Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis
Parasites & Vectors volume 16, Article number: 294 (2023)
The zoonotic nematode Thelazia callipaeda infects the eyes of domestic and wild animals and uses canids as primary hosts. It was originally described in Asia, but in the last 20 years it has been reported in many European countries, where it is mainly transmitted by the drosophilid fruit fly Phortica variegata. We report the autochthonous occurrence of T. callipaeda and its vector P. variegata in Austria. Nematodes were collected from clinical cases and fruit flies were caught using traps, netting, and from the conjunctival sac of one dog. Fruit flies and nematodes were morphologically identified and a section of the mitochondrial cytochrome c oxidase subunit I gene (COI) was analysed. A DNA haplotype network was calculated to visualize the relation of the obtained COI sequences to published sequences. Additionally, Phortica spp. were screened for the presence of DNA of T. callipaeda by polymerase chain reaction. Thelazia callipaeda and P. variegata were identified in Burgenland, Lower Austria, and Styria. Thelazia callipaeda was also documented in Vienna and P. variegata in Upper Austria and South Tyrol, Italy. All T. callipaeda corresponded to haplotype 1. Twenty-two different haplotypes of P. variegata were identified in the fruit flies. One sequence was distinctly different from those of Phortica variegata and was more closely related to those of Phortica chi and Phortica okadai. Thelazia callipaeda could not be detected in any of the Phortica specimens.
Thelazia callipaeda is a parasitic nematode of the order Spirurida that affects the eyes of various mammals and is transmitted by the fruit fly Phortica variegata. Among domestic animals, dogs (Canis lupus familiaris) are frequently affected, but infections in cats (Felis silvestris catus) and rabbits (Oryctolagus cuniculus) have also been reported [1,2,3,4]. As humans can also be infected, though less frequent, T. callipaeda is also a zoonotic nematode and therefore of importance to public health [5, 6]. Among wildlife, T. callipaeda has been found in species of the families Canidae, Felidae, Ursidae, Mustelidae, Procyonidae, Suidae, and Leporidae, which represent many possible potential reservoir hosts [7,8,9,10,11,12].
Clinical signs of infection with Thelazia callipaeda can vary widely and have been divided into four stages, ranging from the absence of clinical signs to corneal ulcers . Thelazia callipaeda is usually found under the eyelid and the nictitating membrane, and is easily distinguishable from other nematodes that can affect the eye, such as Onchocerca lupi, which is embedded in the tissue around the eye and often associated with nodule formation . In combination with the removal of the nematodes from the eye, macrocyclic lactones, such as moxidectin and milbemycin oxime, are useful for both the prevention of infection and its treatment [14,15,16,17].
Thelazia callipaeda is also known as the ‘oriental eye worm’ due to its original distribution in Asia. In Europe, T. callipaeda has been documented in most southern and central countries and is predicted to spread further across the continent [18,19,20]. The commonly used DNA barcode region, a section of the cytochrome c oxidase subunit I gene (COI), is useful for the molecular identification of T. callipaeda and to distinguish different haplotypes. In the European population of T. callipaeda, only one haplotype has been detected, whereas the Asian population is highly diverse, with over 20 different known haplotypes [21, 22].
The sexual reproduction of T. callipaeda takes place in mammals, which act as the definitive hosts, while zoophilic fruit flies of the genus Phortica are the intermediate hosts. Male Phortica spp. feed on lacrimal fluid and take up first-stage larvae of T. callipaeda during feeding . In the intermediate host, the larvae can survive for up to 147 days and develop into third-stage larvae, which can be transmitted to a new definitive host when the fruit fly next feeds on lacrimal fluid .
In Europe, the main vector of T. callipaeda is P. variegata, whereas in Asia it is P. okadai . Phortica oldenbergi is also a competent vector under laboratory conditions, but its vector capacity under field conditions needs to be assessed . Dissection and polymerase chain reaction (PCR) can be used to detect larvae of T. callipaeda in Phortica spp.; however, live fruit flies are necessary for the nematode’s detection through dissection [25, 26].
