This document summarizes an article published in an Elsevier journal about the first reported cases of eye worm infection (Thelazia callipaeda) in dogs and a cat in France. Nematodes were recovered from infected animals and identified as T. callipaeda both morphologically and through DNA sequencing, which matched the haplotype 1 sequence previously identified in Europe. This represents the first reported autochthonous (locally acquired) cases of this parasite in dogs and cats in France, rather than cases in animals that had traveled to infected areas.

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Rapid communication
First reports of autochthonous eyeworm infection by Thelazia
callipaeda (Spirurida, Thelaziidae) in dogs and cat from France
Ph. Dorchies a,*, G. Chaudieu b
, L.A. Sime´on c
, G. Cazalot a
,
C. Cantacessi d
, D. Otranto d
a
Ecole ve´te´rinaire de Toulouse, 23 chemin des Capelles, 31076 Toulouse Cedex, France
b
2 place Beaulieu, 63400 Chamalie`res, France
c
247 route d’Angoule`me, 24000 Pe´rigueux, France
d
Department of Animal Health and Welfare, Faculty of Veterinary Medicine, University of Bari, Italy
Received 27 June 2007; received in revised form 23 July 2007; accepted 1 August 2007
Abstract
Thelazia callipaeda (Spirurida, Thelaziidae) is a small nematode living in the conjunctival sac of domestic and wild carnivores,
rabbits and humans causing lacrimation, epiphora, conjunctivitis, keratitis and even corneal ulcers. The first autochthonous cases of
thelaziosis affecting four dogs and one cat living in South Western France (Dordogne area) are reported and described. Nematodes
recovered from the animals were morphologically identified as T. callipaeda and a partial region of the cytochrome oxidase c
subunit 1 gene (cox1) was amplified by PCR from nematode specimens (from two dogs and the cat). In each case, this was shown to
have an identical sequence to the haplotype 1 (h1) of T. callipaeda. So far, the arthropod acting as intermediate host of T. callipaeda
eyeworms has not been identified in France although it might be Phortica variegata (Steganinae, Drosophilidae) as recently
described in Italy.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Thelazia callipaeda; Phortica variegata; Nematode; Dog; Cat; France; Europe
1. Introduction
Thelaziacallipaeda(Spirurida,Thelaziidae)isa small
nematode living in the conjunctival sac of domestic (i.e.,
dogs and cats) and wild carnivores (i.e., foxes and
wolves), rabbits and humans causing lacrimation,
epiphora, conjunctivitis, keratitis and even corneal ulcers
(reviewed in Anderson, 2000; Otranto et al., 2007). It has
been recently demonstrated, both under laboratory
(Otranto et al., 2005a) and natural conditions (Otranto
et al., 2006b) that in Italy this infection is transmitted by
Phortica variegata (Steganinae, Drosophilidae) feeding
on the lacrimal secretions of the definitive hosts. The
distribution of this nematode, commonly known as
‘‘oriental eyeworm’’ has been considered for a long time
to be confined to the former soviet republics and Asia
(reviewed by Anderson, 2000) where it causes infections
in humans, dogs and cats (reviewed by Shen et al., 2006).
However, in the past decade thelaziosis caused by T.
callipaeda has been demonstrated to be widespread
among dogs and cats from northern to southern Italy with
infection prevalence up to 60.14% in dogs from some
municipalities of southern Italy (Otranto et al., 2003).
Cases of thelaziosis have also been registered sporadi-
cally in France (Chermette et al., 2004) and Germany
(Hermosilla et al., 2004) in dogs which had spent some
time, during summer, in northern Italy.
www.elsevier.com/locate/vetpar
Veterinary Parasitology 149 (2007) 294–297
* Corresponding author. Tel.: +33 5 61 19 38 71;
fax: +33 5 61 19 39 44.
E-mail address: p.dorchies@envt.fr (P. Dorchies).
