Veterinary Parasitology 183 (2012) 330337

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a

canine neglected larial species with dermal microlariae
E. Brianti a , D. Otranto b, , F. Dantas-Torres b , S. Weigl b , M.S. Latrofa b , G. Gaglio a , E. Napoli a ,
G. Brucato a , L. Cauquil c , S. Giannetto a , O. Bain c
a
b
c

Dipartimento di Sanit Pubblica Veterinaria, Facolt di Medicina Veterinaria, Universit degli Studi di Messina, Messina, Italy
Dipartimento di Sanit Pubblica e Zootecnia, Universit degli Studi di Bari, Valenzano, Bari, Italy
Dpartement Systmatique et Evolution, UMR 7205 CNRS, Musum National dHistoire Naturelle, Paris, France

a r t i c l e

i n f o

Article history:
Received 29 March 2011
Received in revised form 9 July 2011
Accepted 15 July 2011
Keywords:
Canine larioids
Rhipicephalus sanguineus
Vector
Dermal microlariae
Cercopithilaria sp.
Cercopithilaria bainae
Cercopithilaria grassii

a b s t r a c t
The life cycles of larioids of dogs presenting dermal microlariae have been little studied. Following the recent retrieval of dermal microlariae identied as Cercopithilaria sp.
in a dog from Sicily (Italy), this study was designed to assess the role of the brown dog
tick Rhipicephalus sanguineus as an intermediate host of this larial species. An experimental 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 molecular analysis at ve time points (i.e., the same day of tick detachment and 10, 20, 30 and
50 days post-detachment) to detect the presence and developmental stage of lariae in the
ticks. A total of 270 engorged nymphs were collected from the dog and developing larioid larvae detected in 10 (5%) out of 200 ticks dissected. Infective third-stage larvae were
observed in 4 (2%) of the all dissected ticks, 30 days post-detachment. Twelve (6.6%) out
of 181 samples molecularly tested were positive for Cercopithilaria sp. This study demonstrates that nymphs of R. sanguineus feeding on a dog naturally infected by Cercopithilaria
sp. can ingest microlariae, which develop up to the third infective stage thus suggesting
that this tick species might act as an intermediate host of this little known canine larioid.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Canine larioids presenting blood microlariae (e.g.,
Dirolaria immitis and Dirolaria repens) are widely studied
due to their veterinarian importance and zoonotic potential (Orihel and Eberhard, 1998), which may increase as a
consequence of vector spreading, as in the case of D. immitis in Italy (Otranto et al., 2009). Recently, more attention
has been paid to other canine larioids with dermal microlariae that cannot be diagnosed in blood stream but only
by examination of fresh skin samples. This is the case of
Onchocerca lupi, identied also as an agent of ocular zoonosis in Turkey (Otranto et al., 2011a) and of Cercopithilaria

Corresponding author. Tel.: +39 080 4679839; fax: +39 080 4679839.
E-mail address: d.otranto@veterinaria.uniba.it (D. Otranto).
0304-4017/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2011.07.031

spp. which parasitize many vertebrate species, including

dogs, and which is transmitted by hard ticks (family Ixodidae) (Bain et al., 2002).
Following the description of Cercopithilaria grassii
(No, 1907) in dogs from central Italy, this larial species
had remained little studied up to the early 1980s when
infective stage larvae were identied in the brown dog tick
Rhipicephalus sanguineus from northern Italy (Pampiglione
et al., 1983) and Switzerland (Bain et al., 1982). A second
species of canine larioid presenting dermal microlariae,
Cercopithilaria bainae (Almeida and Vicente, 1984) was
found in Brazil, but no additional information is available
about this larioid (Almeida and Vicente, 1982, 1984).
Recently, dermal microlariae were detected in a dog
in Sicily (Otranto et al., 2011b). Dermal microlariae were
morphologically and genetically distinct from those of all
the other blood microlariae commonly found in dogs.

