1a. Objectives (from AD-416):
Objective 1. Develop and evaluate potential alternatives to antimicrobials and other intervention products and strategies to control and reduce foodborne pathogens in poultry and swine. Sub-objective 1.a. Select, chemically synthesize and screen antimicrobial peptides (AMP) for ability to kill Campylobacter spp. in vitro. Sub-objective 1.b. Couple the gene for expression of the selected AMP to the gene encoding a well-defined Campylobacter bacteriophage receptor binding protein, Gp047 (short derivative), and express the constructs in yeast. Sub-objective 1.c. Evaluate in vitro and in vivo efficacy of the most active AMP or AMP-receptor binding protein (RBP) products expressed in yeast (purified or as yeast lysate) and, as necessary, develop encapsulation procedures for enhanced stability, site-directed delivery, efficacy and storage of the protein. Sub-objective 1.d. Conduct Campylobacter challenge trials in broiler chickens to determine the ability of oral administration of AMP or AMP-RBP products to reduce Campylobacter colonization and utilize 16S rRNA sequencing to determine the effect of the peptide products on the overall cecal microbiota. Objective 2. Develop, validate and determine the efficacy of a multi-serotype and multi-subunit cross specific vaccine for use in controlling Campylobacter and Salmonella. Sub-objective 2.a. Identify epitopes of the pathogens from peptide microarrays using chicken serum samples from the field. Sub-objective 2.b. Construction and expression of the epitope containing genes in an Escherichia (E.) coli expression system and purification of the recombinant proteins. Sub-objective 2.c. Assay of the immune response in broilers to these recombinant proteins. Sub-objective 2.d. Examination of populations of bacteria in chicken gastrointestinal tract (GIT) after vaccination to determine the effects of vaccines on the microbiota of the GIT.
1b. Approach (from AD-416):
Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) Develop and evaluate antimicrobial peptides (AMP) as potential alternatives to current antibiotics to control and reduce foodborne pathogens in poultry, and 2) Develop, validate and determine the efficacy of a multi-serotype and multi-subunit cross specific vaccine for use in controlling Campylobacter and Salmonella. Specifically, we will select, chemically synthesize, and screen a panel of natural and synthetic AMP for ability to kill Campylobacter spp. in vitro. The genes for expression of the most effective AMP will be coupled to the genes encoding a well-defined bacteriophage receptor binding protein (RBP) to enhance specificity of the AMP for Campylobacters and the AMP-RBP construct will be expressed in a yeast for enhanced production of the protein for evaluation of efficacy. Encapsulation protocols will also be developed for enhanced stability, storage and site-directed delivery of the expressed AMP-RBP product and subsequent Campylobacter colonization challenge trials will be conducted in chicken and swine to evaluate the efficacy of the treatment and determine its overall impact on the gastrointestinal tract (GIT) microbiota. In our second approach (vaccine development) we will identify specific epitopes of the pathogens from peptide microarrays using serum samples from mature commercial chickens. We will then construct and express the epitope containing genes in an Escherichia coli expression system, purify the recombinant proteins, and assay the immune response in broilers to the recombinant proteins. Finally, we will examine the populations of bacteria in the chicken GIT after vaccination to determine the effects of the vaccines on the microbiota.
3. Progress Report:
Progress was made on the development of antimicrobial peptides and poultry vaccines. Under Objective 1.a, 11 unique antimicrobial peptides were synthesized and evaluated for the ability to inhibit growth of 2 strains of Campylobacter (C.) jejuni. Six of the peptides inhibited C. jejuni, and 3 of these peptides were chosen for further investigation because of reduced toxicity to mammalian cells. The peptides were tested against 19 bacteria, and minimum inhibitory concentrations of these peptides against three strains of C. jejuni were determined. Additional peptides have been synthesized and are currently being evaluated. Under Objective 2.a, serum samples from commercial broilers raised in the laboratory from one to six weeks of age were collected and assessed broiler humoral immune response to 330,000 random sequence peptides. Sera from 1 and 2 week-old broilers had the lowest reactivity against the peptides, and sera from 3 week-old broilers had the earliest strong signal. Signals got stronger as broilers matured. Further, ten peptide motifs were identified to have the strongest reaction to the sera, while six other peptide motifs showed decreases in reaction as broilers aged. Also, proteins from the fimbriae of Salmonella were examined for potential for use as antigens for vaccination to reduce Salmonella in the broiler gastrointestinal tract. Bioinformatic searches were carried out to identify the fimbrial genes from the Salmonella genomes, and genes involved in fimbrial proteins were identified. About 15 recombinant fimbrial proteins were cloned, purified and confirmed. Finally, in a preliminary experiment, 7 proteins were used to immunize broilers which were challenged with the same Salmonella serotype. The results indicated that no Salmonella was isolated from the immunized group, while Salmonella were detected in the broiler cecal contents.
1. Use of mass spectrometry-based proteomics in conjunction with chicken sera to map the epitope of the Salmonella enterica FlgK protein. ARS researchers in Athens, Georgia, applied immunoprecipitation and mass spectrometry to identify the immunodominant epitopes in FlgK. These epitopes were also recognized by sera collected from the field. These peptides provide a rational for further evaluation of whether they are able to be utilized as subunit vaccines for chicken immunization. Subsequently, safer poultry products will be provided for human consumption.
2. Use of peptide microarrays to assess broiler humoral immune activity. ARS researchers in Athens, Georgia, collaborated with researchers from Arizona State University in Tempe, Arizona, to assess broiler immune response to 330,000 peptides from broilers aged 1 to 6 weeks. Ten peptides produced a strong reaction and six peptides produced a decreased reaction. These results provide a rationale for further evaluating whether this test can be used for disease diagnostics and/or vaccination monitoring.
Yeh, H., Serrand, K.V., Silvestry, A., Buhr, R.J. 2016. Production of recombinant Salmonella flagellar protein, FlgK, and its uses in detection of anti-Salmonella antibodies in chickens by automated capillary immunoassay. Journal of Microbiological Methods. 122:(3)27-32.