New host record of Thelazia callipaeda (Nematoda: Spirurida) with a notably wide host range and shared zoonotic lineage in Japan.

Thelazia callipaeda (Nematoda: Spirurida: Thelaziidae) parasitizes the eyes of dogs, cats, humans, and various wild mammals, and is transmitted by drosophilid flies. In Japan, T. callipaeda is considered an emerging parasite that has expanded its endemic region northward. However, reports of its detection in mammals other than domestic animals and humans are scarce. This study reports the detection of T. callipaeda in Japanese red fox (Vulpes vulpes japonica), masked palm civet (Paguma larvata), Japanese badger (Meles anakuma), Japanese black bear (Ursus thibetanus japonicus), raccoon (Procyon lotor), Japanese raccoon dog (Nyctereutes viverrinus), domestic dog (Canis lupus familiaris), domestic cat (Felis silvestris catus), and human. Of these, the Japanese red fox, masked palm civet, Japanese badger, and Japanese black bear have been reported as novel host records. Sequence analysis of the cytochrome c oxidase subunit I gene of T. callipaeda revealed two unique lineages specific to Japan, with no regional or host species differences. These results suggest a wide host range for T. callipaeda, highlighting the significant role of wildlife as a reservoir for this parasite in Japan.

The species and abundance of gut bacteria both positively impact Phortica okadai behavior.

BACKGROUND: Gut bacteria, which serve as essential modulators, exert a significant impact on insect physiology and behavior and have substantial application potential in pest management. The dynamics of gut bacteria and their impact on Phortica okadai behavior remain unclear. METHODS: In this study, the dynamics of gut bacteria at different developmental stages in P. okadai were analyzed using 16S ribosomal RNA (rRNA) gene sequencing, and the species and abundance of gut bacteria that affect host behavior were examined via behavioral experiments. RESULTS: A total of 19 phyla, 29 classes, 74 orders, 101 species, and 169 genera were identified. The results of the behavioral experiments indicated that the species Lactiplantibacillus argentoratensis, Acetobacter tropicalis, Leuconostoc citreum, and Levilactobacillus brevis effectively influenced the feeding preference of P. okadai, and the single-bacterium-seeded P. okadai exhibited feeding preferences distinct from those of the germ-free (GF) and wild-type P. okadai. CONCLUSIONS: The species and relative abundance of gut bacteria together positively impact P. okadai behavior. Lactiplantibacillus argentoratensis, as the most attractive bacteria to P. okadai, presents opportunities for novel pest control strategies targeting this vector and agricultural pest.

Human ocular thelaziasis with genetic analysis in Niigata Prefecture, Japan: A case report on an emerging zoonosis.

Purpose: We report the clinical findings and molecular identification of ocular Thelazia callipaeda from Niigata Prefecture in the Hokuriku area of Japan during winter. Observations: A 77-year-old male visited an ophthalmology clinic in Niigata Prefecture in January 2022 after a 2-week-duration of a conjunctival injection in the left eye and foreign body sensation. Slit-lamp microscopy revealed 11 active nematodes in the left conjunctival sac. Morphological characteristics included longer female body length than male, buccal cavity lacking teeth and lips, and serrated striations along the body surface. The specimens were determined to be T. callipaeda. Genetic analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene revealed an h9 haplotype. Conclusions and Importance: T. callipaeda infection, especially the h9 haplotype, commonly occurs in western Japan owing to its higher incidence in warmer climates, suggesting the origin of the case. Here, we report a human case of Thelaziasis diagnosed in a cold region of Japan (the Hokuriku area) during winter. This human case of T. callipaeda infection from a cold, previously unassociated region, raises concerns about the potential geographical widening of its distribution, and further investigation may be warranted to prevent its spread.

Efficacy of a combination of esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) against Thelazia callipaeda in naturally infected cats.

