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Veterinary Parasitology 183 (2012) 330– 337

Contents lists available at ScienceDirect

Veterinary Parasitology

jou rn al h om epa ge: www.elsev ier .com/ locate /vetpar

hipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of aanine neglected filarial species with dermal microfilariae

. Brianti a, D. Otrantob,∗, F. Dantas-Torresb, S. Weiglb, M.S. Latrofab, G. Gaglioa, E. Napoli a,. Brucatoa, L. Cauquil c, S. Giannettoa, O. Bainc

Dipartimento di Sanità Pubblica Veterinaria, Facoltà di Medicina Veterinaria, Università degli Studi di Messina, Messina, ItalyDipartimento di Sanità Pubblica e Zootecnia, Università degli Studi di Bari, Valenzano, Bari, ItalyDépartement Systématique et Evolution, UMR 7205 CNRS, Muséum National d’Histoire Naturelle, Paris, France

r t i c l e i n f o

rticle history:eceived 29 March 2011eceived in revised form 9 July 2011ccepted 15 July 2011

eywords:anine filarioidshipicephalus sanguineusectorermal microfilariaeercopithifilaria sp.

a b s t r a c t

The life cycles of filarioids of dogs presenting dermal microfilariae have been little stud-ied. Following the recent retrieval of dermal microfilariae identified as Cercopithifilaria sp.in a dog from Sicily (Italy), this study was designed to assess the role of the brown dogtick Rhipicephalus sanguineus as an intermediate host of this filarial species. An experimen-tal tick infestation was performed on an infected dog using 300 nymphs of R. sanguineus.Engorged nymphs were collected and examined by both microscopic dissection and molec-ular analysis at five time points (i.e., the same day of tick detachment and 10, 20, 30 and50 days post-detachment) to detect the presence and developmental stage of filariae in theticks. A total of 270 engorged nymphs were collected from the dog and developing filari-oid larvae detected in 10 (5%) out of 200 ticks dissected. Infective third-stage larvae were

ercopithifilaria bainaeercopithifilaria grassii

observed in 4 (2%) of the all dissected ticks, 30 days post-detachment. Twelve (6.6%) outof 181 samples molecularly tested were positive for Cercopithifilaria sp. This study demon-strates that nymphs of R. sanguineus feeding on a dog naturally infected by Cercopithifilariasp. can ingest microfilariae, which develop up to the third infective stage thus suggesting

s migh

that this tick specie

. Introduction

Canine filarioids presenting blood microfilariae (e.g.,irofilaria immitis and Dirofilaria repens) are widely studiedue to their veterinarian importance and zoonotic poten-ial (Orihel and Eberhard, 1998), which may increase as aonsequence of vector spreading, as in the case of D. immi-is in Italy (Otranto et al., 2009). Recently, more attentionas been paid to other canine filarioids with dermal micro-lariae that cannot be diagnosed in blood stream but only

y examination of fresh skin samples. This is the case ofnchocerca lupi, identified also as an agent of ocular zoono-is in Turkey (Otranto et al., 2011a) and of Cercopithifilaria

∗ Corresponding author. Tel.: +39 080 4679839; fax: +39 080 4679839.E-mail address: d.otranto@veterinaria.uniba.it (D. Otranto).

304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2011.07.031

t act as an intermediate host of this little known canine filarioid.© 2011 Elsevier B.V. All rights reserved.

spp. which parasitize many vertebrate species, includingdogs, and which is transmitted by hard ticks (family Ixodi-dae) (Bain et al., 2002).

Following the description of Cercopithifilaria grassii(Noè, 1907) in dogs from central Italy, this filarial specieshad remained little studied up to the early 1980s wheninfective stage larvae were identified in the brown dog tickRhipicephalus sanguineus from northern Italy (Pampiglioneet al., 1983) and Switzerland (Bain et al., 1982). A secondspecies of canine filarioid presenting dermal microfilariae,Cercopithifilaria bainae (Almeida and Vicente, 1984) wasfound in Brazil, but no additional information is availableabout this filarioid (Almeida and Vicente, 1982, 1984).

