Systematic review on infection and disease caused by Thelazia callipaeda in Europe: 2001–2020

Cases of thelaziosis by Thelazia callipaeda have been increasing considerably in Europe throughout the 21st century, with recent emphasis on Eastern Europe. A systematic review was conducted using defined search terms across three major databases and, additionally, with the examination of the references of the 56 articles selected. Available information about epidemiological and clinical features of all cases of thelaziosis by T. callipaeda in companion animals, wildlife and humans was extracted, evaluated and subjected to qualitative and quantitative analysis. In all cross-sectional studies about dogs, cats and red foxes, males were more frequently infected than females (dogs: p = 0.0365; cats: p = 0.0164; red foxes: p = 0.0082). Adult dogs seem to be more prone to infection (p < 0.0001), as well as large-sized dogs (p < 0.0001), and companion animals that live exclusively outdoors (p < 0.0001). Dogs and red foxes involved in these cross-sectional studies harboured significantly more female than male nematodes (p < 0.0001). Thelaziosis by T. callipaeda is far from controlled in Europe. Only through updated epidemiological data, knowledge improvement and awareness can correct diagnosis and appropriate treatment and prevention be ensured to tackle this zoonosis.

Thelazia callipaeda is also known as "the oriental eyeworm" because of its distribution throughout the former Soviet Union and East Asia [1,59,70]. Nevertheless, at the end of the 20th century, autochthonous cases were reported in Italy [50,52] and, since then, T. callipaeda has increasingly been reported in some European countries both in animals and humans [75]. In Europe, the intermediate host of this eyeworm is the male drosophilid fruit fly Phortica variegata Fallén, 1823 (Drosophilidae, Steganinae), which feeds on lachrymal secretions of mammals [53,55,58,60].
There are reported cases of thelaziosis in companion animals (dogs and cats), lagomorphs such as hares (Lepus europaeus) and wild rabbits (Oryctolagus cuniculus), but also in wild carnivores, especially red foxes (Vulpes vulpes), which appear to play an important role in the introduction and geographical dispersion of this eyeworm in non-endemic European regions [15,23,32,44,51,57].
The importance of studying and investigating thelaziosis lies in the fact that T. callipaeda has a broad spectrum of hosts and the number of infected hosts, including humans, in Europe has been increasing since the beginning of the 21st century. Taking into account that thelaziosis is an expanding disease, it becomes necessary to identify the characteristics of all the cases reported, in order to profile this zoonosis. Therefore, the objective of the present study was to carry out a qualitative and quantitative analysis based on a systematic review of the scientific literature. Consequently, it is expected that this compilation of all reported cases will be useful in future investigations on understanding the evolution of thelaziosis by T. callipaeda.

Study design
The present study consisted of a systematic review of the literature in order to answer the following research question: "What are the epidemiological and clinical features as well as the prevalence of thelaziosis by T. callipaeda reported in companion animals, wildlife and humans in Europe in the 21st century?" This study was conducted based on the methodological recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [46].

Article eligibility
Articles published in indexed journals cited in PubMed, ScienceDirect and Web of Science were considered eligible if they consisted of case reports or cross-sectional studies describing clinical and epidemiological features (including species, prevalence, gender, age, country, geographical area, affected eye, and ocular signs). There were other restrictions regarding the eligibility criteria. Only studies published between 1 January 2001 and 10 July 2020, and written in the languages of Western Europe (comprising English, French, German, Italian, Portuguese and Spanish) were included.
The types of publications included original articles, short communications and case reports that addressed issues within the following criteria: (i) information on the clinical presentation of thelaziosis by T. callipaeda in companion animals, wild animals and humans; and (ii) prevalence of the disease in canine, feline and red fox populations.
Reviews of the literature, research notes, editorials, experimental essays, textbook chapters, posters, abstracts, articles with no primary data and dissertations along with unpublished studies and data were excluded.

Information sources and search strategies
The process of identifying articles in indexed journals was developed using the PubMed, ScienceDirect and Web of Science databases. The combination of search terms in English applied included: {Europe AND (Thelazia callipaeda OR thelaziosis OR thelaziasis OR thelazi*)}. To prevent missing data, references of retrieved publications were also checked in order to identify additional papers. The searches were conducted between March and July 2020.

