Update on infections with Thelazia callipaeda in European wildlife and a report in a red fox, Vulpes vulpes, in Portugal

Abstract

Thelazia callipaeda, also known as the “oriental eye worm”, is a zoonotic parasitic nematode with a wide range of hosts, particularly wild and domestic carnivores, but also lagomorphs and humans. Currently, ocular thelaziosis presents an expanding distribution range throughout Europe, including Portugal. This study provides an update on T. callipaeda infection reports (30 studies) in European wildlife comprising 54 host-locality records in 10 host species from nine European countries. The prevalence of T. callipaeda varied widely, with ranges from around 1% in red foxes and European hares to almost 50% in red foxes. The lowest mean intensity was 2.7 nematodes/host in European wildcats and the highest was 38.0 nematodes/host in wolves. In addition, a massive infection with T. callipaeda in a juvenile male red fox from eastern-central Portugal is also described, representing the southernmost report in a wild animal in this country. A total of 188 nematodes (139 females and 49 males) were collected from both eyes and were submitted to morphological and molecular characterization. Collected nematodes were morphologically identified as T. callipaeda. Given the endemicity of T. callipaeda in eastern-central Portugal, surveillance system should be implemented to monitor its presence among wild and domestic animals.

Graphical abstract

Highlights

• •Updated data for Thelazia callipaeda infections in European wildlife.
• •Massive infection (188 worms) detected in a juvenile red fox from central Portugal.
• •Greatest number of T. callipaeda in a single infected wild animal in Europe.
• •The southernmost report of T. callipaeda in a wild animal in Portugal.

1. Introduction

Thelazia callipaeda (Spirurida: Thelaziidae), also called the “oriental eyeworm”, is a vector-borne, zoonotic nematode parasite with a large spectrum of hosts (Anderson, 2000; Otranto and Traversa, 2005; do Vale et al., 2020), both domestic (Rodrigues et al., 2012; Vieira et al., 2012; Pimenta et al., 2013; Soares et al., 2013; Maia et al., 2016; Rolbiecki et al., 2021) and wild animals, notably including red foxes.

This eyeworm infects the conjunctival sac, tear glands, tear ducts and nictitating membrane, and can be responsible for various ophthalmological clinical signs (Otranto and Traversa, 2005; do Vale et al., 2020). In Europe, the large-sized drosophilid fruit fly Phortica variegata Fallén, 1823 (Drosophilidae: Steganinae) was confirmed as the nematode intermediate host (Otranto et al., 2006; Marino et al., 2018). This non-biting fly ingests the larvae whilst feeding on the host’s lachrymal secretions (Otranto et al., 2006; Marino et al., 2018).

Currently, ocular thelaziosis presents an expanding distribution range throughout Europe (do Vale et al., 2019). During the last nearly two decades, human cases of thelaziosis were detected in patients from Italy and France (Otranto and Dutto, 2008), Serbia (Tasić-Otašević et al., 2016), Croatia (Paradžik et al., 2016), Germany (Dolff et al., 2020), Spain (Fuentes et al., 2012; López Medrano et al., 2015; Martínez-Sánchez et al., 2021) and Portugal (Morgado et al., 2022). Wild animals have been involved in the epidemiological cycle of Thelazia spp., contributing to the introduction and dissemination among animals in non-endemic regions. Foxes should be considered the main reservoir of thelaziosis in European wildlife (Otranto et al., 2009; Otranto and Dantas-Torres, 2015).

In Portugal, the first cases of infection with this eyeworm in red foxes (Vulpes vulpes) were reported in the northern and eastern-central regions of the country (Sargo et al., 2014). The present study provides an update on the reports of T. callipaeda in European wildlife and describes a massive infection in a red fox from eastern-central Portugal, representing the southernmost report from a wild animal in this country and one of the greatest worms per host count ever.

2. Materials and methods

2.1. Update on T. callipaeda in European wildlife

An update on studies, reports and/or cases of T. callipaeda infection in European wildlife has been carried out based on six new studies (Diakou et al., 2021; Bezerra-Santos et al., 2022; Cotuțiu et al., 2022; Papadopoulos et al., 2022; Bertos et al., 2023; Deak et al., 2023) published after the most recent review by do Vale et al. (2020). Values for prevalence (proportion of infected hosts in %) and mean intensity (mean number of nematodes per infected individual host) are given as published or calculated (for total sample size ≥ 8 animals and sample size of infected animals ≥ 3) from the data in the original publications whenever possible. European records of T. callipaeda in red foxes were georeferenced according to the World Geodetic System 1984 (WGS 84).

