Nanoluciferase Platform Screens Anticoccidial Compounds Using Transgenic Eimeria

Researchers have developed a scalable in vitro platform that enables high-throughput screening of anticoccidial compounds, addressing a significant bottleneck in the discovery of new treatments for poultry coccidiosis. The study, published in Animal Diseases, introduces a phenotypic screening system based on a transgenic line of Eimeria tenella, a highly pathogenic parasite that causes cecal coccidiosis in chickens. By integrating nanoluciferase and enhanced yellow fluorescent protein (EYFP) into the parasite, the team created a tool that allows for sensitive, quantitative, and rapid assessment of parasite growth in a laboratory setting, moving beyond traditional methods that rely on labor-intensive microscopic counting.

Coccidiosis remains a persistent and economically significant threat to the global poultry industry, with an estimated global cost of approximately £10.4 billion in chickens. The disease is caused by at least seven Eimeria species, each with distinct pathogenicity and tissue tropism in the intestinal tract. Among these, Eimeria tenella is particularly notable for its high pathogenicity and its widespread use as a model organism for studying coccidian biology. Chickens acquire the infection by ingesting sporulated oocysts, which release sporozoites that invade intestinal epithelial cells, undergo asexual replication, and eventually produce new oocysts that are shed in feces.

The development of new anticoccidial agents has been hindered by the lack of suitable high-throughput screening (HTS) platforms. Conventional in vitro models often rely on primary chicken kidney cells, which, while physiologically relevant, require repeated preparation and exhibit limited propagation capacity. This makes it difficult to achieve consistent batch-to-batch reproducibility. Furthermore, traditional methods for assessing parasite growth, such as microscopic counting, are labor-intensive and have low throughput, significantly impeding the identification of new compounds. The new platform aims to overcome these limitations by using a stable cell line and a luminescence-based readout.

The core of the new system involves a transgenic line of E. tenella coexpressing EYFP and nanoluciferase (Nluc). The transgenic parasites were generated using the BJ strain, with the expression cassette controlled by the EtSAG13 promoter and EtActin terminator. The inclusion of TgDHFR facilitates in vivo selection of transgenic parasites propagated in chickens, while EYFP enables visualization and enrichment of fluorescent parasites by flow cytometry. Nluc provides a sensitive quantitative readout of parasite growth, allowing researchers to monitor replication reliably within a 45-hour infection window using Madin-Darby bovine kidney (MDBK) cells as host cells.

To validate the platform, the researchers assessed the relationship between the parasite inoculum and the luminescence signal, observing a strong linear correlation up to at least 2 × 10^5 sporozoites per well. The assay was further validated using two known anticoccidials, monensin and salinomycin, which produced clear dose-response curves with EC50 values of 9.98 nM and 0.10 μM, respectively. The platform demonstrated a large signal window and a Z’ factor of 0.83, which are higher than the recommended acceptance criteria for high-throughput screening, indicating robust performance and reliability.

Using this validated platform, the team screened a library of 1,770 compounds derived from the DiscoveryProbe library, which includes approved drugs and bioactive molecules. The screening was conducted at a concentration of 10 μM, and the assay identified 28 primary hits that inhibited parasite growth by more than 50%. After filtering for cytotoxicity and drug-like properties, three compounds were selected for further evaluation: alendronate, pemetrexed, and torsemide. These compounds represent distinct pharmacological classes, including a bisphosphonate, a folate analog, and a loop diuretic, suggesting diverse potential mechanisms of action against the parasite.

In vitro efficacy and selectivity analyses revealed that all three compounds exhibited micromolar activity against E. tenella, with EC50 values of 10.25 μM for alendronate, 9.65 μM for pemetrexed, and 8.0 μM for torsemide. Cytotoxicity was evaluated in MDBK and HepG2 cells, with alendronate showing moderate cytotoxicity and selectivity indices (SI) of 17.0 and 21.0, respectively. In contrast, pemetrexed and torsemide showed minimal cytotoxicity at concentrations up to 1,000 μM, resulting in selectivity indices greater than 100 in both cell lines, indicating a favorable safety profile for these two compounds.