Selection of studies and data extraction
After a comprehensive systematic searching, a bibliographic manager tool (Zotero version 5.0.88) [65] was used to exclude duplicate records. Then, two independent reviewers selected articles based on their titles and abstracts, followed by a full reading of the text when the title or abstract met the inclusion criteria or could not be rejected with certainty. Any disagreements or divergences were resolved by discussion and consensus.
Two researchers extracted the required data and added the information on an electronic spreadsheet, dividing them into three groups: (i) companion animals (dog, cat, and domestic rabbit), (ii) wildlife, and (iii) humans. The qualitative data about companion animals comprised references (country, authors and year of publication), case report data (host species, breed, age, gender, lifestyle, and affected eye), number of infected animals, number of adult nematodes and ocular signs (Table 1). With regard to wildlife, information was extracted about the authors, year of publication, country, case report (species, gender and affected eye), number of infected animals, number of adult nematodes and ocular signs ( Table 2). For cases of human thelaziosis, references (country, region, data on authors and year of publication), gender, age, affected eye, number of infected hosts, number of adult nematodes and ocular symptoms were collected (Table 3). In turn, the quantitative data extracted from the second group of articles (cross-sectional studies) comprised the references (authors and year of publication), country where the study was conducted, species (dogs, cats and red foxes) and their identification, sample size, number of positive animals, and prevalence (%).

Data analysis
The data collected were analysed using IBM SPSS Statistics 26 statistical software [30]. A Chi-square test was used to test associations between the parameters. Statistical significance was considered if p value was < 0.05.

Results
The identification and study selection are represented in Figure 1. Articles that did not meet the inclusion criteria, as well as duplicates and incomplete articles or studies not available  online or in other sources, were excluded. Thus, out of the 363 studies searched, 56 met the eligibility criteria and were divided according to the subject they addressed: epidemiological and clinical characteristics of thelaziosis (n = 44), which were included in the qualitative analysis; studies with relevant data to both qualitative and quantitative analysis (n = 4); or the prevalence of thelaziosis by T. callipaeda in dogs, cats and red foxes (n = 8), which were considered to be crosssectional (prevalence) studies with data for quantitative analysis.

Qualitative analysis of the epidemiological and clinical aspects of T. callipaeda infection in companion animals
The 31 studies included in the qualitative analysis of thelaziosis in companion animals had been conducted in 19 different countries (Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Czech Republic, France, Germany, Greece, Hungary, Moldova, Portugal, Romania, Serbia, Slovakia, Spain, Switzerland, Turkey and the United Kingdom) ( Table 1). They reported occurrence of T. callipaeda infection in dogs (n = 1343), cats (n = 20), and a rabbit (n = 1), in a total of 1364 animals ( Table 1).
The studies reported the gender of 206 dogs, of which 76 were female (36.9%) and 130 were male (63.1%); 16 cats, of which six were female (37.5%) and 10 were male (62.5%); and the rabbit, which was male.
The studies reported the gender of all animals, except five red foxes. Out of the five beech martens, three were female (3/5, 60%) and two were male (2/5, 40%); the three brown hares were female, the European badger was female and the golden jackal was male; of the three red foxes whose gender was reported, one was female (1/3, 33.3%) and two were male (2/3, 66.7%); one wild rabbit was female and the other one was male; out of the four wildcats, one was female (1/4; 25%) and three were male (3/4; 75%); out of the 14 wolves, four were female (4/14; 28.6%) and the remaining 10 were male (10/14; 71.4%).
A total of 615 specimens of T. callipaeda were collected from the wildlife mentioned in the studies, and the gender of the nematodes was identified in 610 specimens ( 29.0%) in all species, with the exception of wildcats, in which the number of female and male nematodes was equal (9 female and 9 male) and the European badger, in which more males than females were counted (female: 10/33, 30.3%; male: 23/33, 69.7%).
Out of the 11 studies reporting thelaziosis in wildlife, only one study described ocular signs (conjunctivitis) on red foxes ( Table 2).

Qualitative analysis of the epidemiological and clinical aspects of T. callipaeda infection in humans
The seven studies included in the qualitative synthesis of T. callipaeda infection in humans reported 11 cases in six different countries: Croatia (n = 1), France (n = 1), Germany (n = 1), Italy (n = 3), Serbia (n = 1) and Spain (n = 4).
A total of 17 specimens of T. callipaeda were collected from the human cases mentioned in five out of the seven studies. However, the gender of nematodes was only identified in eight specimens, in which four were female (4/8, 50.0%) and four were male (4/8, 50.0%).