For the collection of P. variegata, fruit fly traps can be hand-made cost efficiently from easily available components. Alternatively, netting placed around the eye of a human or dog can be used for this purpose. Although this is a time-consuming and less efficient method than using fruit fly traps, more male specimens can be collected when using this approach . Identification can be done by using morphological features or by analysing the COI barcode region [28,29,30,31].
Suitable habitats for P. variegata, which are mountainous areas at 600–1200 m above sea level, can be found in large parts of Europe, and in particular central Europe. Phortica variegata fruit flies are mainly active at 20–25 °C and their lachryphagous activity increases with air temperature [31, 32].
Few records of P. variegata exist for Austria, but recent autochthonous infections of T. callipaeda have been reported [33,34,35,36]. The few records of P. variegata that were available at the start of this study dated from before 1988, and it is not clear if this drosophilid fruit fly is still endemic in Austria and, if it is, how widespread it is [35, 36].
A first report of P. variegata in Burgenland, Austria, was published only recently , and the genetic diversity of T. callipaeda and that of its vector P. variegata have not yet been investigated in this region. The aims of this study were to examine whether P. variegata and T. callipaeda occur in different parts of Austria, and to assess their genetic diversity. The occurrence of P. variegata and T. callipaeda was also investigated in South Tyrol, which is located in Italy and borders Austria, as this should provide further information on the distribution of these species, which are endemic in some other Italian regions.
A questionnaire designed to identify cases of T. callipaeda infections recorded in private veterinary practices was sent to all of the veterinarians in the database of Boehringer Ingelheim (Vienna, Austria) who had given their consent to receive customer mailings. The questionnaire could be completed online between October and November 2020. The veterinarians had the possibility to send the T. callipaeda specimens that they had collected previously or from a subsequent clinical case to the Institute of Parasitology, University of Veterinary Medicine Vienna. Material of clinical cases was included for 2015 to 2022, and included that from a published case report . Specimens collected from pets that had previously been abroad were excluded from the study.
To collect P. variegata, fruit fly traps built out of disposable plastic bottles were set up near forests, fruit trees, and dog-walking areas, as described in detail by Roggero et al. . Every 2 weeks, the fruit flies were collected from the nets and frozen at − 20 °C until further analysis, and the chopped fruit which was used as the bait was changed. In July and August 2020, two traps in each of four sites were sampled. Two of the sites were in areas where infection with T. callipaeda had been reported (the town of Deutschlandsberg in Styria and the town of Gänserndorf in Lower Austria), and the two other sites were selected independently of known cases (Floridsdorf district, Vienna and Rohr im Kremstal municipality, Upper Austria). In July, August, and September 2021, eighteen traps were set up in Lower Austria, 17 in Upper Austria, 16 in South Tyrol, and 11 at participating veterinary practices that had reported cases of T. callipaeda infections, as well as sites provided by other volunteers (Fig. 1).
Additionally, eight fruit flies were collected from the eyes of a dog, and three by netting. Two P. variegata found during another study, which took place during the same period of time as the present study , were also included.
All of the samples were morphologically identified at the Institute of Parasitology, University of Veterinary Medicine Vienna or at the Department of Veterinary Medicine, University of Bari.
DNA extraction, PCR amplification, and sequencing
DNA was extracted from whole specimens of P. variegata and T. callipaeda using the QIAGEN DNeasy Blood and Tissue kit (QIAGEN, Hilden, Germany). Samples were incubated at 56 °C overnight and processed according to the manufacturer’s protocol. To screen P. variegata samples for the presence of DNA of T. callipaeda and for genetic identification of T. callipaeda samples, PCRs targeting 649-base pair (bp) and 674-bp sections of mitochondrial COI were performed using the primers COIintF/COIintR  and H14FilaCOIFw/H14FilaCOIRv , respectively. DNA barcoding of P. variegata was done with the primers Lep-F1/LepR1  and LCO1490/HCO2198 , which respectively target 665-bp and 658-bp sections of the COI gene. One Phortica sp. sample, which was genetically different from P. variegata, was further analysed by targeting a different region of the COI gene using the primers UEA7/UEA10 . PCR products were analysed by electrophoresis in 1.8% agarose gels stained with Midori Green Advance DNA stain (Nippon Genetics Europe, Germany). PCR-positive samples were sent to a commercial company (LGC Genomics, Germany) for sequencing using the PCR primers.
