Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis.

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine "Hokken" by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.

A rapid assay for detecting antibody against leucocytozoonosis in chickens with a latex agglutination test using recombinant R7 antigen.

A method for detecting antibody against leucocytozoonosis in chickens by latex agglutination (LA) was developed using latex beads coated with recombinant R7 (rR7), an outer membrane antigen of second-generation schizonts of Leucocytozoon caulleryi. Compared with the agar gel precipitation test, which is widely used in Japan, LA could detect antibody induced by L. caulleryi infection with greater sensitivity. No agglutination was detected with sera from specific pathogen free or layer chickens that had not been vaccinated with oil-adjuvanted rR7 antigen, or infected with L. caulleryi, nor with sera from chickens inoculated with other pathogens, establishing the specificity of the assay. The LA test was also able to be used to quantify serum antibody induced by vaccination, which is not possible with the agar gel precipitation test. This study has shown that LA using the rR7 antigen is a simple, quick, and useful method for detecting antibody against L. caulleryi.

Increase of antibody titer against Leucocytozoon caulleryi by oral administration of recombinant R7 antigen.

In this study, we examined oral administration with recombinant R7 (rR7) antigen expressed in Escherichia coli using chicken leucocytozoonosis subunit vaccine (LV)-vaccinated and unvaccinated chickens. Only LV-vaccinated chickens showed re-induction of anti-second-generation schizont (2GS) antibody. Also, LV-vaccinated chickens whose anti-2GS antibody titer was middle-level showed increases of the antibody titer compared to vaccinated-control chickens (P>0.01, >0.05) after oral administration with rR7 antigen.

Field efficacy of recombinant R7 vaccine against chicken leucocytozoonosis.

Effectiveness of vaccine that used recombinant R7 protein (rR7) as antigen that is derived from second-generation schizont (2GS) of Leucocytozoon caulleryi was verified under a field condition against chicken leucocytozoonosis. Chickens reared in a poultry farm where the chickens are attacked by leucocytozoonosis in every year were inoculated with oil-adjuvanted rR7 vaccine (O-rR7), and the immunized chickens were found to have production of antibodies against 2GS at a high level by one shot. Leucocytozoonosis was observed at post-injection. During the epidemic period of leucocytozoonosis, the unique clinical signs of the disease such as discharge of green feces and anemia, and also parasitemia were observed, however, compared to chickens in control group, those in O-rR7 vaccinated group had significantly slight symptoms (P<0.05). In addition to this, immunized chickens had better result of egg production than the unvaccinated chickens did, and the maximum difference of egg production rate, 22%, was observed at the peak of the disease. In conclusion, it is verified that O-rR7 vaccine has efficacy against leucocytozoonosis under field condition, and this vaccine can be put into practical use.

Cellular immune responses in chickens induced by recombinant R7 Leucocytozoon caulleryi vaccine.

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.

The correlation of protective effects and antibody production in immunized chickens with recombinant R7 vaccine against Leucocytozoon caulleryi.

Protective effects of recombinant R7 (rR7) vaccine against Leucocytozoon caulleryi in chickens were studied. After injection of oil-adjuvanted rR7 vaccine into chickens, antibody titers against second-generation schizonts (2GS) antigen of L. caulleryi (anti-2GS antibody) rapidly rose in all the immunized chickens, reached to a peak value 2 weeks after injection, and the titers persisted through 4 or 5 months after injection. Chickens having high levels of the anti-2GS antibody titers (> or = 102,400 ) at pre-challenge completely protected against sporozoites challenge of L. caulleryi. After the challenge inoculation, relatively high parasitemia of L. caulleryi was observed in all the inadequately immunized chickens having low levels of the antibody titers (< or = 3,200) at pre-challenge, although some of them seemed to be clinically normal. Correlation of protective effects in the immunized chickens was observed between both prevention of appearance of clinical signs and parasitemia after parasites challenge and anti-2GS antibody titers of the chickens at pre-challenge. The present study shows that chicken leucocytozoonosis can be prevented by vaccination, and humoral immunity may play an important role in the control of chicken leucocytozoonosis.

Characterization of monoclonal antibodies against the second-generation schizonts of Leucocytozoon caulleryi.

Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L caulleryi.