Morphological and molecular data on the dermal microfilariae of a ...

Veterinary Parasitology 182 (2011) 221– 229
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Veterinary Parasitology
jo u rn al hom epa ge : www.elsevier.com/locate/vetpar
<strong>Morphological</strong> <strong>and</strong> <strong>molecular</strong> <strong>data</strong> on the dermal microfilariae of a
species of Cercopithifilaria from a dog in Sicily
Domenico Otranto a,∗ , Emanuele Brianti b , Filipe Dantas-Torres a ,
Stefania Weigl a , Maria Stefania Latrofa a , Gabriella Gaglio b , Laura Cauquil b ,
Salvatore Giannetto b , Odile Bain c
a Dipartimento di Sanità Pubblica e Zootecnia, Università degli Studi di Bari, 70010 Valenzano, Bari, Italy
b Dipartimento di Sanità Pubblica Veterinaria, Facoltà di Medicina Veterinaria, Università degli Studi di Messina, Messina, Italy
c Département Systématique et Evolution, UMR 7205 CNRS, Muséum National d’Histoire Naturelle, Paris, France
a r t i c l e i n f o
Article history:
Received 14 March 2011
Received in revised form 16 May 2011
Accepted 20 May 2011
Keywords:
Canine filarioids
Dermal microfilariae
Cercopithifilaria bainae
Cercopithifilaria grassii
Acanthocheilonema reconditum
1. Introduction
a b s t r a c t
Filarioids (Spirurida, Onchocercidae) parasitizing wild
<strong>and</strong> domestic mammals can cause zoonosis in tropical <strong>and</strong>
subtropical regions (Orihel <strong>and</strong> Eberhard, 1998; Otranto
<strong>and</strong> Eberhard, 2011). Several of these species are parasites
of dogs, <strong>and</strong> have either blood microfilariae such as of
Dirofilaria spp. (Orihel <strong>and</strong> Ash, 1995; McCall et al., 2008;
Pampiglione et al., 1995, 2009) <strong>and</strong> Acanthocheilonema
reconditum (Huynh et al., 2001), or dermal microfilariae
as Onchocerca lupi (Otranto et al., 2011). Canine filariae
∗ 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.2011.05.043
Dermal microfilariae found in a dog from Sicily, Italy, were characterized morphologically
<strong>and</strong> genetically <strong>and</strong> differentiated from those of all the other blood microfilariae commonly
found in dogs. In particular, the microfilariae were short (mean length of 186.7 m), presented
a body flattened dorso-ventrally <strong>and</strong> a rounded head, bearing a tiny cephalic hook.
The genetic identity of microfilariae herein studied was also assessed by <strong>molecular</strong> amplification,
sequencing <strong>and</strong> analyzing of multiple ribosomal ITS-2 <strong>and</strong> mitochondrial (cox1 <strong>and</strong>
12S) target genes. Both morphologic <strong>and</strong> genetic characterization as well as the <strong>molecular</strong>
phylogenetic history inferred using sequences of a barcoding <strong>data</strong>set were concordant
in supporting the identification of Cercopithifilaria at the genus level. Surprisingly, microfilariae
here examined were well distinct from Cercopithifilaria grassii (Noè, 1907), from
northern Italy, <strong>and</strong> resembled those of a species described in Brazil, Cercopithifilaria bainae
Almeida & Vicente, 1984. This paper provides evidence for the existence of a Cercopithifilaria
species infesting a dog from Sicily <strong>and</strong> also presents a PCR protocol on skin samples as
a tool for further epidemiological studies, which could provide evidence on the aetiology
<strong>and</strong> the natural history of this filarial species.
© 2011 Elsevier B.V. All rights reserved.
with dermal microfilariae are not restricted to O. lupi <strong>and</strong>
two other species have been reported in the genus Cercopithifilaria
(Eberhard, 1980), although they are little known
<strong>and</strong> usually not searched for in dogs (Almeida <strong>and</strong> Vicente,
1984; Bain et al., 1982a).
The genus Cercopithifilaria, originally described as a subgenus
of Dipetalonema by Eberhard (1980), is now well
defined <strong>and</strong> comprises 28 species, either described in or
reclassified to this genus (Bain et al., 2002). Adult worms
are most often tiny, located in subcutaneous tissues <strong>and</strong>
uneasy to detect. Microfilariae are always in the dermis
instead of in the blood circulation (Bain et al., 2002). As
far as it is known, species of Cercopithifilaria are primarily
transmitted by hard ticks (Ixodida, Ixodidae), such as
Rhipicephalus <strong>and</strong> Ixodes (Noè, 1908; Winkhardt, 1980; Bain

222 D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229
et al., 1986; Spratt <strong>and</strong> Haycock, 1988; Petit et al., 1988).
