Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens.

BACKGROUND: Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. RESULTS: Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. CONCLUSION: The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody.

Eimeria tenella is a coccidian parasite of great economical importance for poultry industry. The surface of Eimeria invasive agents, sporozoites and merozoites, is coated with a family of developmentally regulated glycosylphosphatidylinositol (GPI)-linked surface antigens (SAGs), some of them involved in the initiation of the infection process. Using 2D gel electrophoresis followed by mass spectrometry, an antigenic surface protein EtSAG1 (TA4) of E. tenella sporozoites has been identified as a target of neutralizing monoclonal antibody 2H10E3. To clarify the mechanism of invasion inhibition caused by the EtSAG1-specific antibodies, a structural model of EtSAG1 was generated. It appears that "EtSAG fold" does not bear an evolutionary relationship to any known protein structure. The intra- and interchain disulfide bonds could be assigned to certain pairs of six conserved cysteines found in members of the EtSAG protein family. The outward-facing surface of the antigen was found to comprise an expanded positively charged patch, thus suggesting that the parasite invasion process may be initiated by sporozoite attachment to negatively charged sulfated proteoglycans on the surface of the host cell.

Extracellular calcium deficiency and ryanodine inhibit Eimeria tenella sporozoite invasion in vitro.

An in vitro assay system with Eimeria tenella sporozoites was used to study the effects of extracellular calcium and active agents affecting the invasion of parasites into host cells. At concentrations of 900 microM Ca(2+) and less the invasion rates were distinctly decreased. Ryanodine, a herbal alkaloid known for binding to internal Ca(2+) channels (ryanodine receptors), showed an inhibitory effect on E. tenella sporozoite invasion. Preincubation tests and staining with a fluorescent derivative of ryanodine assured that the compound bound specifically to the sporozoites and affected them rather than the host cells.