Quantitative analysis of the epidemiological and clinical aspects of T. callipaeda infection in dogs, cats and red foxes
Regarding canine thelaziosis, seven studies were carried out in Italy, Portugal, Serbia, Spain and Switzerland (Table 4). Of a total of 659 positive dogs, the gender of 366 animals is known (females: 163, 44.5%; and males: 203, 55.5%).
To date, there are only two cross-sectional studies referring to thelaziosis by T. callipaeda in cats. These studies were conducted in Portugal and Switzerland and their prevalences were 23.5% and 0.8%, respectively (Table 4). They reported the occurrence of T. callipaeda infection in 21 cats (females: 5, 23.8%; males: 16, 76.2%).
Regarding wildlife, five cross-sectional studies have already been carried out in order to determine the prevalence of T. callipaeda infection in red foxes. These studies took place in Bosnia and Herzegovina, Italy, Romania, Slovakia and Switzerland (Table 4). Most red foxes came from legal hunts during rabies-monitoring programmes, so their eye examination occurred post-mortem at necropsy. Two hundred and fifty-three positive red foxes were reported, of which 105 were female (105/252, 41.7%) and 147 were male (147/252, 58.3%) (the gender of one red fox was unknown).
Thelazia-positive dogs from quantitative analysis (crosssectional studies) indicated a significantly higher occurrence in male dogs (p = 0.0365). The same scenario was observed in cats (p = 0.0164) and red foxes (p = 0.0082).
The lifestyle of 515 dogs was reported, and those that lived strictly outdoors (482/515, 93.6%) were found to be significantly more infected than those that lived inside and outside the house (33/515, 6.4%) (p < 0.0001). Regarding the 17 cats with information about their lifestyle, all of them lived between indoors and outdoors.
Taking into account the information available for the "infected eye", it was found that in 33 dogs both eyes were infected ( Table 5). Dogs and red foxes involved in these cross-sectional studies harboured significantly more female than male nematodes (p < 0.0001), whereas in cats this difference was not statistically significant (p = 0.2253). It is important to note that the gender of all parasitic specimens collected from red foxes was not determined (total: 391/3925, 10.0%).
All studies reported a sex ratio in favour of females. Also, the intensity of infection with adult forms of T. callipaeda per study shows high variability, as indicated in Table 5.

