Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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. 04 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. 05 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. 06 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.
Impacts
(N/A)
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.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This is the second 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, progress has been made in the development of critical poultry immune reagents that can be used to assess innate and adaptive immune responses of poultry in normal and disease states. Several major chicken cytokine and chemokine genes that mediate host immune response in poultry have been cloned, recombinant proteins have been expressed as immunogens for monoclonal antibody development, and antigen capture sandwich ELISA assays to specifically measure poultry cytokines and chemokines have been developed. Because cytokines and chemokines are major mediators of host innate and adaptive immunities, the availability of these immune reagents and poultry-specific immunoassays will enhance progress in our understanding of poultry immune responses to pathogens including parasites, bacteria, and fungus. Furthermore, these poultry-specific immune reagents developed in this project have been commercialized to address the critical immunological reagent gap for basic and applied immunology research in poultry species to facilitate the development of novel strategies to reduce the use of antibiotics in commercial poultry production. Under Objective 2, significant progress has been made in the development of antigen-specific sandwich ELISA assays for the detection of critical antigens of Eimeria parasites and C. perfringens including NetB toxin and collagen adhesion protein (CNA). Significant progress has also been made in the development of several effective antibiotic alternative strategies against coccidiosis and necrotic enteritis, such as Bacillus subtilis-vectored products, and the recombinant chimeric proteins for subunit vaccines in protection against the Eimeria spp. and C. perfringens challenge infections. B. subtilis has been genetically engineered to carry major immunodominant genes of Eimeria spp., and the expression of parasite proteins has been verified. Bacillus amyloliquefaciens, a subvariant of B. subtilis which inhibits the growth of pathogenic C. perfringens, was genetically engineered to carry C. perfringens genes. This progress may eventually lead to commercializable feed additives that will replace antibiotics to promote food animal welfare and production. Progress on chicken immune reagent development: Several mouse hybridomas secreting monoclonal antibodies specifically detecting chicken-specific cytokines have been characterized and quantitative antigen capture ELISA assays were used to assess host immune response to coccidiosis and Necrotic enteritis (NE) infections in serum, gut secretions and in other biological fluids. The availability of quantitative immunoassays for the chicken cytokines via commercialization will facilitate further understanding of host-pathogen immunobiology of coccidiosis and NE and allow investigation of feed additives and vaccines that will replace antibiotics. Collectively, novel results from in vitro and in vivo studies will enhance our understanding of coccidiosis and NE pathogenesis and will facilitate the development of antibiotic alternative strategies against coccidiosis and NE in commercial poultry. Progress on the development of diagnostic tests for avian coccidiosis. A major immunodominant antigen of Eimeria apicomplexan parasites, profilin, was produced in E. coli, and several mouse monoclonal antibodies detecting profilin were developed. Using these antibodies to 3-1E, a novel diagnostic antigen-capture immunoassay was developed for large- scale screening, and its specificity was validated using serum samples from coccidia-infected chickens of field poultry farms. A better understanding of the host immune response to 3-1E immunodominant antigen of Eimeria parasites will enhance the development of effective vaccines and antibiotic alternative feed additives that will replace antibiotics. Detection of major antigens associated with the pathogenesis of NE. Two major antigens of C. perfringens which are Immunodominant and are being recognized by NE-afflicted chickens have been identified and mouse monoclonal antibodies have been developed against these antigens. Several mouse monoclonal antibodies which specifically detect NetB and CNA antigens of C. perfringens were cloned, and their proteins were expressed to develop mouse monoclonal antibodies. Antigen-specific immunoassays then validated using serum and fecal samples to quantitate the levels of NetB and CNA antigens and antibodies against them in C. perfringens- infected chickens. These assays are the first immunoassays that make it possible to quantitate the two toxin/virulence-associated factors of C. perfringens in biological fluids and will allow early detection of NE outbreaks in commercial broiler farms to minimize economic losses due to NE. Using field samples from a commercial company, the specificity, and validity of these immunoassays to quantitate the two toxin/virulence- associated factors of C. perfringens in biological fluids were confirmed. These immunoassays will allow an early detection of large-scale screening for the NE disease outbreak in broiler farms to minimize economic losses due to NE. Progress on the molecular characterization and detection for major proteins of C. perfringens: New sandwich immunoassay (ELISA) was developed using mouse monoclonal antibodies specific for the detection of two critical antigens: necrotic enteritis B-like toxin (NetB) and collagen adhesin protein (CNA) of pathogenic C. perfringens in infected chickens in the jejunal digesta samples from commercial broiler farms in the United States. The CNA and NetB levels showed a positive correlation with each other, and CNA and NetB levels in jejunal digesta were significantly decreased in certified-organics-used farms compared with those from non-antibiotic farms. These new C. perfringens antigen- specific sandwich ELISAs offer a sensitive and specific means to detect C. perfringens CNA and NetB proteins as biomarkers of early NE occurrence in field samples from commercial broiler chickens. Progress on alternatives to antibiotic strategies using novel phytochemical combinations. ARS scientists and a collaborating 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 using three different