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ARTICLE IN PRESSG ModelETPAR-7286; No. of Pages 7
Veterinary Parasitology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Parasitology
jo u r nal homep age: www.elsev ier .com/ locate /vetpar
ccurrence of Dipylidium caninum in fleas from client-ownedats and dogs in Europe using a new PCR detection assay
rédéric Beugneta,∗, Michel Labuschagneb, Josephus Fourieb, Jacques Guillotc,obert Farkasd, Vasile Cozmae, Lénaïg Halosa, Klaus Hellmannf,artin Knausg, Steffen Rehbeing
Merial S.A.S., 29 Av Tony Garnier, 69007 Lyon, FranceClinVet,Box 11186. Universitas, Bloemfontein, 9321, Republic of South AfricaEcole Vétérinaire de Maisons-Alfort, 94704 Maisons-Alfort Cédex, FranceSzent Istvan University, Faculty of Veterinary Science, Budapest, HungaryVeterinary Faculty, Cluj-Napoca, RomaniaKlifovet AG, Geyerspergerstr. 27, 80689 München, GermanyMerial GmbH, Kathrinenhof Research Center, Walchenseestr. 8-12, 83101 Rohrdorf, Germany
r t i c l e i n f o
rticle history:eceived 18 April 2014eceived in revised form 4 June 2014ccepted 5 June 2014
eywords:ipylidium caninumtenocephalides felistenocephalides canisatsogsCR
a b s t r a c t
Ctenocephalides fleas are not only the most prevalent ectoparasites of dogs and cats butalso the intermediate host of the cestode Dipylidium caninum. Due to the poor sensitivityof coproscopy to diagnose cat and dog infestation by Dipylidium, few epidemiological dataare available on its prevalence among pet populations. A new PCR method was developedto specifically identify D. caninum rDNA inside single fleas. The PCR test was then applied to5529 fleas of Ctenocephalides genus, 2701 Ctenocephalides felis fleas (1969 collected on 435cats and 732 on 178 dogs) and 2828 Ctenocephalides canis fleas collected from 396 dogs.Precisely, 4.37% of cats were infested by a flea population infected with D. caninum. Out ofthe 1969 C. felis from cats, 2.23% were found to be infected with Dipylidium. From the 396dogs infested with C. canis, 9.1%% were infested with the Dipylidium infected fleas, whichis significantly higher than the observation made in cats (p = 0.03). Moreover, 3.1% of the C.canis fleas were found to be infected with Dipylidium, which is not significantly differentthan in C. felis. Looking at the number of infected fleas in the positive samples (at least one
PCR positive flea in a sample), the infestation rate in samples was varied from 3 to 100%with an average of 19.7% which is in favour of easy and regular Dipylidium reinfestations ofboth cats and dogs in households. For the first time, the spread of D. caninum between fleasand dogs and cats is confirmed throughout Europe.. Introduction
Please cite this article in press as: Beugnet, F., et al., Occurrence odogs in Europe using a new PCR detection assay. Vet. Parasitol.
The cat flea, Ctenocephalides felis, is the main flea speciesnfesting both dogs and cats (Bond et al., 2007; Cadiergues
∗ Corresponding author. Tel.: +0033 687748983;ax: +0033 472723298.
E-mail address: [email protected] (F. Beugnet).
