The anticoccidial activity of thymol, carvacrol, saponins and garlic oil was assessed in an in vitro model of coccidiosis. Eimeria spp. sporozoites were collected from field samples, characterized and used for 3 different invasion assays on Madin-Darby Bovine Kidney cells (MDBK). The cells were challenged with 5 x 104 sporozoites without (control) or with various treatments: saponins (10 ppm), thymol and carvacrol (7 ppm each), a combination of saponins, thymol, and carvacrol at 2 doses; MIX 1 (saponins 5 ppm, thymol 3.5 ppm and carvacrol 3.5 ppm), MIX 2 (saponins 10 ppm, thymol 7 ppm and carvacrol 7 ppm). Garlic oil was tested at two concentrations: 10 ppm and 100 ppm. The treated cells were incubated at 37°C for 24 hours (invasion assay 1 and 3) and for 2, 24, and 48 hours (invasion assay 2). The efficiency of invasion was determined by counting the sporozoites left in the supernatant that were not able to invade the cells, while intracellular Eimeria DNA was detected by qPCR to confirm the data. Data were analyzed with ANOVA and differences were considered significant when p-value was ≤ 0.05. Data from invasion assay 1 showed that the thymol and carvacrol-containing blends significantly reduced invasion, especially in combination with saponins at the highest dose. Saponins alone did not have a strong inhibiting activity but acted synergistically with the other molecules. Interestingly, in invasion assay 2 it was found that the effect of the highest dose of the blend of saponins, thymol and carvacrol was already visible at 2 hours post infection, while the other treatments were significantly successful at 24 hours post infection. Data form invasion assay 3 proved the inhibiting power of garlic oil at the lowest dose, while the highest one was cytotoxic for MDBK cells too. The invasion assay protocol was designed to screen molecules in vitro starting from field fecal samples and it can represent a potential tool in Eimeria research. Moreover, this study shows that invasion in MDBK cells by Eimeria sporozoites is inhibited in presence of thymol, carvacrol, saponins and garlic oil, thus highlighting the anticoccidial potential of these compounds.
Felici M., T.B. (2020). ATTIVITÀ ANTICOCCIDICA IN VITRO DI ESTRATTI VEGETALI.
ATTIVITÀ ANTICOCCIDICA IN VITRO DI ESTRATTI VEGETALI
Felici M.;Ghiselli F.;Massi P.;Piva A.;Grilli E.
2020
Abstract
The anticoccidial activity of thymol, carvacrol, saponins and garlic oil was assessed in an in vitro model of coccidiosis. Eimeria spp. sporozoites were collected from field samples, characterized and used for 3 different invasion assays on Madin-Darby Bovine Kidney cells (MDBK). The cells were challenged with 5 x 104 sporozoites without (control) or with various treatments: saponins (10 ppm), thymol and carvacrol (7 ppm each), a combination of saponins, thymol, and carvacrol at 2 doses; MIX 1 (saponins 5 ppm, thymol 3.5 ppm and carvacrol 3.5 ppm), MIX 2 (saponins 10 ppm, thymol 7 ppm and carvacrol 7 ppm). Garlic oil was tested at two concentrations: 10 ppm and 100 ppm. The treated cells were incubated at 37°C for 24 hours (invasion assay 1 and 3) and for 2, 24, and 48 hours (invasion assay 2). The efficiency of invasion was determined by counting the sporozoites left in the supernatant that were not able to invade the cells, while intracellular Eimeria DNA was detected by qPCR to confirm the data. Data were analyzed with ANOVA and differences were considered significant when p-value was ≤ 0.05. Data from invasion assay 1 showed that the thymol and carvacrol-containing blends significantly reduced invasion, especially in combination with saponins at the highest dose. Saponins alone did not have a strong inhibiting activity but acted synergistically with the other molecules. Interestingly, in invasion assay 2 it was found that the effect of the highest dose of the blend of saponins, thymol and carvacrol was already visible at 2 hours post infection, while the other treatments were significantly successful at 24 hours post infection. Data form invasion assay 3 proved the inhibiting power of garlic oil at the lowest dose, while the highest one was cytotoxic for MDBK cells too. The invasion assay protocol was designed to screen molecules in vitro starting from field fecal samples and it can represent a potential tool in Eimeria research. Moreover, this study shows that invasion in MDBK cells by Eimeria sporozoites is inhibited in presence of thymol, carvacrol, saponins and garlic oil, thus highlighting the anticoccidial potential of these compounds.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
