Location: Animal Biosciences & Biotechnology Laboratory
2024 Annual Report
Objectives
Objective 1: Develop immunological tools to evaluate avian immunity.
Sub-objective 1.a. The development of immunological assays and validation of their uses for assessing host immune responses to infectious diseases and vaccination
Sub-objective 1.b. Development of multiplex immunoassays for the simultaneous detection of inflammatory cytokines and chemokines as screening tools to evaluate the efficacy of alternatives to antibiotics feed additives
Objective 2: Develop alternatives to antibiotics for preventing enteric diseases of poultry
Sub-objective 2.a. Discover vaccine platforms that could reduce the use of antibiotics in poultry production and study their modes of action using coccidiosis and necrotic enteritis disease models
Sub-objective 2.a.1. Development of Bacillus subtilis spore-vectored recombinant vaccines against necrotic enteritis using B. subtilis spore surface display technology
Sub-objective 2.a.2. In ovo delivery of recombinant protein vaccines against necrotic enteritis
Sub-objective 2.a.3. Coccidiosis recombinant vaccines delivered with T cell-promoting adjuvants
Sub-objective 2.a.4. Coccidiosis recombinant vaccines, cytokine adjuvants, and Bacillus subtilis spores carrying a chicken antimicrobial peptide, cNK2
Sub-objective 2.b. Develop multi-faceted non-antibiotic approaches for preventing priority enteric diseases of poultry and study their modes of action
Sub-objective 2.c. Determine intestinal metabolites that enhance host immunity, gut health, and growth performance as feed additives and develop antibiotic-free postbiotic strategy to reduce the need for antibiotics in poultry production.
Approach
Develop novel antibiotic alternative strategies to countermeasures against highly resistance pathogens, Eimeria and Clostridium perfringens, to achieve resiliency in antimicrobial resistance challenges in animal agriculture. Develop critical immune reagents(genes, recombinant cytokines, mAbs)and immunoassays for deciphering host Th1, Th2, Th17 and Treg immune responses to better understand host-pathogen interaction in avian coccidiosis and necrotic enteritis. Develop novel strategies such as vaccines and antibiotic alternatives to beneficially modulate innate host response. Identify potential biomarkers of gut health and assess the levels of gut health biomarkers in vivo.
Progress Report
This is the third annual report for the project of 8042-32000-115-000D which started in October 2021. Progress was made on both Objectives and their Sub-objectives, all of which fall under National Program 103, Component 2, Antimicrobial Resistance Problem Statement 2A: Combat the antimicrobial resistance through the development of Alternatives to Antibiotics.
Under Objective 1, significant progress has been made in developing critical poultry immune reagents to assess innate and adaptive immune responses in both normal and disease states. Major chicken cytokine and chemokine genes, which mediate the host immune response, have been cloned, and recombinant proteins have been expressed as immunogens for monoclonal antibody development. Additionally, antigen capture sandwich ELISA assays have been developed to measure these poultry cytokines and chemokines specifically. Given that cytokines and chemokines are key mediators of host immune responses, these newly available immune reagents and poultry-specific immunoassays will enhance our understanding of how poultry respond to pathogens, including parasites, bacteria, and fungi. Furthermore, the poultry-specific immune reagents developed through this project have been commercialized, addressing a critical gap in immunological reagents for both basic and applied poultry immunology research. This commercialization will facilitate the development of novel strategies to reduce antibiotic use in commercial poultry production.
Under Objective 2, significant progress has been made towards finding effective alternatives to antibiotics for combating coccidiosis and necrotic enteritis in poultry. This includes the development of innovative strategies such as Bacillus subtilis-vectored oral vaccines to protect against infections from coccidiosis and necrotic enteritis. Several novel non-antibiotic strategies to mitigate coccidiosis and necrotic enteritis have been developed. For example, the identification of gut-derived metabolites that provide beneficial effects on the host to mitigate the negative effects of parasitic and bacterial infections has been identified. Oral administration of dietary carnosine supplementation induced beneficial effects on intestinal immune responses and gut barrier function in broiler chickens exposed to coccidiosis. Additionally, Bacillus amyloliquefaciens, a subvariant of B. subtilis known for inhibiting the growth of pathogenic C. perfringens, has been characterized by in vitro genetic mining of anti-C. perfringens activities. Furthermore, the multi-epitopes for toxins and virulence factors from C. perfringens have been constructed and refined to promote immune interaction with CD4+ helper T cells, CD8+ Cytotoxic T cells and B cells through bioinformatic tools. These advancements hold promise for the development of commercial feed additives that could potentially reduce or replace the use of antibiotics, thereby promoting animal welfare and enhancing production standards.
