infestation with R. sanguineus remained above 97%
ation with R. sanguineus, was, with the exception of Day over a period of 8 months on regularly shampooed
7 after treatment, above 90%, followed by “rapid killing” dogs, and above 94% on dogs regularly immersed in
acaricidal efficacies ≥98% against the same population of water. Efficacy against C. felis felis remained above
ticks 12 h later (Tables 6 and 7). With the exception of 90% on shampooed dogs for the 8-month duration of
Days 7 and 42, when the 6 h repellent efficacy of the (s)- the study, but declined to below 90% at 6 months in
methoprene/amitraz/fipronil spot-on treatment exceeded the group of dogs that were regularly immersed in
that of the collars, and Days 8 and 43 when the 18 h water [20].
“rapid killing” efficacy of the spot-on treatment exceeded
that of the collars, the effectiveness of the collars Conclusions
exceeded that of the spot-on formulation. The rapidity The 8-month long period of efficacy of medicated col-
with which ticks are killed by the active ingredients of lars incorporating a combination of 10% imidacloprid
the collars and the spot-on treatment, implies that there and 4.5% flumethrin against repeated infestations of
might be significant interference with the transmission of Rhipicephalus sanguineus and Ctenocephalides felis
tick-borne organisms. felis on dogs, provides the wherewithal to overcome
The excellent immediate and medium-term efficacies of the fluctuating medium-term efficacy of spot-on treat-
the spot-on formulations of (s)-methoprene/fipronil, dinote- ments resulting from a lack of pet owner re-treatment
furan/pyriproxyfen/permethrin and (s)-methoprene/ami- compliance. The sustained high level of efficacy of the
traz/fipronil against C. felis felis makes them suitable collars against ticks 6 hours and fleas 24 hours after
candidates for the immediate and medium-term control of infestation, may well interfere with the transmission
fleas and hence also flea allergic dermatitis on dogs [6]. In of vector-borne diseases.
addition to its immediate high efficacy against fleas, the ap-
Competing interests
proximately 8-month long persistent efficacy of the imida- These clinical studies were completely funded by Bayer Animal Health
cloprid/flumethrin collar should control fleas for the whole GmbH, Monheim, Germany, of which D. Stanneck (Germany) is an
flea season from late winter to autumn. The collars could employee. ClinVet is an independent Contract Development
Organisation, which was contracted to manage the conduct of both
thus also constitute an important component in the multi- studies. I.G. Horak is a long-term, contract employee of Clinvet and an
remedy regimen required for the treatment and prevention Extraordinary Professor at the Universities of the Free State and Pretoria.
of flea allergy dermatitis during the entire season of flea ac- All authors voluntarily publish this article and have no personal interest
in these studies other than publishing the scientific findings that they
tivity [6]. have been involved in via planning, setting-up, monitoring and
Although the effectiveness of the various remedies conducting the investigations and analysing the results.
tested in these studies may at various stages after their
Acknowledgements
application be excellent, it is the number of living ticks The authors are sincerely grateful to the staff of the study location at ClinVet
or fleas remaining on treated dogs, when efficacy who took part in the two studies and ensured that the high Good Scientific
decreases or is poor, that are important. Tick burdens Practice standards were adhered to.
exceeding 10 individuals after treatment are quite ad- Author details
equate for the transmission or acquisition of organisms 1
Department of Zoology and Entomology, University of the Free State,
responsible for tick-borne diseases in the field, while a Bloemfontein 9301, South Africa. 2Department of Veterinary Tropical Diseases,
Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110,
few fleas remaining after ineffective treatment are cap- South Africa. 3ClinVet International, P.O. Box 11186Universitas 9321, South
able of inducing severe signs of flea allergy dermatitis. Africa. 4Bayer Animal Health GmbH, 51368, Leverkusen Germany.
This implies that if pet owners do not comply with the
Author’s contributions
recommended time-periods between the administration DS and JJF designed the study and drafted the protocol and JJF conducted
of spot-on remedies serious gaps in efficacy may occur. the study. IGH compiled and analysed the data and wrote the initial drafts of
The long-term, persistently high efficacy of the imidaclo- the manuscript. All authors read and approved the final manuscript.
prid/flumetrin collars would appear to be an excellent Received: 17 January 2012 Accepted: 22 April 2012
counter to these eventualities. Published: 22 April 2012
67

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68

Chapter 3. Prevention of Babesia canis transmission
by ticks
3.1. Jongejan F, Fourie JJ, Chester ST, Manavella C, Mallouk Y, Pollmeier

MG, Baggott D. The prevention of transmission of Babesia canis by
Dermacentor reticulatus to dogs using a novel combination of fipronil,
amitraz and (S)-methoprene. Vet Parasitol. 2011, 179 (4) : 343-350.
69

70

Veterinary Parasitology 179 (2011) 343–350
Contents lists available at ScienceDirect
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
The prevention of transmission of Babesia canis canis by Dermacentor

reticulatus ticks to dogs using a novel combination of fipronil, amitraz
and (S)-methoprene
Frans Jongejan a,b,∗ , Josephus J. Fourie c , S. Theodore Chester d , Coralie Manavella e ,
Yasmina Mallouk e , Matthias G. Pollmeier e , Derek Baggott f
a
Utrecht Centre for Tick-borne Diseases (UCTD), Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands
b
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa
c
ClinVet International (Pty) Ltd, P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa
d
Merial Limited, 3239 Satellite Boulevard, Duluth, Georgia, GA 30096-4640, USA
e
Merial S.A.S., Allée des Cyprès, 01150 Saint-Vulbas, France
f
Merial Animal Health Limited, Sandringham House, Harlow Business Park, Harlow CM19 5TG, UK
a r t i c l e i n f o a b s t r a c t
Keywords: Four groups of seven dogs were treated topically with a novel combination of fipronil,
Babesia canis canis amitraz and (S)-methoprene in a spot-on formulation (CERTIFECTTM , Merial Limited, GA,
CERTIFECTTM
USA) on 28, 21, 14 and 7 days prior to tick infestation, respectively and acaricidal efficacy
Dermacentor reticulatus
and transmission blocking compared with an untreated control group (seven dogs). All dogs
Dogs
Transmission blocking were infested with adult Dermacentor reticulatus ticks harbouring Babesia canis canis.
Babesia canis canis was transmitted by D. reticulatus to all seven untreated control dogs,
confirmed following demonstration of clinical signs, by the detection of B. canis parasites
in thin blood smears and B. canis canis PCR-RLB DNA assay on blood and the development
of B. canis canis antibody titres by 14–21 days after tick infestation. The majority of treated
dogs remained sero-negative for 42 days after infestation. Therefore, the treatment of dogs
with CERTIFECT applied up to 28 days prior to infestation with D. reticulatus harbouring B.
canis canis, successfully prevented the development of clinical signs of canine babesiosis.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction the basis of vector-specificity and cross-immunity and are

now considered to be separate species, B. canis, B. rossi and
Canine babesiosis is a clinically significant tick-borne B. vogeli (Uilenberg et al., 1989; Zahler et al., 1998; Carret
protozoan disease, which occurs worldwide. Historically, et al., 1999). However, both species and sub-species names
Babesia parasites in dogs were divided into two morpholog- remain in use in the current literature. Babesia canis canis
ical distinct groups, the larger Babesia canis and the smaller is widely distributed throughout Europe, where it is trans-
Babesia gibsoni. B. canis has been reclassified into three submitted by adult D. reticulatus and ticks (Matjila et al., 2005;
species (B. canis canis, B. canis rossi and B. canis vogeli) on Bourdoiseau, 2006; Cardoso et al., 2008; Cassini et al., 2009;
Beugnet and Marié, 2009). The clinical signs of babesiosis in
dogs vary from a mild transient illness to acute disease due
∗ Corresponding author at: Utrecht Centre for Tick-borne Diseases to severe haemolysis that rapidly results in death. Clinical
(UCTD), Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, findings include anorexia, pale mucus membranes, icterus,
3584 CL Utrecht, The Netherlands. Tel.: +31 30 253 2568; pyrexia, and splenic and hepatic enlargement (Jorgensen,
fax: +31 30 253 2333.
E-mail address: F.Jongejan@uu.nl (F. Jongejan).
2005; Bourdoiseau, 2006).
0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2011.03.047
71

344 F. Jongejan et al. / Veterinary Parasitology 179 (2011) 343–350
Globally, companion animals, in particular dogs, are 2. Materials and methods

exposed to a broad range of protozoan and bacterial
pathogens transmitted through the bite of infected vec- 2.1. Study design
tor ticks. In particular, canine babesiosis, anaplasmosis and
monocytic ehrlichiosis are the pre-eminent tick-borne dis- The study was conducted according to the International
eases of dogs worldwide (Jongejan and Uilenberg, 2004). Cooperation on Harmonization of Technical Requirements
A recent increase in the incidence of canine tick-borne for Registration of Veterinary Medicinal Products Guideline
diseases is due largely to changes in the ecology (land- 9: Good Clinical Practice (Anon, 2000) and in compliance
scape changes with consequent increased wildlife host with local animal welfare legislation. The study employed
abundance), climate change (increased tick survival and a controlled, blinded, randomized block design and utilized
abundance), human behaviour (recreation and travel with full grown, healthy, mongrel dogs. All dogs were individ-
pets) among other factors (Beugnet and Marié, 2009; ually penned in tick-proof kennels, managed similarly and
Blagburn and Dryden, 2009; Gray et al., 2009). As a result, observed twice daily for health abnormalities through-
there is a strong need for effective ectoparasiticides to con- out the study. When health abnormalities were detected
trol ticks on dogs. between the scheduled physical examinations, additional
The majority of published studies aimed to demon- examinations were conducted. The 13 male and 22 female
strate the utility of tick control compounds on dogs have dogs, all negative for B. canis antibodies by indirect fluores-
been focused on their acaricidal efficacy against the ixo- cent antibody assay (IFA), were randomly divided into five
did tick species. However, relatively little research has equal groups. Group 1 was designated untreated control.
been carried out to determine the ability of such com- The other four groups (treatment groups 2–5) were treated
pounds to prevent transmission of tick-borne diseases to once with the novel combination of fipronil, amitraz and
dogs. Guidelines for evaluating the efficacy of ectopara- (S)-methoprene spot-on at respectively 28, 21, 14 or 7 days
siticides for the treatment, prevention and control of tick before artificial challenge with B. canis canis-infected D.
infestations on dogs do not include suggestions to quan- reticulatus ticks.
tify the dynamics of transmission of tick-borne pathogens Treatments were applied topically to deliver at least
(Marchiondo et al., 2007). However, several studies have 6.7 mg fipronil/kg bodyweight (bw), 8.0 mg amitraz/kg bw
been conducted that suggest that topically applied tick and 6.0 mg (S)-methoprene/kg bw by simultaneously
control compounds such as permethrin and imidacloprid applying two separate formulations from dual pipettes, one
in combination, fipronil and (S)-methoprene in com- containing 10% (w/v) fipronil plus 9% (w/v) (S)-methoprene
bination, fipronil alone, and amitraz alone can aid in and the other 20% (w/v) amitraz. Each formulation was
the prevention of the transmission of specific tick-borne applied directly onto the skin divided equally between two
pathogens including Anaplasma phagocytophilum (granu- spots on the dorsal midline, one in front of the shoulder
locytic anaplasmosis), Borrelia burgdorferi (Lyme disease), blades and one at mid neck. Care was taken when handling
Ehrlichia canis (monocytic ehrlichiosis) and B. canis rossi dogs for study procedures to prevent drug cross contami-
(canine babesiosis) to dogs by ixodid ticks (Elfassy et al., nation between treatment groups by changing protective
2001; Davoust et al., 2003; Spencer et al., 2003; Blagburn clothing and changing/cleaning equipment as necessary.
et al., 2004; Jacobson et al., 2004; Last et al., 2007; Otranto
et al., 2008; Otranto et al., 2010). From this data in the liter- 2.2. Monitoring
ature it is evident that the development of a transmission
blocking model may be feasible. Such a model would have After all treatments were completed physical exam-
to include a sufficient number of treatment groups to test inations of all dogs were conducted on the day before
the duration of preventive activity, plus an un-treated con- infestation and for each dog remaining without signs of
trol group wherein the majority of dogs become infected babesiosis at 14, 21 and 28 days after infestation, as a means
by the tick-borne pathogen. of monitoring the establishment of babesiosis. The physical
In this paper a transmission blocking model is pre- examination included measurement of clinical variables –
sented, wherein topical formulations can be evaluated body temperature, heart rate and respiration rate. In addi-
for their ability to prevent dogs from becoming infected tion, daily body temperature measurements of all dogs
with babesiosis. Specifically we tested the ability of were recorded from 6 to 13 days after infestation, when the
CERTIFECTTM (Merial Limited, GA, USA), a novel combina- risk of clinical signs of babesiosis occurring was considered
tion of fipronil, amitraz and (S)-methoprene in a spot-on highest, in order to rapidly detect the onset of babesiosis
formulation, to prevent transmission of B. canis canis to in individual dogs. When a dog’s body temperature was
dogs artificially infested with D. reticulatus. Fipronil, which recorded above normal (>39.4 ◦ C), a thin blood smear was
belongs to the phenylpyrazole family, and (S)-methoprene, prepared, stained (Kyro-Quick Romanowski stain – Kyron
an insect growth regulator, have been used in combination Laboratories Pty Ltd, Benrose, South Africa), and examined
for several years for the treatment and control of ticks, fleas microscopically for the presence of B. canis species (canis
and lice (FRONTLINE Plus® for dogs or FRONTLINE Combo® canis, canis rossi and canis vogeli) parasites in red blood
Spot-on Dog (Merial, GA, USA)(Dryden, 2005). Amitraz is a cells. Dogs that were positive for B. canis spp. in blood
formamidine which kills ticks by inhibition of monoamine smears were blood sampled for B. canis canis DNA assay
oxidase and it also has been reported as a tick repel- using Polymerase Chain Reaction-Reverse Line Blot (PCR-
lent and tick detachment drug (Folz et al., 1986; Taylor, RLB) to confirm the infection. Any dog positive for B. canis
2005). on blood smear was consequently treated with diminazene
72

F. Jongejan et al. / Veterinary Parasitology 179 (2011) 343–350 345
aceturate to prevent fatal babesiosis, and no further clini- et al., 1999; Matjila et al., 2004). Briefly, QIAAMP® (QIAAMP
cal data was collected from these dogs except for B. canis is a registered trademark of Qiagen GmbH in the United
serology. States of America and elsewhere) blood and tissue extrac-
Blood samples for B. canis antibody serology using B. tion kits were used for DNA extraction, following the
canis canis antigen were collected from all dogs prior to manufacturer’s protocols. PCR was performed with primers
tick infestation on the day of infestation and at 14, 21 and RLB-F2 and RLB-R2 amplifying a fragment of 460–540 bp
28 days and, except for controls, at 42 days after infestation. from the 18S rRNA gene spanning the V4 region (Gubbels
et al., 1999). Reverse line blot hybridization was per-
2.3. Infestation by Dermacentor reticulatus formed on amplified PCR products using an improved
protocol published by Matjila et al. (2005). The protocol
All D. reticulatus were provided from a laboratory- was improved by the addition of an internal quality plas-
maintained population derived from wild ticks collected mid control, which was used to check whether all Babesia
in Europe. All dogs were infested on the same day, either species-specific oligonucleotides were attached correctly
28, 21, 14 or 7 days after treatment administration, while to the RLB membrane and functioning properly. The RLB
under sedation, with 25 male and 25 female adults, unfed probe (TGCGTTGACGGTTTGAC) employed for B. canis canis
ticks of which 33% were harbouring B. canis canis as inferred had been shown previously to be species-specific (Matjila
from other ticks sampled from the same batch. et al., 2005).
2.3.1. Infection of ticks by Babesia canis canis

The D. reticulatus ticks were infected with B. canis canis 2.4. Statistical analysis
by feeding them on dogs infected with a B. canis canis strain
isolated from a D. reticulatus female collected from a dog in The primary variable for determining the ability of the
France. B. canis canis infestation of ticks was determined novel combination to prevent transmission of B. canis canis
by PCR-RLB DNA assay on a sample of ticks (51) taken from to dogs was the antibody titre. At 14 days post-infestation
the infestation batch and ticks collected from study dogs the results were reported as positive (titre ≥1:80 dilution)
on Day 6 post-infestation. or negative, whereas no end-point titres were determined.
Because very few dogs in treatment groups 2–5 devel-
2.3.2. Tick counts oped titres at 21, 28 or 42 days post-infestation, analysis
Following infestation, ticks on all dogs, including of differences in titres between these groups was not
untreated controls, were counted in situ at 2, 3, 4 and 5 days possible.
and removed at Day 6 post-infestation in order to deter- For clinical variables, body temperature, heart rate
mine the efficacy of the combination of fipronil, amitraz and respiration rate measured at 14, 21 and 28 days
and (S)-methoprene at preventing the establishment of tick post-infestation, the pre-infestation (baseline) responses
infestation. Tick counts were performed and recorded by measured the day before infestation were analyzed as an
tick categories (Table 1). analysis of variance using the Mixed procedure with the
effects sex, treatment, and sex-by-treatment interaction
2.3.3. Laboratory analyses as the fixed effects. Replicate was the random effect. To
Blood samples for serology were assayed for B. canis detect responses to B. canis canis infection by each treated
antibodies using an indirect fluorescent antibody (IFA) group (groups 2–5) in the absence of post-infestation data
assay with B. canis canis antigen as the substrate and car- for the untreated controls, the change from pre-infestation
ried out as described by Uilenberg et al. (1989). This test baseline was analyzed for each treated group.
cross reacts with antibodies of B. canis rossi and B. canis In order to determine the effectiveness of the novel
vogeli. However, homologous species antigen and antibody combination of fipronil, amitraz and (S)-methoprene in
combinations (B. canis canis antibodies with B. canis canis preventing establishment of tick infestation, for each dog
antigen) will cause a stronger reaction (higher titre) than at each counting time, the total number of ticks that
heterologous species combinations such as and B. canis rossi were assigned to categories 1, 2, 3, and 6 (live free,
antibodies and B. canis canis antigen where the reaction attached/unengorged and attached/engorged and dead
will be weak. In this study where B. canis sero-negative attached/engorged) were transformed to the natural log-
ticks were infected with B. canis canis it is assumed that arithm of (count + 1) for calculation of geometric means.
all positive B. canis titres reflected B. canis canis antibodies. For the treated groups, the percent reduction in tick counts
For screening purposes the sera were diluted at 1:80, and compared to group 1 (untreated control) was computed
results were expressed as positive (fluorescence at dilu- using the formula 100 × (1 − T/C), wherein T and C were
tion 1:80) or negative (no fluorescence). Positive samples the geometric means of the particular treated and control
collected at 21, 28 and 42 days after infestation were addi- group 1, respectively. For all ticks within each counting
tionally serially two-fold diluted starting at 1:80 through time, the expected tick counts of the treated groups were
1:2560 to determine the B. canis canis antibody titre of each compared with the expected tick count of the control group
one. using the Friedman rank test.
Parasite DNA extraction from blood or ticks, PCR All analyses were conducted using SAS® Version 9.1.3
amplification and reverse line blot (RLB) hybridization (SAS Institute Inc., Cary, NC, USA), and all statistical com-
for simultaneous detection and differentiation of Babesia parisons were made using a (two-sided) 5% significance
species was carried out as described previously (Gubbels level.
73

Table 1
Count categories of live and dead ticks by attachment status.
Categorya General findings Attachment status Interpretation
1 Live Free Acaricidal effect NOT demonstrated

2 Live Attached; unengorged Acaricidal effect NOT demonstrated
3 Live Attached; engorgedb Acaricidal effect NOT demonstrated
4 Dead Free Acaricidal effect demonstrated
5 Dead Attached; unengorged Acaricidal effect demonstrated
6 Dead Attached; engorged Acaricidal effect NOT demonstrated
a
Adapted from Marchiondo et al. (2007).
b
Engorged tick: a tick with a conspicuous enlargement of the alloscutum that has blood in its digestive tract.
3. Results to infestation) (Tables 4 and 5). On Day 42 post-infestation,

the same two dogs were positive again, as well as two addi-
3.1. Treatment tolerance and animal health tional dogs in group 2 (treated 28 days prior to infestation).
The four dogs in treated groups 2 and 5 displayed low titres
The novel combination of fipronil, amitraz and (S)- ranging between 80 and 160 and did not show any clini-
methoprene administered concurrently as topical solu- cal signs of babesiosis including raised body temperature
tions to 28 dogs was well-tolerated by all animals. No (>39.4 ◦ C) throughout the study. The two dogs in group 5
significant health abnormalities, other than babesiosis in were also negative for B. canis on additional blood smear
controls, were detected during the study. examinations and B. canis canis on PCR-RLB DNA assay con-
ducted 43 days post-infestation.
3.2. Tick counts For clinical variables, body temperature, heart rate and
respiration rate, the pre-infestation (baseline) responses
Tick counts for each group by tick category (1–6) and measured the day before infestation showed no significant
day (2, 3, 4, 5, and 6 days after infestation) are summarized (P > 0.05) differences between groups. For body tempera-
in Table 2. The geometric mean of live plus dead engorged ture there was no statistical evidence (P > 0.05) that the
(categories 1, 2, 3 and 6) tick counts per day for each treat- change from pre-infestation baseline interacted with any
ment group is listed in Table 3. Percent reductions in tick treatment effect in the treated groups (2–5). Several varia-
counts for each treated group compared to the untreated tions up and down in heart rate and respiration rate were
control are also listed in Table 3. The treatment rapidly observed that represented significant (P < 0.05) changes
reduced the number of adult D. reticulatus by more than from baseline observations, but these fluctuations were
98% within 2 days after infestation and to 100% within 3 not considered clinically relevant as they were intermittent
days. and not related to treatment interval.
3.3. Challenge with Babesia-infected ticks 4. Discussion
The ticks sampled from the infestation batch contained In order to be able to conduct the study, it was required
17 out of 51 (33.3%) B. canis canis infected tick. For ticks col- to generate a large batch of ticks with an adequate Babesia
lected on Day 6 post-infestation, all from untreated control infection rate. The sample of ticks from the challenge
animals, 55 out of 122 (45.1%) were found to be infected batch of infected ticks taken on the day of infestation con-
with B. canis canis. Of those ticks from the infestation batch, tained 33.3% infected ticks while of those ticks collected
20% of 25 males and 46% of 26 females were infected while on Day 6 post-infestation from untreated control animals,
of ticks collected on Day 6, 50% of 42 males and 42% of 80 45.1% were found positive. On each occasion some male
females were infected. and female ticks were infected although the proportion of
infected males was much lower than females in the sam-
3.4. Babesia canis canis transmission blocking ple from the infestation batch. It is likely that both male
and female ticks facilitated transmission of B. canis .canis,
All dogs were sero-negative for B. canis prior to tick but a separate study with only male or female ticks will be
infestation. All the dogs in group 1 (untreated controls) required to investigate this further. Regardless of which sex
became positive for B. canis on thin blood smears collected or if both sexes transmit the B. canis canis, the infection rate
after body temperatures were measured at >39.4 ◦ C. These in the ticks was sufficiently high to successfully transmit
dogs were consequently treated with diminazene acetu- the infection to 7 out of 7 untreated (control) dogs.
rate to prevent fatal babesiosis. Five of the seven untreated The study was designed to detect exposure to Babesia
control dogs were sero-positive for B. canis canis on Day 14 infection in dogs through an economical use of available
post-infestation, and all seven developed antibody titres diagnostic techniques. The detection and confirmation of
ranging from 160 to ≥2560 on Day 21 and Day 28 post- B. canis canis infection was based on an initial three stage
infestation (Tables 4 and 5). approach: (1) regular monitoring of body temperature of
All treated dogs (groups 2–5) were sero-negative for B. all dogs; (2) thin blood smear examination for B. canis
canis canis at 14, 21, and 28 days post-infestation, except for parasites in red blood cells of pyrexic dogs (>39.4 ◦ C); (3)
two dogs positive at 28 days in group 5 (treated 7 days prior molecular assay for detection of B. canis canis DNA by PCR-
74

Table 2
Tick counts for each treatment group by tick category (1–6) and count day (2, 3, 4, 5, and 6 days after infestation).
Tick category 1. Live free 2. Live attached unengorged 3. Live attached engorged
Days after infestation 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6
Groupa
1 1 1 3 5 3 173 162 121 67 41 0 0 32 61 75
2 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
4 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
5 2 0 0 0 0 8 0 0 0 0 0 0 0 0 0
Tick category 4. Dead free 5. Dead attached unengorged 6. Dead attached engorged
Days after infestation 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6
Groupa
1 1 0 0 0 0 0 0 1 0 1 0 0 0 0 0
2 3 0 0 0 0 0 1 0 0 0 0 0 0 0 0
3 8 0 0 0 0 2 1 0 0 0 0 0 0 0 0
4 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5 3 0 1 1 0 1 5 0 0 0 0 0 0 0 0
a
Group 1 = untreated control; group 4 = treated 14 days before infestation; group 2 = treated 28 days before infestation; group 5 = treated 7 days before
infestation. Group 3 = treated 21 days before infestation.
RLB on blood samples from dogs with a positive B. canis performed on samples collected from all dogs irrespec-
blood smear to confirm the infection. Unfortunately, with tive whether the dogs were pyrexic. The use of serology
this approach the DNA assay was not used on all dogs, a on all dogs to detect specific B. canis antibodies did cor-
shortcoming that may have resulted in subclinical infec- rectly detect infections in all dogs showing clinical signs
tions being undetected. However, serological testing was of babesiosis (i.e. pyrexia). Moreover, serology did detect
Table 3
Geometric mean tick counts (tick categories 1, 2, 3, and 6) by treatment group and percent reductions in counts compared to untreated control (treatment
group 1).
Days after infestation Groupa n Geometric mean Percent reductionb P-valuec
1 7 24.5 – –
2 7 0.1 99.6 0.008
2 3 7 0.1 99.6 0.008
4 7 0.1 99.6 0.008
5 7 0.4 98.3 0.008
1 7 23.0 – –
2 7 0.0 100.0 0.008
3 3 7 0.0 100.0 0.008
4 7 0.0 100.0 0.008
5 7 0.0 100.0 0.008
1 7 21.9 – –
2 7 0.0 100.0 0.008
4 3 7 0.0 100.0 0.008
4 7 0.0 100.0 0.008
5 7 0.0 100.0 0.008
1 7 18.2 – –
2 7 0.0 100.0 0.008
5 3 7 0.0 100.0 0.008
4 7 0.0 100.0 0.008
5 7 0.0 100.0 0.008
1 7 14.6 – –
2 7 0.0 100.0 0.008
6 3 7 0.0 100.0 0.008
4 7 0.0 100.0 0.008
5 7 0.0 100.0 0.008
a
Group 1 = untreated control; group 2 = treated on Day 0; group 3 = treated on Day 7; group 4 = treated on Day 14; group 5 = treated on Day 21, all groups
infested with ticks on Day 28.
b
Percent reduction in tick count compared to untreated control = 100 × (1 − T/C), where T and C are the geometric means of the treated and untreated
control groups, respectively.
c
Two-sided P-values comparing the expected tick counts using Friedman rank test.
75

Table 4
Ratio of sero-positive dogs to the total number of dogs based on Babesia canis canis antibodies according to treatment group.
Days after infestation Group 1 Group 2 Group 3 Group 4 Group 5
−43 0/7a 0/7 0/7 0/7 0/7

0 0/7 0/7 0/7 0/7 0/7
14 5/7 0/7 0/7 0/7 0/7
21 7/7 0/7 0/7 0/7 0/7
28 7/7 0/7 0/7 0/7 2/7
42 –b 2/7 0/7 0/7 2/7
a
Number of sero-positive dogs (=IFA titre of ≥1:80)/total number of dogs.
b
Samples were not collected.
four other dogs with low antibody titres but without clini- not the same dogs positive for B. canis canis antibodies in
cal signs and indeed the two of these that were DNA tested these groups, had relatively small numbers (1 or 8) of live
on Day 43 were in fact negative for B. canis canis DNA using attached, unengorged ticks present at the 2-day tick count
PCR-RLB. Thus the combination of thin blood smear and but none thereafter (Table 2). Although treatment with the
DNA assay on only pyrexic dogs with the use of serology on novel combination did not completely block the transmis-
all dogs was considered an effective means of documenting sion of B. canis canis in a small proportion of dogs (14%),
exposure to B. canis canis in the study dogs (Uilenberg et al., it was 100% successful in preventing the development of
1989). clinical signs of babesiosis in all dogs up to 42 days after
Six out of the seven control dogs, which developed anti- tick infestation.
body titres increasing from 80 on or soon after Day 14 to The results obtained in controlled laboratory trials with
320 up to ≥2560 in 7 days or less, demonstrated strong B. burgdorferi are similar to those obtained in this study
sero-conversion. However, the titre of the remaining con- with B. canis canis. When the challenge infestation was con-
trol dog went up to only 160. The low titre first measured ducted 28 days after treatment, fipronil/(S)-methoprene
at 21 days post-infestation in this control dog was due spot-on prevented all but 2 of 16 dogs from becoming
probably to the early administration of the anti-babesial infected with B. burgdorferi and was 97.6% effective against
treatment at Day 7 post-infestation, despite B. canis canis tick infestation 48 h after challenge (Jacobson et al., 2004). It
infection, as for all other controls, being confirmed by pos- therefore appears feasible to dramatically reduce the trans-
itive blood smear and PCR results on samples collected on mission of tick-borne pathogens with suitable anti-tick
the day a raised body temperature was detected. control compounds, although a 100% transmission blocking
The late stage weak sero-conversion by Days 28–42 may be difficult to achieve.
post-infestation in four of the treated dogs indicated a low The reported study indicates the importance of the
level transmission of B. canis canis sporozoites. Two of these speed of kill of anti-tick compounds with respect to the
dogs (in group 5) and the other two (in group 2) were early removal of feeding ticks in order to successfully block
challenged with ticks at 7 and 28 days post-treatment, transmission. The effect of early removal of feeding ticks
respectively. Transmission is likely to have taken place on pathogen transmission depends on the tick attachment
during a brief attachment by a small number of ticks with- duration that is required for ticks to transmit the specific
out fever or other signs of babesiosis being evident to pathogen concerned. For instance, transmission of Borre-
prompt blood smear collections from these dogs. It was lia spirochetes appears rare within the first 24 h, whereas
noted that one dog in each of the treated groups (2–5), but in the same period most A. phagocytophilum rickettsiae
Table 5
Babesia canis canis antibody titres in individual dogs according to treatment group.
Group Replicate Infestation +21 daysc Infestation +28 days Infestation +42 days
1b 1 ≥1:2560a ≥1:2560 –d
2 1:320 1:320 –
3 1:640 1:320 –
4 ≥1:2560 1:1280 –
5 1:160 1:160 –
6 1:640 1:160 –
7 1:1280 1:640 –
2 1 nege neg 1:80

6 neg neg 1:160
5 2 neg 1:80 1:80

7 neg 1:160 1:80
a
Positive = titre ≥1:80.
b
Group 1 = untreated control; group 2 = treated 28 days before infestation; group 3 = treated 21 days before infestation; group 4 = treated 14 days before
infestation; group 5 = treated 7 days before infestation.
c
B. canis antibody titres were not determined on Day 14 after tick infestation.
d
Samples were not collected.
e
Samples negative for B. canis canis.
76

are already transmitted (Des Vignes et al., 2001). For most Acknowledgement
viral pathogens transmission appears to be rapid; for exam-
ple, Powassan virus can be transmitted within 15 min after We would like to thank Prof. Leon Fourie from Clinvet
attachment of the vector tick (Ebel and Kramer, 2004). International, Republic of South Africa for his contribution
Protozoan parasites require additional time, usually to the development of the transmission blocking models
several days, for their sporoblasts to mature into sporo- and his critical comments on the manuscript.
zoites in the salivary glands of the tick before they can
be secreted into the saliva and transmitted to the mam- References
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78

by ticks
3.2. Fourie JJ, Stanneck D, Jongejan F.