This clinical study assessed the efficacy of a topical combination of esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) in treating cats naturally infected with the eyeworm Thelazia callipaeda (Nematoda, Thelaziidae). On Study Day (SD) 0, sixteen client-owned cats with eyeworm infection were allocated to an untreated control group (G1, 8 cats) or to a NexGard® Combo treated group (G2, 8 cats) and subjected to ocular examination. Cats in G2 received the treatment as per label recommendations. On SD 7 and 14 (±1), cats were examined for the presence of eyeworms and clinical signs. On SD 14, eyeworms were collected and counted. On SD 7 and 14, all cats in G1 were still infected with eyeworms, while G2 cats were free from eyeworms on SD 7 and 14, demonstrating 100% efficacy (p < 0.0001). All collected eyeworms were morphologically and molecularly confirmed to be T. callipaeda. On SD 0, fifteen out of the sixteen cats (7 in G1 and 8 in G2) displayed inflammatory ocular signs. On SD 7, all eight untreated cats and seven treated cats displayed inflammatory ocular signs. On SD 14, five out of eight G2 treated cats had recovered, while the eight untreated cats still displayed inflammatory ocular signs. The treatment significantly reduced lacrimation and conjunctivitis (p = 0.0001). No adverse reactions occurred. This clinical study provides evidence that NexGard® Combo is highly safe and effective for the treatment of T. callipaeda infection in cats under field conditions.

Title: Efficacité d'une association d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard® Combo) contre Thelazia callipaeda chez le chat naturellement infecté. Abstract: Cette étude clinique a évalué l'efficacité d'une association topique d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard® Combo) dans le traitement des chats naturellement infectés par le ver oculaire Thelazia callipaeda (Nematoda, Thelaziidae). Au jour d'étude (JE) 0, seize chats appartenant à des clients et atteints d'une infection par le ver oculaire ont été attribués à un groupe témoin non traité (G1, 8 chats) ou à un groupe traité NexGard® Combo (G2, 8 chats) et soumis à un examen oculaire. Les chats du groupe G2 ont reçu le traitement conformément aux recommandations de l'étiquette. Aux JE 7 et 14 (±1), les chats ont été examinés pour détecter la présence de vers oculaires et de signes cliniques. Au JE 14, les vers oculaires ont été collectés et comptés. Aux JE 7 et 14, tous les chats du G1 étaient toujours infectés par des vers oculaires, tandis que les chats du G2 étaient exempts de vers oculaires aux JE 7 et 14, démontrant une efficacité de 100 % (p < 0,0001). Tous les vers oculaires collectés ont été confirmés morphologiquement et moléculairement comme étant T. callipaeda. Au JE 0, quinze chats sur seize (7 en G1 et 8 en G2) présentaient des signes oculaires inflammatoires. Au JE 7, les huit chats non traités et les sept chats traités présentaient des signes oculaires inflammatoires. Au JE 14, cinq des huit chats traités par G2 s'étaient rétablis tandis que les huit chats non traités présentaient toujours des signes oculaires inflammatoires. Le traitement a réduit de manière significative le larmoiement et la conjonctivite (p = 0,0001). Aucun effet indésirable n'est survenu. Cette étude clinique indique que NexGard® Combo est hautement sûr et efficace pour le traitement de l'infection à T. callipaeda chez les chats dans des conditions de terrain.

Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States.

We report 2 autochthonous feline thelaziosis cases caused by the eyeworm Thelazia callipaeda and discuss the spread among dogs in the northeastern United States. Phylogenetic analysis suggests the parasite was introduced from Europe. Adopting a One Health approach is needed to limit further spread of T. callipaeda eyeworms in North America.

[Epidemiological and clinical characteristics of human ocular helaziasis in China from 2011 to 2022 based on bibliometrics].