Recently, dermal microfilariae were detected in a dogin Sicily (Otranto et al., 2011b). Dermal microfilariae weremorphologically and genetically distinct from those of allthe other blood microfilariae commonly found in dogs.

Parasito

(v.2, Applied Biosystems) in an automated sequencer

E. Brianti et al. / Veterinary

More interestingly, the microfilariae of this Cercopithifi-laria sp. differed morphologically from those of C. grassiibut were close to those of C. bainae (Otranto et al., 2011b).This finding stimulated us to investigate further into thenatural history of Cercopithifilaria sp. Analogously to dataon C. grassii, the competence of R. sanguineus as an inter-mediate host of this recently retrieved Cercopithifilaria sp.was experimentally investigated using the same naturallyCercopithifilaria-infested dog as source of infection for ticks.

2. Materials and methods

2.1. Study design

An experimental controlled tick infestation was per-formed on the same dog from Sicily (Italy) found to benaturally infested with dermal microfilariae of Cercop-ithifilaria sp. (Otranto et al., 2011b). The infestation tickchamber was prepared according to Srivastava and Varma(1964) and fixed in the dorsal region of the dog’s head.At the beginning of the study (T0), dog was infested with300 nymphs of R. sanguineus obtained from a tick colony,as previously described (Dantas-Torres et al., 2011). Thetick chamber was inspected every day starting from 48 hpost-tick infestation. Detached, engorged nymphs werecollected and put in glass tubes (30 nymphs/tube) duringa period ranging from 4 (early detachment of nymphs) upto 6 (late detachment) days post-infestation (p.i.). Collectedticks were maintained in an incubator at 25 ± 1 ◦C of tem-perature and 80% of relative humidity until being dissected.In order to detect the presence and the development stageof filarioid larvae in arthropods, ticks from two tubes wereexamined at different time points by microscopic dissec-tion and by PCR testing. In particular, ticks were dissectedat the same day of their collection from the chamber (T1)and 10, 20, 30 and 50 days pots-detachment (T2–T5).

The study design and the experimental procedureswere submitted to and approved by the Animal EthicsCommittee of the University of Messina. All the exper-imental procedures were conducted according to theEuropean guidelines for the accommodation and care ofanimals used for experimental and other scientific pur-poses (2007/526/EC). Tick rearing procedures were carriedout according to the guidelines for animal experimentationand were approved by the University of Bari (protocol no.1115/10).

2.2. Tick dissection and morphological examination

At each time point, 40 ticks were placed on a micro-scope slide (two ticks per slide) and individually dissectedin few drops of sterile saline solution under a stereomi-croscope. Once dissected, a drop of methylene blue (1%)was added to the dissected tick to visualize the larvalnematode and a coverslip was placed on the prepara-tion. The dissected material was microscopically observedat 40× magnification for larvae presence. All the slides

were checked by two operators and, following the obser-vation, the remaining part of dissected ticks (two for eachslide) was stored in individual vials containing 70% ethanolfor molecular analysis (see below). Filarioid larvae found

logy 183 (2012) 330– 337 331

in the dissected ticks were photographed with a digi-tal camera (Zeiss Axiocam MRc, Carl Zeiss AG, Germany)mounted on the microscope (Zeiss Axioscop 2 plus, CarlZeiss AG, Germany) and measurements (in micrometers)were made with the AxioVision rel. 4.8 software (Carl ZeissAG, Germany). Infective larvae (L3) were morphologicallyanalyzed after clarification in lactophenol and drawingswere made with an optic microscope equipped with acamera lucida. Two infective larvae were deposited in thecollection of the Muséum National d’Histoire Naturelle,Paris, France (MNHM), under the accession numbers 232YU and 233 YU.

The morphological diagnostic characters of develop-ing larval stages and infective larvae were compared withthose of C. grassii (Noè, 1908; Bain et al., 1982; Pampiglioneet al., 1983) as well as to the three other described infec-tive larvae of Cercopithifilaria species, from the roe deer(Winkhardt, 1980; Bain and Chabaud, 1986), from a rodent(Bain et al., 1986) and from cattle (Bain and Denké, 1986).