2.2. New record of T. callipaeda

A juvenile male red fox (V. vulpes), approximately 6 months old, was admitted to the Wildlife Study and Rehabilitation Centre of the National Association for Nature Conservation (CERAS – Quercus), located in the municipality of Castelo Branco, in eastern-central Portugal. The wild animal was found severely debilitated on October 11, 2021, in Covilhã (Gibaltar, Teixoso) (40.3°N, 7.4°W).

Physical examination revealed poor body condition, weight of 1.686 kg, high degree of dehydration (9–12%), prostration, and ectoparasites (Ctenocephalides felis fleas). Both eyes were closed, and the eyelids were covered with an abundant purulent discharge (Fig. 1). Traces of blood were found in the left eye, on the medial corner. Both eyes presented a large number of transparent to whitish filamentous and mobile adult worms under the third eyelid and in the conjunctival sac. Conjunctival hyperemia was present in both eyes, with a small corneal scar on the left eye. The eyes were cleaned with sterile physiological saline solution (NaCl 0.9%) and all worms removed manually once daily for 3 days, and then stored in 70% ethanol.

Fig. 1.

Admission at the Wildlife Study and Rehabilitation Centre (CERAS) of a juvenile male red fox (Vulpes vulpes).

The therapeutic approach included topic application of 1% fusidic acid (Fucithalmic®; Amdipharm, Dublin, Ireland) BID for 7 days and diclofenac (Voltaren®; Novartis, Baden, Germany) BID for 3 days, external deworming with fipronil and permethrin (Frontline Tri-act®; Boehringer Ingelheim Animal Health, Ingelheim, Germany) plus the combination of imidacloprid 10% and moxidectin 2.5% (Advocate®; Elanco Animal Health, Leverkusen, Germany) pour on, and systemic treatment with enrofloxacin (Baytril®; Elanco Animal Health, Germany; and Enroxal®; HCS bvba, Edegem, Belgium) 5 mg/kg, SID, firstly subcutaneous (SC) and then per os (PO) for 7 days, meloxicam (Melovem®; Dopharma, Raamsdonksveer, The Netherlands) 0.2 mg/kg, SID, firstly SC and then PO for 3 days, and intravenous fluids specifically lactated Ringers solution, 10% dextrose solution and a multivitamins and minerals solution (Duphalyte®; Parsippany, NJ, USA).

2.3. Molecular analysis

Eight nematodes were sent to the Department of Genetics and Biotechnology, University of Trás-os-Montes e Alto Douro (Vila Real, Portugal). These worms were used for molecular identification. DNA was extracted from the eight nematodes using DNA NZY Tissue gDNA Isolation Kit (NZYtech, Lisbon, Portugal). A partial sequence (689 bp) of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified by polymerase chain reaction (PCR) using the primers NTF and NTR (Otranto and Traversa, 2005). The fragment was successfully amplified from seven worms and the four with the most intense bands were selected for sequencing. Amplified products from the PCR were purified using ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Sequencing was performed in an Applied Biosystems 3730xl DNA Analyzer (Stab Vida, Caparica, Portugal). Amplicons were sequenced in both directions and sequence alignment was made using MEGA version 11 (Tamura et al., 2021). The consensus sequences were obtained with BioEdit software and sequence similarity was studied using the BLASTN program available on the National Center for Biotechnology Information database (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi).

3. Results and discussion

To date, infections with T. callipaeda have been reported in 30 studies for ten wild vertebrate host species, i.e. red fox, wolf, beech marten, European hare, European wildcat, golden jackal, wild rabbit, Eurasian badger, brown bear and a lion in a zoological garden in Romania, comprising 54 host-locality records in nine European countries (Table 1).

Table 1.

Reports on Thelazia callipaeda infection in European wildlife.