OBJECTIVE: To understand the clinical and epidemiological characteristics of human ocular thelaziasis patients in China. METHODS: Case reports regarding human ocular thelaziasis cases in China were retrieved in international and national electronic databases, including CNKI, VIP, CBM, Traditional Chinese Medical Literature Analysis and Retrieval System, Wanfang Database, PubMed and Web of Science from 2011 to 2022. Patients' gender, age, clinical symptoms, treatment, recurrence, site of infections, time of onset, affected eye, affected sites, number of infected Thelazia callipaeda, sex of T. callipaeda and source of infections were extracted for descriptive analyses. RESULTS: A total of 85 eligible publications were included, covering 101 cases of human ocular thelaziasis, including 57 males (56.44%) and 44 females (43.56%) and aged from 3 months to 85 years. The main clinical manifestations included foreign body sensation (56 case-times, 22.49%), eye itching (38 case-times, 15.26%), abnormal or increased secretions (36 case-times, 14.46%), tears (28 case-times, 11.24%) and eye redness (28 case-times, 11.24%), and conjunctival congestion (50 case-times, 41.67%) was the most common clinical sign. The most common main treatment (99/101, 98.02%) was removal of parasites from eyes using ophthalmic forceps, followed by administration with ofloxacin and pranoprofen. In publications presenting thelaziasis recurrence, there were 90 cases without recurrence (97.83%) and 2 cases with recurrence (2.17%). Of all cases, 51.96% were reported in four provinces of Hubei, Shandong, Sichuan, Hebei and Henan, and ocular thelaziasis predominantly occurred in summer (42.19%) and autumn (42.19%). In addition, 56.45% (35/62) had a contact with dogs. CONCLUSIONS: The human thelaziasis cases mainly occur in the continental monsoon and subtropical monsoon climate areas such as the Yellow River and the Yangtze River basin, and people of all ages and genders have the disease, with complex clinical symptoms and signs. Personal hygiene is required during the contact with dogs, cats and other animals, and individual protection is required during outdoor activities to prevent thelaziasis.

Eight cases of canine thelaziosis found in two localities in Korea.

Thelazia callipaeda, a parasitic nematode that causes thelaziosis in various mammals, including humans, is known to be endemic in Korea. However, life cycle-related information on the parasite, primarily from human infection and a few dog cases, is limited. This study reports additional cases of T. callipaeda infections in dogs from both rural and urban areas in Korea, indicating the potential for transmission to humans and other animals. We collected 61 worms from 8 infected dogs from Paju and Cheongju Cities and observed their morphological characteristics under a light microscope. The findings indicate that T. callipaeda infections in animals in Korea may be underestimated and are distributed close to human environments. Our results contribute to the growing knowledge of the reservoir hosts of T. callipaeda in Korea and highlight the importance of continued surveillance and research to prevent and control this emerging zoonotic disease.

First report of Thelazia callipaeda Railliet & Henry, 1910 in a zoo lion (Panthera leo) (Linnaeus, 1758) from Romania.

Thelazia callipaeda, the zoonotic "oriental eye worm", is an emerging nematode known to infect a wide range of hosts, particularly carnivores (wild and domestic canids and felids, mustelids, and ursids), but also other mammal groups (suids, lagomorphs, monkeys, and humans), across a wide geographical area. New host-parasite associations and human cases have mostly been reported in endemic areas. A less studied group of hosts is represented by zoo animals that may harbor T. callipaeda.A 23-year old male lion (Panthera leo) that was born in a public zoo in Reșița (Caraș-Severin County, Romania) was submitted to the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca for investigations after death from unknown reasons in November 2022. Four nematodes were collected from the right eye during the necropsy and submitted to morphological and molecular characterization.Collected nematodes were morphologically identified as three females and one male T. callipaeda. The BLAST analysis revealed 100% nucleotide identity to numerous isolates of T. callipaeda haplotype 1.The present paper presents a new host-parasite association between T. callipaeda and P. leo from a zoo in Romania.

Prevention of Thelazia callipaeda Reinfection among Humans.

Thelazia callipaeda is a zoonotic vector-borne nematode that infects and causes eye disease among a wide range of domestic and wild mammals, including humans. We describe an unusual case of reinfection by this nematode in Serbia and call for a focus on preventive measures in endemic areas.

Molecular characterization of oriental eyeworm (Thelazia callipaeda) detected from raccoon (Procyon lotor) and Japanese raccoon dog (Nyctereutes viverrinus) in Kanto region, Japan.

