Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 1.a. Generate and apply chicken egg-yolk antibodies to reduce Salmonella and Campylobacter populations in broiler chickens. 1.b. Enhance the mucus-binding ability of Lactobacillus reuteri and L. salivarius cultures by growing them in mucin-containing media and compare the ability of enhanced and original cultures to reduce colonization of Salmonella and Campylobacter isolates in vivo. 1.c. Investigate the effects of Bacillus subtilis and Lactobacillus reuteri delivered in feed for reduction of Salmonella and Campylobacter colonization in the broiler chicken�s gastrointestinal system. 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). 2.a. Identify and clone bacteriophage and prophage lytic enzymes using genomics analyses. 2.b. Express lytic enzymes, bacteriocins and holins in yeast. 2.c. Identify and clone bacteriophage lytic enzymes followed by expression in yeast as a feed additive for swine to reduce bacterial pathogens during production. Approach (from AD-416): Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) evaluate novel biocontrol strategies to reduce bacterial pathogen GI tract colonization of chickens, and 2) identify and characterize the biophysical properties of anti-bacterial peptides and lytic enzymes. Our approach includes application of specific egg-derived immunoglobulin, enhancing probiotic lactobacilli and determining synergism of probiotic treatments. This will be accompanied by isolation and assay of enzymes capable of lysing food-borne pathogens. The lytic enzymes and previously described bacteriocins will also be cloned for enhanced expression in yeast which can be readily incorporated into chicken feed. In vitro bacterial growth inhibition and in vivo chicken trials will be used to determine practical intervention approaches applicable to the poultry industry. By providing novel alternatives to antibiotic usage in poultry, the overall impact of this research will be a reduction in bacterial pathogens associated with chickens. This project terminated during this annual report cycle therefore this report serves as a final summary. Salmonella is the leading pathogen for human food-borne disease associated with poultry. Salmonella proteins involved with bacterial colonization or motility have the potential for use as antigens for vaccination to reduce Salmonella in the chicken gastrointestinal tract. Consequently, bioinformatic searches were performed to select genes encoding proteins potentially involved in colonization and motility from various Salmonella serovar genomes. Ten recombinant flagellar proteins were expressed, purified and confirmed by detection of the His sequence tag and mass spectrometry. Among them, the FlgK protein (flagellar hook- associated protein) was further analyzed by immunoassay. The result shows that (1) this purified recombinant FlgK protein was immunogenic in broilers, (2) FlgK could be used as a probe for antibody detection in broiler sera using an automated capillary immunoassay, and (3) FlgK reacted strongly to sera from commercial broiler chickens older than four weeks, indicating that this anti-FlgK antibodies may be prevalent in the poultry population. In addition, the relationship of the presence of anti- Campylobacter antibodies and Campylobacter isolation from broiler cecal contents was investigated. The results show all bacterial cultures were negative for Campylobacter jejuni. However, sera from 22 out of 34 broilers reacted to the Campylobacter jejuni FliD protein. Due to an increase in reports of antibiotic resistant bacteria, there has been resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium (C.) perfringens is a Gram-positive, spore-forming anaerobic bacterium that is the third leading cause of human foodborne bacterial disease and is also the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among commercial broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated necrotic enteritis in commercial poultry. ARS researchers in Athens, Georgia, isolated two types of bacteriophage from poultry processing plants, those that had long non-contractile tails, members of the family Siphoviridae, and those with short non-contractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain not previously reported in viral genomes, that can potentially digest the cell wall of C. perfringens. Additionally, researchers in Athens, Georgia, expressed a synthetic gene encoding the enzymatic lytic domain of a thermophilic bacteriophage encoded lysin in series with the cell-wall binding domain from two C. perfringens-specific bacteriophage lysins. The protein was able to withstand temperatures up to 42�C without noticeable loss of an ability to lyse C. perfringens in a species-specific manner. This