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Doctoral Thesis
DOI
https://doi.org/10.11606/T.64.2023.tde-31102023-094720
Document
Author
Full name
Poliana Araujo Pacheco
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2023
Supervisor
Committee
Katiki, Luciana Morita (President)
Amarante, Alessandro Francisco Talamini do
Gallo, Sarita Bonagurio
Júnior, Livio Martins Costa
Title in Portuguese
Incremento da atividade anti-helmíntica da Ivermectina pela ação sinérgica de compostos fitoquímicos sob isolados de Haemonchus contortus susceptível e resistente a anti-helmínticos
Keywords in Portuguese
Compostos fenólicos
Haemonchus contortus
Ivermectina
Limoneno
Óleos essenciais
PgP
Abstract in Portuguese
Os fitoquímicos podem aumentar o efeito da ivermectina (IVM) através da modulação da atividade da glicoproteína P (PgP), uma proteína de transporte transmembrana que livra as células de nematóides como o Haemonchus contortus de moléculas tóxicas e considerado um dos principais mecanismos de resistência à IVM. Uma triagem completa foi proposta para verificar a interação entre IVM e fitoquímicos. A primeira etapa deste estudo foi avaliar e comparar a atividade anti-helmíntica de IVM isolada ou combinada com os principais componentes de óleos essenciais (alfa terpineno, beta citronelol, beta pineno, citronelal, limoneno, mentol e terpinoleno) e compostos fenólicos (epicatequina, epigalocatequina, gallocatequina pentagaloilglicose, procianidina e quercetina). Todos os compostos foram testados contra isolados suscetíveis (HcS) e resistentes (HcR) de H. contortus através do teste de desenvolvimento larval (TDL), teste de motilidade de adultos (TMA) e análise de expressão de PGP de vermes adultos por RT-PCR, usando verapamil (VP), conhecido modulador de PgP, como controle em todos os testes. No TDL, a concentração letal de IVM necessária para matar 50% dos nematóides (LC50) foi 10 vezes maior para HcR do que para HcS. IVM + VP inibiu a atividade de PgP em HcR. IVM + limoneno e IVM + quercetina mostraram-se semelhantes a IVM+VP e foram escolhidos para testes subsequentes. No AMT, IVM + VP em HcR foram semelhantes a IVM + limoneno e superaram IVM + quercetina. Na expressão gênica de vermes HcR expostos a IVM, IVM+VP e IVM + limoneno, para a maioria das concentrações avaliadas, PGP-9 foi significativamente mais expresso em vermes tratados com IVM isolada do que em vermes tratados com IVM + VP ou IVM + limoneno. Em conclusão, o limoneno está envolvido na modulação do gene PGP-9 e restaurou a atividade de IVM no isolado HcR. Na última etapa foi proposto um experimento in vivo para verificar a interação entre o limoneno fornecido na dieta com o concentrado e uma etapa de vermifugação com dose única de ivermectina oral (1 mL/4kg/PV) em 24 cordeiros Dorper com aproximadamente 5 meses de idade infectados artificialmente com uma isolado resistente de H. contortus e distribuídos em 4 tratamentos: CONTROLE, LIMO (limoneno - 200 mg/kg/PV/dia), LIVM (limoneno + ivermectina - 200 mg/kg/PV/dia + dose única 1 mL de Ivermectina /4kg PV), IVM (1 mL/4kg PV). Foram coletadas amostras de fezes e sangue durante o período experimental e após 14 dias da administração da IVM aos respectivos tratamentos, todos os animais foram eutanasiados para contagem do total de vermes presentes no abomaso, avaliação de medidas morfométricas de machos e fêmeas, fertilidade das fêmeas. Não foram observadas diferenças nas contagens de vermes e parâmetros de comprimento de machos e fêmeas, nos tratamentos. No entanto, a fertilidade das fêmeas foi afetada, pois observamos diferenças estatísticas entre CONTROLE e LIMO e LIMO e LIVM. Sobre o FEC, o LIVM e o LIMO diferiram entre si e o IVM foi igual ao CONTROLE. LIVM diferiu do controle e IVM, IVM diferiu apenas de LIVM. O uso do limoneno associado a ivermectina pode ser uma alternativa de controle ao uso único de anti-helmínticos na pecuária, sendo importante detacar a necessidade de mais pesquisas de validação
Title in English
Anthelmintic activity increase of Ivermectin by the sinergistic action of phytochemical compounds in isolates of Haemonchus contortus susceptible and resistant to anthelmintics
Keywords in English
Essential oil
Haemonchus contortus
Ivermectin
Limonene
Pgp
Phenolic compounds
Abstract in English
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
 
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Publishing Date
2023-11-21
 
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