BACKGROUND: The oriental eyeworm Thelazia callipaeda (Spirurida: Thelaziidae) is an emerging parasitic ocular nematode of carnivores and humans. In domestic animals and humans, the infection causes varying degrees of inflammation and lacrimation, and wild carnivores represent an important reservoir. In this study we examined the infection status and molecular characterization of T. callipaeda in two urban carnivores, raccoons Procyon lotor and wild Japanese raccoon dogs Nyctereutes viverrinus, in the Kanto region of Japan. METHODS: From January 2020 to December 2021, 193 carcasses including 178 raccoons and 15 raccoon dogs were examined for the presence of worms in the eye. The worms from infected animals (one worm per host) were morphologically identified as T. callipaeda. Worms (1-5 worms per host) were subjected to genetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences. RESULTS: The prevalence of T. callipaeda in raccoons and Japanese raccoon dogs was 20.2% (36/178) and 13.3% (2/15), respectively. The cox1 sequences from 56 worms from 38 animals revealed three haplotypes (h9, h10, and h12). Analysis of multiple worms for five raccoons showed co-infection of two different haplotypes (h9 and h10) in a single host. Comparing our data with published sequences, three sequences obtained from raccoons and raccoon dogs shared the same haplotypes as those reported in humans, dogs, and cats in Japan. CONCLUSIONS: Our findings show a high prevalence of T. callipaeda in raccoons, suggesting that this invasive carnivore species serves as an important natural reservoir of T. callipaeda in the Kanto region of Japan, an area with the highest human population of the country.

Epidemiological and clinical characteristics of human ocular helaziasis in China from 2011 to 2022 based on bibliometrics / 中国血吸虫病防治杂志

Objective To understand the clinical and epidemiological characteristics of human ocular thelaziasis patients in China. Methods Case reports regarding human ocular thelaziasis cases in China were retrieved in international and national electronic databases, including CNKI, VIP, CBM, Traditional Chinese Medical Literature Analysis and Retrieval System, Wanfang Database, PubMed and Web of Science from 2011 to 2022. Patients’ gender, age, clinical symptoms, treatment, recurrence, site of infections, time of onset, affected eye, affected sites, number of infected Thelazia callipaeda, sex of T. callipaeda and source of infections were extracted for descriptive analyses. Results A total of 85 eligible publications were included, covering 101 cases of human ocular thelaziasis, including 57 males (56.44%) and 44 females (43.56%) and aged from 3 months to 85 years. The main clinical manifestations included foreign body sensation (56 case-times, 22.49%), eye itching (38 case-times, 15.26%), abnormal or increased secretions (36 case-times, 14.46%), tears (28 case-times, 11.24%) and eye redness (28 case-times, 11.24%), and conjunctival congestion (50 case-times, 41.67%) was the most common clinical sign. The most common main treatment (99/101, 98.02%) was removal of parasites from eyes using ophthalmic forceps, followed by administration with ofloxacin and pranoprofen. In publications presenting thelaziasis recurrence, there were 90 cases without recurrence (97.83%) and 2 cases with recurrence (2.17%). Of all cases, 51.96% were reported in four provinces of Hubei, Shandong, Sichuan, Hebei and Henan, and ocular thelaziasis predominantly occurred in summer (42.19%) and autumn (42.19%). In addition, 56.45% (35/62) had a contact with dogs. Conclusions The human thelaziasis cases mainly occur in the continental monsoon and subtropical monsoon climate areas such as the Yellow River and the Yangtze River basin, and people of all ages and genders have the disease, with complex clinical symptoms and signs. Personal hygiene is required during the contact with dogs, cats and other animals, and individual protection is required during outdoor activities to prevent thelaziasis.

Identification and Functional Characterization of CYP4D2 Putatively Associated with ß-Cypermethrin Detoxification in Phortica okadai.

Phortica okadai, a polyphagous pest, serves as a vector for Thelazia callipaeda in China. Currently, there are no effective control strategies for this vector. Agricultural pest control may cause P. okadai to become a threat due to the development of pesticide resistance. Cytochrome P450s (CYP450) plays a significant role in detoxifying xenobiotics in insects. In this study, we performed RNA sequencing of P. okadai exposed to ß-cypermethrin for 0 and 1 h and then gene cloning of the five up-regulated CYP450 genes. Three CYP450 genes were successfully cloned, and their expression patterns in different developmental stages and in different tissues were analyzed by RT-qPCR. Pocyp4d2 was observed to have the highest expression in the midgut (fold change 2.82 for Pocyp4d2, 2.62 for Pocyp49a1, and 1.77 for Pocyp28d2). Functional analysis was carried out according to overexpression in S2 cells from the pfastbac1 vector and RNAi with siRNA. The results of the CCK8 assay indicated that the overexpression of the recombinant protein PoCYP4D2 suppressed the decrease in S2 cell viability due to ß-cypermethrin. The expression levels of PoCYP4D2 decreased significantly, and the mortality rates increased from 6.25% to 15.0% at 3 h and from 15.0% to 27.5% at 6 h after Pocyp4d2-siRNA injection. These results suggest that Pocyp4d2 may be an essential key gene in the metabolism of ß-cypermethrin in P. okadai. This study constitutes a foundation to explore further the functions of P. okadai CYP450 genes in insecticide metabolism.