Positivity rates (number of positive ticks/number of dis-sected ticks) at different time points were compared usingFisher’s exact test (Hawkins, 2005). Significance level wasset at 5% (0.05) and all tests were performed two-sided.Statistical analyses were performed using the statisticalpackage SPSS v. 13.0.

2.3. Molecular examination

Molecular analysis were performed on the remainingparts of dissected ticks fixed in 70% ethanol after the micro-scopic examination and, when it was possible, on singularlive ticks exceeding the pre-determined number of ticksdissected at each time point (i.e., 40 ticks). All ticks founddead before dissection were also fixed in 70% ethanol andmolecularly examined. In addition, for negative controlpurpose, a pool of 25 unfed nymphs originated from thesame colony employed in the experimental tick infestationwas fixed in 70% ethanol and stored for molecular analysis.All these samples were molecularly processed for specificamplification and sequencing of genes targeting Cercopithi-filaria spp. as described elsewhere (Otranto et al., 2011b).Briefly, the genomic DNA was extracted by using DNeasyBlood & Tissue Kit (Qiagen, GmbH, Hilden, Germany) inaccordance with the manufacturer’s instructions. A par-tial cox1 (∼304 bp) gene fragment was amplified usingspecific primers CbCox1F/NTR following reaction proce-dures and amplification protocol in Otranto et al. (2011b).R. sanguineus (laboratory colony), blood and skin samplesfrom laboratory-reared beagles employed in a previousstudy (Otranto et al., 2010) were used as negative con-trols and, along with a DNA-free sample, were includedin each reaction to test the specificity of the reaction.All amplicons were purified using ultrafree-DA columns(Amicon, Millipore, Bedford, USA) and sequenced directlyusing the Taq DyeDeoxyTerminator Cycle Sequencing Kit

(ABI-PRISM 377). Sequences were aligned using ClustalWprogram (Larkin et al., 2007) and compared among themand with those available in GenBankTM dataset by BLASTanalysis.

332 E. Brianti et al. / Veterinary Parasitology 183 (2012) 330– 337

Table 1Results of tick dissection. Number, stages and percentage of Rhipicephalus sanguineus infected by Cercopithifilaria sp. larvae and dissected at different time-points (i.e., 0, +10, +20, +30 and +50 days, T1–T5) following the drop-off as nymphs from the infected dog. The different stages of larvae are also reportedand indicated as developing first stage larvae (DL1), second stage larvae (L2), late second stage larvae (LL2) and third stage (L3) infective larvae.

Day of study Tick stage Positive/total (%) Stage and number of Cercopithifilaria sp. larvae

DL1 L2 LL2 L3

T1 Engorged nymphs 3/40 (7.5) 3 – – –T2 (+10 days) Engorged nymphs 1/40 (2.5) 1 – – –

3

tereT(t(

dIe2(

fbtospdodpw(b(

Fv

T3 (+20 days) Adults 2/40 (5)T4 (+30 days) Adults 2/40 (5)T5 (+50 days) Adults 2/40 (5)

. Results

A total of 270 engorged nymphs were collected fromhe chamber between day 4 and day 6 p.i. resulting in anstimated recovery rate of 90%. Thirty ticks were lost (e.g.,emained attached to the glue used to fix the chamber,scaped from the chamber or died before the blood meal).he overall survival rate was high (91.5%), as only 23 ticksi.e., 2 nymphs and 21 adults) died before dissection. Moulto the adult stage occurred 15 ± 3 days after the drop-offi.e., between T2 and T3).

At the dissection, different stages of filarioid larvae wereetected in exposed R. sanguineus at different time points.

ndeed, the presence of Cercopithifilaria sp. larvae at differ-nt developmental stages was recorded in 10 (5%) out of00 dissected ticks with a prevalence ranging from 2.5%T2) up to 7.5% (T1) (Table 1).