http://dx.doi.org/10.1016/j.vetpar.2014.06.008304-4017/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
et al., 2000; Dryden and Rust, 1984; Franc et al., 1998;Mircean et al., 2010; Rust and Dryden, 1997). Neverthe-less, the prevalence of Ctenocephalides canis appears to begreater than previously believed in many regions, espe-cially in central and eastern Europe where C. canis is the
f Dipylidium caninum in fleas from client-owned cats and(2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.008
predominant flea species infesting dogs (Xhaxhiu et al.,2009; Farkas et al., 2009). Even in areas where C. felis felisappears to be predominant, the prevalence of C. canis in dogpopulations may still vary from 10 to 12.5% like in France
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(Beck et al., 2006; Durden et al., 2005; Franc et al., 1998;Gracia et al., 2007). Due to the frequency of parasitism andthe medical consequences, regular flea control is advised onboth dogs and cats (Beugnet and Franc, 2012; Otranto et al.,2009a,b). Ctenocephalides fleas is primarily responsible forflea bite allergy dermatitis in both dogs and cats (Carlottiand Costargent, 1994; Dryden and Blakemore, 1989;Guaguère and Beugnet, 2008; Plant, 1991; Prélaud, 2008).C. felis can also transmit Rickettsia felis and some Bartonellaspecies, such as Bartonella henselae (Azad et al., 1997;Beugnet and Marie, 2009; Bouhsira et al., 2013; Just et al.,2008). The Ctenocephalides fleas are also the primary inter-mediate host of Dipylidium caninum, the common intestinalcestode of dogs and cats (Boreham and Boreham, 1990;Chappell and Pen, 1990; Dunn, 1978; Guzman, 1984; Pugh,1987; Venard, 1938). Larval stage of fleas ingest the eggs ofD. caninum; the eggs hatch and the hexacanth embryos stayalive inside the flea larvae and pupae. Once the adult fleaemerges and infests a host, the hexacanth embryo devel-ops into an infective cysticercoid stage in the flea within2–3 days (Beugnet et al., 2013; Hinaidy, 1991; Pugh andMoorhouse, 1985). Carnivores become infested throughthe ingestion of infected fleas containing mature cysticer-coid larvae. Adult D. caninum typically develops in the smallintestine and begins to shed proglottids within 2–3 weeks.
It is difficult to estimate the real prevalence of D.caninum in cats and dogs due to the poor sensitivityof coproscopical methods (Boreham and Boreham, 1990;Bourdeau and Beugnet, 1993). Proglottids are mobileand often excreted between defecation, therefore Dipy-lidium eggs are usually not present in the faeces. Besidescoproscopy, the other possibility to estimate the preva-lence of Dipylidium would be to assess the infection rateof the main intermediate hosts, i.e. C. felis and C. canis(Hinaidy, 1991). The microscopical examination of fleasfor the presence of D. caninum cysticercoids is a routinemethod of detection in experimental research labs, but istime consuming and requires technical expertise (Beugnetet al., 2013; Fourie et al., 2012). An alternative detectionmethod should include a high level of sensitivity and speci-ficity, the acceptance of a wide range of sample formatsas well as a quick turnaround time from sample submis-sion to results. A PCR detection assay would allow this kindof detection and would permit to conduct epidemiologicalsurvey on D. caninum based on flea collection.
The authors describe here the development of a sensi-tive PCR detection assay for the presence of D. caninum in C.felis and its use on fleas collected throughout Europe from2009 to 2013.
2. Materials and methods
2.1. Flea collection
Fleas were collected from 2009 to 2013 on bothdomestic owned cats and dogs with a flea comb. Thesecollections were organized during five European field
Please cite this article in press as: Beugnet, F., et al., Occurrence odogs in Europe using a new PCR detection assay. Vet. Parasitol.
studies (Study 1, 2, 3, 4 and 5) concerning pet parasitesand efficacy assessment of new ectoparasiticides, con-ducted by veterinary universities and contract researchorganizations. Dogs and cats should not have been treated
PRESStology xxx (2014) xxx–xxx
with an anti-flea product 3 months before the assessmentof flea infestation. The prevalence of flea infestation is notknown because only flea infested cats or dogs were keptfor inclusion in these studies. In all studies, fleas werecollected per animal in alcohol and placed in sample tubes,giving one to three samples per cat or dog. Each flea wasmorphologically identified.
One study (study 1) was conducted on 426 flea infestedcats in Czech Republic, Slovenia, Portugal, Hungary andGermany. Nine additional flea infested cats collected inFrance, Romania and Hungary were added (study 2). A totalof 1969 fleas from 435 flea infested cats were included.
Three studies (studies 3, 4 and 5) were conductedon dogs and allowed to collect fleas. Study 3 was con-ducted in France, allowing to collect 327 fleas on 67 dogs(Table 2). Study 4 was conducted in Sicily, allowing tocollect 139 fleas on 51 dogs (Table 2). Study 5 was a dog-multicentre survey conducted in rural zones of Albania,Bulgaria, France, Hungary and Romania, where 3094 Cteno-cephalides fleas were collected from 449 flea infested dogs(Tables 3 and 4).