For phylogenetic analysis, nucleotide sequences available from GenBank (National Center for Biotechnology Information) and Barcode of Life Data System (BoldSystems) databases were searched with the Basic Local Alignment Search Tool (BLAST) function, using one of the sequences obtained for each organism. In GenBank, the organism group was specified as Thelazia (taxid 103826) for the T. callipaeda sequences and Phortica (taxid 462262) for the P. variegata sequences, with the number of maximum target sequences set to 5000. For P. variegata, only the species belonging to Phortica sensu stricto were included. The sequences were aligned and sorted using the default option (FFT-NS-2) in multiple alignment using fast Fourier transform (MAFFT) v.7.311  and sequences not covering the fragment of the sequences obtained in this study were excluded. All sequences featuring obvious sequencing errors and ambiguity characters were removed from the alignment and were excluded from the analysis.
To provide an overview of the diversity of haplotypes, maximum likelihood and Bayesian inference trees were calculated for each organism based on alignments, and included 110 sequences (617 nucleotide positions) for T. callipaeda (Additional file 1) and 280 sequences (647 nucleotide positions) for P. variegata (Additional file 2). The sequences were collapsed to haplotypes using data analysis in molecular biology and evolution (DAMBE) v.188.8.131.52 , leaving 42 haplotypes for T. callipaeda and 188 haplotypes for P. variegata. A sequence of Mastophorus muris (GenBank accession number MK867476) was used as an outgroup for T. callipaeda, and a sequence of Anopheles gambiae (GenBank accession number MG753768) was used as an outgroup for P. variegata. Maximum likelihood bootstrap consensus trees (1000 replicates) were calculated using the W-IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/; ) applying the models TIM2 + F + I + G4 for T. callipaeda and TIM + F + I + G4 for P. variegata, which were suggested as the best fit for the data sets in the model test according to the corrected Akaike information criterion. The Bayesian inference trees were calculated using MrBayes v.3.2.7 , applying the next complex model GTR+G+I because the same models were not available in this program. The analyses were run for 106 generations (number of chains, 4), sampling every thousandth tree. The first 25% of the trees were discarded as burn-in and 50% majority-rule consensus trees were calculated based on the remaining 7500 trees.
Based on the results of the consensus tree, clades were selected for the calculation of median-joining haplotype networks using Network 10.2.0.0 (Fluxus Technology, Suffolk, UK), applying the default settings. Networks were graphically prepared and provided with information on the countries and hosts in Network Publisher v.184.108.40.206 (Fluxus Technology) and finalized with CorelDRAW 2021 (Corel, Ottawa, ON).
In total, the questionnaire was filled out by 183 participating veterinarians. Of these, 16 practitioners stated that they had detected T. callipaeda and specified the hosts as dogs (n = 11), cats (n = 2), and a horse (n = 1), from Burgenland (n = 5), Lower Austria (n = 1), Salzburg (n = 1), Styria (n = 5), and Vienna (n = 1). The report of Thelazia callipaeda in a horse came from Carinthia and was assumed to be a misidentification since horses have never been reported as hosts of T. callipaeda but rather as hosts of Thelazia lacrymalis [47, 48]. In total, 12 T. callipaeda specimens from six dogs were collected during the period 2015–2022. The dogs had not travelled abroad prior to diagnosis and originated from Styria (n = 2), Lower Austria (n = 1), Vienna (n = 1), and Burgenland (n = 2) (Fig. 1).
Phortica variegata (n = 45) was detected in five of the seven investigated provinces (Table 1; Fig. 1). Thirty-two specimens were caught in the fruit fly traps; of these, 17 were females, eight were males and seven were unidentified (Table 1). Eight fruit flies were found in the eye of a 2-year-old male Doberman Pinscher from Burgenland (Fig. 1; Table 1). Both eyes of this dog showed ocular discharge, which had started 2 weeks previously. It was treated with a combination compound containing moxidectin (2.5 mg/ kg BW) and imidacloprid (10 mg/kg BW) Spot-On (Advocate; Bayer, Leverkusen, Germany) as well as with a topical ointment containing tobramycin and dexamethasone (Tobradex; Novartis, Basel, Switzerland). The dog continued to show ocular discharge and was presented at the surgery after another week. Further examination revealed the presence of dead fruit flies in the conjunctival sac of both eyes. The flies were removed using cotton swabs, after which the ocular discharge resolved.