Host	Country	Locality (n)	n/N	Prevalence (%)	No. of worms	Intensity		Reference
						Range	Mean ± SD
Red fox (Vulpes vulpes)	Switzerland	Ticino canton	7/126	5.6	27	1–10	[3.9]	Malacrida et al. (2008)
	Italy	Basilicata region	37/75	49.3	139	1–13	3.8 ± 2.0	Otranto et al. (2009)
	Spain	Cáceres Province	2/–	–	14	2–12	[7.0 ± 7.1]	Calero-Bernal et al. (2013)
	Bosnia and Herzegovina	Eastern, northeastern, northern and central Bosnia and Herzegovina	51/184	27.7	364	1–50	8.08 ± 9.41	Hodžić et al. (2014)
	Portugal	Montalegre municipality (n = 1); Sabugal municipality (n = 2)	3/–	–	7, 10	–	[5.7]	Sargo et al. (2014)
	Serbia	Belgrade (n = 2); Požarevac (n = 1)	3/–	–	7, 11, 5	[5–11]	[7.7 ± 3.1]	Pavlović et al. (2017)
	Slovakia	Košice (n = 6); Trenčín (n = 1)	7/523	1.3	45	2–12	6.4 ± 4.4	Čabanová et al. (2018)
	Romania	Arad, Bihor, Cluj, Gorj, Hunedoara, Maramureș, Mureș, Sălaj, Satu-Mare and Timiș counties	151/514	29.4	3350	1–192	23.2	Ionică et al. (2018)
	Portugal	Covilhã	1/–	–	188	–	–	Present study
Wolf (Canis lupus)	Italy	Basilicata region	3/–	–	3, 15, 96	[3–96]	[38.0 ± 50.6)]	Otranto et al. (2007)
	Italy	Basilicata region	1/2	–	2	–	–	Otranto et al. (2009)
	Romania	Pianu de Sus (Alba County)	1/13	7.7	4	–	–	Mihalca et al. (2016)
	Serbia	Negotin (n = 1); Sjenica (n = 1); Jagodina (n = 1); Braničevo (n = 2); Ražanj (n = 1); Despotovac (n = 1); Svrljig (n = 1)	8/21	38.1	21, 12, 7, 132, 70, 35, 8, 4	[4–132]	[36.1 ± 44.4]	Gajić et al. (2019)
	Spain	Guadalajara Province	1/–	–	42	–	–	Nájera et al. (2020)
	Greece	Florina Prefecture	13/–	–	9, 10, 7, 16, 13, 6, 8, 13, 10, 15, 8, 7, 9	[6–16]	[10.1 ± 3.2]	Papadopoulos et al. (2022)
	Italy	Piedmont region (n = 1); Tuscany region (n = 2)	3/–	–	41, 8, 7	–	[18.7 ± 19.3]	Bezerra-Santos et al. (2022)
Beech marten (Martes foina)	Italy	Basilicata region	3/22	13.6	15	3–6	5.0 ± 1.7	Otranto et al. (2009)
	Portugal	Vila Real municipality	1/–	–	1	–	–	Seixas et al. (2018)
	Romania	Eastern Romania	1/13	7.7	1	–	–	Ionică et al. (2019)
European hare (Lepus europaeus)	Italy	Basilicata region	3/13	23.1	17	3–11	5.7 ± 4.6	Otranto et al. (2009)
	Romania	Continental (n = 3) and steppic (n = 1) ecoregions	4/326	1.2	1, 4, 9, 70	[1–70]	[21.0 ± 32.8]	Cotutiu et al. (2022)
European wildcat (Felis silvestris)	Italy	Basilicata region	3/8	37.5	16	3–7	5.3 ± 2.1	Otranto et al. (2009)
	Romania	Mesteacanu (Sălaj County)	1/9	11.1	2	–	–	Mihalca et al. (2016)
	Greece	Mainland and Crete	2/23	8.7	–	–	–	Diakou et al. (2021)
	Spain	Sierra Arana (Granada Province)	3/17	17.6	2, 2, 3	[1–3]	[2.7 ± 0.6]	Bertos et al. (2023)
Golden jackal (Canis aureus)	Romania	Ostrovu Mare (Mehedinți County)	1/64	1.6	70	–	–	Mihalca et al. (2016)
Wild rabbit (Oryctolagus cuniculis)	Portugal	Vilar de Ossos (Vinhais municipality)	2/2	–	6	3–3	–	Gama et al. (2016)
Eurasian badger (Meles meles)	Romania	Western Romania	1/55	1.8	33	–	–	Ionică et al. (2019)
Brown bear (Ursus arctos)	Greece	Florina prefecture	8/30	26.7	11, 3, 4, 5, 7, 2, 3, 5	[2–11]	[5.0 ± 2.9]	Papadopoulos et al. (2022)
Lion (Panthera leo)	Romania	Reșița Zoo (Caraș-Severin County)	1/–	–	4	−/−	–	Deak et al. (2023)

Note: The mean ± SD values calculated from the original data for the present update are displayed in square brackets.

