In vitro synergy analysis of the combination of Citrus aurantium bergamia (Risso & Poiteau, 1826), ivermectin, and nitroxinil against gastrointestinal nematodes of sheep.

Nematodes are diverse parasites affecting humans and animals, with resistance to anthelmintics becoming a major concern due to prolonged drug use. Essential oils (EOs) from plants offer a promising alternative for parasite control. This study evaluated the preclinical efficacy of Citrus aurantium bergamia essential oil (BEO) combined with nitroxynil (NTX) or ivermectin (IVM) against Haemonchus contortus and Trichostrongylus spp., using the larval migration inhibition test (LMIT). Gas chromatography-mass spectrometry identified (+)-limonene (50.5 %), linalyl acetate (21.0 %), and linalool (6.9 %) as BEO's main components. Fecal samples from naturally infected sheep were analyzed via flotation, larval culture, and microscopy to quantify nematode eggs and infective-stage larvae (L3). The Nadi reagent was used to assess histochemical changes in L3 post-migration. BEO exhibited a lethal concentration (LC50) of 24.5 mg/mL, showing concentration-dependent activity. While BEO alone achieved 93.6 % efficacy, combinations with NTX (99.1 %) and IVM showed some synergy effects. At 70.16 mg/mL, BEO efficacy was 92.2 %, increasing to 95.6 % with IVM (P = 0.0802). However, at 160 mg/mL, BEO in combination with IVM showed a small antagonistic interaction, reducing efficacy to 93.87 % (P = 0.6029) when compared to IVM alone (100 %). Nadi reagent staining also revealed concentration-dependent coloration changes in nematodes treated with BEO, suggesting internal compound action. However, further histological analysis is needed to confirm cellular effects and vacuole formation. In conclusion, BEO demonstrated activity against L3 in ruminant nematodes. Three drug combinations exhibited synergy effects, while IVM + BEO exhibited antagonistic effects. These findings highlight BEO's potential as a standalone and as an anthelmintic candidate when used in combination. There is still a need for further research into BEO mechanisms of action, histological and toxic effects in preclinical experiments.