New Cases of Thelazia callipaeda Haplotype 1 in Dogs Suggest a Wider Distribution in Romania

Introduction

T helazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic helminth infecting at both adult and larval stages the conjunctival sac and associated tissues of a broad spectrum of definitive hosts, such as dogs, cats, rabbits, wild carnivores, and humans (Anderson 2000, Otranto et al. 2009). The adult and preadult parasites can cause injuries to the conjunctival and corneal epithelium, causing mild (e.g., ocular discharge, epiphora, and conjunctivitis) to severe (e.g., keratitis and corneal ulcers) ocular manifestations in animals (Otranto and Traversa 2005), as well as in humans (Shen et al. 2006).

In Europe, T. callipaeda is transmitted by the zoophilic drosophilid Phortica variegata (Diptera, Drosophilidae, and Steganinae), a small secretophagous, nonbiting fly that feeds on fruits and vegetables, but also on lacrimal secretions of various mammals (Otranto et al. 2005). As an original model of vector-borne diseases, males of P. variegata feed on lacrimal secretions of the definitive host, ingesting first-stage larvae. They develop in the insect's testes into infective third-stage larvae during summer. Late-stage larvae migrate to the proboscis before the final host infestation and are released into the conjunctival sac of a receptive host. The viviparous adult worms can also be found feeding on lacrimal secretions under the eyelids and nictitating membrane, at the conjunctival surfaces, in the conjunctival sac, and in the nasolacrimal ducts (Otranto et al. 2006b).

Owing to its wide geographical distribution in many far eastern countries including India, Thailand, Indonesia, China, Japan, and former Soviet Union, this nematode has been known as the “oriental eye-worm.” However, there are many relatively recent evidences that T. callipaeda infection is spreading, as it is being reported in increasing numbers in several European countries (Otranto et al. 2013, Mihalca et al. 2015). Over the past 10 years, T. callipaeda has been accounted as an emergent vector-borne helminth of animals and humans in several European regions (Colwell et al. 2011). Therefore, after the first report in Italy (Rossi and Bertaglia 1989), T. callipaeda has been reported in many European countries, that is, France (Dorchies et al. 2007, Ruytoor et al. 2010), Switzerland (Malacrida et al. 2008), Germany (Hermosilla et al. 2004, Magnis et al. 2010), Spain (Miró et al. 2011), Portugal (Maia et al. 2014), the Balkans (Bosnia and Herzegovina, Croatia and Serbia) (Gajić et al. 2014, Hodžić et al. 2014), Romania (Mihalca et al. 2015), and Greece (Diakou et al. 2015). Cases of human thelaziosis have also been recently diagnosed in northwestern Italy, southeastern France, and Spain (Otranto and Dutto 2008, Fuentes et al. 2012). All these records and the very clear clinical picture are suggestive of a fast spread of this zoonotic nematode in Europe and the distribution range is expected to increase rapidly in the coming years.

Romania is a relatively large EU country, with heterogeneous climatic and geographic conditions. However, so far the only published record of T. callipaeda is at the western border of the country, in a lowland area. The aim of this study was to document the more extensive distribution of this nematode and to provide additional molecular data on the haplotype of T. callipaeda present in Romania.

Materials and Methods

Results

A total of 22 adult nematodes (8 males and 14 females) were collected from the two dogs (Table 1). Light microscopy examination of the nematodes revealed specific features of T. callipaeda in all specimens: transversally serrated cuticle; presence of a buccal capsule and mouth opening with a hexagonal profile; presence of five pairs of postcloacal papillae in the ventral position in male; position of the vulva, located anterior to the esophagus–intestinal junction in female.

The BLAST analysis of our sequences (GenBank^® accession nos. KT716012, KT716013) showed a 100% similarity to a sequence (GenBank accession no. AM042549) of T. callipaeda haplotype h1.

Discussion

Over the past two decades, canine and feline thelaziosis caused by T. callipaeda eye worm has gained the attention of the veterinary community because of the spread of this ocular infestation in areas previously regarded as nonendemic (Otranto et al. 2013). In this study, two new clinical cases of ocular infection of dogs caused by T. callipaeda were confirmed, extending the known distribution range of this nematode in Romania to almost 300 km southeast. According to the Genetic Algorithm for Rule-Set Prediction model (Otranto et al. 2006a), both originating sites of infested dogs in Romania are also environmentally suitable for the development of P. variegata, the only confirmed vector of T. callipaeda in Europe (Otranto et al. 2005, 2006b). Moreover, detection of the eyeworm in dogs that had never traveled abroad indicates the spread of autochthonous canine thelaziosis in Romania, as it was previously recorded by Mihalca et al. (2015).

There are several factors that can explain the geographical expansion of T. callipaeda in previously nonendemic countries of Europe. Wild carnivores (especially red foxes), which are suitable hosts for this parasite and may easily move in neighboring regions, may have a role to the dispersal of the infection (Hodžić et al. 2014). Such is the case of Serbia, where the emergence and establishment of endemicity of the parasite have been associated with wildlife (Gajić et al. 2014). In contrast, the increasing number of cases of canine thelaziosis in Europe may also be a consequence of the increased mobility of dogs and the absence of applying prophylactic measures in carnivores (Mota et al. 2012). In addition, the growing awareness of parasitologists and veterinary practitioners has most likely also contributed to the increased number of reports of thelaziosis by T. callipaeda in Europe (Otranto et al. 2013).

In both animals originating from two different localities, the haplotype 1 of T. callipaeda was detected. This confirms the known low genetic variability of this nematode in Europe and indicates the wide circulation of a single haplotype in Europe, in contrast to seven haplotypes in Asia and a likely coevolution between the helminth and its European vector (Otranto et al. 2005).

Our current and previous (Mihalca et al. 2015) epidemiological data and molecular findings support the hypothesis that the infection has been introduced into Romania recently, most likely by dogs traveling from southern and western Europe, where thelaziosis is endemic. This is further supported by social factors, as many Romanian families work in Italy and Spain and travel with their dogs for summer holidays to their home localities in Romania. In some of these regions, T. callipaeda might have found appropriate conditions to complete its life cycle, favored by the presence of both arthropod vector and definitive hosts.

Based on the current knowledge, where the infection is endemic in domestic (dogs and cats) and wild carnivores (foxes, beech martens, and wild cats), cases of human thelaziosis may occur. Such situations are reported in France, Italy, and Spain (Otranto and Dutto 2008, Fuentes et al. 2012), indicating that infected animals act as reservoirs for the human infection (Otranto et al. 2013). Therefore, the expansion of this parasite in Romania, as well as in other European countries, has the potential in the near future to pose increased infection risks for both animals and humans living in areas with appropriate ecological conditions for T. callipaeda.

Conclusion

This study describes the occurrence of new autochthonous cases of thelaziosis in dogs in western and central Romania, reinforcing the spreading trend of this zoonotic eyeworm and highlighting the need for increased awareness among medical and veterinary practitioners. Moreover, we provide additional molecular evidence for the wide and exclusive distribution of haplotype 1 of T. callipaeda in Europe.