European Hares, Lepus europaeus, Represent a Reservoir Host for Thelazia callipaeda in Romania.

Thelaziosis caused by Thelazia callipaeda is an emerging disease in Europe. Only two reports of naturally infected lagomorphs have been published so far. The aim of this study was to evaluate the status of the Romanian populations of European brown hares, Lepus europaeus as reservoir hosts for T. callipaeda. Between November 2019 and November 2021, the eyes of 326 L. europaeus carcasses were examined for the presence of ocular parasites. Nematodes were stored in plastic vials with physiological saline, followed by morphological and molecular identification. QGis 3.20 and EpiInfoTM 7 were used for mapping and statistical analysis. Four (1.23%) hares harbored T. callipaeda infection, with a total of 84 nematodes collected (mean intensity 21 nematodes/host), with 45 males, 39 females (two sexually immature, seven with only eggs, and 30 with eggs and larvae). One specimen from each host was successfully sequenced resulting in a 100% similarity with several other sequences of T. callipaeda haplotype 1. Statistical analysis revealed no significant results. The current study represents a first report of T. callipaeda in the European brown hare in Romania, and the second in Europe, also reiterating the role of lagomorphs as reservoir hosts for this zoonotic ocular nematode.

Efficacy of a formulation of sarolaner/moxidectin/pyrantel (Simparica Trio®) for the prevention of Thelazia callipaeda canine eyeworm infection.

BACKGROUND: For a long time known as the oriental eyeworm, Thelazia callipaeda is a zoonotic nematode that infects the eyes of a wide range of vertebrate hosts including dogs, cats, wildlife carnivores, lagomorphs, and humans. The high occurrence of this infection in Europe and the first cases in the United States have increased scientific interest in the parasite, as it also represents a risk for people living in endemic areas. Therefore, treatment and prevention of thelaziosis in canine population are advocated to reduce the risk of human infection as well. Here, we assessed the efficacy of a formulation containing sarolaner/moxidectin/pyrantel (Simparica Trio®) administered orally at monthly intervals, for the prevention of establishment of infection with T. callipaeda in naturally infected dogs. In this formulation, moxidectin is expected to have efficacy against eyeworms, whereas sarolaner and pyrantel are not. METHODS: The study was conducted in eyeworm endemic areas of Italy and France, where dogs (n = 125) were assigned into two groups consisting of a negative control group (G1; n = 62), in which animals were treated monthly with a control product (sarolaner; Simparica®), and a treatment group (G2; n = 63) in which animals were treated monthly with Simparica Trio (sarolaner/moxidectin/pyrantel) from day 0 to day 150. In total, nine animals were withdrawn from the study (two animals became positive at day 30, and seven for reasons unrelated to eyeworm infection), resulting in 116 animals (n = 58 for G1; n = 58 for G2). RESULTS: In G1, 16 out of 58 animals (27.6%) were observed with eyeworms during the study, and none of the animals from G2 were ever observed with eyeworms, resulting in 100% efficacy (P < 0.0001) in the prevention of establishment of T. callipaeda infection. Adult nematodes and fourth-instar (L4)-stage larvae were recovered from the eyes of positive animals, counted, and morphologically identified as T. callipaeda. In addition, specimens from Italy were molecularly confirmed as belonging to the haplotype 1 (i.e., the only one circulating in Europe so far). CONCLUSIONS: Data presented herein demonstrated 100% efficacy of Simparica Trio for the prevention of T. callipaeda eyeworm infection in dogs from highly endemic areas of France and Italy. The use of this formulation is advantageous, as it is a licensed product in Europe with a wide efficacy spectrum against other nematodes, multiple tick species, and fleas. In addition, preventing the development of infection in dogs could also be a prophylaxis measure for zoonotic T. callipaeda infection in humans inhabiting endemic areas.

