First report of canine thelaziosis caused by Thelazia callipaeda in Bhutan

Abstract

Thelazia callipaeda is a vector-borne zoonotic nematode causing ocular infection in animals and humans, with an expanding geographical range across Asia, Europe, and North America. Although the parasite is endemic in neighbouring countries China and India, no cases of canine thelaziosis have previously been reported in Bhutan. Here we describe the first case of canine ocular thelaziosis, in a 7-year-old mixed-breed dog from Punakha District presenting with bilateral lacrimation and epiphora, conjunctival hyperemia, and blepharospasm. Thirty-two whitish, thread-like nematodes were recovered from the conjunctival sacs of both eyes and identified morphologically as T. callipaeda based on cuticular striae, buccal capsule morphology, and characteristic male and female reproductive structures. Molecular confirmation was achieved by sequencing a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, which showed 99.6% identity to an isolate of T. callipaeda from India. Phylogenetic analysis placed the Bhutan isolate within the Asian clade, clustering most closely with the Indian isolate. This report describes the diagnosis and successful management of the first confirmed case of canine ocular thelaziosis in Bhutan, underscoring the need for veterinary and public health surveillance of thelaziosis in this region.

Graphical abstract

Highlights

• •First confirmed case of canine ocular thelaziosis reported in Bhutan.
• •Thirty-two Thelazia callipaeda nematodes recovered from a dog’s conjunctival sacs.
• •Morphology and cox1 sequencing confirmed the identification of T. callipaeda.

1. Introduction

Thelazia callipaeda (Rhabditida, Thelaziidae) is a zoonotic nematode that infects the conjunctival sac of domestic and wild carnivores, lagomorphs, and humans, causing ocular disease (Otranto et al., 2021; Taweethavonsawat et al., 2026) with a range of clinical signs from lacrimation and foreign body sensation to conjunctivitis, redness, epiphora, blepharospasm, and even keratitis and corneal ulceration (Otranto et al., 2003a, 2021, 2026; Taweethavonsawat et al., 2026). The parasite is transmitted by male lachryphagous drosophilid flies of the genus Phortica, which act as vectors and intermediate hosts, with Phortica okadai and Phortica variegata being the principal vectors in East Asia and Europe, respectively (Otranto et al., 2005). Adult T. callipaeda in a host’s eye release first-stage larvae (L1) into the lacrimal secretion, which are ingested by tear-feeding male Phortica spp. flies. In the fly, the larvae develop into infective third-stage (L3) larvae, which migrate to the mouthparts and are released into the conjunctival sac of a new host (Otranto et al., 2005, 2006).

Once considered the “oriental eye-worm” and confined to East Asia, T. callipaeda has expanded its geographical range markedly over the past three decades. After its first European report from Italy in 1989 (Rossi and Bertaglia, 1989), autochthonous cases of the infection have been reported not only in dogs and cats, but also in many wildlife species across France, Germany, Portugal, Spain, Switzerland and many countries in the Balkans (Otranto et al., 2003a, 2021, 2026; do Vale et al., 2020; Otranto et al., 2021). The parasite has more recently been reported in autochthonous cases of dogs and cats from the USA (Manoj et al., 2024).

In India, human thelaziosis has been recorded in many states (e.g. Manipur, Assam, and Himachal Pradesh) (Purnima et al., 2019), as well as from Uttar Pradesh, Karnataka, and Chennai (Kasarla et al., 2021) and in a dog from Nagaland (Northeast India) (Sangwan et al., 2021). In neighbouring countries, human cases have also been reported near the India-Nepal border (Pokhrel et al., 2021) as well as in Bangladesh (Akhanda et al., 2013) and in Vietnam (De et al., 2012). Overall, a recent systematic review documented 134 human thelaziosis cases across 18 countries globally, with 82.8% reported from Asia, notably from China, India, Japan, and South Korea, underscoring the One Health relevance of this parasite (Bonilla-Aldana et al., 2025; Otranto et al., 2026).