A total of 181 samples (i.e., 111 from dissected ticks, 47rom singular live ticks and 23 from dead ticks) were testedy PCR for Cercopithifilaria sp. Twelve samples were posi-ive to Cercopithifilaria sp., thus giving an overall positivityf 6.6%. In particular, 9 (8.1%) out of the 111 specimens dis-ected and 3 (6.4%) out of 47 live ticks (not dissected) wereositive but none of the dead ticks. No statistical significantifference (P = 0.396) was detected between the two meth-ds used to detect Cercopithifilaria sp. in ticks. Sequenceseposited in GenBank (i.e., AN: JF925148–JF925151) dis-layed a nucleotide homology varying from 99.7 to 100%ith that of Cercopithifilaria sp. available in Genbank

JF461457) with variable nucleotide positions representedy transitions occurring at 78, 96, 112 (C ↔ T) and 152A ↔ G) residue. The first two substitutions occurred in sec-

ig. 1. Developing first-stage larvae (DL1) of Cercopithifilaria sp. found in a disseiew. Note that the body of the larvae is flattened dorso-ventrally being wider in

1 5 5 –– – – 3– – – 2

ond position leading to amino acid alteration from Leu toPro and Phe to Ser. The variation at the 152 occurred in thirdposition and lead to an amino acid alteration from Ile to Val,whereas the latter substitution at third codon position didnot lead to any amino acid alteration.

Based on their morphometrical features larvae wereclassified into four different developing types. The firsttype were small larvae (n = 5), slightly longer than thoseretrieved in the infected dog and classified as developingfirst stage larvae (DL1). These larvae presented the mor-phology of a microfilaria, with a mean length of 191.4 �m(±9.1) and a width of 5.5 �m (±1) in lateral view, withrounded apical end, short tail and smooth transversal stri-ated cuticle (Fig. 1). Three DL1 were retrieved at T1, and atT2 and T3 (i.e., one larva in each infected tick; see Table 1).Even if they did not move, all DL1 did not show any signof deterioration. Larvae identified as second larval stage(i.e., L2) presented the first moult exuvium at their ante-rior and posterior ends (Fig. 2). It included five larvae witha relevant increasing in body size and a mean body lengthof 797.2 �m (±105.3) and width of 26 �m (±2.5). Theselarvae presented a rounded apical end and a conical tail.Oesophagus, excretory cell, intestine, rectal cells and analplug were identified. L2 were only retrieved at T3 in twoinfected ticks (Table 1). Other larvae (n = 5) differed from L2being only bigger in size with a mean length of 1051.8 �m(±63.9) and width of 31.2 �m (±4.7) (Fig. 3). These secondstage larvae were considered in the late phase of develop-ment (LL2) before moult to the third infective stage. LL2

were recorded only at T3 in two ticks inside which weresimultaneously present L2 larvae (Table 1). Larvae of thefourth type showed vigorous movements and they were

cted nymph of Rhipicephalus sanguineus. A, dorso-ventral view. B, lateraldorso-ventrally than in laterally. Bars = 50 �m.

E. Brianti et al. / Veterinary Parasitology 183 (2012) 330– 337 333

Fig. 2. Second-stage larvae (L2) of Cercopithifilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, general view of L2 (bar = 100 �m). B,y and a

in the caw). Larv

cephalic region (bar = 50 �m); note the presence of a tubular mouth cavitregion (bar = 50 �m); note the rudimentary anus (head of arrow) and as

making more evident the different shape between new and old tails (arro

infective third stage larvae (L3). They had a mean length of1707 �m (±70.5) and width of 27.1 �m (±0.9) (Fig. 4). Thebuccal cavity was extremely shallow and no buccal cap-sule was identified; in some specimens the anterior liningof oesophagus was extruded. The oesophagus was dividedin anterior muscular and longer glandular posterior part.The tail, 81.5 �m (±9.3) long, was slightly bent ventrally,with rounded extremity ornate with two lateral conical lap-pets slightly longer than wide and one dorsal conical point(Fig. 4). In one larva studied in detail (Fig. 5), the nerve ringwas 82 �m from apex, oesophagus was 330 �m long withthe muscular part of 110 �m; the tail was 70 �m. A totalof five L3 were retrieved at T4 (3) and T5 (2), respectively(Table 1).