A total of 1969 C. felis were collected from 435 cats, 732C. felis from 178 dogs and 2828 C. canis from 396 dogs. Atotal of 5529 Ctenocephalides fleas were individually iden-tified and PCR processed.
2.2. Molecular detection of D. caninum DNA in cat fleas
2.2.1. Primer selection and designThe 28S rDNA region in the genome of D. caninum was
selected as possible target for amplification. The 28S rDNAregion from D. caninum (AF023120) was used to as a queryusing BLAST to search for similar sequences available inGenBank. Homologous sequences were selected and pair-wise aligned. A 5 bp insertion could be detected that wasunique for D. caninum, allowing the design of a D. caninumspecific reverse primer (DC28S-1R: 5′-CACATTCAACGCCC-GACTCCTGTAG-3′; insertion underlined) with the 3′-end ofthe primer covering the 5 bp insertion region. Alignmentdata was also used to design a forward primer (DC28S-1F:5′-GCATGCAAGTCAAAGGGTCCTACG-3′) based on a con-served sequence region. The primer pair would yield anexpected size of 653 bp. Genomic DNA was isolated froma D. caninum proglottid using a commercial DNA isolationkit. The isolated DNA (85 ng) served as template in a PCRduring evaluation of the primer pair to amplify the specificproduct from D. caninum.
2.2.2. Internal amplification control for D. caninum PCRAn artificial internal amplification control (IAC) was
constructed by using PCR to introduce the DC28S-1F andDC28S-1R primer flanking a piece of lambda DNA. Theresultant PCR product was ligated into a pSMART-HC Kanvector and isolated plasmid DNA (10 fg; ∼4600 copies) wasused as internal amplification control to verify PCR success.The presence of a 228 bp fragment post-PCR will serve as averification of the PCR components and conditions used inthe reaction.
f Dipylidium caninum in fleas from client-owned cats and(2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.008
2.2.3. DNA extraction and PCR amplificationGenomic DNA was extracted from 94 single fleas
infected by D. caninum, as confirmed microscopically on
IN PRESSG ModelV
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Fig. 1. PCR amplification of the expected products using primers Dc28S-
ARTICLEETPAR-7286; No. of Pages 7
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he same batch. These fleas were a C. felis strain main-ained at ClinVet (South Africa) which were artificially fedt larval stage on a feeding material containing Dipylidiumggs (Beugnet et al., 2013; Fourie et al., 2012). Individ-al fleas were placed into wells of a standard 96-well PCRlate. These wells contained two ceria stabilized zirconiumxide beads (2 mm diameter) and 19.5 �l dilution bufferupplemented with 0.5 �l DNA release additive (Phire Ani-al Tissue Direct PCR kitTM, Thermo Scientific). Samplesere homogenized by vigorous shaking in a homogenizer.
amples were incubated at 25 ◦C for 5 min, followed byncubation at 98 ◦C for 2 min. Crude extract containing DNA2 �l, equal to 10% of the total extract) was used as tem-late in a subsequent PCR. A total of 4 �l of the PCR volumeas resolved using electrophoresis in a 1% (w/v) agarose
el and documented. The percentage D. caninum positiveeas were calculated and compared to the data obtainedhrough microscopic analysis of 100 single fleas for theresence of D. caninum cysticercoids.
.2.4. Minimum detection limitIsolated D. caninum genomic DNA was serially diluted
or use as template in determining the minimum detec-ion limit of the assay. The Phire Animal Tissue Direct PCRit (Thermo Scientific) was used for amplification usinghe manufacturer recommended reaction setup. The dilu-ion protocol was followed using a total of 500 nM of eachrimer, 10 fg IAC and 2 �l template in a 20 �l final reactionolume. Thermal cycling entailed initial denaturation of at8 ◦C for 5 min followed by 45 cycles of 98 ◦C for 5 s, 68 ◦C for
s, 72 ◦C for 30 s and a final elongation of 72 ◦C for 1 min. total of 4 �l of the reaction volume was resolved usinglectrophoresis in a 1% (w/v) agarose gel and documented.
