Introduction
Chicken coccidiosis, caused by the protozoan parasite Eimeria, is an enteric disease that can occur in any type of production setting, from large commercial operations to small backyard flocks. It has wide-ranging impacts at the farm level, affecting not only the health of the birds but also the economic stability of poultry operations. Coccidiosis damages the intestinal lining of infected birds, which suffer from symptoms like diarrhoea, dehydration, weight loss and lethargy. These health issues reduce the overall health status of the flock, directly impacting on productivity and, in severe cases, infected chickens can die, leading to direct loss of stock. In addition to this, farmers often need to administer anticoccidial drugs or antibiotics to control the disease, leading to increased veterinary costs and growing problems of drug resistance, which result in reduced efficacy and more complex management programmes (Chapman and Rathinam, Reference Chapman and Rathinam2022).
Studying the avian intestinal tract and its pathogens, particularly Eimeria spp. protozoa, has been hindered by the lack of suitable, cost effective and reproducible cell culture models. One of the major challenges in studying coccidiosis is understanding how Eimeria interacts with the chicken intestinal tract at a molecular, cellular and tissue levels. Traditional animal models (live chickens) are valuable but can be expensive, can be time-consuming and may not always provide detailed insights into the specific molecular and cellular mechanisms of infection at cellular level.
The use of in vitro and ex vivo models for coccidiosis research (Table 1) offers a simplified and accurate way to study Eimeria infections and host–parasite interactions compared to animal models. In particular, chicken-derived intestinal organoids and enteroids (following the definition by Taelman et al., Reference Taelman, Diaz and Guiu2022) could provide an in vitro model that mimics the structure and function of the chicken intestine, offering a controlled environment for studying how Eimeria invades, modifies and damages intestinal cells. An overview of the different cell culture models already used or offering promise for Eimeria cultivation is provided in Figure 1 and Table 2.
Existing and potential cell culture models for Eimeria spp. Cultivation. Cells or tissues can be extracted from adult chicken intestinal tissue or chicken embryos. These can then be used to develop various models, including primary cell cultures, immortalized cell lines, intestinal organoids or intestinal explants (Tables 1 and 2). Each of these models can be developed in 2D, 2.5D or 3D formats (scaffold-, spheroid- or organoid-based), with the most advanced models – organoids – closely resembling the architecture of the real chicken intestine. Figure created using BioRender.com.
Definitions of existing and potential cell culture models for Eimeria spp. cultivation
a In vitro: System developed outside of a living organism.
b Ex vivo: Tissues or organs removed from a living organism that maintain their original architecture.
Summary of existing and potential cell culture models for Eimeria spp. cultivation
Current developments in chicken organoid are limited to models including intestinal epithelial cells and not more complex systems, and their use for research in coccidiosis has been limited to study sporozoite infection and development. Future more complex organoid models could be used to observe immune cell recruitment, inflammatory responses and how the parasite modulates the host immune system in response to Eimeria infection. They could also serve as a valuable platform for testing anticoccidial drugs or new treatments prior to in vivo trials, allowing researchers to evaluate the effectiveness of various compounds in preventing or reducing infection. This approach offers a more cost-effective and controlled alternative to conventional in vivo studies, with potentially greater predictive value than current in vitro models (Arias-Maroto et al., Reference Arias-Maroto, Aguiar-Martins, Regidor-Cerrillo, Ortega-Mora and Marugan-Hernandez2024).
Finally, organoids provide an ethical advantage by potentially reducing the need for live animal testing, which is particularly important in an era where animal welfare is a growing concern. By providing an alternative to some in vivo testing, organoids could help minimize the use of chickens in research, aligning with ethical and regulatory guidelines to promote the 3Rs (Replacement, Reduction, Refinement) in animal research.
In this review, we have extensively examined the literature on in vitro and ex vivo systems so far used for research in chicken coccidiosis (Table 2; Figure 1) and explored how these models, including intestinal organoids and explants, could benefit research in this problematic disease. Additionally, we reviewed recent advances in culturing related apicomplexan protozoa, such as Cryptosporidium spp. and Toxoplasma gondii, and discussed how these developments could inform research on Eimeria.
Specific considerations for Eimeria species and their life cycle stages
The parasite genus Eimeria comprises thousands of species that specifically infect a wide range of hosts, from mammals to reptiles, meaning that each parasite species infects only a single, specific host (monoxenous parasite). In chickens, 7 Eimeria species are recognized as causing coccidiosis (Burrell et al., Reference Burrell, Tomley, Vaughan and Marugan-Hernandez2020), with 3 additional cryptic species recently described whose implications are still under investigation (Blake et al., Reference Blake, Vrba, Xia, Jatau, Spiro, Nolan, Underwood and Tomley2021). When studying and aiming to control coccidiosis, it is crucial to consider that the 7 Eimeria species affecting chickens infect distinct regions of the intestinal tract (Figure 2), each producing different pathological effects. The main molecular mechanisms underlying why each species targets a specific niche remain unknown (Lai et al., Reference Lai, Bumstead, Liu, Garnett, Campanero-Rhodes, Blake, Palma, Chai, Ferguson, Simpson, Feizi, Tomley and Matthews2011), complicating the application of models designed to study these species concurrently.