Bhutan has a recorded dog population of 105,644 dogs, of which approximately 70% are free-roaming dogs (Nationwide Accelerated Dog Population Management and Rabies Control Programme, 2024). This huge number of free-roaming dogs and irregular deworming of pet dogs increase the risk of zoonotic parasitoses in the country (Otranto et al., 2017). Although zoonotic parasitic diseases such as cystic echinococcosis and neurocysticercosis have been documented in Bhutan (Tenzin et al., 2024), information on ocular zoonotic parasites such as T. callipaeda is lacking.

Despite the documented presence of T. callipaeda in both neighbouring India and China, canine thelaziosis has never been previously reported in Bhutan. We describe the first confirmed case of canine ocular thelaziosis in Bhutan, diagnosed by morphological examination with molecular confirmation, and describe its clinical course.

2. Material and methods

2.1. Case presentation

A 7-year-old, mixed-breed, owned male dog weighing 15 kg was presented to the District Veterinary Hospital, Punakha, Bhutan, in May 2024 with a 3-day history of excessive lacrimation and epiphora, conjunctival hyperemia, and blepharospasm affecting both eyes. The dog had been resident in Punakha district since birth, with no history of travel outside the district. It was housed outdoors at the owner’s premises and had an irregular vaccination and deworming history. The owner reported no other dogs in the household or immediate neighbourhood showing similar ocular signs.

Following topical anaesthesia with 4% lidocaine (LOX 4% Topical, Neon Laboratories, Mumbai, Maharashtra, India), ophthalmic examination revealed multiple whitish, thread-like nematodes in the conjunctival sac and beneath the nictitating membrane of both eyes, moving with the characteristic serpentine motion (Fig. 1). A total of 32 worms were recovered from the two eyes. The number recovered from each eye, and the sex and maturity of the worms, were not recorded at the time of collection. The visible worms were extracted using sterile cotton swabs, and the conjunctival sacs were subsequently flushed with sterile 0.9% physiological saline (NaCl) to collect remaining nematodes. The recovered parasites were morphologically examined and preserved in 70% ethanol for molecular confirmation.

Fig. 1.

Ocular examination of the affected dog showing multiple whitish, thread-like Thelazia callipaeda nematodes within the conjunctival sac.

After removal of adult worms from both conjunctival sacs, the dog received a single subcutaneous dose of ivermectin at 0.2 mg/kg (0.3 ml of 1% w/v solution; Neomac, Intas Pharmaceuticals Ahmedabad, Gujarat, India) administered in the dorsal cervical region. A topical ophthalmic preparation containing polymyxin B sulphate (5000 IU/ml), chloramphenicol (4 mg/ml), and dexamethasone sodium phosphate (1 mg/ml) (Ocupal Dx, Centaur Pharmaceutical, Mumbai, India) was instilled into both eyes, 2–3 drops three times daily for 5 days. A fluorescein staining test was not performed, so corneal ulceration or other corneal damage could not be excluded before a corticosteroid-containing ophthalmic preparation was administered. No systemic adverse effects were noted following ivermectin administration, based on clinical examination and owner report. The injection site was not specifically evaluated for local reactions. Ocular clinical signs resolved within a week of treatment. The dog was re-examined every 2 weeks for 1 month following treatment to assess the conjunctival sac and the area beneath the nictitating membrane for nematodes; no nematodes were recovered and no recurrence of clinical signs was noted at either follow-up examination.

2.2. Morphological identification

For morphological identification, specimens were cleared with lactophenol solution, mounted on glass slides under coverslips, and examined under a light microscope (10× and 40× magnifications). The nematodes were identified to the species level using main morphological characters (Otranto et al., 2003b). Briefly, T. callipaeda is characterised by a whitish aspect, with transverse cuticular striations on the entire body surface and a buccal capsule with a typically hexagonal mouth opening and a slender body (Otranto et al., 2003b). Males have a ventrally curved caudal end, 15 pairs of papillae on the ventral surface and two spicules, which differ greatly from each other in shape and size. Females present the vulva opening typically anterior to the esophago-intestinal junction. Immature eggs or germ cells are frequently observed in the distal portion of the uterus (Otranto et al., 2004). The first-stage larvae (L1), measuring 382–400 μm in length, are observed in a row in the distal uterus of adult females (Otranto et al., 2003b).