protein could potentially be utilized as a more stable feed additive to control the bacterium during poultry production. Future investigations will examine the ability of these phage lytic proteins to control these pathogens in the chicken gastrointestinal system during poultry production. Reducing populations of pathogens associated with poultry during production will lead to fewer pathogens entering the processing plant and reaching the subsequent consumer and will reduce the risk of human food-borne illness. Portions of this line of research are being continued in a subsequent project: Novel Pre-harvest Interventions and Alternatives to Antibiotics to Reduce Foodborne Pathogens #6040-32000-071- 00D. Accomplishments 01 Detection of anti-Salmonella FlgK antibodies in chickens by automated capillary immunoassay. Western blot is a very useful tool to identify specific protein, but is tedious, labor-intensive and time-consuming. An automated "Simple Western" assay has recently been developed that enables the protein separation, blotting and detection in an automatic manner. However, this technology has not been used in clinical diagnosis. The flagellar hook protein (FlgK) is required for flagellar filament formation. Further, FlgK is an important virulence factor that plays a role in intestinal inflammation and is the most immune-reactive flagellar protein. Therefore, it is a potential target for host immune response. In this communication, researchers in Athens, Georgia, evaluated whether the Simple Western system could be used to detect Salmonella FlgK antibodies from chicken sera. Salmonella FlgK was successfully expressed in and purified from the E. coli expression system. This rFlgK protein induced strong immune response in chickens. This automated immunoassay was successfully used for detection of antibodies in chicken sera against Salmonella rFlgK. These results provide a rational for further evaluation of this automated immunoassay as a tool using Salmonella rFlgK in Salmonella epidemiological investigation. 02 Survey of antibodies in broiler Sera against Salmonella flagellar proteins (FlgK and FliD). Salmonella is a causative pathogen of human acute bacterial gastroenteritis worldwide. Chickens are regarded as one of major reservoirs of this microorganism. Bacterial flagella are involved in motility, adhesion, quorum sensing and other virulence activities. Also, the flagella are immunogenic. In this communication, researchers in Athens, Georgia, used two Salmonella flagellar proteins - FliD and FlgK as probes to survey the prevalence of Salmonella antibodies in broilers. The fliD and flgK genes were amplified by PCR, and the proteins were over-expressed in an Escherichia coli Expression System. The recombinant proteins were purified by a nickel-chelating affinity chromatography, and confirmed by nucleotide sequencing of the plasmid, SDS-PAGE analysis, and the His tag detection. Sera from the FlgK immunized broilers reacted strongly to FlgK, indicating that this protein is immunogenic. The ELISA results show 66% of broiler sera reacted to FlgK, while about 38% to FliD. The results implicating that these anti-FlgK antibodies may be prevalent in the poultry population. These results provide a rationale for further evaluation of these proteins as vaccine candidates for broiler chickens so that food safety for poultry can be improved. These proteins may also hold important insights for Salmonella commensalism in chickens and pathogenesis in humans. 03 Presence of antibodies against Campylobacter flagellar capping proteins versus Campylobacter jejuni isolation in broilers. Campylobacter jejuni is the leading foodborne pathogen that causes human acute bacterial gastroenteritis worldwide. Human cases have been linked to consumption and/or handling of contaminated poultry products. The ARS scientists at Athens, Georgia, previous studies found seroprevalence of antibodies against the Campylobacter jejuni flagellar capping protein (FliD) in older broilers from commercial poultry farms. However, whether Campylobacter jejuni could be isolated from the antibody- positive broilers was not known. Further, researchers in Athens, Georgia, used broilers that were raised in an environmentally controlled house according to the standard brooding and growing guidelines. At seven weeks of age, both blood for immunoblot analysis and cecal content for direct microbiological plating were collected from broilers according to the standard protocols. All bacterial cultures were negative for Campylobacter jejuni. However, sera from 22 out of 34 broilers reacted to the Campylobacter jejuni FliD protein. These findings in this study provide us with a rationale for further evaluation of the roles of antibodies in excretion of Campylobacter jejuni.