Sites of infection and specific stages of Eimeria spp. parasites. (A) Seven Eimeria species (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella) specifically infect distinct regions of the chicken intestinal tract. (B) In the environment, unsporulated oocysts become infective (sporulated oocysts) through the process of sporogony. Within the chicken intestinal tract, sporozoites are released from the oocysts and infect epithelial cells in the gut, where they undergo multiple rounds of schizogony and gametogony, producing different parasite stages at each round. Figure created using BioRender.com.
The life cycle of Eimeria has been extensively reviewed elsewhere (Burrell et al., Reference Burrell, Tomley, Vaughan and Marugan-Hernandez2020; López-Osorio et al., Reference López-Osorio, Chaparro-Gutiérrez and Gómez-Osorio2020), so a detailed description is omitted here. Briefly, sporozoites excysted from oocysts, invade the host intestinal epithelium and develop into first-generation schizonts, which then progress to second-generation schizonts, and so on – typically through 3 rounds – before culminating in the sexual stages. A critical phase of the life cycle involves the fertilization of macrogametes by microgametes, leading to the formation of diploid zygotes. These zygotes mature into oocysts, which are essential for the faecal-oral transmission of the parasite between hosts. It is important to note that each replication cycle of the parasite results in the development of distinct stages (Figure 2), each exhibiting significant variations in morphology and gene expression, leading to distinct protein profiles (Sandholt et al., Reference Sandholt, Wattrang, Lilja, Ahola, Lundén, Troell, Svärd and Söderlund2021). These differences complicate the characterization of host–parasite interactions.
Therefore, when aiming to develop novel in vitro models to study coccidiosis, it is essential to account for the distinct species and developmental stages of Eimeria. A ‘universal’ model may not be feasible, as each species exhibits unique intestinal region preferences and life cycle characteristics.
Traditional and current cultivation models for Eimeria research
Cultivating Eimeria species in vitro has long been challenging due to their complex life cycles and host specificity. Traditional methods, such as embryo derived cells or primary chick kidney cells, have allowed partial reproduction of the Eimeria life cycle, but these models (2D) often yield low efficiency (Table 2). More recent advancements, including multilayered (3D) cell culture systems, have yielded promising results advancing our understanding of Eimeria biology and host–pathogen interactions, as they better reflect the micro-anatomical and physiological milieu (Figure 1). However, challenges remain in achieving full life cycle completion in vitro, and concerns about reproducibility and scalability must be addressed before these models can replace animal use in research. Among the 7 Eimeria species infecting chickens, Eimeria tenella has emerged as a model organism for the genus due to its enhanced adaptability to cultivation and genetic manipulation. Consequently, most recent studies developing in vitro and ex vivo models are based on this specific species.
Primary cell cultures and immortalized cell lines (2D)
In the 1970s, the whole life cycle of E. tenella was achieved in vitro for the first time, evidenced by visual observation of oocysts, using a primary chicken kidney cell culture (Doran and Augustine, Reference Doran and Augustine1973). More recently, in 2010 the completion of the life cycle by presence of oocyst was reported again using caecal epithelial cells from chicken embryos in cell culture (Gu et al., Reference Gu, Hou, Chen, Li and Zheng2010). Later, in 2018, Bussière et al. (Reference Bussière, Niepceron, Sausset, Esnault, Silvestre, Walker, Smith, Quéré and Laurent2018) were able to observe gametes after infecting a lung chicken epithelial cell line (CLEC213) with second-generation merozoites. Other than in these studies, the complete biological life cycle of Eimeria with visual evidence of sexual stages or oocysts has not been successfully reproduced in vitro to the best of the authors’ knowledge.
Several immortalized cell lines have supported sporozoite invasion and partial development of E. tenella to first-generation schizonts (including bovine kidney cells (MDBK), human epithelial cells (Caco-2), chicken hepatoma cells (LMH), baby hamster kidney cells (BHK), chicken fibroblast cells (DF-1) and chicken macrophage-like cells (HD11) (Tierney and Mulcahy, Reference Tierney and Mulcahy2003; Table 2). Interestingly, none of these cell lines originate from chicken intestine, the primary site of infection and reproduction for Eimeria spp., indicating reduced specificity of the parasite during the initial life cycle stages. Unlike primary cell cultures, immortalized cell lines can remain viable through multiple passages while maintaining their morphological and physiological characteristics. This capability provides a consistent supply of target cells, enhancing the reproducibility of experiments. Immortalization of avian cells is, however, inherently more challenging compared to mammalians ones, as avian species have naturally lower mutation rates.
