DOI: 10.5061/dryad.mw6m90605
title: Reduction of chickens use to perform in vitro pre-screening of novel anticoccidials by miniaturisation and increased throughput of the current Eimeria tenella compound-screening model

description: 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.
  
description: 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. 
description: Bio-Rad CFX Manager software (Bio-Rad)