Combined effects of limonene and ivermectin on p-glycoprotein-9 gene expression of lambs infected with Haemonchus contortus

Authors: KATIKI, LUCIANA - Instituto De Zootecnia; GIGLIOTI, RODRIGO - Instituto De Zootecnia; Ferreira, Jorge; PACHECO, POLIANA - Universidade De Sao Paulo; BARBOSA, HENRIQUE - Instituto De Zootecnia; RODRIGUES, LEANDRO - Instituto De Zootecnia; VERISSIMO, CECILIA - Instituto De Zootecnia; BRAGA, PATRICIA - Universidade De Campinas (UNICAMP); AMARANTE, ALESSANDRO - Universidade Estadual Paulista (UNESP); LOUVANDINI, H - Universidade De Sao Paulo

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2023
Publication Date: 10/29/2023
Citation: Katiki, L.M., Giglioti, R., Ferreira, J.F., Pacheco, P.A., Barbosa, H.Z., Rodrigues, L., Verissimo, C.J., Braga, P.A., Amarante, A.F., Louvandini, H. 2023. Combined effects of limonene and ivermectin on p-glycoprotein-9 gene expression of lambs infected with Haemonchus contortus. Veterinary Parasitology. 324. Article 110069. https://doi.org/10.1016/j.vetpar.2023.110069.
DOI: https://doi.org/10.1016/j.vetpar.2023.110069

Interpretive Summary: Ruminant (goats, sheep, and cattle) farmers suffer economic losses each year due to gastrointestinal parasites. The barber-pole worm is the most economically important of all ruminant parasites. Ivermectin (IVM) is a widely used medicine for destroying parasitic worms, but its effectiveness is decreasing because the barber-pole worm has developed multi-drug resistance to ivermectin. Limonene (LIM) is a major component of citrus peel oil and when used in combination with IVM in laboratory studies has been found to regulate genes in a way that should combat parasitic drug resistance and restore the effectiveness of ivermectin. The current animal study investigated the use of LIM to combat parasite drug resistance in sheep. Sheep infected with IVM-resistant barber-pole worm that received LIM combined with IVM had significantly lower fecal egg counts but no reduction in adult parasite numbers. The use of LIM also did not affect the expression of the gene that controls the enzyme that removes IVM from parasite cells. The use of LIM in sheep was safe and caused no toxicity when provided with food. The use of the LIM citrus oil in infected sheep may be a safe and effective way to reduce pasture contamination and prevent the reinfection of sheep with gastrointestinal parasites.

Technical Abstract: Although ivermectin (IVM) has a wide spectrum and long half-life, its frequent use as an anthelmintic led to worldwide tolerance of Haemonchus contortus to IVM. To try to restore IVM anthelmintic activity, the interaction between IVM and limonene (LIM), a P-glycoprotein (pgp) modulator, was evaluated in lambs infected with multi-drug resistant H. contortus. Twenty-four male Dorper lambs were artificially infected with two doses (seven days apart) of 8,000 infective larvae of a multidrug-resistant isolate of H. contortus. Infection was patent 25 days later. Fifteen days before treatment with IVM (DAY -15), animals were divided in 4 groups: Untreated control-not drenched (CTL), IVM, LIM, and LIM+ IVM. From DAY -15 to DAY +14, groups LIM and LIM+IVM received 200 mg/kg of body weight/day of LIM via oral. On DAY 0 a single dose of IVM 200 µg/kg of body weight was administered orally to groups IVM and LIM+IVM. On DAY +7 and DAY +14 fecal egg counts (FEC) were performed and on DAY +14 animals were euthanized for total worm count (TWC), worms length, fecundity of females and Pgp-9 gene expression. Group LIM+IVM had 96.29% efficacy based on Fecal Egg Count Reduction TEST (FECRT) and a highly significant reduction in FEC (P=0.0005) on DAY+7 when compared to CTL. On DAY+14 the efficacy of LIM+IVM was 82.87% on FECRT, although no differences were found between groups for FEC, TWC, worm length, or Pgp-9 gene expression. Female worms from CTL group had higher egg counts in their uterus when compared to LIM. No differences were found for hematological or biochemical parameters, body weight, or weight gain between groups. In conclusion, LIM given daily at 200 mg/kg or combined with IVM was safe to animals but not able to kill H. contortus or had any interaction with Pgp-9 gene expression. LIM was safe and effective in decreasing egg shedding and pasture contamination.