chicken cell lines using 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 with this unique phytochemical mixture showed enhanced growth performance and reduced disease severity following coccidiosis. In conclusion, the combination of GT, CO, and PO in the diet of broiler chickens infected with Eimeria maxima induced enhanced the host 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. Progress on alternatives to antibiotic strategies using B subtilis and its subvariant-vectored vaccines: B. subtilis subvariant Bacillus amyloliquefaciens (B. amylo) has been used as a probiotic in the poultry industry. B. amyloliquefaciens was shown to inhibit the growth of pathogenic Listeria monocytogenes and C. perfringens and is a potential candidate as a mucosal vaccine vector to deliver vaccines encoding bacterial virulence factors against enteric infections such as coccidiosis and necrotic enteritis, which are two leading enteric diseases responsible for substantial economic loss of more than $ 20 billion in the global poultry industry. Significant progress was made in bioengineering recombinant B. amylo strain carrying immunogenic antigens of C. perfringens such as the fructose-1,6-bisphosphate aldolase (FBA) gene. Developing an effective and practical oral delivery strategy to express vaccine antigens of Eimeria and C. perfringens will effectively reduce the gut damage and reduces economic losses resulting from these enteric infections. Development of recombinant vaccine to protect against coccidiosis in commercial broiler chickens. Progress in recombinant vaccination strategy against coccidiosis based on recombinant vaccine mixture composed of the immunodominant antigen of Eimeria, T-cell promoting chicken immune- modulating cytokine and chicken antimicrobial peptide have been formulated. In in vivo trials, recombinant rEF-1a vaccination with rchIL- 7 or simultaneous oral administration of cNK-2 induced an enhanced protective immunity against E. maxima infection compared to the rEF-1a vaccination alone. Furthermore, 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, EF-1a-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. ACCOMPLISHMENTS 01 Commercialization of mouse monoclonal antibodies detecting chicken immune molecules. Ten hybridoma cell lines that secrete mouse monoclonal antibodies detecting chicken chemokine Tumor Necrosis Factor- alpha (TNF-apha) have been commercialized by ARS scientists in Beltsville, Maryland. These mouse monoclonal antibodies detect poultry- specific TNF-alpha cytokines which are involved in the early initiation phase of host immune responses to pathogens such as parasites and viruses, and their levels in the blood provide predictive values in determining the quality of host immune responses. These cell lines address the critical immunological reagent gap for basic immunology research in poultry species, and the commercialization of these immune reagents will enhance basic and applied research to facilitate our understanding of host immune responses to pathogens and vaccines in poultry. Furthermore, these immune reagents will facilitate the development of effective immunity-based strategies to reduce antibiotics in commercial poultry production. 02 Development of large-scale immunoassay detection kits for biomarkers of necrotic enteritis infection in commercial broiler chickens. Necrotic enteritis (NE) is an enteric bacterial disease caused by Gram-positive toxicogenic Clostridium perfringens (CP) and inflicts more than $6 billion annual economic cost to global poultry industry. Antibiotic- free production of commercial poultry has been associated with increasing incidence of NE, with many current commercial poultry productions becoming more dependent on using antibiotic-alternative feed additives. ARS scientists in Beltsville, Maryland, and collaborators at the University of Georgia developed ELISAs to detect Collagen Adhesion Antigen and NetB toxin proteins which are two major virulence factors involved in NE pathogenesis for early detection of NE outbreaks in susceptible flocks in commercial broiler farms. Immunoassays were based on two novel mouse monoclonal antibodies which detect specific biomarkers associated with CP. These assays showed that pathogenic strains of CP can be identified in fecal samples from commercial farms within 1-2 weeks post-hatch allowing timely intervention of NE outbreak in the poultry farms. The use of these antigen-capture sandwich ELISAs will facilitate early detection of NE- associated biomarkers in disease susceptible poultry populations in commercial farms enhancing effective management of NE in commercial poultry production. 03 Development of orally deliverable Bacillus subtilis spores carrying chicken NK-2 antimicrobial peptide to protect against coccidiosis and to stabilize gut microbiota in commercial broiler chickens. Avian Coccidiosis, a ubiquitous intestinal disease caused by several distinct Eimeria species, inflicts major economic losses to global poultry industry due to intestinal damages including inflammation, bloody lesions, high morbidity and mortality, and poor nutrition absorption. Due to intensifying public/legislative pressure to reduce the use of antibiotics in poultry production and to develop safe alternatives to reduce the economic losses due to coccidiosis, ARS scientists in Beltsville, Maryland, in collaboration with a biotechnology company, developed a stable Bacillus subtilis spores carrying NK-lysin antimicrobial peptide (B. subtilis-cNK-2) as an effective oral therapeutic against Eimeria parasites in broiler chickens. In vivo trial showed that a higher dose oral B. subtilis-cNK-2 treatment improved disease protection and intestinal health. Therefore B. subtilis-cNK2 spores exerted protective effects against coccidiosis by reducing fecal oocyst shedding, enhancing local protective immunity, and restoring t gut microbiota homeostasis. 04 Dietary phytochemical mitigates coccidiosis infection in commercial broiler chickens. With increasing governmental restriction of growth promoting antibiotics in commercial poultry production, there is a timely need to develop novel feed additives to enhance gut health and to mitigate negative disease impacts of coccidiosis to increase animal resilience on animal agriculture. ARS scientists in Beltsville, Maryland, and a collaborating 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 based on three different chicken cell lines using unique biomarker combination associated with different cell functions. Both in vitro and in vivo studies confirmed the beneficial effects of three phytochemicals, green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In vivo studies confirmed that chickens fed a diet supplemented with this unique phytochemical mixture showed enhanced growth performance and reduced disease response following coccidiosis. These findings provide 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. 