The host range of Cercopithifilaria is made of a diversity
of ruminants, primates, carnivores, rodents, lagomorphs,
marsupials <strong>and</strong> monotremes. However, species currently
placed within this genus are well supported by traditional
<strong>and</strong> <strong>molecular</strong> <strong>data</strong> (Bain et al., 2008).
In 1907, Noè found a filarial nematode with dermal
microfilariae in samples collected from a dog in Rome
(Italy). This species was originally described as Filaria
grassii <strong>and</strong> later transferred to Cercopithifilaria by Bain
et al. (1982b). This filaria presents characteristic “gigantesche”
(from Italian, giant) microfilariae, as Noè stated
in his original description for this species (Noè, 1907).
These microfilariae developed in ticks (Noè, 1908). C. grassii
species remained ignored until two interesting reports
of this parasite, dated back to nearly 30 years ago, in
Switzerl<strong>and</strong> (Bain et al., 1982a) <strong>and</strong> in northern Italy
(Pampiglione et al., 1983).
In south-eastern Brazil, the species was reported twice,
by Costa <strong>and</strong> Freitas (1962) <strong>and</strong> Almeida <strong>and</strong> Vicente
(1982). However, two years later, the last authors examined
additional material of this rare filarioid <strong>and</strong> concluded
that it represented a new species, Cercopithifilaria bainae
Almeida & Vicente, 1984, distinguished for its much smaller
microfilariae (Almeida <strong>and</strong> Vicente, 1984).
The primary aim of the present investigation was to
reassess the occurrence of C. grassii in Italy. Dermal microfilariae
were retrieved from skin samples of a dog from Sicily,
southern Italy, <strong>and</strong> coupled morphological <strong>and</strong> <strong>molecular</strong>
analyses confirmed that this filarial nematode belonged
to the genus Cercopithifilaria. However, the morphological
study indicated that this species was not C. grassii, but
seemed related morphologically to the Brazilian filarioid
described by Almeida <strong>and</strong> Vicente (1984).
2. Materials <strong>and</strong> methods
2.1. Dermal microfilariae collection
On July 2010, one mongrel dog was found positive for
A. reconditum microfilariae in blood during a survey carried
out in the municipal dog shelter in Messina (38 ◦ 11 ′ N;
15 ◦ 33 ′ E), Sicily, Italy (<strong>data</strong> not shown). These larvae were
identified on the basis of their length (260 m), body width
(4 m), caudal filament, <strong>and</strong> characteristic prominent
cephalic hook. In the same shelter, other dogs were found
to be positive for A. reconditum (<strong>data</strong> not shown). None of
the animals had received any anthelmintic or ectoparasiticide
treatment during the months before <strong>and</strong> almost all of
them were heavily infested by fleas (Ctenocephalides spp.)
<strong>and</strong> by R. sanguineus ticks (Brianti et al., 2010). At the clinical
examination a subcutaneous nodule was retrieved on
the dog’s right thigh (Fig. 1) <strong>and</strong> thus a biopsy of 3 mm was
taken from skin (sample 1; s1). Skin samples were collected
using a disposable scalpel after shaving the hair over an
area of about 0.5 cm × 0.5 cm × 0.6 cm. Dermal sample was
soaked in saline solution for 10 min at 37 ◦ C <strong>and</strong> thereafter
removed <strong>and</strong> stored at −20 ◦ C. The sediment was observed
under light microscopy after adding a drop of methylene
blue (1%). Following the retrieval of motile microfilariae
(see Section 3), five other skin biopsies (s2–s5) were per-
Fig. 1. Subcutaneous nodule on the animal’s right thigh.
formed (i.e., from the left thigh, s2; from both right <strong>and</strong> left
temporal areas, s3 <strong>and</strong> s4; <strong>and</strong> from armpits, s5 <strong>and</strong> s6).
