Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2.

Sixin Zhang,Feifei Bi,Xun Suo,Chaoyue Wang,Fangyun Shi,Xinming Tang,Chunhui Duan,Jie Liu,Xianyong Liu,Si Wang,Dandan Hu,Jingxia Suo

doi:10.3390/vaccines9070791

Sixin Zhang, Feifei Bi + Show 10 more

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https://doi.org/10.3390/vaccines9070791

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Abstract

The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.

Highlights

The EaM2e reached over 95% fluorescent rate without drug selection and fluorescent-activated cell sorting (FACS) after the
E. acervulina parasitizes in the duodenum of the chicken, so the inoculation route via the cloaca applied to E. tenella and E. mitis was not feasible in E. acervulina
We found that the transgenic parasites produced more oocysts than the wild type, which was consistent with previous study on transgenic

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Coccidiosis is one of the most severe parasitic diseases in chickens, which causes. EUR 7.7–13.0 billion losses to the global poultry industry annually [1]. The strategy to control coccidiosis relies on the administration of anticoccidial drugs and live anticoccidial vaccines, the latter of which have been used for 70 years [2,3]. With the development of genetic manipulation technology, genetically modified live vector vaccines are promising vaccines for both humans and animals [4,5]. Since live oocyst-based anticoccidial vaccines are being widely used in the poultry industry, whether

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