In vitro models have supported important advances in biomedical sciences and have significantly contributed to reduce the use of experimental animals in different disciplines. We have developed an in vitro model for the evaluation of potential anticoccidial properties of novel compounds aimed to control chicken coccidiosis, a costly disease for the poultry industry. This disease is caused by protozoan parasites of the genus Eimeria (Apicomplexa), and it is mainly controlled by chemoprophylaxis with ionophors and chemical anticoccidials; however, there is an overall agreement about the limitation of these classical drugs and the need to improve current methods of control. Anticoccidial activities of novel compounds is currently evaluated by expensive experiments that involve large numbers of chickens. The use of our in vitro model for the pre-screening of essential oils led to a reduction of 67% of the chickens used in the vivo trials for validation. In this study, we describe how further optimisation of this in vitro model by miniaturisation can have an additional impact on the number of chickens used for the generation of parasite stocks for provision of the in vitro model (which cannot be done in vitro). We have estimated that the use of one chicken could support the evaluation of ten compounds with a 96-well plate format vs. only two with a 24-well plate format, which means an 80% of chicken use reduction. In this study, we have proved that the miniaturisation into a 96-well plate format has perfectly mimicked the invasion and replication observed before in the 24-well plate format. In addition, this format has allowed the simultaneous pre-screening of higher numbers of anticoccidial drugs at different concentrations following streamlined protocols in a more cost-effective way, factors that are beneficial for a wider uptake of the model by other researchers investigating anticoccidial compounds.  

Real time quantitative PCR (qPCR) was performed in a CFX96 Touch® Real-Time PCR Detection System (Bio-Rad, Hertfordshire, UK) using DNA-binding dye SsoFastTM EvaGreen® Supermix (Bio-Rad). The PCR mixture contained 1 μl DNA template (gDNA), SsoFast EvaGreen supermix (10 μl), forward and reverse primers (gDNA specific primers for Eimeria tenella 5S rDNA) (250 nM) and RNase/DNase-free water in a final volume of 20 μl. Amplification was performed according to the manufacturer’s protocol, with one cycle at 95 °C for 1 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s.

All samples were processed in triplicate and a no template negative control. A standard curve of sporozoite genomic DNA (gDNA) was used (gDNA equivalent to 107 followed by serial dilution until 101).

Data were analysed with the Bio-Rad CFX Manager software (Bio-Rad).

The number of sporozoites for each data point is automatically determined for each well by a regression analysis using Bio-Rad CFX Manager software (Bio-Rad). Outliers showing a standard deviation of more than 0.5 cycles were excluded from the analysis. 

Bio-Rad CFX Manager software (Bio-Rad)