Nonetheless, Ghiselli et al. (Reference Ghiselli, Felici, Piva and Grilli2023) and, more recently, Chen et al. (Reference Chen, Li, Pan, Hu, Cai, Xia, Qi, Liao, Spritzer, Bai and Sun2024), have immortalized a primary cell culture originated from chicken intestinal epithelial cells using the SV40 large T antigen transduction methodology. Ghiselli et al. studies demonstrated the enterocyte nature of the cell line as these expressed markers and enzymatic activity of mature enterocytes. The ability of sporozoites to invade these cells was explored; however, the replicative capacity of E. tenella was not studied. Chen et al. model consisted in immortalization of epithelial that did not develop into enterocytes. When cells were infected with sporozoites, second-generation schizonts and merozoites were observed at 96 h post-infection, but not further stages. These 2 immortalized systems could be a more suitable model for high throughput anticoccidial drug screening trials. Nevertheless, the use of immortalized intestinal epithelial cell lines still presents several drawbacks, including a lack of cellular heterogeneity, partial reproduction of tissue functionality and the presence of genomic abnormalities, as previously discussed by Beaumont et al. (Reference Beaumont, Blanc, Cherbuy, Egidy, Giuffra, Lacroix-Lamandé and Wiedemann2021).
Multidimensional cell culture models (3D)
As mentioned earlier, traditional culture systems, based on bidimensional cell monolayers (2D), have failed to replicate the complex cellular interactions present in vivo, limiting the completion of Eimeria life cycle. Alternatively, multidimensional (3D) culture systems are considered more physiologically relevant, offering better representation of the chicken intestinal microenvironment. These systems could facilitate more accurate studies of cell-to-cell and cell-to-matrix interactions, which are essential for understanding the biology and interactions of E. tenella with the intestinal mucosa, including the local innate immune system.
Aguiar-Martins et al.’s (Reference Aguiar-Martins, Tomley, Blake and Marugan-Hernandez2024) study has demonstrated the potential of adding collagen-based extracellular matrices (CBEM) to monolayered 2D cultures, enabling the multilayering (3D) of epithelial cells, for studying the growth and development of E. tenella. These culture systems have been trialled in multiple cell lines known to support E. tenella infection and replication, such as MDBK, HD11, and the human COLO-680N, and HCC4006 (Table 2), enabling parasite replication up to the first schizogony stage as early as 48 h post-infection. In this same study, scaffold-free cell spheroids (3D) were generated using HD11 cells, which showed lower viability rates compared to the CBEM system. Despite this, the spheroids still supported E. tenella invasion and the release of first-generation merozoites.
While these culture systems described here have supported invasion and development of early stages of E. tenella, they have not yet been able to replicate the full life cycle of the parasite. The progression to the sexual stages, including the development of gametocytes and oocysts, remains elusive in these models. Hence, further research is needed to develop more robust culture systems that can support the complete life cycle of E. tenella.
Introduction to intestinal organoids
Intestinal organoids are 3-dimensional (3D) structures that can be generated by differentiation from embryonic pluripotent stem cells, induced pluripotent stem cells or adult intestinal epithelial stem cells (IESCs), the latter usually referred as ‘enteroids’ (Taelman et al., Reference Taelman, Diaz and Guiu2022; Table 1). Organoids offer a revolutionary platform for biological research (Sato et al., Reference Sato, Vries, Snippert, van de Wetering, Barker, Stange, van Es, Abo, Kujala, Peters and Clevers2009; In et al., Reference In, Foulke-Abel, Estes, Zachos, Kovbasnjuk and Donowitz2016) as they self-organize into complex structures that mimic the architecture, cellular diversity and physiological functions of the native intestine (Stelzner et al., Reference Stelzner, Helmrath, Dunn, Henning, Houchen, Kuo, Lynch, Li, Magness, Martin, Wong and Yu2012). They usually include various epithelial cell types such as enterocytes, goblet cells, Paneth cells and enteroendocrine cells, arranged in a polarized, villi-like configuration with crypt-like domain (Clevers, Reference Clevers2013). In contrast, 2D cultures restrict cell–cell interactions to lateral contact and typically lack spatial organization, extracellular matrix, cell polarity and dynamic microenvironments. These limitations can reduce their ability to replicate in vivo processes and may hinder accurate modelling of pathogen invasion and replication. Immortalized cell lines as those referred to in the previous section fail to replicate the epithelial complexity and innate immune responses essential for studying Eimeria infection dynamics within chicken intestine. Organoids have emerged to address these limitations, providing a more physiologically relevant model that simulates the intestine to study host–parasite interactions (Hares et al., Reference Hares, Tiffney, Johnston, Luu, Stewart, Flynn and Coombes2021). By better mimicking intestinal structure and function, they serve as a bridge between conventional cell culture and whole-animal studies (Ramírez-Flores et al., Reference Ramírez-Flores, Tibabuzo Perdomo, Gallego-López and Knoll2022).