05 Development of a new immunodiagnostic tool for poultry coccidiosis. Avian coccidiosis, an intestinal disease caused by parasites of the genus Eimeria is an economically important disease for the poultry industry worldwide and costs more than $ 13 billion in annual losses. Currently, seven distinct species of Eimeria are known to infect different intestinal sites. There is a timely need for sensitive detection methods for early diagnosis of coccidiosis in commercial poultry production systems for more effective disease management to reduce economic losses associated with coccidiosis. ARS scientists in Beltsville, Maryland, developed a novel antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E, which is present in all Eimeria species. This assay uses a set of 3-1E-specific mouse monoclonal antibodies which were developed by ARS scientists and provides a sensitive diagnostic tool to monitor coccidiosis in commercial poultry farms before clinical symptoms develop. The availability of a sensitive quantitative immunoassay that can specifically detect coccidiosis-infected chickens in large commercial poultry flocks will reduce the economic cost associated with coccidiosis. 06 Development of orally deliverable recombinant vaccine formula to protect chickens against coccidiosis. Avian Coccidiosis, a ubiquitous intestinal disease caused by several distinct Eimeria species (spp.), is one of the costliest diseases in commercial poultry production worldwide. Several distinct species of Eimeria infect young chickens resulting in more than $ 13 billion in annual economic losses due to intestinal damages including inflammation, high morbidity and mortality, and poor nutrition absorption. Although antibiotics are effective in controlling coccidiosis, intensifying public/legislative pressure reduced the use of antibiotics in poultry production, and there is a timely need to develop safe alternatives to control coccidiosis. ARS scientists in Beltsville, Maryland, developed a recombinant vaccination strategy against coccidiosis using a novel recombinant vaccine mixture composed of immunodominant antigen of Eimeria, T-cell promoting chicken cytokine and chicken antimicrobial peptide which targets Eimeria parasites. In vivo, chicken trial showed that rEF-1a vaccine formulated with chicken IL-7 or simultaneous oral administration of chicken NK-2 induced an enhanced protective immunity against E. maxima infection compared to the rEF-1a vaccination alone. Furthermore, EF-1a-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. More importantly, co-administration of EF-1a vaccine with chIL-7 enhanced protection against coccidiosis and mitigated negative gut response. 07 Dietary organic selenium improves growth performance, gut health, and disease response in broiler chickens. During the early stage of the newly hatched chickens, before they develop full adaptive immunity, the occurrence of enteric diseases such as coccidiosis and necrotic enteritis contributes to high levels of oxidative stress leading to lower antioxidant gene expression in broiler chickens. As an essential element for the antioxidant enzyme system, selenium (SE) is vital for detoxifying lipid peroxide and reactive oxygen species which are generated from oxidative stress response. Coccidiosis is a major broiler disease condition caused by Eimeria protozoan parasites resulting in poor nutrient absorption and hindering growth performance via gut epithelial damage. To further understand the impact and interactions of dietary SE and the Eimeria infection on broiler gut health, ARS scientists in Beltsville, Maryland, assessed many biomarkers associated with gut health, and investigate the effect of dietary organic SE on growth performance in coccidiosis-infected broiler chickens. The results of this study showed improved body weight and confirmed the beneficial effect of dietary SE and the efficiency of organic SE compared with inorganic SE for growth improvement. Specifically, the addition of organic selinized yeast improved growth performance and enhanced SE accumulation in the tissue, and therefore, organic SE supplementation is beneficial to produce SE -enriched organic broiler chickens, which may help provide guidance for poultry nutrition. 08 Characterization of collagen binding activity of Clostridium perfringens strains isolated from commercial broiler chickens. Necrotic enteritis (NE) caused by Clostridium perfringens type A/G is one of the leading enteric diseases which negatively impact the global poultry industry by affecting the growth performance, production cost, animal health, and welfare of poultry. Collagen adhesion antigen (CNA) protein is a major virulence factor involved in the pathogenesis of NE that plays an important role in early bacteria colonization. ARS scientists in Beltsville, Maryland, compared the abilities of collagen binding of several C. perfringens field isolates and showed that the cna gene was detected in high copies in pathogenic isolates carrying netB gene, and the most pathogenic strains exhibited potent binding activities to Collagen Type III. This study showed that the detection of cna gene and its protein product could be valuable factors to determine the virulence and pathogenicity of C. perfringens isolates from poultry farms. 09 Development and characterization of monoclonal antibodies specific for chicken interleukin-7 receptor a (CD127). Chicken cytokine and chemokine genes such as CD127 that mediate host immune responses in poultry have been cloned, recombinant proteins have been expressed as immunogens for monoclonal antibody development, and these monoclonal antibodies have been further used to develop sensitive immunoassays to characterize host immune responses in poultry. Several mouse hybridomas secreting monoclonal antibodies specifically detecting chicken-specific cytokines including CD127 have been developed and characterized by ARS scientists Beltsville, Maryland, to establish antigen capture ELISA assays to specifically measure them. CD127 plays a critical role in the differentiation and activation of T lymphocytes. Availability of these new sets of chCD127- specific monoclonal antibodies will facilitate the immunological studies on CD127 in poultry, especially in understanding effector and memory T immune cell responses in normal and diseased states.