2.3. Molecular analysis

Genomic DNA was extracted from three individual nematodes using the DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. A fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified with the primers COIintF and COIintR (Casiraghi et al., 2001). PCR amplification was carried out in a final reaction volume of 25 μl containing 12.5 μl of 2× Qiagen Taq PCR Master Mix, 0.25 μM of each primer, 1 μl of genomic DNA template and nuclease-free water to volume. The PCR cycling program was as follows: initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 52 °C for 2 min, extension at 72 °C for 90 s, and a final extension step at 72 °C for 5 min.

Amplified PCR products were visualised under ultraviolet (UV) illumination using E-gel imager system (Thermo Fisher Scientific, Waltham, MA, USA) after resolution on an agarose gel (1.5%) containing ethidium bromide. PCR amplicons of the expected size were purified by GeneJET PCR Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Purified PCR amplicons were subjected to library preparation using the Oxford Nanopore Rapid Barcoding Kit 96 V14 (SQK-RBK114.96; Oxford Nanopore Technologies, Oxford, UK) following the manufacturer’s protocol for amplicon sequencing from DNA. Briefly, PCR products were barcoded, pooled, and prepared for sequencing according to the kit instructions. The prepared library was loaded onto an Oxford Nanopore flow cell and sequenced using the MinION Mk1D platform. Sequencing runs were controlled using MinKNOW software.

Raw sequencing reads generated from the Oxford Nanopore MinION Mk1D platform were basecalled and demultiplexed using the wf-amplicon EPI2ME (v.1.2.1) workflow (Oxford Nanopore Technologies). Consensus sequences were generated from quality-filtered reads and exported in FASTA format. The resulting sequences were compared against reference sequences available in the NCBI GenBank database using the BLASTn algorithm for species identification. The resulting cox1 consensus sequence was queried against the NCBI nucleotide database using BLASTn. Molecular identification was assessed by examining the taxonomic identities of matching reference sequences, nucleotide sequence identity, query coverage, and E-values, together with the morphological findings. Sequence editing, alignment, and genetic analyses were performed using MEGA X software (Kumar et al., 2018). Representative mitochondrial cox1 gene sequences of T. callipaeda were retrieved from the NCBI GenBank database based on BLASTn results. Multiple sequence alignment was performed using MAFFT v.7 employing the L-INS-i algorithm with default parameters. The resulting alignment was manually inspected and used for phylogenetic analysis.

Phylogenetic relationships were inferred using the Maximum Likelihood (ML) method implemented in MEGA X. The Tamura-Nei model with Gamma-distributed rate variation among sites (TN93+G, 5 categories, parameter = 0.2427) was selected as the best-fit nucleotide substitution model based on the Bayesian information criterion (BIC). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and the topology with the superior log-likelihood value was selected. Statistical support for tree topology was assessed using 1000 bootstrap replicates. Thelazia rhodesi (GenBank: OR673501.1) was included as an outgroup to root the phylogenetic tree. The analysis included 23 nucleotide sequences (Supplementary Table S1) and codon positions 1st+2nd+3rd + noncoding. Positions with less than 95% site coverage (i.e. > 5% alignment gaps, missing data, or ambiguous bases) were excluded using the partial deletion option, leaving a final dataset of 558 positions.

3. Results

The main morphological characteristics of T. callipaeda are depicted in Fig. 2. In particular, in the anterior region of the female specimen, the vulval opening was located anteriorly to the oesophago-intestinal junction (Fig. 2A). The cuticle presented the characteristic transverse cuticular striations in the anterior region (Fig. 2B). The digestive tract comprised a hexagonal buccal capsule followed by the oesophagus (Fig. 2B). The uterus possessed numerous mature, coiled larvae in the distal part (Fig. 2C). The caudal extremity of a male specimen showed unequal spicules, with a longer right spicule and a shorter left spicule (Fig. 2D).

