This work was supported by the National Key Research and Development Program of China (2017YFD0501200) and the National Natural Science Foundation of China (31572507, 31772728 and 31873007).

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)
<ol>
	<li>Release of sporocysts
	<ol>
		<li>Ce...

<ol>
	<li>Suo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+and+economic+benefits+of+Supercox,+a+live+anticoccidial+vaccine+in+a+commercial+trial+in+broiler+chickens+in+China.">The efficacy and economic benefits of Supercox, a live anticoccidial vaccine in a commercial trial in broiler chickens in China.</a> Veterinary Parasitology. 142 (1-2), 63-70 (2006).</li><li>Kim, K., Soldati, D., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+replacement+in+Toxoplasma+gondii+with+chloramphenicol+acetyltransferase+as+selectable+marker.">Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker.</a> Science. 262 (5135), 911-914 (1993).</li><li>Sibley, L. D., Messina, M., Niesman, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+DNA+transformation+in+the+obligate+intracellular+parasite+Toxoplasma+gondii+by+complementation+of+tryptophan+auxotrophy.">Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (12), 5508-5512 (1994).</li><li>Donald, R. G., Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+molecular+transformation+of+Toxoplasma+gondii:+a+selectable+dihydrofolate+reductase-thymidylate+synthase+marker+based+on+drug-resistance+mutations+in+malaria.">Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (24), 11703-11707 (1993).</li><li>Soldati, D., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+and+expression+in+the+obligate+intracellular+parasite+Toxoplasma+gondii.">Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii.</a> Science. 260 (5106), 349-352 (1993).</li><li>Goonewardene, R., Daily, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+the+malaria+parasite+and+expression+of+firefly+luciferase.">Transfection of the malaria parasite and expression of firefly luciferase.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (11), 5234-5236 (1993).</li><li>Kelleher, M., Tomley, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+of+beta-galactosidase+in+differentiating+sporozoites+of+Eimeria+tenella.">Transient expression of beta-galactosidase in differentiating sporozoites of Eimeria tenella.</a> Molecular and Biochemical Parasitology. 97 (1-2), 21-31 (1998).</li><li>Clark, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+facilitating+stable+transfection+of+Eimeria+species.">A toolbox facilitating stable transfection of Eimeria species.</a> Molecular and Biochemical Parasitology. 162 (1), 77-86 (2008).</li><li>Yan, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Eimeria+tenella:+Constitutive+expression+of+the+YFP-YFP+molecule+throughout+the+life+cycle.">Stable transfection of Eimeria tenella: Constitutive expression of the YFP-YFP molecule throughout the life cycle.</a> International Journal for Parasitology. 39 (1), 109-117 (2009).</li><li>Qin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+Eimeria+mitis+with+Yellow+Fluorescent+Protein+as+Reporter+and+the+Endogenous+Development+of+the+Transgenic+Parasite.">Transfection of Eimeria mitis with Yellow Fluorescent Protein as Reporter and the Endogenous Development of the Transgenic Parasite.</a> PloS One. 9 (12), e114188 (2014).</li><li>Duan, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Eimeria+necatrix+through+nucleofection+of+second+generation+merozoites.">Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites.</a> Molecular and Biochemical Parasitology. , 1-5 (2019).</li><li>Li, Z. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Eimeria+mitis+expressing+chicken+interleukin+2+stimulated+higher+cellular+immune+response+in+chickens+compared+with+the+wild-type+parasites.">Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites.</a> Frontiers in Microbiology. 6, 533 (2015).</li><li>Clark, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eimeria+species+parasites+as+novel+vaccine+delivery+vectors:+anti-Campylobacter+jejuni+protective+immunity+induced+by+Eimeria+tenella-delivered+CjaA.">Eimeria species parasites as novel vaccine delivery vectors: anti-Campylobacter jejuni protective immunity induced by Eimeria tenella-delivered CjaA.</a> Vaccine. 30 (16), 2683-2688 (2012).</li><li>Eckert, J., Braun, R., Shirley, M. W., Coudert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eimeria+species+and+strains+of+chickens.">Eimeria species and strains of chickens.</a> Biotechnology: Guidelines on techniques in coccidiosis research. Part. I: Eimeria and Isospora, 1-24 (1995).</li><li>Kurth, M., Entzeroth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene+expression+in+cell+culture+stages+and+oocysts+of+Eimeria+nieschulzi+(Coccidia,+Apicomplexa).">Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa).</a> Parasitology Research. 104 (2), 303-310 (2009).</li><li>Tao, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Eimeria+magna+Perard,+1925+Displays+Similar+Parasitological+Properties+to+the+Wild-type+Strain+and+Induces+an+Exogenous+Protein-Specific+Immune+Response+in+Rabbits+(Oryctolagus+cuniculus+L.).">Transgenic Eimeria magna Perard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.).</a> Frontiers in Immunology. 8, 2 (2017).</li><li>Shi, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Transfection+of+Eimeria+intestinalis+and+Investigation+of+Its+Life+Cycle,+Reproduction+and+Immunogenicity.">Stable Transfection of Eimeria intestinalis and Investigation of Its Life Cycle, Reproduction and Immunogenicity.</a> Frontiers in Microbiology. 7, 807 (2016).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+telomerase-interacting+OTU+protein+of+Eimeria+tenella+and+its+telomerase-regulating+activity.">A novel telomerase-interacting OTU protein of Eimeria tenella and its telomerase-regulating activity.</a> Acta Biochimica et Biophysica Sinica. 49 (8), 744-745 (2017).</li><li>Li, J. N., Zou, J., Yin, G. W., Liu, X. Y., Suo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid+DNA+could+be+delivered+into+Eimeria+maxima+unsporulated+oocyst+with+gene+gun+system.">Plasmid DNA could be delivered into Eimeria maxima unsporulated oocyst with gene gun system.</a> Acta Polytechnica Hungarica. 60 (4), 431-440 (2012).</li></ol>