Impacts
(N/A)
Publications
- Wickramasuriya, S.S., Park, I., Lee, Y., Richer, L.R., Przybyszewski, C., Gay, C.G., Van Oosterwijk, J.G., Lillehoj, H.S. 2023. Orally delivered bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens. Poultry Science. https://doi.org/10. 1016/j.psj.2023.102590.
- Lee, Y., Park, I., Wickramasuriya, S., Ben Arous, J., Koziol, M., Lillehoj, H.S. 2022. Co-administration of chicken IL-7 or NK-lysin peptide 2 enhances the efficacy of Eimeria elongation factor-1a vaccination against Eimeria maxima infection in broiler chickens. Poultry Science. 101:102013. https://doi.org/10.1016/j.psj.2022.102013.
- Sun, Z., Lu, M., Lillehoj, H.S., Lee, Y., Doo, D., Yuan, B., Yan, X., Li, C.Z. 2023. Characterization of collagen binding capability of clostridium perfringens isolate collections from broiler chickens . Pathogens. 12:778. https://doi.org/10.3390/pathogens12060778.
- Li, C.Z., Wang, L., Zhang, S. 2023. Editorial; immunosuppressive diseases in poultry. Frontiers in Immunology. 14:1215513. https://doi.org/10.3389/ fimmu.2023.1215513.
- Goo, D., Park, I., Nam, H., Lee, Y., Sawall, J., Smith, X., Li, C.Z., Lillehoj, H.S. 2023. Collagen adhesin protein and necrotic enteritis B- like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens. Poultry Science. https://doi.org/10.1016/j. psj.2023.102647.
- Lee, Y., Park, I.N., Wickramasuriya, S.S., Lillehoj, H.S. 2023. Short communication: Bacillus subtilis expressing chicken NK-2 peptide enhances the efficacy of EF-1a vaccination in Eimeria maxima-challenged broiler chickens. Animals. https://doi.org/10.3390/ani13081383.
- Truong, A., Tran, H., Nguyen, H., Chu, N., Hong, Y., Lillehoj, H.S., Dang, H., Song, K. 2023. Molecular characterization and functional analysis of chicken interleukin 1 receptor 2 (chIL-1R2). Poultry Science. https://doi. org/10.1016/j.psj.2022.102399.
- Lee, Y., Lillehoj, H.S. 2023. Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria. Poultry Science. https://doi.org/10.1016/j.psj.2023.102790.
- Wickramasuriya, S.S., Park, I., Lee, Y., Lillehoj, H.S. 2023. Effect of dietary organic selenium on growth performance, gut health, and coccidiosis response in broiler chickens. Animals. https://doi.org/10.3390/ ani13091560.
- Park, I., Nam, H., Wickramasuria, S.S., Lee, Y., Wall, E.H., Sripathy, R., Lillehoj, H.S. 2023. Beneficial effects of tea extracts, cinnamon oil, and pomegranate on avian coccidiosis. Frontiers in Immunology. https://doi.org/ 10.3389/fimmu.2023.1145367.