2.2. <strong>Morphological</strong> analysis
<strong>Morphological</strong> analysis was done with fixed microfilariae
cleared in lactophenol. The cover-slide was unsealed
in order to orient the microfilariae in dorso-ventral or
lateral views, as previously described (Bain et al., 1988;
Uni et al., 2001). Drawings were made with an optic
microscope equipped with a camera lucida <strong>and</strong> measurements
were made on drawings. Microscopic images
were acquired using a digital camera (Zeiss Axiocam MRc,
Carl Zeiss, Germany) mounted directly on the microscope
(Zeiss Axioscop 2 plus, Carl Zeiss, Germany). The software
AxioVision rel. 4.8 (Carl Zeiss, Germany) was used for
the image analysis process including measuring of larvae,
which are provided in micrometers. Slide-mounted microfilariae
were deposited in the collection of the Muséum
National d’Histoire Naturelle, Paris, France (MNHM), under
the accession numbers 194 YU <strong>and</strong> 284 YU.
2.3. Molecular amplification <strong>and</strong> phylogenetic analyses
The <strong>molecular</strong> identification was performed by extracting
genomic DNA from microfilariae isolated by the larval
sediment of s1, using a commercial kit (DNeasy Blood
& Tissue Kit, Qiagen, GmbH, Hilden, Germany) in accordance
with the manufacturer’s instructions. In addition, the
remaining skin saline-soaked sample (s1) was extracted
with the remaining samples (s2–s6) as above.
A cox1 (∼689 bp) <strong>and</strong> 12S (∼330 bp) gene fragments,
which are usually employed for barcoding of filarioids
(Ferri et al., 2009) were amplified. In particular, cox1 was
amplified by using filarioid-generic primers (Casiraghi
et al., 2004, 2006) whereas 12S was amplified by a set of
primers (Fila12SF: 5 ′ -CGGGAGTAAAGTTTTGTTTAAACCG-

3 ′ <strong>and</strong> Fila12SR: 5 ′ -CATTGACGGATGGTTTGTACCAC-3 ′ )
designed on the 12S conserved regions of Acanthocheilonema
spp., Cercopithifilaria spp., <strong>and</strong> Dirofilaria
spp. sequences available in GeneBank (Table 2). A third
fragment, the ITS-2 rDNA <strong>and</strong> flanking sequences of the
5.8S <strong>and</strong> 28S rRNA genes were amplified by using the
primers NC1 (forward 5 ′ -ACGTCTGGTTCAGGGTTGTT-3 ′ )
<strong>and</strong> NC2 (reverse 5 ′ -TTAGTTTCTTTTCCTCCGCT-3 ′ ) (Gasser
et al., 1993).
In order to assess a diagnostic <strong>molecular</strong> method
to differentiate A. reconditum <strong>and</strong> Cercopithifilaria
sp., two forward internal primers (i.e., ArCox1F:
5 ′ -ATCTTTGTTTATGGTGTATC-3 ′ <strong>and</strong> CbCox1F: 5 ′ -
CGGGTCTTTGTTGTTTTTATTGC-3 ′ ) were used to amplify a
partial cox1 (pcox1) of A. reconditum (589 bp) <strong>and</strong> Cercopithifilaria
sp. (304 bp), both coupled with reverse primer
described in Casiraghi et al. (2004). The pcox1 specific
primers were designed using the criteria of Sharrocks
(1994), on the basis of the cox1 complete sequences
obtained by the amplification using the filarioid-generic
primers <strong>and</strong> by the multiple alignments of sequences
available in GenBank TM . The amplification reaction of the
pcox1 consisted of 2 l genomic DNA <strong>and</strong> 23 l of PCR
mix containing 2.5 mM MgCl2, 10 mM Tris–HCl, pH 8.3
<strong>and</strong> 50 mM KCl, 250 M of each dNTP, 50 pmol of each
primer <strong>and</strong> 1.25 U of Ampli Taq Gold (Applied Biosystems)
using a thermal cycler (2720, Applied Biosystems). The
amplification reaction of pcox1 fragment was optimized by
a serial of amplification conditions yielding the following
best results: 95 ◦ C for 10 min (first polymerase activation
<strong>and</strong> denaturation), followed by 40 cycles of 95 ◦ C for 1 min
(denaturation); 58 ◦ C for 1 min (annealing), <strong>and</strong> 72 ◦ C for
1 min (extension) <strong>and</strong> a final extension of 72 ◦ C for 7 min.
Approximately 100 ng of genomic DNA were added to each
PCR. In addition, in order to test the specificity of both
primers in amplifying A. reconditum <strong>and</strong> Cercopithifilaria
sp. pcox1, all primer combinations were tested with D.
repens, D. immitis, A. reconditum <strong>and</strong> Cercopithifilaria sp.
here detected. DNA from R. sanguineus, blood <strong>and</strong> skin
samples from laboratory-reared beagles (see Otranto et al.,
2010) were used as negative controls <strong>and</strong>, along with a
no DNA sample, were included in each PCR run to test the
specificity of the reaction.
