Coccidiosis, caused by protozoan parasites of the genus Eimeria, is one of the most economically significant diseases affecting poultry worldwide. Among them, Eimeria tenella is recognized as one of the most pathogenic species, responsible for severe lesions in the ceca, that eventually lead to reduced weight gain and deaths in chickens. Furthermore, its suitability for in vitro cultivation makes it a valuable model organism for the evaluation of anticoccidial compounds (Ball et al., 1990). The economic burden of this disease is substantial, estimated at 13 billion US dollars per year, due to direct losses in poultry production and costs associated with prevention and treatment (Blake et al., 2020).

The control of coccidiosis in broilers relies on the use of ionophores and chemical anticoccidials, however, the spreading of resistance to these treatments has emphasized the urgency of improving research on alternative therapeutic strategies (Noack et al., 2019). In this context, botanicals have gained increasing attention as promising alternatives due to their natural origin, diverse composition, and potential to reduce parasite load while supporting gut health (Muthamilselvan et al., 2016; Noack et al., 2019). These compounds comprehend various classes of molecules and their efficacy can vary depending on various factors, so the assessment of their efficacy needs continuous screening (Felici et al., 2024).

In recent years, anticoccidial research has seen important advances in the development of rapid, sensitive, and well-standardized in vitro methods to quantify parasite invasion and proliferation, with several new approaches now available. Nevertheless, conventional techniques based on in vivo experimentation, such as oocyst counting, histological analysis, or quantitative PCR (qPCR) remain widely used. These methods, while informative, can still be time-consuming, labor-intensive, and in some cases require animal sacrifice, with assay-to-assay variability arising from long sample processing (Felici et al., 2021). Evaluation of alternative anticoccidial compounds through in vitro approaches could allow high throughput screening, reducing the use of animals and related costs. Current in vitro methods rely on the use of microscopy-based assays or quantification of the intracellular parasite DNA by qPCR (Jenkins et al., 2014; Thabet et al., 2015; Marugan-Hernandez et al., 2020; Felici et al., 2021).

While sporozoite staining and manual counting have long been employed to estimate anticoccidial efficacy (Burt et al., 2013; Felici et al., 2020), these approaches are intrinsically subjective and prone to operator bias, therefore, automated systems for quantification offer a more accurate and reproducible method to assess treatments (Marugan-Hernandez et al., 2020). Stably-transfected strains of E. tenella expressing reporter genes (i.e. fluorescent proteins) have been used to track the development of the parasite in cell culture. For instance, Bussiére et al. (2018) (Bussière et al., 2018) used a recombinant strain expressing the yellow-fluorescent protein (YFP) under the mic1 promoter to visualize parasites in invasion and schizont development assays, while another strain was created by transfecting E. tenella with the mCherry gene under the actin promoter to facilitate visualization of sexual stages of the parasite in cell culture (Bussière et al., 2018). Later, Marugan-Hernandez et al. (2020) (Marugan-Hernandez et al., 2020) used E. tenella YFPmYFP (EtdYFP) (Clark et al., 2008) and a custom ImageJ tool to perform a semi-automatic quantification of the parasite development in cells, correlating the data to qPCR analysis. This method was also used to screen anticoccidial activity in vitro and subsequently to evaluate natural compounds, validating its applicability to evaluate the anticoccidial effects of botanical compounds (Felici et al., 2024).

Further innovations in drug screening include the use of bioluminescence systems, as these offer a dynamic method to quantify viability and replication, and has been increasingly applied to develop in vitro models for infectious diseases (Azevedo et al., 2014; Vinayak et al., 2015; England et al., 2016; Berry et al., 2018; Smith et al., 2023). Among the various luciferases available, NanoLuc (NLuc) has gained increasing attention due to its small size, high stability, and exceptional luminescent signal intensity with minimal background noise (England et al., 2016). NLuc is an artificial coelenterazine-dependent luciferase generated from the organism Oplophorum gracilirostris. Its long-lasting bright bioluminescence triggered with the synthetic substrate furimazine, has made this enzyme popular as a reporter in a variety of analytical systems (England et al., 2016). NLuc-based systems have been successfully applied in several protozoan models, enabling high-throughput screening and real-time monitoring of infection dynamics in vitro. For example, Azevedo et al. (2014) produced a luminescent strain of Plasmodium falciparium, causative parasite of malaria, using Nluc to create sensitive and affordable growth assays to understand the mode of action of antimalarian drugs (Azevedo et al., 2014). A similar approach was adopted by Vinayak et al. (2015), who optimized a Nluc luminescent strain of Cryptosporidium parvum to evaluate the basis of drug susceptibility by gene knock out in vitro (Vinayak et al., 2015). Additionally, other research studying Toxoplasma gondii, and Leishmania mexicana has used modified approaches of Nluc assays to evaluate parasite viability in an in vitro cell model during drug testing (Berry et al., 2018; Smith et al., 2023).