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. tenella8,9 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 ...

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, transfection has been applied successfully for apicomplexan parasites such as Plasmodium and Toxoplasma gondii2,3,4,5,6. A study using &#946;-gal as a reporter for the transfection in E. tenella piloted such work in Eimeria7. The transfection of E. tenella8,9, E. mitis10, and E. acervulina (Zhang et al., unpublished data) was successful in chickens. Recently, we achieved transfection using merozoites of E. necatrix through nucleofection11.
Studies showed that Eimeria expressing a heterologous antigen has the potential to be developed as a recombinant vaccine, such as those expressing Campylobacter jejuni antigen A (CjaA) or chicken interleukin 2 (chIL-2)12,13. Therefore, this protocol describes a nucleofection study of Eimeria spp. in chickens. The procedure describes purification of sporozoites or merozoites, nucleofection with plasmid DNA, cloacal inoculation/intravenous injection and in vivo propagation to help researchers starting studies on transgenic Eimeria parasites.

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

nucleofection and in vivo propagation of chicken eimeria parasites

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.

This protocol has been used to transfect eimerian parasites. In this study, the 2nd 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...

Watch this Scientific Journal Video about Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites at JoVE.com

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

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.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

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 ...

Establishment of transfection protocols for Eimeria species would help to advance the study of gene functions, developing novel vaccines, and screening new drug targets for Eimeria. Using this technique, we achieved the transfection of several species of Eimeria, and nucleofection is a useful tool to facilitate the application of genetic manipulation in eimerian parasites. The nucleofection of Eimeria provides a reference for the transfection of other parasites that can't be cultured in vitro and will accelerate the study of their genetic manipulation.If you try this technique for the first time, you should prepare well all the reagents and their instruments before the start of transfection to avoid scramble during the experiment. The realization of this method helps people who focus on the transfection of Eimeria to see the intricate details of this experiment. To start with release of sporocysts, centrifuge one times 10 to the seven sporulated oocysts in potassium dichromate solution at 2, 300 times g for five minutes.Wash them with PBS three times. Resuspend the pellet with one milliliter of PBS, and transfer the mixture to a 15-milliliter tube. Add an equal volume of one-millimeter-diameter glass beads, and oscillate the oocyst suspension using a vortex mixer to release the sporocysts.Every minute, monitor the release of sporocysts by microscopy. When more than 90%of the oocysts are broken, stop vortexing. Transfer the sporocyst suspension to new 1.5-milliliter tubes, and centrifuge at 1, 600 times g for five minutes.Resuspend the precipitate with one milliliter of 50%density gradient solution, and centrifuge at 10, 000 times g for one minute. To release sporozoites, resuspend the precipitate with excystation buffer, and incubate in a 42-degree Celsius water bath for 40 to 60 minutes to release the sporozoites. Shake the tubes once every five minutes during the excystation.When more than 90%of the sporozoites are released, stop incubating. Centrifuge at 600 times g for 10 minutes. Resuspend the precipitation with one milliliter of 55%density