Impacts
(N/A)
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 1.a. Generate and apply chicken egg-yolk antibodies to reduce Salmonella and Campylobacter populations in broiler chickens. 1.b. Enhance the mucus-binding ability of Lactobacillus reuteri and L. salivarius cultures by growing them in mucin-containing media and compare the ability of enhanced and original cultures to reduce colonization of Salmonella and Campylobacter isolates in vivo. 1.c. Investigate the effects of Bacillus subtilis and Lactobacillus reuteri delivered in feed for reduction of Salmonella and Campylobacter colonization in the broiler chicken�s gastrointestinal system. 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). 2.a. Identify and clone bacteriophage and prophage lytic enzymes using genomics analyses. 2.b. Express lytic enzymes, bacteriocins and holins in yeast. 2.c. Identify and clone bacteriophage lytic enzymes followed by expression in yeast as a feed additive for swine to reduce bacterial pathogens during production. Approach (from AD-416): Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) evaluate novel biocontrol strategies to reduce bacterial pathogen GI tract colonization of chickens, and 2) identify and characterize the biophysical properties of anti-bacterial peptides and lytic enzymes. Our approach includes application of specific egg-derived immunoglobulin, enhancing probiotic lactobacilli and determining synergism of probiotic treatments. This will be accompanied by isolation and assay of enzymes capable of lysing food-borne pathogens. The lytic enzymes and previously described bacteriocins will also be cloned for enhanced expression in yeast which can be readily incorporated into chicken feed. In vitro bacterial growth inhibition and in vivo chicken trials will be used to determine practical intervention approaches applicable to the poultry industry. By providing novel alternatives to antibiotic usage in poultry, the overall impact of this research will be a reduction in bacterial pathogens associated with chickens. Campylobacter jejuni and Salmonella are the leading causes of human food- borne diseases associated with poultry. Proteins involved with bacterial colonization or motility have the potential for use as antigens for vaccination to reduce these pathogens in the chicken gastrointestinal tract. Consequently, bioinformatic searches were performed to select genes encoding proteins potentially involved in colonization and motility from these pathogen genomes. Recombinant flagellar proteins were expressed, purified and confirmed by detection of the His sequence tag and mass spectrometry. Among them, the FliD protein (flagellar capping protein) of C. jejuni was further analyzed by an immunoassay. The result shows that Peptides 24, 91 and 92 had relatively high reactivity among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. Also, these peptides were also recognized by sera from chickens immunized with the purified recombinant flagellar capping protein. In addition, a battery of Salmonella flagellar proteins were cloned and purified in and from an E. coli expression system. The immunoblot analyses show that 61 out of 66 chicken sera reacted to the FlgK protein. These antibody screening results provide rationales for further evaluation of these proteins as potential vaccine candidates for broiler chickens to improve food safety for poultry. Additional research showed that bacteriocins and bacteriophage lytic proteins can be utilized to inhibit the growth or kill bacteria such as Campylobacter jejuni and Clostridium perfringens, agents that cause human food-borne disease. Two bacteriophage lysin genes (PlyCP39O; PlyCP26F) were codon optimized for expression in yeast. Both proteins have been expressed in yeast by collaborators at the ARS-USDA National Center for Agricultural Utilization Research and demonstrated to species-specifically lyse Clostridium perfringens. These recombinant proteins will be utilized during experimentation to reduce the levels of Clostridium perfringens in the chicken gastrointestinal tract. Efforts to conduct in vivo trials in chickens have been delayed this FY by unexpected state incinerator permitting requirements for our facility. We applied for a new state permit which was recently issued and in vivo trials are finally underway. Accomplishments 01 Identification of epitopes of Campylobacter flagellar capping protein as a potential vaccine candidate. ARS researchers at Athens, GA screened synthetic peptides of the flagellar protein of Campylobacter with sera from chickens. Peptides exhibited reactivity among serum samples, indicating that peptides shared immunodominant epitopes on the C. jejuni a protein. These peptides were also recognized by sera from chickens immunized with purified recombinant flagellar protein. Results indicate that these peptides may be vaccine candidates for reducing Campylobacter colonization of poultry. 02 Reactions of broiler chicken sera to recombinant flagellar proteins of Salmonella serovars. Bacterial flagella involved in motility, adhesion, quorum sensing and other virulence activities. The ARS scientists at Athens, GA cloned and purified a battery of Salmonella flagellar proteins in and from an E. coli expression system. The immunoblot analyses show that 61 out of 66 chicken sera reacted to the FlgK protein. More studies are needed to further evaluate whether this protein has potential as novel targets for multi-subunit vaccine development.
Impacts
(N/A)
Publications
- Yeh, H., Hiett, K.L., Line, J.E. 2015. Reactions of Chicken Sera to Recombinant Campylobacter jejuni Flagellar Proteins. Archives Of Microbiology. doi: 10.1007/s00203-014-1062-3.
