Thyme essential oil and its phenolic derivatives are effective anticoccidial and antibacterial against Eimeria tenella and Clostridium perfringens in vitro

Coccidiosis, caused by protozoan parasites of the genus Eimeria, is one of the main threats for aviculture, as it causes severe hemorrhagic diarrhea and acts as a predisposing factor for necrotic enteritis, whose causative agent is Clostridium perfringens. The aim of this study was to investigate in vitro the anticoccidial and anticlostridial efficacy of thyme essential oil and its main phenolic bioactive compounds (thymol, carvacrol and eugenol) against E. tenella and C. perfringens, respectively by invasion and minimal inhibitory concentration (MIC) assays, in comparison with conventional antibiotics. For the invasion assay, confluent Madin-Darby bovine kidney cells were challenged with E. tenella sporozoites (5x104/cm2), without (control) or with various treatments: salinomycin (5 ppm), thyme oil (40 ppm, thymol content 50%), thymol, carvacrol or eugenol (20 ppm each). After 2 and 24 h at 37°C the efficiency of invasion was determined by counting free sporozoites left in the medium outside cells. For the MIC assay, a broth microdilution method on 96-well microtiter plates was used to test a range of concentrations (2-fold dilutions) of bacitracin (64-0.5 ppm), thyme essential oil (2000-62.5 ppm, thymol content 50%), thymol, carvacrol or eugenol (7.5-0.23 mM). Clostridium perfringens ATCC® 13124 strain (105 CFU/mL) was incubated with the substances under anaerobic conditions in Reinforced Clostridial Medium at 37°C for 24 h. After incubation, the absorbance at 630 nm was read with a spectrophotometer to measure bacterial growth. The MIC was defined as the lowest concentration that resulted in null absorbance. Data were analyzed with one-way ANOVA and differences considered significant at p<0.05. The invasion assay showed that all the treatments significantly inhibited E. tenella invasion efficiency compared to the control already after 2 hours by 55% on average and similarly at 24 h with the highest reduction for salinomycin (-54% compared to control) and an average reduction of 35% for all the botanical treatments (p<0.001). Against C. perfringens the MIC values were found for bacitracin at 2 ppm, for thymol and carvacrol at 1.87 mM, for eugenol at 3.75 mM and for thyme oil at 500 ppm (equivalent to 1.66 mM of thymol). In conclusion, both thyme essential oil and its main phenolic bioactive compounds demonstrated anticoccidial and bactericidal activity against E. tenella and C. perfringens in vitro. Thus, these botanicals could be valuable tools to counteract the causative agents of avian coccidiosis and necrotic enteritis.