Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid.

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

Conservation of chicken male germline by orthotopic transplantation of primordial germ cells from genetically distant donors†.

Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

Simultaneous detection of chicken cytokines in plasma samples using the Bio-Plex assay.

A chicken multiplex cytokine assay (Bio-Plex) to detect four different cytokines (IL-2, IL-12, IL-10, and interferon gamma) simultaneously in plasma samples was designed. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL, except for IFNγ with the range of 50 to 25,000 pg/mL. Such a chicken multiplex assay proved to be fast and reliable, and comparable in sensitivity, accuracy, and reproducibility to conventional enzyme-linked immunosorbent assays. Comparison of the multiplex assay with the ELISA technique using the same clones of detection and capture antibodies resulted in correlation coefficients for all cytokines ranging from 0.95 to 0.99. Lower limit of detection and limit of quantification values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. To reduce the risk of cross-reaction with other proteins, the Bio-Plex system was used, combining the principle of sandwich immunoassay with the Luminex bead-based technology. The cytokine standard recoveries for each cytokine varied between 86 and 118% in dynamic concentration ranges. A chicken multiplex cytokine assay (Bio-Plex) provided a more complete picture of differences between the Th1/Th2 cytokine profiles of the immunized via a new system of antigen delivery into chicken antigen-presenting cells and control groups. This multiplexed fluorescent-bead-based detection assay can be used as a quantitative or comparative tool for the study of the chicken ex vivo cellular immune response.

Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken.

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G0 roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G1 offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G0 roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines.

Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl.

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa. Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.

Dependence of the immune response to coccidiosis on the age of rabbit suckling.

To study the immune response to coccidiosis, the suckling rabbits were inoculated with 2,000 oocysts of either Eimeria intestinalis or Eimeria flavescens at 19, 22, 25, 29, and 33 days of age (DA) and in the case of E. intestinalis at 14 and 16 DA as well and sacrificed 14 days later. Another group served as an uninfected control and the rabbits were killed at the same age as their infected counterparts. Unlike the antibody response, the parameters reflecting cellular immunity (total number of leukocytes in mesenteric lymph nodes, lymphocyte proliferation upon stimulation with specific antigen and the dynamics of CD4+ and CD8+ cell proportions in the intestinal epithelium at the specific site of parasite development) were significantly changed from about 25 DA onwards. In contrast to the rabbits infected with weakly immunogenic coccidium E. flavescens, the proportions of CD4+ and CD8+ lymphocytes in intraepithelial lymphocytes from the specific site of parasite development were considerably changed after infection with highly immunogenic species E. intestinalis. As the immune system of sucklings from about 25 DA reacts to the infection, this age may be considered in terms of vaccination against coccidiosis.

Immune response to rabbit coccidiosis: a comparison between infections with Eimeria flavescens and E. intestinalis.

Seven- to eight-week-old rabbits were infected with Eimeria intestinalis Cheissin, 1948, a highly immunogenic coccidium, or Eimeria flavescens Marotel et Guilhon, 1941, which is weakly immunogenic. Immune response was investigated at 7, 14 and 21 days post inoculation (DPI). The level of serum immunoglobulins, lymphocyte proliferation stimulated by parasite antigens and weight of mesenteric lymph nodes (MLN) showed similar dynamics in rabbits inoculated with both coccidia species. The amount of serum IgG and IgM, but not IgA, was increased from 14 DPI. The lymphocytes from MLN of infected animals significantly reacted to stimulation with parasite antigen 14 and 21 DPI and MLN were enlarged at 14 DPI. Thus, both parasite species elicited immune response characterized by these parameters in a similar manner despite of their different immunogenicity. The only apparent difference in the responses was in the percentage of CD8+ lymphocytes in the specific site of parasite development (the last third of the small intestine in E. intestinalis, caecum in E. flavescens), which increased in rabbits infected with E. intestinalis but not with E. flavescens. This parameter reflects the status of local immunity and hence the results suggest that the local reaction plays an important role in induction of protective immunity to coccidia in rabbits.

Retrovirus-mediated in vitro gene transfer into chicken male germ line cells.

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated gamma-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.

Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken.

Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.