Progress on chicken immune reagent development. Several mouse hybridomas secreting monoclonal antibodies specifically detecting chicken-specific IL-12 and IL-23 cytokines have been characterized. The specificity and significance of 2 newly developed antigen-capture immunoassays for chIL-12 and chIL-23 were validated which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These monoclonal antibodies for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.
Progress on the molecular characterization of recombinant Bacillus subtilis-vectored vaccines for C. perfringens Six recombinant Bacillus subtilis-vectored vaccines against C. perfringens have been constructed and characterized in both spore-surface display and plasmid-secretion systems. These targets represented 5 virulence factors, including NetB, alpha-toxin, including a fusion of NetB and alpha-toxin, fructose-1,6-bisphosphate aldolase (FBA) and a zinc metalloprotease (Zm), collagen adhesion serving (CNA). The efficacy of these recombinant vaccine candidates for necrotic enteritis (NE) prevention will be evaluated using our animal NE disease challenge model.
Progress on alternatives to antibiotic strategies by construction and refinement of multiepitope vaccine candidates using 5 virulence factors of C. perfringens. Necrotic enteritis (NE) is a multifactorial enteric infectious disease primarily caused by pathogenic C. perfringens infection, which inflicts significant economic losses estimated at around 6 billion annually on the global poultry industry. The escalating incidence of NE has been linked to the voluntary reduction or elimination of antibiotic growth promoters from animal feed in recent years. Consequently, the development of effective vaccines targeting NE has become a top priority for the poultry sector. Clostridium perfringens, a Gram-positive bacterium, poses a substantial threat to both human and animal health, with alpha-toxin and NetB, fructose-1,6-bisphosphate aldolase (FBA) and a zinc metalloprotease (Zm), collagen adhesion serving (CNA), as critical virulence factors in NE pathogenesis among commercial poultry. Focusing on these factors as prime targets for vaccination represents a promising strategy to mitigate the impact of C. perfringens. A comprehensive strategy for constructing and refining multi-epitope vaccines against 5 factors, harnessing CD4+ helper T cell, CD8+ cytotoxic T lymphocyte (CTL), and B cell epitopes, was developed. Through rigorous utilization of bioinformatics tools and algorithms, potential epitopes were identified, optimized and selected to bolster immunogenicity and antigenicity, and avoid allergenicity. Condensed peptides representation encompassing most of these epitopes have been devised. These multi-epitope vaccine candidates confer substantial promise in eliciting protecting immunity against C. perfringens/NE infections.
Progress on alternatives to antibiotic strategies using novel phytochemical combinations ARS scientists and scientists from a commercial company have identified a phytochemical combination that mitigates coccidiosis response and reduces gut damage. A beneficial combination of three phytochemicals that enhanced host immunity was identified using in vitro screening system with several different chicken cell lines and unique biomarker combinations associated with different cell functions. Both in vitro and in vivo studies confirmed the beneficial effects of green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In vivo studies confirmed that chickens fed with a diet that is supplemented by this unique phytochemical mixture showed enhanced growth performance and reduced disease severity following coccidiosis challenge infection. Animal feeding trials using the combination of GT, CO, and PO in broiler chickens infected with Eimeria maxima showed enhanced disease resistance including innate immunity and gut health, which contributed to improved growth and reduced disease responses. These findings will provide the scientific rationale to develop a science-based antibiotic-independent strategy to mitigate coccidiosis response in poultry production to reduce the economic cost associated with commercial broiler production.
Development of recombinant vaccine to protect against coccidiosis in commercial broiler chickens. P recombinant vaccination strategy against coccidiosis based on Bacillus subtilus delivery system composed of the immunodominant antigen of Eimeria, 3-1E protein, which is the Apicomplexa profilin has been made. In vivo trials, Bacillus-3-1E immunization by oral administration induced an enhanced protective immunity against E. maxima infection compared to the rEF-1a alone. Additionally, EF-1a vaccination of newly hatched broiler chickens protected them with a significant level of protective immunity against E. maxim and with reduced fecal oocyst output. Furthermore, profilin-vaccinated chickens showed mitigated pro-inflammatory cytokine profiles in the gut, where parasites underwent intracellular development following E. maxima challenge infection, compared to non-immunized chickens.
