Project Outline

Coccidiosis, caused by Eimeria species (Phylum Apicomplexa) is a pervasive and economically significant parasitic disease affecting the poultry industry in the UK and worldwide. This highly contagious protozoan parasite causes severe enteric infections leading to mortality and poor growth in flocks, while predisposing affected birds to secondary infections. The control of coccidia becomes highly problematic due to its complex life cycle extending between the host and environment. Despite advancements in good production practices that doubled poultry production worldwide during the last two decades, the losses due to coccidiosis over the years escalated. In the UK, industry loss increased from £38 million in 1999 to £99.2 million in 2016, whereas it was £10.4 billion worldwide. While financial loss remains a major concern, the use of anticoccidial drugs also called coccidiostat known to develop drug resistance has remained a longstanding concern in terms of food, and environmental safety adding further complexity to the issue. Addressing the growing economic impact of coccidiosis, underscores the need for research and innovation, especially in the field of developing novel treatments and management strategies that are sustainable, cost-effective, and have a minimal environmental impact. The present research methodology exists to assess host-pathogen interaction and response to treatment often requires in vivo pathogen challenge studies, which are costly, time-consuming, and raise ethical concerns. Overcoming these limitations, the research proposed in this PhD aims to leverage chicken intestinal enteroid cultures to study host-pathogen interaction at the mucosal surface in the presence of novel nutraceuticals.

Enteroids, also known as intestinal organoids or enteric organoids, are three-dimensional cell culture models that mimic the structure and function of the intestinal epithelium as an alternative and more ethically sound platform that abides 3R principle. These culture systems are successfully used as a tool for studying various aspects of the digestive system, including nutrient absorption, gut diseases, interactions with pathogens, and the development of new therapeutic and control solutions. Intestinal organoids are successfully used to study apicomplexan parasites (e.g. Crypsoporedia spp) that affect both livestock and humans¹. In our laboratory, we have successfully used cultured intestinal cells to study host-pathogen and nutritional absorption studies. In this PhD, we will explore the following objectives to fulfill our aim;

1. Establish a caecal enteroid using 18-day-old embryonated chicken eggs: The cells for the enteroid will be isolated using an established method¹. We supplement cells with use of natural growth promotors known to improve intestinal cell integrity ² and will be assessed using imaging and cell-culture techniques available in the lab.

2. use cecal enteroids to study the infection dynamics: the parasites will be collected as described and sporulated in vitro before infecting enteroids to map parasite invasion, sextual maturation and cell modulation² using histology, electron microscopy and immunohistochemistry. We will employ cell culture and gene-expression techniques that are already established in our lab to elucidate the host response. From this, we aim to understand potential innate immune mechanisms to define therapeutic targets.

3. assess the suitability of the model for screening studies: to use as a platform to study anticoccidial therapeutics, a panel of novel nutraceuticals to anticoccidial drugs with the potential to be used as feed additives. We will study potential therapeutics that are known to modulate the innate immune capacities of the enteroids.

In completion. The result of this project will establish an in vitro platform that can be used directly in screening feed additives and drug targets in addition to use in studying host-pathogen interaction. The student will benefit from engaging in cutting-edge research with applications to improve the health and welfare of poultry.

References

¹Bhalchandra S, Lamisere H, Ward H. Intestinal organoid/enteroid-based models for Cryptosporidium. Curr Opin Microbiol. 2020 Dec;58:124-129. doi: 10.1016/j.mib.2020.10.002.

²L. Berriman, S.P. Rose, V. Pirgozliev (2017) Effect of dietary phytonutrients in ameliorating coccidiosis challenge, WPSA Annual UK Branch meeting, Chester 25 -27 April, British Poultry Abstracts, p.4

Techniques

• Isolation of Caecal Enteroids: This technique involves isolating caecal enteroids from 18-day-old embryonated chicken eggs, likely utilising established methods.
• Imaging: These techniques will be employed to assess the integrity of intestinal cells, applying microscopy.
• Electron Microscopy: Electron microscopy will provide high-resolution images to study cellular and subcellular structures.
• Parasite culture: parasites will be grown in vitro before infecting enteroids, aiding in the study of infection dynamics.
• Histology and cytology: Histological techniques will be utilised to examine tissue structures and changes in response to infection.
• Immunohistochemistry: This technique involves using antibodies to visualise specific proteins or molecules in tissue sections.
• Gene Expression Analysis: Molecular techniques will be employed to analyse gene expression patterns in response to infection and treatment.