Species diversity, benzimidazole resistance, and phytochemical synergy in gastrointestinal nematode nemabiomes from sheep farms in Portugal.

Anthelmintic resistance in gastrointestinal nematodes (GINs) threatens small ruminant production. This study assessed ovicidal activity of eight natural aromatic phytochemicals and interactions with synthetic benzimidazoles (BZs: albendazole [ABZ], thiabendazole [TBZ]) against nemabiome eggs of 14 BZ-resistant sheep farms across Trás-os-Montes, Beira Interior, Estremadura, and Alentejo regions of Portugal. Trans-cinnamaldehyde (TCL), octyl gallate (OGA), carvacrol (CRV), thymol (THY), and salicylaldehyde (SAL) achieved >90 % egg hatch inhibition (EHI) at 250 μg/mL in egg hatch tests (EHTs), unlike trans-cinnamic acid, gentisic acid, and curcumin (<90 % EHI). Effective concentrations (EC10-EC90) were determined for each compound against farm nemabiomes. TCL (EC90 ≈ 27.0 μg/mL) and OGA (≈54.1 μg/mL) showed highest ovicidal efficacy, followed by CRV (≈120.3 μg/mL), THY (≈171.3 μg/mL), and SAL (≈155.2 μg/mL), requiring 25-220-fold higher concentrations than BZs. EC10-EC75 concentrations in checkerboard assays determined fractional inhibitory concentration indices (FICIs) to assess interactions. Compound interactions (FICI: 0.20-0.49; all ≤0.5) reduced ABZ mean EC90 by 0.73 μg/mL (89.6 %) and TBZ by 0.72 μg/mL (94.7 %), with TCL and OGA showing strongest effects (FICI ≤0.25). ITS2 metabarcoding identified six GIN species, with Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus colubriformis most prevalent. Nemabiome diversity (H': 0.09-1.17) and species prevalence showed no correlation with EHI EC90 values (|r| < 0.418, p > 0.05), suggesting ovicidal efficacy or BZ-phytochemical interactions are not species-driven. Synergistic mechanisms involving aromatic phytochemical-induced eggshell damage, complementing BZ β-tubulin disruption, are discussed. BZ-phytochemical combinations offer a promising strategy for sustainable GIN control, warranting in vivo validation.