Prevention of transmission of Babesia canis by Dermacentor reticulatus
ticks to dogs treated with an imidacloprid/flumethrin collar. Vet Parasi-
tol. 2013, 192
(1-3) : 273-278.
79

80

Contents lists available at SciVerse ScienceDirect
Short communication
Prevention of transmission of Babesia canis by Dermacentor reticulatus

ticks to dogs treated with an imidacloprid/flumethrin collar
Josephus J. Fourie a,∗ , Dorothee Stanneck b , Frans Jongejan c,d
a
ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa
b
Bayer Animal Health GmbH, BHC Business Group Animal Health, Clinical Development, Building 6700, 51368 Leverkusen, Germany
c
d
Article history: A group of 8 dogs was treated with an imidacloprid/flumethrin collar (Seresto® ) 28 days
Received 13 June 2012 prior to infestation with adult Dermacentor reticulatus ticks, infected with Babesia canis.
Received in revised form 17 October 2012
The ability of the collar to prevent transmission of B. canis in the treated group was com-
Accepted 18 October 2012
pared to an untreated control group. All 8 dogs in the untreated control group became
infected with B. canis parasites, which were detected in blood smears as early as day 6 post
Keywords:
tick-application. All control dogs developed clinical signs of babesiosis and were rescue-
Imidacloprid
Flumethrin treated with imidocarb dipropionate. These dogs also developed specific B. canis antibodies
Collar as identified by serology (IFA test) and were confirmed PCR/RLB positive.
Dermacentor reticulatus None of the 8 dogs treated with the imidacloprid/flumethrin collar became infected with
Ticks B. canis, which was confirmed by the absence of specific B. canis antibodies and babesial
Babesia canis DNA as confirmed by PCR/RLB.
Transmission blocking The collar caused 96.02% of the ticks to die within 48 h post challenge and this increased
to 100% within 4 days. Although a high percentage of 44% of the Dermacentor ticks were
infected with B. canis, they were unable to transmit the infection to the treated group.
Hence, the imidacloprid/flumethrin collar effectively prevented transmission of B. canis 1
month after application onto the dogs.
© 2012 Elsevier B.V. Open access under CC BY-NC-ND license.
1. Introduction 2009). Moreover, over the past two decades several novel
tick-borne infections have been identified, whereas oth-
Worldwide, dogs are exposed to a broad range of proto- ers are regarded as re-emerging diseases of dogs (Chomel,
zoan and bacterial pathogens transmitted by infected ticks. 2011).
Canine babesiosis, monocytic ehrlichiosis and granulocytic The vectorial capacity of ticks infesting dogs varies
anaplasmosis are the pre-dominant tick-borne diseases of widely. For instance, the cosmopolitan tick, Rhipicephalus
dogs (Jongejan and Uilenberg, 2004). Ticks and tick-borne sanguineus, transmits Babesia vogeli, Babesia gibsoni, Hep-
diseases that affect dogs in different regions of the world atozoon canis, Ehrlichia canis and Rickettsia conorii, and
are expanding (Chomel, 2011). Since international travel probably also Anaplasma platys, the cause of throm-
with companion animals is now quite common, ticks and bocyic anaplasmosis. Ixodes ricinus ticks are widely
associated diseases are also crossing borders and are caus- distributed throughout Europe, where they transmit Bor-
ing novel emerging disease threats (Beugnet and Marié, relia burgdorferi and Anaplasma phagocytophilum, both
zoonotic pathogens, which are affecting a broad range of
hosts, including dogs.
In this paper we have studied the ornate dog tick, Der-
∗ Corresponding author. Tel.: +27 51 445 2424; fax: +27 51 445 2421. macentor reticulatus, which is a vector of a broad spectrum
E-mail address: Josephus.Fourie@clinvet.com (J.J. Fourie). of pathogens, including B. canis in dogs, Babesia caballi
0304-4017 © 2012 Elsevier B.V. Open access under CC BY-NC-ND license.

http://dx.doi.org/10.1016/j.vetpar.2012.10.017
81

274 J.J. Fourie et al. / Veterinary Parasitology 192 (2013) 273–278
in horses, Anaplasma marginale in cattle and a number adult dogs of mixed breed mainly mongrel. All dogs were
of zoonotic rickettsial species. D. reticulatus is expanding dewormed and did not harbour any ticks at the initiation
its distributional range within Europe into, for instance, of the study. The dogs had not been treated with an aca-
Hungary, The Netherlands, Norway and elsewhere (Sréter ricide/insecticide spot-on/spray for 12 weeks prior to tick
et al., 2005; Matjila et al., 2005; Øines et al., 2010). This has infestation (day 0). All were serologically negative for B.
led to cases of canine babesiosis being detected in regions canis prior to day −35. The study animals were acclimatised
where the disease had previously not been encountered, for 7 days prior to the treatment. The study animals were
which adds to the relevance of practicing acaricidal tick kept individually in pens in compliance with local animal
control on dogs. welfare regulation.
Therefore, acaridical treatment that kills ticks and thus All the animals were observed daily for general health
reduces the effective tick population capable of transmiting conditions and clinical signs of adverse reactions to treat-
disease-inducing pathogens, seems evident. Importantly, it ment. All dogs were subjected to a clinical examination
would be a significant step forward, if it could be demon- on days −35, +7, +14, +21 and +27. Additionally clinical
strated that the rapid killing of infected ticks on dogs could examinations were conducted on all dogs displaying clini-
actually prevent tick-borne disease to develop at all. cal signs (elevated body temperature, anaemia, haematuria
Acaricidal control of ticks on dogs is a nessessity in many and/or icterus) associated with babesiosis. The dogs were
parts of the world. The active compounds are predomi- observed on an approximately hourly basis for 4 h after
nantly applied as shampoos, powders, spot-ons, sprays and fitting the collar for possible adverse events. Rectal body
impregnated collars. Acaricide impregnated collars have temperatures were recorded daily from days +6 to +13.
been widely used against ticks on companion animals (Van Clinical examinations were conducted and two blood
den Bos and Curtis, 2002; Fourie et al., 2003; Estrada- smears were prepared for dogs displaying abnormally high
Pena and Reme, 2005). For instance, flumethrin, a synthetic body temperatures (>39.4 ◦ C).
pyrethroid, has been extensively used in livestock as an Blood for PCR was collected from all dogs diagnosed
acaricide over the past 25 years and has also been used with babesiosis as confirmed on a blood smear and from
in a collar preparation in combination with propoxur to all dogs not yet diagnosed with babesiosis on days +14,
control ticks on dogs (Fourie et al., 2003). Furthermore, +21 and +28. Blood was collected in EDTA tubes and stored
imidacloprid is a highly effective insecticide, registered for at −30 ◦ C prior to DNA extraction. PCR amplification and
use in dogs and cats as a spot-on product and is also avail- reverse line blot hybridisation (RLB) was carried out as
able in combination with permethrin (another pyrethroid) described by Matjila et al. (2005), employing an improved
for spot-on use in dogs (Advantix ® ) (Hellmann et al., laboratory protocol for detecting and differentiation of
2003). canine Babesia species. The protocol was improved by the
Recently, a combination of imidacloprid (10%) with addition of an internal quality plasmid control to check
flumethrin (4.5%) has been formulated in a collar matrix whether all probes were functioning properly. PCR-RLB
which proved safe to dogs and cats. Moreover, this col- tests were only conducted on blood samples collected from
lar exhibited a sustained high level of efficacy against ticks dogs that were diagnosed with B. canis infection based on
(Stanneck et al., 2012). Consequently, this collar may have blood smears or IFA test.
the potential to prevent the transmission of vector-borne Blood for serology (IFA test) was collected on day +14,
diseases. +21 and +28 and frozen at −20 ◦ C until assayed for B. canis
This study was designed with the aim to evaluate the antibodies. An indirect fluorescent antibody (IFA) test was
efficacy of an imidacloprid/flumethrin collar formulation in used with B. canis antigen as substrate (Uilenberg et al.,
preventing the transmission of B. canis to dogs by infected 1989). All sera were diluted at 1:80. Sera were recorded as
D. reticulatus ticks. positive if specific fluorescence was observed at dilution
1:80 or negative (no fluorescence).
2. Materials and methods A laboratory-bred D. reticulatus tick strain infected with
Babesia canis-was used in the artificial infestations. A sam-
The study was conducted according to International ple of D. reticulatus ticks was taken from the batch of ticks to
Cooperation on Harmonization of Technical Requirements be used for infestation and the infection rate determined.
for Registration of Veterinary Medicinal Products Guide- 16 out of 36 ticks (44%) were found to be infected with
line 9: Good Clinical Practice (Anonymous, 2000). The study Babesia canis by PCR-RLB analysis. RLB analysis also con-
was a parallel group design, randomised, unicentre, confirmed that the ticks were not infected with any other
trolled efficacy study using two groups of eight dogs each. known tick-borne pathogen. Adult ticks, which were used
Group 1: negative control (n = 8); Group 2: dogs fitted with in the artificial infestations, were unfed, at least 1 month
the collar on day −28 (n = 8). The negative control group old and had a balanced sex ratio (50% female: 50% male).
was not treated with a placebo collar making blinding of Each dog was artificially infested with approximately 50
the study impossible. On day −30 the 16 dogs were ranked ticks on day 0. The time of infestation was recorded for all
according to body weight, within gender (9 male and 7 animals.
female), in descending order from 16.6 to 10.8 kg indi- The time of tick counting was recorded. Day +2, +3,
vidual body weight. Animal ID’s were used to break ties. +4 and +5 tick counts were conducted in situ. During the
Within each gender animals were then blocked into blocks in situ thumb counts, ticks were categorised according to
of two dogs each. Within each block, dogs were randomly categories 1, 2, 4 and 5. The ticks counted and removed
allocated to Groups 1 or 2. The dogs were sub adult and on day +6 were categorised within gender (male/female)
82

J.J. Fourie et al. / Veterinary Parasitology 192 (2013) 273–278 275
Table 1
Categorization of ticks for counting.
Attached;
engorged
Category General findings Attachment status
1 Live Free
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2 Live Attached; unengorgeda
3 Live Attached; engorgedb
unengorged
4 Killed Free
Free Attached;
5 Killed Attached; unengorgeda
6 Killed Attached; engorgedb
0
0
0
2
0
0
0
0
0
0
0
0
1
0
0
a
No filling of the alloscutum evident.
Dead
b
Obvious or conspicuous filling of the alloscutum evident.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
according to categories 1–6 (see Table 1), adapted after
Marchiondo et al. (2007).
Attached;
engorged
SAS Version 8 (release 8.02 TS Level 02M0) was used
for all the statistical analyses. Efficacy against ticks was
0
10
0
18
0
9
0
5
0
0
5
0
10
0
8
calculated for each treatment group at each assessment
day according to the formulas given below (EMEA, 2007).
unengorged
Due to the fact that small and even zero tick counts were
Free Attached Free Attached Free Attached Free Attached Free Attached Free Attached Free Attached Free Attached Free Attached;
Study day +6
recorded, it was expected that the tick counts would not
follow a normal distribution. It was therefore decided
12
0
17
0
23
0
12
0
6
0
0
4
0
6
0
15
that the primary efficacy calculations would be based on
Live
geometric means rather than arithmetic means. The cal-
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
culations were based on the geometric means of the tick
(count + 1) data. One (1) was subsequently subtracted from
the result to obtain a meaningful value for the geomet-
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
Dead
ric mean of each treatment group. Percent efficacy against
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ticks was calculated as follows:
Study day +5
Gmc − Gmt
Efficacy (%) = 100 × ,
Gmc
18
0
26
0
34
0
22
0
14
0
0
16
0
18
0
20
Tick counts for each treatment group by tick category and count day (2, 3, 4, 5 and 6 days after infestation).
where Gmc is the geometric number of live ticks (categories

Live
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1–3) on dogs in the negative control group (Group 1) at a
specific time point. Gmt is the geometric mean number of
live ticks and dead ticks (categories 1–3 and 6; (see Table 1)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
residual efficacies) in the treatment group (Group 2) at a

Dead
specific time point (EMEA, 2007).

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Descriptive statistics (mean, minimum, maximum,

Study day +4
standard deviation, CV%, geometric mean and median) on

tick counts for the various assessment days were calcu-
20
0
28
0
35
0
21
0
16
0
0
22
0
22
0
20
lated.
Live
The groups were compared using an ANOVA with a

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
treatment effect after a logarithmic transformation on the

tick (count + 1) data. The level of significance of the formal
tests was set at 5%, all tests were two sided.
0
2
0
0
0
1
0
0
0
2
0
0
0
0
0
0
Dead
A protection failure (successfully infected) was regarded

0
0
0
2
0
0
0
1
0
0
1
0
0
0
0
0
when a dog in the treatment group tested serologically pos-

Study day +3
itive for Babesia canis antibodies as well as positive for B.

canis DNA in PCR-RLB assay.
19
0
28
0
34
0
19
0
15
0
0
21
1
22
1
21
The percentage protection for the treatment group was

Live
calculated as follows:
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Tc − Tt
Protection (%) = 100 × ,
Tc
0
3
0
1
0
1
0
0
0
3
1
0
5
0
0
0
where Tc is the total number of infected dogs in the negative

Dead
control Group 1 and Tt is the total number of infected dogs

0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
in the treatment Group 2.

ID Study day +2
21
3
26
1
34
0
20
0
23
0
3
20
1
24
0
18
3. Results
Group 1
Group 2
Live
Table 2
1 0
1 0
2 0
2 0
3 0
3 0
4 0
4 0
5 0
5 0
6 2
6 1
7 0
7 0
8 1
8 0
The tick counts for the two study groups are given
in Table 2. The geometric mean tick counts recorded for
83

Table 3
Mean tick counts by treatment group and percentage efficacies.
Day Geometric mean tick counts
Group 1 – negative control Group 2 – dogs fitted with collar
+2b 23.1 0.9a

+3b 21.8 0.2a
+4b 22.4 0.0a
+5b 20.3 0.0a
+6 18.7 0.0a
Day Efficacies (%)

Group 2 – (dogs fitted with collar)
+2b 96.02
+3b 99.1
+4b 100.0
+5b 100.0
+6 100.0
a
Geometric mean tick count differs statistically significantly (p < 0.05) from that recorded for the negative control Group 1.
b
In situ counts.
Table 4
A summary of clinical observations recorded.
Dog number Group Day Fever Blood smear Other observations
3 40.5 ◦ C Positive for Babesia canis Not observed

1 +6
7 39.8 ◦ C Positive for B. canis Not observed
2 39.9 ◦ C B. canis + Haemoglobinuria

3 39.4 ◦ C B canis + Haemoglobinuria
5 39.5 ◦ C B. canis + Not observed
1
6 +7 40.3 ◦ C B. canis + Haemoglobinuria
8 39.7 ◦ C B. canis + Not observed
7 40.5 ◦ C B. canis + Haemoglobinuria
1 2 38.7 ◦ C Negative for B. canis Tremors – nervousness
6E1 208 1 +9 No fever; blood smear positive for B. canis. Depressed; enlarged spleen; pale mucous membranes
both groups are summarised in Table 3. A mean tick count Eight of 8 untreated control dogs developed specific anti-
of 23.1 was recorded for the untreated negative control bodies to B. canis on one or more post-challenge time
group on day +2 indicating a vigorous tick challenge. Effi- points (Table 5). None of the treated dogs tested positive
cacy values (%) based on geometric mean tick counts for for B. canis antibodies on any of the post challenge time
the treated group are also summarised in Table 3. The points (Table 5). PCR-RLB tests that were conducted on dogs
collar was highly effective against challenges with D. retic- that were diagnosed with B. canis based on blood smear
ulatus ticks 1 month after application. Tick mortality as
high as 96.02% was recorded 48 h post challenge with
100% of all infested ticks being dead within 4 days post Table 5
Babesia canis antibodies in individual dogs according to treatment group.
challenge.
The clinical signs observed in the untreated control Dog number Group Day −35 Day 0 Day 14 Day 21 Day 28
group were correlated with onset and progress of babesio- 1 – – POS POS POS
sis and are summarised in Table 4. On day 6 post tick 2 – – POS POS POS
application, two dogs reacted with fever and Babesia para- 3 – – POS POS POS
4 – – POS POS POS
sites were detected in blood smears. The next day both dogs 1
5 – – POS POS POS
displayed fever again and parasites were again detected. 6 – – – POS –
Both were treated with imidocarb dipropionate (12%) and 7 – – – POS POS
recovered. Four other dogs developed fever on day 7 8 – – POS POS POS
and were positive on blood smears (Table 4). The two 1 – – – – –
remaining dogs in the untreated group did not develop 2 – – – – –
fever, but were depressed with pale mucous membranes 3 – – – – –
and enlarged spleen upon palpation with blood smears 4 – – – – –
2
5 – – – – –
revealing the presence of Babesia parasites. All 8 dogs 6 – – – – –
were rescue treated with imidocarb dipropionate and fully 7 – – – – –
recovered. 8 – – – – –
All dogs included in the study tested negative for B. POS: positive for Babesia canis antibodies; −, negative for Babesia canis
canis antibodies in the IFA assay prior to tick infestation. antibodies.
84

Table 6
Detection of Babesia canis DNA by PCR/RLB in individual dogs.
Dog number Group Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13
1 – – – POS – – – –
2 – POS – – – – – –
3 POS – – – – – – –
4 – – – POS – – – –
1
5 – POS – – – – – –
6 – POS – – – – – –
7 POS – – – – – – –
8 – POS – – – – – –
1 – – – – – – – –
2 – – – – – – – –
3 – – – – – – – –
4 – – – – – – – –
2
5 – – – – – – – –
6 – – – – – – – –
7 – – – – – – – –
8 – – – – – – – –
POS: confirmed positive for Babesia canis DNA; −, tested negative.
evaluation were all confirmed B. canis positive at differ- 8 months (Stanneck et al., 2012). This proven sustainable
ent time points from day 6 to day 9 after tick challenge high efficacy of the collar over 8 months strongly suggests
(Table 6). that the prevention of B. canis transmission may have the
The level of protection (100%) of the imidaclo- same duration.
prid/flumethrin collar in preventing transmission of B. canis Transmission blocking has become a novel standard in
by infected Dermacentor ticks is summarised in Table 7, the evaluation of the capacity of anti-tick compounds to
together with the 95% confidence interval (69–100%) and prevent transmission of tick-borne pathogens to dogs: The
p-value (0.0002) from the Fisher’s exact test. transmission blocking model for D. reticulatus ticks as used
in the present study was recently developed and used suc-
4. Discussion and conclusion cessfully for the first time with a novel combination of
fipronil, amitraz and (S)-methoprene (Certifect® ) (Jongejan
The imidacloprid/flumethrin collar, which has recently et al., 2011). The advantage of this model is that the chal-
been registered (Seresto® ) by Bayer Animal Health GmbH lenge load with infected ticks can be pre-determined and
(Leverkusen, Germany) was well tolerated by the dogs. standardised. Several other studies have been carried out
The efficacy of 96.02% of ticks killed 48 h post challenge, to ascertain the capability of various anti-tick compounds
which increased to 100% mortality within 4 days, was in to prevent transmission of tick-borne pathogens to dogs.
line with the reported efficacy above 97% against adult D. In these studies, dogs were either exposed to infected
reticulatus ticks (Stanneck et al., 2012). Moreover, the speed ticks under field conditions or infected ticks were collected
of kill by the collar against infesting ticks is of particular from the field and tested on dogs under controlled lab-
importance with respect to the ability to block transmis- oratory conditions (Spencer et al., 2003; Jacobson et al.,
sion of pathogens. Evidently, this speed was sufficient in 2004; Blackburn et al., 2004; Last et al., 2007; McCall et al.,
order to prevent transmission of mature Babesia sporo- 2011).
zoites into the treated dogs, whereas they were readily For instance, the capacity of an amitraz-impregnated
transmitted to the untreated control group. Development collar to block transmission of Babesia rossi by Haema-
of Babesia sporoblasts into mature sporozoites within the physalis elliptica ticks has been evaluated in the field in
tick’s salivary gland was therefore prevented by the treat- South Africa. Eight of 30 control dogs (26.6%) became
ment, despite the fact that 44% of the ticks were infected. PCR/RLB positive during the trial period, which lasted
Moreover, it has been demonstrated that this collar has a for 6 months. None of the treated dogs became infected
sustained acaricidal (48 h killing) efficacy over a period of with babesiosis during the same period (Last et al., 2007).
Furthermore, in a laboratory study targetting B. burgdor-
feri, when challenge infestation was conducted 28 days
Table 7 after application of fipronil/(S)-methoprene, the treatment
Comparison of treated and control dog group for Babesia canis infection. prevented all but 2 of 16 dogs from becoming infected
and was 97.6% effective against tick infestation 48 h after
Group 1 Group 2
(n = 8) (n = 8)
tick challenge (Jacobson et al., 2004). In addition, imi-
dacloprid/permethrin spot-on prevented transmission of
Infected 8 0
A. phagocytophilum from field collected Ixodes scapularis
Non-infected 0 8
% Infected 100 0 ticks reported by Blagburn et al. (2004). Recently, the
% Protection – 100 ability of the topical combination of fipronil, amitraz
95% Confidence interval: % protection 69–100 and (S)-methoprene protected dogs from B. burgdorferi
p-Value – 0.0002
and A. phagocytophilum using dual infected field collected
85

I. scapularis ticks (McCall et al., 2011). The question about Beugnet, F., Marié, J.L., 2009. Emerging arthropod-borne diseases of
companion animals in Europe. Vet. Parasitol. 163, 298–305.
the speed of transmission and the early events after tick
Blagburn, B.L., Spencer, J.A., Billeter, S.A., Drazenovich, N.L., Butler, J.M.,
attachment may be easier to address by using in vitro tick Land, T.M., Dykstra, C.C., Stafford, K.C., Pough, M.B., Levy, S.A., Bledsoe,
feeding methods (Kröber and Guerin, 2007). D.L., 2004. Use of imidacloprid–permethrin to prevent transmission of
As discussed above, blocking of transmission of vector Anaplasma phagocytophilum from naturally infected Ixodes scapularis
ticks to dogs. Vet. Ther. 5, 212–217.
borne diseases is currently becoming a standard evaluation Chomel, B., 2011. Tick-borne infections in dogs – an emerging infectious
of highly effective acaricides. As a consequence, guidelines threat. Vet. Parasitol. 179, 294–301.
for evaluating efficacy of ectoparasiticides for treatment, EMEA, 2007. Guideline for the testing and evaluation of the efficacy of
antiparasitic substances for the treatment and prevention of tick and
prevention and control of tick infestations on dogs are flea infestation in dogs and cats. In: EMEA/CVMP/EWP/005/2000-
expected to be revised. This is important in order to accom- Rev.2.
modate the testing of their ability to block transmission Estrada-Pena, A., Reme, C., 2005. Efficacy of a collar impregnated
with amitraz and pyriproxyfen for prevention of experimen-
of tick-borne pathogens and prevent disease (Marchiondo tal tick infestations by Rhipicephalus sanguineus, Ixodes ricinus,
et al., 2007). and Ixodes scapularis in dogs. J. Am. Vet. Med. Assoc. 226 (2),
Finally, similar models have now been developed for 221–224.
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In conclusion, the imidacloprid/flumethrin collar was formulation of imidacloprid and permethrin (advantix) in dogs nat-
highly effective against challenge with D. reticulatus ticks urally infested with ticks and/or fleas. Parasitol. Res. 90 (Suppl. 3),
1 month after application. The high acaricidal efficacy S125–S126.
Jacobson, R., McCall, J., Hunter III, J., Alva, R., Irwin, J., Eschner, A., Jeannin,
resulted in a 100% protection level (95% confidence inter- P., Boeckh, A., 2004. The ability of fipronil to prevent transmission
val: 69–100%) of the collar to prevent transmission of of Borrelia burgdorferi, the causative agent of lyme disease to dogs. J.
Babesia canis by infected D. reticulatus ticks applied 1 month Appl. Res. Vet. Med. 2, 39–45.
Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitol-
after the dogs were treated with the collar. ogy 129 (Suppl.), 3–14.
Jongejan, F., Fourie, J.J., Chester, S.T., Manavella, C., Mallouk, Y., Pollmeier,
Author’s contributions M.G., Baggott, D., 2011. The prevention of transmission of Babesia canis
canis by Dermacentor reticulatus tick to dogs using a novel combi-
nation of fipronil, amitraz and (S)-methoprene. Vet. Parasitol. 179,
DS and JJF designed the study protocol and design, 343–350.
whereas JJF carried out the study. DS and JJF compiled and Kröber, T., Guerin, P.M., 2007. In vitro feeding assays for hard ticks. Trends
analysed the data. FJ wrote the first draft of the manuscript, Parasitol. 23, 445–449.
Last, R.D., Hill, J.M., Matjila, P.T., Rème, C.A., 2007. A field trial evaluation
which was subsequently revised and the final version of the prophylactic efficacy of amitraz-impregnated collars against
approved by all authors. canine babesiosis (Babesia canis rossi) in South Africa. J. S. Afr. Vet.
Assoc. 78, 63–65.
Competing interests Marchiondo, A.A., Holdsworth, P.A., Green, P., Blagburn, B.L., Jacobs,
D.E., 2007. World Association for the Advancement of Veteri-
nary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy
This clinical study was completely funded by Bayer of parasiticides for the treatment, prevention and control of
Animal Health GmbH Monheim, Germany, of which D. flea and tick infestation on dogs and cats. Vet. Parasitol. 145,
332–344.
Stanneck (Germany) is an employee. ClinVet, of which J.J. McCall, J.W., Baker, C.F., Mather, T.N., Chester, S.T., McCall, S.D., Irwin, J.P.,
Fourie is an employee, is an independent, South African, Young, S.L., Cramer, L.G., Pollmeier, M.G., 2011. The ability of a topical
Contract Research Organisation contracted to manage the novel combination of fipronil, amitraz and (S)-methoprene to pro-
tect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum
conduct of the study. F. Jongejan is PhD supervisor of JJF infections transmitted by Ixodes scapularis. Vet. Parasitol. 179,
and Professor at the Universities of Pretoria and Utrecht. 335–342.
All authors voluntarily publish this article and have no Matjila, P.T., Nijhof, A.M., Taoufik, A., Houwers, D., Teske, E., Penzhorn,
B.L., de Lange, T., Jongejan, F., 2005. Autochthonous canine babesiosis
personal interest in these studies other than publishing
in The Netherlands. Vet. Parasitol. 131, 23–29.
the scientific findings that they have been involved in via Øines, Ø., Storli, K., Brun-Hansen, H., 2010. First case of babesiosis caused
planning, initiating, monitoring and conducting the inves- by Babesia canis canis in a dog from Norway. Vet. Parasitol. 171,
350–353.
tigations and analysing the results.
Spencer, J.A., Butler, J.M., Stafford, K.C., Pough, M.B., Levy, S.A., Bledsoe,
D.L., Blagburn, B.L., 2003. Evaluation of permethrin and imidacloprid
Acknowledgement for prevention of Borrelia burgdorferi transmission from blacklegged
ticks (Ixodes scapularis) to Borrelia burgdorferi-free dogs. Parasitol. Res.
90 (Suppl.), 106–107.
The authors acknowledge the contributions of technical
Sréter, T., Széll, Z., Varga, I., 2005. Spatial distribution of Dermacentor
staff at Clinvet International (Republic of South Africa) in reticulatus and Ixodes ricinus in Hungary: evidence for change? Vet.
executing the study. Parasitol. 128, 347–351.
Stanneck, D., Kruedewagen, E.M., Fourie, J.J., Horak, I.G., Davis, W., Krieger,
K.J., 2012. Efficacy of an imidacloprid/flumethrin collar against fleas,
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86

by ticks
3.3. Beugnet F, Halos L, Larsen D, Labuschagné M, Erasmus H, Fourie JJ.

The ability of an oral formulation of afoxolaner to block the transmis-
sion of Babesia canis by Dermacentor reticulatus ticks to dogs. Parasit
Vectors. 2014, 7 (1) : 283.
87

88

Beugnet et al. Parasites & Vectors 2014, 7:283
RESEARCH Open Access
The ability of an oral formulation of afoxolaner

to block the transmission of Babesia canis by
Dermacentor reticulatus ticks to dogs
Frederic Beugnet1*, Lenaig Halos1, Diane Larsen1, Michel Labuschagné2, Heidi Erasmus2 and Josephus Fourie2
Abstract
Background: Canine babesiosis due to Babesia canis is an endemic disease in many European countries. A vaccine
is available in some countries, but it does not prevent the infection and just helps in reducing the gravity of clinical
signs. Therefore, the major way to help preventing the disease is by controlling tick infestations on dogs.
To assess the preventive efficacy of afoxolaner (NexGard®), a new oral anti- flea and tick product, against Babesia
canis infected adult Dermacentor reticulatus in an experimentally controlled study.
Methods: Sixteen healthy mixed breed adult dogs, negative for Babesia canis antibodies were included in a single
centre, randomized, blinded and controlled study to evaluate the impact of treatment with afoxolaner on the
transmission of Babesia canis to dogs exposed to Dermacentor reticulatus. The dogs were randomly allocated into
two groups of 8 dogs each. One group remained untreated. In the other group, dogs were treated orally with a
novel formulation of afoxolaner (NexGard®) on day 0. All dogs were infested each by 50 adult Dermacentor
reticulatus ticks (equal sex ratio) at days 7, 14, 21 and 28. The Dermacentor reticulatus ticks were confirmed to
harbour Babesia canis by Polymerase Chain Reaction (PCR).
Results: The treatment was well tolerated by all dogs without any adverse effects. Babesia canis was transmitted
by D. reticulatus to all untreated control dogs, confirmed following demonstration of hyperthermia, detection of
B. canis parasites in blood smears and PCR assay from blood and serology. These confirmed infected dogs were
subsequently treated with imidocarb and diminazene. The treated dogs remained negative based on all criteria
until the last study, Day 56, confirming that the oral treatment of dogs with NexGard® prevented transmission of
Babesia canis and development of clinical babesiosis for up to 28 days.
Conclusion: This is the first demonstration that an oral acaricidal treatment may prevent the transmission of a
pathogen despite the need for the tick to attach and start feeding before being killed by the acaricide.
Keywords: Babesia canis, Dogs, Dermacentor reticulatus, Transmission blocking, Afoxolaner
Background with areas of tick concentration dependent upon local

Ticks are endemic throughout Europe with more than environmental conditions [3]. Its distribution has been
twelve different species, of varying biology and geo- expanding in Europe [3-6]. The period of tick activity is
graphic distribution [1]. Ixodes ricinus and Dermacentor also increasing in Europe. For example, the total duration
reticulatus commonly infest dogs and are vectors of of Dermacentor questing activity over the year as well as
various canine vector borne pathogens including Bor- its presence in winter was shown to increase in Belgium,
relia burgdorferi and Anaplasma phagocytophilum, both Switzerland, Poland, Germany, Slovenia and Slovakia
transmitted by I. ricinus, and Babesia canis transmitted [1,7-9]. Climate change is one of the factors which could
by D. reticulatus [1,2]. D. reticulatus is widely distributed explain the change in distribution and in activity amongst
many others such as land use and host distribution [8,9].
* Correspondence: Frederic.beugnet@merial.com
Canine babesiosis is a clinically significant tick-borne
1
Merial S.A.S., 29 Av Tony Garnier, 69007 Lyon, France protozoan disease, which occurs worldwide. Historically
Full list of author information is available at the end of the article
© 2014 Beugnet et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
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89