- Lee, Y., Park, I., Lillehoj, H.S. 2023. Oral administration of chicken NK- lysin or recombinant chicken IL-7 improves vaccine efficacy of Eimeria tenella Elongation Factor-1a (EF-1a) against coccidiosis in commercial broiler chickens. Poultry Science. 102:102611. https://doi.org/10.1016/j. psj.2023.102611.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This is the first annual report for the project of 8042-32000-115-00D 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, progress has been made in the development of critical poultry immune reagents that can be used to assess innate immune response of poultry in normal and disease states. Several major chicken cytokine and chemokine genes that mediate host immune response in poultry have been cloned, recombinant proteins have been expressed as immunogens for monoclonal antibody development, and antigen capture ELISA assays to specifically measure poultry cytokines and chemokines have been developed. Because cytokines and chemokines are major mediators of host innate and adaptive immunity, the availability of these immune reagents and poultry-specific immunoassays will enhance progress in our understanding of poultry immune response to pathogens including parasites, bacteria, and fungus. Furthermore, these poultry-specific immune reagents developed in this project have been commercialized to address the critical immunological reagent gap for basic and applied immunology research in poultry species to facilitate the development of novel strategies to reduce the use of antibiotics in commercial poultry production. Under Objective 2, significant progresses have been made in the development of antigen-specific sandwich ELISA assays for the detection of critical antigens of C. perfringens including NetB toxin and collagen adhesion protein (CNA). Significant progresses have also been made in the development of several effective antibiotic alternative strategies against coccidiosis and necrotic enteritis, such as Bacillus subtilis-spore surface display, the recombinant chimeric proteins for subunit vaccines and hyperimmune egg yolk antibody production in protection against the pathogenic C. perfringens challenge infection. These progresses may eventually lead to commercializable feed additives that will replace antibiotics to promote food animal welfare and production. Progress on chicken immune reagent development: Several mouse hybridomas secreting monoclonal antibodies specifically detecting chicken-specific cytokines including CSF, TNF and IFN-kappa have been developed and quantitative antigen capture enzyme-linked immunosorbent assay (ELISA) assays to measure these cytokines in biological fluids have been validated. Compared to their mammalian counterparts, the immunological properties of chicken CSF, TNF and IFN-kappa have not been clearly defined. Interferon (IFN) is a type I IFN that plays a central role in antiviral host defense response. Tumor necrosis factor-a (TNF-a) is a type II transmembrane protein with either membrane-bound or soluble forms and is a pleiotropic cytokine associated with the regulation of systemic inflammation. Macrophage colony-stimulating factor-1 (M-CSF-1 or CSF-1) is a hematopoietic growth factor that stimulates the survival, proliferation, and differentiation of the mononuclear phagocytes and is involved in inflammatory processes. Availability of quantitative immunoassays for these three cytokines will enhance fundamental and applied studies on innate host immune response in the normal and diseased states in poultry. Using antigen-capture ELISA for chicken TNF-a, in- depth functional characterization and immunomodulatory properties of TNF- a were conducted and the levels of native TNF-a in the circulation of Clostridium perfringens-, Eimeria-, or dual Eimeria/C. perfringens- infected chickens were determined. Collectively, new results of our studies will enhance our understanding of the functional characteristics of CSF, TNF, and IFN-kappa cytokines and new immunoassays will serve as valuable tools to decipher inflammatory responses and disease pathogenesis in poultry. Progress on the development of diagnostic tests for important proteins of C. perfringens: Several mouse monoclonal antibodies which specifically detect two critical antigen epitopes of pathogenic C. perfringens, were developed to construct new sandwich immunoassays to quantitate the levels of NetB and CNA antigens of C. perfringens in infected chickens, specifically in the serum, gut contents, and fecal samples. These assays are the first immunoassays that make it possible to quantitate the two toxin/virulence-associated factors of C. perfringens in biological fluids and will allow an early detection large-scale screening for the NE disease outbreak in broiler farms to minimize economic losses due to NE. Progress on the production and protection using hyperimmune egg yolk IgY antibodies against Eimeria- and C. perfringens-mediated inflammatory enteric infections: Passive immunity from hyperimmune egg yolk IgY is an antibiotic alternative strategy which utilizes pathogen-specific antibodies to reduce pathogen burden on chickens. Antigen-specific hyperimmune IgY antibodies against protective antigens of Eimeria and C. perfringens were developed using whole protein or synthetic protein fragments of these two pathogens, and the effects of orally-fed hyperimmune IgY antibodies were evaluated for their protective effects in coccidiosis- and NE-afflicted broiler chickens. These key antigens chosen were 3-1E, EF1alpha, NetB, and various synthetic protein fragments of NetB toxoid and Alpha toxoid (Plc), Fructose-1,6-bisphosphate aldolase (FBA), a Zinc metalloprotease (Zm), and one pilus subunit (Collagen adhesion protein (CNA). In addition, two recombinant chimeric proteins (NetB-Plc-Cna and FBA-Zm) targeting these toxins and virulence-associated factors were also used to immunize the hens of layer chickens to obtain therapeutic hyperimmune egg yolk IgY antibodies for oral treatment. Preliminary trial results showed significant protection against coccidiosis and NE in chickens fed with various combinations of these IgY antibodies based on body weight gains using two disease models developed at ARS. Furthermore, hyperimmune IgY antibodies against wo chimeric C. perfringens proteins conferred protection to the broiler chicks