First report of Thelazia callipaeda infection in wild European rabbits (Oryctolagus cuniculus) in Portugal
© Gama et al. 2016
Received: 15 March 2016
Accepted: 21 April 2016
Published: 10 May 2016
Thelazia callipaeda is a zoonotic nematode that affects the eyes of domestic and wild animals, including dogs, cats and red foxes. This parasitic eye worm is transmitted by Phortica variegata, which is a zoophilic fruit fly spread in Europe. Two wild European rabbits (Oryctolagus cuniculus) found dead in north-eastern Portugal were submitted to necropsy.
Both animals presented gross lesions compatible with haemorrhagic viral disease. Eye examination revealed the presence of six worms (three in each animal, on both eyes). Out of the six nematodes, five females and one male were morphologically and molecularly identified as T. callipaeda.
This is the first report of T. callipaeda in wild rabbits from Portugal, which reveals a new host for this parasite in southern Europe and emphasizes the importance of including thelaziosis in the differential diagnosis of ocular alterations in both animals and humans from areas where the eye worm is endemic.
KeywordsThelazia callipaeda Eye worm Oryctolagus cuniculus European rabbit Portugal Wild Zoonosis cox 1
Thelazia callipaeda (Spirurida, Thelaziidae), also known as the “Oriental eye worm”, is a zoonotic nematode first described in East Asia and in the eastern end of the former Soviet Union . Originally recognized in Far Eastern countries, it is now acknowledged that the parasite is also widespread in western European countries . Following its description in dogs, cats and foxes in Italy , T. callipaeda has been reported in either domestic or wild hosts from France [4, 5], Switzerland , Spain , Bosnia and Herzegovina, Croatia  and Romania .
Thelazia callipaeda is transmitted by Phortica variegata (Diptera, Drosophilidae, Steganinae), a drosophilid insect that feeds on lachrymal secretions of mammals. The eye worm is larviparous and transmitted exclusively by those secretophagous flies that, during daytime and after landing on the eyes, release the infective larvae on the host’s conjunctiva [10–12]. Due to the presence of this nematode in the conjunctival sac, infection may lead to ocular manifestations ranging from mild conjunctivitis, epiphora and ocular discharge to severe keratitis and corneal ulcers in animals and humans [13, 14].
Infection in wild carnivores, including red foxes (Vulpes vulpes), beech martens (Martes foina), wolves (Canis lupus) and wildcats (Felis silvestris), may play a role in the geographical distribution of this nematode, by maintaining and spreading the parasite into previously non-endemic countries and regions of Europe [15, 16].
In Portugal, cases of infection have been described in domestic animals (i.e. dogs and cats) and red foxes from inland areas of the north and central regions of the country [17–22]. To the best of our knowledge, no human cases have been reported in Portugal, though they were reported in western Spain near the Portuguese-Spanish border . In spite of the fact that leporids may be potential hosts for this parasite , reported cases of T. callipaeda infection in those vertebrate hosts in Europe are scarce, with just one reference of infection in brown hares (Lepus europaeus) . Here we described the first cases of T. callipaeda infection in wild European rabbits (Oryctolagus cuniculus) in Portugal.
Two wild rabbits (one female and one male) found dead at Vilar de Ossos, in the municipality of Vinhais, north-eastern Portugal (41°88′25.40″ N, -07°02′18.62″ W, and 41°86′14.06″ N, -07°01′96.44″ W), were sent frozen by the local authorities to the Laboratory of Histology and Pathology of UTAD, Vila Real, in order to have their cause of death determined.
Morphological identification of worms was done according to the keys proposed by Otranto et al. . Briefly, T. callipaeda females have a vulva anterior to the oesophagus-intestinal junction, whereas males possess five pairs of postcloacal papillae. Collected worms were soaked in tap water for 24 h and then stored in 70 % ethanol.
