The first feline and new canine cases of Thelazia callipaeda (Spirurida: Thelaziidae) infection in Hungary

Background In Europe, the first Thelazia callipaeda infections were found in the eyes of some dogs in Italy three decades ago. Since that time, this vector-borne nematode species has been diagnosed in domestic and wild carnivores and humans in some western European countries. During the last few years, autochthonous thelaziosis of dogs, red foxes, cats and humans has also been reported from eastern Europe. The first cases of ocular infections caused by T. callipaeda have been described in dogs living in the eastern and southern part of Slovakia and Hungary. Methods Whitish parasites found in the conjuctival sac and/or under the third eyelid of one or both eyes of animals were removed and morphologically identified according to species and sex. To confirm the morphological identification with molecular analysis a single step conventional PCR was carried out. Results A total of 116 adult worms (1–37 per dog, median: 7, IQR: 14.5 and 7 from a cat) were collected from the eyes of 11 animals. Nematodes were identified as T. callipaeda according to the morphological keys and molecular analysis. The sequences of a portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were identical to those representing T. callipaeda haplotype 1, previously reported in neighbouring and other European countries. Since the infected cat and dogs had never travelled abroad, all of the cases were autochthonous thelaziosis. Conclusions The present study reports the first case of thelaziosis in a cat and new cases in 10 dogs found in the southern and northern region of Hungary, respectively. Further studies are needed to clarify whether wild carnivores (e.g. red foxes, golden jackals) may act as reservoirs of this eyeworm species in the country.

The present paper reports the first autochthonous thelaziosis in a cat and new ocular worm infections of ten dogs caused by T. callipaeda found in the northern area of Hungary.

Sample collection
Between October 2015 and December 2017, 11 cases of ocular worm infection in a cat and 10 dogs were signalled by practitioners to the Department of Parasitology and Zoology, University of Veterinary Medicine, Budapest. The cat was a one-year-old male of the European shorthair breed. Affected dog breeds were German Shepherd, Rottweiler, Collie, Hanoverian Hound, Hungarian Vizsla and crossbreeds, aged from 0.8 to 14 years, mostly males except for one (Table 1). Each animal was born in Hungary. All animals examined spent most or all of their time outdoors. The cat and dogs' owners confirmed that their pets had never travelled abroad.

Clinical examination, parasite collection and treatment
On clinical examination after administration of a local anaesthetic, threadlike motile whitish parasites were observed in the conjunctival sac and/or under the third eyelid of one or both eyes of each animal. The worms were removed using sterile cotton swabs or fine forceps and the conjunctival sacs were thoroughly flushed with sterile physiological saline solution. The parasites were collected into 70% ethanol and sent to the Department of Parasitology and Zoology for morphological and molecular identification.
The dogs were treated with a spot-on application of 10% imidacloprid and 2.5% moxidectin (Advocate Spot-On; Bayer HealthCare, Leverkusen, Germany) or Milprazon tablets (milbemicin-oxim 2.5 mg and praziquantel 12.5 mg, KRKA, Novo mesto, Slovenia). The cat did not receive any anti-parasitic treatment.

Morphological and molecular identification of the worms
Nematodes collected from the eyes of each animal were morphologically identified according to species and sex under a light microscopy combined with a camera (EURmex HD II with IS Capture software) using the previous descriptions [29]. To confirm the morphological identification with molecular analysis a single step conventional PCR was carried out. Genomic DNA was extracted from one or more specimen of worms from each animal, with the NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer's instructions. An approximately 689 bp partial fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified with the primer pair NTF (5'-TGA TTG GTG GTT TTG GTA A-3') and NTR (5'-ATA AGT ACG AGT ATC AAT ATC-3') [30].

Results
Nine out of ten infected dogs were kept in five settlements of Borsod-Abaúj-Zemplén county and one animal in Budapest (Fig. 1). Except for two dogs, the others were taken by the owners many times for walking to Bükk National Park located in the north-eastern part of Hungary.
For the cat and all dogs, conjunctival hyperaemia and ocular purulent discharge due to conjunctivitis were observed in one or both eyes. The third eyelid protrusion occurred in one dog. The ophthalmological examination Fig. 1 Thelazia callipaeda found in a cat (circle), dogs (squares) and red foxes (triangles) in Hungary and close to its borders with neighbouring countries revealed the presence of motile nematodes on the eye surface, in the conjunctival sac and/or under the third eyelids of one or both eyes. A total of 116 adult worms (1-37 per dog, median: 7, IQR: 14.5 and 7 from a cat) were collected from 11 infected animals (Table 1). All specimens recovered were morphologically identified as T. callipaeda adults. The eyes of four and six dogs were infected with females and parasites of both sexes, respectively (Table 1). Four weeks after removing the worms and starting treatment, the infection had resolved in each animal. Their eyes appeared normal, and no parasites were observed.

