Prevention of Transmission of Babesia Canis by Dermacentor Reticulatus

Veterinary Parasitology 192 (2013) 273–278

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Short communication

Prevention of transmission of Babesia canis by Dermacentor reticulatus

ticks to dogs treated with an imidacloprid/ﬂumethrin collar

a,∗ b c,d

Josephus J. Fourie , Dorothee Stanneck , Frans Jongejan

ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa

Bayer Animal Health GmbH, BHC Business Group Animal Health, Clinical Development, Building 6700, 51368 Leverkusen, Germany

Utrecht Centre for Tick-borne Diseases (UCTD), Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa

a r t i c l e i n f o a b s t r a c t

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.

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-

∗ macentor reticulatus, which is a vector of a broad spectrum

Corresponding author. Tel.: +27 51 445 2424; fax: +27 51 445 2421.

E-mail address: [email protected] (J.J. Fourie). of pathogens, including B. canis in dogs, Babesia caballi

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, con- ﬁrmed 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)

J.J. Fourie et al. / Veterinary Parasitology 192 (2013) 273–278 275

Table 1

Categorization of ticks for counting.

Category General ﬁndings Attachment status

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Live Free Attached; engorged

2 Live Attached; unengorged

3 Live Attached; engorged 4 Killed Free a

5 Killed Attached; unengorged

6 Killed Attached; engorged

0 0 0 0 0 0 0 0 0 0 0 0 0

a Attached; unengorged 2 2 1

No ﬁlling of the alloscutum evident.

Obvious or conspicuous ﬁlling of the alloscutum evident. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Dead

Free

according to categories 1–6 (see Table 1), adapted after

Marchiondo et al. (2007).

SAS Version 8 (release 8.02 TS Level 02M0) was used

0 0 0 0 0 0 0 0 for all the statistical analyses. Efﬁcacy against ticks was 5 9 5 5 8 10 10

Attached; engorged

calculated for each treatment group at each assessment

day according to the formulas given below (EMEA, 2007).

Due to the fact that small and even zero tick counts were

recorded, it was expected that the tick counts would not

0 0 0 0 0 0 0 0

6 4 6

follow a normal distribution. It was therefore decided day

Attached; unengorged 12 17 23 12 15

that the primary efﬁcacy calculations would be based on

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Study Live Free

geometric means rather than arithmetic means. The cal-

culations were based on the geometric means of the tick

(count + 1) data. One (1) was subsequently subtracted from

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Attached 2

the result to obtain a meaningful value for the geomet-

ric mean of each treatment group. Percent efﬁcacy against

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Dead Free

ticks was calculated as follows:

− G +5 G

mc mt

= × ,

Efﬁcacy (%) 100 0 0 0 0 0 0 0 0

G day

20 mc Attached 18 26 34 22 14 16 18

where Gmc is the geometric number of live ticks (categories 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Study Live Free

1–3) on dogs in the negative control group (Group 1) at a

speciﬁc time point. Gmt is the geometric mean number of infestation).

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 Attached

after

residual efﬁcacies) in the treatment group (Group 2) at a

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 speciﬁc time point (EMEA, 2007). Dead Free days

Descriptive statistics (mean, minimum, maximum, +4

and

standard deviation, CV%, geometric mean and median) on

0 0 0 0 0 0 0 0 day

20 20 Attached 28 35 21 16 22 22

tick counts for the various assessment days were calcu- 4,

3, lated.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Study Live Free (2,

The groups were compared using an ANOVA with a

day

treatment effect after a logarithmic transformation on the

tick (count + 1) data. The level of signiﬁcance of the formal

0 0 0 0 0 0 0 0 0 0 0 0 0 Attached 2 1 2 count

tests was set at 5%, all tests were two sided.

A protection failure (successfully infected) was regarded and

0 0 0 0 0 0 0 0 0 0 0 0 0 Dead Free 2 1 1

when a dog in the treatment group tested serologically pos-

itive for Babesia canis antibodies as well as positive for B.

category

0 0 0 0 0 0

1 1

canis DNA in PCR-RLB assay. day

Attached 19 28 34 19 15 21 22 21

tick

The percentage protection for the treatment group was

by 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Study Live Free

calculated as follows:

Tc − Tt

group

= × , Protection (%) 100 T

c 0 0 0 0 0 0 0 0 0 0 Attached 3 1 1 3 1 5

where Tc is the total number of infected dogs in the negative

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Dead Free 1

control Group 1 and T is the total number of infected dogs treatment

in the treatment Group 2. +2

each

0 0 0 0 3 1 3 1 for day

20 20

Attached 21 26 34 23 24 18

3. Results

1 2

0 0 0 0 0 0 0 0 0 Study Live Free 2 counts

The tick counts for the two study groups are given 10 1 ID Group 2 3 4 5 6 7 80 Group 2 3 4 50 61 70 81

Table

in Table 2. The geometric mean tick counts recorded for Tick

276 J.J. Fourie et al. / Veterinary Parasitology 192 (2013) 273–278

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

b a

+2 23.1 0.9 b a

+3 21.8 0.2 b a

+4 22.4 0.0 b a

+5 20.3 0.0

+6 18.7 0.0

Day Efﬁcacies (%)

Group 2 – (dogs ﬁtted with collar)

+2b 96.02 +3b 99.1 +4b 100.0 +5b 100.0

+6 100.0

Geometric mean tick count differs statistically signiﬁcantly (p < 0.05) from that recorded for the negative control Group 1.