Amplicons were purified using Ultrafree-DA columns
(Amicon, Millipore; Bedford, USA) <strong>and</strong> then sequenced
directly using the Taq DyeDeoxyTerminator Cycle Sequencing
Kit (v.2, Applied Biosystems) in an automated
sequencer (ABI-PRISM 377). Sequences were determined
from both str<strong>and</strong>s (using the same primers individually as
for the PCR) <strong>and</strong> the electropherograms verified by eye.
D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229 223
In order to ensure open reading frames, all nucleotide
sequences of the cox1 fragment were conceptually translated
into amino acid sequences using the invertebrate
mitochondrial code by MEGA 4.0 (Tamura et al., 2007).
Sequences were aligned using ClustalW program (Larkin
et al., 2007) <strong>and</strong> compared with those available in Gen-
Bank <strong>data</strong>set by BLAST analysis. In order to investigate the
relationships among filarioids of the Onchocercidae family,
sequences of both genes were analyzed with those
available in GenBank TM . The evolutionary history was
inferred by MEGA 4.0, using cox1 <strong>and</strong> 12S sequences, under
Neighbor-Joining methods using 8000 replicates bootstrap
values. Thelazia callipaeda (Spirurida, Thelaziidae) was chosen
as an out-group. The nucleotide sequences analyzed
in this paper are available in the GenBank TM <strong>data</strong>base
JF461457, JF461461.1 <strong>and</strong> JF501396.
3. Results
Two types of microfilariae were found on the same
slide (MNHM, 284 YU) as well as in other slides prepared
from the sediment of skin samples soaked in saline solution.
Some dermal microfilariae presented morphological
characteristics compatible with Cercopithifilaria <strong>and</strong> the
others were indistinguishable from blood microfilariae of A.
reconditum. The morphological diagnostic characters were
compared with those available in the literature (Table 1)
(Fülleborn, 1912; Bernard <strong>and</strong> Bausche, 1913; Lent <strong>and</strong>
Freitas, 1937; Olmeda-Garcia <strong>and</strong> Rodriguez-Rodriguez,
1994), by comparing measurements <strong>and</strong> features with
those of the filarial nematodes most commonly retrieved in
dogs in Italy, namely, D. immitis, D. repens <strong>and</strong> A. reconditum
(Otranto <strong>and</strong> Dantas-Torres, 2010) <strong>and</strong> Acanthocheilonema
dracunculoides, with blood microfilariae, as well as C. grassii
<strong>and</strong> C. bainae with dermal microfilariae (Table 1). These
dermal microfilariae here detected were also clearly distinct
from the blood microfilariae of the above filarioid
species (Table 1).
Dermal microfilariae (n = 15) were short with a mean
length of 186.7 ± 3.9 m (182–190 m); the body was flattened
dorso-ventrally, 8.5–11 m but 3–3.5 wide in lateral
view; microfilariae mostly laid on the dorso-ventral side.
No sheath was present <strong>and</strong> the body cuticle was thick with
transverse striations, interrupted in lateral plane. A 30 m
long cephalic anterior part was slightly attenuated <strong>and</strong>
identified in dorso-ventral view only; the main part of body
presented constant width (Fig. 2). As for the arrangement of
body nuclei, on a transverse line, approximately 4–5 angular
nuclei were detectable in dorso-ventral view <strong>and</strong> one
rounded nucleus in lateral view. The head was rounded,
Table 1
Measurements (in micrometers) <strong>and</strong> morphological features of microfilariae of filarioids affecting dogs (references in the text).
Species Length Width Posterior end Cephalic hook
Acanthocheilonema reconditum 230–290 4.5 Filiform Prominent
Acanthocheilonema dracunculoides 246–258 4–6 Non filiform Sharp, extended
Dirofilaria immitis 218–273 5–7 Filiform Tiny
Dirofilaria repens 300–360 6–8 Filiform Tiny
Cercopithifilaria grassii 567–660 12.2–15.5 Non filiform ?
Cercopithifilaria bainae 185.18 6.59 Non filiform ?
Cercopithifilaria sp. (present study) 182–190 3–3.5 Non filiform Tiny

224 D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229
mouth <strong>and</strong> plain axis of anterior oesophagus were identified;
the cephalic space was short; on the left side, a slight
protuberance bearing the tiny cephalic hook was identified.
