Laboratory Growth and Genetic Manipulation of Eimeria tenella

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.

In vitro Anticoccidial Study of Oregano and Garlic Essential Oils and Effects on Growth Performance, Fecal Oocyst Output, and Intestinal Microbiota in vivo

“…MDBK cells were incubated for over 24 h with a concentration of 100 μg/ml (the maximum to which cells were subsequently exposed during the invasion experiments) of oregano or garlic essential oils, modifying the approaches described in Pastor-Fernandez et al ( 29 ). No morphological changes were observed compared with the control groups (DMEM/DMSO).…”

“…Eimeria tenella sporozoites (0.5 × 10 6 /well) were pre-treated for 1 h at 41°C−5% CO 2 with essential oils of garlic or oregano at different concentrations (100, 50, 20, 5 μg/ml from the stocks) in Advanced DMEM, as described in Pastor-Fernandez et al ( 29 ). DMSO (10 μl/ml) and robenidine (anticoccidial; 5 μg/ml) were used as controls for E. tenella infection.…”

The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture

“…The E. tenella Wisconsin (Wis) strain (McDougald and Jeffers, 1976 ) and a transgenic population, E. tenella YFPmYFP, derived from this strain (Clark et al, 2008 ) were propagated in 3-week-old White Leghorn chickens reared under specific pathogen free conditions as previously described (Shirley, 1995 ). Oocyst purification, excystation and sporozoite purification were performed as described previously (Pastor-Fernandez et al, 2019 ). Freshly purified sporozoites were used to infect cell monolayers.…”

Vaccination with transgenic Eimeria tenella expressing Eimeria maxima AMA1 and IMP1 confers partial protection against high-level E. maxima challenge in a broiler model of coccidiosis

“…This suggests that antigen load should be always considered as it may influence the presence or absence of a protective response, at least in terms of BWG. However, since the parasite populations used for immunisation were not clonal it is extremely difficult to determine the exact quantity of transprotein that was effectively delivered in each vaccine formulation, even employing indirect methods such as the q-PCR described earlier [15]. It is also worth highlighting that the variation observed in BWG in the non-vaccinated and challenged control group could have interfered with data interpretation, since performance of a quarter of those chickens was comparable to birds from the non-challenged (H 2 O-H 2 O) and the vaccinated (Emax-Emax) control groups.…”

“…Four weeks old Lohmann Selected Leghorn (LSL) chickens reared under specific pathogen-free conditions were used to propagate oocysts of the Wisconsin (Wis) strain of E. tenella and the Weybridge (W) strain of E. maxima as previously described [14]. Standard methods were used to recover and sporulate oocysts and to purify sporozoites through nylon wool and DE-52 columns [15,16].…”