The sequences obtained in this study were uploaded to BoldSystems (process identifiers PAVEA165-22–PAVEA176-22, PAVEA183-22, PAVEA184-23–PAVEA227-23) and GenBank (accession numbers OP620892–OP620903, OQ507612, OQ359791–OQ359834, and OQ689078).
All T. callipaeda corresponded to haplotype 1, which is the only haplotype that has been found in Europe so far (Fig. 2). In the fruit flies, 43 sequences could be assigned to P. variegata, with a total of 22 different haplotypes. In the case of one fruit fly from a dog’s eye, it was not possible to obtain a sequence of sufficient quality and it was therefore excluded from the phylogenetic analysis. One sequence was different from those of P. variegata and more similar to those of species previously found in Asia, such as Phortica chi and Phortica okadai (Fig. 3). Analysis of this sample using a different region of the COI gene showed 99.67% similarity to one P. okadai (GenBank accession number EU431942), 92.16% similarity to another P. okadai (GenBank accession number: EF576924), and only 93.46% and 92.32% similarity to Phortica variegata and Phortica semivirgo, respectively (GenBank accession numbers MK659848 and EF576935, respectively). Thelazia callipaeda could not be detected in any of the Phortica specimens.
Thelazia callipaeda and its vector P. variegata were found in different parts of Austria in the present study. Although P. variegata had been previously reported from Burgenland, Lower Austria, Styria, and Vienna [35,36,37], it was detected for the first time here in Upper Austria. New areas in the distributions of P. variegata and T. callipaeda were identified in the present study, but the presence and absence of the parasite in Austria could not be seamlessly mapped, as not all Austrian provinces were sampled and the locations of the traps were based on the presumed suitability of the habitats for the host species and not according to a systematic grid.
Female P. variegata were mainly caught in the fruit fly traps, and males predominantly around the eyes of the dogs. The preference of male P. variegata for the eye was not unexpected as this has also been observed in other studies, and only male P. variegata are considered to act as vectors of T. callipaeda [25, 27, 49]. To the best of our knowledge, clinical signs caused by the presence of P. variegata fruit flies in the conjunctival sac of a dog have not been reported up until now. It is likely that the obtained fruit flies were caught in the eyes while feeding on lacrimal fluid.
As expected, only T. callipaeda haplotype 1, which is the only haplotype detected in Europe to date, was found in Austria. In contrast, there is a high haplotype diversity in Asia [21, 50]. It is presumed that T. callipaeda is not native to Europe and that its introduction into Europe occurred as a single event. This hypothesis is also supported by the fact that T. callipaeda was first reported in Italy in 1989, after which it spread to other European countries due to the presence of P. variegata, which acts as an intermediate host [25, 51,52,53].
In both Europe and Asia, T. callipaeda is commonly found in dogs, although several wild animals have also been reported as hosts in Europe. Interest in this parasite has increased since its presence in Europe was first reported, and reports of it presence in new hosts are probably partly due to increased research efforts. Recent reports indicate that it is likely that wild animals in Asia are also frequently infected [10, 11]. Although there are case reports of human infections with T. callipaeda in Europe, these are more common in Asia. The infection rate in animals in Europe is probably not yet high enough to lead to many human cases. However, this may change in the future if this parasite becomes more prevalent in Europe [3, 18]. That the current prevalence of T. callipaeda in Austria is probably low was indicated by the low number of reported clinical cases of thelaziosis in the present study and the fact that none of the investigated Phortica fruit flies were positive for this parasite.
Many species within the P. variegata complex are not monophyletic at the COI barcoding region. However, P. variegata sensu stricto was shown to be monophyletic in both a previous study  and in the present one. Two haplotypes of P. variegata have been reported in the USA and 23 different haplotypes in Europe, including the 20 new ones reported in this study. The diversity of haplotypes found in Austria can be attributed to the fact that this fruit fly has long been native to Europe .