0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2007.08.005

3. Author's personal copy
A molecular investigation employing a mitochon-
drial DNA marker (Otranto et al., 2005b) demonstrated
that, despite a relatively high degree of genetic
variability among T. callipaeda isolates from Asia,
no genetic variation occurs between individual nema-
todes collected from different host species (i.e., dogs,
cats and foxes) and localities within Europe (i.e., Italy,
Germany and the Netherlands). This finding suggested a
strict affiliation of this nematode to the intermediate
host rather than to the definitive hosts and that the
distribution of the parasite would be expected to
coincide with that of the vector (Otranto et al., 2005b).
In a recent work the ecological niche model suitable for
P. variegata flies was predicted both for Italy and
Europe, and the most suitable areas have been found to
be concentrated in central Europe, particularly in
France (Otranto et al., 2006a).
The present paper reports the first French auto-
chthonous cases of thelaziosis caused by T. callipaeda
in four dogs and one cat. Nematode larvae from
these cases were morphologically and molecularly
identified.
2. Materials and methods
2.1. Case reports
All the animals referred and described below had
spent their lives in the Dordogne region, or had spent
some time there during holidays, and they had never
travelled abroad. Most of the cases came from a
strawberry production area around the village of Vergt
(latitude: 458010
4100
N; longitude: 08430
0900
E). This
area is close to the Atlantic Ocean and it is included
in Aquitaine (latitude: 44–458N and longitude close
to 08). Its altitude ranges from 112 to 246 mt above
sea level and it has an oceanic climate with an
average of 800 mm annual rainfall. The vegetation is
mixed.
Since March 2006, four dogs referred to veterinary
practitioners with unilateral conjunctivitis were found
on clinical examination to have adult nematodes in the
conjunctival sac. In September 2006, a similar finding
was made in the conjunctival sac of a 2-year-old male
adult farm cat (Brown Tabby European) suffering
from unilateral conjunctivitis (left eye) with severe
chemosis and a wound on the 3rd eyelid (without
corneal ulcer).
After removal of the worms by flushing with saline
solution, all dogs and the cat were treated topically and
received milbemycin per os (Milbemax1
) as per label
recommendations. No recidives were registered.
2.2. Morphological and molecular identification
Nematodes collected from the eyes of the above
animals were stored in 70% ethanol and sent to the
Parasitological Unit of the Faculty of Veterinary
Medicine (University of Bari, Italy) for the morpho-
logical and molecular identification.
All nematodes collected were identified according the
keys of Skrjabin et al. (1967) and Otranto et al. (2004). To
confirm the morphological identification and to analyse
the haplotype sequence, a total number offour specimens
(from two dogs and the cat) were molecularly processed
viathespecificamplificationbyPCRofapartialsequence
ofthemitochondrialcytochromecoxidasesubunit1gene
(cox1—689 bp) as previously described (Otranto et al.,
2005b). Briefly, genomic DNA from single nematodes
was isolated using the QIAamp Tissue Kit (Qiagen
GmbH, Germany) and eluted in 50 ml H2O. A partial
sequence of the mitochondrial cox1 was amplified using
primers NTF (50
-TGATTGGTGGTTTTGGTAA-30
) and
NTR(50
-GATATTGATACTCGTACTTAT-30
)(Casiraghi
et al., 2001). Genomic DNA (4 ml) was added to the PCR
reaction mix (46 ml) containing 2.5 mM MgCl2, 10 mM
Tris–HCl, pH 8.3 and 50 mM KCl, 250 mM of each
dNTP, 50 pmol of each primer and 1.25 U of Ampli Taq
Gold (Applied Biosystems). PCR was performed in a
Applied Biosystems 2700 thermal cycler using the
following cycling protocol: 94 8C for 12 min (polymer-
ase activation), followed by 40 cycles of 95 8C for 1 min
(denaturation); 48 8C for 1 min (annealing); 72 8C for
1 min (extension), followed by 7 min at 72 8C (final exte-
nsion). Negative and positive control reactions were also
carried out by substituting the Thelazia DNA template
with sterile water and T. callipaeda DNA, respectively.
Amplicons were purified using Ultrafree-DA columns
(Amicon, Millipore; Bedford, MA, USA) and sequenced
inaABI-PRISM377usingtheTaqDyeDeoxyTerminator
Cycle Sequencing Kit (Applied Biosystems).