Commercial licensing of the first novel hyperimmune egg yolk IgY antibodies protecting against coccidiosis and necrotic enteritis. Coccidiosis and necrotic enteritis cause major economic losses to commercial poultry with a combined economic loss of over $19 billion annually for global poultry industry. ARS scientists in collaboration with the Arkion Life Sciences commercial partner successfully developed hyperimmune egg yolk antibodies which were generated against critical protective epitopes of Eimeria and Clostridium perfringens and demonstrated significant protection against challenge infections with coccidiosis and necrotic enteritis. This is the first demonstration that passive immunization of egg yolk antibodies was successfully used therapeutically to mitigate coccidiosis and necrotic enteritis, two major intestinal infections which cost industry more than $19 billion combined annual losses worldwide.
Accomplishments
1. Commercialization of immunodiagnostic mouse monoclonal antibodies detecting chicken Colony Stimulating Factor 1. Understanding poultry immunity is crucial for developing new disease intervention strategies. USDA-ARS researchers in Beltsville, Maryland, have developed three cell lines (hybridomas) that secrete mouse monoclonal antibodies (mAbs) targeting the chicken immune molecule Colony Stimulating Factor 1 (CSF1). CSF1 is a key mediator in the early immune response to pathogens, such as parasites and viruses, and its blood levels can predict the quality of host immune responses. Using these new mAbs, USDA-ARS researchers in Beltsville, Maryland, developed a novel immunodiagnostic assay. This assay enables the quantitative measurement of CSF1 in biological samples from numerous diseased chickens on commercial farms. The hybridoma cell lines producing CSF1 fill a critical gap in immunological reagents for basic poultry immunology research and they have been patented and licensed. Their commercialization will enhance both basic and applied research, improving our understanding of poultry immune responses to pathogens and vaccines. Moreover, these immune reagents will facilitate the development of immunity-based strategies to reduce antibiotic use in commercial poultry production.
2. Development of subunit-specific mouse monoclonal antibodies detecting chicken interleukin 12 and 23. There is a need to develop various immunodiagnostic tools for poultry that can be used to assess host immune response to various infectious agents. Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of immune factors that play crucial roles in both protective host immune response against intracellular parasites and mediating inflammatory responses. USDA-ARS researchers in Beltsville, Maryland, developed sets of mouse antibodies that can identify and distinguish between IL-12 and IL-23 cytokines. Using these antibodies, they can now monitor the changes in IL-12 and IL-23 cytokine levels in the serum of chickens infected with Eimeria parasites which cause major damage in the gut. These valuable immune reagents are expected to provide insights into the function of IL-12 and IL-23 in inflammatory responses and disease pathogenesis in poultry.
3. Changes in gut microbiota populations in chickens orally treated with B. subtilis spores carrying chicken NK lysin 2 (B. subtilis/cNK-2). Antimicrobial peptides (AMPs) are natural defense proteins effective against a range of microorganisms. These molecules, abundant in hydrophobic cationic residues, disrupt bacterial membranes. USDA-ARS researchers in Beltsville, Maryland, developed a novel oral delivery system using the spores of the bacterial strain Bacillus subtilis that produce a chicken protein (NK-lysin peptide) which kills Eimeria pathogens. Recent trials showed that chickens receiving B. subtilis-cNK-2 spores showed improved growth, immunity, gut health, and changes in gut bacteria populations following challenge infection with Eimeria, parasites which damages the intestine of broiler chickens. Although further studies are needed to better understand how the changes in the chicken gut bacterial population due to B. subtilis-cNK-2 treatment can enhance broiler performance, the documented beneficial effects of B. subtilis-cNK-2 in these studies indicate potential application of Bacillus bacteria chicken antimicrobial peptide as an antibiotic alternative to improve gut health and reduce gut damage caused by avian coccidiosis, a common intestinal parasitic infection in commercial chickens.
4. Use of selected plant extracts in controlling and neutralizing toxins and sporozoites associated with necrotic enteritis (NE) and coccidiosis. Due to concerns about contamination of animal food products with antibiotic-resistant bacteria, plant-derived feed additives have been explored as antibiotic alternative feed additives in broiler chickens. USDA-ARS scientists in Beltsville, Maryland, in collaboration with North Carolina Agricultural and Technical State University, investigated the effects of extracts from various plant sources such as ginger root extract, green tea extract, and a combination of onion peel extracts on bacteria (Clostridium perfringens) and parasites (Eimeria tenella) that cause intestinal diseases in chickens. Beneficial combinations of various phytochemicals were shown to enhance host immunity using an in vitro screening system with three different chicken cell lines, and also in live populations of chickens. These novel findings provide a scientific basis for developing antibiotic-independent strategies to mitigate intestinal diseases in poultry, reducing the economic costs associated with commercial broiler production.