Four nematodes (three females and one male) were subjected to specific PCR amplification of a portion (689 bp) of the cytochrome c oxidase subunit 1 (cox1) gene . Amplicon sequences were determined in both directions (using the same primers individually as for the PCR) by visual inspection of the individual electropherograms. Sequences were aligned using the ClustalW program  and then compared with those available in public databases by BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The morphological identification was confirmed by molecular analysis. A representative sequence (GenBank accession no. KX033489) obtained from all the nematodes examined was identical to T. callipaeda haplotype 1 (GenBank accession no. AM042549).
This is the first report of T. callipaeda infection in wild rabbits in Portugal. To our knowledge, only Skrjabin et al.  (cited by Otranto et al. ) described Thelazia infection in rabbits. Otranto et al.  investigated the occurrence of this infection in wildlife species from Italy and reported for the first time T. callipaeda in brown hares (L. europaeus), a species that belongs to the same family (Leporidae) of the European rabbit.
This report of T. callipaeda on the eyes of wild European rabbits indicates this species as a novel recognized host for this nematode in southern Europe. Thus, it confirms and amplifies the wide range of definitive hosts of T. callipaeda, as previously stated by other authors . Although their population sizes are not accurately ascertainable at the regional and local levels (N. Fernandes, personal communication), wild rabbits are fairly common animals across the country in Portugal. In the near future, it will be important to investigate the prevalence of this infection in wildlife in Portugal at the population level, by more thoroughly assessing wild rabbits and red foxes , which have been found infected and could play a role in the dissemination of T. callipaeda in this country .
The infected wild rabbits described in this study were found in a geographical area which is environmentally comparable to other European areas where thelaziosis is currently considered as endemic, such as Spain , France  and Italy [16, 28]. Ocular T. callipaeda infection has also recently been described in dogs from the same municipality (Vinhais) and other contiguous municipalities (Chaves and Bragança) . All these findings indicate that this region provides suitable habitats for the vector P. variegata . However, further studies are necessary to investigate the ecology of P. variegata in northern Portugal.
Although no cases of human thelaziosis have so far been reported in Portugal, an increasing number of case reports in domestic and wild animals suggests the likely risk also for humans, especially in the north and central regions of Portugal, where several animal cases of infection have been described [17–22]. Thelazia callipaeda infection may cause lacrimation, epiphora, conjunctivitis, keratitis and even corneal ulcers , and should always be considered in the differential diagnosis of ocular alterations in both animal and human species, especially in endemic regions. The cases described in this report had no ocular alterations that could be associated with the presence of the eye worm; however, if and when present, potential ocular alterations may hamper the vision of the affected animals rendering them more vulnerable to predators.
Preventive measures to avoid eye worm transmission should be investigated in wild animals. With regard to canine species, the administration of systemic macrocyclic lactones (e.g. milbemycine oxime and moxidectin) may be recommended, whereas the use of slow-release insecticide collars do not protect against thelaziosis . Finally, the European rabbit might constitute an experimental model for the study of potential therapeutic and preventive agents against T. callipaeda.
The present findings reveal a new host for T. callipaeda in southern Europe and provide new clues to the epidemiology of Thelazia infection in wildlife species. Considering the growing number of cases described in domestic and wild animals, frequently in close contact with humans, thelaziosis should be included in the differential diagnosis of ocular diseases both in animals and humans.
All the procedures in this study were in accordance with the Portuguese legislation for the protection of animals (Decree-Law n° 113/2013).
To Mr. Nuno Fernandes, chairman of the board of Hunting and Fishing Association “Horas Selvagens”, Vilar de Ossos, Vinhais, Portugal.