Beugnet et al. Parasites & Vectors 2014, 7:283 Page 2 of 7
Babesia parasites in dogs were divided into two morpho- with South African animal welfare legislation. The study
logically distinct groups, the larger Babesia canis and the employed a controlled, blinded, randomized block de-
smaller Babesia gibsoni. B. canis has been reclassified sign and utilized adult, healthy, mongrel dogs. All dogs
into three sub-species (B. canis canis, B. canis rossi and were individually penned in tick-proof kennels, were
B. canis vogeli) on the basis of vector-specificity and managed similarly and observed once daily for health
cross-immunity and are now considered to be separate abnormalities throughout the study. When health ab-
species, B. canis, B. rossi and B. vogeli [10-12]. However, normalities were detected between the scheduled physical
both species and sub-species names remain in use in the examinations, additional examinations were conducted. In
current literature. Babesia canis is widely distributed order to control bias, the animals were not treated by a
throughout Europe, where it is transmitted by adult person involved in performing the post-administration as-
Dermacentor reticulatus ticks [13-17]. France is the sessments and observations.
most endemic country in Europe [18,19]. The clinical Two groups of 8 dogs were included, one untreated
signs of babesiosis in dogs vary from a mild transient ill- control and one NexGard® (Merial) treated group. The
ness to acute disease due to severe haemolysis that rap- treatment was administered on Day 0. All dogs were
idly results in death. Clinical findings include anorexia, infested by ticks on Days 7, 14, 21 and 28. The ticks were
pale mucous membranes, icterus, pyrexia, and splenic not removed and counted until Day 30. The dogs were
enlargement. blood sampled on Days −7, 14, 21, 28, 35, 42, 49, 56. At
Published studies demonstrating the utility of tick con- Day 56, the last study day, dogs were removed from their
trol compounds on dogs have been focused on their boxes and put back to their original runs. In order to check
acaricidal efficacy against a broad range of ixodid tick the maintenance of seropositivity in the infected dogs, a
species [20]. Some experimental studies have shown blood sample was taken for serology on Day 93.
the protective effect provided by topical insecticide/
acaricide. Efficacy is based on repellent/irritant effect by Animals
contact, inhibition of attachment and blood meal, and/or On Day −1, body weight of each dog was used for rank-
a quick speed of kill [21]. The protective effect of an acar- ing and group allocation purposes. The study followed
icidal molecule given orally to dogs and acting systemic- a randomised block design. The 16 dogs included on
ally is less obvious as the ticks must attach and start to Day −7 were ranked, within sex, in descending order
feed before being killed. Nevertheless, it has been demon- of individual body weight. Within each block, dogs
strated that pathogens need some time to be transmitted were randomly allocated to groups 1 or 2. The dogs were
[1]. Nevertheless, an acaricide may cause rapid death clinically healthy as verified by a veterinarian on Day −7;
after ingestion and or block feeding physiology of the they were older than 2 months; they weighed 11.97 kg to
tick. The present study describes the results of an experi- 21.43 kg; the females were not pregnant; and they had not
mental study to assess the efficacy of afoxolaner, a new been treated with a long acting topical or systemic acari-
insecticide-acaricide administered orally in a soft chew- cide/insecticide during the 12 weeks preceding Day −7. All
able formulation (Nexgard®, Merial), against D. reticula- the animals were observed daily from Days −7 to 56.
tus infected with Babesia canis in dogs. Afoxolaner They were tested PCR negative and sero-negative
belongs to a new class of insecticides-acaricides, the (IFAT) for Babesia canis on Day −7 at the start of
isoxazoline [22], acting systemically through oral for- acclimatization. They were then maintained individually in
mulation [23]. Afoxolaner is highly bound to plasmatic cages in a controlled tick free zone where all experimental
proteins and it is ingested by the hematophagous arthro- tick studies are conducted, in order to avoid any external
pods during their blood meal. It acts as a ligand to a spe- contamination.
cific receptor on both GABA and glutamate receptors on During the study, the dogs were examined clinically
the ion chloride channel in the neuron synapses. It in- daily to detect any signs of canine babesiosis including
duces hyperexcitation before death of both fleas and ticks. body temperature > 39.4°C.
The new oral formulation, NexGard®, has been proven to In addition to scheduled blood samples, a tem-
control flea and tick infestation (Ixodes, Dermacentor and perature > 39.4°C was the trigger for collection of a
Rhipicephalus) for a month on dogs [24]. blood sample, an immediate blood smear and a PCR.
All dogs confirmed positive for Babesia by blood smear
Methods received the appropriate treatment with imidocarb
Study design (Forray® 65) and diminazene (Berenil® RTU). By experience,
The study was conducted according to the International the combination of imidocarb and diminazene is chosen to
Cooperation on Harmonization of Technical Require- allow a single treatment without repeat treatment 14 days
ments for Registration of Veterinary Medicinal Products later, and to minimize the risk of relapse. Tick challenges
Guideline 9: Good Clinical Practice, and in compliance were discontinued for these animals.
90

Dog cages were part of an indoor animal unit in a tick Tick removal and count
free kennel, environmentally controlled for temperature The ticks were removed and counted on dogs diagnosed
(20 ± 4°C). The study animals were kept individually in for babesiosis and on all remaining dogs on Day 30.
cages and no direct contact between dogs was possible They were categorized as free or attached and dead or
during the study. alive, following WAAVP recommendation [25].
Treatment Blood collection and blood smear for Babesia canis

Eight dogs were treated on Day 0 with NexGard®, an oral detection
formulation of 2.27% w/w afoxolaner. They were dosed In addition to scheduled blood samples, a blood sam-
orally by giving a palatable chew by hand. They all ple was collected and a blood smear prepared in case
ingested the chew spontaneously when offered. All dogs of clinical suspicion of Babesia infection (body tem-
weighed between 10.1 to 25 kg and received the 3 g perature > 39.4°C). Blood sampling was realized on the
chew containing 68 mg afoxolaner. No vomiting was ob- cephalic vein of the arm. Blood was collected for PCR ana-
served and none of the animals was redosed. lysis and serology from all dogs diagnosed with babesiosis
(on the day of diagnosis, before rescue treatment) based
Tick challenges on blood smears and from all dogs on Days - 7, 14, 21,
A laboratory-bred Dermacentor reticulatus tick strain 28, 35, 42, 49, 56. A Day 93 post-study sample was
infected with Babesia canis was used for the chal- taken on all dogs to check the maintenance of the sero-
lenges. The Dermacentor reticulatus strain is main- logical status.
tained on dogs and derived from wild ticks collected Three-milliliter blood samples were collected in EDTA
in Europe. The D. reticulatus ticks were infected tubes for PCR analysis. Prior to starting the procedure,
with B. canis by feeding them on dogs previously in- approximately 1 mL of blood was taken from the 3 mL
fected intravenously by with a B. canis strain originated whole blood sample and stored in a cryo tube in a −80°C
from France. The Babesia strain was firstly isolated freezer (<70°C), which served as a secondary sample for
from a D. reticulatus female collected from a dog in PCR analysis. The remaining 2 mL whole blood sam-
France. It is conserved in frozen EDTA dog blood and ples were transferred to the ClinVet molecular laboratory
injected to dogs when needed for maintenance or to for analysis. Total DNA was isolated from individual ticks
initiate studies. (for batch infectivity determination and verification) and
A sample of 50 D. reticulatus ticks was taken weekly 200 μl whole blood (for diagnostic purposes) using a
from the batch of ticks to be used for challenges and the commercially available DNA isolation kit. Up to 400 ng
infectivity was tested and confirmed by PCR analysis be- isolated DNA served as template in a PCR assay based
fore the infestation of the dogs (Table 1). All dogs were on targeting 18 S rRNA ITS-1 gene regions originally
infested with 50 ticks each on Days 7, 14, 21 and 28. identified by Duarte et al. [26]. The following primers:
Adult ticks, which were used in the challenges, were un- Babesia canis 2F (5′-GGAAGGAGAAGTCGTAACAA
fed, at least one week old and had a balanced sex ratio GGTTTCC-3′) and B.canis 2R (5′-CAGTGGTCACA
(50% female: 50% male). For logistic reason, the tick in- GACCGGTCG-3′) were used. They are species specific
festations of the second week were split between Day 14 and there is no cross-reaction with Babesia vogeli and
and Day 15. Babesia rossi. The PCR method was developed and tested
Ticks were applied directly on the dog by tapping prior to the study at ClinVet. Babesia species have 2 to 5
the vial to dislodge the ticks from the container so copies of the rDNA targeted region present per genome
that they could be placed or spread directly over the [27]. Each set of species specific primers was tested
dog’s hair coat. Gloved fingers were used to facilitate against two individually confirmed cases each for B. canis
hair penetration by ticks once placed on the animal. and B. vogeli and one confirmed and one suspected case
Dogs were placed for approximately four hours in an of B. rossi. During this study, each PCR run included a
infestation crate to enhance tick attachment, then they positive control, negative control and a no template con-
were placed again in their individual box. Ticks dis- trol. An internal amplification control was employed to
lodged during the first 10 minutes were placed back on validate each PCR reaction success in order to eliminate
the dog. false negatives. The presence of an approximately 300 bp
PCR product, subsequent to agarose gel electrophoresis,
confirmed detection of Babesia canis DNA in the sample.
Table 1 Results of the weekly Babesia canis PCR on 50 ticks
from the batch used for dog infestations
The obtained PCR products were processed for sequence
analysis. Both strands were completely sequenced and as-
Day 7 Day 14 Day 21 Day 28
sembled sequence data was subjected to BLAST analysis
PCR results 10% 10% 8% 10%
where a 100% identity was obtained towards known
91

B. canis sequence data (GenBank accession number: Babesia infected (Table 1). The arithmetic mean tick
AF394533) as well as the Babesia canis originally used for counts recorded for the untreated control group 1 was
donor animal infection and then to infect the D. reticulatus 15.0 at Day 9 and 41.1 on Day 16, indicating a vigorous
tick vector. tick challenge.
Another 3 mL of blood was collected from each dog
for serology. Serum was recovered from the plain tubes Blood smear evaluation results
and divided into primary and duplicate aliquots. Primary In addition to scheduled blood smears at the start of the
aliquots were stored at 2°C to 8°C for two days until study, blood smears were prepared and examined for the
assayed for Babesia canis antibodies, using a commercial presence of B. canis sporozoites for all dogs with pyrexia
IFA test carried out as described by Uilenberg et al. [10]. (>39.4°C) from Day 7 onwards after first tick infestation
Duplicate aliquots were frozen at < −35°C. For screening (Table 2). Babesia canis sporozoites were observed on
purposes the sera were diluted at 1:80 and results were blood smears from all dogs in the untreated control group
expressed as positive (fluorescence at dilution 1:80) or on at least one occasion. No parasites were observed on
negative (no fluorescence). One positive IFA result was any of the blood smears from the treated dogs.
considered sufficient and therefore testing of serum was
discontinued after a first positive result on a dog. Never- Tick counts and tick count efficacy data
theless a post-study Day 93 serology was performed on a Live ticks were present on all control dogs at the time
blood sample from each dog. they were diagnosed positive for Babesia (Day 15 – 16)
(Table 2). Infestations were relatively high (8 – 67 ticks).
Effectiveness assessment In contrast, only a few dead ticks (0 – 6) were observed
The primary assessment criterion was the number of on treated dogs on Day 30 at the end of tick phase of
dogs infected with Babesia canis in the control and the study. This reinforces the efficacy of afoxolaner
treated group. against Dermacentor reticulatus ticks (Table 3). The
count and the categorization of ticks on the treated
Statistical analysis dogs showed that only a few dead attached ticks were
An efficacy failure (successful infection with Babesia) observed.
was regarded as a dog in the Nexgard® treated group that
was tested serologically positive for Babesia canis anti- Babesia canis infection
bodies or tested positive for Babesia canis by PCR analysis All dogs in the untreated control group were positive for
or tested positive by blood smear direct examination. babesiosis based on blood smear and PCR analysis.
The infection rate was calculated in the treated group Seven out of the eight control dogs became serologically
at the end of the study, Day 56, and was compared sta- positive on Day 21 and remained positive until the last
tistically with that of the control group. study Day 56 (Table 4). All control dogs were Babesia
The percentage blocking efficacy was calculated as infected after the two tick infestations on Day 7 or
follows: Day 14/15. None of the dogs in the treated group was
positive for babesiosis on blood smear, IFAT or PCR
Efficacy ð%Þ ¼ 100 � ðTc−TtÞ=Tc; analysis during the 56 days of the study. Nexgard®
treatment was therefore regarded to be fully effective
where: Tc = Total number of infected dogs in the nega- in preventing the transmission of Babesia canis by in-
tive control groupTt = Total number of infected dogs in fected Dermacentor reticulatus ticks (100% preventive
the NexGard® administration group. efficacy, p = 0.0014).
The proportion of animals infected in each group was At Day 93, the 7 seropositive control dogs were still
also compared using the chi-square test or Fisher’s exact positive. Surprisingly, one previously negative treated
test as applicable. SAS® Version 9.3 TS Level 1 M2 was dog was found slightly positive by IFAT (considered
used for all the statistical analyses. The level of signifi- positive by one examiner and negative by another). PCR
cance of the formal tests was set at 5%, all tests were and blood smear were negative. This particular dog was
two sided. negative for blood smear, PCR and serology throughout
the study until day 56 and did not show any clinical
Results signs of infection.
Tick infectivity
A sample of 50 D. reticulatus ticks was taken from the Discussion
batch of ticks to be used for each artificial challenge and Conduct of this study required the generation of a large
the infectivity confirmed by PCR analysis. Results of batch of ticks with an adequate Babesia infection rate.
the PCR analysis indicated that 8 – 10% of ticks were The rates of infection calculated by PCR on ticks were
92

Table 2 Results of blood smear examination of dogs having a temperature > 39.4°C on a daily observation
Body temp range (°C) Blood smear preparation and examination day
Group
Min Max 14 15 20 21 25 26 28 29 35 42 49
38.0 40.8 NEG POS - - - - - - - - -
37.7 39.7 - POS - - - - NEG - NEG - -
38.0 39.6 POS NEG - - - - - - NEG NEG -
38.1 39.8 POS NEG - - - - - - - NEG -
1
38.2 39.8 - POS - - - - - - - - -
37.0 40.4 NEG POS - - - - - - - - -
38.2 39.5 - POS - - - - - - - NEG -
37.5 40.4 NEG POS - - - - - - - - -
37.6 39.5 - - - NEG NEG - - - - NEG -
37.5 39.2 - - - - - - - - - NEG -
37.9 40.0 NEG NEG NEG - - - NEG - - NEG NEG
38.0 39.3 - - - - - - - - - - -
2
37.8 41.3 NEG NEG NEG NEG - - NEG - NEG NEG NEG
38.1 39.4 - - - - - - NEG - NEG NEG -
37.9 39.4 - - - - - - NEG - - - -
37.6 40.1 NEG NEG NEG - - NEG NEG NEG NEG NEG NEG
Positive results are in bold.
8-10% during one month. This is similar to what was de- was sufficiently high to successfully transmit the infection
scribed by Fourie et al. [28] assessing the efficacy of to 8 out of 8 untreated (control) dogs. All untreated dogs
the combination of fipronil-amitraz-(S)-methoprene were infected and were rescue treated immediately after
(Certifect®). Regardless of which sex or if both sexes diagnosis. The early treatment can explain the absence
transmit the B. canis, this infection rate in the ticks of the classical clinical signs of babesiosis, including fever
Table 3 Results of tick counts on dogs

Study Live: male Live: female Live: male Live: female Dead: male Dead: female Dead: male Dead: female
Group
day* free free attached attached free free attached attached
15 Control 0 0 32 35 0 0 0 0
15 Control 0 0 28 31 0 0 0 0
16 Control 1 0 16 15 2 0 0 0
16 Control 0 0 25 24 1 0 1 0
16 Control 0 0 17 18 0 0 0 0
16 Control 0 0 24 24 0 0 2 0
16 Control 0 0 6 10 2 0 1 1
16 Control 0 0 16 16 0 1 0 0
30 Treated 0 0 0 0 2 3 0 0
30 Treated 0 0 0 0 1 1 0 0
30 Treated 0 0 0 0 2 2 0 1
30 Treated 0 0 0 0 0 1 0 0
30 Treated 0 0 0 0 0 0 2 0
30 Treated 0 0 0 0 3 2 1 0
30 Treated 0 0 0 0 0 0 1 0
30 Treated 0 0 0 0 0 0 0 0
*Study day where the dogs were removed due to canine babesiosis; or Day 30, 48 h after the last tick infestation.
Tick numbers are indicated in bold when it is >0.
93

Table 4 Summary of assessment for B. canis infection

-7 14/15 +21 +28 +35 +42 +49 +56 (last study Day) + 93
Group
IFAT IFAT PCR IFAT PCR IFAT PCR IFAT PCR IFAT PCR IFAT PCR IFAT PCR IFAT
NEG NEG POS POS POS POS NEG NEG NEG NEG POS
NEG NEG POS POS NEG NEG NEG NEG NEG NEG POS
Control
NEG NEG POS POS POS NEG NEG NEG NEG NEG POS
NEG NEG POS NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG +/− POS*
NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
Treated
*This dog did not show any clinical signs, and remained Blood smear, PCR and serology negative until the end of the study (Day 56). He was found to be positive
by one examiner at Day 93, showing a diffuse fluorescence including dog cells and some Babesia canis, which was considered as negative by another examiner.
Positive results and final check at Day 56 and Day 93 are indicated in bold.
syndrome, anemia, hemolysis and hemoglobinuria. It also Fipronil-amitraz-(S)-methoprene demonstrated 86% effi-
possibly explains that one control dog, despite a positive cacy against transmission using a similar protocol [21].
blood smear and PCR remained serologically negative. This topical formulation is known to induce a quick
Regarding the treated dog that possibly seroconverted on death, in less than 24 h and a detachment or a preven-
Day 93 post-study, as all dogs were moved back to their tion of attachment of ticks [30].
community runs on Day 56, it is not known whether it is a
late seroconversion, a more recent infection that appeared Conclusion
after the study, or a false positive fluorescence due to an- The oral formulation of afoxolaner demonstrated in this
other reason. experimental study a complete efficacy in preventing the
Protozoan parasites require additional time, usually transmission of Babesia canis.
several days, for their sporoblasts to mature into sporo-
Competing interest
zoites in the salivary glands of the tick before they can The work reported herein was funded by Merial SAS, France. The authors are
be secreted into the saliva and transmitted to the mam- current employees or contractors of Merial.
malian host. For instance, Babesia microti is transmitted ®CERTIFECT and NEXGARD are registered trademarks of Merial. All other marks
are the property of their respective owners.
by Ixodes scapularis between 36 and 48 hours after tick This document is provided for scientific purposes only. Any reference to a
attachment [29]. The treatment assessed here is an oral brand or trademark herein is for informational purposes only and is not
formulation, meaning that ticks need to attach and start intended for a commercial purpose or to dilute the rights of the respective
owner(s) of the brand(s) and trademark(s).
to feed before being killed. Afoxolaner efficacy against
Dermacentor reticulatus has been demonstrated for a Authors’ contributions
full month [24] with ticks evaluated at 48 hours after All authors read and approved the final version of the manuscript.
each infestation. The protection against the transmission
Acknowledgment
of Babesia canis demonstrated in this study is a conse- The authors would like to thank all the technicians from ClinVet who take
quence of the time needed for ticks to transmit Babesia. care of the dogs during the study.
It may also be due to the action of afoxolaner on the tick
Author details
feeding physiology or on the rapid tick death. The fact 1
Merial S.A.S., 29 Av Tony Garnier, 69007 Lyon, France. 2ClinVet International
that no live ticks were found at Day 28 and very few at- (Pty) Ltd, P.O. Box 11186, Universitas, Bloemfontein 9321, Republic of South
tached ticks is suggestive of a quick death but also de- Africa.
tachment, which may also impact on the transmission Received: 7 April 2014 Accepted: 3 June 2014
of Babesia. Another study involving the combination of Published: 23 June 2014
94

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www.biomedcentral.com/submit
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transmission by ticks
4.1. Fourie JJ, Ollagnier C, Beugnet F, Luus HG, Jongejan F.

Prevention of transmission of Ehrlichia canis by Rhipicephalus sangui-
neus ticks to dogs treated with a combination of fipronil, amitraz and
(S)-methoprene (CERTIFECT©). Vet Parasitol. 2012, 193 : 223-228.
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98

Contents lists available at SciVerse ScienceDirect
Prevention of transmission of Ehrlichia canis by Rhipicephalus

sanguineus ticks to dogs treated with a combination of fipronil, amitraz
and (S)-methoprene (CERTIFECT® )
Josephus J. Fourie a , Catherine Ollagnier b , Frederic Beugnet b,∗ , Herman G. Luus a ,
Frans Jongejan c,d
a
ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa
b
Merial SAS, 29 Avenue Tony Garnier, 69007 Lyon, France
c
d
Article history: The ability of CERTIFECT® (a combination of fipronil, amitraz and (S)-methoprene) to pre-
Received 3 October 2012 vent transmission of Ehrlichia canis was studied in two groups of eight dogs. One group
Received in revised form
was treated with CERTIFECT while the other group remained untreated. All dogs were
29 November 2012
exposed to E. canis-infected Rhipicephalus sanguineus ticks on Days 7, 14, 21 and again
Accepted 5 December 2012
on day 28 post-treatment by releasing ticks into the kennels of the dogs to simulate the
natural way of infestation. Animals were examined in situ for ticks on Days 9, 16 and 23
Keywords:
Fipronil–amitraz–(S)methoprene and any ticks present were counted and removed on Day 30. The efficacy of CERTIFECT
Rhipicephalus sanguineus against R. sanguineus was 100%, since no ticks were found on the treated dogs at any time.
Ehrlichia canis Clinical examinations (including monitoring body temperature and blood collections for
Dog PCR analysis and serology) were performed at intervals throughout the study until Day 56.
Transmission Five out of 8 untreated control dogs (62.5%) became infected with E. canis, as demonstrated
Prevention by detection of specific E. canis antibodies and the presence of E. canis DNA in blood sam-
ples by PCR. These dogs displayed fever and thrombocytopenia and were rescue-treated
with doxycline. None of the 8 dogs treated with CERTIFECT became infected with E. canis,
in comparison to the 5/8 control dogs, as confirmed by the lack of specific antibodies and
absence of any ehrlichial DNA in blood samples by PCR. CERTIFECT prevented transmission
of E. canis and effectively provided protection against monocytic ehrlichiose for at least 4
weeks post treatment.
1. Introduction anaplasmosis (Anaplasma phagocytophilum), Lyme Disease

(Borrelia burgdorferi) and canine monocytic ehrlichiosis
Globally, dogs are exposed to a broad range of pro- (Ehrlichia canis) are the most common tick-borne diseases
tozoan and bacterial pathogens transmitted through the of dogs (Shaw et al., 2001; Jongejan and Uilenberg, 2004).
bite of infected vector ticks (Chomel, 2011). In par- Relatively little research has been carried out to deter-
ticular, canine babesiosis (Babesia spp.), granulocytic mine the ability of tick control products to prevent the
transmission of tick-borne diseases to dogs. Most pub-
lished studies are aimed at demonstrating the acaricidal
∗ Corresponding author at: Merial, 29 Av Tony Garnier, 69007 Lyon,
product’s efficacy against the range of ixodid tick species.
Guidelines for evaluating the efficacy of ectoparasiticides
France.
E-mail address: frededric.beugnet@merial.com (F. Beugnet). on dogs and cats focus on treatment, prevention and
99

control of ticks only (EMA, 2007; Marchiondo et al., 2007). tick-proof kennels, and, throughout the study, they were
Hence, the development of models wherein the blocking of observed twice daily for health abnormalities. When
pathogen transmission can be demonstrated is a relatively health abnormalities were observed between the sched-
new area of research. Studies that have been conducted uled physical examinations, additional full examinations
suggest that topically applied tick control compounds can were conducted. The dogs, all negative for E. canis, prior
aid in the prevention of the transmission of specific tick- to treatment and/or first tick challenge, as shown by the
borne pathogens. For instance, the ability of fipronil to absence of specific antibodies in the indirect fluorescent
prevent transmission of B. burgdorferi by field-collected assay (IFA), were divided randomly into two equal groups.
Ixodes scapularis ticks was reported several years ago by Group 1 was designated the untreated controls and Group
Jacobson et al. (2004). 2 dogs were treated once with the combination of fipronil,
Recently, CERTIFECT® (Merial, GA, USA), a combination amitraz and (S)-methoprene spot-on on Day 0.
of fipronil, amitraz and (S)-methoprene in a spot-on for- CERTIFECT was topically applied on each treated dog
mulation. It has been published that fipronil and amitraz in two spots, as per the label to deliver at least 6.7 mg
act synergistically in this formulation, providing a high fipronil/kg bodyweight (bw), 8.0 mg amitraz/kg bw, and
speed of kill but also a good prevention of tick attachment 6.0 mg (S)-methoprene/kg bw. The pipette size was based
(Pfister, 2011; Prullage et al., 2011). These two properties on the weight of the dogs as per product labelling.
may explain a good indirect prevention of pathogen trans-
mission by the ticks. This formulation was therefore shown 2.2. Infection of ticks by E. canis
to protect dogs from B. burgdorferi and A. phagocytophilum
infections transmitted by field-collected I. scapularis ticks A laboratory-bred R. sanguineus strain of French origin
(Pfister, 2011; McCall et al., 2011). Moreover, a labora- was used as a source to create an Ehrlichia infected pop-
tory transmission blocking model was developed recently ulation and subsequently to do the artificial challenges
to evaluate the ability of topical formulations to prevent in a kennel environment. The ticks had been maintained
Babesia canis transmission in dogs. In this model, treat- for several generations under laboratory conditions. The
ment of dogs with CERTIFECT applied up to 28 days prior to immature and adult stages of the ticks in the breeding
infestation with adult Dermacentor reticulatus ticks infected program were fed on rabbits, not previously treated with
with B. canis successfully prevented the development of any acaricides. E. canis was isolated from a local case of
clinical signs of canine babesiosis (Jongejan et al., 2011). canine monocytic ehrlichiosis (Bloemfontein, South Africa)
This paper focuses on the prevention of canine mono- by subinoculation of blood into a susceptible laboratory-
cytic ehrlichiosis, which is caused by the rickettsial bred Beagle dog. R. sanguineus nymphs fed on this recipient
pathogen E. canis, is of worldwide importance, and orig- dog acquired the infection, and, after moulting, the adult
inally reported from Algeria (Donatien and Lestoquard, ticks were used as a basis for the current study. The iden-
1935). E. canis is transmitted transstadially and intrasta- tity of E. canis was confirmed by comparing the partial
dially by the brown dog tick, Rhipicephalus sanguineus sequence of the p36 gene of E. canis with that of a number
(Bremer et al., 2005). The majority of cases of monocytic of other E. canis isolates, whereby the South African isolate
ehrlichiose are seen in sub-tropical regions hospitable to appeared closely related and formed a clade with several
R. sanguineus ticks. Due to the host preference of R. san- Asian isolates (Fourie, unpublished data).
guineus, dogs serve both as a reservoir and a domestic Prior to the study a sample of ticks from the challenge
animal host for E. canis (Stich et al., 2008). In dogs, E. canis batch of infected ticks was tested for E. canis, and 38% of
develops in monocytes and macrophages, whereas in ticks ticks were found infected. In addition, a further sample of
the infection can be localized in the midgut and salivary 50 ticks (25 males and 25 females) from the same batch
glands. Monocytic ehrlichiosis is characterized by throm- was pre-tested by feeding on a susceptible naive dog and
bocytopenia, leukopenia, fever, depression and bleeding resulted in transmission of E. canis. Hence, a rate of 38% of R.
tendencies (Harrus et al., 1999; de Castro et al., 2004). sanguineus ticks infected with E. canis was considered ade-
Here, we tested the ability of a spot-on formulation quate under the conditions of the model and represented
combining fipronil, amitraz and (S)-methoprene, to pre- a realistic challenge.
vent transmission of E. canis to dogs exposed to infected R.
sanguineus ticks. 2.3. Infestations by R. sanguineus ticks
2. Material and methods On Days 7, 14, 21 and 28, 50 ticks were released into each
individual kennel of each dog to simulate the natural expo-
2.1. Study design sure to ticks. The potentially Ehrlichia infected adult ticks,
which were used in the artificial challenges, were unfed,
This study was carried out according to the International at least one week old, and had a balanced sex ratio of 50%
Cooperation on Harmonization of Technical Requirements female and 50% male ticks.
for Registration of Veterinary Medicinal Products Guide-
line: Good Clinical Practice (EMA, 2000) and in compliance 2.4. Monitoring of dogs
with appropriate animal welfare requirements. The study
employed a controlled, blinded, randomized block design All dogs were observed daily (from Days −7 to 56)
with 6 males and 10 females fully grown, healthy, Bea- for general health condition and clinical signs or adverse
gle dogs. All dogs were managed similarly in individually reactions to the treatment. The dogs (both groups) were
100

observed approximately every hour for 4 h after initiation 5 = killed, attached and unengorged; 6 = killed, attached and
of the treatment. Rectal body temperatures were recorded engorged.
daily from Days 17 to 51 and from Days 54 to 56. All During the thumb counts on dogs, genders were not dis-
dogs were subjected to a clinical examination on Days tinguished, but ticks were categorized. The ticks counted
−7, 21, 28, 35 42, 49 and 56. Additional clinical examina- and removed on Day 30 were categorized within gender
tions were conducted on dogs displaying an abnormally (male/female) according to categories 1–6. Additionally,
high body temperature (>39.5 ◦ C). Clinical examinations the pens of the dogs were inspected daily from Day 14 up
included general appearance, respiration rate, heart rate to Day 30 for engorged detached ticks. For each dog these
and body temperature. Particular attention was given to ticks were collected and preserved in 70% ethanol.
clinical manifestations of monocytic ehrlichiosis, which
include fever, depression, anorexia, haemorrhages and 2.7. Statistical analysis
epistaxis.
Blood samples for serology, platelet counts and PCR Any dog serologically positive for E. canis antibodies
were collected on Days −7, 7, 21, 28, 35, 42, 49 and Day and positive for E. canis by PCR analysis was regarded as
56. All samples collected, including those taken from dogs infected. To determine the effectiveness of the treatment,
with suspected ehrlichiosis (e.g., abnormally low platelet the total number of ticks that were assigned to categories
count) and/or dogs that tested positive for E. canis antibod- 1, 2, 3, and 6 were transformed to the natural logarithm
ies using the IFA, but also asymptomatic dogs from both of (count + 1) for calculation of geometric means. For the
groups, were PCR assayed (Table 3). treated group, the percent reduction in tick counts com-
Dogs with abnormally low platelet counts or fever pared to the untreated control was computed using the
(>39.5 ◦ C) were suspected of starting clinical ehrlichiosis formula 100 × (1 − T/C), wherein T and C were the geo-
and rescue treated with doxycycline per os for 21 days. metric means of the particular treated and control group,
respectively. The proportions of the animals infected in
each group were compared by a Chi-square test. SAS® Ver-
2.5. Laboratory assays
sion 8 (Release 8.02 TS Level 02M0) was used for all the
statistical analyses. The level of significance of the formal
Sera were assayed for E. canis antibodies using a com-
tests was set at 5%.
mercial IFA test (IGG IFA, Fuller Laboratory) performed
according to the manufacturer descriptions at the Depart-
3. Results
ment of Veterinary Tropical Diseases (DVTD), Faculty of
Veterinary Science, University of Pretoria, South Africa.
3.1. Tick counts
EDTA blood samples for platelet counts were exam-
ined at Pathcare Veterinary Laboratory, Bloemfontein,
The arithmetic and geometric mean tick counts
South Africa. In addition, DNA was extracted from EDTA
recorded for the two study groups are summarized in
blood samples using a commercial genomic DNA iso-
Table 1. On all assessment days, statistically significant
lation kit. Isolated DNA was used as a template for a
(p < 0.0006) fewer ticks were recorded 48 h after each
PCR that employed sequence specific primers for the
weekly exposure on the treated dogs compared to the
detection of E. canis target DNA. Primer pair ECAdsbF
untreated control dogs. Efficacy values (%) based on arith-
(5� -GCAAGTGCGGGCAGAGAATGAAG-3� ) and ECAdsbR (5� -
metic mean tick counts for the group treated once with the
GTATCCCCTACTATGATAGCAGGAGTGC-3� ) was used to
combination of fipronil, amitraz and (S)-methoprene spot-
amplify the disulphide oxidoreductase gene (AF403710).
on are summarized in Table 1. The combination fipronil,
PCR mixtures were separated electrophoretically using
amitraz and (S)-methoprene was fully effective (100%)
agarose gel electrophoresis and visualized under UV exci-
against all weekly exposures to R. sanguineus ticks up to
tation after staining with ethidium bromide. Necessary
at least four weeks post treatment.
controls (positive control, negative control, no template
control, as well as an internal amplification control to
3.2. Transmission blocking of E. canis
validate the reaction) were included for each batch. An
amplified fragment of 500 bp indicated the presence of E.
There were no adverse effects of the treatment admin-
canis DNA in the sample.
istered as a topical solution. During the study, no health
abnormalities, other than signs of ehrlichiosis in controls,
2.6. Tick counts were observed. Five untreated control dogs developed
fever (>39.5 ◦ C) between Day 28 and Day 35 and received
Tick thumb counts were performed on dogs 48 hours doxycycline 10 mg/kg treatment for 21 days starting on
(h) after each exposure, except on Day 30 when all ticks Day 30 (4 dogs) or on Day 35 (one dog). All of these dogs
were counted and removed. To simulate natural expo- recovered fully. Low platelet counts (<200 × 109 /L) were
sure to the ticks, it was decided not to remove ticks from observed in the same five untreated control dogs but not
dogs until the end of the study. Tick counts were recorded in any of the treated dogs. Thrombocytopenia was evi-
according to the following six categories: 1 = live and free; dent with platelets counts as low as 2 and 4 × 109 /L were
2 = live, attached and unengorged (no filling of the alloscu- recorded on Day 28 and Day 35, respectively. These values
tum evident); 3 = live, attached and engorged (obvious or returned to normal between 200 and 500 × 109 /L on Day 42
conspicuous filling of the alloscutum); 4 = killed and free; as a result of the doxycycline treatment (Table 2). None of
101