Discussion
In Europe, the first documented T. callipaeda infections were found in the eyes of some dogs in Italy three decades ago [2]. In the following decades our knowledge about the distribution of this vector-borne eyeworm species in Europe has greatly expanded. Autochthonous and/or imported thelaziosis affecting domestic and/or wild carnivores has been reported in Germany [16], France [17], Portugal [31][32][33], Switzerland [18,34], Spain [19] and Italy [3]. Thelazia callipaeda infection has also been found in wild European rabbits [5]. The human ocular thelaziosis reported from Italy and Spain [6,20] confirms the medical importance of this nematode species in Europe. The occurrence of this spirurid nematode species has also been reported in Eastern Europe. The adult worms have been found in one or both eyes of dogs, cats, foxes and/or humans in Croatia [7,21], Serbia [8,22,35], Romania [23,24], Bosnia and Herzegovina [21], Bulgaria [25] and Slovakia [26,27]. Before this study, only a single canine ocular thelaziosis was reported in Hungary [28]. In January 2014, a ten-year-old, male poodle dog with unilateral chronic conjunctivitis and epiphora was presented to a veterinary practice in Pécs, Baranya county, located in the southern part of the country (Fig. 1). The worms were identified as T. callipaeda both morphologically and molecularly [25,28]. However, there has been no explanation of how this dog acquired this eyeworm infection. A year later, in October 2015, seven adult worms were removed from the right conjunctival sac of a one-year-old male cat in Mohács (Fig. 1). This town is 36 km away from Pécs where the infected dog was examined. Ocular worm infections caused by T. callipaeda were found in nine dogs originating from five settlements (Table 1 and Fig. 1) of Borsod-Abaúj-Zemplén county. The specimens of this spirurid worm species were found in both eyes of a dog kept in Budapest at the end of 2017. The cat and all the dogs had autochthonous thelaziosis because they had no history of travelling abroad. The infections presented here confirm the parasite's establishment in two areas of the country. Like in other European countries [12,19,32] most Hungarian cases have been diagnosed in late summer and autumn relating to the maximum activity of the vector [15,36]. The dogs found to be infected with T. callipaeda adults in December became infected in summer or earlier because the third stage larvae released by flies in the spring and/or late summer will reach the adult stage many weeks later [9]. It has been experimentally demonstrated that T. callipaeda survives at least nine months in definitive hosts [1,12].
Considering the sporadic cases of feline thelaziosis reported in Europe [17,21,22,32,35] (except for Switzerland [34]) it is not surprising that only one cat was found in Hungary with a confirmed T. callipaeda infection. Scientists related the occurrence of rare feline eyeworm infections with the intensive cleaning habits of cats resulting in less contact with the vectors [34]. On the other hand, difficulties in inspecting cats' eyes by practitioners are also assumed [37]. According to the former dipterological studies, Phortica variegata, the vector species of T. callipaeda occurs in Hungary [38,39] where it finds suitable habitat predicted earlier by a geoclimatic model [15]. It is unknown how long T. callipaeda has been present in Hungary. This vector-borne nematode species was probably imported to the country in the last decade. It is unlikely that the parasites arrived from abroad with vectors because fruit flies are not good flyers, and they are not known to disperse by wind [40]. Considering the first cases of canine thelaziosis found close to the southern and northern borders of Hungary, we assume that T. callipaeda may have been introduced recently or arrived in the country several years ago by infected host(s) from neighbouring countries where the occurrence of this nematode species has been reported [21,22,26,27]. Thelazia callipaeda seems more likely to be transmitted to Hungary by wild carnivores, which play an important role in the sylvatic cycle of this eyeworm species [3,21,24,37]. It has been reported that the specimens of this nematode were found in the eyes of red foxes in the northern regions of Balkans [21] and in the southern part of Slovakia close to the Hungarian border [27] (Fig. 1). Another possible explanation is also plausible. Thelazia callipaeda might have arrived in Hungary by infected dogs, such as hunting dogs that spent time in a thelaziosis-endemic area. Although there have been some hypotheses of how this eyeworm species could have arrived in the country, the origin and spreading of T. callipaeda remains unclear because the haplotype 1 reported in the neighbouring [22][23][24] and other [19,21,[31][32][33] countries has also been found in this study. Our findings also confirm the low genetic variability of this parasite species in Europe [30].
Based on the results of this study, the most urgent need is for improving the knowledge of the local veterinarians who have been largely unaware of this eyeworm infection of dogs and cats. Besides preventative measures, correct diagnosis and appropriate treatment of infected dogs and cats, the veterinarians are also responsible for preventing this parasite infection of pet owners with education. Due to the potential risk of human infestation, physicians and medical ophthalmologists must be informed about the occurrence of this zoonotic parasite in the endemic regions. To date, we have no information about T. callipaeda infection of wild canids in Hungary. Therefore, research is needed to clarify whether red foxes and golden jackals act as reservoirs and spreaders of this eyeworm species.

Conclusions
To the best of our knowledge, this is the first report on the detection of T. callipaeda in a cat in Hungary. After reporting the first case of thelaziosis in a dog living in the southern part of the country, ten autochthonous canine thelaziosis have been found in the northern region of Hungary. Further studies are needed to clarify whether wild carnivores (e.g. red foxes, golden jackals) may act as reservoirs of the parasite species in the country.