Discussion
The occurrence of T. callipaeda infection was significantly higher in male dogs, cats and red foxes from cross-sectional studies, and an equal outcome was observed in the qualitative analysis. In addition, there is no evidence of a host sex predisposition, but there are contradictory results concerning this, as has been shown in previous reports [40,45,52]. In relation to cats, it is known that in the samples from the study by Motta et al. [47] all the hosts were male and had outdoor access, which could be risk factors for T. callipaeda infection, possibly due to their territorial and hunting behaviour, but also because of a wider roaming area. Perhaps a similar situation regarding the possible male predisposition in red foxes may be assumed, given that the quantitative analysis has shown that male foxes were significantly more affected than female foxes.
It was observed that the age group corresponding to the adult phase (1-8 years old) was the most affected, having significant differences between the three age groups of dogs. This may be due to the fact that adults potentially had more access to outdoor spaces than juvenile and elderly dogs, a circumstance that predisposed them to contact with P. variegata. This result is in line with that observed in cats [47], as well as in the qualitative analysis of the present study. Nevertheless, some studies have not reported significant differences in infected animals when ages were compared [45,52]. In relation to the possible breed effect, on the quantitative analysis, there were statistical significant differences regarding the breed, with crossbred dogs being more frequently infected than purebred dogs. Nonetheless, given that the sample consisted of only 43 dogs, it is not possible to confirm that crossbred dogs are more susceptible to infection than purebred dogs. In addition, there is no article that points out in that direction and whose qualitative analysis shows a different perspective. Likewise, in previous reports, no effect of breed was detected [45,52]. Nevertheless, when it comes to aptitude/ management, there seems to be a greater tendency for crossbred or purebred dogs, whose aptitude is shepherd or hunting, being infected by T. callipaeda, since they have a higher possibility for physical contact with the vector while outside in the forest environment [5,8,39].
Another feature that appears to play an important role in infection with Thelazia is body size. Small-sized dogs have been found to be less frequently infected than medium-sized dogs, followed by large-sized dogs. This can be explained by the fact that most large-sized dogs are usually housed outdoors, a circumstance that increases their exposure to the intermediate hosts [40,45,75]. Additionally, it is accepted that a larger body surface also favours physical contact with P. variegata [40]. In contrast, the lower prevalence in small-sized dogs and in cats may be due to their small body mass index, which apparently makes them less attractive to the intermediate host. Moreover, cats eliminate eye discharges through their intensive cleaning habits, therefore losing their decoy to flies [40,47,52]. In addition, feline infection seems to be underestimated because of difficulties experienced by veterinarians inspecting cats' eyes [15,47].
As already reported, lifestyle seems to be an important feature in the occurrence of Thelazia infection. In fact, animals that frequently (or exclusively) live outdoors are more highly exposed to P. variegata flies. Similarly, places where there is physical contact between animals, which are potential hosts, seem to attract the intermediate host [39,45].
In the three species submitted to a quantitative analysis, it was found that the left eye was the most frequently affected. However, these results come from few sources and represent a small sample [6,25,28,31,40,47,57]. Consequently, it is not possible to compare or infer about eye predisposition. Moreover, to the best of our knowledge, there is no scientific article that has identified the existence of a pattern with respect to the most affected eye by T. callipaeda.
Thelaziosis has already been described, based on cases reports and prevalence studies, in several European countries, having as the common denominator the zoophilic fruit fly P. variegata, which is the intermediate host of T. callipaeda. All cases reported have occurred in regions characterised by similar altitude (800-1000 a.s.l) and climate and habitat conditions: continental Mediterranean climate, and cultivated areas and deciduous woods, which fall within the geoclimatic model for the distribution of P. variegata [54].
A higher prevalence of T. callipaeda infection, especially in dogs and red foxes, as described in Spain [41,45] or Italy [57], suggests a stable endemic condition [42] (Table 4). A lower prevalence of T. callipaeda, such as in Portugal [39] or Slovakia [6], could be associated with the recent emergence of the infection at the local level [42] (Table 4). However, this outcome is expected to be underdiagnosed and/or underreported. The number of companion animals and wildlife (not only red foxes) positive to T. callipaeda might be considerably higher in the mentioned countries, but also in other European countries that have not yet performed prevalence studies. It is important to raise awareness about the need to perform these studies in order to understand the European reality of this zoonosis.
Canine thelaziosis was firstly reported in southern Europe, in Italy, and then in western Europe and the Balkan area. Subsequently, more and more cases appeared in eastern Europe, and it is evident that this zoonosis is already established in Central Europe [5,10,29,34,35,38,51]. The spread of Table 5. Gender identification and intensity of T. callipaeda infection in the eyes of dogs, cats and red foxes from the cross-sectional studies.

Species
References Positive animals

Thelazia callipaeda
Intensity of infection thelaziosis in endemic regions in Europe, but also to previously non-endemic areas, has been linked to the migration of infected wild animals, especially red foxes as well as wolves, jackals and other wildlife. This demonstrates the role of free-ranging wild carnivores as reservoirs of T. callipaeda and highlights the importance of the sylvatic cycle, especially in rural areas, where transmission to humans and domestic animals is facilitated [6,28,34,44,57]. However, the role of pets, especially dogs, in spreading the disease, should not be neglected [35,36]. The movement of pets when traveling with their owners within the European Union, but also the adoption and import of dogs from shelters from endemic regions are a crucial driver of Thelazia, but also for other pathogenic agents and their vectors [16,34]. Undoubtedly, these risk factors highlight the importance of preventive programmes [35,36] and surveillance polices to restrict cross-border spread of the nematode [34,75].

Conclusions
In this work, isolated cases of thelaziosis were summarized and an in-depth analysis of all cross-sectional studies was conducted. The reduced number of prevalence studies and the small sample per study were the main disadvantages found, as this made it difficult or even impossible to infer or determine the situation for certain features. However, this work shows the expansion potential of T. callipaeda and the urgent need for additional large-scale studies in order to provide information on the current situation in the European Union. Given the scarcity of papers on human health, the need to stress the importance of the One Health approach is sustained. Only through updated epidemiological data, knowledge improvement, and awareness can correct diagnosis and appropriate treatment and prevention of thelaziosis be ensured.