Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Summary

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Abstract

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Introduction

Eimeria spp. causes coccidiosis, which leads to substantial economic losses in the livestock and poultry industry. Although anticoccidial drugs, and to an extent, attenuated anticoccidial vaccines, have been used widely for the control of coccidiosis, there are still shortcomings regarding their drug resistance, drug residues, and the potential diffusion of vaccine strains that regain virulence¹. With the development of molecular biology, transfection has become a vital tool for studying gene functions, developing novel vaccines, and screening new drug targets for Eimeria.

In the last decades, trans....

Protocol

Chickens for all animal experiments were housed and maintained according to the China Agricultural University Institutional Animal Care and Use Committee guidelines and followed the International Guiding Principles for Biomedical Research Involving Animals. The experiments were approved by the Beijing Administration Committee of Laboratory Animals.

1. Extraction and purification of sporozoites of Eimeria spp. (e.g., E. tenella)

1. Release of sporocysts
   1. Centrifuge 1 x 10⁷sporulated oocysts in potassium dichromate solution (2.5%, m/v) at 2,300 x g for 5 min. Wash them with PBS (phosph....

Results

This protocol has been used to transfect eimerian parasites. In this study, the 2^nd generation meronts and merozoites of E. necatrix were shown in Figure 2A and Figure 2B, while Figure 2C and Figure 2D showed the sporocysts and sporozoites of E. tenella after using the densit.......

Discussion

In the 1990s, a transfection system was developed for apicomplexan parasites, and it was used for studies on eimerian parasites. Recently, stable transfection was conducted in E. tenella^8,9 and E. nieschulzi¹⁵. We achieved the stable transfection of E. necatrix by transfecting second-generation merozoites¹¹. Inoculation of transfected sporozoites of E. acervulina through the wing .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ATP-disodium	Sigma	A26209
Cellulose DE-52	Solarbio	C8350
Constant Flow Pump	SHANGHAI JINGKE INDUSTRIAL CO., LTD.	HL-2B
DMEM	MACGENE	CM15019
Glass beads	Sigma	Z250473-1PAK
Glucose	Sigma	No. V900116
Glycine	Biotopped	G6200
HBSS	MACGENE	CC016
KH2PO4	Sigma	No. V900041
Low Speed Centrifuge	BEIJING ERA BEILI CENTRIFUGE CO., LTD.	DT5-2
Magnetic Mixer	SCILOGEX	MS-H280-Pro
MgCl2	Sigma	449164
MoFlo cell sorter	BeckMan Coulter, US	201309995
NaHCO3	Sigma	144-55-8
Nucleofection device	LONZA/amaxa	90900012 (Nucleofector II)
PBS	Solarbio	P1010
Percoll (DG gradient stock solution)	GE Healthcare	17-0891-09
Sodium taurodeoxycholate hydrate	Sigma	T0875
Sorvall Legend Micro 17 Microcentrifuge	ThermoFisher Scientific	75002430
The composition of DMEM: 4.5 g/L glucose with sodium pyruvate, L-glutamine, and 25 mM HEPES.
Trypsin	Solarbio	T8150
Vortex Mixer	Beijing North TZ-Biotech Develop.co.	HQ-60-II
Water Bath Thermostat	Grant Instruments (Cambridge), Ltd.	GD120,GM0815010