The Eimeria-host cell interaction in broiler chickens

Abstract

Coccidiosis is an enteric infection of chickens caused by protozoan parasites of the genus Eimeria. Coccidiosis is a worldwide disease with an economic impact on broiler chicken production. An outbreak of disease can reduce weight gain and feed digestion in the entire flock, reducing the production of processed meat for market. The major characteristics of Eimeria species are the invasion of specific sites in the intestine of chickens and specificity of the immune response. To date, prophylaxis and vaccination are used to control coccidiosis. However, the continuous use of chemotherapeutics has led to increased drug resistance by Eimeria. In the case of vaccination, immunity against Eimeria is species-specific, hence, there is a need to vaccinate chickens against all species of Eimeria for complete protection. The Eimeria-host cell interaction is the first stage in the reproductive cycle in chickens that produces the damage in the chicken intestine. A more complete understanding of the enviromnental factors within the intestinal tract that influence this interaction will be useful to control the disease. The lack of a suitable method to study the interaction between Eimeria and host cells derived from different areas of intestine has hampered our understanding of the disease. The cell type of interest for this study was the chicken enterocyte. A layer of mucus is secreted by goblet cells in the intestinal epithelial to protect the enterocytes. Eimeria sporozoites have to cross the mucus layer in order to invade the epithelial cells. It is reasonable to assume that this mucus may have some involvement in Eimeriaenterocyte attachment. The objectives of this study were to investigate the roles of the enterocyte and intestinal mucus in the attachment process and the subsequent penetration of host cells by Eimeria sporozoites. Newly hatched, and 3-week-old chickens, were killed and intestinal segments were collected for developing an in vitro method ex vivo ( organ culture system, isolated enterocytes and a frozen section method) to study the Eimeria interaction with intestinal epithelial cells. Eimeria sporozoites were .extracted from oocysts and then labelled with a fluorescent dye (PKH-67). The frozen section model was found to be superior to the ~se of isolated enterocytes and organ culture systems, and was used for subsequent experiments in this project. This method was used to investigate the Eimeria-enterocyte attachment at preferred and non-preferred sites on the surface of enterocyte membranes. Indeed, the use of this method demonstrated that D-galactose on the surface of sporozoites had an important role in the attachment of E. tenella sporozoites to caecal enterocytes, with caecal and duodenal mucus both functioning as a physical barrier to E. tenella. In addition, two other major developments resulted from this project, these being; the development of a PCR protocol that can specifically identify different Eimeria species in a mixed sample containing at least 0.05 ng/μl of Eimeria DNA and a propidium iodide method that is a suitable indicator tool to assess the viability of oocysts and sporocysts. Finally, the inclusion of MgCh in the extraction buffer increases the hatchability of sporozoites from sporocysts. In conclusion, this study led to development of a frozen section method which can be used ex vivo to investigate further the role of mucus from vaccinated and non-vaccinated chickens, diets with different compositions, anticoccidial drugs, and the identification of the specific receptors in different areas of the chicken intestine. Finally, the propidium iodide method in combination with the PCR protocols can be used as a quality assurance tool in the production of Eimeria vaccines.