Publication of this paper has been sponsored by Bayer Animal Health in the framework of the 11th CVBD World Forum Symposium.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Anderson RC. Nematode parasites of vertebrates: their development and transmission. 2nd ed. Wallingford: CABI Publishing; 2000.View ArticleGoogle Scholar
- Otranto D, Dantas-Torres F, Brianti E, Traversa D, Petrić D, Genchi C, et al. Vector-borne helminths of dogs and humans in Europe. Parasit Vectors. 2013;6:16.View ArticlePubMedPubMed CentralGoogle Scholar
- Otranto D, Ferroglio E, Lia RP, Traversa D, Rossi L. Current status and epidemiological observations of Thelazia callipaeda (Spirurida, Thelaziidae) in dogs, cats and foxes in Italy: a “coincidence” or a parasitic disease of the Old Continent? Vet Parasitol. 2003;116:315–25.View ArticlePubMedGoogle Scholar
- Dorchies P, Chaudieu G, Siméon LA, Cazalot G, Cantacessi C, Otranto D. First reports of autochthonous eyeworm infection by Thelazia callipaeda (Spirurida, Thelaziidae) in dogs and cat from France. Vet Parasitol. 2007;149:294–7.View ArticlePubMedGoogle Scholar
- Ruytoor P, Déan E, Pennant O, Dorchies P, Chermette R, Otranto D, et al. Ocular thelaziosis in dogs, France. Emerg Infect Dis. 2010;16:1943–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Malacrida F, Hegglin D, Bacciarini L, Otranto D, Nägeli F, Nägeli C, et al. Emergence of canine ocular thelaziosis caused by Thelazia callipaeda in southern Switzerland. Vet Parasitol. 2008;157:321–7.View ArticlePubMedGoogle Scholar
- Miró G, Montoya A, Hernández L, Dado D, Vázquez MV, Benito M, et al. Thelazia callipaeda: infection in dogs: a new parasite for Spain. Parasit Vectors. 2011;4:148.View ArticlePubMedPubMed CentralGoogle Scholar
- Hodžić A, Latrofa MS, Annoscia G, Alić A, Beck R, Lia RP, et al. The spread of zoonotic Thelazia callipaeda in the Balkan area. Parasit Vectors. 2014;7:352.View ArticlePubMedPubMed CentralGoogle Scholar
- Mihalca AD, D’Amico G, Scurtu I, Chirilă R, Matei JA, Ionică AM. Further spreading of canine oriental eyeworm in Europe: first report of Thelazia callipaeda in Romania. Parasit Vectors. 2015;8:48.View ArticlePubMedPubMed CentralGoogle Scholar
- Otranto D, Cantacessi C, Testini G, Lia RP. Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: evidence for pathogen transmission by a male arthropod vector. Int J Parasitol. 2006;36:1167–73.View ArticlePubMedGoogle Scholar
- Máca J, Otranto D. Drosophilidae feeding on animals and the inherent mystery of their parasitism. Parasit Vectors. 2014;7:516.View ArticlePubMedPubMed CentralGoogle Scholar
- Otranto D, Filipe Dantas-Torres F. Transmission of the eyeworm Thelazia callipaeda: between fantasy and reality. Parasit Vectors. 2015;8:273.View ArticlePubMedPubMed CentralGoogle Scholar
- Otranto D, Traversa D. Thelazia eyeworm: an original endo- and ectoparasitic nematode. Trends Parasitol. 2005;1:1–4.View ArticleGoogle Scholar
- Shen JL, Gasser RB, Chu D, Wang Z, Cantacessi C, Otranto D. Human thelaziosis – a neglected parasitic disease of the eye. J Parasitol. 2006;92:872–5.View ArticlePubMedGoogle Scholar
- Otranto D, Cantacessi C, Mallia E, Lia RP. First report of Thelazia callipaeda (Spirurida, Thelaziidae) in wolves (Canis lupus) in Italy. J Wildl Dis. 2007;43:508–11.View ArticlePubMedGoogle Scholar