.2.5. SpecificityAll PCR products obtained from assay development and
valuation of the collected fleas were subjected to DNAequence analysis and were confirmed in GenBank to be. caninum. We also analyzed anal swabs from Dipylid-
um infested dogs (confirmed with a positive coproscopy)ll over central South Africa (77 samples from individualogs). All positive PCR products were subjected to DNAequence analysis, and were confirmed to be D. caninum.nitial primer design was based on multiple alignment of allvailable DNA sequence data in GenBank showing homol-gy to the region targeted. The primer set was designedo exclude any of the known cestodes based on multiplevailable DNA sequences. Primers were evaluated againstaenia taeniaeformis collected from infested cats in order toheck specificity. No product could be amplified from the. taeniaeformis DNA when using the D. caninum specificrimers.
. Results
.1. Primer evaluation and minimum detection level
Please cite this article in press as: Beugnet, F., et al., Occurrence odogs in Europe using a new PCR detection assay. Vet. Parasitol.
The DC28S-1F forward primer was used in combinationith reverse primer DC28S-1R to evaluate amplification
f serially diluted genomic DNA from D. caninum. Thexpected target region was amplified without yielding
1F and DC28S-1R. Lanes 2 to 10 represent PCR products obtained fromserially diluted Dipylidium caninum genomic DNA. The presence of the D.caninum specific product as well as the IAC is also indicated.
non-specific background amplification (Fig. 1). Seriallydiluted genomic DNA from D. caninum ranging from 8.5 ngto 10.6 fg was used as template for the optimized PCR.Result obtained from the minimum detection limit PCRassay indicate that 21.3 fg of template could reliably bedetected using the assay. The genome size of D. caninum isunknown, but genome databases suggest that the genomesizes of parasitic flatworm vary from 60 fg per genome to20.52 pg per genome, with the closest related genome sizesto D. caninum being Taenia solium (280 fg) and Echinococ-cus multilocularis (450 fg) (http://www.genomesize.com).Results obtained indicate the assay is sensitive enough todetect D. caninum DNA to single copy genome level makingthis assay sensitive enough for detection of D. caninumDNA present in cysticercoids inside fleas. The presence ofthe unique IAC did not have a negative effect on sensitivityand did not contribute to significantly more complexity inthe reaction due to the fact that a single primer pair wasused to amplify both the D. caninum target and the IAC.
3.2. DNA extraction and amplification
Heat extracted crude DNA was used as template in anoptimized PCR to evaluate the DNA extraction and detec-tion methods and compare it to results independentlyobtained after microscopic examination of D. caninuminfected fleas from the same batch. Results obtained fromthe microscopic examination of 100 individual fleas indi-cated that 42% of the fleas were positive for cysticercoids.Total genomic DNA was extracted from 91 homogenizedfleas to allow for inclusion of an extraction control, neg-ative control, positive control as well as a no templatecontrol 96-well PCR plate format. Any detected fragment
f Dipylidium caninum in fleas from client-owned cats and(2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.008
of the expected size for each sample was determined tobe positive. A total of 36 out of 91 (40%) samples werepositive for the presence of the D. caninum PCR targetregion (Fig. 2). Analysis of the agarose gel images revealed
Please cite this article in press as: Beugnet, F., et al., Occurrence of Dipylidium caninum in fleas from client-owned cats anddogs in Europe using a new PCR detection assay. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.008
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Fig. 2. Agarose gel after electrophoretic separation of the 91 single flea samples analyzed. DNA Markers, sample numbers as well as the appropriate controlshave been indicated.
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Table 1PCR results for the detection of Dipylidium caninum DNA in Ctenocephalides felis fleas collected on cats.
Country Number of cats Cats with + fleas Number of fleas PCR positive fleas % PCR+
Czech Republic 20 1 (5%) 32 1 3.13France 83 3 (3.6%) 285 3 1.05Germany 20 1 (5%) 139 2 1.44Hungary 111 5 (4.5%) 311 18 5.79Portugal 69 2 (2.89%) 440 3 0.68Romania 48 4 (8.33%) 48 8 16.67Slovenia 84 3 (3.57%) 714 9 1.26
Total 435 19 (4.37%) 1969 44 2.23
Table 2PCR results for the detection of Dipylidium caninum DNA in Ctenocephalides felis fleas collected on dogs.