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

Table 5

100% of all infested ticks being dead within 4 days post

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

3 – – POS POS POS

application, two dogs reacted with fever and Babesia para-

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 – – – – –

4 – – – – –

and enlarged spleen upon palpation with blood smears 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.

J.J. Fourie et al. / Veterinary Parasitology 192 (2013) 273–278 277

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 – – – –

5 – POS – – – – – –

6 – POS – – – – – –

7 POS – – – – – – –

8 – POS – – – – – –

1 – – – – – – – –

2 – – – – – – – –

3 – – – – – – – –

4 – – – – – – – –

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-

cessfully for the ﬁrst time with a novel combination of

4. Discussion and conclusion

ﬁpronil, amitraz and (S)-methoprene (Certifect ) (Jongejan

et al., 2011). The advantage of this model is that the chal-

The imidacloprid/ﬂumethrin collar, which has recently

® lenge load with infected ticks can be pre-determined and

been registered (Seresto ) by Bayer Animal Health GmbH

standardised. Several other studies have been carried out

(Leverkusen, Germany) was well tolerated by the dogs.

to ascertain the capability of various anti-tick compounds

The efﬁcacy of 96.02% of ticks killed 48 h post challenge,

to prevent transmission of tick-borne pathogens to dogs.

which increased to 100% mortality within 4 days, was in

In these studies, dogs were either exposed to infected

line with the reported efﬁcacy above 97% against adult D.

ticks under ﬁeld conditions or infected ticks were collected

reticulatus ticks (Stanneck et al., 2012). Moreover, the speed

from the ﬁeld and tested on dogs under controlled lab-

of kill by the collar against infesting ticks is of particular

oratory conditions (Spencer et al., 2003; Jacobson et al.,

importance with respect to the ability to block transmis-

2004; Blackburn et al., 2004; Last et al., 2007; McCall et al.,

sion of pathogens. Evidently, this speed was sufﬁcient in

2011).

order to prevent transmission of mature Babesia sporo-

For instance, the capacity of an amitraz-impregnated

zoites into the treated dogs, whereas they were readily

collar to block transmission of Babesia rossi by Haema-

transmitted to the untreated control group. Development

physalis elliptica ticks has been evaluated in the ﬁeld in

of Babesia sporoblasts into mature sporozoites within the

South Africa. Eight of 30 control dogs (26.6%) became

tick’s salivary gland was therefore prevented by the treat-

PCR/RLB positive during the trial period, which lasted

ment, despite the fact that 44% of the ticks were infected.

for 6 months. None of the treated dogs became infected

Moreover, it has been demonstrated that this collar has a

with babesiosis during the same period (Last et al., 2007).

sustained acaricidal (48 h killing) efﬁcacy over a period of

Furthermore, in a laboratory study targetting B. burgdor-

feri, when challenge infestation was conducted 28 days

after application of ﬁpronil/(S)-methoprene, the treatment

Table 7

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

tick challenge (Jacobson et al., 2004). In addition, imi-

(n = 8) (n = 8)

dacloprid/permethrin spot-on prevented transmission of

Infected 8 0

A. phagocytophilum from ﬁeld collected Ixodes scapularis

Non-infected 0 8

ticks reported by Blagburn et al. (2004). Recently, the

% Infected 100 0

% 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

278 J.J. Fourie et al. / Veterinary Parasitology 192 (2013) 273–278

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

Anaplasma phagocytophilum from naturally infected Ixodes scapularis

As discussed above, blocking of transmission of vector

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.

EMEA, 2007. Guideline for the testing and evaluation of the efﬁcacy of

for evaluating efﬁcacy of ectoparasiticides for treatment,

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.

Estrada-Pena, A., Reme, C., 2005. Efﬁcacy of a collar impregnated

modate the testing of their ability to block transmission

with amitraz and pyriproxyfen for prevention of experimen-

of tick-borne pathogens and prevent disease (Marchiondo

tal tick infestations by Rhipicephalus sanguineus, Ixodes ricinus,

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221–224.

Finally, similar models have now been developed for

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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

Parasitol. 23, 445–449.

analysed the data. FJ wrote the ﬁrst draft of the manuscript,

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.

Marchiondo, A.A., Holdsworth, P.A., Green, P., Blagburn, B.L., Jacobs,

Competing interests

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

ﬂea and tick infestation on dogs and cats. Vet. Parasitol. 145,

Animal Health GmbH Monheim, Germany, of which D.

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

novel combination of ﬁpronil, amitraz and (S)-methoprene to pro-

Contract Research Organisation contracted to manage the

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.

Matjila, P.T., Nijhof, A.M., Taouﬁk, A., Houwers, D., Teske, E., Penzhorn,

All authors voluntarily publish this article and have no

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,

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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.

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