Toxocariasis is one of the most widespread zoonotic parasitic diseases on the planet. Adult worms live in the intestines of dogs and other canids, each female shedding up to 200,000 eggs per day into feces that contaminate soil, sand, water and food. Humans are infected accidentally when they ingest embryonated eggs, which hatch in the small intestine and release larvae that migrate through the lungs, liver, eyes, brain and muscles while evading the immune system. This wandering can cause visceral larva migrans, ocular larva migrans and neurotoxocariasis, conditions capable of lasting damage when not diagnosed and treated early. Global seroprevalence estimates range from 5% to 15%, and the disease is associated with roughly 91,714 disability-adjusted life years and an estimated annual cost of $2.5 billion.

Diagnosis currently relies on antigen-based detection methods such as ELISA and Western blot, but these suffer from a persistent problem: cross-reactivity with antigens from other parasitic helminths, particularly Ascaris species that cause ascariasis, as well as strongyloidiasis. This overlap makes it difficult to estimate true worm burdens and raises the risk of misdiagnosis. Identifying novel, highly specific candidate antigens has therefore become a central goal for researchers hoping to improve immunodiagnostic methods, and the new adult-stage protein catalog is a direct step toward that goal.

The experimental approach was meticulous. Adult worms, roughly equal numbers of males and females, were collected from the intestine of a stray dog, washed in phosphate-buffered saline and cultured overnight in serum-free RPMI-1640 medium with antibiotics at 37 degrees Celsius and 5% carbon dioxide. The culture supernatant was filtered through a 0.45-micrometer membrane to remove debris and eggs, concentrated with 3-kilodalton molecular-weight-cutoff centrifugal filters and quantified by Bradford assay. Proteins were then purified by acetone and trichloroacetic acid precipitation, digested with trypsin and analyzed on an Orbitrap Eclipse Tribrid mass spectrometer coupled to a Vanquish Neo UHPLC system, with peptide searches run against the complete T. canis protein database using Mascot software.

The analysis yielded 2,546 peptides matched to 468 proteins, which ranged in size from 62 to 8,647 amino acids. Relative abundance was quantified using normalized spectral abundance factor values, which adjust spectral counts for protein length. The secretome was dominated by a small number of protein families. The single most abundant ESP was a C-type lectin, accounting for 5.2% of all detected protein, but the list of top proteins was crowded with 26-kilodalton secreted antigens known as TES-26, seven of which appeared among the top 15, and Collectin-12 proteins, another class of C-type lectins, six of which ranked among the top 10. Together, TES-26 proteins, Collectin-12 lectins and superoxide dismutases accounted for 34 ESPs and 49.3% of the total detected protein by mass.

Functional enrichment analysis revealed what these proteins are built to do. Among 85 significantly enriched Gene Ontology molecular function terms, the strongest signal was catalytic activity, annotated to 43.2% of ESPs, followed by carbohydrate binding, a hallmark of lectins that help parasites recognize and invade host tissues. Cellular component analysis confirmed enrichment for the extracellular region and extracellular matrix, as expected for secreted molecules. KEGG pathway analysis added a striking finding: the exosome pathway was the most significantly enriched of all, with 69 of the 468 ESPs assigned to it. These included cytoskeletal actins that serve as structural components of extracellular vesicles and triosephosphate isomerase, a glycolytic enzyme routinely exported as non-classical vesicle cargo.

That exosome signal points to a secretion strategy beyond the classical route. While 26.5% of detected ESPs carried classical signal peptides for secretion, compared with only 9.9% of all T. canis proteins, an additional 62 exosome-pathway proteins lacked signal peptides altogether. This suggests the parasite uses exosome-facilitated, non-classical secretion to deliver a subset of its molecular payload to host tissues. Extracellular vesicles from related nematodes are known to carry microRNAs, proteins, lipids and nucleic acids that can suppress host Type 2 innate immune responses, and the new data indicate T. canis adults likely deploy a similar mechanism during their long residence in the intestine.

To distinguish what makes T. canis unique from what it shares with relatives, the team compared its ESPs against proteomic data from the closely related Clade III roundworm Ascaris suum and the more distant Clade V hookworm Ancylostoma ceylanicum. Orthologs could be identified for 77.0% of T. canis proteins in A. suum and 60.1% in A. ceylanicum, but the secreted fractions told a different story: 331 of the 468 T. canis ESPs, or 70.7%, were detected only in T. canis, compared with 48.3% species-specific ESPs in A. suum and 57.8% in A. ceylanicum. Just 35 ESPs were shared across all three species, including six TES-26 proteins, six transthyretin-like proteins and ABA-1, a known major allergen in both T. canis and A. suum. The authors interpret this predominance of species-specific ESPs as reflecting rapid adaptation to each parasite’s particular host niche and tropism.

The species-specific fraction contains some of the most promising diagnostic leads. The four most abundant T. canis-specific proteins, with no orthology to either comparison species, were all TES-26 paralogs from a single orthologous protein family, and together they make up 14.8% of the total ESP proteome. One of them, Tcan_15190, also known as TES-120, was previously detected among the 19 proteins identified in larval ESPs, suggesting it is produced consistently across life cycle stages while remaining specific to T. canis, a combination the authors highlight as potentially optimal for diagnosis. The cross-reactivity problem also gained a possible molecular explanation: among the 58 ESPs shared with A. suum, a galectin of 54.1 kilodaltons and a phosphoglycerate kinase of 66.2 kilodaltons fall within the 55 to 66 kilodalton band previously implicated in serological cross-reactivity between the two species.

Several other protein families illuminate how the adult worm survives host defenses. Superoxide dismutases and glutathione transferases, abundant in both T. canis and A. suum ESPs, are typically induced during immune challenge and likely detoxify the reactive oxygen species that host immune cells deploy against parasites; in the filarial worm Onchocerca volvulus, an ESP superoxide dismutase is known to provoke strong human IgG responses. The TES-26 family itself carries phosphatidylethanolamine-binding domains and ShK toxin-like motifs with hydrophobic lipid-binding regions, and these proteins have previously been identified as key antigens for diagnosis and potential vaccine development. One monosaccharide-binding TES-26 ortholog, recombinantly expressed in earlier work, improved disability scores in a mouse model of autoimmune encephalomyelitis. With the complete annotation database now publicly available, the study’s authors position this catalog as a foundation for comparative parasitology and for the rational design of more specific diagnostics, vaccine candidates and targeted control strategies against a parasite that has long hidden in plain sight.

Subject of Research: Proteomic characterization of excretory-secretory proteins from adult Toxocara canis worms

Article Title: Proteomics analysis of excretory-secretory proteins in mixed-sex adult stage Toxocara canis

Article References: Suksomboon, P., Rosa, B. A., Singh, K. S., Martin, J., Zhan, B., Bottazzi, M. E., & Mitreva, M. (2026). Proteomics analysis of excretory-secretory proteins in mixed-sex adult stage Toxocara canis. Frontiers in Parasitology, 5, 1965955. https://doi.org/10.3389/fpara.2026.1965955

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1965955

Keywords: Toxocara canis, toxocariasis, proteomics, excretory-secretory proteins, LC-MS/MS, TES-26, C-type lectins, superoxide dismutase, exosomes, diagnostics, vaccine candidates, zoonotic parasites