Development of chicken intestinal organoids: from human to avian models
The journey of intestinal organoids began with human and mouse models. In 2009, Sato et al. (Reference Sato, Vries, Snippert, van de Wetering, Barker, Stange, van Es, Abo, Kujala, Peters and Clevers2009) established the first intestinal organoids from LGR5+ stem cells in mouse crypts, cultured in Matrigel with Wnt agonists (R-spondin 1), epidermal growth factor (EGF) and Noggin (WRN). Each of these components have essential roles in maintaining stemness and supporting the self-renewal of organoids by modulating key signalling pathways: Wnt antagonists will promote expansion of stem cells preventing premature differentiation, with R-spondin 1 exhibiting a synergistic role to amplify stem cell maintenance, with particular importance for LGR5+ stem cells, which are present in intestinal tissue; EGF is associated to intestinal proliferation; Noggin prevents differentiation into mature cell types by inhibiting Bone Morphogenetic Protein signalling. Organoids with budding crypts were able to produce and maintain stability for dozens of passages. These advancements laid the groundwork for species-specific adaptations in the generation of intestinal organoids (reviewed by Sato and Clevers, Reference Clevers2013).
Chicken intestinal organoids emerged later (reviewed by Nash and Vervelde, Reference Nash and Vervelde2022), adapting mammalian protocols to avian physiology (Table 2, Figure 1). In 2012, Pierzchalska et al. developed the first chicken intestinal organoid from embryonic chicken intestines using a method previously developed for isolation of mice crypts and villi fractions (Barker et al., Reference Barker, van Es, Kuipers, Kujala, van den Born, Cozijnsen, Haegebarth, Korving, Begthel, Peters and Clevers2007), and medium containing EGF, R-spondin 1 and Noggin in Matrigel-coated plates. These organoids were ‘basal-out’ with the enclosed lumen, with crypt-like structure only occasionally seen. Similar results were obtained when Prostaglandin E2 was used instead R-spondin 1 and Noggin. It was 5 years later when Powell and Behnke (Reference Powell and Behnke2017) generated enteroids for several animal species using the L-WRN cell line, which secrete the 3 WNR signalling factors (Wnt3a, R-spondin 3 and Noggin; Miyoshi and Stappenbeck, Reference Miyoshi and Stappenbeck2013), including an enteroid model derived from chicken caecal crypts, that were propagated up to 125 days. Soon after, in 2018, Li et al. developed chicken enteroids from small intestine. In this study, characteristic microvilli and crypts typical of mature enterocytes were observed by transmission electron microscopy (TEM), and the model seemed responsive to specific stimuli (CHIR99021, a glycogen synthase kinase 3β inhibitor), which supported comparable physiological function to the animal epithelium. Later, in 2022, Zhao et al. collected crypts from the small intestine of chicken embryos and adult chickens (layers and broilers). These crypts were then embedded in a basement membrane matrix and cultured with organoid growth media containing standard growth factors. The resulting organoids formed spherical structures with a polarized epithelium composed of enterocytes, where the presence of Paneth cells, goblet cells, fibroblasts and mesenchymal cells was confirmed by identification of specific markers by RT-PCR and immunoblotting.
Alternatively, Orr et al. (Reference Orr, Sutton, Christian, Nash, Niemann, Hansen, McGrew, Jensen and Vervelde2021) developed a 2D enteroid model, comprising enterocytes, Paneth cells, goblet cells, enteroendocrine cells and leukocytes. Interestingly, this system self-organized into an exposed epithelial and mesenchymal sub-layer, and polarized into luminal, apical and basal aspects, mimicking more reliably the intestinal mucosa morphology (i.e. epithelial lining and lamina propria). Although Eimeria infections have not been assessed in this model, innate immune responses against lipopolysaccharide (LPS) from Salmonella enterica serotype Typhimurium were evidenced by an upregulation of inflammatory cytokines IL-6 and IL8. Additionally, the morphological and physiological properties of this system were preserved under freezing and thawing conditions.
Pierzchalska et al. (Reference Pierzchalska, Panek, Czyrnek, Gielicz, Mickowska and Grabacka2017) studied for first time the effects of stimulation of Toll-like receptors (TLRs) of chicken intestinal organoids adding specific antagonists to TLR4 and TLR2 (types 1 and 2) or co-culturing them with Lactobacillus acidophilus. Interestingly, the TLR2 Pam3CSK4 and LA-5 ligand was able to modulate organoids by promoting growth and increasing prostaglandin (PGE2 and PGD2) levels. A major improvement came with studies (Oost et al., Reference Oost, Ijaz, van Haarlem, van Summeren, Velkers, Kraneveld, Venema, Jansen, Pieters, Ten and Klooster2022), who discovered that supplementing cultures with chicken-derived RSPO1 and WNT3 (instead of mammalian ones), along with Prostaglandin E2 (PGE2) and a Forkhead box O1 (FOXO1) inhibitor, significantly enhanced their longevity (both in 2D and 3D cultures). Increased levels of PGE2 and PGD2 in longer-lived organoids had previously been observed by Pierzchalska et al. (Reference Pierzchalska, Panek, Czyrnek, Gielicz, Mickowska and Grabacka2017), further supporting the role of prostaglandins in the survival of chicken intestinal organoids. These avian-specific factors stabilized stem cell signalling, extending organoid cultures to 15 passages and allowing the differentiation into different intestinal cell types (goblet cells, endocrine cells and enterocytes).
