Recipient Organization
APPLIED BIOTECHNOLOGY INSTITUTE
(N/A)
SAN LUIS OBISPO,CA 93407
Performing Department
(N/A)
Non Technical Summary
Avian coccidiosis caused by the parasite Eimeria is a widespread disease of poultry causing enormous losses to the industry. It is controlled with a combination of antibiotic drugs and live vaccines derived from the parasite. Development of antibiotic resistant pathogens and changing consumer preferences for meat raised without antibiotics have created an urgent need for new approaches to vaccination. Existing practices for poultry vaccination can also cause detrimental stress to the animals. A vaccine relying only on proteins from the pathogen that could be easily administered in animal feed has the potential to greatly reduce losses to this disease. Existing research has shown that several proteins from Eimeria separately show some protection as candidate vaccines. This project will extend this work by combining these three proteins, as the simultaneous use of multiple proteins is likely to have a greater protective effect, but a simple and cost effective method of production and administration would facilitate development of an effective vaccine.Plant systems can supply large amounts of vaccine proteins with minimal equipment and cost compared to other systems. Production of various candidates in plants results in an immune response and confers protection from infection. A corn-based system has emerged as a viable platform for production that can overcome barriers to commercialization, and vaccines in minimally processed corn grain are heat-stable and suitable for direct feeding. The purpose of this project will be to prepare transgenic corn lines producing three key proteins from Eimeria as a candidate vaccine for chickens. The proteins will be produced together specifically in the kernels of one corn line. Ground meal from seed containing these proteins will be fed to chickens to assess immune response and protection from infection. Successful completion of the work will lead to future development of a low-cost heat-stable effective oral vaccine for avian coccidiosis that should reduce reliance on antibiotics and losses to this disease. Commercialization directed to major poultry producers will be undertaken with our partner Mazen Animal Health.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Goals / Objectives
Avian coccidiosis is a highly communicable disease that causes poor nutrient absorption, reduced growth, and sometimes death in many farmed and domestic animals. There are three circulating strains which cause the majority of infections, Eimeria acervulina, Eimeria tenella, and Eimeria maxima. The disease is currently controlled by a combination of drug treatment and vaccines based on Eimeria oocysts, but new vaccines are needed to help combat drug resistance.Purpose: To bioencapsulate Eimeria antigens in maize grain which, when delivered orally, will lead to a more efficacious vaccine with a practical method of delivery. This vaccine will protect vulnerable poultry from this highly transmissible and destructive infection.Goal: The goal for this project is to produce, optimize, and characterize maize lines expressing the highest levels of 3-1e, EF-1 alpha, and Gam82 to induce an immune response to Eimeria acervulina, Eimeria tenella, and Eimeria maxima, respectively.The specific objectives are to:1. Optimize the codon sequence of the three antigens for expression in maize.2. Design a construct containing all the three antigens encoded in three transcription units for expression in maize.3. Produce two variations of the construct using different intracellular targeting locations.4. Transform maize, obtain at least 10 independent transformants for each construct, and analyze transformants for expression of all three antigens.5. Analyze antigens from transformants by gel electrophoresis and western blotting to ensure integrity of the antigens.6. Identify and propagate transformants containing the highest level of expression of the antigens.A secondary goal, conducted using matching commercial partner funding, is to demonstrate that bioencapsulated Eimeria antigens in maize grain when delivered orally will lead to a more efficacious vaccine with a practical method of delivery. Our eventual commercial target for this potential product is a vaccine that will provide >85% protection in chickens against an Eimeria spp. challenge.The primary goal of this project will be accomplished in a 6-month time frame using 0.6 FTE of scientists and technical staff.
Project Methods
Methods used in this project:1. Molecular biology: Standard molecular biology technology will be used for synthesizing DNA sequences with optimized maize codon usage, construction of plasmids incorporating all three optimized antigen transcription units. Two versions of the constructs will be made, targeting the antigens to different intracellular locations.2. Transformation and propagation of maize: The constructs will be transformed into Agrobacterium. These Agrobacterium will be used to transform maize by standard tri-parental mating, with a target of 10 independent transformation events for each construct. The transformed maize will be selected and grown by standard methods.3. Biochemistry: Maize expression of the antigens will be assessed using standard biochemistry procedures. The levels of antigen will be estimated by using antibodies specific for the antigens in western blotting. This will allow both the levels and the integrity of the antigens to be evaluated. Initially four seed pools from six plants from each independent transformation event will be tested to assess relative levels of expression with a target of at least 0.2% tsp for each antigen.4. Immunology: The immunogenicity of the antigens will be established by feeding maize grain and then evaluating the immune response in chickens using well-established assays used by our Agricultural Research Service collaborators. This step will be funded by an outside partner.