The COI barcoding sequence that differed from the sequence of P. variegata was more closely related to those of P. chi and P. okadai, which have not yet been reported from Europe. These latter two species are not monophyletic or clearly separated from their closely related morphospecies or cryptic species, and therefore delineating them through use of the COI gene is limited [54, 55]. Phortica chi and P. okadai have only been reported from Asia, but P. semivirgo, another species of the P. variegata complex, has been found in Europe [29, 56, 57]. Since no reference sequence of the COI barcode region was available at the time of analysis, another region of the COI gene was additionally analysed to determine whether the sample might be from a P. semivirgo specimen. While the sequence was 99.67% similar to one reported P. okadai sequence (GenBank accession number EU431942) it was not closely related to one reported for P. semivirgo (GenBank accession number EF576935), and was only 92.16% similar to the P. okadai sequence (GenBank accession number EF576924) used in a phylogenetic study comparing European Phortica spp. .
Further analysis of Phortica spp. with the use of additional genetic markers is needed to clarify the significance of the new sequence found in the present study and to assess its occurrence in other parts of Europe. Thelazia callipaeda, as well as its vector P. variegata, can be considered endemic in Austria.
Availability of data and materials
Miterpáková M, Trbolová A, Hurníková Z, Balicka A, Čabanová V, Valentová D, et al. Thelazia callipaeda in Slovakia—from sporadic cases to endemic areas. Parasitol Int. 2022;87:102495. https://doi.org/10.1016/j.parint.2021.102495.
Gama A, Pires I, Canado M, Coutinho T, Lopes AP, Latrofa MS, et al. First report of Thelazia callipaeda infection in wild European rabbits (Oryctolagus cuniculus) in Portugal. Parasit Vectors. 2016;9:236. https://doi.org/10.1186/s13071-016-1526-1.
Marino V, Gálvez R, Montoya A, Mascuñán C, Hernández M, Barrera JP, et al. Spain as a dispersion model for Thelazia callipaeda eyeworm in dogs in Europe. Prev Vet Med. 2020;175:104883. https://doi.org/10.1016/j.prevetmed.2020.104883.
Otranto D, Mendoza-Roldan JA, Dantas-Torres F. Thelazia callipaeda. Trends Parasitol. 2021;37:263–4. https://doi.org/10.1016/j.pt.2020.04.013.
Morgado ACT, Vale B, Ribeiro P, Coutinho T, Santos-Silva S, de Sousa Moreira A, et al. First report of human Thelazia callipaeda infection in Portugal. Acta Trop. 2022;231:106436. https://doi.org/10.1016/j.actatropica.2022.106436.
Shen J, Gasser RB, Chu D, Wang Z, Yuan X, Cantacessi C, et al. Human thelaziosis—a neglected parasitic disease of the eye. J Parasitol. 2006;92:872–5. https://doi.org/10.1645/GE-823R.1.
Papadopoulos E, Komnenou A, Karamanlidis AA, Bezerra-Santos MA, Otranto D. Zoonotic Thelazia callipaeda eyeworm in brown bears (Ursus arctos): a new host record in Europe. Transbound Emerg Dis. 2022;69:235–9. https://doi.org/10.1111/tbed.14414.
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. https://doi.org/10.1016/j.vetpar.2009.08.027.
Bezerra-Santos MA, Moroni B, Mendoza-Roldan JA, Perrucci S, Cavicchio P, Cordon R, et al. Wild carnivores and Thelazia callipaeda zoonotic eyeworms: a focus on wolves. Int J Parasitol Parasites Wildl. 2022;17:239–43. https://doi.org/10.1016/j.ijppaw.2022.03.005.
Jin Y, Liu Z, Wei J, Wen Y, He N, Tang L, et al. A first report of Thelazia callipaeda infection in Phortica okadai and wildlife in national nature reserves in China. Parasit Vectors. 2021;14:13. https://doi.org/10.1186/s13071-020-04509-0.
Doi K, Tokiwa T, Imoto M, Chou S, Yamasaki F, Kato T, et al. Molecular characterization of oriental eyeworm (Thelazia callipaeda) detected from raccoon (Procyon lotor) and Japanese raccoon dog (Nyctereutes viverrinus) in Kanto region, Japan. Parasit Vectors. 2023;16:116. https://doi.org/10.1186/s13071-023-05736-x.
