The objective of the present study was to evaluate the antiviral properties of Thymoquinone (TQ), an active component of Nigella sativa, on avian infectious bronchitis virus (IBV) using embryonated eggs.
120 conventional embryonated eggs aged between 9 and 11 d were randomly divided into 8 groups (G1 to G8) and inoculated via the allantoïc route as follows: G1 with Argan oil (0.2ml), G2, G3 and G4 with TQ, diluted in Argan oil, at concentrations of 40 µg/0.2 ml, 20 µg/0.2 ml and 10 µg/0.2 ml respectively. G5, G6 and G7 were inoculated simultaneously, in addition to the TQ in Argan oil at the above concentrations, with the IBV Italy 02 strain at the 10-4 DIE50 load. The G8 acted as a negative control.
This protocol was previously developed to select the appropriate solvent and non-embryotoxic concentrations of TQ. For all assays, eggs were incubated at 37°C and 54% relative humidity from day 11 to day 18 and were examined (candled) daily during the incubation period.
As a result, tests with the following solvents: DMSO, Argan oil, kerosene oil and PBS revealed that only DMSO was embryotoxic. The choice of the solvent to be used for the other experiments was therefore Argan oil, since no study has shown any interaction with the immune system.
As for the concentrations of TQ used in these experiments, the dose of 200µg/0.2ml was found to be toxic, while the doses of 100µg/0.2ml and 40µg/0.2ml did not cause high mortality or external abnormalities in the embryos. However, both pure fixed oil and ½ diluted fixed oil of black cumin caused 100% mortality, revealing very acute toxicity.
The results obtained showed that the survival rates were 100% for G1 (HA) and G2 (40 µg TQ), 60% for G3 (20 µg), 80% for G4 (10 µg), 37.5% for G5 (40 µg + virus), 62.5% for G6 (20 µg + virus) and G7 (20 µg + virus) and 100% for G8 (negative control). Macroscopic study revealed no lesions or alterations in the external appearance of the embryos, and real-time RT-PCR gave negative results demonstrating that TQ neutralized the virus.