- Swift, S., Seal, B.S., Garrish, J.K., Oakley, B., Yeh, H., Woolsey, R., Schegg, K.M., Line, J.E., Donovan, D.M., Hiett, K.L. 2015. A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability. Viruses and Bacteriophages. 7(6):3019-3034.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 1.a. Generate and apply chicken egg-yolk antibodies to reduce Salmonella and Campylobacter populations in broiler chickens. 1.b. Enhance the mucus-binding ability of Lactobacillus reuteri and L. salivarius cultures by growing them in mucin-containing media and compare the ability of enhanced and original cultures to reduce colonization of Salmonella and Campylobacter isolates in vivo. 1.c. Investigate the effects of Bacillus subtilis and Lactobacillus reuteri delivered in feed for reduction of Salmonella and Campylobacter colonization in the broiler chicken�s gastrointestinal system. 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). 2.a. Identify and clone bacteriophage and prophage lytic enzymes using genomics analyses. 2.b. Express lytic enzymes, bacteriocins and holins in yeast. 2.c. Identify and clone bacteriophage lytic enzymes followed by expression in yeast as a feed additive for swine to reduce bacterial pathogens during production. Approach (from AD-416): Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) evaluate novel biocontrol strategies to reduce bacterial pathogen GI tract colonization of chickens, and 2) identify and characterize the biophysical properties of anti-bacterial peptides and lytic enzymes. Our approach includes application of specific egg-derived immunoglobulin, enhancing probiotic lactobacilli and determining synergism of probiotic treatments. This will be accompanied by isolation and assay of enzymes capable of lysing food-borne pathogens. The lytic enzymes and previously described bacteriocins will also be cloned for enhanced expression in yeast which can be readily incorporated into chicken feed. In vitro bacterial growth inhibition and in vivo chicken trials will be used to determine practical intervention approaches applicable to the poultry industry. By providing novel alternatives to antibiotic usage in poultry, the overall impact of this research will be a reduction in bacterial pathogens associated with chickens. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. Campylobacter jejuni is the leading cause of human food-borne disease associated with poultry. Campylobacter proteins involved with bacterial colonization or motility have the potential for use as antigens for vaccination to reduce C. jejuni in the chicken gastrointestinal tract. Consequently, bioinformatic searches were performed to select genes encoding proteins potentially involved in colonization and motility from the C. jejuni genome. Twelve recombinant Campylobacter jejuni flagellar proteins were expressed, purified and confirmed by detection of the His sequence tag and mass spectrometry. Among them, the FliD protein (flagellar capping protein) was further analyzed by an immunoassay. The result shows that this purified recombinant FliD protein reacted strongly to sera from broiler chickens older than four weeks, indicating that this anti-FliD antibody may be prevalent in the poultry population. In addition, C. jejuni isolates without this protein at the distal end of the flagella greatly reduced their motility and abilities of adhesion and invasion in in vitro studies. In addition, a set of chemotactic proteins of C. jejuni were cloned and expressed. These recombinant proteins were tagged with six-His and hemagglutinin short sequences at amino and carboxyl termini, respectively. Chicken sera reacted to various numbers of recombinant proteins, but all sera reacted to the Cjj0473 protein (annotated as methyl-accepting chemotaxis protein). These antibody screening results provide rationales for further evaluation of the FliD and Cjj0473 proteins as potential vaccine candidates for broiler chickens to improve food safety for poultry. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). Bacteriocins and bacteriophage lytic proteins can be utilized to inhibit the growth or kill bacteria such as Campylobacter jejuni and Clostridium perfringens, agents that cause human food-borne disease. Two bacteriophage lysin genes (PlyCP39O; PlyCP26F) were codon optimized for expression in yeast. Both proteins have been expressed in yeast by collaborators at the ARS-USDA National Center for Agricultural Utilization Research and demonstrated to species-specifically lyse Clostridium perfringens. These recombinant proteins will be utilized during experimentation to reduce the levels of Clostridium perfringens in the chicken gastrointestinal tract. Many enzymes are added to feeds during food-animal production and these proteins are subjected to high- heat stress due to cooking of feed stuffs and environmental factors. Our laboratories have been developing bacteriophage lysin proteins for potential feed additives as alternative antimicrobials to control