The in vivo evaluation was conducted in a chicken infection model to validate the predictive value of the in vitro screening platform. Chickens were infected with E. tenella and treated with the selected compounds beginning at two days post-infection. The dosages were based on pilot experiments for drug tolerance, with alendronate and pemetrexed administered at 100 mg/kg/day and torsemide at 200 mg/kg/day. A vehicle control, a healthy control, and a diclazuril-treated group were included for comparison. Mortality occurred in the vehicle control group (33.3%) and the alendronate-treated group (16.7%), whereas no deaths occurred in the other treatment groups.

Compared with the vehicle control, all three test compounds significantly reduced oocyst shedding, with reductions of 66.3% for pemetrexed, 43.7% for torsemide, and 39.4% for alendronate. The diclazuril group showed a 77.5% reduction in oocyst shedding. In terms of weight gain, the diclazuril group performed comparably to the healthy group, while torsemide showed the best performance among the test compounds. Cecal lesion scores were markedly reduced in the diclazuril group, followed by torsemide, pemetrexed, and alendronate. Gross examination revealed that torsemide treatment resulted in mild lesions with limited hemorrhage, whereas alendronate-treated ceca exhibited pronounced pathological changes comparable to the infected control.

Overall efficacy, summarized as the anticoccidial index (ACI), indicated that diclazuril was highly effective with an ACI of 188.6. Among the test compounds, torsemide showed the greatest efficacy with an ACI of 163.3, followed by pemetrexed with an ACI of 153.5. Alendronate showed only marginal activity with an ACI of 110.8. The authors note that the in vivo experiments are exploratory and primarily intended to validate the screening platform, and that further studies with larger sample sizes and optimized dosing regimens are needed to better assess the therapeutic potential of these compounds.

The study highlights the importance of developing robust and scalable screening platforms to accelerate the discovery of new anticoccidial agents. The use of nanoluciferase provides a sensitive and quantitative readout, while the incorporation of EYFP allows for direct visualization and quality control of transgenic parasites. The use of a stable MDBK cell line offers improved reproducibility compared with primary chicken kidney cells, which are commonly used but suffer from variability and limited scalability. Although parasite development is less efficient in MDBK cells than in primary avian cells, the system supports at least one complete merogonic cycle, which is sufficient for identifying compounds that inhibit early parasite development.

The identified hits, alendronate, pemetrexed, and torsemide, represent distinct pharmacological classes and may act through different mechanisms. Alendronate, a bisphosphonate used for osteoporosis, showed measurable in vitro activity but limited in vivo efficacy, which may reflect suboptimal pharmacokinetic properties. Pemetrexed, a folate analog that targets thymidylate synthase, demonstrated moderate in vivo efficacy, suggesting that folate metabolism may represent a viable target in Eimeria. Torsemide, a loop diuretic targeting Na+/K+/2Cl− cotransporters, showed the highest in vivo efficacy among the tested compounds, suggesting that ion transport systems in coccidia could serve as novel therapeutic targets.

This study establishes a practical and scalable phenotypic screening platform that addresses a key limitation in anticoccidial drug discovery. By enabling efficient identification of candidate compounds, this system provides a foundation for accelerating the development of new anticoccidial agents. The platform is robust, quantitative, and compatible with medium-throughput screening workflows, effectively addressing a major bottleneck in the field. The findings highlight several approved drugs as potential leads for further optimization and structural modification to improve their potency and selectivity against Eimeria species.

Subject of Research: Development of a nanoluciferase-based in vitro high-throughput screening platform for anticoccidial compounds using transgenic Eimeria tenella

Article Title: A nanoluciferase-based in vitro platform for high-throughput screening of anticoccidial compounds using transgenic Eimeria tenella

Article References: Li, M., Zhang, X., Meng, X., Zou, B., Yin, J., Suo, X., Liu, X., & Zhu, G. (2026). A nanoluciferase-based in vitro platform for high-throughput screening of anticoccidial compounds using transgenic Eimeria tenella. Animal Diseases, 6(1), Article 23. https://doi.org/10.1186/s44149-026-00238-6

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00238-6

Keywords: Eimeria tenella, Anticoccidial, High-throughput screening, Nanoluciferase, Poultry disease, Drug discovery, Transgenic parasites, In vitro assay, Torsemide, Pemetrexed, nanoluciferase-based, vitro