Bertos E, Sánchez-Cerdá M, Virgós E, Gil-Sánchez JM, Moleón M. Thelazia callipaeda as a potential new threat to european wildcats: insights from an eco-epidemiological study. Vet Res Commun. 2023. https://doi.org/10.1007/s11259-023-10071-8.
Otranto D, Giannelli A, Scotty Trumble N, Chavkin M, Kennard G, Latrofa MS, et al. Clinical case presentation and a review of the literature of canine onchocercosis by Onchocerca lupi in the United States. Parasit Vectors. 2015;8:89. https://doi.org/10.1186/s13071-015-0699-3.
Otranto D, Solari Basano F, Pombi M, Capelli G, Nazzari R, Falsone L, et al. Effectiveness of the spot-on combination of moxidectin and imidacloprid (Advocate®) in the treatment of ocular thelaziosis by Thelazia callipaeda in naturally infected cats. Parasit Vectors. 2019;12:25. https://doi.org/10.1186/s13071-018-3262-1.
Motta B, Schnyder M, Basano FS, Nägeli F, Nägeli C, Schiessl B, et al. Therapeutic efficacy of milbemycin oxime/praziquantel oral formulation (Milbemax®) against Thelazia callipaeda in naturally infested dogs and cats. Parasit Vectors. 2012;5:85. https://doi.org/10.1186/1756-3305-5-85.
Otranto D, Colella V, Crescenzo G, Solari Basano F, Nazzari R, Capelli G, et al. Efficacy of moxidectin 2.5% and imidacloprid 10% in the treatment of ocular thelaziosis by Thelazia callipaeda in naturally infected dogs. Vet Parasitol. 2016;227:118–21. https://doi.org/10.1016/j.vetpar.2016.07.035.
Bezerra-Santos MA, Mendoza-Roldan JA, Sgroi G, Lia RP, Venegoni G, Solari Basano F, et al. Efficacy of a formulation of sarolaner/moxidectin/pyrantel (Simparica Trio®) for the prevention of Thelazia callipaeda canine eyeworm infection. Parasit Vectors. 2022;15:370. https://doi.org/10.1186/s13071-022-05501-6.
Palfreyman J, Graham-Brown J, Caminade C, Gilmore P, Otranto D, Williams DJL. Predicting the distribution of Phortica variegata and potential for Thelazia callipaeda transmission in Europe and the United Kingdom. Parasit Vectors. 2018;11:272. https://doi.org/10.1186/s13071-018-2842-4.
Vale B, Lopes AP, Conceição Fontes M, Silvestre M, Cardoso L, Coelho AC. Thelaziosis due to Thelazia callipaeda in Europe in the 21st century-a review. Vet Parasitol. 2019;275:108957. https://doi.org/10.1016/j.vetpar.2019.108957.
Vale B, Lopes AP, Conceição Fontes M, Silvestre M, Cardoso L, Coelho AC, et al. Systematic review on infection and disease caused by Thelazia callipaeda in Europe: 2001–2020. Parasite. 2020;27:52. https://doi.org/10.1051/parasite/2020048.
Zhang X, Shi YL, Han LL, Xiong C, Yi SQ, Jiang P, et al. Population structure analysis of the neglected parasite Thelazia callipaeda revealed high genetic diversity in eastern Asia isolates. PLoS Negl Trop Dis. 2018;12:e0006165. https://doi.org/10.1371/journal.pntd.0006165.
Otranto D, Lia RP, Cantacessi C, Testini G, Troccoli A, Shen JL, et al. Nematode biology and larval development of Thelazia callipaeda (Spirurida, Thelaziidae) in the drosophilid intermediate host in Europe and China. Parasitology. 2005;131:847–55. https://doi.org/10.1017/S0031182005008395.
Arme TM, Lia RP, Annoscia G, Casalino E, Pombi M, Otranto D. Survival of Phortica variegata experimentally and naturally infected with Thelazia callipaeda. Med Vet Entomol. 2020;34:201–6. https://doi.org/10.1111/mve.12419.