specific pathogens that afflict commercial food-producing animals. Consequently, the predicted N-terminal enzymatic active domain of a thermophilic bacteriophage lysin gene was synthesized to also encode the C-terminal cell-wall binding domain of a Clostridium perfringens-specific lysin protein. Significant Activities that Support Special Target Populations: Hosted a summer student from the Peach State Louis Stokes Alliance for Minority Participation (Peach State LSAMP). Accomplishments 01 Identification of Campylobacter flagellar capping protein as a potential vaccine candidate. Vaccination of food animals is one of the best means to control infectious diseases in food production. The ARS researchers at Athens, Georgia screened a battery of the recombinant Campylobacter jejuni proteins, and found the flagellar capping protein reacted strongly to sera from broiler and breeder chickens from selected areas in the U.S., suggesting that this antibody may be prevalent in the poultry population. Because of its strong antigenicity, and because the flagellar capping protein is highly homologous among Campylobacter species, this protein may be a potential vaccine candidate for the reduction of Campylobacter spp. in poultry. 02 Expression of a thermophilic bacteriophage lysin protein with a Clostridium perfringens-specific cell wall binding domain. Clostridium perfringens is the third leading cause of human foodborne bacterial disease and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among commercial broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated necrotic enteritis in commercial poultry. Researchers in Athens, Georgia expressed a synthetic gene encoding the enzymatic lytic domain of a thermophilic bacteriophage encoded lysin in series with the cell- wall binding domain from two C. perfringens-specific bacteriophage lysins. The protein was able to withstand temperatures up to 42�C without noticeable loss of an ability to lyse C. perfringens in a species-specific manner. This protein could potentially be utilized as a more stable feed additive to control the bacterium during poultry production. 03 Expression of Campylobacter jejuni chemotactic proteins and their reactivity to broiler sera. Bacterial chemotaxis requires a group of specialized proteins to perform a cascade of signal transduction for guiding microorganisms to the colonization sites and invasion in the host cells. The ARS scientists at Athens, Georgia cloned and purified a battery of chemotactic proteins in and from an Escherichia coli expression system. The immunoblot analyses show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product. More studies are needed to further evaluate whether these proteins have potential as novel targets for multi-subunit vaccine development.
Impacts
(N/A)
Publications
- Yeh, H., Hiett, K.L., Line, J.E., Seal, B.S. 2014. Characterization and Antigenicity of Recombinant Campylobacter jejuni Flagellar Capping Protein FliD. Journal of Medical Microbiology. 63(4):602-609.
- Yeh, H., Hiett, K.L., Line, J.E., Seal, B.S. 2014. Characterization and Reactivity of Broiler Chicken Sera to Selected Recombinant Campylobacter jejuni Chemotactic Proteins. Archives Of Microbiology. 196:375-383.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 1.a. Generate and apply chicken egg-yolk antibodies to reduce Salmonella and Campylobacter populations in broiler chickens. 1.b. Enhance the mucus-binding ability of Lactobacillus reuteri and L. salivarius cultures by growing them in mucin-containing media and compare the ability of enhanced and original cultures to reduce colonization of Salmonella and Campylobacter isolates in vivo. 1.c. Investigate the effects of Bacillus subtilis and Lactobacillus reuteri delivered in feed for reduction of Salmonella and Campylobacter colonization in the broiler chicken�s gastrointestinal system. 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). 2.a. Identify and clone bacteriophage and prophage lytic enzymes using genomics analyses. 2.b. Express lytic enzymes, bacteriocins and holins in yeast. Approach (from AD-416): Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) evaluate novel biocontrol strategies to reduce bacterial pathogen GI tract colonization of chickens, and 2) identify and characterize the biophysical properties of anti-bacterial peptides and lytic enzymes. Our approach includes application of specific egg-derived immunoglobulin, enhancing probiotic lactobacilli and determining synergism of probiotic treatments. This will be accompanied by isolation and assay of enzymes capable of lysing food-borne pathogens. The lytic enzymes and previously described bacteriocins will also be cloned for enhanced expression in yeast which can be readily incorporated into chicken feed. In vitro bacterial growth inhibition and in vivo chicken trials will be used to determine practical intervention approaches applicable to the poultry industry. By providing novel alternatives to antibiotic usage in poultry, the