All these studies have supposed a major step forward, transforming chicken intestinal organoids from short-lived spheres into functionally relevant models.
Advances in chicken organoids: towards Eimeria research
Early chicken intestinal organoids faced significant hurdles in infection studies. While Oost et al. (Reference Oost, Ijaz, van Haarlem, van Summeren, Velkers, Kraneveld, Venema, Jansen, Pieters, Ten and Klooster2022) developed stable cultures that grew as organoids in a gel-like protein matrix (i.e. Matrigel), these models were not tested for pathogen susceptibility. Additionally, the absence of immune system components limited their ability to mimic closer host–parasite interactions. Insights from human organoid research could help explain these challenges. In gel-embedded organoids, the basal side faces outward while the lumen remains enclosed; therefore, microinjection is usually required to introduce pathogens in human and livestock intestinal organoids (Heo et al., Reference Heo, Dutta, Schaefer, Iakobachvili, Artegiani, Sachs, Boonekamp, Bowden, Hendrickx, Willems, Peters, Riggs, O’Connor and Clevers2018; Beaumont et al., Reference Beaumont, Blanc, Cherbuy, Egidy, Giuffra, Lacroix-Lamandé and Wiedemann2021). Alternatively, mechanical disruption or reversion to 2D cultures could expose the apical epithelium for parasite invasion (Derricott et al., Reference Derricott, Luu, Fong, Hartley, Johnston, Armstrong, Randle, Duckworth, Campbell, Wastling and Coombes2019; Luu et al., Reference Luu, Johnston, Derricott, Armstrong, Randle, Hartley, Duckworth, Campbell, Wastling and Coombes2019; Oost et al., Reference Oost, Ijaz, van Haarlem, van Summeren, Velkers, Kraneveld, Venema, Jansen, Pieters, Ten and Klooster2022). These studies underscore the importance of organoid polarity in infection research. Shin et al. (Reference Shin, Tsai, Hsu, Liang and Wu2025) expanded on this concept by demonstrating how polarity – apical-out vs basal-out configurations – affects pathogen replication in chicken intestinal organoids. Using low-pathogenic avian influenza as a model, they showed that apical-out organoids, with lumens exposed, better mimic natural gut infections. In contrast, reversing polarity to expose basolateral surfaces altered the dynamics of infection.
Nash et al. (Reference Nash, Morris, Mabbott and Vervelde2021) marked a transformative shift by developing ‘inside-out’ chicken enteroids with an innate leukocyte component. By replacing traditional gel-embedded organoids to floating ones, these exposed their apical surfaces to the outside, in this way, pathogens could directly access epithelial targets, by simply adding them into the culture media, without supplementary techniques for inoculation. Caecal enteroids supported the infection by E. tenella sporozoites, revealing sporozoite invasion, intracellular replication (schizogony) and potential development into gametes supported by the presence of transcripts of etgam56 gene. Validation of high level of reproducibility of this enteroid model came from Nash et al. (Reference Nash, Morris, Mabbott and Vervelde2023), who profiled floating apical-out chicken enteroids using temporal transcriptomics. Gene transcription demonstrated that these enteroids possessed the cellular diversity that is found in the chicken intestinal epithelium (Paneth cells, goblet cells, enterocytes, enteroendocrine cells, transit-amplifying cells, intestinal stem cells and tuft cells). There was also presence of transcripts for brush border enzymes and genes under the GO term ‘cell projection organisation’, which reflected microvilli development, which were further confirmed by TEM. There were also genes that represented several populations of lymphocytes (T cells, B cells, macrophages, dendritic cells and natural killer cells). One drawback of this model is the impossibility to propagate ‘apical-out’ floating enteroid cultures, unlike for gel-embedded ones, where continuous passage is possible. The reasons for this remain uncertain and not related to epithelial mesenchymal transitions or growth factor deficiencies.
Finally, it is important to consider that region-specific organoids may help refining infection models for each of the chicken Eimeria species, which infect different regions of the intestinal track. For example, Powell and Behnke (Reference Powell and Behnke2017) developed cecum-derived organoids, which could better match E. tenella’s natural infection site, as Nash et al. (Reference Nash, Morris, Mabbott and Vervelde2021) demonstrated, suggesting that intestinal region-specific organoids could replicate infection patterns more accurately.
Intestinal chicken explants
Intestinal explants also offer an alternative physiologically relevant ex vivo model for studying intestinal health, inflammation and host responses to external stimuli. Randall et al. (Reference Randall, Turton and Foster2011) reviewed the methods and applications of intestinal explants from humans and a few animal species, particularly for the study of inflammatory intestinal diseases, as well as their use in toxicology and drug development, with the potential to reduce animal use in human medicine. Explants are prepared by isolating small sections of intestinal tissue and maintaining them in culture conditions that preserve their native architecture. Different to cultures of cell monolayers, explants retain the full cellular complexity and spatial organization of the intestine, including polarized epithelial cells, lamina propria and resident immune populations. This structural integrity allows for a more accurate representation of tissue-level responses to microbial, dietary or environmental stimuli (reviewed by Russo et al., Reference Russo, Zeppa, Iovino, Del Giorno, Zingone, Bucci, Puzziello and Ciacci2016).