Table 1
Tick counts on dogs 48 h after exposure in crates.
Day Group 1 – Untreated control Group 2 – (CERTIFECT® spot-on dogs)
Arithmetic mean Geometric mean Arithmetic mean Geometric mean (% efficacy)
9a 10.1 9.5 0.0 0.0 (100%)

16a 12.9 11.5 0.0 0.00 (100%)
23a 13.0 10.7 0.0 0.00 (100%)
30 23.0 20.4 0.0 0.00 (100%)
a
In situ counts.
the CERTIFECT treated animals developed illness, although sanguineus ticks during a full month after treatment,
one dog displayed a transient fever on Day 21 but platelet which indirectly reduce the risk of pathogen transmission
counts were normal. No rescue treatment was started on (Prullage et al., 2011).
this dog as the temperature subsided and its serological and As far as the prevention of transmission of tick-borne
PCR status remained negative during the whole study. pathogens is concerned, field studies conducted in West
The IFA assay results are summarized in Table 3. All dogs Africa revealed that fipronil by itself could reduce the
tested negative for E. canis antibodies prior to the first tick transmission of E. canis to dogs (Davoust et al., 2003).
challenge. Five untreated control dogs (Group 1) developed Furthermore, it has been demonstrated that the applica-
specific E. canis antibodies detected first on Day 21 (3 dogs) tion of a combination of 10% imidacloprid/50% permethrin
or Day 28 (2 dogs) and remained positive through the end of reduced E. canis infection in dogs under field conditions
the study. The same untreated control dogs that developed in southern Italy (Otranto et al., 2008). McCall et al. (2011)
specific E. canis antibodies were confirmed PCR positive. studied the transmission blocking capacity of fipronil, ami-
None of the treated dogs showed seroconversion neither traz and (S)-methoprene under laboratory conditions using
PCR positive result (Table 3). field-collected I. scapularis ticks. The latter authors demon-
In total, 5 of 8 dogs (62.5%) became infected with E. canis strated that CERTIFECT was able to protect dogs against B.
in the untreated control group and none in the CERTIFECT burgdorferi and A. phagocytophilum infections. In addition,
treated group (p = 0.007). a transmission blocking model of B. canis was developed
recently based on experimental batches of infected D.
4. Discussion reticulatus ticks. In this study it was shown that CERTI-
FECT applied up to 28 days prior to infestation with B.
The control of infestations of R. sanguineus using CER- canis infected D. reticulatus ticks successfully prevented the
TIFECT on dogs has been reported previously, whereby an development of clinical signs of canine babesiosis (Jongejan
efficacy of (98.4–100%) was achieved in the first 48 h post et al., 2011).
treatment (Hunter et al., 2011). In addition, the improved Here we show that using a tick exposure study model
speed-of-kill for this combination of fipronil and amitraz is successful in demonstrating prevention of transmis-
has been demonstrated on dogs experimentally infested sion of E. canis by infected R. sanguineus ticks. Both
by R. sanguineus ticks with an acaricidal efficacy > 96% as of these transmission blocking study models could be
early as 18 h following infestations for at least 35 days used to test other tick control compounds. Furthermore,
(Hunter et al., 2011). It has also been demonstrated that this adaptations of these studies to measure the transmis-
combination was able to prevent the attachment of R. sion blocking of other tick-borne pathogens, such as
Table 2
Thrombocyte counts in treated and control dogs.
Group Animal ID Day 21 (×109 /L) Day 28 (×109 /L) Day 35 (×109 /L) Day 42 (×109 /L)
Control * CC3 3F3 226 7 234 365

9AF 5EA 336 327 332 304
958 EB5 301 2 448 496
8AF 142 345 18 293 408
6DF 0EF 458 210 4 367
958 B91 216 2 214 308
B8C B85 600 557 518 442
CCF C02 361 376 376 366
CERTIFECT treated E9E E23 399 341 375 383

group E9F F45 261 348 360 354
CD0 108 315 279 324 291
9AE D8D 333 292 365 367
DF4 8A3 370 359 276 414
9B0 374 395 391 446 384
954 C83 544 538 550 462
DF6 ADF 450 289 460 407
A rescue treatment was started on all dogs with a platelet count <200 × 109 /L.
*
Platelet counts normal range 200–500 × 109 /L.
102

Table 3
Ehrlichia canis antibodies determined by IFA test and PCR results.
Group Animal ID <Day −7 Day 7a Day 21 Day 28 Day 35 Day 42 Day 49 Day 56
Control CC3 3F3 neg/neg neg/neg neg/neg POS/neg POS/POS POS/POS POS/neg POS/neg
9AF 5EA neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
958 EB5 neg/neg neg/neg POS/neg POS/neg POS/POS POS/neg POS/neg POS/neg
8AF 142 neg/neg neg/neg POS/neg POS/neg POS/POS POS/POS POS/neg POS/neg
6DF 0EF neg/neg neg/neg neg/neg POS/POS POS/POS POS/POS POS/neg POS/neg
958 B91 neg/neg neg/neg POS/neg POS/neg POS/POS POS/neg POS/POS POS/neg
B8C B85 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
CCF C02 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
CERTIFECT treated E9E E23 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
group E9F F45 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
CD0 108 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
9AE D8D neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
DF4 8A3 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
9B0 374 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
954 C83 neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
DF6 ADF neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg neg/neg
a
Prior to tick challenge. POS, positive for E. canis antibodies; neg, negative for E. canis antibodies; /POS, positive for PCR; /neg = negative for PCR.
Babesia vogeli transmitted by R. sanguineus ticks become Frederic Beugnet) are currently employees of Merial,
feasible. however, there were no conflicting interests that may
A prerequisite for the present study was the availabil- have biased the work reported in this paper.
ity of a large batch of R. sanguineus ticks with an adequate
level of infection with E. canis. An infection rate of 38% of Acknowledgements
the ticks was considered adequate under the conditions of
the model and represented a realistic challenge. This con- The authors gratefully acknowledge the expert contri-
clusion is further supported by the fact that infection rates butions of all technical staff at ClinVet International Ltd. in
of R. sanguineus for E. canis in the field are usually lower conducting the study to high standards.
than 38%. For instance, in northeastern Brazil the preva- CERTIFECT® is a registered trademark of Merial. All
lence of E. canis in R. sanguineus ticks removed from dogs other marks are the property of their respective owners.
and analysed by PCR was 21.9% (Souza et al., 2010).
Reliable laboratory tests are required to confirm the References
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dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infec- Stich, R.W., Schaefer, J.J., Bremer, W.G., Needham, G.R., Jittapalapong, S.,
tions transmitted by Ixodes scapularis. Vet. Parasitol. 179, 335–342. 2008. Host surveys, ixodid tick biology and transmission scenarios as
Otranto, D., Paradies, P., Testini, G., Latrofa, M.S., Weigl, S., Cantacessi, C., related to the tick-borne pathogen, Ehrlichia canis. Vet. Parasitol. 158,
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4.2. Fourie JJ, Luus HG, Stanneck D, Jongejan F.

The efficacy of Advantix© to prevent transmission of Ehrlichia canis to
dogs by Rhipicephalus sanguineus ticks. Parasite. 2013, 20 : 36.
105

106

Parasite 2013, 20, 36
J. J. Fourie et al., published by EDP Sciences, 2013
DOI: 10.1051/parasite/2013037
Available online at:
www.parasite-journal.org
RESEARCH ARTICLE OPEN ACCESS
The efficacy of Advantix to prevent transmission of Ehrlichia

canis to dogs by Rhipicephalus sanguineus ticks
Josephus Johannes Fourie1,*, Herman Gerhardus Luus1, Dorothee Stanneck2, and Frans Jongejan3,4
1
ClinVet International (Pty) Ltd, PO Box 11186, Universitas, Bloemfontein 9321, South Africa
2
Bayer Animal Health GmbH, BHC Business Group Animal Health, Clinical Development, Building 6700, 51368 Leverkussen,
Germany
3
Utrecht Centre for Tick-Borne Diseases (UCTD), Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht,
The Netherlands
4
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04,
Onderstepoort 0110, South Africa
Received 7 June 2013, Accepted 5 October 2013, Published online 21 October 2013
Abstract – The capacity of a topical combination of imidacloprid and permethrin (Advantix) to prevent transmission
of Ehrlichia canis was studied in two groups of six dogs. One group served as controls, whereas the other group was
treated. All dogs were exposed to E. canis-infected Rhipicephalus sanguineus ticks on Days 7, 14, 21 and Day 28 post
acaricidal treatment. The adult R. sanguineus ticks were released into the individual kennels of the dogs to simulate
natural tick exposure. In situ tick counts were conducted on Day 9, 16 and 23 and any remaining ticks were counted
and removed on Day 30. The efficacy of the acaricidal treatment against R. sanguineus ranged between 96.1% and
98.9% at 48 h post-application and lasted up to 4 weeks. Four out of six control dogs became infected with E. canis,
as demonstrated by the presence of specific E. canis antibodies and the detection by PCR of E. canis DNA in blood
samples. These dogs became thrombocytopenic and displayed fever and were consecutively rescue-treated by doxycy-
cline. None of the six treated dogs became infected with E. canis, as confirmed by the lack of specific antibodies and
absence of E. canis DNA in blood samples. Advantix prevented transmission of E. canis and provided protection
against monocytic ehrlichiosis for 4 weeks post acaricidal treatment.
Key words: Imidacloprid, Permethrin, Ehrlichia canis, Rhipicephalus sanguineus, Ehrlichiosis, Transmission.
Résumé – Efficacité d’Advantix pour prévenir la transmission d’Ehrlichia canis aux chiens par les tiques
Rhipicephalus sanguineus. La capacité d’une association locale d’imidaclopride et perméthrine (Advantix) pour
prévenir la transmission d’Ehrlichia canis a été étudiée dans deux groupes de six chiens. Un groupe a servi de
témoin, tandis que l’autre groupe a été traité. Tous les chiens ont été exposés à des tiques Rhipicephalus
sanguineus infectées par E. canis aux jours 7, 14, 21 et 28 jours après traitement acaricide. Les R. sanguineus
adultes ont été lâchés dans les niches individuelles des chiens pour simuler une exposition naturelles aux tiques.
Des comptages de tiques in situ ont été menés aux jours 9, 16 et 23 et les tiques restantes ont été comptées et
enlevées au jour 30. L’efficacité du traitement acaricide contre R. sanguineus a varié entre 96,1 % et 98,9 % à 48 h
après l’application et a duré jusqu’à quatre semaines. Quatre des six chiens témoins ont été infectés avec E. canis,
comme en témoigne la présence d’anticorps spécifiques contre E. canis et la détection par PCR d’ADN d’E. canis
dans les échantillons de sang. Ces chiens sont devenus thrombocytopénique et fiévreux et ont été consécutivement
traités jusqu’à guérison par la doxycycline. Aucun des six chiens traités n’a été infecté par E. canis, comme le
confirme l’absence d’anticorps spécifiques et d’ADN d’E. canis dans les échantillons de sang. Advantix a
empêché la transmission d’E. canis et a fourni une protection contre l’ehrlichiose monocytaire pendant quatre
semaines après le traitement acaricide.
*Corresponding author: josephus.fourie@clinvet.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
107

2 J.J. Fourie et al.: Parasite 2013, 20, 36
Introduction Cooperation on Harmonization of Technical Requirements for

Registration of Veterinary Medicinal Products Guideline 9:
Ticks and tick-borne diseases that affect the health of dogs Good Clinical Practice [6]. It was a randomised, blinded effi-
are expanding in different regions of the world [5]. Changes in cacy study conducted with two groups of six dogs, which were
human behaviour (increased outdoor recreation and interna- males and females of mixed breed (mongrel), with a weight
tional travel with companion animals) and changes in landscape range of 13.8–18.8 kg. All dogs were kept individually in
ecology with increased wildlife host abundance for ticks are tick-proof kennels and were observed twice daily for health
among the factors contributing to the increased incidence of abnormalities. The dogs, all negative for E. canis specific anti-
canine tick-borne diseases [2]. Worldwide, canine monocytic bodies in the indirect fluorescent antibody test (IFA), were
ehrlichiosis, babesiosis and granulocytic anaplasmosis are the ranked according to weight and divided randomly into two
most common tick-borne diseases of dogs [13, 17]. equal groups. Group 1 dogs were designated control and Group
The capacity of ticks to transmit these protozoan or bacte- 2 dogs were treated with 10% w/v imidacloprid and 50% w/v
rial pathogens varies widely. For instance, the cosmopolitan permethrin on Day 0. The product was administered according
tick, Rhipicephalus sanguineus, is vector of Ehrlichia canis, to label instructions (topical spot-on at four spots along the back
which is the cause of canine monocytic ehrlichiosis. Moreover, from the shoulder to the base of the tail) to each of the dogs in
R. sanguineus transmits a broad range of other pathogens, the acaricidal treatment group.
including Babesia vogeli, Babesia gibsoni, Hepatozoon canis,
Rickettsia conorii, Rickettsia rickettsii and probably also Ana-
plasma platys, the cause of thrombocytic anaplasmosis [5]. Infection of R. sanguineus ticks with E. canis
Effective acaricidal control of ticks is a necessity in many
Rhipicephalus sanguineus ticks, originating from France and
parts of the world. Acaricidal treatment that kills ticks reduces
maintained for several generations on rabbits under laboratory
the number of ticks capable of transmitting tick-borne pathogens.
conditions, were used as a source to generate a batch of ticks
In addition, the effectiveness of an acaricide that acts suffi-
infected by E. canis. Rhipicephalus sanguineus nymphs were
ciently fast to prevent transmission of tick-borne pathogens car-
fed on a susceptible laboratory-bred Beagle dog, previously inoc-
ries an important added value that needs to be demonstrated
ulated with blood derived from a local case of canine monocytic
empirically. Studies that have been conducted thus far suggest
ehrlichiosis, identified in Bloemfontein, South Africa. After
that topically applied acaricides can assist in the prevention of
moulting, the adult ticks were used as a basis for the study. A sam-
the transmission of specific tick-borne pathogens. For instance,
ple taken from the challenge batch of ticks was tested for E. canis
the ability of imidacloprid/permethrin to prevent transmission
by PCR and confirmed to be infected at a rate of 17%. The identity
of Anaplasma phagocytophilum, the cause of granulocytic ana-
of this novel strain of E. canis was confirmed by its partial gene
plasmosis, from naturally infected Ixodes scapularis ticks to
sequence (GenBank Accession No. KC935387) of E. canis
dogs was reported several years ago [3].
gp36 with a number of other E. canis isolates. It was found that
A relatively new area of research is the development of
E. canis (Bloemfontein) is closely related and formed a clade with
transmission-blocking models, wherein the ability of tick con-
several Asian isolates [11].
trol compounds to prevent transmission of pathogens can be
effectively measured under controlled laboratory conditions
[14]. First models were established for the blocking of transmis- Tick infestation of dogs
sion of Babesia canis by infected Dermacentor reticulatus ticks
[9, 14]. Recently, further developments in this area have been On Days +7, +14, +21 and +28, fifty adult ticks were
the creation of a tick exposure laboratory model, wherein trans- released into the individual tick-proof kennel of each dog to
mission of E. canis by infected R. sanguineus ticks can be stud- simulate natural exposure to ticks, instead of applying the ticks
ied [10]. Since the mode of action of compounds differs, it is directly onto the dogs. The E. canis-infected ticks, which were
necessary to evaluate each product’s ability to prevent transmis- used in the study, were unfed, at least one week old and had a
sion of pathogens. For example, some products may have an balanced sex ratio of 50% male and 50% female ticks.
irritant and toxic effect, such as permethrin and other substances
such as amitraz present an effect which includes both an expel-
Monitoring of dogs
lant effect, along with acaricidal properties [4, 8].
Here, this model was used to evaluate the efficacy of the The study animals were observed on a daily basis between
topical product Advantix, which combines imidacloprid Day 7 and Day +56 for general health purposes. The dogs
10% w/v with permethrin 50% w/v, in preventing the transmis- were observed on an approximately hourly basis for 4 h after
sion of E. canis by infected R. sanguineus ticks to dogs. initiation of the acaricidal treatment to detect any adverse
events. Full clinical examinations were conducted on Days
7, +21, +28, +35 +42, +49 and +56. Rectal body tempera-
Material and methods tures were recorded daily from Day +17 to Day +56. Additional
clinical examinations were conducted on all dogs displaying an
Study design abnormally high body temperature (>39.4 C). Clinical exam-
inations included general appearance, heart rate, respiration rate
This study was in compliance with the animal welfare and body temperature. The examinations focussed on possible
requirements and carried out according to International clinical manifestations of monocytic ehrlichiosis, which include
108

J.J. Fourie et al.: Parasite 2013, 20, 36 3
fever, depression, anorexia, weight loss, haemorrhages and epi- category 1, 2, 3 and 6 was transformed to the natural logarithm
staxis. To prevent fatal ehrlichiosis, dogs with abnormally high of (count + 1) and then corrected by subtracting one (1) for the
body temperatures (>39.4 �C) for at least two consecutive calculation of the geometric means. The categories used to cal-
days and an abnormally low platelet count were treated with culate effectiveness were according to the recommendations
10 mg/kg doxycycline per os for 21 consecutive days. made by the ‘‘Guidelines for the Testing and Evaluation of
Blood samples for serology were collected on Days 7, +7, the Efficacy of Antiparasitic Substances for the Treatment and
+21, +28, +35, +42, +49 and Day +56 from all dogs. Blood Prevention of Tick and Flea infestation in Dogs and Cats’’
samples for platelet counts and PCR were only collected post adopted on 7 November 2007 by the Committee for Veterinary
tick challenge on Days +21, +28, +35, +42, +49 and Day Medicinal Product of the European Agency for the Evaluation
+56 from all dogs. In dogs with suspected ehrlichiosis (e.g., of Medicinal Products (EMEA/CVMP/005/2000-Rev.2). In the
due to low platelet count), additional samples were taken on acaricide-treated group, percentage reduction in tick counts
the day of diagnosis, before rescue treatment. All samples col- compared to the control group was calculated using the formula
lected were tested by PCR (Table 4). 100 · (1 T/C), wherein T and C were the geometric means
of the acaricide-treated and control group, respectively. Effec-
Laboratory tests tiveness was also calculated based on the arithmetic group
means. Furthermore, the groups were compared by an ANOVA
Blood samples in EDTA for platelet counts were examined with a treatment effect after a logarithmic transformation on the
by Pathcare Veterinary Laboratory, Bloemfontein, South Africa. (count + 1) tick data.
Serum samples were frozen at 20 �C until assayed for Dogs which displayed E. canis antibodies and were also
E. canis antibodies using a commercial IFA test (IGG IFA, positive for E. canis DNA by PCR analysis were regarded as
Fuller Laboratory, USA). The tests were performed according infected. The proportions of dogs infected in each group were
to the manufacturer’s descriptions at the Department of Veteri- compared by using Fisher’s Exact Test. In addition, the exact
nary Tropical Diseases (DVTD), Faculty of Veterinary Science, 95% confidence interval for the blocking effect in Group 2
University of Pretoria, South Africa. was calculated. Version 8 of SAS (Release 8.02 TS Level
A further blood sample collected in EDTA was centrifuged 02M0) was used for all statistical analyses, whereby the level
at 3,000 rpm for 15 min and the buffy coat stored in a 80 �C of significance of the tests was set at 5%.
freezer, until PCR assayed. DNA extraction and PCR analysis
of all buffy coat samples were performed in the molecular lab-
oratory of ClinVet International Ltd. DNA extractions were per- Results
formed using Qiagen DNeasy Blood and Tissue kit according
to the instructions of the manufacturer. A novel primer set for Tick counts
PCR was designed based on the disulphide oxidoreductase gene
of E. canis, as previously described [10]. Both arithmetic and geometric mean tick counts recorded
for the acaricidal treatment and control groups are provided
Tick counts in Table 1. Statistically significantly (p < 0.05) less ticks were
recorded on the treated dogs compared to the control dogs on
In situ tick thumb counts were carried out on all dogs 48 h all assessment days. Efficacy values (%) based on mean tick
after each exposure (Day +9, +16 and +23), but on Day +30 all counts for the group treated once are summarised in Table 1.
ticks were counted and removed. The counts were recorded into The acaricidal treatment was highly effective (between 96.1%
six categories specified by the ‘‘Guidelines for the Testing and and 98.9%, based on geometric means) against infestations with
Evaluation of the Efficacy of Antiparasitic Substances for the R. sanguineus ticks up to four week post acaricidal treatment.
Treatment and Prevention of Tick and Flea infestation in Dogs
and Cats’’ adopted on 7 November 2007 by the Committee for
Veterinary Medicinal Product of the European Agency for the Ehrlichia canis transmission blocking
Evaluation of Medicinal Products (EMEA/CVMP/005/2000-
Rev.2). These six categories were: 1 = live, free; 2 = live, There were no adverse effects observed on the dogs with
attached, unengorged (no filling of the alloscutum); 3 = live, respect to the topical administration of the acaricidal treatment.
attached, engorged (obvious or conspicuous filling of the allos- Three dogs of the control group (CC5 CDA, E46 0EE and CC4
cutum); 4 = killed, free; 5 = killed, attached, unengorged; 90E) with abnormally high body temperatures (>39.4�C)
6 = killed, attached and engorged. Ticks counted and removed received doxycycline at 10 mg/kg per os for 21 days starting
on Day +30 were also categorised within gender (male/female) on Day +23 (CC5 CDA), Day +31 (E46 0EE) and Day +38
in addition to recording them according to categories 1–6. (CC4 90E). Low platelet counts were observed in the same
Furthermore, each animal kennel was inspected daily from three dogs with elevated body temperature, but also in a fourth
Day +14 up to Day +30 for any engorged ticks. dog of the same group (9B4 937) (Table 2). Thrombocytopenia
was evident in all four dogs; as a result of doxycycline treat-
Statistics ment, values returned to normal between 200 and 500 ·
109/L towards the end of the study on Day +56 (Table 2). In
In order to determine the effectiveness of the acaricidal all other animals platelet counts were within the normal range
treatment, the total number of ticks assigned to counting (Table 2).
109

The IFA test results are summarised in Table 3. All dogs
on arith. mean (geo. mean#)
*In situ counts. #The geometric means of Group 2 differed statistically significantly (p < 0.05) from those in Group 1 in all cases. Att = Attached; Ue = Unengorged; E = Engorged;
included in the study tested negative for E. canis antibodies
Efficacy (%) based

prior to the first tick infestation. Four control dogs (Group 1)
92.9 (96.1)
98.6 (98.9)
97.3 (98.3)
97.6 (98.3)
developed specific antibodies against E. canis first detected
on Day +21 (2 dogs), Day +28 (one dog) and Day +35 (one
dog) and remained positive throughout to the end of the study
(Table 3). The same control dogs that developed specific
E. canis antibodies were confirmed PCR positive. None of
the acaricide-treated dogs became seropositive neither PCR
positive (Table 4).
In total, four out of six dogs became infected with E. canis
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
Att; E
in the control group and none in the acaricide-treated group

(p = 0.0606).
Dead ticks
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
Att; Ue
Discussion
Group 2 – Advantix-treated group
Advantix was highly effective (between 96.1% and

0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
98.9%) against infestations of R. sanguineus ticks up to four

Free
week post acaricidal treatment (Table 1). Previous studies have

demonstrated that the efficacy of the 10% w/v imidacloprid/
50% w/v permethrin combination at 48 h is above 90%, depen-
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
dent upon the tick species tested. The acaricidal efficacy of

Att; E
imidacloprid/permethrin spot-on against R. sanguineus has

Table 1. Tick counts on dogs 48 h after environmental challenges by treatment group and percentage efficacies.
been reported to range between 91.5% and 97.6% for up to

37 days [7].
Live ticks
0.5 (0.3)
0.2 (0.1)
0.3 (0.2)
0.5 (0.3)
Att; Ue
The first study which indicated that the combined action of

10% w/v imidacloprid/50% w/v permethrin could reduce path-
ogen transmission was published almost a decade ago (3), and
reported the prevention of transmission of A. phagocytophilum
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
from field-collected I. scapularis ticks to dogs treated with

Free
Advantix. Another study conducted in southern Italy indicated

that the application of 10% w/v imidacloprid/50% w/v permeth-
rin as a topical spot-on reduced E. canis infection in dogs [15].
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.8 (0.7)
Att; E
In another study using the same model a fipronil, amitraz and

(S)-methoprene combination successfully prevented transmis-
sion of E. canis by R. sanguineus to dogs [10].
Dead ticks
Although there was no statistical difference observed

0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.5 (0.3)
Att; Ue
between the number of infected dogs in the two study groups,

the fact that no infection was observed in Advantix treated
dogs clearly demonstrated that a single administration of
Group 1 – Control group
Advantix was able to prevent transmission of E. canis by

0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
Free
R. sanguineus ticks for a duration of 4 weeks. The model that

was employed did simulate natural exposure to ticks by releas-
ing them into each individual dog kennel rather than applying
0.0 (0.0)
2.8 (2.1)
1.8 (1.6)
2.3 (2.1)
them directly onto each dog.

Att; E
Four out of six control dogs became infected with

Geo = geometric; Arith = Arithmetic.
E. canis, as demonstrated by thrombocytopenia, development

of specific E. canis antibodies and the presence of ehrlichial
18.5 (17.7)
10.3 (11.1)
Live ticks
DNA in blood samples. A possible reason why two out of

7.0 (6.7)
9.5 (8.0)
Att; Ue
six control dogs did not develop monocytic ehrlichiosis may

have been due to an insufficient number of ehrlichial organisms
carried by those ticks that actually fed on these animals, or not
all ticks being infected. Although one has to aim for an exper-
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
Free
imentally infected batch of ticks capable of infecting all control

animals, the challenge has to be realistic and comparable with
infection rates in field-collected ticks. In this study, 17% of ticks
from the batch used to challenge the dogs was infected with
+16*
+23*
Day
+9*
+30
E. canis as determined by PCR. This infection rate was
110

Table 2. Platelet counts in treated and control dogs.

Platelet counts · 109/L
Group Dog no. Day 21 Day 22 Day 28 Day 30 Day 35 Day 37 Day 42 Day 49 Day 56
Control CC5 CDA 119 49 – 291 – – 378 339 372
CD2 B63 370 – 469 – 343 – 386 394 340
E46 0EE 337 – 183 5 206 – 370 382 335
CC4 90E 240 – 284 – 144 97 192 78 256
CC2 21F 267 – 325 – 259 – 310 190 334
9B4 937 143 – 36 167 – – 601 406 508
Treated group E17 E19 363 – 386 – 295 – 393 395 223
DF5 A66 338 – 363 – 219 – 344 307 430
964 441 364 – 411 – 307 – 388 388 451
CC2 1BD 319 – 391 – 264 – 357 337 353
CC2 25E 374 – 334 – 263 – 363 321 394
CC4 55E 262 – 311 – 234 – 321 309 298
Table 3. Ehrlichia canis antibodies determined by IFA.

Group Dog no. Day 7 Day 71 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56
Control CC5 CDA – –2 POS POS POS POS POS POS
CD2 B63 – – – – – – – –
E46 0EE – – – POS POS POS POS POS
CC4 90E – – – – POS POS POS POS
CC2 21F – – – – – – – –
9B4 937 – – POS POS POS POS POS POS
Treated group E17 E19 – – – – – – – –
DF5 A66 – – – – – – – –
964 441 – – – – – – – –
CC2 1BD – – – – – – – –
CC2 25E – – – – – – – –
CC4 55E – – – – – – – –
1
Prior to tick challenge; 2negative; POS = positive.
Table 4. Detection of Ehrlichia canis DNA by PCR in blood samples from individual dogs.
PCR
Group Dog no. Day 21 Day 22 Day 23 Day 28 Day 30 Day 35 Day 37 Day 42 Day 49 Day 56
Control CC5 CDA POS POS POS nd nd nd nd nd nd nd
CD2 B63 –2 nd1 nd – nd – nd – – –
E46 0EE – nd nd POS POS nd nd nd nd nd
CC4 90E – nd nd – nd POS POS nd nd nd
CC2 21F – nd nd – nd – nd – – –
9B4 937 POS nd nd nd nd nd nd nd nd nd
Treated group E17 E19 – – – – – – – – – –
DF5 A66 – – – – – – – – – –
964 441 – – – – – – – – – –
CC2 1BD – – – – – – – – – –
CC2 25E – – – – – – – – – –
CC4 55E – – – – – – – – – –
1
nd = Not done; 2negative; POS = positive.
111

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Competing interests 8. Folz SD, Ash KA, Conder GA, Rector DL. 1986. Amitraz: a
tick and flea repellent and tick detachment drug. Journal of
The work reported herein was funded by Bayer Animal Veterinary Pharmacology and Therapeutics, 9, 150–156.
Health GmbH, Leverkusen, Germany of which D. Stanneck 9. Fourie JJ, Stanneck D, Jongejan F. 2013. Prevention of
is an employee. ClinVet International Ltd., of which J.J. Fourie transmission of Babesia canis by Dermacentor reticulatus ticks
is an employee, is a South African Contract Research Organi- to dogs treated with an imidacloprid/flumethrin collar. Veteri-
zation contracted to carry out the study. F. Jongejan is PhD nary Parasitology, 192, 273–278.
supervisor of JJF and Professor at the Universities of Pretoria 10. Fourie JJ, Ollagnier C, Beugnet F, Luus HG, Jongejan F. 2013.
and Utrecht. None of the authors have any personal interest Prevention of transmission of Ehrlichia canis by Rhipicephalus
sanguineus ticks to dogs treated with a combination of fipronil,
in these studies other than publishing the scientific results that
amitraz and (S)-methoprene (CERTIFECT). Veterinary Para-
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Health GmbH, Leverkusen, Germany. cephalus sanguineus ticks feeding on dogs and on artificial
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study to high standards. ahead of print].
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analysed the data. FJ wrote the first draft of the manuscript, Parasitology, 129(Suppl.), 3–14.
which was subsequently revised and the final version approved 14. Jongejan F, Fourie JJ, Chester ST, Manavella C, Mallouk Y,
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Cite this article as: Fourie JJ, Luus HG, Stanneck D & Jongejan F: The efficacy of Advantix to prevent transmission of Ehrlichia
canis to dogs by Rhipicephalus sanguineus ticks. Parasite, 2013, 20, 36.
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4.3. Fourie JJ, Stanneck D, Luus HG, Beugnet F, Wijnveld M, Jongejan F.

Transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks fee-
ding on dogs and on artificial membranes. Vet Parasitol. 2013, 197 (3-4) :
595-603.
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116

Contents lists available at ScienceDirect
Transmission of Ehrlichia canis by Rhipicephalus sanguineus

ticks feeding on dogs and on artificial membranes
Josephus J. Fourie a,∗ , Dorothee Stanneck b , Herman G. Luus a ,
Frederic Beugnet c , Michiel Wijnveld d , Frans Jongejan d,e
a
ClinVet International (Pty) Ltd, P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa
b
Bayer Animal Health GmbH, BHC Business Group Animal Health, Clinical Development, Building 6700, 51368 Leverkussen, Germany
c
Merial, 29 Av Tony Garnier, 69007 Lyon, France
d
Utrecht Centre for Tick-borne Diseases (UCTD), Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The
Netherlands
e
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110,
South Africa
Article history: A South African strain of Ehrlichia canis was isolated and used to infect a laboratory-bred
Received 28 May 2013 Beagle dog. Rhipicephalus sanguineus nymphs, which fed on this dog, moulted to adult ticks
Received in revised form 7 July 2013
which carried infection rates of E. canis between 12% and 19% and were used in a series of
Accepted 15 July 2013
in vivo and in vitro experiments.
Five groups of 6 dogs were challenged with the infected R. sanguineus ticks, which were
Keywords:
removed 24 h, 12 h, 6 h or 3 h after the ticks had been released onto the dogs. The ani-
Speed of transmission
Ehrlichia canis mals were monitored for fever and thrombocytopenia and were considered infected if they
Rhipicephalus sanguineus ticks became serologically positive for E. canis antibodies as well as PCR positive for E. canis DNA.
Dogs Seven dogs became infected with E. canis in the following groups: Group 1 (24 h tick chal-
In vitro lenge) 1 out of 6; Group 2 (12 h) 1 of 6; Group 3 (6 h) 2 of 6; Group 4 (6 h) 2 of 6 and Group 5
Feeding (3 h) 1 out of 6. Six of those 7 infected dogs developed fever and a significant thrombocyto-
penia. One dog did not show any symptoms, but seroconverted and was found PCR positive
on several occasions. Five additional dogs were PCR positive on one test sample only but
were not considered infected because they did not develop any specific E. canis antibodies.
In vitro, R. sanguineus ticks attached and fed on bovine blood through silicone membranes
with attachment rates up to 72.5% after 24 h increasing to 84.2% at 72 h. The ticks transmit-
ted E. canis as soon as 8 h post application as demonstrated by E. canis DNA found in the
nutritive blood medium.
In conclusion, transmission of E. canis by R. sanguineus ticks starts within a few hours
after attachment, which is earlier than previously thought. These findings underpin the
need for acaricides to provide either a repellent, an anti-attachment and/or a rapid killing
effect against ticks in order to decrease the risk of transmission of E. canis.
1. Introduction
Worldwide, there is a growing recognition of the impor-

∗ Corresponding author. Tel.: +27 51 445 2424; fax: +27 51 445 2421. tance of rickettsial pathogens in dogs and humans, which
E-mail address: Josephus.Fourie@clinvet.com (J.J. Fourie). are transmitted by a number of different ixodid tick
117

species (Nicholson et al., 2010). Rhipicephalus sanguineus, Borrelia infections after the ticks were allowed to feed for
the brown dog tick, is the most widespread tick in the world 48 h (Kahl et al., 1998). The speed of E. canis transmission
and is a well-recognized vector of tick-borne pathogens by infected R. sanguineus ticks has not been determined
affecting dogs and occasionally humans (Dantas-Torres, in any laboratory study thus far. Importantly, there is no
2010). Rhipicephalus sanguineus transmits a broad range report published wherein R. sanguineus has been adapted to
of pathogens to dogs such as Babesia vogeli, Babesia feed on artificial membranes in order to study the pathogen
gibsoni, Hepatozoon canis, Rickettsia conorii, Rickettsia rick- transmission dynamics in vitro.
ettsii, Ehrlichia canis and Anaplasma platys (Jongejan and Here, the speed of E. canis transmission by infected R.
Uilenberg, 2004; Beugnet and Marié, 2009). sanguineus ticks to dogs was determined in a controlled
Rhipicephalus sanguineus is the primary vector of E. laboratory study. In addition, R. sanguineus ticks were suc-
canis, which is the cause of monocytic ehrlichiosis (Groves cessfully adapted to feed in vitro by modifying the methods
et al., 1975). Ehrlichia canis has a worldwide distribution originally developed for in vitro feeding of I. ricinus ticks
(Asia, Africa, Europe and the Americas), where the major- (Kröber and Guerin, 2007a). The speed of transmission of
ity of clinical cases occur in sub-tropical and Mediterranean E. canis by infected R. sanguineus ticks attached for defined
regions where the vector is abundant. Canine mono- periods on dogs was compared with the transmission of E.
cytic ehrlichiosis is characterized by thrombocytopenia, canis by R. sanguineus ticks attached and feeding through
leukopenia, fever, depression and bleeding tendencies artificial membranes.
(epistaxis) (Harrus et al., 1999). Ehrlichia canis is transmit-
ted transstadially and intrastadially by R. sanguineus ticks 2. Materials and methods
(Bremer et al., 2005). In dogs, E. canis develops in mono-
cytes and macrophages, whereas in ticks the infection is 2.1. Dog study design
localized in midgut and salivary glands.
Effective control of ticks on dogs is a necessity in many The in vivo component of the study was conducted with
parts of the world. In addition to the killing effect on ticks, five groups of six dogs, which were adult males (19) and
acaricides which can prevent transmission of tick borne- females (11) of mixed breed. Prior to the experiments the
pathogens bring an important added value. The decrease dogs were all negative for E. canis as demonstrated by the
of the transmission rate can be explained by several prop- absence of specific antibodies in the indirect fluorescent
erties of the acaricidal molecule: a repellent/irritant effect antibody test (IFA). They had not been treated with any
inhibiting tick infestation and attachment; a neurohor- acaricidal spot-on or spray for 12 weeks prior to the first
monal disruption of tick attachment and intake of the blood tick challenge. There were two study phases with an assess-
meal; and/or a quick speed of kill before transmission can ment time point for the second study phase (Groups 4 and
occur (Halos et al., 2012). These properties can be com- 5) based on the outcome of the first study phase (Groups
bined together depending on the acaricidal molecules and 1–3). The study employed a randomized block design with
their concentrations. Several studies have demonstrated 18 dogs allocated to Groups 1–3 and a further 12 dogs
the capability of anti-tick products based on either amitraz, allocated to Groups 4 and 5. The dogs were sedated and
fipronil and/or permethrin to help in preventing pathogen infested with 100 adult ticks each. The infection rate of
transmission. Field studies conducted thus far suggested the ticks ranged between 12% and 19%. In the first phase
that topically applied acaricides can assist in the preven- of the study, dogs were challenged with ticks followed by
tion of the transmission of specific tick-borne pathogens removal at 24 h post challenge (Group 1), at 12 h post chal-
(Davoust et al., 2003; Otranto et al., 2008). Controlled lab- lenge (Group 2) and at 6 h post challenge (Group 3). In
oratory studies have also focussed on these prophylactic the second study phase dogs were challenged with ticks,
efficacies (Jongejan et al., 2011; Fourie et al., 2013a,b). A which were removed at 6 h post challenge (Group 4) and
transmission blocking model was recently used to evaluat 3 h post challenge (Group 5). The study was in compli-
ate the efficacy of two topical products, CERTIFECT® spot ance with the South African animal welfare regulations and
on (fipronil 6.26% w/v, amitraz 7.48% w/v, (S)-methoprene carried out according to International Cooperation on Har-
5.63% w/v) and SERESTO® collar (imidacloprid, flumethrin) monization of Technical Requirements for Registration of
in preventing transmission of Babesia canis by Dermacen- Veterinary Medicinal Products Guideline 9: Good Clinical
tor reticulatus to dogs (Jongejan et al., 2011; Fourie et al., Practice (EMA, 2000).
2013a). Another transmission blocking model was used to
demonstrate the efficacy of (CERTIFECT® ) in preventing the 2.2. Ehrlichia canis strain
transmission of E. canis by R. sanguineus to dogs (Fourie
et al., 2013b). A laboratory-bred Beagle dog, inoculated with blood
In order to successful prevent pathogen transmission, derived from a local clinical case of canine monocytic ehrli-
the speed of kill asserted by a particular acaricidal com- chiosis, identified in Bloemfontein, South Africa served as
pound is of prime importance. The time elapsed from tick the source of the infectious material. EDTA blood collected
attachment to pathogen transmission varies according to from this animal was frozen at −80 ◦ C with 10% DMSO as
the nature of the pathogen. For instance, in one study on the a cryoprotectant and thereafter stored in liquid nitrogen.
transmission of Borrelia burgdorferi, Ixodes ricinus nymphs Gp36, an immunodominant glycoprotein-encoding gene,
were allowed to feed on gerbils during limited time inter- was PCR amplified from this blood and used as a tool for
vals. It was found that 50% of gerbils became infected within the classification of E. canis isolates as previously published
17 h of tick attachment, whereas all gerbils developed (Doyle et al., 2005; Hsieh et al., 2010).
118

PCR amplification of gp36 was carried out with for- respiration rate and body temperature and focussed on
ward primer EC36-F3 (5� -AGA AGA TGC GGA TGG AGT possible clinical manifestations of monocytic ehrlichiosis,
GGG ATT CG-3� ) and reverse primer EC36-R3.1 (5� - which included fever, anorexia, haemorrhages and epis-
GTTGAACCTGTTGCTGCAGTAGCTGGAG-3� ). A 25-l reac- taxis. Blood was collected from all dogs for platelet counts,
tion volume consisted of 5 l of 5× Phusion® HF Buffer serology and PCR on days −2, +14, +21, +28, +35 and +42.
(Thermo scientific), 0.4 M of each primer, 200 M dNTPs, To prevent fatal ehrlichiosis, dogs with body temperatures
0.25 l Phusion® Hotstart II polymerase, 2.5 l of the tem- >39.4 ◦ C for at least two consecutive days and low platelet
plate DNA. The PCR program used was 30 s at 98 ◦ C; and 35 count were rescue treated with doxycycline 10 mg/kg per
cycles of 98 ◦ C for 10 s, 69 ◦ C for 10 s, and 72 ◦ C for 20 s, os for 21 consecutive days.
final extension step was performed at 72 ◦ C for 10 min.
PCR products for sequencing were cloned using the Clone- 2.5. Laboratory assays
Jet PCR Cloning kit (Thermo scientific). Plasmid DNA was
extracted using the GeneJet Plasmid Miniprep kit (Thermo Serum samples from the dogs were frozen at −20 ◦ C
scientific) and resulting plasmids sequenced by Baseclear until assayed for E. canis antibodies using a commercial
(Leiden, The Netherlands). The nucleotide sequence of E. IFA test (IGG IFA, Fuller Laboratory, USA). The tests were
canis gp36 has been deposited in Genbank under accession performed at the Department of Veterinary Tropical Dis-
number KC935387. eases (DVTD), Faculty of Veterinary Science, University
Sequence alignment and phylogenetic analysis of E. of Pretoria, South Africa according to the manufacturer
canis was conducted and its partial gene sequence com- descriptions.
pared with those of 27 other E. canis isolates (Hsieh et al., Additional EDTA blood samples were examined for
2010; Kamani et al., 2013). The BLAST program was used platelet counts by Pathcare Veterinary Laboratory in
for comparison of sequence data obtained in this study with Bloemfontein, South Africa.
those previously deposited in GenBank. The phylogenetic EDTA blood samples from the dogs were centrifuged at
tree of gp36 was constructed using the neighbour-joining 3000 rpm for 15 min and the buffy coat stored at −80 ◦ C
method as implemented by MEGA software version 4 until PCR assayed in the molecular laboratory of ClinVet
(Tamura et al., 2004). A bootstrap resampling technique International Ltd. DNA extractions were carried out using
of 1000 replications was conducted to statistically support Qiagen DNeasy Blood and Tissue kit. A primer set for PCR
the reliability of the phylogenetic tree. was designed based on the disulphide oxidoreductase gene
of E. canis, as previously reported (Fourie et al., 2013b).
2.3. Infection of R. sanguineus ticks with E. canis Blood samples used in the in vitro feeding part of the
study were frozen at −20 ◦ C prior to DNA extraction. DNA
Rhipicephalus sanguineus ticks were derived from a was extracted from whole blood using the NucleoSpin®
laboratory colony, originally established from ticks col- Blood kit (Macherey-Nagel, Düren, Germany). A reverse
lected in France, and fed for several generations on rabbits. line blot assay (RLB) developed for simultaneous detec-
Nymphs were used for acquisition feeding on a suscepti- tion of Ehrlichia and Anaplasma species was used (Bekker
ble laboratory-bred Beagle dog, previously inoculated with et al., 2002). To this end, the hypervariable V1 region
a sample of frozen blood infected with E. canis (Bloem- of the 16S rRNA gene was PCR amplified as previously
fontein). After moulting, DNA was extracted from 50 adult described (Bekker et al., 2002). Amplified products were
ticks and tested for E. canis by PCR, as described recently hybridized onto a membrane containing species-specific
(Fourie et al., 2013b). A sample of ticks (50) was tested for oligonucleotide probes essentially as described previously
infectivity and were shown capable of transmitting E. canis (Matjila et al., 2005).
to another susceptible laboratory-bred dog. Ticks from the
same batch were used to challenge the dogs enrolled in 2.6. Tick removal
the study. The ticks used for the artificial infestations, were
unfed, at least one week old and had a balanced sex ratio Ticks were removed according to the schedule by visual
of 50% female and 50% males. One hundred potentially E. examination of the dogs and through palpation. The dogs
canis-infected ticks were applied directly onto each dog. were combed twice to ensure all ticks were removed. Addi-
The infection rate of the ticks ranged between 12% and 19%. tionally all dogs were dipped in a Bayticol® (flumethrin,
100 ml/5 l) solution immediately following the tick count
2.4. Monitoring of dogs procedure to ensure that no live ticks remained on the dogs.
The dog kennels were sprayed with Bayticol® to ensure that
All dogs were kept individually in tick-proof kennels no live ticks remained in the environment.
and were observed twice daily for health abnormalities.
The animals were observed on a daily basis between day 2.7. Statistical analysis
−2 and day +42 for general health purposes. Full clinical
examinations were conducted on days −8, +7, +14, +21, Descriptive statistics (mean, minimum, maximum,
+28, +35 and +42. Rectal body temperatures were recorded standard deviation, geometric mean and arithmetic mean)
daily from days +4 to +42. Additional clinical examinations on tick counts for the five study groups were calculated and
and platelet counts were conducted on all dogs displaying tabulated. A dog was considered positive if tested serolog-
an abnormally high body temperature (>39.4 ◦ C). Clini- ically positive for E. canis antibodies as well as PCR positive
cal examinations included general appearance, heart rate, for E. canis DNA.
119

2.8. In vitro feeding of ticks Table 1

Tick counts (arithmetic means) per treatment group.
In vitro feeding of ticks was conducted according to Day Group Arithmetic mean
methods published for I. ricinus by Kröber and Guerin +1 (24 h)a 1 46.2
(2007a). A number of alterations were made to accommo- 0 (12 h) 2 44.5
date other hard tick species, including R. sanguineus. Bovine 0 (6 h) 3 67.5
blood was collected weekly from cattle maintained at the 0 (6 h) 4 66.5
0 (3 h) 5 70.0
Farm Animal Department, Faculty of Veterinary Medicine
a
at Utrecht University. Blood donors were screened for tick- Hours post tick infestation.
borne pathogens by PCR/RLB to ensure that they were
free from any blood parasites. Approximately 200 ml blood 24 h intervals, whereas blood samples for PCR/RLB were
was manually defibrinated by stirring with a sterile plas- collected at 8–12 h intervals during 72 h.
tic pipette, 2 g/l glucose and gentamycine (5 g/ml) were
added and this nutritive medium stored at 4 ◦ C. 3. Results
Silicone membranes were prepared by using lens clean-
ing paper (Tiffen, USA) as a matrix. A layer of transparent 3.1. Ehrlichia canis and transmission to dogs
plastic kitchen film was fixed onto a glass sheet with adhe-
sive tape and pieces of lens paper were fixed on top of The identity of the Bloemfontein strain of E. canis was
this plastic film. A thin layer of a mixture of 15 g silicone confirmed by comparing its partial gene sequence (Gen-
glue RTV-1 Elastocil E4 (Wacker, Germany), 4.5 g (30%) Bank accession number KC935387) of E. canis gp36 with
silicone oil DC 200 (Sigma, Germany), 2.9 g 15% hexane 27 other geographically widely separated E. canis isolates.
(Sigma, Germany) and a few drops of white colour paste It was found that this novel isolate is closely related and
RAL 9010 (Wacker, Germany) was spread evenly over the formed a clade with several Asian E. canis isolates (Fig. 1).
fixed pieces of lens paper. Membranes were left to polymer- The arithmetic means for the tick counts per group are
ize for 12 h at room temperature. They were subsequently given in Table 1. The mean tick number ranged between
gently rubbed over the predilection sites of the tick on 46.2 and 70, depending on the group. The clinical and
dogs to give the membranes a typical dog odour. The plas- haematological observations are summarized in Table 2.
tic film remained attached to the membranes to prevent Six dogs from different groups reacted with fever ranging
contamination of the surface of the membrane that would between 39.9 ◦ C and 40.7 ◦ C and the animals displayed
later come into contact with blood. The thickness of the severe thrombocytopenia at a rate of 2–65 × 109 platelets
membrane was measured with a micro-calipers and those per litre (normal platelet counts are between 200 and
between 70 and 90 m were used. 500 × 109 /l). All six dogs required rescue treatment with
Feeding units of Plexiglas tubing, suitable to sink into doxycycline and recovered.
the wells of a six-well cell culture plate (Greiner), were The IFA assay results are summarized in Table 3. All dogs
manufactured according to Kröber and Guerin (2007a). were tested negative for E. canis antibodies prior to the tick
Membranes were fixed to the feeding tube with silicone challenge (day −2). Seven dogs seroconverted: 1 out of 6
glue and left to dry for 3 h. Feeding units were examined in Group 1 (24 h tick challenge), 1 of 6 in Group 2 (12 tick
for leakages by immersion for 20 min in Petri dishes filled challenge), 2 of 6 in Group 3 (6 h tick challenge), 2 of 6 in
with sterile distilled water. The membranes were not rein- Group 4 (6 h tick challenge) and one dog in Group 5 (3 h
forced by mosquito fibre netting as originally described tick challenge).
by Kröber and Guerin (2007a). Moreover, the perforated The PCR assay results are summarized in Table 4. It was
lid was replaced by a stopper, which could be inserted found that all 7 dogs that were tested seropositive for E.
into the feeding units until a few millimetres above the canis were confirmed positive by PCR. Since dogs were
membrane, thus forcing the ticks to stay close to the mem- considered infected with E. canis if they were tested sero-
brane. The stopper was wrapped with organza fabric for logically positive as well as PCR positive, 7 out of 30 dogs
easy removal. acquired the ehrlichial infection. Six of those 7 dogs devel-
At the start of each experiment, finely cut pieces of oped fever and a significant thrombocytopenia (Table 2).
dog hair were included to cover the membrane, to rein- One dog did not show any symptoms, but nevertheless
force the impregnated dog odour. Ticks, typically 5 males seroconverted and was PCR positive on several occasions.
and 5 female ticks were included and assembled feeding Five additional dogs were found PCR positive on one test
units were inserted into pre-warmed blood (3.1 ml) dis- sample only, but were not considered infected because they
tributed over the wells of a six-well cell culture plates. did not carry any specific E. canis antibodies through day 42
During an experiment, the membrane surface facing the (Table 4).
blood was rinsed with sterile saline solution before placing
the feeding unit in a well containing fresh blood. A typical 3.2. In vitro transmission of E. canis
experiment consisted of 40 adult ticks distributed over 4
feeding units placed into one cell culture plate. Plates were The components of the redesigned feeding unit are
left floating on the water surface in a 37 ◦ C water bath cov- shown in Fig. 2a, whereas the assembled feeding unit is
ered with a cloth to create a dark and humid environment. shown in Fig. 2b. Rhipicephalus sanguineus ticks attached
The blood was changed twice daily at approximately 12 h and fed on bovine blood through the silicone membranes
intervals for a maximum of 5 days. Ticks were counted at (Fig. 3a) and produced faeces, which proved that they were
120

Fig. 1. Phylogenetic tree based on the gp36 gene of E. canis. DNA sequences from 28 strains of E. canis from geographically widely separated areas were
compared with the neighbour-joining method, operated by MEGA software version 4. Scale bar indicates the number of mutations per sequence position.
The numbers at the nodes represent the percentage of 1000 bootstrap re-samplings.
feeding (Fig. 3b). An example of a typical in vitro feeding with an adequate Ehrlichia infection rate. Rhipicephalus
experiment is given in Table 5. Tick attachment rates of sanguineus nymphs feeding on a clinically reacting dog suc-
72.5% were reached within 24 h post application, while the cessfully acquired the infection and were able to transmit
rates slightly increased to over 80% attachment at 72 and E. canis after moulting to the adult stage. Although ticks
96 h post application (Table 5). Some ticks were able to carried infection rates ranging between 12 and 19%, the
fully engorge, which took approximately 7 days, but usually infectivity of the ticks used for infestations in phase 1 of
experiments were terminated after 5 days. Blood samples the in vivo study was lower than expected. This resulted in
examined for E. canis by PCR/RLB were negative prior to tick a lower number of infected dogs, in particular in Group 1,
exposure, but samples taken during blood exchange con- wherein ticks were allowed to attach for 24 h (only 1 of 6
tained E. canis DNA. It was found that E. canis DNA could dogs became positive). However, since dogs on which ticks
be detected 8 h post tick application in the in vitro feeding only fed for 6 h did become infected (2 of 6 dogs positive) it
units, which was the first time point when the nutritive was decided to confirm this timing of transmission and to
blood was changed. extend the study with a second phase in which ticks were
only allowed to attach for 6 and 3 h. Results obtained with
4. Discussion the dogs in Group 4 (2 of 6 became positive) confirmed the
findings obtained in Group 3. Furthermore, transmission
The identification and characterization of a South can even take place earlier than 6 h post tick infestation as
African isolate of E. canis from a local dog enabled us to it was clearly demonstrated in Group 5. In this group, one
conduct both in vivo and in vitro transmission studies. dog seroconverted and was PCR positive on day 35 and day
To conduct transmission studies with E. canis, it is 42 (Table 3) confirming that transmission of E. canis had
required to generate large batches of R. sanguineus ticks taken place (Table 4).
Table 2
Clinical and haematological observations of the 6 sick dogs.
Animal number Group number Highest temperature (day) Onset of treatmenta (day) Platelet countb (×109 /l) (day)
CC3 20D 3 40.2 ◦ C (19) 22 65 (23)

CD4 71D 3 39.9 ◦ C (20) 22 8 (21)
954 9CC 2 40.2 ◦ C (23) 23 37 (23)
EA1 6B1 4 40.4 ◦ C (22) 23 42 (21)
CC1 B76 4 40.3 ◦ C (23) 23 2 (21)
DF5 399 5 40.7 ◦ C (36) 37 58 (35)
a
10 mg/kg body weight doxycycline per os daily for 21 consecutive days.
b
Normal platelet counts range between 200 and 500 × 109 /l.
121

Table 3
Ehrlichia canis antibodies determined by IFA in dog sera.
Group Dog no. Day −2 Day +14 Day +21 Day +28 Day +35 Day +42
960 012 − − − − − −
CD6 879 − − − − − −
1 CC3 EF0 − − − − POS POS
(24 h) CC5 B0A − − − − − −
CDC 59F − − − − − −
CD3 0A7 − − − − − −
CC0 726 − − − − − −
CD4 74B − − − − − −
2 CC7 1F7 − − − − − −
(12 h) 954 9CC − − POS POS POS POS
9B2 C9A − − − − − −
B8D 805 − − − − − −
CC3 20D − POS POS POS POS POS

CD0 D7 C − − − − − −
3 CD4 71D − − POS POS POS POS
(6 h) CC1 633 − − − − − −
956 6D3 − − − − − −
CC1 F8F − − − − − −
CC1 F13 − − − − − −
CC1 5A3 − − − − − −
4 CD4 7BE − − − − − −
(6 h) CC1 9CA − − − − − −
EA1 6B1 − POS POS POS POS POS
CC1 B76 − POS POS POS POS POS
DF6 C93 − − − − − −
CC0 334 − − − − − −
5 DF5 399 − − − − POS POS
(3 h) E44 358 − − − − − −
CC0 B32 − − − − − −
CD4 ED0 − − − − − −
POS: positive for E. canis antibodies with a titre of ≥80; −: negative for antibodies against E. canis.
The risk of acquiring tick-borne diseases after an membranes with attachment rates up to 72.5% after 24 h.
infected tick bite depends on a number of factors, such In order to achieve this, the method originally designed by
as the geographical distribution of the ticks, pathogeni- Kröber and Guerin (2007a) was adapted to meet the specific
city and most importantly the duration of tick attachment. requirements of ticks, which have a preference for dogs.
For instance, virus can be transmitted within minutes, Compared to Ixodes, one of the major limiting factors is
whereas it usually takes several days for protozoans to the length of the mouthparts of the ticks. For instance, lar-
develop within the salivary gland of the ticks before the vae of R. sanguineus failed to attach because of their short
ticks become infective (Ebel and Kramer, 2004; Konnai mouthparts (less than 50 m) (data not shown). However,
et al., 2007). Since there were no data available on E. canis larvae of I. ricinus did attach with their longer mouth-
speed of transmission, data concerning the transmission of parts and fed to repletion on membranes impregnated with
Anaplasma phagocytophilum by nymphal Ixodes scapularis cattle odour (unpublished data). The perforated lid was
were considered as similar. They indicated that transmis- replaced by a stopper, which could be inserted into the
sion takes place between 24 and 36 h post tick application feeding units until a few millimetres above the membrane,
(Des Vignes et al., 2001; Katavolios et al., 1998). It is now thus forcing the ticks to stay close to the membrane (Fig. 2).
clear that E. canis can be transmitted by adult Rhipicephalus This greatly improved the attachment rates of the ticks to
as soon as 3–6 h after dog infestation. the membranes.
Silicone membranes have been successfully adapted This is the first study wherein ticks feeding in vitro did
from the in vitro feeding of adult I. ricinus ticks by actually transmit a pathogen through a silicone membrane.
Kröber and Guerin (2007a), who suggested in a subse- The ticks were able to transmit E. canis in the first 8 h
quent review article that in vitro feeding would be suitable post application as demonstrated by E. canis DNA in blood
to accommodate other hard tick species and also ideal samples by PCR. In order not to disturb the ticks, it was
to study tick–pathogen interactions without host interfer- decided to take blood samples only when the blood was
ence (Kröber and Guerin, 2007b). Recently, the first report exchanged twice daily which limited the timing assess-
describing the successful in vitro feeding of two impor- ment of the nutritive medium. Eight hour was the first time
tant ixodid tick ticks of the genus Hyalomma was published point, therefore it was not possible to check more precisely
(Tajeri and Razmi, 2011). when the pathogen was transmitted into the nutritive
In our in vitro feeding unit system, adult R. sanguineus medium. A follow-up study wherein real-time PCR will be
ticks attached and fed on bovine blood through silicone used in order to more accurately quantify the transmission
122

Table 4
Detection of Ehrlichia canis DNA by PCR in the blood of individual dogs.
Group ID Day −2 Day +14 Day +21 Day +28 Day +35 Day +42
960 012 − − − − − −
CD6 879 − − − − − −
1 CC3 EF0 − POS − POS − −
(24 h) CC5 B0A − − − − − −
CDC 59F − − − − − −
CD3 0A7 − − − − − −
CC0 726 − − − − − −
CD4 74B − − − − − −
2 CC7 1F7 − − − − − POS
(12 h) 954 9CC − − POS POS − −
9B2 C9A − − − POS − −
B8D 805 − − − POS − −
CC3 20D − − POS POS − −

CD0 D7 C − − − − − −
3 CD4 71D − − POS POS − −
(6 h) CC1 633 − − − POS − −
956 6D3 − − − − − −
CC1 F8F − − − − POS −
CC1 F13 − − − − − −
CC1 5A3 − − − − − −
4 CD4 7BE − − − − − −
(6 h) CC1 9CA − − − − − −
EA1 6B1 − POS POS − − −
CC1 B76 − − POS POS POS −
DF6 C93 − − − − − −
CC0 334 − − − − − −
5 DF5 399 − − − − POS POS
(3 h) E44 358 − − − − − −
CC0 B32 − − − − − −
CD4 ED0 − − − − − −
POS: positive for Ehrlichial DNA; −: no E. canis DNA detected.
dynamics and determine the infective dose required to tick attachment. Likewise, the in vitro data also indicate
infect a dog is ongoing. that E. canis is transmitted within hours after attachment,
In conclusion, it has been demonstrated both on dogs which is earlier than presumed. These findings highlight
and in vitro that transmission of E. canis starts quickly after the need for acaricides to assert a rapid effect against ticks
Table 5
Attachment rates of Rhipicephalus sanguineus ticks feeding in vitro.
Time Feeding unit Attached Total no. attached (%) Unattached Mortality Total no. of ticks
Males Females Males Females Males Females
U1 2 4 6 3 0 0 1 10
U2 5 3 8 0 1 0 1 10
(24 h) U3 4 5 9 1 0 0 0 10
U4 3 3 6 2 2 0 0 10
% 72.5
U1 2 4 6 3 0 0 0 9
U2 5 3 8 0 1 0 0 9
(48 h) U3 4 4 8 1 1 0 0 10
U4 3 3 6 2 2 0 0 10
% 73.7
U1 4 4 8 1 0 0 0 9
U2 5 3 8 0 1 0 0 9
(72 h) U3 5 3 8 0 2 0 0 10
U4 4 4 8 1 1 0 0 10
% 84.2
U1 5 4 9 0 0 0 0 9
U2 4 4 8 0 0 1 0 9
(92 h) U3 4 3 7 0 2 0 0 10
U4 4 3 7 0 0 0 1 9
% 83.8
123

Fig. 2. (a) Flexiglas feeding unit, plus a perforated stopper wrapped by a Fig. 3. (a) Rhipicephalus sanguineus ticks attached to the silicone mem-
piece of fabric. (b) Assembled feeding unit. brane in a feeding unit. (b) Rhipicephalus sanguineus ticks covered with
dog hair and producing faeces, which demonstrated their actual feeding.
in order to prevent transmission of tick-borne pathogens
to dogs.
Pretoria and Utrecht. None of the authors have any personal
Authors’ contributions interest in these studies other than publishing the scientific
results that they have been involved in via planning, initi-
JJF, FB and DS designed the protocol for the clinical ating, monitoring and conducting the investigations and
study, which was carried out by JJF. The data were compiled analysing the scientific outcome.
and analysed by JJF and HGL. Phylogenetic and sequence
analysis of E. canis and reverse line blots were carried out Acknowledgements
MW. In vitro feeding experiments were carried out by FJ,
who also wrote the first draft of the manuscript, which was The authors acknowledge the contributions of all tech-
subsequently revised and the final version approved by all nical staff at ClinVet International Ltd., by carrying out
authors. the clinical part of the study. Anne-Marie Rhebergen, Jesse
Lenssen, Marise Bonga, Nathanie van Dijk, Milou Veld-
Competing interests huizen, Jorinda Schellekens, Sabrina Riekerk en Isaura
Wayop are thanked for their contributions to the in vitro
ClinVet International Ltd., of which J.J. Fourie is an feeding experiments as part of their research internship
employee, is a South African Contract Research Organiza- at the Utrecht Centre for Tick-borne Diseases, Faculty of
tion contracted to carry out the in clinical part of the study. Veterinary Medicine, Utrecht University.
This part was funded by Bayer Animal Health GmbH, Lev-
erkussen, Germany of which D. Stanneck is an employee.
The artificial feeding experiments were conducted at the Appendix A. Supplementary data
Utrecht Centre for Tick-borne Diseases (UCTD) of Utrecht
University. This part was funded by Merial, Lyon, France Supplementary material related to this article can be
of which F. Beugnet is an employee. F. Jongejan is PhD found, in the online version, at http://dx.doi.org/10.1016/j.
supervisor of JJF and Professor at the Universities of vetpar.2013.07.026.
124

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126

Chapter 5. Prevention of Dipylidium caninum trans-
mission by fleas
5.1. Fourie JJ, Crafford D, Horak IG, Stanneck D.