In this study, the generation of a genetically modified E. tenella population stably expressing NLuc (EtNLuc) is reported, with a focus on development for rapid screening of potential anticoccidial compounds. This transgenic parasite population enabled robust luminescent detection of intracellular parasite stages in cultured cells. This system also provided a highly sensitive, rapid, and reproducible method to quantify E. tenella invasion and replication in vitro, suitable to perform anticoccidial compound screenings. The development of this luminescence-based platform represents a valuable advancement in the field of coccidiosis research, reducing the number of animals used for the generation of oocysts, and contributing to more efficient discovery pipelines and screening accuracy.

This study describes the generation of a transgenic population, EtNLuc, derived from the reference strain EtWis, that incorporates both fluorescent and bioluminescent reporter cassettes. To verify whether the bioluminescence produced by Nluc was suitable to detect replication, the parasite was taken through a time course assay with luminescence measured at 2, 24, 48, 72 hpi. The increase of signal detected over time was comparable to observations from previous work using quantification by qPCR or fluorescence measurements using the EtdYFP strain (Marugan-Hernandez et al., 2020; Arias-Maroto et al., 2022). Between 2 and 24 hpi there was no significant change in the levels of luminescence, consistent with the qPCR data using an equivalent infection set up (Arias-Maroto et al., 2022). This period is representative of sporozoites rounding up forming a trophozoite, without the nuclear replication occurring (Pacheco et al., 1975; Marugan-Hernandez et al., 2020). Between 24 and 48 hpi there was a significant increase in luminescence readings, consistent with the increase in genome copy number previously detected by qPCR, representing the development from trophozoites into immature schizonts and subsequent nuclear replication (Pacheco et al., 1975; Marugan-Hernandez et al., 2020). Between 48 and 72 hpi the signal kept increasing, correlating with the increasing genome copy numbers described between 44 and 52 hpi by Arias-Maroto et al. (2022) (Arias-Maroto et al., 2022). These time points correspond to the period of development representing maturing schizonts and the release of merozoites (Pacheco et al., 1975; Marugan-Hernandez et al., 2020). Thus, the new EtNLuc bioluminescent strains reliably reflect parasite replication at the selected time points corresponding to specific developmental processes.

Previous studies have shown that the 4:1 parasite to cell infection ratio allowed the supported a higher number of infected cells on the monolayer in comparison to 2:1 or 1:1 ratios (73%, 60% and 39% respectively) (Arias-Maroto et al., 2022), however, how these initial proportions affect replication rates has not been studied before. At 2 hpi and 24 hpi, luminescence readings were not significantly different between the three MOIs, however, a clear trend of lower reading at lower MOI was observed. At 48 hpi a significant increase was observed between 4:1 and the other MOIs. Similarly, at 72hpi, an analogous pattern was observed. The comparison of growth curves for each ratio showed that lower number of parasites led to a slower replication rate (slope), supporting a direct relationship between initial number of intracellular sporozoites and their replication rates. Since the primary aim of the EtNluc strain is to be used for the assessment of potential anticoccidial therapies, the MOI 4:1 was selected as this provided a wider dynamic range to evaluate inhibitory effects in parasite replication. Additionally, the higher goodness of fit value (R²) observed at a MOI 4:1, indicates that this dose of infection not only produced the highest signal, but also the most predictable and reproducible growth kinetics, making it the optimal condition for quantitative parasite proliferation assays.

The initial time course experiment for the bioluminescence-based assay standardization discussed above was performed by doing single readings per plate. To evaluate the impact of performing continuous measurements on the same infected monolayer, only 2 and 72 hpi points were selected, as based on the single readings, they would provide enough significance for studies evaluating inhibition of invasion and replication. The same three MOIs were evaluated, showing equivalent significance and trends that in the single measurement experiments. This supported the adaptability of the method to repeated reading over the same plate. This repeated measurement system reduces the need of oocysts for each tested compound and, consequently, the number of experimental chickens needed to produce these oocysts, in accordance with the 3R (Replacement, Reduction and Refinement) principle of animal experimentation (Russel and Burch, 1959).

When comparing the results obtained from the EtNluc strain to those generated by the qPCR method using EtWis parasites, as well as imaging with the fluorescent EtdYFP strain, the outcomes were comparable (Marugan-Hernandez et al., 2020; Arias-Maroto et al., 2022). This highlights the bioluminescence-based assay as a valuable and reliable tool. While fluorescent reporters are ideal to track the presence and localization of parasites in biological samples and cell culture, they still present certain limitations. For instance, their moderate sensitivity can be hindered by sample autofluorescence, and their fluorescence signal is prone to photobleaching over time. Moreover, proper visualization often requires cell fixation with paraformaldehyde and washing steps, which restricts the use of these methods for real-time or live-cell quantification (Haugwitz et al., 2008). Hence, the bioluminescence-based system is less labour-intensive, requires fewer steps, and produces results more quickly, enabling the simultaneous screening of a larger number of compounds.