Several studies have demonstrated the utility of chicken explants for short-term inflammatory modelling. Zhang et al. (Reference Zhang, Eicher, Ajuwon and Applegate2017) established a chicken ileal explant model to study responses to LPS, showing that explants remain over 90% viable for up to 2 h. LPS exposure induced nitric oxide release and upregulated inflammatory markers, confirming the tissue responsiveness and application for short-term studies. Similarly, Duarte et al. (Reference Duarte, Mallmann, Liberalesso, Simões, Gressler, Molossi, Bracarense and Mallmann2021) used jejunal explants in Ussing chambers to assess epithelial damage caused by deoxynivalenol, a mycotoxin commonly found in chicken feed. They reported histopathological changes including size reduction of enterocytes, denudation of villi and increased levels of apoptosis evidenced by accumulation of caspase-3. Application of an antimycotoxin additive ameliorated the deoxynivalenol harmful effects. This study demonstrated the explant model’s relevance for toxicological screening.
More recently, Mátis et al. (Reference Mátis, Tráj, Hanyecz, Mackei, Márton, Vörösházi, Kemény, Neogrády and Sebők2024) used an ileal chicken explant to evaluate cathelicidin-2, as potential replacement to antibiotics treating intestinal infections. Cathelicidin-2 exhibited anti-inflammatory effects. A year later, these same researchers developed a miniature chicken ileal explant system using biopsy punches to optimize viability over time and experimental consistency (Mátis et al., Reference Mátis, Sebők, Horváth, Márton, Mackei, Vörösházi, Kemény, Neogrády, Varga and Tráj2025). Explants retained epithelial architecture and lining for at least 12 h and responded to stimulation with pathogen-associated molecular patterns, showing variations of expression levels for different cytokines. This validated the model for assessing intestinal innate immune signalling. The smaller explant format allowed medium-throughput screening while maintaining essential tissue-level features. Studies on explants infected with Eimeria have not been reported. However, our preliminary results (unpublished) demonstrate the ability of sporozoites to infect caecal explants generated by precision cut tissue slicing, as well as supporting parasite development in explants derived from previously in vivo-infected caeca. Nevertheless, completion of the life cycle evidenced by visualization of oocysts was not achieved.
In comparison to explants, intestinal organoids require extended culture and external signals to reach differentiation. While organoids offer advantages in scalability, long-term culture and genetic manipulation, they typically lack immune components unless co-cultured and require polarity modification for pathogen access (Nash and Vervelde, Reference Nash and Vervelde2022). Explants, by contrast, retain luminal orientation and immune elements natively, making them more suited for rapid, physiologically grounded assessments. Therefore, organoids and explants could serve complementary roles depending on the experimental timescale and complexity required.
Development of cell culture and intestinal organoids in related apicomplexa
There are Eimeria closely related apicomplexan parasites for which cell cultures and organoid models have been used to study the invasion and replication (Hares et al., Reference Hares, Tiffney, Johnston, Luu, Stewart, Flynn and Coombes2021) and that this review examined for their potential application to coccidiosis research.
Cryptosporidiosis is a major cause of diarrheal disease, particularly in young children, immunocompromized individuals and neonatal ruminants. Similarly to Eimeria spp., understanding the pathogenesis of Cryptosporidium spp., monoxenus parasites, has been historically hindered by the absence of physiologically relevant in vitro models that replicate the complex architecture of the small intestinal epithelium. Conventional cell lines, such as HCT-8 and Caco-2, derived from human intestinal carcinoma cells, have supported partial parasite development but fail to recapitulate the complete life cycle or accurately mimic the intestinal niche (Yu et al., Reference Yu, Choi and Kim2000). However, in 2016, Morada et al. (Reference Oost, Ijaz, van Haarlem, van Summeren, Velkers, Kraneveld, Venema, Jansen, Pieters, Ten and Klooster2016) reported that the adaptation of hollow fibre technology using HCT-8 cell line allowed the continuous production of oocysts Cryptosporidium parvum for 6 months. In 2019, Wilke et al. (Reference Wilke, Funkhouser-Jones, Wang, Ravindran, Wang, Beatty, Baldridge, VanDussen, Shen, Kuhlenschmidt, Kuhlenschmidt, Witola, Stappenbeck and Sibley2019) described the use of an air–liquid interface (ALI) cultivation system based on IESC derived (Wang et al., Reference Wang, Yamamoto, Wilson, Zhang, Howitt, Farrow, Kern, Ning, Hong, Khor, Chevalier, Bertrand, Wu, Nagarajan, Sylvester, Hyams, Devers, Bronson, Lacy, Ho, Crum, McKeon and Xian2015) to allow the completion of the life cycle and long-term cultivation of C. parvum, providing an accessible model to study the biology and host–parasite interactions. Application of organoid systems derived from human LGR5+ intestinal stem cells has also enabled completion of the life cycle and oocyst production (DeCicco, RePass et al., Reference DeCicco, Chen, Lin, Zhou, Kaplan and Ward2017). These systems have allowed to expand host–pathogen interaction studies under more in vivo-like conditions (reviewed by Bhalchandra et al., Reference Bhalchandra, Lamisere and Ward2020; Marzook and Sateriale, Reference Marzook and Sateriale2020). Techniques such as microinjection and apical surface exposure – achieved through organoid fragmentation followed by monolayer culture – have facilitated infection and life cycle completion in these models, as well as in lung organoids (Heo et al., Reference Heo, Dutta, Schaefer, Iakobachvili, Artegiani, Sachs, Boonekamp, Bowden, Hendrickx, Willems, Peters, Riggs, O’Connor and Clevers2018). Additionally, murine colonic explants were developed by Baydoun et al. (Reference Baydoun, Vanneste, Creusy, Guyot, Gantois, Chabe, Delaire, Mouray, Baydoun, Forzy, Chieux, Gosset, Senez, Viscogliosi, Follet and Certad2017) as an ex vivo model for studying cryptosporidiosis.