Prophylactic treatment of flea-infested dogs with an imidacloprid/flu-
methrin collar to preempt infection with Dipylidium caninum. Parasitol
Res. 2013, 112 : S33-S46.
127

128

Parasitol Res (2013) 112:S33–S46 DOI 10.1007/s00436-013-3279-5 ECtoparaSItES
Prophylactic Treatment of Flea-Infested

Dogs with an Imidacloprid / Flumethrin
Collar (Seresto®, Bayer) to Preempt
Infection with Dipylidium caninum
Josephus J Fourie1, Dionne Crafford1 (*), Ivan G Horak2,3, Dorothee Stanneck4
1 ClinVet International, P.O. Box 11186, Universitas, Bloemfontein, 9321, South Africa
2 Department of Zoology and Entomology, University of the Free State, Bloemfontein,
9301, South Africa
3 Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria,
Onderstepoort, 0110, South Africa
4 Bayer Animal Health GmbH, 51368 Leverkusen, Germany
Corresponding author:
Dionne Crafford
* E-mail: dionne.crafford@clinvet.com
Abstract
The objective of the study was to determine the sus- metacestodes. Prior to each post-treatment infesta-
tained effectiveness of 10 % imidacloprid and 4.5 % tion the D. caninum infection rate for the fleas was
flumethrin, incorporated in a slow-release matrix determined by microscopically examining 100 fleas
collar, in preventing Dipylidium caninum infection for D. caninum metacestodes. The D. caninum
in dogs after repeated laboratory infestations with prevalence in the fleas used for infestations ranged
fleas infected with metacestodes of this tapeworm. from 23 % to 52 %. Medicated collars were fitted to
Efficacy against infection with D. caninum was 8 of the dogs on study day 0. The weight of the IVP
evaluated by infesting 16 dogs with cat fleas (Cteno- collars varied between 35.48 g and 38.48 g (aver-
cephalides felis) on study days 7, 14, 21, 28, 35 and age 37.16 g), whilst animal weight varied between
42, from batches suitably infected with D. caninum 12.20 kg and 17.98 kg (treated group, n = 8, average
S33
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EctoparasitEs
14.79 kg). Seven days later infestation of each of of veterinary and zoonotic importance, including
the 16 dogs with 250 fleas commenced. Infestations three protozoan species and the metacestode stage
continued at weekly intervals until Day 42 with of the dog and cat tapeworm, Dipylidium caninum
efficacy against fleas evaluated 24 hours after each (Pugh 1987; de Avelar et al. 2007).
infestation. From Days 21 to 74, infection of the Dogs and cats acquire infection with D. caninum by
dogs with D. caninum was verified (daily examina- ingesting infected fleas, most often during groom-
tion of faeces and cages for the presence of expelled ing. Infection of dogs and cats with D. caninum is
proglottids). Calculation of prophylactic effective- a global phenomenon. The parasite is widely dis-
ness of the collars in preventing infection with tributed in Italy (Oranto and Dantas-Torres 2010)
D. caninum was based on the difference in geomet- and 38 of 63 adult dogs selected for an anthelmin-
ric mean numbers of scoleces between groups at tic efficacy trial were infected (based on faecal
necropsy on Day 75. Effective prevention of infec- examination for proglottids) (Genchi et al. 1990).
tion with D. caninum was found to be 96.6 %. Effi- Dipylidium caninum was also the most common
cacy of the collars against fleas was ≥ 99.9 % for the helminth parasite in 156 dogs examined (either
duration of the assessment period. after treatment with arecoline hydrobromide or at
Newly acquired infestations of fleas are rapidly necropsy) in Israel, with 97 of the dogs infected
eliminated by the insecticidal components of the (Furth and El-On 1990). Twelve of 15 dogs belong-
medicated collars over a period of several months. ing to an aboriginal community on the South coast
In the event of fleas being infected with metaces- of New South Wales, Australia, were infected, with
todes, infection with D. caninum can be prevented the intensity of infection ranging between 1 and
in collared dogs, concurrently reducing the likeli- 65 tapeworms (Jenkins and Andrew 1993). In an
hood of transmission to humans. anthelmintic study conducted on dogs in Texas,
USA, 18 noticeably flea-infested dogs were also all
infected with D. caninum based on faecal examina-
Introduction tion (Craig et al. 1991). At necropsy, scoleces (num-
bers ranging from 2 to 44) were recovered from all
The cat flea, Ctenocephalides felis, infests both dogs but one of the 8 untreated control animals (Craig
and cats, is widespread throughout most regions of et al. 1991). Examination of 55 stray cats in Iraq
the world (Beaucournu and Ménier 1998; Ménier yielded a D. caninum infection prevalence of 64 %
and Beaucournu 1999; Beck et al. 2006) and is con- (Al-Obaidi 2012). In Gauteng Province, South Afri-
sidered to be one of the most important ectopar- ca, 27 of 69 dogs (belonging to a resource-limited
asites of dogs and cats (de Avelar et al. 2007). community) were found to be infected at necropsy.
Infestation often results in itching and scratch- The number of scoleces recovered varied between
ing, often progressing to hair loss and skin lesions 1 and 288 (Minnaar and Krecek 2001). Boreham
caused by continuous grooming in more sensitive and Boreham (1990) reviewed some of the litera-
animals. The development of flea allergy dermati- ture prior to 1990 and list Australia, India, Iraq,
tis provoked by the saliva of feeding fleas has also Jordan, Malaysia, Morocco, Nigeria, Pakistan, the
been reported (Genchi et al. 2000). Consequently USA and Zambia as countries in which infection
effective control of C. felis not only eliminates fleas, with D. caninum has been encountered in dogs.
but also alleviates discomfort caused to its hosts. In The near global occurrence of D. caninum infec-
addition, fleas are considered to be of considerable tion can be considered an indication of the extent
importance as vectors of pathogens in many parts of the problem.
of the world (Bitam et al. 2010). More specifically, It is generally accepted that infection with D. ca-
C. felis plays host to a number of endosymbionts ninum produces few if any clinical signs in dogs
S34
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EctoparasitEs
Fig. 1 Dissected Ctenocephalides felis showing the inner organs and metacestodes of Dipylidium caninum (arrows)
S35
131

EctoparasitEs
(Boreham and Boreham 1990). These may include family dog “more or less” frequently is not sufficient
mild gastrointestinal signs and scratching (Mani to provide reliable long-term protection against
and Maguire 2009). However, infection of pets is human exposure to infection.
usually a cause of considerable distress and often Besides changes in human behaviour (Jithendran
embarrassment to their owners (e.g. when a dog and Bhat 2001) to mitigate the risk of accidental
drags its anus over an indoor carpet). More impor- ingestion of fleas, biological control measures such
tantly, humans may also become infected with as nematophagous fungi that have an effect on
this cestode (Chappell et al. 1990; Raitiere 1992). D. caninum eggs in the environment (and would
Infections are most often diagnosed in small chil- hence forestall infection of fleas) may be a preventa-
dren as they are generally in closer contact with tive option (Araujo et al. 2009). Both options, how-
family pets (and hence the fleas that infest them) ever, pose logistical challenges and the last one is
(Chappell et al. 1990; Raitiere 1992; Jithendran scarcely effective. According to Mani and Maguire
and Bhat 2001) and because of their propensity for (2009): “Precautionary measures are necessary to
pica (Mani and Maguire 2009), but adults can also prevent zoonotic transmission of pathogens while
be infected (Adam et al. 2012). A stool passed by a keeping a pet. Routine and regular veterinary
6-month-old infant contained 13 intact tapeworms care of companion animal pets with appropriate
and several short strands of proglottids on the day preventative medicine is extremely important for
after treatment (Chappell et al. 1990). prevention of transmission”. Chemical control thus
The most obvious recourse to reduce the risk of appears to be an unavoidable option and when
infection with D. caninum in the human environ- directed at breaking the D. caninum life cycle, may
ment is certainly regular deworming of dogs with be efficacious both against fleas and tapeworms.
a praziquantel containing drug. However, the It is thus obvious that a formulation offering sus-
required frequency of treatment may be underes- tained efficacy against fleas on dogs may aid great-
timated by the dog owner, especially as the dog can ly in preventing the zoonotic transfer of parasites,
be re-infected at any time after successful anthel- including D. caninum.
mintic treatment by the ingestion of an infected Imidacloprid was introduced in 1996 and has
flea. Accordingly, simply deworming the infected since become one of the most successful and
Fig. 2 SEM photograph showing a closer view of a Fig. 3 SEM photograph showing the surface detail of a
metacestode of Dipylidium caninum (bar = 50 μm, metacestode of Dipylidium caninum (bar = 5 μm)
box indicating the area shown in Fig. 3)
S36
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EctoparasitEs
largest selling veterinary products for flea control Materials and methods
(Schroeder et al. 2003). Due to the rapid mode of
action of imidacloprid, it decreases flea feeding This parallel, group-designed, randomised, uni-
periods and hence reduces the risk of transmission centre, controlled efficacy study was conducted in
of flea-derived diseases (Mehlhorn et al. 2001). It South Africa and involved two groups of dogs each
is often combined with actives such as permethrin comprising eight animals. Initially 20 mixed-breed
(e.g. Mehlhorn et al. 2003) or moxidectin (Mehlhorn domestic dogs ranging in age from sub-adults to
et al. 2005; Schmahl et al. 2007) to also provide effi- adults were enrolled in the study. These dogs had
cacy against ticks, mites and nematodes. As such, not been treated with an acaricide, or insecticide,
products containing imodacloprid were shown to or a compound with an insect growth-regulating
be effective against C. felis on hosts as diverse as activity during the previous 12 weeks and were
dogs (Epe et al. 2003; Hellman et al. 2003), cats not infected with D. caninum (as confirmed by
(Arther et al. 2003), mink (Larsen et al. 2005) and visual inspection of faeces and surrounds during
ferret (Wenzel et al. 2008). In all these instances acclimatisation). As an added precaution, all dogs
an efficacy exceeding 90 % was obtained for a period were dewormed with Triworm-D® (praziquantel
of at least 28 days post-treatment. These findings 50 mg; pyrantel pamoate 144 mg; febantel 150 mg;
correspond well to that recorded against Tunga Cipla-Vet; South Africa) seven days prior to the
penetrans following treatment with an imidaclo- commencement of the study.
prid / permethrin combination (Klimpel et al. 2005). The dogs were housed individually in pens for
The rapid and sustained efficacy of a slow-release the duration of the investigation, and no contact
matrix collar formulation of 10 % (m/m) imidaclo- between dogs was possible. The dog pens con-
prid and 4.5 % (m/m) flumethrin (Seresto®) against sisted of a 1.69 m x 0.7 m enclosed sleeping area
laboratory infections of dogs with the cat flea and an outside run of 1.69 m x 3.0 m. A large roof
C. felis has recently been demonstrated by Stan- covered all the pens and the dogs were therefore
neck et al. (2012). In addition, the efficacy of these not exposed to rain, but were exposed to ambient
collars in preventing infection with the cestode temperature and sunlight. The pens had concrete
D. caninum in cats repeatedly infested with fleas floors to facilitate cleaning, and no bedding was
infected with the metacestodes of the tapeworm provided. The accommodation was in compliance
has also been reported (Fourie et al. 2012). Con- with the South African National Standard (SANS
trol of the metacestode-infected fleas resulted in a 10386:2008. The care and use of animals for sci-
99.7 % reduction in the number of scoleces recov- entific purposes). The animals were fed once daily
ered from collared cats compared to untreated cats with commercially available dog pellets according
(Fourie et al. 2012). to the manufacturer’s recommendation. The pel-
The aim of the present investigation was twofold. lets, and fresh, clean water were provided in stain-
Firstly to assess the efficacy of the 10 % (m/m) imi- less steel bowls and the water was replenished at
dacloprid/4.5 % (m/m) flumethrin collars against least twice daily. The animals were maintained
fleas that were infected with metacestodes of and handled with due regard for their welfare,
D. caninum, and concurrently evaluate the effec- and were acclimatised to the pen environment for
tiveness of the formulation in preventing infection 7 days prior to the commencement of the study.
with the tapeworm in dogs. On Day –6, all the dogs were infested with 100 fleas
that were not infected with D. caninum and that
originated from a laboratory-bred strain of C. felis
(ClinVet European strain; routinely fed on dogs).
The flea count of each animal 24 h after infestation
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was used for ranking and group allocation pur- proglottids. We have accepted this reasoning and
poses. Three dogs with the lowest flea counts, and terminology and consequently refer to the develop-
a dog that did not comply with the required study mental stages of D. caninum in the fleas as meta-
criteria were excluded from the remainder of the cestodes. The fleas were infected with metacestodes
study. The remaining 16 dogs were ranked in by incubating thousands of their eggs, as well as
descending order on their individual pre-treatment the larvae that hatched, on flea-rearing medium
flea counts, and their IDs were used to break ties. mixed with proglottids and eggs of the tapeworm
They were then blocked into blocks of two animals at temperatures varying between 24 °C and 28.5 °C.
each, and within each block, dogs were randomly Prior to each post-treatment flea infestation of
allocated to two groups. The 16 animals included the dogs, the infection rate of D. caninum in the
in the study weighed between 12.2 and 19.5 kg and fleas was determined by microscopically dissecting
their hair-lengths varied between 9.5 and 34.5 mm. 100 specimens and examining them for metaces-
On Day 0, the medicated collars were fitted to the todes. The prevalence of D. caninum metacestodes
necks of the dogs allocated to the treatment group. in the fleas used for infestation varied between 23 %
Each collar was adjusted by means of the buckle and 52 %. Each dog was infested with 250 of these
to achieve a comfortable fit and any excess was cut fleas on the days indicated in Table 1. The fleas
off approximately 2 cm beyond the retaining loop. used for infestation were unfed and of mixed sex,
All collars were marked with the dog’s ID number and were not placed on or near the site of the fit-
so that in case a collar was accidentally dislodged, ted collar. Dogs were restrained by hand during
it could easily be identified and immediately re- infestation and during flea recovery.
applied. The weight of the IVP collars fitted in this A fine-toothed flea comb was used to recover fleas
manner, varied between 35.48 g and 38.48 g (aver- from the animal’s hair coat and its skin surface.
age 37.16 g), whilst animal weight varied between Combing was performed by several strokes of the
12.20 kg and 17.98 kg (treated group, n= 8, average comb over each body part of the dog, each time mov-
14.79 kg). At pre-determined time intervals on the ing in the same direction and following the pattern
day that the collars were fitted all animals were of the hair coat. Movement, from one part of the
carefully observed for adverse signs that could be animal’s hair to the next, was via strokes overlap-
ascribed to the collars or to the active ingredients ping each other, so that no area of the body or hair
that they contained. Thereafter they were observed was missed. After completion of the combing of all
at daily intervals for clinical signs that could be body areas, the whole procedure was repeated so
associated with the collars or for any other concur- that all sites were combed at least twice. If neces-
rent conditions. sary, the combing was performed for a third time or
Ctenocephalides felis, infected with a South Afri- more until no live fleas were found. Fleas collected
can strain (isolated from resident ClinVet animals) in this manner were quickly counted and live fleas
of D. caninum, were used for all post-treatment placed back on the dog from which they had come.
infestations. Chervy (2002) defines a cysticercoid Counting of fleas was not blinded since the control
as a metacestode with a primary lacuna, retracted dogs were not fitted with placebo collars, thus ma-
scolex with a cercomer or reduced cercomer. Pugh king blinding impossible.
(1987) argued that D. caninum metacestodes can- The groups were compared on their flea counts by
not be considered cysticercoids as the primary lacu- a one-way ANOVA (analysis of variance performed
na develops but then disappears. He suggested the using the Proc GLM procedure in SAS Version 8,
use of the term metacestode to ecompass all growth Release 8.02, released 2001 TS Level 02M0) with a
forms following the metamorphosis of D. cani- treatment effect on the flea counts (count +1) after
num oncospheres and before the development of logarithmic transformation of the data.
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Efficacy of the collars against C. felis was calcu- examination of fresh faeces, the anal and perineal
lated as follows: regions of the animals, their hair and their cages.
If no proglottids were found, freshly excreted fae-
Efficacy (%) = 100 x (N1 – N2) / N1
N1 = geometric mean number of live fleas on dogs in ces were washed through steel-mesh sieves with an
the untreated control group aperture size of 0.15 mm. The residues in the sieves
N2 = geometric mean number of live fleas on dogs in
the treated group
were collected and suspended in a small amount of
water, which was then examined macroscopically
The dogs were observed daily from Day 21 to Day for the presence of proglottids. The proglottids
74 in order to detect the presence of expelled pro- or fragments of worms recovered were examined
glottids (Table 1). This involved visual, macroscopic microscopically to ensure that identification was
Table 1 Design of a study aimed at determining the effectiveness of imidacloprid/flumethrin collars in the prevention of
Dipylidium caninum infection in dogs repeatedly infested with infected fleas
Study day Activity
–7 to – 1 Acclimatisation to cage environment
–6 Infestation with 100 non-infected fleas
Flea counts, 3 dogs with lowest counts excluded from remainder of study; 1 dog excluded as it
–5
did not comply with the inclusion criteria
–2 Ranking and allocation to 2 groups of 8 dogs each
0 Imidacloprid/flumethrin collars fitted to treated group
7 Infestation with 250 infected fleas
8 Flea counts and re-infestation with the fleas that had been counted
21 Infestation with 250 infected fleas; daily examination of faeces for expelled proglottids commences
43 Flea counts
74 Daily examination of faeces for expelled proglottids ceases
75 Necropsy and collection and counting of scoleces
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correct. All proglottids were preserved and retained Prophylactic effectiveness against infection with
until the completion of the study as a record of the D. caninum by infected fleas was calculated as
diagnoses. Once a dog had shed proglottids on two follows:
separate occasions, no further faecal examinations
Prophylactic effectiveness (%) = 100 x (N1 – N2) / N1
or other examinations for proglottids were conduct-
N1 = geometric mean number of D. caninum scoleces
ed on that dog. recovered from the untreated control group of
All the dogs were euthanised on Day 75 by intra- dogs
venous injection of EuthapentTM (sodium pento- N2 = geometric mean number of D. caninum scoleces
recovered from the group of dogs fitted with
barbitone 200 mg/ml; Kyron laboratories) at an
medicated collars
approximate dose of 1 ml/kg. Food was removed
from the cages during the afternoon prior to eutha-
nasia in order to reduce the volume of ingesta in
the gastrointestinal tract at necropsy. At necrop- Results
sy, a ligature was applied at the ileo-caecal junc-
tion between the small and large intestines and The geometric mean flea counts of the two groups
the digestive tract from the stomach to the rec- of dogs on the various assessment days are sum-
tum was removed from the abdominal cavity. The marised in Table 2. The mean flea counts of the
small intestine, including the stomach, and the untreated control group (from Day 8 to Day 43)
large intestines were treated separately. They were varied between 139.9 and 226.5, indicating a
carefully cut open and their contents flushed with robust flea challenge on all post-treatment assess-
water, and their mucosa thoroughly scraped. All ment days. The geometric mean flea counts of the
material that had been flushed, washed or scraped collared group of dogs differed statistically signif-
from the small intestines and stomach were then icantly (p < 0.05) from those of the untreated con-
washed over a sieve with 0.15 mm apertures. The trol group on all post-treatment assessment days.
contents of the large intestines and their mucosal The efficacy of the collars against infestation with
scrapings were also washed over sieves with an C. felis was ≥ 99.9 % for the 42-day duration of that
aperture size of 0.15 mm. The residues in the part of the study devoted to fleas.
sieves were collected and preserved with formalin Based on the collection of expelled D. caninum
in labelled bottles. The bottles were coded for each proglottids, 87.5 % (7/8) of the dogs in the untreat-
dog in order to blind the counting of D. caninum ed control group and 25 % (2/8) of the dogs fitted
scoleces. with collars were infected with D. caninum. The
The effectiveness of the imidacloprid/flumethrin time between fitting the collars and detection of
collars in the prevention of D. caninum infection D. caninum proglottids in the dogs’ faeces or in
was based on the difference between the geomet- their immediate surroundings is summarised in
ric mean numbers of scoleces recovered from the Table 3. All but one of the dogs in the untreated
control and treated groups of dogs. The geometric control group and two dogs in the collared group
mean was calculated following logarithmic trans- shed proglottids and consequently were considered
formation. In cases where a scolex count was zero, to be infected with D. caninum. The first proglot-
all counts were modified by adding one (1) to each tids to be detected were present in the faeces on
count prior to transformation. Thereafter one (1) Days 24 and 25 of two dogs in the treated group.
was subtracted from the antilog value to meaning- The majority of untreated dogs had started shed-
fully represent the geometric mean for each group. ding proglottids by Day 38, but one dog only started
SAS Version 8 (Release 8.02, released 2001, TS shedding proglottids on Day 41 and another on Day
Level 02M0) was used for all statistical analyses. 43 after the collars had been fitted.
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The numbers of D. caninum scoleces recovered infected and each harboured 1 scolex. Each of the
from the intestinal tracts of the dogs at necropsy latter dogs had shed proglottids during the period
are summarised in Table 4. Scoleces were recov- prior to euthanasia.
ered from all but one of the untreated control The geometric mean number of scoleces recovered
dogs and numbers varied between 1 and 126. No from the negative control group of dogs (5.5) dif-
proglottids had been recorded for dog A2D in the fered statistically significantly (p < 0.05) from that
untreated control group prior to euthanasia, yet of the collared group of dogs (0.2). Based on the
it was found to be positive at necropsy and har- geometric mean number of scoleces recovered, the
boured 3 scoleces. On the other hand dog 150 shed collars were 96.6 % effective in preventing infection
proglottids on Days 41 and 48, but harboured no with D. caninum in the dogs.
scoleces at necropsy. Two of the collared dogs were
Table 2 Efficacy of an imidacloprid/flumethrin collar applied on study day 0 against Ctenocephalides felis on repeatedly
infested dogs
Geometric mean number of fleas recovered

Study day Efficacy (%)
Untreated control dogs Dogs fitted with collars*
8 139.9 0.0 100
15 205.4 0.3 99.9
22 201.5 0.2 99.9
29 220.7 0.1 99.9
36 226.5 0.0 100
43 200.5 0.0 100
* The flea counts of the collared group of dogs differed statistically significantly (p < 0.05) from those of the untreated
control group of dogs on all post-treatment assessment days
Table 3 The presence of Dipylidium caninum proglottids in the faeces and surroundings of untreated dogs and of dogs
fitted with imidacloprid/flumethrin collars
Untreated control dogs Dogs fitted with collars
Dog ID Days proglottids detected Dog ID Days proglottids detected
420 34 and 35 7F8 None
059 37 and 38 937 None
E9A 43 and 44 219 None
A2D None B54 24 and 39
177 38 and 39 E80 None
D08 37 and 39 3EA 25 and 40
71E 37 and 39 931 None
150 41 and 48 539 None
Infection (%) 87.5 Infection (%) 25.0
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Table 4 Dipylidium caninum scoleces recovered from untreated control dogs and from dogs fitted with imidacloprid/
flumethrin collars
Untreated control dogs Dogs fitted with medicated collars
Dog ID Number of scoleces Dog ID Number of scoleces
420 126 7F8 0
059 2 937 0
E9A 1 219 0
A2D 3 B54 1
177 86 E80 0
D08 1 3EA 1
71E 5 931 0
150 0 539 0
Total 224 Total 2
Geometric mean 5.5 Geometric mean 0.2
Prophylaxis (%) 96.6
Discussion extraordinary grooming efficiency of these animals

as reported by Hinkle et al. (1998).
The efficacy of 99.9 % to 100 % of the imidacloprid/ Differences in faecal worm egg counts between
flumethrin collar (Seresto®) against repeated infes- treated and control groups of animals may in some
tations with C. felis during the first 42 days of the instances prove to be valid for the determination of
study confirms the results of earlier investigations anthelmintic efficacy against nematodes, yet they
on the efficacy of the collars against fleas on dogs are more often considered as supportive of results
(Stanneck et al. 2012). The geometric mean num- obtained during necropsy. No such quantitative
ber of 139.9 fleas counted on the untreated dogs methods (with regard to faecal egg count evalu-
on Day 8 of the investigation is probably the most ation) exist for efficacy studies against cestodes.
reliable indication of the number of C. felis that This is particularly true for D. caninum as the
became established after each infestation with shedding of proglottids is neither quantitative nor
250 fleas. All subsequent counts exceeded 200 fleas, consistent. This phenomenon is evident for one dog
with the arithmetic mean number collected on Day in the control group in which there was a break of
36 exceeding 226, only 24 less than used for infesta- 7 days between the first and the next appearance
tion. These high burdens probably result from the of proglottids and two dogs in the treated group
fact that once fleas had been counted they were where this gap was 14 days. Furthermore, one
replaced on the dog from which they had been col- of the dogs in the control group shed no proglot-
lected. This increase in flea numbers on the dogs tids, but harboured 3 scoleces at necropsy. These
differs from the flea counts of cats in a similar scoleces, however, appeared to have resulted from
study in which the greatest mean number of fleas a recent infection as each had developed only a few
was collected on Day 8 and lesser numbers there- segments beyond its neck.
after (Fourie et al. 2012). The lack of an increase The shedding of proglottids by two dogs in the treat-
in flea numbers on the cats was ascribed to the ed group, 24 and 25 days after the commencement
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of the study represents prepatent periods of 17 and not blood meals taken by the flea, then enables the
18 days after infestation with the first batch of metacestodes to mature and become infective for
infected fleas on Day 7. These prepatent periods are the definitive host (Pugh 1987). The latter obser-
a day or two shorter than the 19 days observed in vation is of particular significance in the present
a cat similarly infected in an earlier study (Fourie study during which the fleas in the pool used for
et al. 2012), and a few days less than the three infestation were maintained at temperatures
weeks quoted by numerous authors without stating ranging from 24 °C to 28.5 °C. This is in agreement
their source. The short prepatent periods in these with maximum temperature ranges encountered
two dogs suggests that metacestodes in the fleas in microhabitats in many households and loca-
with which they were infested had completed their tions around homes, where temperatures ranging
development to infectivity off-host, and that the between 13 and 27 °C favour the survival of flea lar-
infected fleas were ingested shortly after release vae (Guardis et al. 1992). At this temperature range
onto the dogs. Proglottids were detected in one of (i.e. 13 °C to 27 °C) many of the metacestodes in the
the dogs in the untreated group on Days 41 and 48, approximately 14-day old groups of fleas used for
but this dog appears to have lost its infection as no infestation would not have developed to infectiv-
scoleces were recovered at necropsy 27 days later. ity. These metacestodes would thus have required
A similar instance of self-cure has been recorded in that their flea hosts infested a mammalian host
a naturally infected dog, selected for inclusion in an for a few days before they became infective. It was
anthelmintic trial on the presence of proglottids in precisely for this reason that once fleas had been
its faeces, yet at necropsy it was found to harbour counted they were released back onto the same dog
no D. caninum (Craig et al. 1991). from which they had come, so as to ensure that
There is a 10-fold difference in the geometric mean any metacestodes with which they were infected
number of scoleces collected from the 8 untreated could develop to infectivity and thus more closely
dogs in this study and from 8 untreated cats in resemble conditions pertaining in the field. Apart
a similar study (Fourie et al. 2012). A geometric from temperature, the diet available to flea larvae
mean of 5.5 scoleces was recovered from the dogs (yeast content) may also have a slight influence on
compared to 58.3 from the cats. Infection in only metacestode development (Benesh 2010), poten-
2 of the dogs, one harbouring 126 scoleces and the tially resulting in additional variability in parasite
other 86 scoleces, fell within the range of 19 to development under field conditions.
349 scoleces recovered from individual cats (Fourie Within a home environment the temperature may
et al. 2012). It is difficult to explain the disparity in fluctuate considerably, but the average daily tem-
scolex numbers between the dogs and cats. It could perature is unlikely to exceed 25 °C, as demon-
be due to spontaneous loss of infection as appears strated by Guardis et al. (1992). In sheds, stables,
to have occurred in the dog that shed proglottids, kennels, yards, outside rooms or verandahs fre-
but harboured no scoleces at necropsy. It could quented by dogs the average daily temperature is
possibly also be due to an immune reaction to the liable to be even lower. Under these circumstances
large number of metacestodes with which the dogs infected fleas would have to spend a few days on a
were infected over a period of 6 weeks resulting in dog before the metacestodes become infective. It
self-cure. is during this pre-infective period on the host that
Pugh (1987) has demonstrated that at tempera- infected fleas must be killed to prevent infection of
tures below 30 °C metacestodes are unable to com- the host animal with D. caninum. Studies prior to
plete their development to infectivity in adult fleas the present one have shown that the imidacloprid/
unless the fleas are placed on a mammalian host flumethrin collars killed > 99 % of fleas that access
for a few days. The host’s surface temperature, and a dog within 24 h after the collars had been applied
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EctoparasitEs
(Stanneck et al. 2012). Furthermore, the collars nematode, Acanthocheilonema reconditum, which
also proved to be > 94 % effective within 24 h of has a worldwide distribution and is the cause of
subsequent infestations with C. felis over a period canine subcutaneous filariosis (Brianti et al. 2012).
of 8 months (Stanneck et al. 2012). It is thus the The rapidity with which the imidacloprid/flume-
rapidity with which the active ingredients of the thrin collars eliminate fleas and their sustained
collars kill fleas that prevents them spending suf- efficacy make them an excellent candidate for the
ficient time on the host for the metacestodes with prevention of this condition.
which they may be infected to develop to maturity
and capable of infecting a host.
Dog owners who detect fleas on their animals, Conclusion
and who at the same time notice single or chains
of proglottids in their dogs’ faeces or on their hair The rapidity with which the insecticidal compo-
coats, are likely to seek advice from their local vet- nents of 10 % imidacloprid/4.5 % flumethrin collars
erinarians. The animals should then be treated eliminate newly acquired infestations of fleas and
for tapeworms and the owners informed that fleas the collars’ sustained high level of efficacy imply
must be controlled on the dogs in the future. Should that should fleas that access dogs be infected with
imidacloprid/flumethrin collars be used for flea con- the metacestodes of D. caninum, infection of the
trol the prolonged period of protection they afford dogs can be prevented by application of the col-
against flea infestation will play an integral role lars. Because of their sustained release kinet-
in the prevention of re-infection with D. caninum. ics and hence efficacy against fleas over a period
The logic behind this approach is that provided of 8 months the collars can also be regarded as
temperature and moisture are adequate, D. cani- a means of protecting humans from D. caninum
num proglottids that had been shed several weeks infection in preference to regular short-term treat-
or even months previously, remain viable (Craig ment applications by the animal owner otherwise
et al. 1991), and consequently flea larvae, that were needed for rigorous flea management.
already present in the dogs’ environment before
treatment, are still likely to become infected. The Ethical standards
sustained efficacy of the collars will then eliminate All institutional and national guidelines for the
the resultant infected adult fleas that access the care and use of laboratory and study animals were
dogs long after the collars have been fitted. followed.
The imidacloprid/flumethrin collars are not only
effective against adult fleas, but also effective Conflict of interest
against flea larvae in the dogs’ immediate sur- This clinical study was completely funded by
roundings (Stanneck et al. 2012). Thus if larvae are Bayer Animal Health GmbH, Monheim, Germany,
eliminated in the collared dog’s favourite sleeping, of which D Stanneck (Germany) is an employee.
resting or loafing places in the home or beyond its ClinVet, of which JJ Fourie and D Crafford are
confines, the numbers available to ingest D. cani- employees, is an independent, South African, Con-
num eggs will be drastically reduced. This in turn tract Research Organisation contracted to manage
will result in a significant decline in the number of the conduct of the study. IG Horak is a long-term,
infected adult fleas, of which the vast majority will contract employee of Clinvet and an Extraordinary
in turn be killed on the dog. Professor at the Universities of the Free State and
In the context of preventing dipylidiasis, another Pretoria. All authors voluntarily publish this arti-
flea-transmitted helminth is worth mentioning. cle and have no personal interest in these studies
Fleas are also the recognised vectors of the filarial other than publishing the scientific findings.
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Acknowledgements They are also grateful to all other ClinVet staff

The authors express their sincere thanks to DJ Kok members who took part in the study and ensured
(Jr) who bred and provided various batches of fleas that Good Clinical Practice standards were adhered
infected with D. caninum and who, with P Swart, to. IG Horak acknowledges funding received from
diagnosed infection with D. caninum in the dogs. the National Research Foundation of South Africa.
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imidacloprid, imidacloprid/permethrin and phoxim for flea
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S46
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Chapter 6. General Discussion
143