Fig. 2.

Thelazia callipaeda.A-C Female, anterior region. A Vulva opening anterior to the oesophago-intestinal junction (arrow). B Buccal capsule with a hexagonal shape (arrow) and oesophagus (arrowhead). Transverse cuticular striations (asterisk) are present in the anterior part of the body. C Numerous mature, coiled larvae in the uterus are present in the distal part. D Male, with posterior extremity bearing two unequal spicules, the right spicule being longer (arrowhead) and the left spicule shorter (arrow).

The newly generated partial cox1 sequence of T. callipaeda was deposited in the GenBank database under the accession number QB110476. BLASTn analysis of the partial cox1 sequence showed 99.6% nucleotide identity with an isolate of T. callipaeda (GenBank: PX482520.1), supporting the molecular identification of the recovered nematode as T. callipaeda.

Maximum-likelihood phylogenetic analysis based on 558 nucleotide positions from 23 sequences, including the outgroup, placed the Bhutan isolate (QB110476) within the Asian T. callipaeda clade (Fig. 3). The Bhutan isolate clustered with the Indian isolate PX482520.1, with 92% bootstrap support, indicating a close genetic relationship between the two isolates.

Fig. 3.

Maximum likelihood phylogenetic tree based on 558 aligned nucleotide positions of the mitochondrial cox1 gene from 23 sequences, inferred using the Tamura-Nei model with gamma-distributed rate variation (TN93+G) in MEGA X. Numbers at nodes indicate bootstrap support (%) from 1000 replicates. The Bhutan isolate (GenBank: QB110476), highlighted in bold and marked with a red triangle, clusters with the Indian isolate (GenBank: PX482520) with 92% bootstrap support. Thelazia rhodesi (GenBank: OR673501) was used as the outgroup. The scale-bar represents 0.02 substitutions per site. Details for the reference sequences used in the analysis are provided in Supplementary Table S1.

The relatively short branch lengths among T. callipaeda sequences suggest limited genetic divergence within the analysed cox1 region. Overall, the phylogenetic findings were consistent with the BLASTn results and supported the morphological and molecular identification of the Bhutan isolate as T. callipaeda.

4. Discussion

To the best of our knowledge, this report describes the diagnosis and successful management of the first confirmed case of canine ocular thelaziosis in Bhutan. The finding is consistent with Bhutan’s geographical position between two highly endemic neighbouring countries, China and India (Otranto et al., 2021). The clinical signs herein reported (i.e. lacrimation and epiphora, conjunctival hyperemia, and blepharospasm) are characteristic of the ocular injury induced by the mechanical irritation caused by adult T. callipaeda nematodes within the conjunctival sac (Miró et al., 2011; Otranto et al., 2021).

Reported management of canine ocular thelaziosis varies in its reliance on systemic versus topical treatment. While manual removal of adult worms is generally considered curative on its own, adjunct treatment is commonly used to eliminate larvae or worms not retrieved during examination and to reduce recurrence risk. In this case, ivermectin was used because it was the only antiparasitic agent effective against T. callipaeda available at the clinic. Nonetheless, topical spot-on formulations such as moxidectin 2.5%/imidacloprid 10% (Otranto et al., 2016), or oral administration of sarolaner/moxidectin/pyrantel (Bezerra-Santos et al., 2022) and milbemycin oxime in combination with praziquantel (Motta et al., 2012) have demonstrated high efficacy against T. callipaeda in naturally infected animals. Mechanical extraction combined with a single subcutaneous dose of ivermectin and topical antibiotic-corticosteroid eye drop achieved full clinical resolution. However, where alternative antiparasitic treatments are accessible, particularly in regions where the genotyping of MDR1 (a gene encoding the p-glycoprotein pump, which is crucial for preventing toxic accumulation of avermectins and other medications in the central nervous system) cannot be readily performed, a topical macrocyclic lactone-based regimen (Geyer and Janko, 2012) or oral treatment with mebendazole (Calero-Bernal et al., 2014) may represent a safer first-line adjunct to manual removal than injectable ivermectin.