At 72 m from apex, the excretory cell (but not excretory
pore) was identified in dorso-ventral view (Fig. 2). The posterior
part of the body was conical, 30 m long; the anal
pore was not visible. The last caudal nucleus was elongated,
at 15 m to tip tail <strong>and</strong>, at its level, the tail became slightly
more attenuated in dorso-ventral view only; the tip tail was
blunt (Fig. 2).
The morphological differentiation between A. reconditum
<strong>and</strong> the dermal short microfilaria was confirmed by
nucleotide differences in cox1, 12S, <strong>and</strong> ITS-2 sequences
here examined <strong>and</strong> those available in GenBank TM <strong>data</strong>base.
The PCR amplification of each target gene from individual
DNA samples resulted in amplicons of the expected
size. No intraspecific differences were detected within each
species for each gene sequence examined. cox1 mitochondrial
sequences of the dermal microfilariae had a typical
base composition of AT content (62.1%) <strong>and</strong> a bias at the
third codon position to AT (69.8%) compared with the
first <strong>and</strong> second positions (58.1%). The conceptual translation
at second codon position of cox1 sequence led to
216 amino acids without stop codons. The mean inter-
specific aminoacidic difference ranged from 3.2% to 7.9%
in D. immitis vs. D. repens <strong>and</strong> in dermal microfilariae vs.
A. reconditum, respectively. By comparing the nucleotide
sequences of the most frequent species of filarioids affecting
dogs (i.e., A. reconditum, D. immitis, <strong>and</strong> D. repens) <strong>and</strong>
those of dermal microfilariae, the mean level of interspecific
pairwise (Pwc) distance (%) was of 12.9%, 13.4% <strong>and</strong>
61.9% for cox1, 12S <strong>and</strong> ITS-2, respectively. In particular,
cox1 mean interspecific difference ranged from 9.7% to
15.1% in D. immitis vs. D. repens <strong>and</strong> dermal microfilariae
vs. A. reconditum, respectively (Table 3). Analogously, 12S
mean interspecific difference ranged from 9.2% to 17.3% in
D. immitis vs. D. repens <strong>and</strong> dermal microfilariae vs. A. reconditum,
respectively (Table 3). The alignment of the ITS-2
sequences ranged from 390 bp in D. repens to 485 bp in A.
reconditum with interspecific difference ranged from 41.2%
to 69.3% in D. immitis vs. D. repens <strong>and</strong> A. reconditum vs. D.
repens, respectively (Table 3).
The BLAST analysis of the Cercopithifilaria sp. sequences
showed the highest nucleotide similarity (i.e., 86.5% <strong>and</strong>
93.6%) with the cox1 <strong>and</strong> 12S sequences of C. tumidicervicata
<strong>and</strong> C. roussilhoni available in GenBank TM . ITS-2
sequence did not display any significant similarity with any
of those available in GenBank TM . Analogously, the phyloge-
Table 2
GenBank accession numbers (AN) of the Onchocercidae <strong>and</strong> Thelazia callipaeda (outgroup) for the mitochondrial cytochrome c oxidase subunit 1 (cox1)
gene sequences used herein. Sequences obtained in this study are marked with an asterisk.
Species AN Host
Acanthocheilonema reconditum (Grassi, 1890) JF461456* Canis familiaris
Acanthocheilonema reconditum (Grassi, 1890) AJ544876 Canis familiaris
Acanthocheilonema viteae (Krepkogorskaya, 1933) AJ272117 Meriones libycus
Brugia malayi (Brug, 1927) AF538716 Homo sapiens
Brugia pahangi (Buckley & Edeson, 1956) AJ271611 Felis catus
Cercopithifilaria sp. JF461457* Canis familiaris
Cercopithifilaria bulboidea Uni & Bain, 2001 AM749247 Capricornis crispus
Cercopithifilaria crassa Uni, Bain & Takaoka, 2002 AM749260 Cervus nippon
Cercopithifilaria japonica Uni, 1983 AM749261 Ursus thibetanus
Cercopithifilaria longa Uni, Bain & Takaoka, 2002 AM749246 Cervus nippon
Cercopithifilaria minuta Uni & Bain, 2001 AM749253 Capricornis crispus
Cercopithifilaria multicauda Uni & Bain, 2001 AM749255 Capricornis crispus
Cercopithifilaria roussilhoni Bain, Petit & Chabaud, 1986 AM749264 Atherurus africanus