Country Number of dogs Dogs with + fleas Number of fleas PCR positive fleas % PCR+
Albania 22 0 41 0 0Bulgaria 12 0 12 0 0France 80 (67 + 24)* 11 (13.75%) 496 (327 + 29)* 18 3.63Germany 5 0 23 0 0Hungary 5 0 10 0 0Sicily (Italy) 51 18 (35.29%) 139 20 14.39Romania 3 0 11 0 0
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Total 178 29 (16.3%)
* In study 3 and study 5, respectively.
ighly specific amplification of the target region. Theesults obtained from the PCR detection for the presencef D. caninum DNA in infected fleas indicate that 40% ofhe fleas tested positive. This is in accordance with the 42%eas containing D. caninum cysticercoids. Results from PCRased studies can be expressed as a minimum, since someamples could contain target region that is not detected byhe assay. The appropriate assay validations and inclusionf the controls were performed during this assay devel-pment to yield results that would realistically reflect onhat is happening in the sample set being analyzed.
.3. PCR based occurrence of D. caninum intenocephalides fleas collected in Europe
Out of the 435 cats infested with fleas, 19 (4.37%) werenfested by a flea population infected with D. caninum
Please cite this article in press as: Beugnet, F., et al., Occurrence odogs in Europe using a new PCR detection assay. Vet. Parasitol.
Table 1). Within the 1969 fleas, Ctenocephalides felis washe only identified species and 44 (2.23%) were foundhrough the PCR test to be infected by Dipylidium. Lookingt the number of infected fleas in the positive samples (at
able 3dentification of fleas collected on dogs in some European countries during a sing
Country No. dogs No. dogs infested with (%)*
C. canis C. felis P. irritans A. er
Albania 100 99 (99%) 22 (22%) 1 (1%) 0
Bulgaria 147 130 (88.4%) 12 (8.2%) 66 (44.9%) 1
France 24 3 (12.5%) 13 (54.2%) 11 (45.8%) 0
Germany 14 3 (21.4%) 5 (35.7%) 8 (57.1%) 2
Hungary 91 90 (98.9%) 5 (5.5%) 2 (2.2%) 2
Lithuania 8 8 (100%) 0 (0%) 1 (12.5%) 0
Romania 58 56 (96.5%) 3 (5.2%) 8 (13.8%) 0
Slovakia 7 7 (100%) 0 (0%) 0 (0%) 1
Total 449 396 (88.2%) 60 (13.4%) 97 (21.6%) 6 (1.
* A few co-infestations were found (7 dogs with both C. felis and C. canis).
732 38 5.2
least one PCR positive flea in a cat sample), the infestationrate was 20.27%.
In regard to dogs, several flea species were found instudy 5 conducted in France, Germany and Central Europe(Table 3) whereas only Ctenocephalides felis fleas werefound in Studies 3 (France) and 4 (Sicily) (Table 2).
From the dogs infested with C. felis, 16.3% (29/178) wereinfested by at least one PCR+ fleas, but the results varied sig-nificantly between the sample batches with, respectively,13.75% of dogs carrying infected fleas in France comparedto 35.29% in Sicily (p < 0.05) (Table 2).
Concerning C. canis, this flea species was found to becommon in central Europe on rural dogs with an occur-rence in 88.2% of flea-infested dogs, compared to 13.4% forC. felis, 21.6% for Pulex irritans and 1.3% for Archeophsyllaerinacei (Study 5, Table 3). Seven dogs were found to beco-infested with C. felis and C. canis. Surprisingly, P. irritans
f Dipylidium caninum in fleas from client-owned cats and(2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.008
was more common than C. felis on these dogs. From the 396dogs infested with C. canis, 36 (9.09%) were infested withat least one Dipylidium infected flea, which is significantlyhigher than the 4.37% of cats carrying at least a positive flea
le Merial epidemiological survey.
Total count of fleas
inacei C. canis C. felis P. irritans A. erinacei
490/92.1% 41/7.7% 1/0.2% 01313/70.7% 51/2.7% 491/26.5% 1/0.1%29/14.0% 140/67.6% 38/18.4% 04/6.6% 23/37.7% 32/52.5% 2/3.3%538/97.1% 10/1.8% 4/0.7% 2/0.4%53/98.1% 0 1/1.9% 0321/91.2% 11/3.1% 20/5.7% 080/98.8% 0 0 1/1.2%
3%) 2828/76.4% 276/7.5% 587/15.9% 6/0.2%
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Table 4PCR results for the detection of Dipylidium caninum DNA in Ctenocephalides canis fleas collected on dogs.