- Otranto D, Dantas-Torres F, Mallia E, DiGeronimo PM, Brianti E, Testini G, et al. Thelazia callipaeda (Spirurida, Thelaziidae) in wild animals: Report of new host species and ecological implications. Vet Parasitol. 2009;166:262–7.View ArticlePubMedGoogle Scholar
- Pimenta P, Cardoso L, Pereira MJ, Maltez L, Coutinho T, Alves MS, et al. Canine ocular thelaziosis caused by Thelazia callipaeda in Portugal. Vet Ophthalmol. 2012;16:312–5.View ArticlePubMedGoogle Scholar
- Rodrigues FT, Cardoso L, Coutinho T, Otranto D, Diz-Lopes D. Ocular thelaziosis due to Thelazia callipaeda in a cat from northeastern Portugal. J Feline Med Surg. 2012;14:952–4.View ArticlePubMedGoogle Scholar
- Vieira L, Rodrigues FT, Costa A, Diz-Lopes D, Machado J, Coutinho T, et al. First report of canine ocular thelaziosis by Thelazia callipaeda in Portugal. Parasit Vectors. 2012;5:124.View ArticlePubMedPubMed CentralGoogle Scholar
- Soares C, Sousa SR, Anastácio S, Matias MG, Marquês I, Mascarenhas S, et al. Feline thelaziosis caused by Thelazia callipaeda in Portugal. Vet Parasitol. 2013;196:528–31.View ArticlePubMedGoogle Scholar
- Maia C, Catarino AL, Almeida B, Ramos C, Campino L, Cardoso L. Emergence of Thelazia callipaeda infection in dogs and cats from east-central Portugal. Transbound Emerg Dis. 2014. doi:https://doi.org/10.1111/tbed.12284.PubMedGoogle Scholar
- Sargo R, Loureiro F, Catarino AL, Valente J, Silva F, Cardoso L, et al. First report of Thelazia callipaeda in red foxes (Vulpes vulpes) from Portugal. J Zoo Wildl Med. 2014;45:458–60.View ArticlePubMedGoogle Scholar
- Fuentes I, Montes I, Saugar JM, Latrofa S, Gárate T, Otranto D. Thelaziosis in humans, a zoonotic infection, Spain, 2011. Emerg Infect Dis. 2012;18:2073–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Otranto D, Lia RP, Traversa D, Giannetto S. Thelazia callipaeda (Spirurida, Thelaziidae) of carnivores and humans: morphological study by light and scanning electron microscopy. Parassitologia. 2003;45:125–33.PubMedGoogle Scholar
- Otranto D, Testini G, De Luca F, Hu M, Shamsi S, Gasser RB. Analysis of genetic variability within Thelazia callipaeda (Nematoda: Thelazioidea) from Europe and Asia by sequencing and mutation scanning of the mitochondrial cytochrome c oxidase subunit 1 gene. Mol Cell Probes. 2005;19:306–13.View ArticlePubMedGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–8.View ArticlePubMedGoogle Scholar
- Skrjabin KI, Sobolev AA, Ivashkin VM. Spirurata of animals and man and the diseases caused by them. Part 4. Thelazioidea. In: Skrjabin KI, editor. Essentials of Nematodology, vol. 16. Moscow: Academy of Sciences of the USSR; 1967. p. 1–54 [ in Russian; Israel Program for Scientific Translation].Google Scholar
- Otranto D, Dantas-Torres F. Canine and feline vector-borne diseases in Italy: current situation and perspectives. Parasit Vectors. 2010;3:2.View ArticlePubMedPubMed CentralGoogle Scholar
- Otranto D, Brianti E, Cantacessi C, Lia RP, Máca J. The zoophilic fruitfly Phortica variegata: morphology, ecology and biological niche. Med Vet Entomol. 2006;20:358–64.View ArticlePubMedGoogle Scholar
- Lechat C, Siméon N, Pennant O, Desquilbet L, Chahory S, Le Sueur C, et al. Comparative evaluation of the prophylactic activity of a slow-release insecticide collar and a moxidectin spot-on formulation against Thelazia callipaeda infection in naturally exposed dogs in France. Parasit Vectors. 2015;8:93.View ArticlePubMedPubMed CentralGoogle Scholar