144

6.1. Introduction
A prerequisite for the testing of a topically applied combination of cyphenothrin and

pyriproxyfen against Haemaphysalis elliptica ticks and Ctenocephalides felis fleas
on dogs as well as slow-release collar formulations of imidacloprid/flumethrin and
deltamethrin and of spot-on formulations of fipronil/(s)-methoprene, dinotefuran/
pyriproxyfen/permethrin and (s)-methoprene/amitraz/fipronil against Rhipicepha-
lus sanguineus ticks and C. felis, was the availability of colonies of fleas and ticks
(Chapter 2). Similarly, the studies included in other chapters of this thesis required
the breeding of significant numbers of ticks and fleas, which is an essential prereq-
uisite for these types of clinical veterinary studies. Moreover, the ectoparasites used
need to satisfy the requirements of various guidelines.
The current European Medicines Agency (EMEA) guideline recommends that

established tick strains that originate from recent field collections and cycled for
at least two generations under controlled conditions, be used for laboratory effica-
cy studies (EMEA/CVMP/005/2000-Rev2). In a draft guideline (published on 12
March 2015), EMEA proposed that laboratory tick strains should be genetically en-
riched approximately every three years (EMEA/CVMP/005/2000-Rev3). Although
no such guidance with regard to laboratory bred flea strains is given in the EMEA
guideline, it can be assumed that the best practice principles recommended for
ticks will apply also to flea colonies. In the guideline published by the World Asso-
ciation for the Advancement of Veterinary Parasitology (WAAVP), no guidance
is provided with regard to strain origin and maintenance of ticks or fleas, but a clear
differentiation is made between efficacy studies using laboratory bred ticks and
fleas and field studies wherein efficacy is assessed under natural conditions (Mar-
chiondo et al, 2013).
In addition to the breeding of ticks in order to comply with guideline

requirements, the establishment of laboratory strains has many advantages. It
allows for the year-round conduct of laboratory efficacy studies, without being
influenced by the seasonal abundance of ticks in the field. Furthermore, specific
pathogen-free (SPF) laboratory tick and flea strains eliminate the risk of study
animals, and more importantly study personnel, becoming infected with unwanted
vector-borne pathogens. Maintaining SPF tick and flea colonies also allows for the
145

artificial infection of ticks with specific pathogens for use in different tick-borne
disease transmission blocking models. These models constitute an important part
of this thesis (Chapter 3, 4 and 5).
To successfully breed ticks and fleas, species-specific breeding plans needs to

be developed and implemented. Important considerations when developing a breed-
ing plan for a specific tick or flea species are the duration of the life cycle, host spec-
ificity, environmental conditions and biosafety. Any breeding plan involves two dis-
tinct processes, namely the on- host and off-host management of ticks and fleas. The
most important considerations when developing the on-host management plan for
ticks and fleas are the housing of host animals, infestation techniques and collection
of the fed life cycle stages. Off-host the storage containment of different life cycle
stages, environmental control and record keeping must be taken into account.
When deciding on a suitable host, the host specificity of the tick or flea species
should be evaluated first. Some tick species, such as H. elliptica (Chapter 2.1)
and R. sanguineus (Chapter 2.3) are more host-specific than D. reticulatus (Chapter
2.2), which will parasitize a range of different hosts. Once possible hosts have been
identified, the decision on which host species will be the most suitable must be
based on host capacity, ease of maintenance and availability. Host capacity is the
tick or flea burden that can be maintained on a specific host with careful consider-
ation for the welfare of the animal. For example large hosts such as cattle can
be infested with more (usually adult) ticks than a smaller animal such as a rabbit,
without significant adverse effects on the animal’s health. A larger host capacity
may also result in a reduction in the number of animals to be used, which is another
important consideration. Housing and maintaining large host animals is, however,
more difficult than housing smaller hosts. All the advantages and disadvantages of
the various host options need to be considered before an efficient tick or flea breed-
ing program can be established.
Host capacity is however not only influenced by the size of the host but also
by the infestation techniques used. Whole-body infestation increases host capacity
but lowers yield because of host grooming. Confined infestations in chambers or
stockinets are only possible on certain body areas of the host, thereby reducing
capacity, but increasing percentage yield since the parasites are less affected by
host grooming. Both these approaches have their advantages and disadvantages
pertaining to the collection of dislodged life cycle stages, biosafety, housing and
labour requirements.
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To successfully manage off-host ticks and fleas in the laboratory, one must
be able to safely handle the different life cycle stages and securely store them
within a set range of environmental conditions. Safe handling of ticks and fleas
can be achieved by using special handling containers in dedicated areas and by
personally wearing protective clothing such as gloves and coveralls. Primary stor-
age containers should be non-breakable, and fitted with a screen of mesh of which
the apertures should prevent escape. Primary containers should also be of an ap-
propriate size to avoid over-crowding of the particular life cycle stage. The
environmental conditions requiring control when rearing ticks and fleas are tem-
perature, relative humidity and photoperiod. These environmental conditions can be
created as well as controlled in a single system unit such as an incubator cabinet or
acaridarium/insectarium, or a multiple control approach may be followed in which
humidity, temperature and photoperiod are controlled in custom-fitted rooms. Cau-
tion should, however, be exercised when choosing an environmentally controlled
system regarding the risk of system failure when using a single control unit versus
a multiple control approach.
Good scientific execution of clinical efficacy trials on dogs also requires

the use of standardised and accurate infestation and parasite counting methodology.
The WAAVP guidelines give some assistance with choosing appropriate scientific
techniques for infesting dogs with ticks and fleas. The guidelines recommend that
newly emerged fleas should be used and that ticks should be of a uniform age.
Moreover they stipulate that the gender distribution of the ticks and fleas used for
infestation should be approximately equal, except when using Ixodes species, where
more females than males may be used. The recommended number of ticks and fleas
that can be used for infestation range between 25 to 100 ticks and 50 to 100 fleas.
Flea and tick infestations may be performed by spreading the parasites over the
dog’s body but avoiding product application sites (Marchiondo et al, 2013). An-
aesthesia and/or Elizabethan collars may be used during the infestation procedure.
It is however important that the technique used is appropriate for addressing the
objectives of the trial, especially for ticks. The choice of infestation technique
will therefore be based on what acaricidal activity is to be assessed. Moreover, it
is important to realise that acaricidal activity may be progressive and can even be
a combination of different activities as illustrated in Figure 7.
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Fig 7: Progression of acaricidal effect
The first aspect of acaricidal activity to consider is whether repellence efficacy needs
to be assessed or not. Repellency sensu stricto is characterized by an irritant effect,
causing the tick to avoid or move away from the treated animal leading to its
falling off soon after contact (Halos et al, 2012). Placing ticks directly on the host
will therefore result in an artificially induced contact with the acaricidal compound,
whereby repellency can be assessed but not avoidance, which is also a component
of repellency efficacy. The more appropriate infestation technique is therefore to
confine a dog in a suitable cage or crate with the intended number of ticks, thereby
allowing the ticks to seek the host naturally. We recently used this approach in
a study designed to ascertain the combined repellent effect of fipronil and perme-
thrin on dogs (Dumont et al, 2015). This approach can also be modified to simulate
a more natural state of affairs similar to that which will happen in the field. A good
example of this modified approach can be found in Chapter 4.1 and 4.2, where R.
sanguineus, a tick commonly found in kennels, was placed in the sleeping kennels of
the dogs enrolled in the study, thereby simulating a more natural scenario.
A further important consideration pertinent to the infestation methodology is

the use of anaesthesia. A fully mobile dog may dislodge ticks more easily than a se-
dated dog, thereby influencing repellence efficacy. Irrespective of whether the dog is
sedated or not, it is important that the repelled ticks are contained in a suitable crate
or cage for collection at predefined time points. If repellency is not to be assessed,
148

ticks can be placed directly on a dog and sedation is recommended, except if there
is a potential risk that the anaesthetics may affect the metabolism of systemic
acaricidal compounds. For this reason, we chose not to sedate the dogs enrolled
in the study in which the efficacy of the systemic compound (Nexgard) was evalu-
ated (Chapter 3.3). Furthermore, since evaluation of repellence efficacy was not an
objective of any of the studies using topical acaricidal products in Chapters 2 and 3,
infestations were performed on sedated dogs confined in crates. In general, the same
principles apply for infestations with fleas, except that sedation is not required,
since a dog can easily be restrained for the short period it takes for fleas to
disperse over the animal (Chapters 2.1, 2.3 and 5.1).
In addition to using appropriate infestation techniques, tick and flea counting

methodology must also be appropriate in order to address the specific objec-
tives of the efficacy studies conducted as part of this thesis. Moreover, the
time of assessment after treatment or infestation including the categorisation of
enumerated ticks and fleas should be accurate. The EMEA guideline provides little
help on the techniques of tick and flea counting, but does, however, elaborate on
assessment time points and tick categorisation. In this guideline a clear differentia-
tion is made in respect of acaricidal/insecticidal or repellence efficacy. Assessments
done at 48 hours after treatment or infestation are classified as acaricidal/insecticidal
assessments, whereas those performed at 24 hours post infestation are considered
assessments of repellency. Moreover, enumerated ticks must be classified based
on viability (dead or live), attachment status (attached or free) and feeding status
(engorged or unengorged) as performed in the efficacy trials in Chapter 2. Depend-
ing on categorisation, enumerated ticks are considered either an efficacy failure
or success, while engorged dead ticks are considered an efficacy failure (Table 4).
Table 4: Definition of parameters suggested by the European Medicines Agency guideline
Category General Findings Attachment status Effect
1 Live Free No
2 Live Attached; unengorged No (Except single ticks)
3 Live Attached; engorged No (Except single ticks)
4 Killed Free Yes
5 Killed Attached; unengorged Yes
6 Killed Attached; engorged No (Except single ticks)
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The EMEA guideline for evaluating engorgement of ticks by the presence of blood
in the digestive tract as determined visually by squeezing the tick or by weighing is
problematic. These methods are only reliable if the tick has ingested a certain
volume of blood. For instance, a recent study using qPCR to assess tick feeding
has shown that ticks start ingesting host fluids as soon as 2 hours post infestation,
which is impossible to detect accurately by the methods recommended in the
EMEA guideline (Fourie et al, 2014). If the study aims to correlate acaricidal
efficacy based on engorgement with a reduction of tick-borne disease risk of infec-
tion by infected ticks, specific transmission blocking models have been developed
to address this aspect (Chapters 3 and 4). Moreover, the use of specific models to
evaluate a compound’s ability to prevent transmission of tick-borne pathogens, has
been recommended in the WAAVP guidelines. In these guidelines the engorgement
status of ticks is not considered when evaluating acaricidal efficacy, but only the
final outcome (dead or alive). The methods for evaluating acaricidal efficacy sug-
gested by Halos et al (2012) also do not take into account tick engorgement status,
but instead rely on assessment methods and times to determine different acaricidal
efficacies. Consequently the evaluation of engorgement should not be considered
when assessing acaricidal efficacy, although when using qPCR, it can give interest-
ing insights into the anti-feeding effect of different compounds.
Accurate efficacy results can only be generated if tick and flea counts are
accurate and reliable. Various techniques are described in the WAAVP guidelines of
which comb-counts can be regarded as the most accurate technique for enumer-
ating fleas. Consequently this technique was used to count fleas in the efficacy
studies included in Chapters 2.1, 2.3 and 5.1, and in which a fine-toothed flea comb
(~11-13 teeth per centimetre) was used to recover fleas present in the dog’s haircoat.
Flea removal and counting that is facilitated by spraying a dog thoroughly with an
alcohol-based pyrethrin or dosed orally with nitenpyram, as described by Marchion-
do et al (2013), can also be very accurate, but inappropriate when animals need to
be re-infested with fleas.
Effective combing requires several strokes of the comb, each time moving in
the same direction following the ly of the hair coat. Movement, from one part of
the animal’s coat to the next, must be via strokes overlapping each other, so that
no area of hair is missed. Areas to be combed, not necessarily in this order, are the
outsides of the hind legs including feet, the tail and peri-anal area, lateral areas, not
including shoulders, abdominal area, from the chest to the inner surface of the hind
legs, the fore legs and shoulders including feet, the neck and head surfaces and the
150

dorsal strip from the shoulder blades to the base of the tail. After completion of
the combing procedure on all body regions, the whole procedure is repeated once
more so that all areas are combed twice. If fleas are still present, the combing pro-
cedure is repeated for a third time or more until no live fleas are found. Alternatively
combing can be done for a specific duration of time (~ 10 min) and continued for
predefined strokes or time increments if live fleas are still found after the minimum
combing time.
Tick count assessments are performed by a thorough and systematic exam-

ination of the whole body, with special attention to the ears, pinna, pinnal fold and
interdigital spaces. This can be accomplished through combing or palpation with
gloved fingers combined by visual examination and confirmation (Marchiondo et
al, 2013). Using the combing process as described for fleas is, however, not desir-
able for removing ticks since it can cause unnecessary dermal irritation. Palpation
and visual examination on the other hand allows the researcher to find the ticks and
carefully remove them with an appropriate device (e.g. forceps), thus causing the
least dermal harm and ensuring that the whole tick, including its mouthparts is
removed. Moreover palpation and visual examination also enables the researcher to
count the ticks in situ if the objective is to follow the efficacy progression of a prod-
uct over time, as was the case when we evaluated the efficacy of a slow release
collar formulation (imidacloprid/flumethrin) in Chapter 2.3. It is however advisable
to comb a dog after the whole body has been examined to ensure that all ticks
are removed. It is for this reason that all tick removal counts in Chapters 2, 3 and 4
were done by combining these two methods, first removing and counting all ticks
found by palpation and visual examination, followed by combing the dog to ensure
all ticks were removed.
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6.2. Pros and cons of spot-on, collars and oral formulations of acaricides
Acaricidal and insecticidal product development has been driven by a number

of factors. Development is aimed at delivering products with a broad spectrum of
fast acting efficacy, which persists for at least one month. Moreover a product
must be safe for the animal and the environment, easy to administer and with as
little as possible cosmetic impact. Currently the three main administration routes
for these products can be summarised as topical spot-ons, collars and oral tablets
or chews, each one of which has certain advantages and disadvantages.
The spectrum of activity of spot-ons is determined by the active substances

included in the formulation (see Chapter 1.8). Moreover, several active substances
can be combined to broaden the spectrum of activity. This is clearly illustrated
in Chapter 2.1 for combinations for cyphenothrin/pyriproxyfen, in Chapter 2.2 for
fipronil/amitraz/(S)-methoprene and in Chapter 2.3 for combinations of imidaclo-
prid/permethrin, and dinotefuran/pyriproxyfen/- permethrin against ticks and fleas.
In these studies it was demonstrated that cyphenothrin provided a good, per-
sistent acaricidal and insecticidal efficacy on its own. Moreover, it was shown that
by combining a pyrethroid (permethrin) with a neonicotinoid (imidacloprid or
dinotefuran) the spectrum of activity of the formulation can be increased from just
insecticidal to include acaricidal effects. The same is true when combining a potent
insecticide such as fipronil with a formamidine such as amitraz, which only has an
acaricidal activity. Furthermore, some active substances may display a synergistic
effect when combined, as is the case for the acaricidal effect of amitraz and fipronil
when administered in combination (Prullage et al, 2011). Not only can combin-
ing active substances in spot-on formulations broaden the spectrum of activity,
but also improve the onset of efficacy. This was recently demonstrated for perme-
thrin based formulations, which provided a consistently more rapid onset of residual
protection against the brown dog tick (R. sanguineus), compared to a formulation
based only on fipronil (Varloud et al, 2015). Furthermore, new combination spot-
ons containing both fipronil and permethrin can have improved repellency against
ticks and provide persistent effectiveness against fleas even when the dogs are ex-
posed to environmental factors that are believed to adversely affect efficacy, such
as water exposure or shampooing (Dumont et al, 2015; Fankhauser et al, 2015).
The rapid onset of acaricidal efficacy of permethrin based spot-ons and synergistic
152

combinations like fipronil/amitraz also have the ability to prevent infections with
tick-borne pathogens such as B. canis and E. canis, as demonstrated in Chapters 3.1,
3.3, 4.1 and 4.2. Not only can fast acting spot-ons protect dogs against tick-borne
pathogens, such combination products can also pre-empt flea-borne infections of D.
caninum (Beugnet et al, 2013). Spot-on formulations have great flexibility with
regard to combining different active substances to improve the spectrum of ac-
tivity, speed of kill and persistent effectiveness. Moreover, spot-on formulations are
relatively easy to administer and are generally well tolerated by dogs. The acaricidal
and insecticidal persistent efficacy of spot-ons remains, however, limited and rarely
exceeds one month because of the safety margins of the active substances used.
Moreover the efficacy of spot- ons can be negatively affected by environmental
factors, such as water exposure or shampooing. Spot-ons may also have transient
cosmetic effects on dogs such as spiking of hair at the site of application and pre-
cautions must be taken not to come into contact with the application site until it is
dry and the product has spread.
Medicated collars rely on the slow release of the active substance from
the collar matrix to provide long lasting efficacy. As is the case with spot-ons, the
spectrum of activity relies on the nature of the active substances included in the
collar matrix. The duration of efficacy is, however, dependent on the constant slow
release of the active substances from the collar matrix. The collar matrix therefore
plays a critical role in the efficacy performance of any medicated collar. In Chapter
2.3, the acaricidal and insecticidal efficacy of two medicated collars were compared,
one containing a pyrethroid (flumethrin) and neonicotinoid (imidacloprid) and the
other collar contained only a pyrethroid (deltamethrin). Moreover, the collar con-
taining the flumethrin/imidacloprid combination represented the latest development
in polymer matrix design (Stanneck et al, 2012a). In this study the collar matrix
containing flumethrin/imidacloprid was fast acting and had a much more consistent
acaricidal and insecticidal efficacy combined with a longer persistent duration of
efficacy than the deltamethrin collar (Chapter 2.3). Moreover, the synergistic ac-
tion of flumethrin/imidacloprid combined with their persistent slow release from
the collar matrix resulted in an unsurpassed eight month duration of acaricidal and
insecticidal efficacy (Stanneck et al, 2012a). Regular exposure to water or sham-
pooing also had little impact on efficacy since the active substances are continuously
released, as demonstrated by Stanneck et al (2012b). The fast acting properties of
the flumethrin/imidacloprid collar combined with its long persistent acaricidal
efficacy enables it to protect dogs against infection with B. canis (Chapter 3.2)
and also E. canis (Stanneck et al, 2013). Moreover the fast acting properties of
153

this specific medicated collar not only protect against tick-borne pathogens, but can
also pre-empt infections with D. caninum (Chapter 5.1). Medicated collars therefore
have the potential for an unsurpassed duration of efficacy as well as protection
against various vector-borne diseases. Although collars are relatively easy to apply,
dogs should, however, be accustomed to wearing a collar and mechanical irritation
may occur in isolated cases due to the collar rubbing against the dog’s skin.
Systemically active substances (e.g. moxidectin, selamectin) with an insec-

ticidal activity and administered as topical spot-ons have been available for some
years. More recently, however, orally administered systemically active products
with an insecticidal activity (spinosad) and insecticidal/acaricidal activity (afoxol-
aner and fluralaner) have been developed. The onset of activity of these products
against fleas is very rapid (Blagburn et al, 2010; Beugnet et al, 2014; Taenzler
et al, 2014). Moreover, these oral formulations also possess a good persistent
effectiveness against fleas (Snyder et al, 2007; Beugnet et al, 2015b; Hunter et
al, 2014). In addition to their insecticidal activity, afoxolaner and fluralaner are both
very effective against a range of tick species, including D. reticulatus, I. ricinus and
R. sanguineus (Wengenmayer et al, 2014; Dumont et al, 2014; Beugnet et al, 2015a).
Moreover, afoxolaner is sufficiently fast acting to prevent transmission of B. ca-
nis by D. reticulatus (Chapter 3.3). This new generation of oral formulations there-
fore provide a broad spectrum of activity, and are fast acting with good persistent
efficacy against ticks and fleas. Moreover, environmental factors such as water ex-
posure and shampooing that may adversely affect the efficacy of topically applied
products, have no impact on these oral products. In addition, dermal safety issues
are of no concern. However, since ticks and fleas must ingest these compounds to
be effective, there is no repellency effect, which may increase the risk for infection
with vector-borne borne pathogens, in particular those that are very rapidly transmit-
ted after tick attachment (e.g. E. canis).
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6.3. Speed of pathogen transmission
Pathogen transmission by feeding ticks depends on the duration of tick attach-

ment required by ticks to transmit the specific pathogen concerned. Reliable data on
the duration of tick attachment necessary for pathogen transmission is therefore es-
sential in order to evaluate whether the speed of kill of a specific acaricidal product
is adequate to prevent infection. For instance, the protozoan parasite Babesia mi-
croti usually requires several hours and even days (36 to 48 hours) for sporoblasts
to mature into sporozoites in the salivary glands of the tick before they can be
transmitted and infect the host (Piesman et al, 1980). These data on the rapidity
with which infectious organisms can be transmitted were compared with data
on acaricidal speed of kill before testing the efficacy of the products in Chapter
3 to prevent infection with B. canis. Furthermore, bacterial pathogens such as Ana-
plasma phagocytophilum require 24 to 36 hours to be transmitted by nymphal Ixodes
scapularis (Des Vignes et al, 2001; Katavolos et al, 1998). In contrast, very little
was known about the speed of transmission of E. canis by R. sanguineus. In Chapter
4.3 the time that an infected tick had to be attached before it could transmit E. canis
was determined in vitro as well as in vivo. The study revealed that transmission of E.
canis by R. sanguineus commences within a few hours (3 hours on dogs and 8 hours
on artificial membranes), an interval considerably shorter than previously presumed.
These findings underpin the need for further research on the speed of transmission
of tick-borne pathogens.
155

6.4. Transmission blocking models
The majority of veterinary clinical studies have focussed on demonstrating acari-

cidal or insecticidal efficacy against ticks and fleas. Consequently relatively little re-
search has been done on ascertaining the ability of compounds to prevent the trans-
mission of vector- borne diseases, especially under laboratory conditions. The main
constraint to research along these lines was the availability of reliable challenge
models. As a result the 2007 WAAVP guidelines did not include recommendations
to quantify the dynamics of transmission of tick- borne pathogens (Marchiondo et
al, 2007). More recently, however, the WAAVP recognised the research that we had
done and acknowledged that reliable challenge models have been developed that
could be used for specific claims regarding the prevention or reduction of tick- borne
pathogen transmission (Marchiondo et al, 2013).
The successful development of a challenge model is dependent on the avail-

ability of infected vectors. The establishment of B. canis (Chapter 3) and E. canis
(Chapter 4) challenge models were therefore dependent on infecting laboratory bred
ticks with these pathogens. Moreover, large numbers of ticks had to be infect-
ed in order to conduct the number of tick challenges required by these studies.
The establishment of B. canis and E. canis-infected batches of ticks relied on
the acquisition of infection by feeding ticks on an infected dog. Furthermore, the
final engorgement phase of the ticks must coincide with the acute infection phase
of a parasitaemic dog in order to increase the success of infecting the ticks. The
timing of host infection as well as tick infestation is therefore critical to ensure a
high proportional yield of infected ticks, before the infected donor dog is rescue
treated to ensure minimal impact on its welfare. Since B. canis is transmitted transo-
varially and transstadially in ticks, adult ticks can be fed on an infected dog, and
the detached engorged female ticks allowed to oviposit and the following generation
of adult ticks can be tested for infection. Using this approach, the adults of D.
reticulatus used in Chapter 3 were infected with B. canis by feeding them on dogs
infected with a B. canis strain isolated from an infected female tick collected from a
dog originating in France. To confirm the percentage of infected ticks in each batch,
infection was determined by PCR/RLB (Chapters 3.1 and 3.2) or by a Babesia-spe-
cific PCR assay (Chapter 3.3) in a representative sample of ticks from each batch.
156

Infection with E. canis is only transmitted transstadially in ticks and not
transovarially as in Babesia species. In order to infect ticks, R. sanguineus nymphs
were fed on a naïve dog, previously inoculated with blood derived from a local case
of canine monocytic ehrlichiosis, identified in Bloemfontein, South Africa. After
moulting, the percentage of infectivity of adult ticks was determined in a rep-
resentative sample by PCR assay. The identity of this novel E. canis strain was
confirmed by its partial gene sequence (GenBank Accession No. KC935387) of E.
canis gp36 with a number of other E. canis isolates and found to be closely related
to several Asian isolates (Chapter 4.3).
Once B. canis or E. canis infected tick batches were established, the appropri-
ate tick challenge design must be used to address the study objectives. Moreover
the criteria for an efficacy success or failure must be clearly defined. The timing
of tick challenges to dogs can be designed to determine the onset of protection,
exact duration of protection within the claimed acaricidal effectiveness period or
total protection over the claimed acaricidal effectiveness period. To determine the
onset of protection, challenges with infected ticks need to be done within a pre-de-
termined time after treatment, normally not later than 48 hours as done by Navarro
et al (2015) for B. canis. The duration of protection within the claimed acaricidal
effectiveness period can be determined by challenging different sub-sets of naïve
dogs at specific time points after treatment (Chapters 3.1 and Navarro et al, 2015).
The advantage of this approach is that it allows for the individual determination of
blocking efficacy at each specific challenge time point thereby determining the exact
duration of protection. The disadvantage is that more dogs have to be used since
each challenge time point requires a sub-set of naïve dogs. An alternative ap-
proach is to challenge the dogs repeatedly with infected ticks over the claimed aca-
ricidal effective period, thereby assessing the protection over the full period as done
for B. canis in chapters 3.2 and 3.3 and for E. canis by Stanneck et al (2013) and in
Chapters 4.1 and 4.2. The advantage of this approach is that fewer dogs are required,
but the disadvantage is that if a product does not completely prevent infection over
the total assessment period, the duration of protection up to the efficacy failure
cannot be exactly determined since the treated dogs are repeatedly challenged. It is
therefore difficult to attribute an infection to a specific time of challenge due to the
variability in onset of clinical signs and subsequent positive diagnosis.
Another important consideration in transmission blocking models is the defi-

nition of an efficacy success or failure. Per definition transmission blocking implies
the prevention of any ehrlichial organisms or babesial sporozoites passing from
157

the tick vector to the host. A dog that has sero-converted and/or tested positive
for B. canis or E. canis DNA by PCR is therefore regarded as an efficacy failure,
irrespective of any clinical manifestation of disease. This definition of efficacy was
used in Chapters 3.1 and 3.3. It can however also be argued that a successful
transmission of a pathogen is the one that results in a clinical case of disease. It is
potentially possible that a dog that seroconverted or tested positive for E. canis or
B. canis DNA by PCR did not develop any clinical symptoms due to an insufficient
challenge. An acaricidal product can therefore potentially disrupt the feeding
process sufficiently to prevent the transmission of a viable infection load, as was
the case in Chapter 3.1 for B. canis. Consequently, an alternative definition of
a successfully infected B. canis dog was used in Chapter 3.2 and by Navarro et
al (2015). In this case a dog that tested positive for B. canis DNA by PCR and that
sero-converted, confirming that the dog was parasitaemic enough to develop spe-
cific antibodies was regarded as an efficacy failure (Navarro et al, 2015). This
definition differed from our work wherein a blocking efficacy failure was defined as
a dog that sero-converted (Chapter 3.1) or that tested positive for B. canis DNA or
that was found positive on a blood smear (Chapter 3.3).
In addition to B. canis and E. canis, the transmission blocking concept can

be applied to infections with Dipylidium caninum and its intermediate host,
Ctenocephalides felis as well (Chapter 5.1). In this chapter fleas were infected with
metacestodes by incubating their eggs and hatched larvae on flea-rearing medi-
um mixed with proglottids and eggs of the tapeworm at temperatures varying
between 24 °C and 28.5 °C. The infection rate of D. caninum in the fleas was
subsequently determined by microscopically dissecting specimens and examining
them for metacestodes (Chapter 5.1: Fig 1, 2 and 3). Moreover, since these me-
tacestodes are unable to complete their development to infectivity in adult fleas at
temperatures below 30 °C unless the fleas are placed on a mammalian host for a few
days, a grace period exists during which infected fleas can be killed on a treated
dog thereby pre- empting infection (Pugh, 1987). The ability of an insecticidal
product to forestall infections with D. caninum can therefore be evaluated over the
claimed efficacy period by challenging dogs with fleas infected with metacestodes
during this period (Chapter 5.1 and Beugnet et al, 2013).
158

6.5. Concluding remarks and future directions
The combination of different topical active compounds and development of different

delivery systems for those compounds has resulted in an improved spectrum of ac-
tivity, speed of kill and persistent effectiveness against ticks and fleas on dogs.
By combining topical compounds into a single formulation, synergistic effects were
discovered for fipronil/amitraz and imidacloprid/flumethrin (Prullage et al, 2011;
Stanneck et al, 2012). Moreover, advances made in respect of improved delivery
systems, in particular the slow release collar matrix, resulted in improved onset
of activity and duration of efficacy against both ticks and fleas. Further advances
were, however, not limited to improved formulations and delivery systems, but
were also due to the discovery of novel systemic compounds (fluralaner and
afoxolaner) (Shoop et al, 2014; Gassel et al, 2014). These compounds have for the
first time made the persistent control of ticks and fleas by a single broad spectrum
oral treatment possible. The advances made with regard to tick and flea control on
dogs should, however, not be limited to the examples studied in this thesis. Fur-
ther research is therefore needed to identify additional synergistic effects, improved
delivery systems and novel classes of active substances.
Future research can also be aimed at the development of additional challenge

models. Although we were able to establish reliable challenge models for B.
canis, E. canis and D. caninum, there remain many other vector-borne diseases
of importance without a reliable laboratory model. For instance, laboratory models
are required for small piroplasms of canines such as Babesia gibsoni and for the
bacterial pathogen Anaplasma phagocytophilum. B. gibsoni is a pathogenic parasite
affecting dogs in Asia, where it is transmitted by a number of different tick species.
Anaplasma phagocytophilum causes granulocytic anaplasmosis in a wide range of
hosts including dogs. Since strain differences have been reported, it is imperative
to identify a strain that is pathogenic for dogs. Moreover, A. phagocytophilum has
been detected in questing D. reticulatus collected from the vegetation in Eastern
Europe suggesting a possible role for this tick as a vector (Paulauskas et al, 2012).
In conclusion, the development of laboratory tick transmission models for tick-
borne pathogens will not only make research possible on the blocking capacity of
acaricidal products, but also on the vectorial capacity of different tick species as well
as on the life cycles of infective organisms.
159

160

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164

Summary
In Chapter 2 the efficacy of several compounds was evaluated against Haemaph-

ysalis elliptica, Dermacentor reticulatus and Rhipicephalus sanguineus as well as
Ctenocephalides felis on dogs in controlled laboratory studies. This research was
done to determine whether 1) broad spectrum active substances can provide sus-
tained effective control against ticks and fleas, 2) the combination of active
substances can improve sustained speed of kill and 3) alternative administration
routes can improve onset of action and persistent effectiveness against ticks and
fleas.
In these studies it was found that the broad spectrum active substance cy-
phenothrin, had a sustained persistent efficacy against both H. elliptica and C. felis
infestations on dogs for 5 weeks. This sustained persistent efficacy against ticks
and fleas confirms its appropriateness for use during the commencement of the flea
season when the numbers of C. felis are rapidly increasing and also thereafter to keep
flea numbers down (Chapter 2.1).
Based on the 24-hour efficacy of fipronil/amitraz/(S)-methoprene, which was

significantly higher (p < 0.05) than that of imidacloprid/permethrin against D. re-
ticulatus at all time points, it was concluded that combinations of certain active
substances have an improved sustained speed of kill against ticks (Chapter 2.2).
A combination of 10% imidacloprid and 4.5% flumethrin in a collar matrix

provided an 8-month long period of efficacy against repeated infestations of R. san-
guineus and C. felis on dogs. Moreover, this improved delivery system had a supe-
rior onset of action and surmounted the fluctuations in the medium-term efficacy of
spot-on treatments (Chapter 2.3).
In Chapter 3 the ability of certain compounds to prevent the transmission of

the tick- borne pathogen B. canis by D. reticulatus was evaluated in controlled lab-
oratory studies. This research was undertaken to determine whether the onset of
acaricidal efficacy was rapid enough to prevent transmission. Using a transmission
blocking model for D. reticulatus infected with B. canis it was demonstrated that the
combination of fipronil, amitraz and (S)- methoprene as a spot-on topical formulation
165

applied up to 28 days prior to challenge, successfully prevented the development
of clinical signs of canine babesiosis for up to 42 days after infestation despite
a low level of B. canis transmission in a small proportion of dogs (Chapter 3.1).
It was also demonstrated that the high acaricidal efficacy of the imidacloprid/
flumethrin collar for 1 month after application resulted in a 100% protection
level (95% confidence interval: 69–100%) of the collar to prevent transmission of
B. canis by infected D. reticulatus ticks (Chapter 3.2). Similarly the oral formula-
tion of afoxolaner demonstrated complete efficacy in preventing the transmission of
B. canis (Chapter 3.3).
In Chapter 4 the ability of various compounds to prevent the transmission of