In contrast to Eimeria spp. and Cryptosporidium spp., T. gondii exhibits broader host range and tissue tropism, infecting most warm-blooded animals and a wide range of cell types, with a particular affinity for muscle and nervous tissues, with infections starting though the oral route. However, the development of asexual intestinal and sexual stages is restricted to feline intestines (where the parasite develops a life cycle equivalent to Eimeria and Cryptosporidium), and studies on these stages have been neglected compared to studies in tachyzoites and bradyzoites due to the lack of suitable models for cultivation. Classic in vitro models, including feline epithelial intestinal cells, as well as murine and human immortalized cell lines such as Caco-2, SW480 and T84, have supported advances in understanding invasion and replication mechanisms in the intestine; however, they lack the tissue complexity necessary to fully mimic in vivo conditions (Sena et al., Reference Sena, Cancela, Bollati-Fogolín, Pagotto and Francia2023). Recent advances include the development of 3D intestinal organoids can offer more spatially and physiologically relevant platforms for studying host–pathogen intestinal interactions (Luu et al., Reference Luu, Johnston, Derricott, Armstrong, Randle, Hartley, Duckworth, Campbell, Wastling and Coombes2019). Intestinal organoid models facilitate early-stage infection studies and enable investigation of animal species-specific responses (reviewed by Hares et al., Reference Hares, Tiffney, Johnston, Luu, Stewart, Flynn and Coombes2021; Sena et al., Reference Sena, Cancela, Bollati-Fogolín, Pagotto and Francia2023). Regarding the specific cat intestinal stages, murine-derived organoids treated with a delta-6-desaturase inhibitor to mimic feline intestinal environments supported sexual development in vitro (Martorelli Di Genova et al., Reference Martorelli Di Genova, Wilson, Dubey and Knoll2019). Importantly, the transcription factors leading the transition from tachyzoite to merozoites have recently been identified (Antunes et al., Reference Antunes, Shahinas, Swale, Farhat, Ramakrishnan, Bruley, Cannella, Robert, Corrao, Couté, Hehl, Bougdour, Coppens and Hakimi2024), which can significantly help to studies in these stages without relying on cats to obtain oocysts to release sporozoites to commence infections. Interestingly, a human intestine-on-a-chip model has recently been developed by Humayun et al. (Reference Humayun, Ayuso, Park, Martorelli, Genova, Skala, Kerr, Knoll and Beebe2022), incorporating a microfluidic system that simulates local vascularization. Infections of this model with T. gondii have allowed the study of specific immunoresponses and immunomodulatory roles of specific cytokines.
Conclusions and future directions
Cultivation of apicomplexan parasites has been crucial over the past 50 years for advancing our understanding of their biology and molecular mechanism of important processes such as host cell invasion and replication (Feix et al., Reference Feix, Cruz-Bustos, Ruttkowski and Joachim2023). In particular, the ability to maintain early stages of Eimeria spp. (i.e. sporozoites and first-generation schizonts) within host cells has enabled the detailed study of these molecular mechanisms for these specific parasites (Marugan-Hernandez et al., Reference Marugan-Hernandez, Sanchez-Arsuaga, Vaughan, Burrell and Tomley2021; Burrell et al., Reference Burrell, Marugan-Hernandez, Wheeler, Moreira-Leite, Ferguson, Tomley and Vaughan2022). This in vitro cultivation has also facilitated the development of pre-screening assays for identifying novel compounds with anticoccidial activity against Eimeria (Thabet et al., Reference Thabet, Zhang, Alnassan, Daugschies and Bangoura2017; Arias-Maroto et al., Reference Arias-Maroto, Aguiar-Martins, Regidor-Cerrillo, Ortega-Mora and Marugan-Hernandez2024). However, despite the promising results from the 1970s regarding the observation of sexual stages and oocyst production in cell culture using primary cell cultures (Table 2), there has been little to no progress in this area since. Eimeria first-generation merozoites appear to be reluctant to reinvade host cell, not only non-chicken cells but also chicken-derived cell lines. This limitation prevents progression to the second-generation and further stages, which is critical for completing the life cycle. This reinvasion failure represents a major bottleneck in Eimeria in vitro cultivation.