against pathogenic C. perfringens challenges. These results indicate a potential application of these combined IgY antibodies specific for chimeric proteins of Eimeria and C. perfringens to prevent field coccidiosis and NE disease outbreak in commercial poultry farms. Further trials are planned to verify the lab results in the field trials. Hyperimmune egg IgY strategy is a promising non-antibiotic strategy to reduce the clinical impacts of necrotic enteritis in commercial broilers at the early stage. Progress on alternatives to antibiotic strategies using B subtilis spore- vectored vaccines: B. subtilis has been approved as Generally Regards As Safe (GRAS) by FDA and may be used as a probiotic. Bioengineered B subtilis is a practical mucosal vector to deliver key genes encoding beneficial proteins that can reduce pathogenic effects in enteric infections such as coccidiosis and NE. Coccidiosis which is caused by several distinct species of Eimeria parasites and NE that is commonly associated with co-infection with E. maxima and pathogenic strains of C. perfringens, are two leading enteric diseases responsible for significant economic loss of more than $ 20 billion in the global poultry industry. Significant progress was made in constructing transgenic Bacillus strains carrying immunogenic antigens of Eimeria and C. perfringens. Recombinant vaccine candidates using NetB and Plc fragment of Alpha toxin, FBA and Zm genes have been successfully constructed using Bacillus subtilis to be tested against coccidiosis and NE. Developing an effective and practical oral delivery strategy to express vaccine antigens of Eimeria and C. perfringens will effectively reduce the gut damage and reduces economic losses due to these enteric infections. ACCOMPLISHMENTS 01 Commercialization of monoclonal antibodies to detect chicken immune molecules. Eleven mouse monoclonal antibodies (mAbs) detecting chicken chemokines (mediators of cellular immunity) have been commercialized by ARS scientists in Beltsville, Maryland. These mAbs detect poultry- specific cytokines which are involved in the early initiation phase of host immune responses to pathogens such as parasites and viruses, and their levels in the blood provide predictive values in determining the quality of host immune responses. These accomplishments address the critical immunological reagent gap for basic immunology research in poultry species, and the availability of these immune reagents will enhance basic and applied research to facilitate our understanding of host immune responses to pathogens and vaccines in poultry that will promote the development of novel immunity-based strategies to reduce antibiotics in commercial poultry production. 02 Development of bioassays to detect poultry immunity. ARS scientists in Beltsville, Maryland, cloned several major chicken cytokine and chemokine genes that mediate poultry immune responses to pathogens, their corresponding recombinant proteins were expressed as immunogens for mAb development, and these mAbs further used to develop sensitive bioassays to measure poultry immunity in chickens after infection or vaccination. The availability of these new immunological tools will be valuable for advancing fundamental and applied studies in avian species to enhance our understanding of host immunity in poultry. 03 Elucidated function of CSF-1 during coccidiosis. Colony-stimulating factor-1 (CSF-1) stimulates the survival, proliferation, and differentiation of macrophages and is involved in bone metabolism, fertility, pregnancy, inflammatory processes, and homeostasis. Five monoclonal antibodies which specifically detect chicken CSF-1 were developed and characterized by ARS scientists in Beltsville, Maryland, and used to develop a bioassay to detect CSF-1 in chickens undergoing infections. Circulating serum CSF-1 was detected in chickens following infection with E. acervulina, E. maxima, and E. tenella, demonstrating an important role of CSF-1 in the immune response to intracellular coccidia parasite infection and suggesting CSF-1 is a biomarker for coccidiosis infection. This mAb-based bioassay will be a valuable tool for the assessment of CSF-1 levels during various poultry infections, and these new antibodies will facilitate fundamental and applied research related to CSF-1 function in normal and disease states in chickens. 04 Biological function of TNF-a during coccidiosis. One of important chicken cytokines called tumor necrosis factor alpha, TNF-a, is well known for its essential and far-reaching regulatory functions in facilitating inflammatory responses in host defense against microbial infections. ARS scientists in Beltsville, Maryland, developed monoclonal antibodies to measure the levels of chicken TNF-a in the circulation of diseases chickens. Augmented serum TNF-a levels were observed at varying time points following Eimeria parasitic infections. Overall, E. tenella-infected chickens showed the highest level of TNF-a suggesting significant damage to the intestinal epithelial cells and systematic inflammatory responses induced by E. tenella infection. The availability of the new TNF-a assay to measure this cytokine in coccidiosis during the early phase of infection will enable the development of counteracting strategies to ameliorate severe gut tissue damage and inflammation in enteric infections. 05 Commercial licensing of hyperimmune egg yolk technology for coccidiosis and necrotic enteritis prevention. In collaboration with a private company, ARS scientists in Beltsville, Maryland, prepared egg yolk antibodies from hens hyper-immunized with Eimeria and C. perfringens antigens and used these antibodies to enhance the immature immune response of newly-hatched chickens and to prevent coccidiosis and necrotic enteritis (NE)(license No. 1916-001 1.2 on hyperimmune eggs and hyperimmune egg supplements to deal with coccidiosis and necrotic enteritis in poultry as encompassed within the claims of the Licensed Patents Serial No. 10,450,364, issued on October 22, 2019). This novel strategy will help to reduce the incidence and economic losses of coccidiosis and NE that are currently estimated to cost around $20 billion annually in global poultry production. 