2011 Annual Report
1a.Objectives (from AD-416)
The purpose of the Poultry Microbiological Safety Research Unit is to further reduce or eliminate bacterial pathogen contamination in poultry operations and the following objectives are to:.
1)Assess the effectiveness and further development of bacteriocins (anti-bacterial peptides) and bacteriophage by in vitro bacterial growth inhibition in culture and in vivo experimentation via challenge in chickens;.
2)Reduce bacterial populations in chicken litter by monitoring poultry houses for bacterial pathogens carried by chickens during use of intervention technologies. Decontamination techniques, such as in-house foam applied disinfectants will be examined for reduction of Campylobacter spp., Salmonella spp., Clostridium perfringens and Listeria monocytogenes; and.
3)Improve cultural methods for Campylobacter spp. in poultry in further support of FSIS needs, including the improvement of recovery. Use microarray expression analysis under various cultural conditions to identify nutrients necessary for optimal growth, colonization and culture of Campylobacter spp.
1b.Approach (from AD-416)
New antimicrobial agents will be developed that are usable by the poultry farmer on
a large scale to reduce Campylobacter spp. and salmonellae by applying in feed at
the appropriate times before slaughter. Also, new means for the drug-free
production sector to control clostridial disease without relying on antibiotics are
the major expected approaches of Objective 1. Providing a science based source of
data to better dispose of spent poultry litter without major energy or financial
inputs is the approach for Objective 2. A more reliable and quantitative method to
routinely culture Campylobacter spp. from food and environmental samples is the
approach of Objective 3.
Progress relating to the previous research project #6612-32000-046-00D, has occurred in this bridging period (primarily publication of data) and will be documented here. Recent research has been directed primarily toward newly approved objectives to be addressed in the new research project #6612-32000-060-00D.
Bacteriocin/phage discovery and applications. A new bacteriocin, L-1077, was isolated which appears to hold promise in controlling C. jejuni/S. Enteritidis among commercial broiler chickens during production. This accomplishment is outlined below and in the recent publication. In addition, work was recently published detailing the comparative genomics of four closely related Clostridium perfringens bacteriophages. These results, which reveal variable evolution among core genes with therapeutic potential, have also been published recently. Studies to determine the molecular mechanisms of bacteriocin resistance in Campylobacter were completed and the work recently published. Patent #7,988,958 “Novel Enterococcus and Streptococcus Strains and Bacteriocins” was issued on August 2, 2011 based on work completed earlier in the research project #6612-32000-046-00D. Collaborative research with colleagues at Cambridge University resulted in a recent publication on the design of dose response experiments for studies involving poultry; and the effects of a novel plant-based antimicrobial, lupulone, was detailed in a separate collaborative effort and recently published.
Bacteriocin/phage discovery and applications. A new bacteriocin, L-1077 appears to hold promise in controlling C. jejuni/S. Enteritidis among commercial broiler chickens during production. Lactobacillus salivarius 1077 (NRRL B-50053) was isolated from poultry intestinal materials, and in vitro anti-Campylobacter jejuni activity was demonstrated. The isolate was then used for bacteriocin production and its enrichment. A typical bacteriocin-like response of sensitivity to proteolytic enzymes and resistance to lysozyme, lipase, and 100°C was observed with this preparation. The polypeptide was further purified by gel filtration, ion-exchange, and hydrophobic-interaction chromatography. Bacteriocin L-1077 contained the class IIa bacteriocin signature N-terminal sequence YGNGV and the therapeutic benefit of bacteriocin L-1077 was demonstrated in market-age broiler chickens to reduce both gastrointestinal C. jejuni and Salmonella enterica serovar Enteritidis. These results were recently published.
Simmons, I.M., Donovan, D.M., Siragusa, G.R., Seal, B.S. 2010. Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal domain. Journal of Agricultural and Food Chemistry. 58(19):10330-10337.
Volozhantseva, N.V., Verevkina, V.V., Bannova, V.A., Krasilnikovaa, V.M., Popovaa, A.V., Zhilenkova, E.L., Svetocha, E.A., Seal, B.S., Stern, N.J., Oakley, B. 2010. The Genome Sequence of Bacteriophage CPV1 Virulent for Clostridium perfringens. Virus Research. 155(2):433-439.
Oakley, B., Morales, C., Seal, B.S., Hiett, K.L., Talundzic, E., Volozhantsev, N.V. 2011. Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable rates of evolution within a core genome. Biomed Central (BMC) Genomics. 12:212.
Zsak, L., Day, J.M., Oakley, B., Seal, B.S. 2011. The complete genome sequence and genetic analysis of phiCA82 a novel uncultured microphage from the turkey gastrointestinal system. Virology Journal. 8:331.
Svetoch, E.A., Eruslanov, B.V., Levchuk, V.P., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Stepanshin, J., Dyatlov, I., Seal, B.S., Stern, N.J. 2011. Isolation of Lactobacillus salivarius 1077 (NRRL B-50053) and characterization of its bacteriocin and spectra of antimicrobial activity. Applied and Environmental Microbiology. 77(8):2749-2754.