Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites

Abstract

Chicken coccidiosis, caused by Eimeria sp., occurs in almost all poultry farms and causes huge economic losses in the poultry industry. Although this disease could be controlled by vaccination, the reduced feed conservation ratio limits the widespread application of anticoccidial vaccines in broilers because some intermediate and/or low immunogenic Eimeria sp. only elicit partial protection. It is of importance to enhance the immunogenicity of these Eimeria sp. by adjuvants for more effective prevention of coccidiosis. Cytokines have remarkable effects on the immunogenicity of antigens. Interleukin 2 (IL-2), for example, significantly stimulates the activation of CD8+ T cells and other immune cells. In this study, we constructed a transgenic Eimeria mitis line (EmiChIL-2) expressing chicken IL-2 (ChIL-2) to investigate the adjuvant effect of ChIL-2 to enhance the immunogenicity of E. mitis against its infection. Stable transfected EmiChIL-2 population was obtained by pyrimethamine selection and verified by PCR, genome walking, western blotting and indirect immunofluorescence assay. Cellular immune response, E. mitis-specific IFN-γ secretion lymphocytes in the peripheral blood mononuclear cells, stimulated by EmiChIL-2 was analyzed by enzyme-linked immunospot assay (ELISPOT). The results showed that EmiChIL-2 stimulated a higher cellular immune response compared with that of the wild-type parasite infection in chickens. Moreover, after the immunization with EmiChIL-2, elevated cellular immune response as well as reduced oocyst output were observed These results indicated that ChIL-2 expressed by Eimeria sp. functions as adjuvant and IL-2 expressing Eimeria parasites are valuable vaccine strains against coccidiosis.

Keywords: ELISPOT; cellular immune response; chicken interleukin 2; reproductive potential; transgenic Eimeria mitis.

FIGURE 1

Construction of transgenic Eimeria mitis expressing secreted chicken IL-2 (ChIL-2). (A) Schematic of double-cassette expression vectors. The selection gene [DHFR-Ts-enhanced yellow fluorescent gene (EYFP)] and ChIL-2 were driven by the histone 4 and actin promoter, respectively. Signal sequences (ss) from T. gondii GRA 8 regulated the secretion of ChIL-2. (B) Both the transiently transfected E. mitis sporozoites (a) and the stable transfected EmiChIL-2 (b) were expressing EYFP. (C) Genomic DNA from EmiChIL-2 was amplified with the primers ChIL-2-F and ChIL-2-R (giving a 432 bp product) to verify the recombination of ChIL-2, and the primers EYFP-F and EYFP-R (giving a 726 bp product) to confirm the recombination of EYFP as a positive control, genomic DNA from wild type E. mitis was used as a control. (D) Genomic DNA from EmiChIL-2 was amplified with arbitrary degenerate primers (AP 1, AP 2, AP 3, and AP 4) and specific primers [SP 1, SP 2, and SP 3 (Table 2)] from histone 4 promoter by thermal asymmetric interlaced PCR, and the products from the third-round PCR were cloned into pEasy-T1 vector for sequencing. (E) One integration site (Emh_scaff1365) was confirmed by BLAST from more than 50 clones in the E. mitis GeneDB. (F) Oocysts antigens extracted from EmiChIL-2 reacted with the poly antibody against ChIL-2 producing a clear band with a size of approximately 14 kd to verify ChIL-2 expression by WB. Recombinant ChIL-2 (with two His 6 tag) and the wild-type E. mitis oocysts antigens were used as a positive and negative control, respectively. (G) EmiChIL-2 sporozoites reacted with the poly antibody against ChIL-2 to confirm the localization of ChIL-2 by IFA, and the wild-type E. mitis sporozoites were utilized as a control. Bar = 10 μm.

FIGURE 2

Oocysts shedding of EmiChIL-2. (A) Oocyst shedding was measured every 24 h from 3 to 11 dpi, and the mean value was estimated from four individuals. The wild-type E. mitis infection was used as a control. (B) Mean total oocyst output per chicken of EmiChIL-2 was three times lower than that of the wild-type E. mitis (p < 0.05) 3 to 11 days after the infection.

FIGURE 3

Gamma interferon (IFN-γ) secretion lymphocytes in peripheral blood mononuclear cells (PBMCs) after immunization. (A) 10⁶ PBMCs from PBS (Ctrl, upper), wild-type E. mitis (middle) and EmiChIL-2 (bottom) immunized birds (10⁴ oocysts/bird) were stimulated for 24 h with PBS (negative control, left), E. mitis oocysts antigen (middle) and PMA plus ionomycin (positive control, right). The number of IFN-γ secretion lymphocytes (spots) was determined as described in the section of materials and methods. (B) The mean amount of IFN-γ secretion lymphocytes in PBMCs in EmiChIL-2 immunized birds was significantly higher (p < 0.05) than that of the birds immunized with the wild-type E. mitis (n = 4). (C) Mean total oocyst output per chicken from 4 to 8 days after the immunization with EmiChIL-2 was much lower (p < 0.05) than after that, done with the wild-type E. mitis.

FIGURE 4

IgY (IgG) antibody titer in the serum and oocyst output post primary and boost immunization. (A) The OD₄₅₀ value was similar between the birds immunized with EmiChIL-2 and those, immunized with the wild-type E. mitis 2 weeks after both the primary and the boost immunization (p > 0.05). (B) Mean total oocyst output per chicken of EmiChIL-2 was significantly lower than that of the wild-type E. mitis after the primary immunization (p < 0.05) 4–8 days, whereas, there was no significant difference between the EmiChIL-2 and the wild-type E. mitis-immunized birds after the boost immunization (p > 0.05) 4 to 8 days.

FIGURE 5

Oocyst output following challenge infection in birds vaccinated with or without EmiChIL-2 or its wild type. (A) Oocysts in fecal and litter samples (n = 10) every 2 days post vaccination with transgenic or wild type E. mitis and challenge with wild-type E. mitis (10⁴ oocysts/bird). (B) Mean total oocyst output per chicken between 4 and 8 days post challenge.