gradient solution, and centrifuge the tube at 10, 000 times g for one minute.Resuspend the precipitation with one milliliter of PBS, and count the sporozoites using a hemocytometer. To collect the second-generation merozoites of Eimeria necatrix, cut the chicken intestine longitudinally from the yolk stalk to the ileocecal orifice, and wash it with PBS or HBSS gently three times in a Petri dish. Cut the intestine into pieces, and place them in a conical flask with a digestion buffer.Place the flask on a magnetic mixer at 37 degrees Celsius with a stirring bar for 30 to 60 minutes to release merozoites. After 30 minutes of incubation, monitor the release of merozoites by microscopic examination every five minutes. To purify the merozoites, pour the suspension containing digested merozoites over four layers of gauze to filter into 50-milliliter tubes, and centrifuge the tubes at 600 times g for 10 minutes.After centrifugation, discard the supernatant containing intestine debris. Transfer the precipitation with purified merozoites to 1.5-milliliter tubes. Resuspend the precipitation with one milliliter of PBS, and count the merozoites using a hemocytometer.First, centrifuge the sporozoite or merozoite suspension at 600 times g for 10 minutes. Then discard the supernatant. In the following order, add 85 microliters of nuclear transfection buffer, 10 micrograms of plasmid, and 25 units of restriction enzyme into the 1.5-milliliter tube containing sporozoites or merozoites.Transfer the suspension to a nuclear transfection cup. Put the cup into a nuclear transfer groove. Turn on the nucleofection device by using the power button, and select the transfection procedure U-033.When the program finishes, press the X button of the nucleofection device, and the screen displays OK, indicating that the nucleofection is successful. Add 0.5 to one milliliters of Dulbecco's Modified Eagle's Medium to the nucleofection cup to stop the reaction. After mixing gently, transfer the suspension to a 1.5-milliliter tube.Inoculate the nucleofected parasites into seven-day-old chickens. Inoculate the merozoites of Eimeria necatrix or the sporozoites of Eimeria tenella via the cloacal route, but inoculate Eimeria acervulina sporozoites via intravenous injection. After collecting oocysts from feces, use fluorescein-activated cell sorting and 150 milligrams per kilogram pyrimethamine to increase the transgenic population ratio.This protocol has been used to transfect eimerian parasites. In this study, the second-generation meronts and merozoites of Eimeria necatrix are shown here, as well as the sporocysts and sporozoites of Eimeria tenella after using the density gradient solutions. The images of the oocysts of Eimeria necatrix after infecting with the nucleofected merozoites and the oocysts of Eimeria tenella after infecting with nucleofected sporozoites indicate successful nucleofection.The most important thing while attempting this procedure is to make sure the purification of sporozoites and merozoites are successful and to use the 50%density gradient solution to remove the OS2 while making sure the sporocysts and the sporozoites are more purified. Following this procedure, you can optimize this protocol, such as transfecting oocyst or sporocyst to simplify the transfection procedures of Eimeria parasites in the future. With the development of transfection in Eimeria, CRISPR-Cas9 technology in Eimeria will be expectable to significantly put forward the cautious genetic manipulation of Eimeria and to be applied as a suitable vaccine delivery vehicle.

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6.7K 조회수.  China Agricultural University. 아이메리아 종에 대한 경질 프로토콜의 설립은 유전자 기능의 연구를 진행하는 데 도움이 될 것입니다, 새로운 백신을 개발, 아이메리아에 대한 새로운 약물 목표를 선별. 이 기술을 사용하여, 우리는 Eimeria의 몇몇 종의 전염을 달성하고, 핵은 eimerian 기생충에 있는 유전 조작의 적용을 용이하게 하기 위하여 유용한 공구입니다. Eimeria의 핵은 시험관에서 배양될 수 없는 그밖 ?...