Recent advancements in cell culture models, particularly the development of immortalized avian epithelial cell lines (Ghiselli et al., Reference Ghiselli, Felici, Piva and Grilli2023; Chen et al., Reference Chen, Li, Pan, Hu, Cai, Xia, Qi, Liao, Spritzer, Bai and Sun2024) and the use of 3D culture models based on immortalized lines (scaffold- or spheroid-based; Aguiar-Martins et al., Reference Aguiar-Martins, Tomley, Blake and Marugan-Hernandez2024), initially offered promise as they provide a more accurate representation of in vivo conditions. However, despite these improvements, the parasite appears to behave similarly to how it does in traditional 2D culture systems, halting development at the stage of first-generation merozoites. Therefore, important challenges remain to be addressed, particularly the ability to support the parasite’s complete life cycle in vitro.
In the past decade, there has been significant progress in the development of ‘basal-out’ chicken intestinal organoids (Table 2), providing improved culture conditions, enhanced differentiation and extended lifespans. These developments culminated in an ‘apical-out’ model with significant potential for studying avian infectious diseases, including Eimeria spp. (Nash et al., Reference Nash, Morris, Mabbott and Vervelde2021). Although research on Eimeria infection in chicken organoids is still in its early stages, initial findings are promising. Nash et al. (Reference Nash, Morris, Mabbott and Vervelde2021) provided the most compelling evidence to date, demonstrating that inside-out enteroids can successfully support E. tenella infection and intracellular replication. In our opinion, this model represents a critical step towards the development of physiologically relevant Eimeria infection systems.
Adapting these advanced systems for Eimeria cultivation holds great potential for future breakthroughs. Such models could enable the study of previously neglected life cycle stages, such as merozoites and sexual stages, which play crucial roles in disease pathogenesis but remain poorly studies, even in other relevant Apicomplexa. Examining cultivation systems for the intestinal and sexual stages of Cryptosporidium spp., the adaptation of the ALI cultivation system (Wang et al., Reference Wang, Yamamoto, Wilson, Zhang, Howitt, Farrow, Kern, Ning, Hong, Khor, Chevalier, Bertrand, Wu, Nagarajan, Sylvester, Hyams, Devers, Bronson, Lacy, Ho, Crum, McKeon and Xian2015) to chicken enteroids could be of interest for coccidiosis research. Moreover, the application of hollow fibre technology to 2D chicken enteroid models (Orr et al., Reference Orr, Sutton, Christian, Nash, Niemann, Hansen, McGrew, Jensen and Vervelde2021) could support the scaling up of these organoid systems to produce oocysts in vitro, potentially reducing the large number of chickens currently required for oocyst production in vaccine manufacturing. Advancing research on the intestinal and sexual stages of T. gondii is limited by the parasite’s definitive host, the cat. Research involving this species is typically subject to strict regulations, and both oocyst production and the development of cat intestinal organoids from feline tissues rely on live animals. In this context, significant progress has been made in manipulating tachyzoites to differentiate into merozoites, thereby bypassing the oocyst/sporozoite stage (Antunes et al., Reference Antunes, Shahinas, Swale, Farhat, Ramakrishnan, Bruley, Cannella, Robert, Corrao, Couté, Hehl, Bougdour, Coppens and Hakimi2024). Additionally, efforts have been made to ‘felinise’ mice and mouse-derived cells to render them susceptible to sporozoite and merozoite infections (Martorelli Di Genova et al., Reference Martorelli Di Genova, Wilson, Dubey and Knoll2019).
Looking ahead, chicken intestinal organoids hold strong potential for drug screening, providing a gut-like environment for testing novel compounds, as well as for vaccine development, enabling the assessment of antigen responses in immune-enhanced organoids (Mitchell et al., Reference Mitchell, Sutton, Elango, Borowska, Perry, Lahaye, Santin, Arsenault and Vervelde2024). However, current models still lack adaptive immune components, which limits their ability to accurately replicate infections and systemic host responses (Lahree and Gilbert, Reference Lahree and Gilbert2024). Future advancements could focus on integrating microbiota or microfluidic systems to better replicate the gut ecosystem, addressing these limitations and further enhancing the model’s physiological relevance (Holthaus et al., Reference Holthaus, Delgado-Betancourt, Aebischer, Seeber and Klotz2021).
Author contributions
P.Y.K.C. and B.M.-G. wrote the original draft. V.M.-H. conceived and designed the article, and reviewed and edited the final version. P.Y.K.C. and B.M.-G. contributed equally.
Financial support
P.Y.K.C. is funded by the BBSRC London Interdisciplinary Doctoral Programme (LIDo).
Competing interests
The authors declare no commercial or financial relationships that could be interpreted as a potential conflict of interest.
Ethical standards
Not applicable.
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