Sequences were determined in both directions (using
the same primers individually as for the PCR) and the
electro-pherograms verified by eye. Sequences were
aligned using the ClustalX program (Thompson et al.,
1997). The alignments were verified by eye and
compared with the sequences available for the cox1 of
T. callipaeda (Gen Bank accession nos. AM042549,
AM04550,AM04551,AM04552,AM04553,AM04554,
AM04555, AM04556; Otranto et al., 2005b).
3. Results and discussion
Five worms from dogs and one adult female from a
cat have been morphologically identified as three male
P. Dorchies et al. / Veterinary Parasitology 149 (2007) 294–297 295

4. Author's personal copy
and three female T. callipaeda. The cox1 sequences
obtained were identical to the sequence representing
haplotype 1 (i.e., h1) (available in Gen Bank accession
no. AM042549, see Otranto et al., 2005b). The clinical
cases here described represent the first reports of
autochthonous thelaziosis by T. callipaeda affecting
French dogs and cats which had never travelled abroad.
Previously Chermette et al. (2004) had described cases
of canine thelaziosis in France, but this was in animals
that had spent time during the summer in northern Italy
where, the authors argued, infection by T. callipaeda
eyeworms had occurred. The occurrence of adult T.
callipaeda specimens on the conjunctiva of dogs and
cats implies that third stage larvae, released by the
insect intermediate host, had developed to adult
nematodes in the ocular cavity, thus suggesting the
presence of the arthropod vector in the area that
infection occurred. In a paper dealing with the
molecular delineation of isolates of T. callipaeda from
different European and Asiatic localities, Otranto et al.
(2005b) recorded an infected dog from the Netherlands
that had earlier spent 3 months in the Dordogne region
of southern France. The above report, along with the
autochthonous cases here described, lend credit to the
hypothesis that T. callipaeda is present in the Dordogne.
This region is at the same latitude as Piedmont in
northern Italy where canine thelaziosis has been
previously reported (Rossi and Bertaglia, 1989). It also
fits into the putative areas in Europe in which P.
variegata flies (which act as vectors for T. callipaeda
eyeworms in southern Italy) could be endemic based on
a predictive geoclimatic model (Otranto et al., 2006a).
In addition, the natural environment in which P.
variegata flies have been collected in Italy matches
to some extent with that of Dordogne region.
Interestingly, all autochthonous cases of thelaziosis
here reported came from an environment close to
strawberry farms where the intermediate host P.
variegata, a drosophilid fruitfly, may develop (Otranto
et al., 2006b). It has been reported that male P. variegata
feed on tears and other animal secretions, while females
grow and develop on fruit and other vegetable matter,
thus making the environment in which T. callipaeda
infections have been reported, highly suitable for the
vector. However, further studies are needed in order to
identify the species of arthropod acting as intermediate
host in France.
The results of the present work indicate that T.
callipaeda infection is present in France and that h1 is
the only haplotype found in Europe. This raises
questions about the origin of thelaziosis in France. It
has been recently argued that wild fauna (i.e., foxes and
wolves) play a major role in maintaining and spreading
this nematode disease among pets in rural areas
(Otranto et al., 2007). Considering the characteristics
of the environment in which T. callipaeda has been
found in France, the possibility cannot be excluded that
canine thelaziosis has been introduced into this region
by wild animals moving from northern Italy, from
where fox thelaziosis has been previously reported
(Rossi et al., 2002). Therefore, the autochthonous
population of red foxes, which are spread across the
whole Dordogne region (reviewed by Aubert, 1999),
may have contributed to the establishment of the
infection by T. callipaeda in pets from that area. Despite
the high number of cases of T. callipaeda infection
registered across all Europe (Otranto et al., 2003;
Chermette et al., 2004; Hermosilla et al., 2004), no
human case has yet been reported. This may be because
a poor awareness of the zoonotic potential of this
parasite among physicians across Europe could lead to
underdiagnosis of the human disease, which is easily
confused with bacterial and viral conjunctivitis
(reviewed by Shen et al., 2006).
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