06 Development of bioassays for early detection of necrotic enteritis outbreaks in commercial poultry farms. ARS scientists in Beltsville, Maryland, developed a sensitive bioassay based on the C. perfringens collagen adhesion antigen (CNA) protein and validated its utility using large field screening of necrotic enteritis-afflicted chickens from commercial farms. The results of these screenings indicated that this new assay would serve as a valuable early warning test to allow the accurate measurement of necrotic enteritis outbreak caused by pathogenic C. perfringens strains to monitor flock health status at the farm level or to conduct early detection of necrotic enteritis outbreaks in commercial poultry farms. 07 Dietary sopholipid feed additives as antibiotic alternatives against coccidiosis and necrotic enteritis. Sophorolipids (SPL) are biodegradable, eco-friendly compounds that enhance the growth performance and gastrointestinal functionality in chickens infected with coccidiosis and NE. In collaboration with a private industry research team, ARS scientists in Beltsville, Maryland, developed a novel strategy to use SPL as alternative feed additives to antibiotics to reduce financial losses due to these gut pathogens. The animal studies showed that SPL enhanced growth of chickens infected with these enteric pathogens and decreased gut damage, and suppressed harmful local inflammatory response mediated by cytokine release, therefore enhancing gut integrity. Overall, dietary SPL maintains growth performance, positively modulates gut immunity, and promotes intestinal barrier integrity of young broiler chickens during coccidiosis, demonstrating the potential for using sophorolipids as an alternative to antibiotics for commercial poultry production. 08 Gut-microbiome-derived indole compounds enhance host immunity and increase gut health in broiler chickens. Identification of natural gut compounds that provide beneficial effects to the host may potentially offer a new antibiotic alternative as a feed additive. ARS scientists in Beltsville, Maryland, identified indole compounds as potent immunomodulators which directly contributed to enhanced poultry gut health and growth performance of young chickens afflicted with coccidiosis. Dietary supplementation with indole alleviated intestinal tissue damage of young chickens infected with coccidiosis, reduced inflammation, decreased harmful cytokine expression, and increased expression of genes associated with nutrient transport. These data provide the foundation for future strategies to use natural biochemicals associated with improving poultry growth performance in the absence of antibiotic growth promoters. 09 Development of multivalent recombinant protein vaccines for necrotic enteritis in broiler chickens. ARS scientists in Beltsville, Maryland, developed 3 recombinant C. perfringens proteins and evaluated their protection efficacies with a challenge model of E. maxima/C. perfringens dual infection. Young chickens vaccinated with these C. perfringens proteins demonstrated higher serum antibody levels to the recombinant proteins compared with unvaccinated controls. Following the challenge infection, the pooled antigen-immunized group demonstrated no mortality and the least lesion scores against virulent challenge. These data indicate that the vaccination with these antigens conferred protection against necrotic enteritis challenge in broiler chickens. 10 Deciphering Clostridium perfringens-induced host-pathogen gene expression profiles in the small intestine of broiler chickens. The molecular mechanisms of C. perfringens-induced pathogenesis in the gut and how C. perfringens interacts with other gut microbes remain to be investigated. ARS scientists in Beltsville, Maryland, compared the gene expression profiles of both the host intestine and gut microbiome in C. perfringens-infected and uninfected chickens using RNA sequencing technology. In total, 13,473 genes were differentially expressed between the infected and uninfected groups, of which a subgroup of 96 genes achieved statistical significance. These latter genes were involved in immunity, energy production, and metabolism. Additionally, the levels of 4 foodborne pathogens (C. jejuni, E coli O157, L. monocytogenes and C. perfringens) were significantly elevated in C. perfringens. This study provides new insights into the mechanism of host-pathogen interactions in necrotic enteritis in broiler chickens. 11 Protein analysis of Eimeria necatrix. Eimeria necatrix is a causative pathogen causing coccidiosis that can reduce the growth performance of poultry. The protein structure of this parasite at the sporozoite stage is unknown, and better understanding of this structure is important for the development of effective biological controls. ARS scientists in Beltsville, Maryland, in collaboration with scientists in China, elucidated the complete protein profile of E. necatrix sporozoites. A total of 680 protein were identified, of which 98 were found to react with E. necatrix-specific antibodies. These new findings will enhance our understanding of parasite immunogenicity and immune evasion mechanisms of E. necatrix and may facilitate the discovery of highly effective recombinant vaccine candidates.
Impacts
(N/A)
Publications
- Lu, M., Yuan, B., Yan, X., Sun, Z., Lillehoj, H.S., Lee, Y., Li, C.Z. 2021. Clostridium perfringens-Induced Host-Pathogen Transcriptional Changes in the Small Intestine of Broiler Chickens. Pathogens. 10(12):1607. https:// doi.org/10.3390/pathogens10121607.
- Lee, K., Lillehoj, H.S. 2020. Role of Clostridium perfringens necrotic enteritis (NE) B-like toxin in NE pathogenesis. Avian Pathology. https:// doi.org/10.1637/0005-2086-64.4.490.
- Teng, P., Choi, J., Tompkin, Y.H., Lillehoj, H.S., Kim, W. 2021. Impacts of increasing challenge of E. maxima on gut integrity and gene expression of nutrient transporters in broiler chickens. International Journal of Parasitology. https://doi.org/10.1186/s13567-021-00949-3.
- Hong, Y., Lee, J., Vu, T.H., Lillehoj, H.S., Hong, Y.H. 2021. Immunomodulatory effects of poly (I:C)-stimulated exosomes derived from chicken macrophages. International Journal of Molecular Sciences. https:// doi.org/10.1016/j.psj.2021.101247.
- Lee, Y., Lu, M., Lillehoj, H.S. 2021. Development of antigen-capture ELISA to analyze the immunological functions of chicken interferon-kappa using specific monoclonal antibodies. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2021.104204.
