GENETICS, EVOLUTION, AND PHYLOGENY - SHORT COMMUNICATION First report of eyeworm infection by Thelazia callipaeda in gray wolf (Canis lupus) from Serbia Gajić Bojan 1 & Bugarski-Stanojević Vanja 2 & Penezić Aleksandra 3 & Kuručki Milica 3 & Bogdanović Neda 3 & Ćirović Duško 3 Received: 6 May 2019 /Accepted: 22 October 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Thelazia callipaeda, originally known as an “Oriental eyeworm,” is a small nematode parasitizing the conjunctival sacs of domestic and wild animals and humans. Previous studies conducted in Serbia have reported the eyeworm infections in dogs, cats, and foxes, as well as in a human patient. As the data regarding thelaziosis from wildlife is still scarce, the aim of this study was to investigate the presence of T . callipaeda in gray wolf (Canis lupus) from Serbia. All collected nematodes were morpho- logically identified as T . callipaeda males (n = 64) or females (n = 225). Molecular characterization, conducted by PCR amplification followed by sequence analysis of partial cytochrome c oxidase subunit 1 gene (cox1), revealed only haplotype 1 of T . callipaeda. The overall prevalence of thelaziosis was 38.1% (8/21). In all positive animals, both eyes were affected, with a total parasitic load ranging from four to 132 worms per animal. Our results indicate the important epidemiological role of wolves as wildlife reservoirs of T . callipaeda, expanding geographic range of infection, as well as intra- and interspecies contact rates, although the role of other wild carnivore species (i.e., foxes and jackals) should be investigated in future studies. Keywords Cox1 . h1 haplotype . Reservoir . Thelaziosis . Wildlife Introduction Thelazia callipaeda (Spirurida, Thelaziidae) is an insect-borne nematode living in the conjunctival sac of domestic and wild carnivores, rabbits, and humans (Anderson 2000). Regardless of the definitive host species, the only confirmed vector for T . callipaeda under natural conditions in Europe is a zoophilic fruitfly Phortica variegata (Drosophilidae, Steganinae) (Otranto et al. 2006). The nematode was traditionally known as “Oriental eyeworm” due to its occurrences in some Asian countries and the former Soviet Union (summarized in Otranto et al. 2009). After the first detection in dogs from Northern Italy (Rossi and Bertaglia 1989), T . callipaeda has been in- creasingly reported in dogs and cats from other European countries including France (Dorchies et al. 2007), Switzerland (Malacrida et al. 2008), Germany (Magnis et al. 2010), Spain (Miró et al. 2011), and Portugal (Vieira et al. 2012). More recent findings indi- cate that thelaziosis has been spreading in both domestic and wild carnivores across the Balkan countries (Colella et al. 2016), such as Bosnia and Herzegovina and Croatia (Hodžić et al. 2014) and Greece (Papadopoulos et al. 2018). Although ocular infection was detected in several species of wild animals (Otranto et al. 2009), only a few cases of infection were confirmed in wolves (Otranto et al. 2007, 2009; Mihalca et al. 2016). In Serbia, thelaziosis was initially reported in dogs and cats (Gajić et al. 2014), with the first human case confirmed only a few years later (Tasić-Otašević et al. 2016). Despite the recent detection of eyeworm infection in foxes (Pavlović et al. 2017), data regarding wildlife from Serbia is still scanty. Therefore, the aim of this study was to investigate the occurrence and prevalence of zoonotic T . callipaeda in a population of gray wolf (Canis lupus) from different locations in Serbia. Section Editor: Hiroshi Sato * Bugarski-Stanojević Vanja vanjabs@ibiss.bg.ac.rs 1 Faculty of Veterinary Medicine, University of Belgrade, Bulevar Oslobođenja 18, Belgrade 11000, Serbia 2 Institute for Biological Research “Siniša Stanković”, University of Belgrade, Despota Stefana Blvd.142, Belgrade 11060, Serbia 3 Faculty of Biology, University of Belgrade, Studentski Trg 16, Belgrade 11000, Serbia Parasitology Research https://doi.org/10.1007/s00436-019-06519-z