Laboratory Culture and Life Cycle of Thelazia callipaeda in Intermediate and Definitive Hosts.

Human thelaziasis caused by Thelazia callipaeda is being increasingly reported worldwide. Notably, an epidemic trend is observed in Southwest China. Whether Phortica okadai found in Southwest China can act as a vector of T. callipaeda and human-derived T. callipaeda animal infections has not been widely reported. Here, P. okadai was maintained in a laboratory and experimentally infected with first-stage larvae collected from adult T. callipaeda that were isolated from infected human subjects. Dead P. okadai were subjected to PCR assay and dissected every two days to detect T. callipaeda. Subsequently, live flies were used to infect a rabbit. The infection procedures were performed once a day (20 min) for two weeks. The results show that L1 collected from the adult T. callipaeda could successfully parasitize P. okadai captured in Zunyi, a city in Southwest China, and developed into L3, and a rabbit was successfully infected with T. callipaeda using P. okadai as the intermediate host. The present study demonstrates a human-derived T. callipaeda infection in rabbits, through P. okadai, under laboratory conditions for the first time. These results provide insights into the transmission cycle of T. callipaeda and constitute a foundation to develop an effective treatment protocol for T. callipaeda infection.

Human Ocular Thelaziasis: A Case Report from Indonesia.

Thelaziasis is a parasitic disease caused by a nematode of genus Thelazia, which is rare in the world, including Indonesia. The definitive hosts for Thelazia are canids, felids, mustelids, and other mammals, while the vector is drosophila flies. Consequently, this study reported an uncommon occurrence of human ocular thelaziasis in Indonesia. Based on the patient's complaints and physical examination, we found a living worm that move actively in the anterior chamber; then documentation is carried out both during the examination at the polyclinic and in the operating room. The surgery was performed using topical anaesthesia, clear corneal incision, and removing worm through the main port. Morphological examination from the parasitology laboratory showed that the worm was Thelazia callipaeda species. Following this intervention, the patient was given an oral anti-helminthics drug, topical and oral antibiotics, topical steroid, and surgical treatment. There was no recurrence or appearance of any other symptoms reported in 2 months of follow-up.

Laboratory breeding of two Phortica species (Diptera: Drosophilidae), vectors of the zoonotic eyeworm Thelazia callipaeda.

BACKGROUND: Some species of drosophilid flies belonging to the genus Phortica feed on ocular secretions of mammals, acting as biological vectors of the zoonotic eyeworm Thelazia callipaeda. This study describes an effective breeding protocol of Phortica variegata and Phortica oldenbergi in insectary conditions. METHODS: Alive gravid flies of P. oldenbergi, P. variegata and Phortica semivirgo were field collected in wooded areas of Lazio region (Italy) and allowed to oviposit singularly to obtain isofamilies. Flies were maintained in ovipots (200 ml) with a plaster-covered bottom to maintain high humidity level inside. Adult feeding was guaranteed by fresh apples and a liquid dietary supplement containing sodium chloride and mucin proteins, while larval development was obtained by Drosophila-like agar feeding medium. The breeding performances of two media were compared: a standard one based on cornmeal flour and an enriched medium based on chestnut flour. All conditions were kept in a climatic chamber with a photoperiod of 14:10 h light:dark, 26 ± 2 °C and 80 ± 10% RH. RESULTS: From a total of 130 field-collected Phortica spp., three generations (i.e. F1 = 783, F2 = 109, F3 = 6) were obtained. Phortica oldenbergi was the species with highest breeding performance, being the only species reaching F3. Chestnut-based feeding medium allowed higher adult production and survival probability in both P. oldenbergi and P. variegata. Adult production/female was promising in both species (P. oldenbergi: 13.5 F1/f; P. variegata: 4.5 F1/f). CONCLUSIONS: This standardized breeding protocol, based on controlled climatic parameters and fly densities, together with the introduction of an enriched chestnut-based feeding medium, allowed to investigate aspects of life history traits of Phortica spp. involved in the transmission of T. callipaeda. Obtaining F3 generation of these species for the first time paved the road for the establishment of stable colonies, an essential requirement for future studies on these vectors in controlled conditions.