Bezerra-Santos MA, Bernardini I, Lia RP, Mendoza-Roldan JA, Beugnet F, Pombi M, et al. Phortica oldenbergi (Diptera: Drosophilidae): a new potential vector of the zoonotic Thelazia callipaeda eyeworm. Acta Trop. 2022;233:106565. https://doi.org/10.1016/j.actatropica.2022.106565.
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. https://doi.org/10.1016/j.ijpara.2006.06.006.
Marino V, Gálvez R, Colella V, Sarquis J, Checa R, Montoya A, et al. Detection of Thelazia callipaeda in Phortica variegata and spread of canine thelaziosis to new areas in Spain. Parasit Vectors. 2018;11:195. https://doi.org/10.1186/s13071-018-2773-0.
Roggero C, Schaffner F, Bächli G, Mathis A, Schnyder M. Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland. Vet Parasitol. 2010;171:58–67. https://doi.org/10.1016/j.vetpar.2010.03.012.
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.
Otranto D, Stevens JR, Testini G, Cantacessi C, Máca J. Molecular characterization and phylogenesis of Steganinae (Diptera, Drosophilidae) inferred by the mitochondrial cytochrome c oxidase subunit 1. Med Vet Entomol. 2008;22:37–47. https://doi.org/10.1111/j.1365-2915.2008.00714.x.
Cao H, Wang X, Gao J, Prigent SR, Watabe H, Zhang Y, et al. Phylogeny of the African and Asian Phortica (Drosophilidae) deduced from nuclear and mitochondrial DNA sequences. Mol Phylogenet Evol. 2011;61:677–85. https://doi.org/10.1016/j.ympev.2011.08.002.
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. https://doi.org/10.1111/j.1365-2915.2006.00643.x.
Pombi M, Marino V, Jaenike J, Graham-Brown J, Bernardini I, Lia RP, et al. Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations. Parasit Vectors. 2020;13:89. https://doi.org/10.1186/s13071-020-3955-0.
Hinney B. Thelazia callipaeda bei Hunden aus Österreich. KompaktVET. 2016:12.
Hodžić A, Payer A, Duscher GG. The first autochthonous case of feline ocular thelaziosis in Austria. Parasitol Res. 2019;118:1321–4. https://doi.org/10.1007/s00436-019-06275-0.
Basden EB. Type collections of Drosophilidae (Diptera). I. The Strobl Collection. Contrib Entomol 1961. https://doi.org/10.21248/contrib.entomol.11.1-2.160-224.
Bächli G. Die Drosophiliden-Arten (Diptera) in der Sammlung des Naturhistorischen Museums Wien. Ann Naturhist Mus Wien. 1988;90:131–48.
Schlüsslmayr G. Erstnachweise und bemerkenswerte Funddaten von Fliegen (Diptera, Brachycera) für Österreich und seine Bundesländer III. Linzer Biol Beitr. 2021;53:293–412.
Casiraghi M, Anderson TJ, Bandi C, Bazzocchi C, Genchi C. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology. 2001;122:93–103. https://doi.org/10.1017/s0031182000007149.
Hodžić A, Alić A, Fuehrer H-P, Harl J, Wille-Piazzai W, Duscher GG. A molecular survey of vector-borne pathogens in red foxes (Vulpes vulpes) from Bosnia and Herzegovina. Parasit Vectors. 2015;8:88. https://doi.org/10.1186/s13071-015-0692-x.
Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W. Ten species in one: DNA barcoding reveals cryptic species in the Neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci U S A. 2004;101:14812–7. https://doi.org/10.1073/pnas.0406166101.
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3:294–9.
Lunt DH, Zhang DX, Szymura JM, Hewitt GM. The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol Biol. 1996;5:153–65. https://doi.org/10.1111/j.1365-2583.1996.tb00049.x.
Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80. https://doi.org/10.1093/molbev/mst010.
Xia X, Xie Z. DAMBE: software package for data analysis in molecular biology and evolution. J Hered. 2001;92:371–3. https://doi.org/10.1093/jhered/92.4.371.
Trifinopoulos J, Nguyen L-T, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016;44:W232–5. https://doi.org/10.1093/nar/gkw256.
