Avian coccidiosis is a disease causing considerable economic losses in the poultry industry. It is caused by Eimeria spp., protozoan parasites characterized by an exogenous–endogenous lifecycle. In vitro research on these pathogens is very complicated and lacks standardization. This review provides a description of the main in vitro protocols so far assessed focusing on the exogenous phase, with oocyst viability and sporulation assays, and on the endogenous phase, with invasion and developmental assays in cell cultures and in ovo. An overview of these in vitro applications to screen both old and new remedies and to understand the relative mode of action is also discussed.

Coccidiosis poses a significant challenge in poultry production and is typically managed with ionophores and chemical anticoccidials. However, the emergence of drug resistance and limitations on their use have encouraged the exploration of alternative solutions, including botanical compounds and improvements in in vitro screening methods. Prior research focused only on the impact of these alternatives on Eimeria invasion, with intracellular development in cell cultures receiving limited attention. This study assessed the impact of thyme (Thymus vulgaris), oregano (Origanum vulgare), and garlic (Allium sativum) essential oils, as well as their bioactive compounds, on the initial phase of schizogony in Madin–Darby bovine kidney cells, comparing their effectiveness to two commercially used anticoccidial drugs. Using image analysis and quantitative PCR, the study confirmed the efficacy of commercial anticoccidials in reducing invasion and schizont formation, and it found that essential oils were equally effective. Notably, thymol and carvacrol exhibited mild inhibition of intracellular replication of the parasite but significantly reduced schizont numbers, implying a potential reduction in pathogenicity. In conclusion, this research highlights the promise of essential oils and their bioactive components as viable alternatives to traditional anticoccidial drugs for mitigating coccidiosis in poultry, particularly by disrupting the intracellular development of the parasites.

Primary chicken intestinal epithelial cells or 3D enteroids are a powerful tool to study the different biological mechanisms that occur in the chicken intestine. Unfortunately, they are not ideal for large-scale screening or long-term studies due to their short lifespan. Moreover, they require expensive culture media, coatings, or the usage of live embryos for each isolation. The aim of this study was to establish and characterize an immortalized chicken intestinal epithelial cell line to help the study of host–pathogen interactions in poultry. This cell line was established by transducing into primary chicken enterocytes the SV40 large-T antigen through a lentiviral vector. The transduced cells grew without changes up to 40 passages maintaining, after a differentiation phase of 48 h with epidermal growth factor, the biological properties of mature enterocytes such as alkaline phosphatase activity and tight junction formation. Immortalized enterocytes were able to generate a cytokine response during an inflammatory challenge, and showed to be susceptible to Eimeria tenella sporozoites invasion and generate a proper immune response to parasitic and lipopolysaccharide (Escherichia coli) stimulation. This immortalized cell line could be a cost-effective and easy-to-maintain model for all the public health, food safety, or research and pharmaceutical laboratories that study host–pathogen interactions, foodborne pathogens, and food or feed science in vitro.

Objectives: This study investigated the relationship between the chemical composition of Yucca schidigera saponins and their antimicrobial and anticoccidial activities against Clostridium perfringens and Eimeria tenella . Methods: Extracts of Y. schidigera were obtained using 100% methanol (MeOH), 50% MeOH, and water, and characterized by LC–HRMS to identify saponins. Multivariate analyses (PLS–DA and OPLS-DA) were performed to assess chemical differences among extracts. In vitro assays were conducted to evaluate antimicrobial activity against C. perfringens and anticoccidial activity against E. tenella . The most active extract was fractionated into three saponin-enriched subfractions, and ridge regression was applied to link chemical composition to bioactivity. Results: LC–HRMS identified multiple saponins in all extracts, with chemical differences among solvents. The 100% and 50% MeOH extracts inhibited C. perfringens growth at 750 ppm, while the water extract was inactive. The 100% MeOH extract reduced E. tenella intracellular development by 77%, compared with 48% and 25% inhibition by the 50% MeOH and water extracts. Fractionation of this extract produced three subfractions, of which the first (fraction 1) showed the highest bioactivity (MIC = 500 ppm; 84% inhibition of E. tenella ). Ridge regression highlighted schidigerasaponins D3/D4, smilagenin, sarsasapogenin, and hecogenin as key contributors to bioactivity. Conclusion: Y. schidigera saponins exhibit significant antimicrobial and anticoccidial activity. The study establishes a data-driven framework for predicting structure–activity relationships, providing a rational basis for botanical screening and standardization.