E. Brianti et al. / Veterinary Parasitology 183 (2012) 330337

More interestingly, the microlariae of this Cercopithilaria sp. differed morphologically from those of C. grassii
but were close to those of C. bainae (Otranto et al., 2011b).
This nding stimulated us to investigate further into the
natural history of Cercopithilaria sp. Analogously to data
on C. grassii, the competence of R. sanguineus as an intermediate host of this recently retrieved Cercopithilaria sp.
was experimentally investigated using the same naturally
Cercopithilaria-infested dog as source of infection for ticks.
2. Materials and methods
2.1. Study design
An experimental controlled tick infestation was performed on the same dog from Sicily (Italy) found to be
naturally infested with dermal microlariae of Cercopithilaria sp. (Otranto et al., 2011b). The infestation tick
chamber was prepared according to Srivastava and Varma
(1964) and xed in the dorsal region of the dogs head.
At the beginning of the study (T0), dog was infested with
300 nymphs of R. sanguineus obtained from a tick colony,
as previously described (Dantas-Torres et al., 2011). The
tick chamber was inspected every day starting from 48 h
post-tick infestation. Detached, engorged nymphs were
collected and put in glass tubes (30 nymphs/tube) during
a period ranging from 4 (early detachment of nymphs) up
to 6 (late detachment) days post-infestation (p.i.). Collected
ticks were maintained in an incubator at 25 1 C of temperature and 80% of relative humidity until being dissected.
In order to detect the presence and the development stage
of larioid larvae in arthropods, ticks from two tubes were
examined at different time points by microscopic dissection and by PCR testing. In particular, ticks were dissected
at the same day of their collection from the chamber (T1)
and 10, 20, 30 and 50 days pots-detachment (T2T5).
The study design and the experimental procedures
were submitted to and approved by the Animal Ethics
Committee of the University of Messina. All the experimental procedures were conducted according to the
European guidelines for the accommodation and care of
animals used for experimental and other scientic purposes (2007/526/EC). Tick rearing procedures were carried
out according to the guidelines for animal experimentation
and 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 microscope slide (two ticks per slide) and individually dissected
in few drops of sterile saline solution under a stereomicroscope. Once dissected, a drop of methylene blue (1%)
was added to the dissected tick to visualize the larval
nematode and a coverslip was placed on the preparation. The dissected material was microscopically observed
at 40 magnication for larvae presence. All the slides
were checked by two operators and, following the observation, the remaining part of dissected ticks (two for each
slide) was stored in individual vials containing 70% ethanol
for molecular analysis (see below). Filarioid larvae found

331

in the dissected ticks were photographed with a digital camera (Zeiss Axiocam MRc, Carl Zeiss AG, Germany)
mounted on the microscope (Zeiss Axioscop 2 plus, Carl
Zeiss AG, Germany) and measurements (in micrometers)
were made with the AxioVision rel. 4.8 software (Carl Zeiss
AG, Germany). Infective larvae (L3) were morphologically
analyzed after clarication in lactophenol and drawings
were made with an optic microscope equipped with a
camera lucida. Two infective larvae were deposited in the
collection of the Musum National dHistoire Naturelle,
Paris, France (MNHM), under the accession numbers 232
YU and 233 YU.
The morphological diagnostic characters of developing larval stages and infective larvae were compared with
those of C. grassii (No, 1908; Bain et al., 1982; Pampiglione
et al., 1983) as well as to the three other described infective larvae of Cercopithilaria species, from the roe deer
(Winkhardt, 1980; Bain and Chabaud, 1986), from a rodent
(Bain et al., 1986) and from cattle (Bain and Denk, 1986).
Positivity rates (number of positive ticks/number of dissected ticks) at different time points were compared using
Fishers exact test (Hawkins, 2005). Signicance level was
set at 5% (0.05) and all tests were performed two-sided.
Statistical analyses were performed using the statistical
package SPSS v. 13.0.