Country Number of Dogs Dogs with + fleas Number of fleas PCR Positive fleas % PCR+
Albania 99 6 (6.1%) 490 6 1.22Bulgaria 130 19 (14.6%) 1313 67 5.1France 3 0 29 0 0Germany 3 0 4 0 0Hungary 90 1 (1.11%) 538 1 0.19Lithuania 8 0 53 0 0Romania 56 10 (17.9%) 321 13 4.05
Slovakia 7 0Total 396 36 (9.09%)
(p = 0.03) (Table 4). 3.1% of the C. canis fleas were found tobe infected by Dipylidium (Table 4).
From the fleas collected on dogs, C. canis fleas were sig-nificantly less infected than the C. felis (5.2%) (p = 0.005). TheC. felis from dogs were also significantly more infected thanthe C. felis from cats (2.23%) (p < 10−5) but this difference isdue to the high rate of infection in fleas from Sicily (14.39%).If we consider the C. felis collected on dogs in France, only3.63% were PCR positive which is not significantly differentthan what was observed in C. felis from cats or in C. canisfrom dogs.
No D. caninum DNA was found in A. erinacei.PCR detec-tion was positive in 23/587 (3.91%)Pulex irritans collectedon 2 dogs from Romania and 7 dogs from Bulgaria.
4. Discussion
The PCR test for detection of D. caninum DNA in DNAextracted from fleas is a sensitive and specific method. It isa new tool available to estimate the occurrence of D. can-inum in a flea population, avoiding the time consuming andpoorly sensitive flea dissection (Beugnet et al., 2013).
This first assessment of fleas collected on cats and dogsthroughout Europe confirmed that Dipylidium PCR positivefleas are found in several European countries on both catsand dogs. 4.37% of cats and 11.46% (65/567) of dogs werefound to be infested with at least one Dipylidium infectedflea. In 1991, Hinaidy collected 1934 fleas from 198 cats and182 dogs in Austria. He dissected all the fleas to search forcysticercoid larvae of Dipylidium. He found flea infestationrates of 2.3% in C. felis from cats, 1.2% in C. felis from dogs and3.1% in C. canis from dogs. Except C. felis from dogs whichwere less infected, these results are almost similar to whatwas found in this study using the PCR method. Dogs seem tosignificantly be more at risk of Dipylidium considering thepercentage of dogs carrying infected fleas, but this couldprobably be balanced by the higher grooming activity ofcats. A study conducted in 1998 showed that cats were ableto groom and ingest up to 17.6% of their fleas each day,leading to a lifespan of about 8 days for a flea. It indicatesthat the probability for a cat to ingest a Dipylidium infectedflea is quite high, even if the percentage of infected fleas islow (Hinkle et al., 1998).
Considering fleas, 2.23% of C. felis from cats, 5.2% of C.
Please cite this article in press as: Beugnet, F., et al., Occurrence odogs in Europe using a new PCR detection assay. Vet. Parasitol.
felis from dogs and 3.1% of C. canis from dogs were foundPCR positive. This can probably not be compared becausethe fleas were collected during different surveys and notalways on both dogs and cats in the same country. But
80 0 0
2828 87 3.1%
regarding the rate of infection in positive samples, mean-ing in flea infected populations, it varied from 3 to 100%,with an average of 19.7%, which is in favour of easy andregular Dipylidium reinfestations of both cats and dogs intheir households. The results confirm the spread of D. can-inum in Ctenocephalides fleas collected on owned dogs andcats throughout Europe whatever their treatment statusagainst ecto- and endoparasites. The presence of severalflea species on dogs in Europe indicate that C. felis shouldnot always be considered as the only or the major species.The high rate of P. irritans could be related to the rural originof the dogs in the concerned study.
The circulation of Dipylidium in Ctenocephalides fleashighlights the need for combining regular flea control withDipylidium control in pet populations (Beugnet et al., 2013;Fourie et al., 2012).
Conflict of interest statement
The authors are salary or contractors of MERIAL S.A.S.but they do not declare any conflict of interest with thisepidemiological survey.
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