- Vu, T.H., Hong, Y., Truong, A.D., Lee, J., Lee, S., Song, K., Cha, J., Dang, V.D., Tran, H., Lillehoj, H.S., Hong, Y.H. 2022. Cytokine-cytokine receptor interactions in the highly pathogenic avian influenza H5N1 virus- infected lungs of genetically disparate Ri chicken lines. Journal of Animal Bioscience. https://doi.org/10.5713/ab.21.0420.
- Qu, G., Xu, Z., Tuo, W., Li, C.Z., Wan, G., Lillehoj, H.S., Gong, H., Huang, J., Tian, G., Li, S., Liu, Y., Liu, L. 2021. Clostridium perfringens-induced host-pathogen transcriptional changes in the small intestine of broiler chickens. Frontiers in Veterinary Science. https:// doi.org/10.1016/j.vetpar.2021.109642.
- Hong, Y., Lee, J., Vu, T.H., Lee, S., Heo, J., Truong, A.D., Lillehoj, H.S. , Hong, Y.H. 2022. Influenza a pathway analysis of highly pathogenic avian influenza virus (H5N1) infection in genetically disparate Ri chicken lines. Veterinary Immunology and Immunopathology. 246:110404. https://doi.org/10. 1016/j.vetimm.2022.110404.
- Panebra, A., Lillehoj, H.S. 2022. Development of a sandwich ELISA for the detection of chicken colony stimulating factor 1a. Poultry Science. https:/ /doi.org/10.1016/j.psj.2022.101924.
- Wickramasuriya, S., Park, I., Lee, K., Lee, Y., Kim, W., Nam, H., Lillehoj, H.S. 2022. Role of Physiology, Immunity, Microbiota and Enteric Diseases in Gut Health in Poultry. Vaccines. https://doi.org/10.3390/ vaccines10020172.
- Lee, Y., Lu, M., Lillehoj, H.S. 2022. Coccidiosis: Recent progress in host immunity and alternatives to antibiotic strategies. Vaccine. 10:215. https://doi.org/10.3390/vaccines10020215.
- Kim, W., Min, W., Park, K.I., Lillehoj, H.S., Fernandez-Colorado, C.P., Flores, R.A., Cammayo, P.L., Nguyen, B.T. 2022. Expression of chicken NK- Lysin and its role in chicken Coccidiosis induced by Eimeria necatrix. Korean Journal of Parasitology. 59:439. https://doi.org/10.3347/kjp.2021. 59.5.439.
- Park, I., Oh, S., Goo, D., Celi, P., Lillehoj, H.S. 2022. Effect of dietary sophorolipids on growth performance and gastrointestinal functionality of chickens infected with Eimeria maxima. Animal Feed Science and Technology. https://doi.org/10.1016/j.psj.2022.101944.
- Park, I., Oh, S., Nam, H., Celi, P., Lillehoj, H.S. 2022. Antimicrobial activity of sophorolipids against Eimeria maxima and Clostridium perfringens, and their effect on growth performance and gut health in necrotic enteritis. Poultry Science. 101:101731. https://doi.org/10.1016/j. psj.2022.101731.
- Lu, M., Lee, Y., Li, C.Z., Lillehoj, H.S. 2022. Immunological characterization of chicken tumor necrosis factor-a (TNF-a) using new sets of monoclonal antibodies specific for poultry TNF. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2022.104374.
- Hong, Y., Truong, A.D., Vu, T.H., Lee, S., Heo, J., Kang, S., Lillehoj, H. S., Hong, Y.H. 2022. Exosomes from H5N1 avian influenza virus-infected chickens regulate antiviral immune responses of chicken immune cells. Veterinary Research. 130:104368. https://doi.org/10.1016/j.dci.2022.104368.
- Lee, K., Kim, W., Li, C.Z., Lillehoj, H.S. 2020. "Detection of Necrotic Enteritis B-like toxin secreted by Clostridium perfringens using capture Enzyme-Linked Immunosorbent Assay". Avian Diseases. 64(4):490-495. https:// doi.org/10.1637/0005-2086-64.4.490.
- Park, I., Nam, H., Goo, D., Wickramasuriya, S.S., Zimmerman, N., Smith, A. H., Rehberger, T.G., Lillehoj, H.S. 2022. Gut microbiota-derived indole-3- carboxylate influences mucosal integrity and immunity through the activation of the aryl hydrocarbon receptors and nutrient transporters in broiler chickens challenged with Eimeria maxima. Frontiers in Immunology. 13:867754. https://doi.org/10.3389/fimmu.2022.867754.
- Vu, T.H., Hong, Y., Truong, A.D., Lee, J., Lee, S., Song, K., Cha, J., Dang, H.V., Tran, H.T., Lillehoj, H.S., Hong, Y.H. 2021. Cytokine-cytokine receptor interactions in the highly pathogenic avian influenza H5N1 virus- infected lungs of genetically disparate Ri chicken lines. Journal of Animal Bioscience. 35 (3):367-376. https://doi.org/10.5713/ab.21.0163.
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