Abbreviations: n, number of infected animals; N, number of examined animals; −, not available/not computable; SD, standard deviation.

The relative majority of reports have been from red foxes (Fig. 2), with 25 host-locality records in eight countries, a circumstance that may be related to the fact that this is the most abundant terrestrial wild carnivore in the world (Basto et al., 2016). For wolves, 14 host-locality-time reports (2 reports different in time from the same locality; Otranto et al., 2007, 2009) in five countries have been recorded. Therefore, from a One Health perspective, foxes and also wolves can be considered appropriate sentinels of T. callipaeda infection to both humans and other wild and domestic animals.

Fig. 2.

Map of approximate European locations from where red foxes (Vulpes vulpes) infected with Thelazia callipaeda have been reported. The map was constructed using ArcGIS Desktop v10.8.1 (ESRI, 2020), and records were georeferenced according to the World Geodetic System 1984 (WGS 84). For additional details, see Table 1.

Regarding the ten wild host species, studies/records from Mediterranean and western European countries (Greece, Italy, Portugal, Spain and Switzerland) were slightly more frequent (n = 18 versus n = 12) than from eastern European countries (Bosnia and Herzegovina, Romania, Serbia and Slovakia). For red foxes, four studies have been carried out in Italy, Portugal, Spain and Switzerland, and another four studies in Bosnia and Herzegovina, Slovakia, Romania and Serbia. For wolves, five studies have been carried in Greece, Italy and Spain, and two studies in Romania and Serbia (Table 1).

Whenever computable, the prevalence of T. callipaeda varied widely, ranging from around 1% in red foxes in Slovakia and European hares in Romania to almost 50% in red foxes in Italy (Table 1). Wide prevalence ranges were also observed in most host species as follows: (i) in red foxes, from 1.3% in Slovakia (Košice and Trenčin; Čabanová et al., 2018) to 49.3% in Italy (Basilicata region; Otranto et al., 2009); (ii) in wolves, from 7.7% in Romania (Pianu de Sus, Alba County; Mihalca et al., 2016) to 38.1% in Serbia (seven localities; Gajić et al., 2019); (iii) in beech martens, from 7.7% in Romania (eastern Romania; Ionică et al., 2019) to 13.6% in Italy (Basilicata region; Otranto et al., 2009); (iv) in European hares, from 1.2% in Romania (continental and steppic ecoregions; Cotuțiu et al., 2022) to 23.1% in Italy (Basilicata region; Otranto et al., 2009); and (v) in European wildcats, from 8.7% in Greece (Diakou et al., 2021) to 37.5% in Italy (Basilicata region; Otranto et al., 2009). Other reports have revealed single prevalence values of: (i) 1.6% in golden jackals in Romania (Ostrovu Mare, Mehedinți County; Mihalca et al., 2016); (ii) 1.8% in Eurasian badgers also in Romania (western Romania; Ionică et al., 2019); and (iii) 26.7% in brown bears in Greece (Florina prefecture; Papadopoulos et al., 2022).

Mean intensity values also ranged widely with the lowest mean intensity of 2.7 nematodes/host recorded in European wildcats from Spain and the highest mean intensity of 38.0 nematodes/host in wolves from Italy (Table 1). Mean intensity ranges by host species were as follows: (i) in red foxes, from 3.8 nematodes/host in Italy (Basilicata region; Otranto et al., 2009) to 23.2 nematodes/host in Romania (10 localities, Ionică et al., 2019); (ii) in wolves, from 10.1 nematodes/host in Greece (Florina prefecture; Papadopoulos et al., 2022) to 38.0 nematodes/host in Italy (Basilicata region; Otranto et al., 2007); (iii) in European hares, from 5.7 nematodes/host in Italy (Basilicata region; Otranto et al., 2009) to 21.0 nematodes/host in Romania (continental and steppic ecoregions; Cotuțiu et al., 2022); and (iv) in European wildcat, from 2.7 nematodes/host in Spain (Sierra Arana, Granada province; Bertos et al., 2023) to 5.3 nematodes/host in Italy (Basilicata region; Otranto et al., 2009). The intensity value from a unique host study was 5.0 nematodes/host in eight brown bears from Greece (Table 1; Florina prefecture; Papadopoulos et al., 2022).