Morphological and molecular analysis confirmed the identification of the recovered nematode as T. callipaeda. Furthermore, phylogenetic analysis revealed a close relationship between the Bhutan and Indian isolates, consistent with their high nucleotide similarity. Whether this reflects connected transmission cycles, recent parasite dispersal, or simply a widely conserved regional haplotype cannot be determined from a single isolate; distinguishing between these possibilities would require broader vector and wildlife sampling across the India-Bhutan border region.

This pattern of low divergence is consistent with recent studies demonstrating that T. callipaeda exhibits relatively low mitochondrial diversity across broad geographical regions, with several dominant haplotypes shared among countries in Asia and Europe (Doi et al., 2023; Otranto et al., 2026; Taweethavonsawat et al., 2026). Population genetic analyses have shown that while European populations are dominated by a small number of haplotypes, East and Southeast Asian populations display comparatively higher genetic diversity, supporting the hypothesis that Asia may represent the ancestral distribution range of the parasite (Otranto et al., 2026). The low bootstrap support observed for several internal nodes in the phylogenetic tree is likely attributable to the limited number of phylogenetically informative sites within the analysed cox1 fragment.

The occurrence of T. callipaeda in Bhutan has important One Health implications, as the large range of mammalian hosts, including dogs, cats, and wildlife, facilitates transmission between domestic, wild, and human populations (Otranto et al., 2021). Reservoir hosts such as dogs, foxes, wolves, and raccoon dogs, along with bears and other carnivores, may contribute to parasite maintenance in endemic areas, emphasising the need for integrated surveillance and control strategies (Doi et al., 2023; Taweethavonsawat et al., 2026).

As entomological surveys for Phortica spp. are not available in Bhutan, future studies should be conducted to clarify local transmission cycles. In addition, future investigations incorporating larger sample sizes and broader geographical coverage would provide deeper insight into the evolutionary history, genetic diversity, and transmission dynamics of T. callipaeda in Bhutan and neighbouring countries. Greater awareness of the risk of human infection is also needed to mitigate the public health impact of this zoonosis, particularly in countries with large stray-dog populations.

5. Conclusions

This report describes, to the best of our knowledge, the first confirmed case of canine ocular thelaziosis caused by T. callipaeda in Bhutan. The identification was based on morphology and cox1 sequencing. Manual removal of the nematodes combined with a single subcutaneous dose of ivermectin and a topical antibiotic-corticosteroid preparation was followed by clinical resolution, with no recurrence at follow-up examinations over one month. Given the zoonotic nature of T. callipaeda and the large free-roaming dog population in Bhutan, this finding has One Health implications. Entomological surveys for Phortica spp. and studies with larger sample sizes and broader geographical coverage are needed to clarify local transmission cycles and the genetic diversity of the parasite in Bhutan and neighbouring countries.

Ethical approval

Informed consent was obtained from the dog’s owner for the diagnostic procedures performed and for publication of this case report, including the accompanying clinical images.

CRediT authorship contribution statement

Rinchen Tshering: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Karma Wangchuk: Data curation, Methodology, Resources, Visualization, Writing - review & editing. Domenico Otranto: Methodology, Resources, Supervision, Validation, Visualization, Writing - review & editing.

Data availability

The data supporting the conclusions of this article are included within the article and its supplementary file. The newly generated sequence was submitted to the GenBank database under the accession number QB110476.

Statement on the use of AI-assisted technologies

During the preparation of this work, the authors used Claude (Anthropic) to paraphrase sentences and improve the wording of the text. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Domenico Otranto was partially supported by the European Commission within the National Research, Innovation and Competitiveness Program for the Green and Digital Transition (PN RIC 21-27), Call DD 307 of 18/03/2025, Action 1.1.2 - Support for a limited number of strategic research chains - Project Title: Development of a Research Ecosystem for Monitoring and Analysis (DOMARE) MI2615 [CUP B99H26000280007].

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article: