nucleofection-and-in-vivo-propagation-of-chicken-eimeria-parasites

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

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.

Exogenous melatonin could induce cashmere growth. However, induced growth of cashmere fleece by melatonin implants cannot be combined with the typical growth, resulting in earlier shedding followed by another cycle of cashmere growth. To address this issue, we examine the effects on the cashmere yield, fibre characteristics, and the growth and reproductive performance of cashmere goats of planned administration of melatonin.

Effects of melatonin implantation on cashmere yield, fibre characteristics, duration of cashmere growth as well as growth and reproductive performance of Inner Mongolian cashmere goats.

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.

Coccidiosis is a serious threat to the poultry industry, resulting in substantial economic losses worldwide. The effective development of alternative treatments for coccidiosis that does not involve chemotherapy drugs and does not result in antibiotic resistance relies on gaining a clearer understanding of the interaction between host intestinal microbiota and enteric coccidia. Here, we established an Eimeria tenella infection model in chickens and subsequently monitored the changes in the overall intestinal microbiome using 16S rRNA gene sequencing. We found that the gut (i.e. fecal) microbiota of infected chicken differed from that of uninfected naïve animals. Levels of non-pathogenic bacteria, including Lactobacillus and Faecalibacterium declined, whereas those of pathogenic bacteria, including Clostridium, Lysinibacillus, and Escherichia, increased over time in response to E. tenella infection. Similar dynamic changes of the fecal microbiota were observed in both Arbor Acres broilers and White Leghorn chickens, indicating that the perturbation of the microbiota was directly induced by E. tenella infection. Our findings could be used to further elucidate the serious damage to host health caused by coccidia infection, leading to the development of new effective treatment options for coccidiosis.

Eimeria tenella infection perturbs the chicken gut microbiota from the onset of oocyst shedding.

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.

Coccidiosis, caused by the infection of Eimeria parasites, is one of the most common diseases in domestic rabbits. Live anticoccidial vaccine formulated with attenuated precocious lines of pathogenic eimerian parasites is expected to be valuable for the control of rabbit coccidiosis as a similar strategy to produce anticoccidial vaccines against chicken coccidiosis has being used for several decades. Eimeria media, moderate pathogenic, is widespread in China. Therefore, attenuated anticoccidial vaccines against rabbit coccidiosis should contain vaccine strain(s) of E. media. In this study, a precocious line of E. media (Empre) was selected by collecting and propagating the early excreted oocysts with 16 successive generations. The prepatent period of Empre reduced from 108 h of its parental strain (Emwt) to 70 h. The fecundity of Empre was about 1/10 to 1/3 lower than that of Emwt. Each sporocyst of Empre sporulated oocyst contained only one large refractile body instead of two smaller ones seen in the parental strain. When vaccinated with 1 × 10 or 1 × 10 precocious line oocysts, the rabbits were completely protected against homologous challenge with the parental strain 14 days post challenge by terms of body weight gain and oocyst output counting, indicating the efficacy of Empre. Meanwhile, all immunized rabbits showed no clinical sign post immunization, indicating the safety of Empre. For co-immunization, 1 × 10Empre oocysts and 5 × 10 oocysts of a precocious line of E. intestinalis (EIP8) were inoculated to each rabbit in a trial. No diarrhea or mortality was found after vaccination, and the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group, while the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group (P > 0.05), but significantly higher than that of UCC group (P < 0.01) after challenge, indicating it is safe and effective when using co-immunization. These results together show that Empre, as a precocious line, is a good candidate of precocious line of E. media for anticoccidial vaccine development.

Article	Year
Co-immunization with two recombinant Eimeria tenella lines expressing immunoprotective antigens of E. maxima elicits enhanced protection against E. maxima infection. Parasites & vectors\| PubMed ID: 31300007	2019
Effects of melatonin implantation on cashmere yield, fibre characteristics, duration of cashmere growth as well as growth and reproductive performance of Inner Mongolian cashmere goats. Journal of animal science and biotechnology\| PubMed ID: 26075068	2015
Transgenic Eimeria tenella Expressing Profilin of Eimeria maxima Elicits Enhanced Protective Immunity and Alters Gut Microbiome of Chickens. Infection and immunity\| PubMed ID: 29967093	2018
Eimeria tenella infection perturbs the chicken gut microbiota from the onset of oocyst shedding. Veterinary parasitology\| PubMed ID: 30105975	2018
Comparative transcriptome analysis of Eimeria maxima (Apicomplexa: Eimeriidae) suggests DNA replication activities correlating with its fecundity. BMC genomics\| PubMed ID: 30249186	2018
Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites. Molecular and biochemical parasitology\| PubMed ID: 30658178	2019
Selection and characterization of a precocious line of Eimeria media. Parasitology research\| PubMed ID: 31407118	2019

Chunhui Duan

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