4. Discussion

Our study demonstrates that nymphs of R. sanguineusfeeding on a Cercopithifilaria sp.-infected dog can ingest

dermal microfilariae and allow their development to infec-tive third-stage larvae. The occurrence of developingsecond stage and infective third-stage larvae in R. san-guineus at different time points post-detachment, their

clear visible rudimentary excretory apparatus (head of arrow). C, caudaludal end the old cuticle is partially detached from the new inner cuticleae were stained with methylene blue (1%).

morphological similarity with those of C. grassii described(Noè, 1907; Bain et al., 1982; Pampiglione et al., 1983) andtheir molecular homology (AN JF925147–JF925151) withthe microfilariae of Cercopithifilaria sp. recently describedfrom the same dog (Otranto et al., 2011b) allows us to assessthat this tick species is a likely competent intermediate hostfor the filarial herein studied. Completing the transmissionof infective third-stage larvae to a naïve dog should providedefinitive proof of its vector competence. The competenceof R. sanguineus as a vector of C. grassii was reported byNoè in 1908 and, later on, it was re-discussed by Bain et al.(1982) and Pampiglione et al. (1983) in ticks collected fromdogs in Switzerland and Italy, respectively.

The infection in ticks occurs transstadially from nymphto adult with larvae of Cercopithifilaria reaching the L3infective stage into approximately 30 days after the tickblood meal; however, it was noted that a few micro-filariae could survive without apparent change for 30days (Table 1). The tick infection rate found in this

study is not very high compared to what is known forAcanthocheilonema dracunculoides, a filarioid with bloodmicrofilariae (Olmeda-Garcia and Rodriguez-Rodriguez,1994). At the dissection, only 5% of the exposed ticks were

334 E. Brianti et al. / Veterinary Parasitology 183 (2012) 330– 337

Fig. 3. Late second-stage larvae (LL2) of Cercopithifilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, general view of LL2 (bar = 200 �m);n head of

N methy

ftfecamctbCi

totwcwarl(

ote the presence of parts of tick salivary glands in the top left of picture (ote the similarities with the second stage (L2). Larvae were stained with

ound to harbour larvae of Cercopithifilaria sp. However, theick infection rate did not varied significantly between dif-erent time points and the tick survival rate observed in thexperiment was very high. These findings suggest that Cer-opithifilaria sp. infection is well tolerated by R. sanguineusnd the infection does not impair the survival rate and theoult into adult stage. Nonetheless, this could also be a

onsequence of the low number of larvae in the infectedicks. Indeed, with the exception of one infected tick har-ouring eight developing forms (i.e., 4 L2 and 4 LL2) ofercopithifilaria, there was a mean number of 2 larvae per

nfected tick.As far as the localization of filarial larvae inside the

ick, due to the dissection technique and the preparationf slides, it was not possible to record the exact posi-ion of developing larvae inside the arthropods. Indeed,e found larvae in the hemocoel and always free from

yst or cyst-like structures. It is known that filarial larvae,hatever the genus, develop in a syncitial structure (Bain

nd Babayan, 2003). For instance, larvae of Cercopithifilariaoussilhoni were found in the hypodermis, transformed intoarge gales, which are easily disrupted during dissectionPetit et al., 1988). In one instance, in our study, a L3 larva

arrow). B, cephalic region (bar = 100 �m). C, caudal region (bar = 100 �m).lene blue (1%).

of Cercopithifilaria sp. was found intricate inside the aciniof salivary gland.

Among the young, thin larvae recovered (DL1), none wasas long as the microfilariae of C. grassii, 567–660 �m (Noè,1907, 1908). On the other hand, DL1 found in dissected tickswere similar to dermal microfilariae found in the dog skin(182–190 �m), which resemble in length those of C. bainae(Almeida and Vicente, 1984).