2.3. Molecular examination

Molecular analysis were performed on the remaining
parts of dissected ticks xed in 70% ethanol after the microscopic examination and, when it was possible, on singular
live ticks exceeding the pre-determined number of ticks
dissected at each time point (i.e., 40 ticks). All ticks found
dead before dissection were also xed in 70% ethanol and
molecularly examined. In addition, for negative control
purpose, a pool of 25 unfed nymphs originated from the
same colony employed in the experimental tick infestation
was xed in 70% ethanol and stored for molecular analysis.
All these samples were molecularly processed for specic
amplication and sequencing of genes targeting Cercopithilaria spp. as described elsewhere (Otranto et al., 2011b).
Briey, the genomic DNA was extracted by using DNeasy
Blood & Tissue Kit (Qiagen, GmbH, Hilden, Germany) in
accordance with the manufacturers instructions. A partial cox1 (304 bp) gene fragment was amplied using
specic primers CbCox1F/NTR following reaction procedures and amplication protocol in Otranto et al. (2011b).
R. sanguineus (laboratory colony), blood and skin samples
from laboratory-reared beagles employed in a previous
study (Otranto et al., 2010) were used as negative controls and, along with a DNA-free sample, were included
in each reaction to test the specicity of the reaction.
All amplicons were puried using ultrafree-DA columns
(Amicon, Millipore, Bedford, USA) and sequenced directly
using the Taq DyeDeoxyTerminator Cycle Sequencing Kit
(v.2, Applied Biosystems) in an automated sequencer
(ABI-PRISM 377). Sequences were aligned using ClustalW
program (Larkin et al., 2007) and compared among them
and with those available in GenBankTM dataset by BLAST
analysis.

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E. Brianti et al. / Veterinary Parasitology 183 (2012) 330337

Table 1
Results of tick dissection. Number, stages and percentage of Rhipicephalus sanguineus infected by Cercopithilaria sp. larvae and dissected at different timepoints (i.e., 0, +10, +20, +30 and +50 days, T1T5) following the drop-off as nymphs from the infected dog. The different stages of larvae are also reported
and indicated as developing rst 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 (%)

T1
T2 (+10 days)
T3 (+20 days)
T4 (+30 days)
T5 (+50 days)

Engorged nymphs
Engorged nymphs
Adults
Adults
Adults

3/40 (7.5)
1/40 (2.5)
2/40 (5)
2/40 (5)
2/40 (5)

3. Results
A total of 270 engorged nymphs were collected from
the chamber between day 4 and day 6 p.i. resulting in an
estimated recovery rate of 90%. Thirty ticks were lost (e.g.,
remained attached to the glue used to x the chamber,
escaped from the chamber or died before the blood meal).
The overall survival rate was high (91.5%), as only 23 ticks
(i.e., 2 nymphs and 21 adults) died before dissection. Moult
to the adult stage occurred 15 3 days after the drop-off
(i.e., between T2 and T3).
At the dissection, different stages of larioid larvae were
detected in exposed R. sanguineus at different time points.
Indeed, the presence of Cercopithilaria sp. larvae at different developmental stages was recorded in 10 (5%) out of
200 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, 47
from singular live ticks and 23 from dead ticks) were tested
by PCR for Cercopithilaria sp. Twelve samples were positive to Cercopithilaria sp., thus giving an overall positivity
of 6.6%. In particular, 9 (8.1%) out of the 111 specimens dissected and 3 (6.4%) out of 47 live ticks (not dissected) were
positive but none of the dead ticks. No statistical signicant
difference (P = 0.396) was detected between the two methods used to detect Cercopithilaria sp. in ticks. Sequences
deposited in GenBank (i.e., AN: JF925148JF925151) displayed a nucleotide homology varying from 99.7 to 100%
with that of Cercopithilaria sp. available in Genbank
(JF461457) with variable nucleotide positions represented
by transitions occurring at 78, 96, 112 (C T) and 152
(A G) residue. The rst two substitutions occurred in sec-

Stage and number of Cercopithilaria sp. larvae

DL1

L2

LL2

L3

3
1
1

3
2

ond position leading to amino acid alteration from Leu to

Pro and Phe to Ser. The variation at the 152 occurred in third
position and lead to an amino acid alteration from Ile to Val,
whereas the latter substitution at third codon position did
not lead to any amino acid alteration.
Based on their morphometrical features larvae were
classied into four different developing types. The rst
type were small larvae (n = 5), slightly longer than those
retrieved in the infected dog and classied as developing
rst stage larvae (DL1). These larvae presented the morphology of a microlaria, with a mean length of 191.4 m
(9.1) and a width of 5.5 m (1) in lateral view, with
rounded apical end, short tail and smooth transversal striated cuticle (Fig. 1). Three DL1 were retrieved at T1, and at
T2 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 sign
of deterioration. Larvae identied as second larval stage
(i.e., L2) presented the rst moult exuvium at their anterior and posterior ends (Fig. 2). It included ve larvae with
a relevant increasing in body size and a mean body length
of 797.2 m (105.3) and width of 26 m (2.5). These
larvae presented a rounded apical end and a conical tail.
Oesophagus, excretory cell, intestine, rectal cells and anal
plug were identied. L2 were only retrieved at T3 in two
infected ticks (Table 1). Other larvae (n = 5) differed from L2
being 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 second
stage larvae were considered in the late phase of development (LL2) before moult to the third infective stage. LL2
were recorded only at T3 in two ticks inside which were
simultaneously present L2 larvae (Table 1). Larvae of the
fourth type showed vigorous movements and they were

Fig. 1. Developing rst-stage larvae (DL1) of Cercopithilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, dorso-ventral view. B, lateral
view. Note that the body of the larvae is attened dorso-ventrally being wider in dorso-ventrally than in laterally. Bars = 50 m.

E. Brianti et al. / Veterinary Parasitology 183 (2012) 330337

333

Fig. 2. Second-stage larvae (L2) of Cercopithilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, general view of L2 (bar = 100 m). B,
cephalic region (bar = 50 m); note the presence of a tubular mouth cavity and a clear visible rudimentary excretory apparatus (head of arrow). C, caudal
region (bar = 50 m); note the rudimentary anus (head of arrow) and as in the caudal end the old cuticle is partially detached from the new inner cuticle
making more evident the different shape between new and old tails (arrow). Larvae were stained with methylene blue (1%).

infective third stage larvae (L3). They had a mean length of

1707 m (70.5) and width of 27.1 m (0.9) (Fig. 4). The
buccal cavity was extremely shallow and no buccal capsule was identied; in some specimens the anterior lining
of oesophagus was extruded. The oesophagus was divided
in 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 lappets slightly longer than wide and one dorsal conical point
(Fig. 4). In one larva studied in detail (Fig. 5), the nerve ring
was 82 m from apex, oesophagus was 330 m long with
the muscular part of 110 m; the tail was 70 m. A total
of ve L3 were retrieved at T4 (3) and T5 (2), respectively
(Table 1).
4. Discussion
Our study demonstrates that nymphs of R. sanguineus
feeding on a Cercopithilaria sp.-infected dog can ingest
dermal microlariae and allow their development to infective third-stage larvae. The occurrence of developing
second stage and infective third-stage larvae in R. sanguineus at different time points post-detachment, their

morphological similarity with those of C. grassii described

(No, 1907; Bain et al., 1982; Pampiglione et al., 1983) and
their molecular homology (AN JF925147JF925151) with
the microlariae of Cercopithilaria sp. recently described
from the same dog (Otranto et al., 2011b) allows us to assess
that this tick species is a likely competent intermediate host
for the larial herein studied. Completing the transmission
of infective third-stage larvae to a nave dog should provide
denitive proof of its vector competence. The competence
of R. sanguineus as a vector of C. grassii was reported by
No in 1908 and, later on, it was re-discussed by Bain et al.
(1982) and Pampiglione et al. (1983) in ticks collected from
dogs in Switzerland and Italy, respectively.
The infection in ticks occurs transstadially from nymph
to adult with larvae of Cercopithilaria reaching the L3
infective stage into approximately 30 days after the tick
blood meal; however, it was noted that a few microlariae could survive without apparent change for 30
days (Table 1). The tick infection rate found in this
study is not very high compared to what is known for
Acanthocheilonema dracunculoides, a larioid with blood
microlariae (Olmeda-Garcia and Rodriguez-Rodriguez,
1994). At the dissection, only 5% of the exposed ticks were

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E. Brianti et al. / Veterinary Parasitology 183 (2012) 330337

Fig. 3. Late second-stage larvae (LL2) of Cercopithilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, general view of LL2 (bar = 200 m);
note the presence of parts of tick salivary glands in the top left of picture (head of arrow). B, cephalic region (bar = 100 m). C, caudal region (bar = 100 m).
Note the similarities with the second stage (L2). Larvae were stained with methylene blue (1%).

found to harbour larvae of Cercopithilaria sp. However, the

tick infection rate did not varied signicantly between different time points and the tick survival rate observed in the
experiment was very high. These ndings suggest that Cercopithilaria sp. infection is well tolerated by R. sanguineus
and the infection does not impair the survival rate and the
moult into adult stage. Nonetheless, this could also be a
consequence of the low number of larvae in the infected
ticks. Indeed, with the exception of one infected tick harbouring eight developing forms (i.e., 4 L2 and 4 LL2) of
Cercopithilaria, there was a mean number of 2 larvae per
infected tick.
As far as the localization of larial larvae inside the
tick, due to the dissection technique and the preparation
of slides, it was not possible to record the exact position of developing larvae inside the arthropods. Indeed,
we found larvae in the hemocoel and always free from
cyst or cyst-like structures. It is known that larial larvae,
whatever the genus, develop in a syncitial structure (Bain
and Babayan, 2003). For instance, larvae of Cercopithilaria
roussilhoni were found in the hypodermis, transformed into
large gales, which are easily disrupted during dissection
(Petit et al., 1988). In one instance, in our study, a L3 larva

of Cercopithilaria sp. was found intricate inside the acini

of salivary gland.
Among the young, thin larvae recovered (DL1), none was
as long as the microlariae of C. grassii, 567660 m (No,
1907, 1908). On the other hand, DL1 found in dissected ticks
were similar to dermal microlariae found in the dog skin
(182190 m), which resemble in length those of C. bainae
(Almeida and Vicente, 1984).
Concerning the morphology of infective third-stage larvae observed in this study, all of them clearly presented the
characters of the L3 of the Cercopthilaria genus, such as
the absence of buccal capsule, long tail and caudal extremity with two well-developed lateral lappets and axial point
(Bain and Chabaud, 1986). The fragility of the anterior lining of the oesophagus, which protrudes in some specimens,
was also previously observed in other species of Cercopithilaria. L3 morphology is thus concordant with the generic
diagnosis made previously with the dermal microlariae
found in the same Sicilian dog used as source of infection
for ticks in this study (Otranto et al., 2011b). By the shape of
caudal extremity of L3, Cercopithilaria sp. is distinct from
C. grassii, which has a large axial conical point and twice
smaller conical lappets (Bain et al., 1982) (Table 2).

E. Brianti et al. / Veterinary Parasitology 183 (2012) 330337

335

Fig. 4. Infective third-stage larvae (L3) of Cercopithilaria sp. found in a dissected nymph of Rhipicephalus sanguineus. A, caudal region (bar = 200 m); note
the rounded apical end, short tail, clearly dened gut and absence of old cuticle (exuvium). B, cephalic region (bar = 50 m); note the presence of stamp
coagulum of the oesophagus that leaks from the stoma (arrow) and an obliquely oriented nervous ring (head of arrow). C, caudal region, lateral view
(bar = 50 m). D, caudal region, dorsal view (bar = 25 m); note the presence of three conical lappets, two lateral shorter and one central elongated (head
of arrow). Larvae were stained with methylene blue (1%).