Cercopithifilaria shohoi (Uni, Suzuki & Katsumi, 1998) AM749251 Capricornis crispus
Cercopithifilaria tumidicervicata Uni & Bain, 2001 AM749259 Capricornis crispus
Dipetalonema gracile Rudolphi, 1809 AJ544877 Cebus olivaceus
Dirofilaria immitis Leidy, 1856 AJ271613 Canis familiaris
Dirofilaria immitis Leidy, 1856 AM749228 Canis familiaris
Dirofilaria immitis Leidy, 1856 AM749227 Felis catus
Dirofilaria immitis Leidy, 1856 AM749226 Felis catus
Dirofilaria repens Railliet & Henry, 1911 AM749230 Canis familiaris
Dirofilaria repens Railliet & Henry, 1911 AM749231 Felis catus
Dirofilaria repens Railliet & Henry, 1911 AM749232 Felis catus
Dirofilaria repens Railliet & Henry, 1911 JF461458* Homo sapiens
Dirofilaria repens Railliet & Henry, 1911 AM749234 Homo sapiens
Dirofilaria repens Railliet & Henry, 1911 AM749233 Homo sapiens
Foleyella furcata (Linstow, 1899) AJ544879 Chameleon
Litomosa westi (Gardner & Smith, 1986) AJ544871 Geomys bursarius
Litomosoides sigmodontis Ch<strong>and</strong>ler, 1931 AM749286 Sigmodon hispidus
Loxodontofilaria caprini Uni & Bain, 2006 AM749242 Capricornis crispus
Onchocerca gibboni Clel<strong>and</strong> & Johnston, 1910 AJ271616 Bos taurus
Onchocerca gutturosa Neumann, 1910 AJ271617 Bos taurus
Onchocerca ochengi Bwangamoi, 1969 AJ271618 Bos taurus
Onchocerca lupi Rodonaja, 1967 HQ207644 Homo sapiens
Onchocerca volvulus (Leuckart, 1893) AM749284 Homo sapiens
Setaria equina (Abildgaard, 1789) AJ544873 Equus caballus
Setaria labiatopapillosa (Aless<strong>and</strong>rini, 1848) AJ544872 Bos taurus
Thelazia callipaeda Railliet & Henry, 1910 AJ544882 Canis familiaris

Table 3
Level of interspecific pairwise (Pwc) distance (%) calculated among consensus sequences for cox1, 12S rDNA <strong>and</strong> ITS-2 for the most common species of filarioids affecting dogs (i.e., Acanthocheilonema reconditum,
A.r.; Dirofilaria immitis, D.i.; Dirofilaria repens, D.r.; Cercopithifilaria sp., C. sp.).
Pwc (%) cox1 12S rDNA ITS-2
C. sp.
D. r.
AY693808
D. i.
EU182331
A.r.
AF217801
C. sp.
JF461461
D.r.
JF461462
D.i.
FN391554
A.r.
JF461460
C. sp.
JF461457
D. r.
JF461458
D. i. FN391553
EU169124
A.r.
JF461456
A.r. – –
D.i. 13.1 – 13.5 – 59.2
D.r. 12.6 9.7 – 10.3 9.2 – 65.1 32.3
C.sp. 15.1 14.5 12.9 – 17.3 15.9 14.1 – 58.5 57.1 65.7 –
D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229 225
Fig. 2. Cercopithifilaria sp. microfilaria. (A) Dorso-ventral view. (B–D) Lateral
view of anterior end, mid region <strong>and</strong> posterior end, respectively.
(E–G) Dorso-ventral view of anterior end, mid region <strong>and</strong> posterior end,
respectively. Scale bars in m: (A) 50; others, 20.
netic analysis of the sequences of the dermal microfilariae
here examined <strong>and</strong> those available for onchocercid species
was concordant in clustering it with those of other Cercopithifilaria
spp. available in GenBank TM (Fig. 3). There was
consistency in the topology of the tree inferred by the NJ
(not shown) <strong>and</strong> ME methods (for both target genes). The
phylogenetic analysis of the cox1 sequence of the most
common species of filarioids available in GenBankTM <strong>and</strong>
on the Cercopithifilaria sp. here produced revealed the existence
of two main clades. In particular, Onchocerca spp.,
Setaria spp., <strong>and</strong> Brugia spp. were clustered all together
<strong>and</strong> differentiated by a second group including as Cercopithifilaria
spp. <strong>and</strong> Acanthocheilonema spp. These two
genera were grouped to the exclusion of D. immitis <strong>and</strong>
Litomosoides spp. (Fig. 3). The branches were overall well
differentiated <strong>and</strong> supported by high bootstrap values in
their main nodal points.

226 D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229
Fig. 3. Phylogeny of filarioid Onchocercidae based on cox1 gene sequences under Neighbor-Joining methods using 8000 replicates bootstrap values.
