Abstract
Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host-specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high-density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co-express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.
Highlights
Coccidiosis is a common disease caused by apicomplexan parasites of the genus Eimeria
The expression of these reporters permits assessment of the efficiency of transfection by simple microscopic visualization, and allows the selective isolation of transgenic oocysts by flow-activated cell sorting (FACS) from progeny individuals and verification of the expression and localisation of a protein of interest when it is fused to a reporter protein (Clark et al, 2008; Marugan-Hernandez et al, 2016; Marugan-Hernandez et al, 2017; Pastor-Fernandez et al, 2018; Yan et al, 2009)
Transfection efficiency in Eimeria spp. has been greatly improved by the use of restriction enzyme mediated integration (REMI) techniques (Clark et al, 2008; Kurth & Entzeroth, 2009; Liu et al, 2008). This method relies on linearisation of the transfection plasmid with a restriction enzyme combined with the addition of the same enzyme to the transfection mix before shock, apparently improving plasmid integration into the genome at open sites that have been generated by the endonuclease
Summary
The availability of protocols supporting genetic complementation of Eimeria tenella has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors expressing and delivering heterologous proteins from other Eimeria species, and from other pathogens of veterinary or zoonotic significance which can infect poultry. Current protocols can be used to expand biological understanding about the Eimeria species through reverse genetics. Eimeria tenella; transfection; genetic manipulation; transgenic parasites; vaccine delivery vector
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