In the present study, a total of 188 nematodes were recovered from both eyes of V. vulpes (106 from the left eye and 82 from the right eye), which represent one of the highest numbers found in wild (Table 1) and also domestic animals. Collected nematodes were morphologically identified as 139 females and 49 males of T. callipaeda (Fig. 3). The four partial cox1 sequences selected had 98.1–98.6% nucleotide identity to the corresponding T. callipaeda gene in NCBI Core nucleotide database, and have been deposited in GenBank (accession numbers PQ203220-PQ203223).

Fig. 3.

A Adult female of Thelazia callipaeda: anterior extremity with buccal capsule (arrow 1) and vulva (arrow 2). B Adult male of T. callipaeda: posterior extremity with short spicule (arrow 1) and long spicule (arrow 2).

In Portugal, infections with T. callipaeda in both domestic and wild animals have been reported mainly in the North of the country, above the Douro River (Rodrigues et al., 2012; Vieira et al., 2012; Pimenta et al., 2013; Sargo et al., 2014; Gama et al., 2016; Seixas et al., 2018). The presence of T. callipaeda in the district of Castelo Branco could mean a spread of the nematode. However, cases of thelaziosis caused by T. callipaeda in east-central Portugal have already been reported in red foxes (Sargo et al., 2014), dogs and cats (Maia et al., 2016), and in a human (Morgado et al., 2022). These facts support the endemicity of T. callipaeda in eastern-central Portugal.

The identification of T. callipaeda, but also other Thelazia spp., is important, in epidemiological terms, because the district of Castelo Branco has a large economic component sustained in agriculture, especially in the production of large and small ruminants. Moreover, it is well known for its hunting areas and considerable presence of red foxes, including in urban areas. Therefore, the risk of increased cases occurrence in animals and humans due to the presence of infected hosts (vertebrates and the fruit fly P. variegata) must be considered and evaluated.

4. Conclusions

The present study updated the information on T. callipaeda infections in ten wild species from nine European countries. The prevalence of T. callipaeda varied widely, with ranges from around 1% in red foxes and European hares to almost 50% in red foxes. The lowest mean intensity was 2.7 nematodes/host in European wildcats and the highest was 38.0 nematodes/host in wolves. The first case of thelaziosis in a red fox from the district of Castelo Branco is also reported, highlighting the endemicity of T. callipaeda in the eastern-central area of Portugal. There is a need for a surveillance system that monitors Thelazia spp. in domestic and wild animals. Attention should also be given to infection in humans, due to the zoonotic character of T. callipaeda.

Funding

This work was supported by projects UIDB/00772/2020 (https://doi.org/10.54499/UIDB/00772/2020), UIDB/04033/2020 (https://doi.org/10.54499/UIDB/04033/2020), LA/P/0059/2020 and LA/P/0126/2020, all funded by the Portuguese Foundation for Science and Technology (FCT).

Ethical approval

All procedures complied with the Portuguese legislation for the protection of animals used for scientific purposes (i.e. Decree-Law no. 113/2013, of August 7, 2013), which transposes European legislation (i.e. Directive, 2010/63/EU of the European Parliament and the Council, of September 22, 2010).

CRediT authorship contribution statement

Ana Filipa Lopes: Conceptualization, Investigation, Writing – original draft, Writing – review & editing. Mariana Ribeiro Ferreira: Conceptualization, Investigation, Writing – original draft, Writing – review & editing. Beatriz do Vale: Formal analysis, Writing – review & editing, Visualization. Marlene Santos: Investigation. Inês Silveira: Investigation. Sofia Claudino: Investigation. Manuel Martins: Formal analysis. Telma Brida: Investigation. Luís Figueira: Formal analysis. Luís Cardoso: Formal analysis, Writing – review & editing, Visualization. Ana Patrícia Lopes: Formal analysis, Resources. Ana Cláudia Coelho: Formal analysis, Writing – review & editing, Visualization. Manuela Matos: Methodology, Validation. Ana Cristina Matos: Formal analysis, Visualization, Supervision.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Given their role as Guest Editors, Ana Cláudia Coelho, Ana Patrícia Lopes and Luís Cardoso had no involvement in the peer review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Professor Aneta Kostadinova (Editor-in-Chief).

Data availability

The data collected and analysed during the study are provided within the article. The newly generated sequences were deposited in the GenBank database under the accession numbers PQ203220-PQ203223.