The NLuc reporter expressed by EtNLuc sporozoites provided stable luminescence, enabling sensitive detection of intracellular parasites during treatment with natural compounds and anticoccidial drugs. Using this reporter system, the efficacy of anticoccidials and botanical compounds was evaluated in two distinct assay formats: a pre-incubation protocol, in which sporozoites were exposed to compounds for 1 h prior to invasion and during the first 2 hpi; and a continuous exposure protocol, in which compounds remained present throughout intracellular development, form the addition of sporozoites to the cell monolayers until 72 hpi. In the first assay, the experiment was designed to evaluate specific effects while the parasites remain extracellular; whereas in the second protocol, the potential intracellular activity of the compounds was assessed by maintaining the treatment after the invasion of the parasite.

In both assays, salinomycin and robenidine significantly impaired parasite invasion. Salinomycin consistently achieved higher inhibition, in line with its well-documented ability to disrupt ionic gradients and compromise sporozoite viability prior to cell entry (Noack et al., 2019). The short pre-treatment was also sufficient to significantly inhibit intracellular replication, indicating a lasting effect of the molecule. On the other hand, robenidine showed a different pattern of efficacy. Robenidine is a guanidine derivative whose main action is the inhibition of maturation of 1^st generation schizonts (McDougald and Galloway, 1976). Robenidine displayed higher inhibition of replication during continuous treatment rather than with a short pre-treatment during invasion. This is consistent with other findings which revealed a higher antiparasitic effect when the treatment was left in contact with the infected cells for the whole duration of schizogony, confirming the specific efficacy against intracellular development (Arias-Maroto et al., 2022; Felici et al., 2024).

Among the tested botanicals, TEO exhibited a strong invasion-blocking effect in the pre-incubation exposure assay, reducing entry by 40-50%, but also affecting development, with a significant reduction of the replication rate compared to the control. The other compounds showed similar efficacy. These findings agree with previous research reporting that terpenoids such as THY and CAR, major components of TEO and OEO, can rapidly destabilize the parasite membranes and reduce sporozoite infectivity (Felici et al., 2023). However, a stronger effect of botanicals was observed under continuous exposure, especially on replication, highlighting a possible cumulative impact of these compounds as the parasite develop. These findings further support the notion that essential oils interfere with multiple aspects of the intracellular development and replication, including parasite vacuole integrity, oxidative response, and cell immunity, acting as multi-target anticoccidial agents (Muthamilselvan et al., 2016).

Some variability was observed on the inhibition of invasion between both replicates in the continuous exposure experiments. In the first experimental replicate, none of the treatments showed a reduction in invasion, while in the second the invasion inhibition was more marked, likely reflecting the intrinsic variability of these in vitro assays (Arias-Maroto et al., 2024). Regardless of this less reliable invasion evaluation, the primary aim and most significant outcome of the continuous exposure experiments was on intracellular replication, which was markedly reduced for all treatments.

The EtNluc-based in vitro assay proved to be highly sensitive, reproducible, and well suited for dissecting stage-specific effects of the tested anticoccidial compounds. Unlike fluorescent microscopy or qPCR, this luminescent system offers real-time quantification, enabling higher throughput and lower variability (Vinayak et al., 2015). The correlation between luminescence signal and renowned drug activities validates this approach as a reliable platform for screening both synthetic and natural product. Furthermore, the ability to perform repeated measurements at different time points minimizes the number of parasites required, thereby reducing animal sacrifice and aligning with the 3Rs principle in parasitology research (Russel and Burch, 1959).

This study reports the successful generation of a NLuc-expressing Eimeria tenella strain and demonstrates its application as a sensitive, rapid, and reproducible platform for in vitro anticoccidial screening. By distinguishing the effects of treatments applied during short pre-incubation versus continuous exposure, the assay enabled assessment of stage-specific activity against both invasion and intracellular replication. Commercial anticoccidials such as salinomycin and robenidine displayed the expected inhibitory profiles, thereby validating the approach. Importantly, natural compounds including thyme and oregano essential oils and their constituents thymol and carvacrol, significantly impaired parasite development, particularly under continuous exposure, supporting their potential as alternative anticoccidial agents. Together, these findings highlight the value of the NLuc-based assay in advancing discovery pipelines while reducing animal use, and highlight plant-derived compounds as promising candidates for integrated coccidiosis control strategies.