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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. Simeon c, G. Cazalot a,C. Cantacessi d, D. Otranto d

a Ecole veterinaire de Toulouse, 23 chemin des Capelles, 31076 Toulouse Cedex, Franceb 2 place Beaulieu, 63400 Chamalieres, France

c 247 route d’Angouleme, 24000 Perigueux, Franced 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

Thelazia callipaeda (Spirurida, Thelaziidae) is a 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

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: 4580104100N; longitude: 084300900E). 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 of four specimens

(from two dogs and the cat) were molecularly processed

via the specific amplificationbyPCRofa partial sequence

of the mitochondrial cytochrome c oxidase subunit 1 gene

(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

in a ABI-PRISM377 using the Taq DyeDeoxyTerminator

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

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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