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

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.

Oral delivery of Eimeria acervulina transfected sequentially with two copies of the VP2 gene induces immunity against infectious bursal disease virus in chickens

“…Liu et al constructed transgenic E. tenella expressing the monomer M2 protein of the avian influenza virus that could not elicit immune responses ( 30 ). Moreover, Zhang et al constructed a transgenic E. acervulina line that expresses 12 copies of M2e, and the immune response was not significantly different from the control group ( 9 ). So, the location of immunogen expression may be responsible.…”

“…So far, 10 species of chicken coccidian have been clearly identified, including E. acervulina , E. tenella , E. maxima , E. mitis , E. necatrix , E. brunetti , E. praecox , E. lata , E. nagambie , and E. zaria ( 8 ). Among these, E. acervulina is highly prevalent but has moderate pathogenicity ( 9 ). One Eimeria sporozoite can yield 1,000 merozoites per cycle, repeating two to four times in the chicken host, leading to a rapid surge in oocyst numbers ( 10 ).…”

Vaccination Against Poultry Parasites

“…Better protection in terms of decrease in lesion scores and parasite replication after challenge was achieved when a transformed E. tenella line expressed two E. maxima antigens or when two transformed E. tenella lines, each expressing a different antigen were used as vaccines (117,118). Furthermore, coccidia expressing an avian influenza, Toxoplasma gondii , or Dermanyssus gallinae antigen have been constructed (119,120,121). A different approach was creating an E. mitis line expressing chicken IgG Fc to increase immunogenicity and improve protection (122).…”

Vaccines against chicken coccidiosis with particular reference to previous decade: progress, challenges, and opportunities

“…Recombinant Eimeria vaccines featuring use of biological agents (BCG, fowl pox virus) can act as vaccine vectors, offering protective immunity (Wang et al 2014 ; Tang et al 2018 ). The stable transfection by employing E. acervulina expressing the multiple copies of extracellular domain (M2e) of H9N2 influenza virus has been reported (Zhang et al 2021 ). These studies encourage more research on further exploration of Eimeria as live vaccine vectors.…”