overall impact of this research will be a reduction in bacterial pathogens associated with chickens. Objective 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. Campylobacter jejuni is the leading cause of human food-borne disease associated with poultry. Campylobacter proteins involved with bacterial colonization or motility have the potential for use as antigens for vaccination to reduce C. jejuni in the chicken gastrointestinal (GI) tract. Consequently, bioinformatic searches were performed to select genes encoding proteins potentially involved in colonization and motility from the C. jejuni genome. Twelve recombinant Campylobacter jejuni flagellar proteins were expressed, purified and confirmed by detection of the His sequence tag and mass spectrometry. Among them, the FliD protein (flagellar capping protein) was further analyzed by an immunoassay. The result shows that this purified recombinant FliD protein reacted strongly to sera from broiler chickens older than four weeks, indicating that this anti-FliD antibody may be prevalent in the poultry population. In addition, C. jejuni isolates without this protein at the distal end of the flagella greatly reduced their motility and abilities of adhesion and invasion in in vitro studies. In addition, a set of chemotactic proteins of C. jejuni were cloned and expressed. These recombinant proteins were tagged with six-His and hemagglutinin (HA) short sequences at amino and carboxyl termini, respectively. Chicken sera reacted to various numbers of recombinant proteins, but all sera reacted to the Cjj0473 protein (annotated as methyl-accepting chemotaxis protein). These antibody screening results provide rationales for further evaluation of the FliD and Cjj0473 proteins as potential vaccine candidates for broiler chickens to improve food safety for poultry. Objective 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). Bacteriocins and bacteriophage lytic proteins can be utilized to inhibit the growth or kill bacteria such as Campylobacter jejuni and Clostridium perfringens, agents that cause human food-borne disease. Two bacteriophage lysin genes (PlyCP39O; PlyCP26F) were codon optimized for expression in yeast. Both proteins have been expressed in yeast by collaborators at the ARS-USDA National Center for Agricultural Utilization Research and were demonstrated to species-specifically lyse Clostridium perfringens. These recombinant proteins will be utilized during experimentation to reduce the levels of Clostridium perfringens in the chicken gastrointestinal tract.
Impacts
(N/A)
Publications
- Yeh, H., Hiett, K.L., Line, J.E., Oakley, B., Seal, B.S. 2013. Construction, expression, purification and antigenicity of recombinant campylobacter jejuni flagellar proteins microbiological research. Microbiological Research. 168(4):192-198.
- Seal, B.S. 2013. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium. Poultry Science. 92(2):526-533.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 1.a. Generate and apply chicken egg-yolk antibodies to reduce Salmonella and Campylobacter populations in broiler chickens. 1.b. Enhance the mucus-binding ability of Lactobacillus reuteri and L. salivarius cultures by growing them in mucin-containing media and compare the ability of enhanced and original cultures to reduce colonization of Salmonella and Campylobacter isolates in vivo. 1.c. Investigate the effects of Bacillus subtilis and Lactobacillus reuteri delivered in feed for reduction of Salmonella and Campylobacter colonization in the broiler chicken�s gastrointestinal system. 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). 2.a. Identify and clone bacteriophage and prophage lytic enzymes using genomics analyses. 2.b. Express lytic enzymes, bacteriocins and holins in yeast. Approach (from AD-416): Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) evaluate novel biocontrol strategies to reduce bacterial pathogen GI tract colonization of chickens, and 2) identify and characterize the biophysical properties of anti-bacterial peptides and lytic enzymes. Our approach includes application of specific egg-derived immunoglobulin, enhancing probiotic lactobacilli and determining synergism of probiotic treatments. This will be accompanied by isolation and assay of enzymes capable of lysing food-borne pathogens. The lytic enzymes and previously described bacteriocins will also be cloned for enhanced expression in yeast which can be readily incorporated into chicken feed. In vitro bacterial growth inhibition and in vivo chicken trials will be used to determine practical intervention approaches applicable to the poultry industry. By providing novel alternatives to antibiotic usage in poultry, the overall impact of this research will be a reduction in bacterial pathogens associated with chickens. Campylobacter jejuni is the leading cause of human food-borne disease associated with poultry. Campylobacter proteins involved with bacterial colonization or motility have the potential for use as