The infective third-stage larva of C. grassii was not

described by No (1908) who illustrated a late secondstage larva but larvae identied to C. grassii were described
by Bain et al. (1982) and Pampiglione et al. (1983) who
noticed a short oesophagus. The L3 of Cercopithilaria sp.
here studied differs to those of C. grassii by the wider body
and the shape of caudal extremity since they present a large
axial conical point and smaller conical lappets, instead of
three almost equal conical points. The L3 of Cercopithilaria
sp. also differs from that of the other species of Cercopithilaria from the African porcupine, European roe deer,
and African cattle (Table 2). However, no comparison can
be made with C. bainae described from a Brazilian dog
because its cycle and infective third-stage larva are yet to be
described.
From a biological point of view, knowledge on a new
cycle of a Cercopithilaria species conrms the association
between this larial genus and ixodid ticks which have
probably played an important role in the diversication of
this genus (Bain et al., 2002).

In conclusion, the results reported in this paper as well

as in a previous one (Otranto et al., 2011b) provide a comprehensive evidence for the existence and biology of a
third species of Cercopithilaria infesting dogs. Although
the occurrence of Cercopithilaria spp. in dogs has been
claimed for long time, our studies highlight that Cercopithilaria should be considered among those larioids that can
cause dermal microlarial infection in dog. Although we
do not have epidemiological data on the prevalence and
geographical distribution of the Cercopithilaria species
studied herein, considering the widespread distribution of
its potential vector (R. sanguineus) (Dantas-Torres, 2010),
this larioid may be widespread in dogs living in temperate areas. At the same time, further efforts should be
undertaken in order to dene the taxonomic identity of
this larioid and to elucidate its pathogenic role. Certainly,
further epidemiological studies using PCR-based detection
methods (Otranto et al., 2011b) could provide a better gure about the dissemination of this larioid in dog and tick
populations in Italy and elsewhere in Europe.

Present study
Bain et al. (1982)
Pampiglione et al. (1983)
Bain and Chabaud (1986)
Bain et al. (1986)
Bain and Denk (1986)
Conical
Ear-shaped
Cuspid
Conical, obtuse
Conical
Conical, obtuse
Conical
Larger, conical
Larger, cuspid
Larger, round
Larger, crests at base
Conical, obtuse
7090
6588
77
7078
6578
78
330
220275
270
405420
270360
560
2628
1823
20
2734
17
22
16511810
10501625
1236
19602180
12001480
1600
Dog
Dog
Dog
Roedeer
Porcupine
Cattle
Cercopithilaria sp.
Cercopithilaria grassii
Cercopithilaria grassii
Cercopithilaria rugosicauda
Cercopithilaria roussihoni
Cercopithilaria sp. Bain &
Denk, 1986

Sicily
Switzerland
North Italy
Europe
Gabon
Togo

Body width
Host
Species

Geographic origin

Body length

Oesophagus length

Tail length

Axial point

Two lateral lappets

References

E. Brianti et al. / Veterinary Parasitology 183 (2012) 330337

Table 2
Infective larvae of different species of Cercopithilaria: comparison of the main diagnostic characters as dened in Bain and Chabaud (1986). Measurements are expressed in micrometers. Roedeer: Capreolus
capreolus. Porcupine: Atherurus africanus. Cattle: Bos indicus. Axial point: larger means compared to lateral lappets.

336

Fig. 5. Infective third-stage larva of Cercopithilaria 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, caudal
extremity, sub-ventral view (bar = 10 m).

Acknowledgments
The authors thank Alessandro Fogliazza (Merial S.p.A.,
Italia) and Lnag Halos (Merial SAS, France) for supporting
this research. This study was also partially supported by the
European Community grant INCO-CT-2006-032321 and by
the MNHN grant ATM: Taxonomie molculaire: DNA barcode et gestion des collections. The authors also thank Dr.
Yurii Kuzmin, from Kiev, for editing the drawings.
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