Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

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.