Numbers are GenBank accession numbers. The tree was rooted against Thelazia callipaeda (out-group).
Two different sized pcox1 amplicons were produced
for the dermal microfilariae (300 bp) <strong>and</strong> for A. reconditum
(∼590 bp). Both the PCR reactions with specific
primers amplified exclusively the dermal microfilariae or
A. reconditum, <strong>and</strong> did not amplify negative controls (i.e., R.
sanguineus, skin <strong>and</strong> blood from dogs <strong>and</strong> no DNA sample)
as well as the other species of filarioids tested (i.e., D. immitis
<strong>and</strong> D. repens). Blood sample was only PCR positive for A.
reconditum (Fig. 4). A. reconditum was amplified by specific
pcox1 primers in five out of the six skin samples (but not in
right armpit, s5) whereas all cutaneous samples were PCR
positive for the Cercopithifilaria sp.
4. Discussion
The dermal localization of the microfilariae in the dog
suggested that the species belonged to either Onchocerca
or Cercopithifilaria, genera that include filarioids already
reported in dogs. The gene sequencing <strong>and</strong> comparison
with the sequences available in <strong>data</strong>base (Ferri et al.,
2009) well support the morphological diagnosis at the
genus level as Cercopithifilaria. Indeed, in accordance with
those already suggested by Ferri et al. (2009), the phylogenetic
topology inferred by cox1 <strong>and</strong> 12S was efficacious
in resolving this species of Cercopithifilaria within the
Onchocercidae <strong>and</strong> within the clades of the genus Cercopithifilaria.
In addition, the morphological characteristics
of the microfilariae confirmed this generic identification,
particularly the dorso-ventrally flattened body <strong>and</strong> tiny
left cephalic hook which are common in the genus (Bain
et al., 1982b, 1987, 1988; Bartlett, 1983; Uni et al., 2001,
2002). Without any doubts, the microfilaria of the dog from
Sicily is not C. grassii that measures from 567 ± 12.25 m
(Noè, 1907) to 660 ± 15.5 m (Noè, 1911) in length. Among
the remaining 27 species of Cercopithifilaria, twenty have
distinctly shorter or longer microfilariae <strong>and</strong>, when stud-

D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229 227
Fig. 4. Results of specific PCR amplification of a partial mitochondrial cox1 (pcox1) specific for Acanthocheilonema reconditum (589 bp; lanes 2–15) <strong>and</strong>
Cercopithifilaria sp. (304 bp; lanes 17–30). Genomic DNA skin samples from right <strong>and</strong> left thigh, right <strong>and</strong> left temporal areas <strong>and</strong> right <strong>and</strong> left armpits
(lanes 2–7 <strong>and</strong> 17–22). A. reconditum (lanes 8, 23), Cercopithifilaria sp. (lanes 9, 24), D. immitis (lanes 10, 25), D. repens (lanes 11, 26), Rhipicephalus sanguineus
(lanes 12, 27), blood (lanes 13, 28) <strong>and</strong> skin samples (lanes 14, 29), no-DNA control (lanes 15, 30). Amplicons were sized by comparison with a 100-bp
ladder (Gene RulerTM, MBI Fermentas) (lanes 1, 16).
ied, very distinct gene sequences (C. crassa, C. japonica,
C. longa, C. roussilhoni, C. shohoi, <strong>and</strong> C. tumidicervicata;
Table 2). Among the seven species with microfilariae of
similar length than those examined the first three are distinct
for both sequences (Table 2, Fig. 3) <strong>and</strong> morphological
features (Uni et al., 2001). The microfilaria of C. multicauda
170 m long <strong>and</strong> 5–10 m wide, according to body orientation,
has a cephalic <strong>and</strong> a caudal subterminal constriction.
In C. minuta Uni & Bain, 2001, the microfilaria, 195 m long,
is less flattened (4 <strong>and</strong> 6 m wide, compared to 3–3.5 <strong>and</strong>
8.5–11 m), <strong>and</strong> the posterior extremity is shortly attenuated
with a bent point. In C. bulboidea Uni & Bain, 2001, the
microfilaria is slightly longer <strong>and</strong> thinner (190–208 long,
4–8 m wide), <strong>and</strong> the cephalic space is longer. For the last
species sequences are not available but only morphological
<strong>data</strong> (Bain et al., 1987; Böhm <strong>and</strong> Supperer, 1953; Fain <strong>and</strong>
Herin, 1955). In C. corneti Bain, Chabaud & Georges, 1987,
from an African viverrid, N<strong>and</strong>inia bilobata, the microfilaria
185–190 m long is less flattened (5.5–7 m wide).