Concerning the morphology of infective third-stage lar-vae observed in this study, all of them clearly presented thecharacters of the L3 of the Cercopthifilaria genus, such asthe absence of buccal capsule, long tail and caudal extrem-ity with two well-developed lateral lappets and axial point(Bain and Chabaud, 1986). The fragility of the anterior lin-ing of the oesophagus, which protrudes in some specimens,was also previously observed in other species of Cercopithi-filaria. L3 morphology is thus concordant with the genericdiagnosis made previously with the dermal microfilariaefound in the same Sicilian dog used as source of infection

for ticks in this study (Otranto et al., 2011b). By the shape ofcaudal extremity of L3, Cercopithifilaria sp. is distinct fromC. grassii, which has a large axial conical point and twicesmaller conical lappets (Bain et al., 1982) (Table 2).

E. Brianti et al. / Veterinary Parasitology 183 (2012) 330– 337 335

Fig. 4. Infective third-stage larvae (L3) of Cercopithifilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, caudal region (bar = 200 �m); notethe rounded apical end, short tail, clearly defined gut and absence of old cuticle (exuvium). B, cephalic region (bar = 50 �m); note the presence of stamp

obliquence of th

coagulum of the oesophagus that leaks from the stoma (arrow) and an(bar = 50 �m). D, caudal region, dorsal view (bar = 25 �m); note the preseof arrow). Larvae were stained with methylene blue (1%).

The infective third-stage larva of C. grassii was notdescribed by Noè (1908) who illustrated a late second-stage larva but larvae identified to C. grassii were describedby Bain et al. (1982) and Pampiglione et al. (1983) whonoticed a short oesophagus. The L3 of Cercopithifilaria sp.here studied differs to those of C. grassii by the wider bodyand the shape of caudal extremity since they present a largeaxial conical point and smaller conical lappets, instead ofthree almost equal conical points. The L3 of Cercopithifilariasp. also differs from that of the other species of Cercop-ithifilaria from the African porcupine, European roe deer,and African cattle (Table 2). However, no comparison canbe made with C. bainae described from a Brazilian dogbecause its cycle and infective third-stage larva are yet to bedescribed.

From a biological point of view, knowledge on a new

cycle of a Cercopithifilaria species confirms the associationbetween this filarial genus and ixodid ticks which haveprobably played an important role in the diversification ofthis genus (Bain et al., 2002).

ly oriented nervous ring (head of arrow). C, caudal region, lateral viewree conical lappets, two lateral shorter and one central elongated (head

In conclusion, the results reported in this paper as wellas in a previous one (Otranto et al., 2011b) provide a com-prehensive evidence for the existence and biology of athird species of Cercopithifilaria infesting dogs. Althoughthe occurrence of Cercopithifilaria spp. in dogs has beenclaimed for long time, our studies highlight that Cercopithi-filaria should be considered among those filarioids that cancause dermal microfilarial infection in dog. Although wedo not have epidemiological data on the prevalence andgeographical distribution of the Cercopithifilaria speciesstudied herein, considering the widespread distribution ofits potential vector (R. sanguineus) (Dantas-Torres, 2010),this filarioid may be widespread in dogs living in tem-perate areas. At the same time, further efforts should beundertaken in order to define the taxonomic identity ofthis filarioid and to elucidate its pathogenic role. Certainly,

further epidemiological studies using PCR-based detectionmethods (Otranto et al., 2011b) could provide a better fig-ure about the dissemination of this filarioid in dog and tickpopulations in Italy and elsewhere in Europe.

336 E. Brianti et al. / Veterinary Parasitology 183 (2012) 330– 337

Tab

le

2In

fect

ive

larv

ae

of

dif

fere

nt

spec

ies

of

Cerc

opit

hifil

aria

:

com

par

ison

of

the

mai

n

dia

gnos

tic

char

acte

rs

as

defi

ned

in

Bai

n

and

Ch

abau

d

(198

6). M

easu

rem

ents

are

exp

ress

ed

in

mic

rom

eter

s.

Roe

dee

r:

Capr

eolu

sca

preo

lus.

Porc

up

ine:

Ath

erur

us

afri

canu

s.

Cat

tle:

Bos

indi

cus.

Axi

al

poi

nt:

larg

er

mea

ns

com

par

ed

to

late

ral l

app

ets.

Spec

ies

Hos

tG

eogr

aph

ic

orig

inB

ody

len

gth

Bod

y

wid

thO

esop

hag

us

len

gth

Tail

len

gth

Axi

al

poi

nt

Two

late

ral l

app

ets

Ref

eren

ces

Cerc

opit

hifil

aria

sp.

Dog

Sici

ly16

51–1

810

26–2

833

070

–90

Con

ical

Con

ical

Pres

ent

stu

dy

Cerc

opit

hifil

aria

gras

sii

Dog

Swit

zerl

and

1050

–162

518

–23

220–

275

65–8

8La

rger

, con

ical

Ear-

shap

edB

ain

et

al. (

1982

)Ce

rcop

ithi

filar

ia

gras

sii

Dog

Nor

th

Ital

y12

3620

270

77La

rger

, cu

spid

Cu

spid

Pam

pig

lion

e

et

al. (

1983

)Ce

rcop

ithi

filar

ia

rugo

sica

uda

Roe

dee

rEu

rop

e19

60–2

180

27–3

4

405–

420

70–7

8

Larg

er, r

oun

d

Con

ical

, obt

use

Bai

n

and

Ch

abau

d

(198

6)Ce

rcop

ithi

filar

ia

rous

siho

niPo

rcu

pin

eG

abon

1200

–148

017

270–

360

65–7

8

Larg

er, c

rest

s

at

base

Con

ical

Bai

n

et

al. (

1986

)Ce

rcop

ithi

filar

ia

sp. B

ain

&D

enké

, 198

6C

attl

e

Togo

1600

22

560

78

Con

ical

, obt

use

Con

ical

, obt

use

Bai

n

and

Den

ké

(198

6)

Fig. 5. Infective third-stage larva of Cercopithifilaria sp. from a

Rhipicephalus sanguineus adult. A, cephalic region, left lateral view(bar = 30 �m). B, caudal region, left lateral view (bar = 30 �m). C, caudalextremity, sub-ventral view (bar = 10 �m).

Acknowledgments

The authors thank Alessandro Fogliazza (Merial S.p.A.,Italia) and Lénaïg Halos (Merial SAS, France) for supportingthis research. This study was also partially supported by theEuropean Community grant INCO-CT-2006-032321 and bythe MNHN grant ATM: Taxonomie moléculaire: DNA bar-code et gestion des collections. The authors also thank Dr.Yurii Kuzmin, from Kiev, for editing the drawings.

References

Almeida, G.L.G., Vicente, J.J., 1982. Dipetalonema reconditum (Grassi, 1890),Dipetalonema grassii (Noè, 1907) e Dirofilaria immitis (Lidy, 1856) emcaès na cidade do Rio de Janeiro (Nematoda-Flarioidea). Atas Soc. Biol.Rio de Janeiro 23, 9–12.

Almeida, G.L.G., Vicente, J.J., 1984. Cercopithifilaria bainae sp. n. parasitade Canis familiaris (L.) (Nematoda, Filarioidea). Atas Soc. Biol. Rio deJaneiro 24, 18.

Bain, O., Aeschlimann, A., Chatelanat, P., 1982. Présence, chez des tiques dela région de Genève, de larves infestantes qui pourraient se rapporter

Parasito

E. Brianti et al. / Veterinary

à la filaire de chien Dipetalonema grassii. Ann. Parasit. Hum. Comp. 57,643–646.

Bain, O., Babayan, S., 2003. The behaviour of filariae: morphological andanatomical signatures of their life style within the arthropod and ver-tebrate hosts. Filaria J. 2 (16), 1–13.

Bain, O., Chabaud, A.G., 1986. Atlas des larves infestantes de Filaires. Trop.Med. Parasit. 37, 301–340.

Bain, O., Denké, A.M., 1986. Larves infestantes d’une filaire: Cercopithifi-laria sp. chez des tiques de Bovins au Togo. Ann. Parasit. Hum. Comp.61, 131–135.

Bain, O., Petit, G., Chabaud, A.G., 1986. Une nouvelle filaire Cercopithifilariaroussilhoni n. sp., parasite de l’athérure au Gabon transmise par tiques;hypothèse sur l’évolution du genre. Ann. Parasitol. Hum. Comp. 61,81–93.

