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

Abstract

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 ionophores and chemical anticoccidials; however, there is an overall agreement about the limitation of these traditional 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 in vivo trials for validation. Eimeria parasites can only complete their life cycle in their animal host, therefore chickens are required for their propagation as they cannot be propagated in vitro. In this study, we describe how further optimisation of this in vitro model by miniaturisation can have an additional impact in reduction of the number of chickens used for the generation of parasite stocks for provision of the in vitro model. We have estimated that the use of one chicken could support the evaluation of 10 compounds with a 96-well plate format versus only two compounds with a 24-well plate format, which means an 80% reduction in chicken use. In this study we have proved that the miniaturisation into a 96-well plate format perfectly mimics the invasion and replication observed before in the 24-well plate format. In addition, the 96-well plate 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.

Investigating the effects of essential oils and pure botanical compounds against Eimeria tenella in vitro

“…Furthermore, these molecules could also interfere with cell proliferation and biochemical pathways, thus exhibiting a certain degree of toxicity in animals ( Koutoulis et al, 2013 ; Kaushik et al, 2018 ; Noack et al, 2019 ). Limitations of anticoccidial drugs and the need to improve the current methods to control the disease have pushed research toward the investigation of alternative molecules ( Soutter et al, 2021 ; Arias-Maroto et al, 2022 ).…”

Thyme, Oregano, and Garlic Essential Oils and Their Main Active Compounds Influence Eimeria tenella Intracellular Development

“…As previously reported, a pre-treatment with these molecules, before infection, is already enough to cause sporozoites death, thus stopping any further maturation [ 18 ]. In a similar study, the impact of 1 h of pre-incubation with salinomycin and robenidine on intracellular development was investigated by qPCR [ 25 ]. Pre-treatment with salinomycin 1 ppm lowered the level of invasion by 31%.…”

“…Pre-treatment with salinomycin 1 ppm lowered the level of invasion by 31%. Additionally, both robenidine and salinomycin pre-treatment managed to decrease the rate of replication between 24 and 44 hpi [ 25 ]. In the present study, the treatment with anticoccidials occurred along invasion and continued for the whole time course.…”

“…This quantification was achieved using gDNA-specific primers targeting E. tenella 5S rDNA (Fw_5S: TCATCACCCAAAGGGATT, Rv_5S: TTCATACTGCGTCTAATGCAC) [ 20 ]. A standard curve of sporozoite gDNA, extracted using the same methods, was generated [ 25 ]. This standard curve encompassed gDNA equivalent to 10 7 genomes, followed by serial dilution until 10 2 genomes.…”

“…To compare the replication rates after treatments, excluding the effects of invasion, slopes of the replication lines were calculated between 24 and 72 hpi. The slopes were calculated with the formula below, as previously described by Arias-Maroto et al [ 25 ]: …”