Nucleofection and In Vivo Propagation of Chicken <em>Eimeria</em> 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 ...

China Agricultural University

Ten polymorphic SSR markers of Flammulina velutipes were developed and characterized with FIASCO methods. The polymorphism information content (PIC) ranged from 0.13 to 0.69. This is the first report on development of SSR markers in F. velutipe.

Development and characterization of simple sequence repeat (SSR) markers for the mushroom Flammulina velutipes.

Cystic echinococcosis (hydatid disease), caused by the tapeworm Echinococcus granulosus (class Cestoda; family Taeniidae), is a neglected tropical disease that results in morbidity and mortality in millions of humans, as well as in huge economic losses in the livestock industry globally. Proteins from the tetraspanin family in parasites have recently become regarded as crucial molecules in interaction with hosts in parasitism and are therefore suitable for the development of vaccines and diagnostic agents. However, no information is available to date on E. granulosus tetraspanin.

Molecular insights into a tetraspanin in the hydatid tapeworm Echinococcus granulosus.

Coccidiosis is one of the most serious diseases of livestock and birds in the world. Vaccination with live-parasite anticoccidial vaccines with genetic manipulation improving the immunogenicity of vaccine strains would be the best means for controlling coccidiosis in breeder and layer stocks, even in fast-growing broilers. Profilin from apicomplexan parasites is the first molecularly defined ligand for Toll-like receptor 11 (TLR11) and TLR12 in mice and is a potential molecular adjuvant. Here, we constructed a transgenic  line (Et-EmPro) expressing the profilin of , the most immunogenic species of chicken coccidia, and evaluated the adjuvant effects of EmPro on the immunogenicity of  We found that immunization with the transgenic  parasites, compared with the wild type, elicited greater parasite antigen-specific cell-mediated immunity, characterized by increased numbers of interferon gamma (IFN-γ)-secreting lymphocytes. The transgenic parasite also induced better protective immunity against  challenge than the wild type. In addition, the diversity of the fecal microbiome of the birds immunized with the transgenic parasite differed from that of the microbiome of the wild-type-immunized birds, indicating interactions of  with the gut microbiome of chickens. Our results showing enhanced immunogenicity of  by use of EmPro as a molecular adjuvant derived from the most immunogenic affinis species represent a large step forward in the development of the next generation of coccidiosis vaccines using  as a vaccine platform expressing molecular adjuvants and potentially other pathogen antigens against not only coccidiosis but also other infectious diseases.

Transgenic Eimeria tenella Expressing Profilin of Eimeria maxima Elicits Enhanced Protective Immunity and Alters Gut Microbiome of Chickens.

Chicken coccidiosis, caused by the infection of Eimeria species, leads to important economic losses to the poultry industry. Vaccination with attenuated live parasites seems to be the best way to control this disease. Attenuated eimerian parasites with shortened prepatent times show great changes in intracellular development compared to their parent strains but the mechanisms involved in these biological differences are still unclear.

Comparative transcriptome analysis of Eimeria maxima (Apicomplexa: Eimeriidae) suggests DNA replication activities correlating with its fecundity.

Eimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2 generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocysts was noticed with more than 90% EYFP + oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine-delivery vehicle.

Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites.

Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns and activate downstream signalling pathways, resulting in modulation of host immunity against pathogens. Here, we investigated whether PRR-mediated recognition is involved in host immune responses to the blood-feeding nematode Haemonchus contortus.

Transcriptional profiling of innate immune responses in sheep PBMCs induced by Haemonchus contortus soluble extracts.

Live anticoccidial vaccines have been a tremendous success for disease prevention. The establishment of the reverse genetic manipulation platform has enabled the development of Eimeria parasites, the live anticoccidial vaccine strains, as vaccine vectors. In our previous study, recombinant E. tenella expressing a single immunodominant antigen of E. maxima (Et-EmIMP1) was able to protect chickens against challenge infection with E. maxima. This promising result encouraged us to further explore strategies to improve the protection efficacy of recombinant Eimeria and develop it as a vaccine vector.

Co-immunization with two recombinant Eimeria tenella lines expressing immunoprotective antigens of E. maxima elicits enhanced protection against E. maxima infection.

Dandan Hu

National Animal Protozoa Laboratory,
College of Veterinary Medicine,
National Animal Protozoa Laboratory, College of Veterinary Medicine

China Agricultural University

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Dandan Hu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}National Animal Protozoa Laboratory,College of Veterinary Medicine,National Animal Protozoa Laboratory, College of Veterinary Medicine

China Agricultural University

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

National Animal Protozoa Laboratory,
College of Veterinary Medicine,
National Animal Protozoa Laboratory, College of Veterinary Medicine