Bain, O., Uni, S., Takaoka, H., 2002. A synthetic look at a twenty yearsold taxon, Cercopithifilaria; its probable evolution. In: Proceedings ofthe 10th International Congress of Parasitology – ICOPA X, Vancouver(Canada), August 4–9, Monduzzi Editore, pp. 365–368.

Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipi-cephalus sanguineus. Parasit. Vectors 3, 26.

Dantas-Torres, F., Figueredo, L.A., Otranto, D., 2011. Seasonal variationin the effect of climate on the biology of Rhipicephalus sanguineus insouthern Europe. Parasitology 138 (April (4)), 527–536.

Hawkins, D. (Ed.), 2005. Biomeasurement: Understanding, Analysing, andCommunicating Data in the Biosciences. Oxford University Press Inc.,New York, USA, p. 133.

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A.,McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thomp-

son, J.D., Gibson, T.J., Higgins, D.G., 2007. ClustalW and ClustalX version2. Bioinformatics 23, 2947–2948.

Noè, G., 1907. Contribuzioni alla Sistematica e alla Anatomia del Genere.Filaria. La Filaria grassii. Roma, Istituto di Anatomia Comparata dellaRegia, Università di Roma, pp. 236–252.

logy 183 (2012) 330– 337 337

Noè, G., 1908. Il ciclo evolutivo de la Filaria grassii, mihi, 1907. Atti R. Accad.Lincei, Roma 17, 282–293.

Olmeda-Garcia, A.S., Rodriguez-Rodriguez, J.A., 1994. Stage-specific devel-opment of a filarial nematode (Dipelalonema dracunculoides) in vectorticks. J. Helminthol. 68, 231–235.

Orihel, T., Eberhard, M.L., 1998. Zoonotic filariasis. Clin. Microbiol. Rev. 11,366–381.

Otranto, D., Capelli, G., Genchi, C., 2009. Changing distribution patterns ofcanine vector borne diseases in Italy: leishmaniosis vs. dirofilariosis.Parasit. Vectors 2 (1), S2.

Otranto, D., Sakru, N., Testini, G., Gürlü, V.P., Yakar, K., Lia, R.P., Dantas-Torres, F., Bain, O., 2011a. First evidence of human zoonotic infectionby Onchocerca lupi (Spirurida, Onchocercidae). Am. J. Trop. Med. Hyg.84, 55–58.

Otranto, D., Brianti, E., Dantas-Torres, F., Weigl, S., Latrofa, M.S., Gaglio, G.,Cauquil, L., Giannetto, S., Bain, O., 2011b. Morphological and moleculardata on the dermal microfilariae of a species of Cercopithifilaria froma dog in Sicily. Vet. Parasitol., doi:10.1016/j.vetpar.2011.05.043.

Pampiglione, S., Canestri Trotti, G., Marchetti, S., 1983. Ritrovamento diDiptalonema grassii (Noè, 1907) in Rhipicephalus sanguineus su cane inItalia e descrizione di alcuni suoi stadi larvali. Parassitologia 25, 316.

Petit, G., Bain, O., Cassone, J., Seureau, C., 1988. La filaire Cercopithifi-laria roussilhoni chez la tique vectrice. Ann. Parasitol. Hum. Comp. 63,296–302.

Srivastava, S.C., Varma, M.G.R., 1964. The culture of the tick Rhipicephalussanguineus (Latreille) (Ixodidae) in the laboratory. J. Med. Ent. 1,154–157.

Winkhardt, H.J., 1980. Untersuchungen über den Entwicklungszyklus von

Dipetalonema rugosicauda (syn. Wehrdikmansia rugosicauda) (Nema-toda: Filarioidea). II. Die Entwicklung von Dipetalonema rugosicaudaim Zwischenwirt Ixodes ricinus und Untersuchungen über dasVorkommen der Mikrofilariae im Reh (Capreolus capreolus). Tropen-med. Parasitol. 31, 21–30.