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.
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.
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 β-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 The transfection of E. tenella8,9, E. mitis10, and E. acervulina (Zhang et al., unpublished data) was successful in chickens.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.
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)
2. Collection and purification of merozoites of E. necatrix
NOTE: Use Arbor Acre (AA) broilers aged 7-14 d in the experiment. Coccidia-free chickens (n=3) were inoculated with 2 x 105 oocysts of E. necatrix. At 109 h post-infection, the birds were sacrificed by cervical dislocation. The intestine was removed for the collection of the 2nd generation merozoites. For different Eimeria species, there was a different time for collection of the 2nd generation merozoites: E. necatrix at 109 h, and E. tenella at 112 h post-inoculation. For the transfection of E. necatrix, merozoites are the optimal choice as the second merozoites are easy to purify.
3. Nucleofection of merozoites or sporozoites
4. Cloacal inoculation or intravenous injection
5. Propagation and FACS sorting
6. Optional column purification
NOTE: If more pure sporozoites or merozoites are needed, there is an optional method that purifies them through a diethylaminoethyl-52 cellulose (DE-52 cellulose) column.
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 density gradient solutions. The oocysts of E. necatrix (Figure 3A) and E. tenella (Figure 3B) after nucleofecting the merozoites or sporozoites were also shown. The transfection efficiency of first-generation oocysts after nucleofecting the sporozoites is about 3-10%, in general. However, after nucleofecting the merozoites, the transfection efficiency of second-generation oocysts is only a few thousandths (Transfection efficiency of first-generation oocysts could not be calculated).
Figure 1: Purification of sporozoites. (A) Purify sporozoites through the density-gradient centrifugation with density gradient solutions. (B) Purify sporozoites through the DE-52-cellulose column. Please click here to view a larger version of this figure.
Figure 2: The 2nd generation meronts and merozoites of E. necatrix along with the sporocysts and sporozoites of E. tenella. (A) Mature 2nd generation meronts of E. necatrix. (B) The released 2nd generation merozoites of E. necatrix. (C) Sporocysts of E. tenella after purification. (D) Sporozoites E. tenella after purification. The scale bar is 10 µm. Please click here to view a larger version of this figure.
Figure 3: The oocysts obtained after infecting with the nucleofected merozoites or sporozoites. (A) The oocysts of E. necatrix after infecting with the nucleofected merozoites. (B) The oocysts of E. tenella after infecting with nucleofected sporozoites. The scale bar is 10 µm. Please click here to view a larger version of this figure.
Buffer | Composition |
Digestion buffer | 0.25 g trypsin, 0.5 g sodium taurodeoxycholate hydrate, 100 ml PBS or HBSS |
0.1 M NaOH | 2 g NaOH, 500 ml water |
0.1 M HCl | 4.3 ml concentrated hydrochloric acid, 500 ml water |
Glycine eluent buffer | 0.75 g glycine, 7.9 g NaCl, 500 ml water |
Excystation buffer | 0.75% trypsin, 10% chicken bile, PBS |
1 × DG gradient stock solution(Percoll) | 90% 1 × DG gradient stock solution(Percoll), 10% PBS (10 X) |
Nucleofection buffer I | ATP-disodium 2 g, MgCl2-6H2O 1.2 g, 10 ml water |
Nucleofection buffer II | KH2PO4 6 g, NaHCO3 0.6 g, glucose 0.2 g, 500 ml water |
*water: distilled water |
Table 1: Composition of Buffers.
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 vein resolved the the inability of sporozoites of E. acervulina to be inoculated via the cloacal route (Zhang et al., unpublished data). Here, we described a detailed transfection procedure to help researchers nucleofect eimerian parasites.
Previous studies showed that it was feasible to inject transfected sporozoites into the intestinal lumen of rabbits in a laparotomy for in vivo stable transfection of E. magna16 and E. intestinalis17. According to our experience, there was higher efficiency when sorting sporocysts by FACS instead of oocysts. There were also reports about transfection of unsporulated oocysts of E. maxima using a gene gun system or successful electroporation of sporulated oocysts with eGFP-Ham-OTU RNA18,19. Thus, our future studies explore transfection of oocysts or sporocysts to simplify the transfection procedures in Eimeria parasites.
The transfection success in eimerian parasites could enable genetically modified Eimeria to be used as vaccine vehicles to carry heterologous antigens, such as CjaA from C. jejuni13. Although transfection efficiency in Eimeria has been significantly improved, gene editing technology continues to have limitations in eimerian parasites. With the development of transfection in Eimeria, CRISPR/CAS9 technology in Eimeria (Hu et al., unpublished data) could lead to genetic manipulation of Eimeria.
In conclusion, this protocol provides a detailed procedure for nucleofection in chicken Eimeria. The transfection of sporozoites or merozoites is valuable for the study of gene function in Eimeria.
The authors have nothing to disclose.
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).
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 |