Anthelmintic activity increase of Ivermectin by the sinergistic action of phytochemical compounds in isolates of Haemonchus contortus susceptible and resistant to anthelmintics

Author(s)

Poliana Araujo Pacheco

• Document type: Doctoral Thesis
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Centro de Energia Nuclear na Agricultura (CENA/STB)
• Defense date: 2023-01-10

Examining board members

Luciana Morita Katiki; Alessandro Francisco Talamini do Amarante; Sarita Bonagurio Gallo; Livio Martins Costa Júnior

• Advisor: Helder Louvandini; Luciana Morita Katiki

Abstract

Phytochemicals can increase ivermectin (IVM) effect through the modulation of glycoprotein P (PgP) activity, a trans-membrane transport protein that rids worm cells like Haemonchus contortus from toxic molecules and its considerado of the main IVM-resistance mechanisms. A complete screening was proposed to verify the interaction between IVM and phytochemicals. The first step of this work was to evaluate and compare the anthelmintic activity of IVM alone or combined with essential-oil main components (alpha terpinene, beta citronellol, beta pinene, citronellal, limonene, menthol, and terpinolene) and with phenol compounds (epicatechin, epigallocatechin, gallocatechin pentagalloylglucose, procyanidin, and quercetin). All compounds were tested against susceptible (HcS) and resistant (HcR) isolates of H. contortus through the larval development test (TDL), adult motility assay (AMT) and gene expression of PgP by RT-PCR, using verapamil (VP), a known PgP modulator, as a control in all tests. In TDL the lethal concentration of IVM required to kill 50% of nematodes (LC50) was 10 times greater for HcR than for HcS. IVM + VP inhibited the activity of PgP in HcR. IVM + limonene and IVM + quercetin both showed similar to IVM+VP and were chosen for subsequent tests. In AMT, IVM + VP in HcR were similar to IVM + limonene and outperformed IVM + quercetin. In gene expression of adult worms exposed to IVM, IVM+VP and IVM + limonene, for most concentrations evaluated, PGP-9 was significantly more expressed in worms treated with IVM alone than worms treated with IVM + VP or IVM + limonene. In conclusion, limonene is involved in the modulation of the PGP-9 gene and restored the activity of IVM in the HcR isolate. The last step an in vivo experiment was proposed to verify the interaction between limonene supplied in the diet with the concentrate and a deworming step with single oral ivermectin (1 mL/4kg/PV) in 24 Dorper lambs at approximately 5 months of age artificially infected with a resistant strain of H. contortus distributed into 4 treatments: CONTROL, LIMO (limonene - 200 mg/kg/pv/day supply), LIVM(limonene plus ivermectin - 200 mg/kg/BW/day and a single oral dose of Ivermectin 1 mL/4kg BW), IVM (1 mL/4kg BW). Fecal and blood samples were collected during the experimental period and after 14 days of IVM administration to the respective treatments, all animals were euthanized to count the total worms present in the abomasum, evaluation of morphometric measurements of males and females, fertility of females Worms. No differences were observed in the worm counts and length parameters of males and females, in the treatments. However, female fertility was affected, as we observed statistical differences between CONTROL and LIMO, LIMO and LIVM. About fecal egg count, LIVM and LIMO differed from each other and IVM was equal to CONTROL. LIVM differed control and IVM, IVM differed only LIVM. The use of limonene associated a ivermectin as a control alternative to the single anthelmintics in livestock, being important to highlight the need for more validation research (AU)

• FAPESP's process: 18/02423-0 - Increase anthelmintic activity of Ivermectin by synergistic action of phytochemical compounds in Haemonchus contortus
• Grantee: Poliana Araújo Pacheco
• Support Opportunities: Scholarships in Brazil - Doctorate