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61:539–42. https://doi.org/10.1093/sysbio/sys029.
Beelitz P, Dongus H, Schöl H, Gerhards H, Gothe R. Thelazia lacrymalis (Nematoda, Spirurida, Thelaziidae): report in a horse in Germany and contribution to the morphology of adult worms. Parasitol Res. 1997;83:627–31. https://doi.org/10.1007/s004360050309.
Giangaspero A, Tieri E, Otranto D, Battistini ML. Occurrence of Thelazia lacrymalis (Nematoda, Spirurida, Thelaziidae) in native horses in Abruzzo region (central eastern Italy). Parasite. 2000;7:51–3. https://doi.org/10.1051/parasite/2000071051.
Máca J, Otranto D. Drosophilidae feeding on animals and the inherent mystery of their parasitism. Parasit Vectors. 2014;7:516. https://doi.org/10.1186/s13071-014-0516-4.
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. https://doi.org/10.1016/j.mcp.2005.05.001.
Rossi L, Bertaglia PP. Presence of Thelazia callipaeda Railliet & Henry, 1910, in Piedmont Italy. Parassitologia. 1989;31:167–72.
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. https://doi.org/10.1186/1756-3305-4-148.
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. https://doi.org/10.1016/j.vetpar.2007.08.005.
Huang J, Gong LU, Tsaur S-C, Zhu L, An K, Chen H. Revision of the subgenus Phortica (sensu stricto) (Diptera, Drosophilidae) from East Asia, with assessment of species delimitation using DNA barcodes. Zootaxa. 2019. https://doi.org/10.11646/zootaxa.4678.1.1.
Zhu L, Cao H, Chen H. Phortica eparmata species complex (Diptera, Drosophildae) from the oriental region, with DNA barcoding information of Chinese species. EJT. 2018. https://doi.org/10.5852/ejt.2018.403.
Bächli G, Bystrowski C, Richter VA. Fauna Europaea: Phortica. In: Pape T, (Ed). Fauna Europaea: Drosophilidae; 2013.
Lutovinovas E, Ivinskis P, Rimšaitė J. Phortica semivirgo (Máca, 1977)—new to the fauna of lithuania (Diptera: Drosophilidae). Bulletin Lithuanian Entomol Soc. 2020;4:109–13.
The authors would like to thank Eva Spallinger, Dagmar Grohe, Sandra Utzmann, Hemma Schichl, Tina Jaeger, Jana Schmitt, Christian Zainzinger, Peter Übersberger, Jutta Schlapschy, Angelika Köppel, and Gudrun Immler for their participation in the collection of the fruit flies; Albert Payer and Beatrix Grund for sending in eye worm specimens; and Bita Shahi Barogh for technical support.
This study was partially funded by Boehringer Ingelheim.
Ethics approval and consent to participate
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. No ethical approval was required as the obtained samples were gathered during procedures that were deemed of medical necessity. The pet owners were informed and gave their consent for the publication of the data obtained.
The authors declare that they have no competing interests.
Consent for publication
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Bayesian interference (BI) tree featuring mitochondrial cytochrome c oxidase subunit I gene (COI; 617 nucleotide positions) sequences of Thelazia spp. Nodes are marked with BI posterior probabilities and maximum likelihood bootstrap values. Clades which are marked in red were used for calculation of the median-joining haplotype (hpt) network containing the sequences obtained in this study. Scale bar indicates the expected mean number of substitutions per site according to the model of sequence evolution applied.
BI tree featuring COI (647 nucleotide positions) sequences of Phortica sensu stricto. Nodes are marked with BI posterior probabilities and maximum likelihood bootstrap values. Clades which are marked in red were used for calculation of the median-joining hpt network containing the sequences obtained in this study. Scale bar indicates the expected mean number of substitutions per site according to the model of sequence evolution applied.
About this article
Cite this article
Unterköfler, M.S., Dengg, P., Niederbacher, M. et al. Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis. Parasites Vectors 16, 294 (2023). https://doi.org/10.1186/s13071-023-05913-y
- Oriental eye worm
- Canine thelaziosis
- Zoophilic fruit fly
- Vector-borne disease
- Emerging zoonotic disease
- Cytochrome c oxidase subunit I