Phortica oldenbergi (Diptera: Drosophilidae): A new potential vector of the zoonotic Thelazia callipaeda eyeworm.

Thelazia callipaeda is a zoonotic nematode parasitizing the eyes of many hosts species, primarily dogs. To date Phortica variegata and Phortica okadai are the only known vectors of this nematode in Europe and China, respectively. In this study we investigated the role played by a third species, Phortica oldenbergi, as vector of T. callipaeda in Europe. Drosophilid flies of this species were collected in central Italy and maintained in laboratory. One hundred forty P. oldenbergi were experimentally infected with T. callipaeda L1 recovered from field collected gravid females belonging to haplotype 1, which is that detected in several countries in Europe. Seventy-four (i.e., 60 females and 14 males) specimens died at 5 days post infection (d.p.i.) (±1) and scored negative for T. callipaeda larvae at the dissection. Sixty-six (i.e., 46 females and 20 males) P. oldenbergi survived and were dissected at 21 d.p.i. From those, T. callipaeda L3 were detected in the proboscis of two females (3.0%). Overall, at the molecular analysis, 11.4% (n = 16/140; 13 females and 3 males) scored positive for the presence of T. callipaeda DNA. Data herein reported brings further insights on the biology of T. callipaeda by adding P. oldenbergi as a new potential intermediate host under experimental conditions. The role of this drosophilid in the transmission cycle of T. callipaeda needs to be confirmed under natural conditions.

First report of human Thelazia callipaeda infection in Portugal.

The zoonotic parasitic nematode Thelazia callipaeda, also known as the oriental eye worm, is endemic in several European countries, including Portugal. Infections may result in ocular disease in domestic and wild animals as well as humans, with more or less severe manifestations. We report the first human case of ocular thelaziosis by T. callipaeda in Portugal, a country where the parasite had already been found to infect dogs, cats, red foxes, wild rabbits and a beech marten. An 80-year-old patient from east-central Portugal, who had been suffering from tearing for a few years, had whitish filiform fragments removed from the left eye. Polymerase chain reaction of partial cytochrome c oxidase subunit 1 and 18S small subunit rRNA genes followed by bidirectional sequencing and BLAST analysis confirmed T. callipaeda haplotype 1, the only haplotype previously reported in Europe. The endemicity of T. callipaeda in domestic and wild animals in east-central Portugal makes it very likely that infection of the human patient had occurred locally. In east-central and other geographical areas of Portugal, veterinarians and physicians, especially ophthalmologists, should regard T. callipaeda as a cause of ocular pathology in animals and humans.

Wild carnivores and Thelazia callipaeda zoonotic eyeworms: A focus on wolves.

Thelazia callipaeda is a zoonotic parasite causing ocular disease in domestic dogs, cats, several wild carnivores, hares, and humans. This nematode is widely distributed in Europe, where it is transmitted by the drosophilid fly Phortica variegata. Since the first report of infection in grey wolves (Canis lupus) from southern Italy, other cases of thelaziosis have been recorded in this animal species throughout Europe, raising questions about their role in spreading T. callipaeda. Indeed, for their wandering behavior through long distances and living in woody areas where the vectors thrive, wolves may act as reservoirs and spreaders of thelaziosis. In this study we reviewed the literature about wolves acting as reservoirs of T. callipaeda in Europe. In addition, we report the first detection of T. callipaeda eyeworms in grey wolves in the Italian Alps, discussing its possible implications in the epidemiology of thelaziosis in the Alpine landscape. Animals (n = 3) included in this study were originated from the Italian Alps, one juvenile male wolf was found dead, and the other two were seven-year-old males translocated from Piedmont region to a Zoological Garden, in Tuscany. All animals were infected with eyeworms, which were morphologically and molecularly identified as T. callipaeda. Data herein presented confirm those available in the literature about the circulation of a unique cox1 haplotype in Europe. In addition, the report of T. callipaeda in wolves from the Alps suggests an ecological continuity of habitats which are suitable for the distribution of T. callipaeda from the southern to northern Italy through the Apennine backbone. Retrospectively, it could also explain the spreading of the oriental eyeworm infection in Europe over the last 20 years with many wild carnivores, such as foxes and possibly wolves, playing a pivotal role as reservoirs of the infection for dogs, cats and humans.