antigens for vaccination to reduce C. jejuni in the chicken gastrointestinal (GI) tract. Consequently, bioinformatic searches were performed to select genes encoding proteins potentially involved in colonization and motility from the C. jejuni genome. Twelve C. jejuni flagellar proteins (involved in bacterial movement) have been expressed as recombinants, purified to homogeneity and assayed for biochemical purity. These proteins will be assayed during in ovo vaccine procedures as candidates to reduce C. jejuni in the broiler chicken GI tract. In addition, probiotic bacteria Bacillus subtilis and Lactobacillus reuteri have been developed by an industry partner for use as a feed additive to reduce Campylobacter spp. and Salmonella spp. in the chicken gastrointestinal tract. These bacteria will be utilized in recently designed broiler chicken feeding studies to measure bacterial load in the chicken GI tract and the effect on Campylobacter and Salmonella levels. To further our understanding of changes in the microbial ecology associated with pathogen colonization novel fluoresence in-situ hybridiization (FISH) probes were developed to target Bacillus, Lactobacillus, Salmonella, and Campylobacter. PMSRU researchers have optimized the protocol for utilizing FISH directly with cecal samples and are currently working to optimize hybridization conditions with each of these taxon-specific probes. To rapidly analyze acquired fluorescence images, several high-throughput data analysis pipelines were constructed with scripts written for the programs ImageJ, MatLab, and R. FISH is an essential cultivation-independent tool for microbial ecology that will now allow researchers to evaluate the effects of probiotic treatments on chicken gut microbial ecology. Bacteriocins and bacteriophage lytic proteins can be utilized to inhibit the growth or kill bacteria such as Campylobacter jejuni and Clostridium perfringens, agents that cause human food-borne disease. Synthetic genes optimized for expression in yeast encoding the bacteriocin OR7 and bacteriophage lysin Ply39O have been synthesized by a commercial supplier. The recombinant plasmids encoding the gene for bacteriocin OR7 and Ply39O were transformed into the yeast, Pichia pastoris, and positive clones were identified. Subsequent protein expression was induced by standard procedures and demonstrated that the target proteins were expressed as recombinant proteins. The recombinant proteins will be assayed for the ability to inhibit growth of C. jejuni and C. perfringens in the chicken gastrointestinal system during future feeding trials. Accomplishments 01 Identification and cloning of bacteriophage or prophage lytic enzymes using genomic analyses. Due to an increase in reports of antibiotic resistant bacteria, there has been resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal and poultry diseases. Agricultural Research Service researchers in Athens, Georgia isolated two types of bacteriophage from poultry processing plants, those that had long non-contractile tails, members of the family Siphoviridae, and those with short non-contractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded amidases, lysozyme-endopeptidases, and a zi carboxypeptidase domain not previously reported in viral genomes, that c potentially digest the cell wall of C. perfringens. Additionally, bioinformatic analysis was utilized to identify an amidase lytic enzyme the genome of Listeria monocytogenes, the leading cause of bacterial foo borne death in humans. Lytic proteins were cloned and expressed as recombinant proteins, then utilized to kill C. perfringens and L. monocytogenes in the laboratory. Future investigations will examine the ability of these phage lytic proteins to control these pathogens in the chicken gastrointestinal system during poultry production. Reducing populations of pathogens associated with poultry during production will lead to fewer pathogens entering the processing plant and reaching the subsequent consumer and will reduce the risk of human food-borne illness
Impacts
(N/A)
Publications
- Morales, C., Oakley, B., Garrish, J.K., Seal, B.S., Siragusa, G.R., Ard, M. B. 2012. Complete genome sequence of the podoviral bacteriophage CP24R virulent for Clostridium perfringens. Archives of Virology. 157:769-772.
- Simmons, M., Morales, C., Oakley, B., Seal, B.S. 2012. Recombinant expression of a putative prophage amidase cloned from the genome of Listeria monocytogenes that lyses the bacterium and its biofilm. Probiotics and Antimicrobial Proteins. 4:1-10.
- Volozhantsev, N.V., Oakley, B., Morales, C., Verevkkin, V.V., Bannov, V.A., Popova, A.V., Zhilenkov, E.L., Svetoch, E.A., Garrish, J.K., Schegg, K.M., Woolsey, R., Quilici, D.R., Line, J.E., Hiett, K.L., Siragusa, G.R., Seal, B.S. 2012. Molecular characterization of podoviral bacteriophages virulent for clostridium perfringens and their comparison with members of the picovirinae. PLoS One. 7:e38283.
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