Information on the morphological features of the microfilariae
of the species in the following is scant, there being
only a single value available for the body width <strong>and</strong> thus it
remains unknown whether their body is flattened or not.
The microfilaria of C. rugosicauda (Böhm & Supperer, 1953),
from the European roe deer Capreolus capreolus, is 212–222
long, 6–7 wide (Böhm <strong>and</strong> Supperer, 1953) or 195–205 m
long <strong>and</strong> 6–6.5 m wide (Winkhardt, 1980), moreover the
fourth posterior body part is attenuated (not the last 30 m,
as in the dog microfilaria). In C. ru<strong>and</strong>ae (Fain <strong>and</strong> Herin,
1955), a species from cattle in Africa, the microfilaria is
170–195 m long <strong>and</strong> 6 m wide (Fain & Herin, 1955);
it is improbable that the present material might be this
species. Finally, microfilariae of C. bainae are 185.18 m
<strong>and</strong> 6.59 m in length <strong>and</strong> width, respectively (Almeida
<strong>and</strong> Vicente, 1982), being similar in length to those of the
Sicilian dog.
Among onchocercids, microfilariae are useful to morphologically
differentiate species in absence of other
nematode stages <strong>and</strong> of <strong>molecular</strong> confirmatory results. In
accordance with the natural history of the genus Cercopithifilaria,
microfilariae (first stage larvae) were only detected
by soaking skin in saline solution. Adults of species of
Cercopithifilaria are easily overlooked in the subcutaneous
tissues due to their small size (e.g., in C. bainae: males are
from 7.28 mm to 9.10 mm long <strong>and</strong> 44 m wide; females
are from 13.6 mm to 17.85 mm long <strong>and</strong> 72–100 m wide)
<strong>and</strong> consequently some authors have relied upon the protocol
of soaking carcasses in warm saline soon after death
as proposed by Eberhard (1980). However, this may not
always be efficacious for the detection of either adults
or skin-inhabiting microfilariae. Xenodiagnosis, the detection
of larvae in their vectors, may be more useful in
detecting the developing stages of skin-inhabiting microfilariae.
Alternatively, a simple PCR protocol on dog skin,
as employed in this study, may provide valuable information
on the occurrence of adult or microfilariae of filarioid
species associated with the skin of dogs. Although the
actual adult parasitic load of the dog is unknown, the presence
of positive samples both by skin soaked sediment
examination (5 out of six samples were positive for microfilariae)
<strong>and</strong> by PCR (all samples were positive), might
indicate that these microfilariae are evenly distributed in
the subcutaneous tissue throughout the whole body of animals,
mainly in anatomical regions where R. sanguineus
ticks most likely attach for blood feeding (Lorusso et al.,
2010).
The microfilariae usually reported from dogs are those
found in blood samples <strong>and</strong> the picture of filarial nematodes
is reduced to Dirofilaria spp. <strong>and</strong> Acanthocheilonema
spp. However, in the past decade the presence of skin
microfilariae have been established with an Onchocerca
species (Sréter <strong>and</strong> Széll, 2008), O. lupi. This study provides
evidences that, in addition to the most frequent species
of filarioids infesting dogs, other species are present with
their microfilariae in the dermis of these animals. Evidences
for this are represented both by morphological <strong>and</strong> molec-

228 D. Otranto et al. / Veterinary Parasitology 182 (2011) 221– 229
ular <strong>data</strong> here presented. Thus, this study also emphasizes
that the skin is a site to be investigated towards screening
a complete panel of filarioids. The resemblance of the
presently studied microfilariae with C. bainae, if proven
with gene sequencing <strong>and</strong> detailed morphology of other
parasitic stages, might raise interesting question about the
origin of C. bainae, which might have been introduced from
the Palearctic region, with the importation of domestic
dogs by humans. Further studies on this nematode are
needed to elucidate its true identity, life cycle, <strong>and</strong> potential
pathogenic role for dogs.
Acknowledgements
Authors thank Aless<strong>and</strong>ro Fogliazza (Merial, Italy) <strong>and</strong>
Lénaïg Halos (Merial, Europe) for supporting this research.
It has also been partially supported by the European Community
grant INCO-CT-2006-032321 <strong>and</strong> by the MNHN
grant ATM: Taxonomie moléculaire: DNA barcode et gestion
des collections. Thanks are also to Dr. Yurii Kuzmin,
from Kiev, for editing the drawings.
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