Background

Infections of the small intestine are widespread and can cause significant morbidity and mortality worldwide (Munot and Kotler, 2016). Children and the elderly, due to their weaker immune system, suffer the worst consequences. Gaining a better understanding of how the small intestinal immune system functions is therefore of prime importance. However, many of the pathogens used in mouse models are not natural to the host, can cause systemic inflammation or can persist in the tissues for long periods. This can result in uncharacteristic induction of the immune response and inadequate development of protective immunity.

Eimeria vermiformis is a tissue specific, intracellular single cell protozoan, which infects the small intestinal epithelia. Unlike the related pathogen used in infection models, Toxoplasma gondii, this is a natural, self-limiting, murine parasite, which induces a strong immune response and establishes long-lasting protection against subsequent infections. Furthermore, E. vermiformis is monoxenous, its life cycle is completed without the need of a second host species (see Figure 1). It is therefore an ideal pathogen to study the mechanisms of host protection against primary and secondary infection at the small intestinal barrier.

Mice infected with E. vermiformis excrete a non-infectious, unsporulated form of oocysts into the environment. Once in contact with oxygen and moisture, in a process which takes between 2 to 7 days, these oocysts mature into an infectious, sporulated form, which, upon ingestion, disseminates the infection to other animals (Fayer, 1980).

The E. vermiformis infection model has been used in the past and has yielded valuable information in areas such as the role of αβ and γδ T cells that line the small intestinal epithelia (Roberts et al., 1996) and the influence of age of the host in the development of protective immune responses in the intestine (Ramsburg et al., 2003). More recently we have used the E. vermiformis model to highlight the role of mitochondria in controlling the activation state of epithelial-resident T lymphocytes (Konjar et al., 2018).

Here, we present an adapted version of the original protocol used by Long et al. (1976) to purify and sporulate E. vermiformis. We describe in detail the method used to propagate the parasite, infect mice and accurately assess parasite load.


Figure 1. E. vermiformis life cycle. A single sporulated oocyst can initiate an infection when swallowed by its respective host, the mouse. Each mature oocyst holds four sporocysts, each sporocyst gives rise to two sporozoites, which can infect the enterocytes. Inside the host cell, the parasite develops via trophozoite and schizont stages into merozoites. Merozoites can re-infect the epithelial cells and start a new cycle. Eimeria typically undergoes three rounds of asexual multiplication whereupon merozoites give rise to gametes. Male and female gametes can fuse to form an oocyst, protected by a multi-layered cell wall making them highly resistant to environmental pressures. These oocysts shed with the feces. Unsporulated oocysts undergo sporulation upon contact with oxygen and moisture, undergoing meiosis, which takes between 2 to 7 days.