5. The effect of gut microbiota-derived metabolite on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima. In the post-antibiotic era, developing novel non-antibiotic feed additives have become increasingly important for controlling poultry diseases and improving the intestinal health and growth performance of commercial poultry. While changes in microbial populations can provide information on changes in intestinal bacterial populations, analysis of the by-products of host metabolism reveals the actual alterations in metabolic pathways that exert beneficial effects on gut function. Carnosine, is a gut metabolite which is widely distributed in various tissues of chickens that has an important role in cellular functions and may play a role in mediating the immune response during inflammatory processes. USDA-ARS researchers in Beltsville, Maryland, showed beneficial effects of gut metabolite, called carnosine on disease parameters in broiler chickens challenged with E. maxima parasites. In vivo feeding studies showed that dietary carnosine increased the performance parameters of E. maxima-infected chickens. Studies indicated that carnosine exhibited anti-inflammatory and antioxidant effects in chickens and improved host immunity and gut integrity. These findings highlight the beneficial effects of dietary carnosine on intestinal immune responses and gut health in broiler chickens exposed to E. maxima parasitic infection.
6. In vitro and genomic mining to identify anti-Clostridium perfringens compounds in Bacillus amyloliquefaciens. The ban on in-feed antibiotic growth promoters has driven the search for alternatives to destroy Clostridium perfringens. Pathogenic strains of Clostridium perfringens bacteria cause a devastating intestinal infection called necrotic enteritis (NE) which is characterized by dead tissue patches in the intestine of commercial broiler chickens. There has been increasing incidence of NE in commercial broiler farms associated with the antibiotic ban in poultry production worldwide. ARS Researchers in Beltsville, Maryland, identified two novel bacterial strains of Bacillus amyloliquefaciens which secrete antimicrobial compounds which can kill toxin-producing strains of Clostridium perfringens. Genetic analysis revealed that the Bacillus amyloliquefaciens strains effective against C. perfringens were similar to each other in genetic pattern. Additionally, seven potential antimicrobial compounds, likely secondary metabolites, were identified using specific software which was used to analyze the genomes of B. amyloliquefaciens species. Based on these results, beneficial Bacillus amyloliquefaciens strain, BaD747 strain, was identified as a probiotic bacterium that produces a toxic compound against Clostridium perfringens. These findings indicate a possibility of using these anti-microbial compounds to kill toxin-producing C. perfringens to mitigate NE in commercial broiler farms instead of antibiotics.
Review Publications
Goo, D., Gadde, U., Kim, W., Gay, C.G., Portia, E., Jones, S., Walker, S., Lillehoj, H.S. 2024. Novel hyperimmune egg yolk IgY antibodies developed against protective antigens of Eimeria and Clostridium perfringens protect against necrotic enteritis. Poultry Science. 102. Article e102841. https://doi.org/10.1016/j.psj.2023.102841.
Khan, M., Lillehoj, H.S., Lee, Y.H., Adetunji, A., Omaliko, P.C., Kang, H., Nagabhushanam, K., Fasina, Y.O. 2024. Use of selected plant extracts in controlling and neutralizing toxins and sporozoites associated with Necrotic Enteritis and Coccidiosis. Applied Sciences. V.14/3178. https://doi.org/10.3390/app14083178.
Lee, Y., Kim, W., Nam, H., Lillehoj, H.S. 2024. Differential detection of chicken heterodimeric cytokines, interleukin 12 and 23 using their subunit-specific mouse monoclonal antibodies. Poultry Science. VOL.103/ 103872. https://doi.org/10.1016/j.psj.2024.103872.
Li, C.Z., Yan, X., Yang, Y., Nou, X., Sun, Z., Lillehoj, H.S., Lu, M., Harlow, K., Rivera-Colon, I. 2024. An integrated systematic study for identification of antimicrobial compounds from Bacillus amyloliquefaciences. Poultry Science. 103. Article e103871. https://doi.org/10.1016/j.psj.2024.103871.
Truong, A., Tran, H., Chu, N., Nguyen, H., Phan, L., Phan, H., Vu, T., Song, K., Lillehoj, H.S., Hong, Y. 2023. Comprehensive genome-wide analysis of the chicken heat shock protein family: identification, genomic organization, and expression profiles in indigenous chicken with highly pathogenic avian influenza infection. BMC Genomics. vol.24/793. https://doi.org/10.1186/s12864-023-09908-y.
Vu, T., Heo, J., Kang, S., Kim, C., Lillehoj, H.S., Hon, Y. 2023. Chicken miR-26a-5p modulates MDA5 during highly pathogenic avian influenza virus infection. Developmental and Comparative Immunology. Vol.149/104921. https://doi.org/10.1016/j.dci.2023.104921.