E. canis by R. sanguineus ticks was evaluated in laboratory studies. Moreover,
in this chapter the speed of transmission of E. canis by infected R. sanguineus ticks
was determined and compared in vivo on dogs and in vitro on artificial feeding
membranes. This research was carried out to ascertain whether the onset of aca-
ricidal efficacy of specific compounds is rapid enough to prevent transmission and
to generate reliable data on the speed of transmission of E. canis by R. sanguine-
us. It was found that a fipronil, amitraz and (S)- methoprene combination spot-
on prevented transmission of E. canis and effectively provided protection against
monocytic ehrlichiosis for at least 4 weeks post treatment (Chapter 4.1).
It was also shown that the transmission blocking capacity of a permethrin

and imidacloprid combination spot-on was complete and provided full protection
against monocytic ehrlichiosis for 4 weeks post acaricidal treatment (Chapter 4.2).
In addition it was demonstrated that the transmission of E. canis by R. sanguineus
starts within a few hours after attachment, which is sooner than previously thought
(Chapter 4.3).
Finally, in Chapter 5 the prophylactic effectiveness of a collar formula-

tion against infected fleas was evaluated and its ability to prevent D. caninum
infection in dogs. This research was performed to determine whether the speed
of kill of the collar formulation against metacestode infected fleas was rapid
enough to kill them before the metacestodes became infective, thereby pre-empt-
ing infection. This study demonstrated that infection of dogs can be prevented by
application of the collars and that because of the rapidity and sustained high level of
efficacy with which the insecticidal components of 10 % imidacloprid/4.5 % flu-
methrin kill newly acquired infestations of infected fleas, infection with D.canis
166

could be forestalled (Chapter 5.1). Moreover by pre-empting the infection of dogs
by D. caninum in a household, the risk of zoonosis is effectively eliminated, espe-
cially for children.
167

Afrikaanse opsomming
In Hoofstuk 2 word die doeltreffendheid van verskeie kombinasie aktiewe bestan-

dele teen Haemaphysalis elliptica, Dermacentor reticulatus en Rhipicephalus san-
guineus, sowel as Ctenocephalides felis op honde, in beheerde laboratoriumstudies
geëvalueer. Hierdie navorsing is onderneem om vas te stel of 1) breëspektrum-ak-
tiewe bestandele volgehoue, doeltreffende beheer van bosluise en vlooie kan verskaf,
2) die verskillende kombinasies van aktiewe bestandele die volgehoue spoed van
doodmaak kan verhoog, en 3) alternatiewe toedieningsroetes die aanvang van wer-
king en nawerkende doeltreffendheid teen bosluise en vlooie kan verbeter.
Hierdie studies het bevind dat die breëspektrum- aktiewe bestandele cypheno-
thrin ‘n volgehoue, nawerkende doeltreffendheid van vyf weke teen sowel H. ellip-
tica as C. felis infestasies in honde het. Hierdie volgehoue nawerkende doeltreffend-
heid teen bosluise en vlooie bevestig dat dit geskik is vir gebruik aan die begin van
die vlooiseisoen, wanneer die populasie van C. felis vinnig toeneem, en daarna, om
die vlooi infestasie op honde laag te hou (Hoofstuk 2.1).
Gebaseer op die 24-uur doeltreffendheid van fipronil/amitraz/(S)-methoprene

teen D. reticulatus, wat op alle tydpunte beduidend hoër was as dié van imidacloprid/
permethrin (p < 0.05), is die gevolgtrekking dat kombinasies van sekere aktiewe
bestandele ‘n verbeterde volgehoue doodmaakspoed ten opsigte van bosluise het
(Hoofstuk 2.2).
‘n Kombinasie van 10% imidacloprid en 4.5% flumethrin in ‘n halsband-

matriks het ‘n langtermyn nawerkende doeltreffendheid van agt maande teen her-
haalde infestasies van R. sanguineus en C. felis by honde verskaf. Voorts het die
verbeterde toedieningstelsel ‘n beter aanvang van werking gehad, en dit het die wis-
selvalligheid in die mediumtermyn doeltreffendheid van koltoedieningsbehandel-
ings oorkom (Hoofstuk 2.3).
In Hoofstuk 3 is die vermoë van verskillende kombinasie aktiewe bestandele,

om die oordrag van B. canis deur D. reticulatus na honde te voorkom, in beheerde
laboratorium-studies geëvalueer. Hierdie navorsing is onderneem om vas te stel
of die aanvang van bosluisdodende doeltreffendheid vinnig genoeg was om oor-
168

drag te voorkom. Deur ‘n model te gebruik wat oordragvoorkoming van B. canis
deur D. reticulatus evalueer, is getoon dat die kombinasie van fipronil, amitraz en
(S)-methoprene as ‘n direkte koltoediening 28 dae voor uitdaging, die ontwikkeling
van kliniese tekens van honde-babesiose vir tot 42 dae na besmetting voorkom. Lae
vlakke van B. canis oordrag by ‘n klein proporsie honde is egter waargeneem (Hoof-
stuk 3.1).
Die studie het ook getoon dat die imidacloprid/flumethrin halsband vir ‘n
maand na toediening ‘n hoë bosluisdodende doeltreffendheid het. Die halsband het
‘n 100% beskermingsvlak teen die oordrag van B. canis deur besmette D. reticulatus
bosluise gelewer (95% vertrouensinterval: 69–100%) (Hoofstuk 3.2). Dienooreen-
komstig het die orale toediening van afoxolaner omvattende doeltreffendheid ten
opsigte van die voorkoming van oordrag van B. canis getoon (Hoofstuk 3.3).
In Hoofstuk 4 is die vermoë van verskillende kombinasie aktiewe bestandele

om die oordrag van E. canis deur R. sanguineus bosluise na honde te voorkom, in
laboratorium-studies geëvalueer. Voorts is die spoed van oordrag van E. canis deur
R. sanguineus bosluise bepaal en vergelyk, beide in vivo op honde en in vitro op
kunsmatige voedingsmembrane. Hierdie navorsing is uitgevoer om te bepaal of die
aanvang van die bosluisdodende doeltreffendheid van spesifieke aktiewe bestandele
vinnig genoeg is om oordrag te voorkom, en om betroubare data te genereer oor die
oordragspoed van E. canis deur R. sanguineus. Daar is bevind dat ‘n kombinasie van
fipronil, amitraz en (S)-methoprene as ‘n koltoediening oordrag van E. canis voor-
kom het, en doeltreffende beskerming gebied het teen monositiese ehrlichiose vir ten
minste vier weke na behandeling (Hoofstuk 4).
Die studie het ook getoon dat ‘n kombinasie van permethrin en imidacloprid
as ‘n koltoediening oordrag volledig voorkom het, en dat dit volledige beskerming
gebied het teen monositiese ehrlichiose vir vier weke na behandeling (Hoofstuk 4.2).
Voorts is getoon dat die oordrag van E. canis deur R. sanguineus binne ‘n paar uur na
hegting begin, wat vinniger is as wat voorheen vermoed is.
Laastens, in Hoofstuk 5 is die voorkomende doeltreffendheid van ‘n halsband

behan-deling teen besmette vlooie en die vermoë daarvan om D. caninum infeks-
ie in honde te voorkom, geëvalueer. Hierdie navorsing is uitgevoer om te bepaal
of die doodmaakspoed van die halsband teen metasestode-besmette vlooie vinnig
genoeg is om hulle te dood voor die metasestode infektief raak. Hierdie studie toon
dat infeksie van honde met D. caninum voorkom kan word deur die gebruik van
169

die halsband. Weens die spoed en volgehoue hoë vlak van doeltreffendheid van die
10% imidacloprid/4.5% flumethrin halsband teen nuwe infestasies van vlooie, kan
infeksies van honde met D. canis voorkom word. Voorts deurdat die infeksie van
honde met D. caninum voorkom kan word, word die risiko dat veral kinders besmet
kan word, uitgeskakel.
170

Nederlandse samenvatting
In hoofdstuk 2 is de werkzaamheid van verschillende acaricide/insecticide middel-

en getest tegen Haemaphysalis elliptica, Dermacentor reticulatus en Rhipicepha-
lus sanguineus teken en eveneens tegen Ctenocephalides felis vlooien op honden
in klinische laboratorium studies. Dit onderzoek is uitgevoerd om te bepalen of 1)
verschillende middelen met een breed activiteit spectrum een goede bescherming
bieden tegen teken en vlooien, 2) bepaalde combinaties van verschillende middelen
wellicht kunnen resulteren in een snellere sterfte, en 3) alternatieve wijze van toedi-
ening van de middelen kan leiden tot een verhoogde werkzaamheid tegen teken en
vlooien.
Allereerst bleek dat het breed spectrum middel cyphenothrine een goede
werkzaamheid bezat tegen zowel H. elliptica en C. felis infestaties, die geduren-
de 5 weken aanhield. Door deze langdurige werkzaamheid is dit middel met name
geschikt voor gebruik bij de aanvang van het vlooien seizoen wanneer C. felis snel
in aantal toeneemt, maar ook daarna om het aantal vlooien te beperken (Hoofdstuk
2.1).
Vervolgens kon worden geconcludeerd dat op basis van de werkzaamheid van

een combinatie van fipronil/amitraz/(S)-methoprene, die na 24 uur significant hoger
was (p < 0.05) dan een combinatie van imidacloprid/permethrine tegen D. reticu-
latus op alle meetpunten, dat bepaalde combinaties van middelen resulteren in een
snellere sterfte van de teken (Hoofdstuk 2.2).
De combinatie van 10% imidacloprid met 4.5% flumethrine geformuleerd in

een halsband matirx werkte zelfs 8 maanden lang tegen herhaalde infestaties met R.
sanguineus en C. felis op honden. Bovendien werkte deze verbeterde formulering
sneller en voorkwam de wisselende werkzaamheid van middellange spot-on behan-
delingen (hoofdstuk 2.3).
In hoofdstuk 3 werd in klinische studies met honden het vermogen van ver-
schillende acaricide middelen getest om de transmissie van Babesia canis door
D. reticulatus teken te blokkeren. Dit onderzoek werd uitgevoerd om te bepalen
of acaricide middelen snel genoeg zouden kunnen werken om de transmissie van
171

pathogenen te voorkomen. Uit het transmissie model met D. reticulatus teken die
geïnfecteerd waren met B. canis bleek dat de combinatie van fipronil, amitraz en
(S)-methoprene, toegepast als een spot-on formulering 28 dagen voor infestatie met
teken, met succes de ontwikkeling van klinische babesiose voorkwam tot 42 dagen
ondanks een geringe transmissie van B. canis in een klein aantal van de honden
(Hoofdstuk 3.1).
Het werd eveneens aangetoond dat een imidacloprid/flumethrin halsband tot

1 maand na toepassing 100% bescherming kon bieden (69-100% met een betrou-
wbaarheidsinterval van 95%) tegen transmissie van B. canis door geïnfecteerde D.
reticulatus teken (Hoofdstuk 3.2). Tevens bleek ook het nieuwe orale middel afoxo-
laner in staat om honden volledig te beschermen tegen de transmissie van B. canis
(Hoofdstuk 3.3).
In hoofdstuk 4 werd in klinische studies met honden het vermogen van ver-
schillende acaricide middelen getest om de transmissie van Ehrlichia canis door
geïnfecteerde R. sanguineus teken te blokkeren. Bovendien werd in dit hoofdstuk
voor het eerst de snelheid van transmissie van E. canis bepaald door geïnfecteerde
R. sanguineus teken te voeden op honden maar ook in vitro op kunstmatige mem-
branen. Dit onderzoek werd uitgevoerd om te bepalen of de verschillende acaricide
middelen snel genoeg zouden kunnen werken om transmissie te verhinderen en te-
vens om gegevens te verzamelen omtrent de snelheid van transmissie van E. canis
door R. sanguineus. Het bleek dat een spot-on formulering van fipronil, amitraz en
(S)-methoprene de transmissie van E. canis kon voorkomen en dat de honden volle-
dig beschermd waren tegen monocytaire ehrlichiosis tenminste 4 weken na behande-
ling (Hoofdstuk 4.1).
Het werd ook bewezen dat de transmissie volledig geblokkeerd is door een
spot-on combinatie van permethrine en imidacloprid, hetgeen de honden afdoende
bescherming kon bieden tegen monocytic ehrlichiosis gedurende 4 weken na de be-
handeling (Hoofdstuk 4.2). Bovendien werd aangetoond dat de transmissie van E.
canis door R. sanguineus al van start gaat als de teek slechts een paar uur is vastge-
hecht, hetgeen eerder is dan tot dusver werd aangenomen (Hoofdstuk 4.3).
Tenslotte werd in hoofdstuk 5 de profylactische werking van een halsband

getest om geïnfecteerde vlooien te doden en zodoende de infectie van honden met
Dipylidium caninum te voorkomen. Dit onderzoek werd gedaan om te bepalen of
de snelheid waarmee de vlooien worden gedood afdoende is om de ontwikkeling
172

van de metacestode in de vlo te voorkomen. Het bleek dat infectie van de honden
kon worden voorkomen door de snelle en effectieve wijze waarop de geïnfecteerde
vlooien door de insecticide componenten in de halsband (10 % imidacloprid/4.5 %
flumethrine) werden gedood (Hoofdstuk 5.1). Van belang hierbij is dat door de pre-
ventie van de infectie van D. caninum in honden, het risico op een zoönose met name
bij kinderen, effectief kan worden vermeden.
173

Acknowledgements
This thesis would not have been possible without the contribution of many people
and I would like to take this opportunity to thank them all. Firstly I would like to
thank my promoters, Prof. dr. H. P. Haagsman and Prof. dr. F. Jongejan for giving
me the opportunity to complete my doctorate at Utrecht University. Without your
guidance and valuable input, it would not have been possible. I especially want to
thank Prof. Frans for his mentorship not only working on my thesis but also during
my scientific career and for the great research collaboration we have had. It’s a priv-
ilege working with you.
I would like to thank all the veterinary pharmaceutical companies and their
respective representatives that I collaborated with that sponsored the work present-
ed in this thesis. Without your support this work would not have been possible. Of
these people, I would like to especially thank Dr. Dorothee Stanneck and Prof. Fred
Beugnet. As an inexperienced investigator Dorothee took me under her wing and
mentored me. Thank you Dorothee for all your support and the trust you placed in
me to conduct so many clinical trials for you over the last couple of years, I really
miss the years when we worked so closely together. Fred, thank you for giving me
the opportunity to collaborate with you and trusting me with the conduct of so many
interesting novel research projects. I truly value your mentorship as a scientist and
thoroughly enjoy all our brainstorming sessions.
This thesis would not have been possible without the support of my fellow
ClinVet directors. I would like to thank Prof. L.J. Fourie, Dr. H. G. Luus, Mr T.
Erasmus and Mr. W.Z. Fourie for giving me the opportunity to complete my thesis.
Pa, oom Herman, Boendoes en Wessel, ek waardeur die kans opreg. Furthermore I
would like to thank my world-class research team at ClinVet, without whom none
of the work would have been done. It’s a pleasure and a privilege working each day
with you. Furthermore I would especially like to thank Prof. Dawie and Dr. Peet
whose subject matter knowledge at ClinVet I could always rely on.
I would also like to thank Prof. Ivan Horak who I have known since child-
hood. Prof. has been such an inspiration to me not only with regard to your wealth of
knowledge but also your positive attitude and general enthusiasm in every endeav-
174

our you undertake. Thank you for all your mentorship, patience and for being such
a great role model. It’s a privilege and honour to know such an accomplished scien-
tist personally and a personal goal of mine to still catch a bigger tigerfish than Prof!
My paranimfs, thank you for your moral support and traveling all the way to
Utrecht to share the occasion with me. To have my brother Wessel and sister Rykie
next to me was very special.
My father and mother, thank you for always believing in me and for all your
support. The life principles you taught me and the example you set shaped me into
what I am today. Ma, baie dankie vir al Ma se ondersteuning en dat Ma altyd in my
geglo het. Dit kon nie maklik gewees het om my groot te maak nie. Ma was egter
altyd daar vir my en het altyd net die beste vir my gesoek, selfs deur die moeilike
tye. Om in ‘n huis van so ‘n sterk vrou op te groei is ‘n voorrreg! Pa, dankie vir die
voorbeeld wat Pa vir my gestel het in alle aspekte van my lewe. Pa het diep spore in
ons bedryf getrap en is die rede waarom ClinVet bestaan en ek vandag in die beroep
is. Dankie ook vir al Pa se mentorskap en advies wat my oor die jare gevorm het. Ek
is bevoorreg en trots om ‘n ouer soos Pa te hê!
Finally and most importantly I would like to thank my wife Abby. Thank you
for all your support and for always believing in me. Thank you for your willingness
to always assist me wherever you can and putting up with me through all my work
pressures. Thank you for raising two wonderful boys, Lukas and Liam who I am so
proud of. Without you, none of this would have been possible, you are the love of
my life.
175

Curriculum Vitae
Josephus Johannes Fourie was born in Cradock, Eastern Cape Province, South Afri-
ca on the 15th of January 1981. He graduated with a Master of Science degree from
the University of the Free Sate, South Africa in 2006. During his studies he worked
as a researcher at ClinVet International, a contract research organisation situated in
Bloemfontein, South Africa. During this period he was responsible for conducting
various tasks in clinical research trials. In 2008 he was promoted to Head of the
Department of ectoparasitology at Clinvet with a main research focus on conduct-
ing clinical trials for the development and registration of ectoparasiticides for com-
panion animals. During this period he was responsible for personally conducting
over 330 clinical trials. In 2011 he was promoted to Vice President of Scientific
Operations at ClinVet International and became responsible for the management
of all departments involved in the conduct and support of pre-clinical and clinical
trials.
176

List of Publications
1. Baker C, Tielemans E, Prullage JB, Chester ST, Knaus M, Rehbein S,

Fourie JJ, Young DR, Everett WR, Rosentel JK. Efficacy of a novel topical
combination of fipronil, (S)-methoprene, eprinomectin and praziquantel
against adult and immature stages of the cat flea (Ctenocephalides felis) on
cats. Vet Parasitol. 2014, 202 (1-2) : 54-8.
2. Beugnet F, Fourie JJ, Chalvet-Monfray K. Comparative efficacy on dogs of
a single topical treatment with fipronil/ (S)-Methoprene or weekly physio-
logical hygiene shampoos against Ctenocephalides felis in a simulated flea
infested environment. Parasite. 2012, 19 (2) : 153 – 158.
3. Beugnet F, Delport P, Luus H, Crafford D, Fourie JJ. Preventive efficacy of
Frontline® Combo and Certifect® against Dipylidium caninum infestation
of cats and dogs using a natural flea (Ctenocephalides felis) infestation mod-
el. Parasite. 2013, 20 : 7.
4. Beugnet F, Halos L, Larsen D, Labuschagne M, Erasmus H, Fourie JJ. The abil-
ity of an oral formulation of afoxolaner to block the transmission of Babesia
canis by Dermacentor reticulatus ticks to dogs. Parasit Vectors. 2014, 7 : 283.
5. Beugnet F, Labuschagne M, Fourie JJ, Guillot J, Farkas R, Cozma V, Halos L,
Hellmann K, Knaus, M, Rehbein S. Occurrence of Dipylidium caninum in
fleas from client-owned cats and dogs in Europe using a new PCR detection
assay. Vet Parasitol. 2014, 205 (1-2) : 300-6.
6. Beugnet F, deVos C, Liebenberg J, Halos L, Fourie J. Afoxolaner against
fleas: immediate efficacy and resultant mortality after short exposure on
dogs. Parasite. 2014, 21 : 42.
7. Dumont P, Blair J, Fourie JJ, Chester TS, Larsen DL. Evaluation of the effi-
cacy of afoxolaner against two European dog tick species: Dermacentor
reticulatus and Ixodes ricinus. Vet Parasitol. 2014, 201 (3-4) : 216-9.
8. Dumont P, Chester TS, Gale B, Soll M, Fourie JJ, Beugnet F. Acaricidal effica-
cy of a new combination of fipronil and permethrin against Ixodes ricinus
and Rhipicephalus sanguineus ticks. Parasit Vectors. 2015, 8 : 51.
9. Dumont P, Fourie JJ, Soll M, Beugnet F. Repellency, prevention of attach-
ment and acaricidal efficacy of a new combination of fipronil and perme-
thrin against the main vector of canine babesiosis in Europe, Dermacentor
reticulatus ticks. Parasit Vectors. 2015, 8 : 50.
177

10. Fankhauser B, Dumont P, Halos L, Hunter JS 3rd, Kunkle B, Everett WR,
Chester TS, Fourie JJ, Soll MD. Efficacy of a new combination of fipronil
and permethrin against Ctenocephalides felis flea infestation in dogs. Para-
sit Vectors. 2015, 8 : 62.
11. Fourie JJ, Fourie LJ, Horak IG. Efficacy of orally administered powdered
aloe juice (Aloe ferox) against ticks on cattle and ticks and fleas on dogs.
J S Afr Vet Assoc. 2005, 76 (4) : 193–196.
12. Fourie JJ, Delport P, Fourie LJ, Heine J. Comparative efficacy and safety
of two treatment regimens with a topically applied combination imidaclo-
prid and moxidectin (Advocate®) against generalised demodicosis in dogs.
Parasitology Research. 2009, 105 : S115–S124.
13. Fourie JJ, Fourie LJ, Horak IG and Snyman MG. The efficacy of a topically
applied combination of cyphenothrin and pyriproxifen against the south-
ern African yellow dog tick Haemaphysalis elliptica and the cat flea Cteno-
cephalides felis. J S Afr Vet Assoc. 2010, 81 (1) : 33-36.
14. Fourie JJ, Horak IG, de la Puente Redondo V. Efficacy of a spot-on formula-
tion of pyriprole on dogs infested with Sarcoptes scabiei. Vet Rec. 2010, 167
: 442-445.
15. Fourie JJ, Beugnet F, Ollagnier C, Pollmeier MG. Study of the sustained
speed of kill of the combination of fiproil/amitraz/(S)-methoprene and the
combination of imidacloprid/permethrin against Dermacentor reticulatus,
the European dog tick. Parasite. 2011, 18 (4) : 319-23.
16. Fourie JJ, Ollagnier C, Beugnet F, Luus HG, Jongejan F. Prevention of the
transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs
treated with a combination of fipronil, amitraz and (S)-methoprene (Certi-
fect®). Vet Parasitol. 2012, 193 (1-3) : 223-228.
17. Fourie JJ, Crafford D, Horak IG, Stanneck D. Prophylactic treatment of flea-
infested cats with an imidacloprid/flumethrin collar to forestall infection
with Dipylidium caninum. Parasit Vectors. 2012, 5 : 151.
18. Fourie JJ, Stanneck D, Luus HG, Beugnet F, Wijnveld M, Jongejan F. Trans-
mission of Ehrlichia canis by Rhipicephalus sanguineus ticks feeding on
dogs and on artificial membranes. Vet Parasitol. 2013, 197 (3-4) : 595-603.
19. Fourie JJ, Stanneck D, Jongejan F. Prevention of transmission of Babesia ca-
nis by Dermacentor reticulatus ticks to dogs treated with an imidaclo-
prid/flumethrin collar. Vet Parasitol. 2013, 192 (1-3) : 273-278.
20. Fourie JJ, Luus HG, Stanneck D, Jongejan F. The efficacy of Advantix® to
prevent transmission of Ehrlichia canis to dogs by Rhipicephalus sanguine-
us ticks. Parasite. 2013, 20 : 36.
178

21. Fourie JJ, Liebenberg J, Nyangiwe N, Austin C, Horak I, Bhushan C. The ef-
fects 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.
22. Fourie JJ, Beugnet F, Bubb A, Labuschagné M. Evaluation of the anti-feeding
effect of a new topical combination of fipronil/ amitraz/(S)-methoprene
against ticks (Rhipicephalus sanguineus) on dogs. Comp Immunol Microbiol
Infect Dis. 2013, 37 : 181-187.
23. Fourie J, Dumont P, Halos L, Beugnet F, Pollmeier M. Efficacy of a topical ap-
plication of Certifect® (fipronil 6.26% w/v, amitraz 7.48% w/v, (S)- metho-
prene 5.63% w/v) for the treatment of canine generalized demodicosis.
Parasite. 2013, 20 : 46.
24. Fourie JJ, Horak IG, Crafford D, Erasmus HL, Botha OJ. The efficacy of a
generic doxycycline tablet in the treatment of canine monocytic ehrlichi-
osis. J S Afr Vet Assoc. 2015, 86 : 1.
25. Fourie JJ, Liebenberg JE, Horak IG, Taenzler J, Heckeroth AR, Frénais R. Ef-
ficacy of orally administered fluralaner (BravectoTM) or topically applied
imidacloprid/moxidectin (Advocate®) against generalized demodicosis in
dogs. Parasit Vectors. 2015, 8 : 187.
26. Horak IG, Fourie JJ, Stanneck D. Efficacy of slow-release collar formula-
tions of imidacloprid/flumethrin and deltamethrin and of spot-on formu-
lations of fipronil/(s)-methoprene, dinotefuran/pyriproxyfen/permethrin
and (s)- methoprene/amitraz/fipronil against Rhipicephalus sanguineus
and Ctenocephalides felis felis on dogs. Parasit Vectors. 2012, 5 : 79.
27. Hunter JS3rd, Baggott D, Everett WR, Fourie JJ, Cramer LG, Yoon SS, Col-
lidor N, Mallouk Y, Lee L, Blair J, Prullage JB. Efficacy of a novel topical
combination of fipronil, amitraz and (S)-methoprene for treatment and
control of induced infestations of brown dog ticks (Rhipicephalus san-
guineus) on dogs. Vet Parasitol. 2011, 179 (4) : 318-23.
28. Hunter JS3rd, Dumont P, Chester TS, Young DR, Fourie JJ, Larsen DL. Evalu-
ation of the curative and preventive efficacy of a single oral administration
of afoxolaner against cat flea Ctenocephalides felis infestations on dogs. Vet
Parasitol. 2014, 201 (3-4) : 207-11.
29. Jongejan F, Fourie JJ, Chester ST, Manavella C, Mallouk Y, Pollmeier MG,
Baggott D. The prevention of the transmission of Babesia canis by Der-
macentor reticulatus to dogs using a novel combination of fipronil, amitraz
and (S)- methoprene. Vet Parasitol. 2011, 179 (4): 343-350.
179

30. Kužner J, Turk S, Grace S, Soni-Gupta J, Fourie JJ, Marchiondo AA, Rugg D.
Confirmation of the efficacy of a novel fipronil spot-on for the treatment
and control of fleas, ticks and chewing lice on dogs. Vet Parasitol. 2013,
193 (2013): 245-251.
31. 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 rici-
nus) on cats. Parasitol Res. 2013, 112 (2013) : 365-372.
32. Navarro C, Reymond N, Fourie J, Hellmann K, Bonneau S. Prevention of
Babesia canis in dogs: efficacy of a fixed combination of permethrin and
fipronil (Effitix®) using an experimental transmission blocking model with
infected Dermacentor reticulatus ticks. Parasit Vectors. 2015, 8 (1) : 32.
33. Stanneck D, Kruedewagen EM, Fourie JJ, Horak IG, Davis W, Krieger KJ. Ef-
ficacy of an imidacloprid/flumethrin collar against fleas, ticks, mites and
lice on dogs. Parasit Vectors. 2012, 5 : 102.
34. Stanneck D, Kruedewagen EM, Fourie JJ, Horak IG, Davis W, Krieger KJ. Ef-
ficacy of an imidacloprid/flumethrin collar against fleas and ticks on cats.
Parasit Vectors. 2012, 5 : 82.
35. Stanneck D, Fourie JJ. Imidacloprid 10 % / flumethrin 4.5 % collars
(Seresto®, Bayer) successfully prevent long-term transmission of Ehrlich-
ia canis by infected Rhipicephalus sanguineus ticks to dogs. Parasitol Res.
2013, 1 : 21-32.
36. Taenzler J, Wengenmayer C, Williams H, Fourie J, Zschiesche E, Roepke RK,
Heckeroth AR. Onset of activity of fluralaner (Bravecto™) against Cteno-
cephalides felis on dogs. Parasit Vectors. 2014, 7 : 567.
37. Varloud M, Fourie JJ. One-month comparative efficacy of three topical ecto-
parasiticides against adult brown dog ticks (Rhipicephalus sanguineus sen-
su lato) on mixed-bred dogs in controlled environment. Parasitol Res. 2015,
114 (5) : 1711-9.
38. Varloud M, Fourie JJ, Blagburn BL, Deflandre A. Expellency, anti-feeding
and speed of kill of a dinotefuran-permethrin-pyriproxyfen spot-on (Vec-
tra®3D) in dogs weekly challenged with adult fleas (Ctenocephalides felis)
for 1 month- comparison to a spinosad tablet (Comfortis®). Parasitol Res.
2015, Apr 15 [Epub ahead of print].
39. Varloud M, Fourie JJ. Onset of efficacy and residual speed of kill over one
month of a topical dinotefuran-permethrin-pyriproxyfen combination
(Vectra®3D) against the adult cat flea (Ctenocephalides felis felis) on dogs.
Vet Parasitol. 2015, May 14 [Epub ahead of print].
180

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Integrated control of ticks and fleas on dogs

with particular reference

Thesis Fourie NIEUW.indd 1 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 2 2-9-2015 15:06:40

Geïntegreerde bestrijding van teken en vlooien op honden

(met een samenvatting in the Nederlands)

ter verkrijging van de graad van doctor aan de Universiteit Utrecht

geboren op 15 Januari 1981 te Cradock, Zuid Afrika

Thesis Fourie NIEUW.indd 3 2-9-2015 15:06:40

Prof. dr. H. P. Haagsman

Prof. dr. F. Jongejan

Thesis Fourie NIEUW.indd 4 2-9-2015 15:06:40

Chapter 1. Introduction and literature review 9

Chapter 2. Control of ticks and fleas on dogs 39

Thesis Fourie NIEUW.indd 5 2-9-2015 15:06:40

Chapter 4. Prevention and speed of Ehrlichia canis

Chapter 5. Prevention of Dipylidium caninum transmission

Thesis Fourie NIEUW.indd 6 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 7 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 8 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 9 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 10 2-9-2015 15:06:40

According to Saunders Comprehensive Veterinary Dictionary, third edition (2007)

Thesis Fourie NIEUW.indd 11 2-9-2015 15:06:40

Ticks are obligate, blood-feeding ectoparasites in the phylum Arthropoda. Within

Thesis Fourie NIEUW.indd 12 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 13 2-9-2015 15:06:40

Thesis Fourie NIEUW.indd 14 2-9-2015 15:06:41

Genus Species Zoogeographical Region

Dermacentor D. variabilis (American dog tick) Nearctic

Rhipicephalus R. sanguineus (Brown dog tick) Worldwide

Haemaphysalis H. elliptica Afrotropical

Amblyomma A. americanum (lone star tick) Nearctic

Hyalomma H. truncatum Afrotropical

Thesis Fourie NIEUW.indd 15 2-9-2015 15:06:41

The brown dog tick, Rhipicephalus sanguineus (Latreille, 1806), has

R. sanguineus is a vector of a number of tick-borne pathogens affecting

Although R. sanguineus is in general very host specific, human parasitism

Thesis Fourie NIEUW.indd 16 2-9-2015 15:06:41

The ornate dog tick, Dermacentor reticulatus (Fabricius, 1794), is a Palearctic

Although D. reticulatus infrequently bites humans, it is associated with a

Thesis Fourie NIEUW.indd 17 2-9-2015 15:06:41

1.5. Flea-borne pathogens

Thesis Fourie NIEUW.indd 18 2-9-2015 15:06:41

Thesis Fourie NIEUW.indd 19 2-9-2015 15:06:41

Ctenocephalides felis also serves as a vector of various bacterial pathogens

1.6. Tick control

Thesis Fourie NIEUW.indd 20 2-9-2015 15:06:41

1.7. Flea control

The effective control of flea infestations on dogs involves a combination of vari-

Thesis Fourie NIEUW.indd 21 2-9-2015 15:06:41

Thesis Fourie NIEUW.indd 22 2-9-2015 15:06:41

Juvenile hormone analogues

Thesis Fourie NIEUW.indd 23 2-9-2015 15:06:41

Route of Main spectrum

Prevents closure of Na+

Binding to GABA and

Binds to octopamine re-

Affects fecundity, egg

Thesis Fourie NIEUW.indd 24 2-9-2015 15:06:41

Prevents closure of Na+

Affects fecundity, egg

Prevents closure of Na+

Topical Nicotinic acetylcholine re-