2007 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..
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.
Bacteriocin isolation and production. Campylobacter antagonistic bacteria have been identified and their corresponding bacteriocins isolated and characterized. Control of both Campylobacter and Salmonella has been demonstrated in live poultry. We have reduced the level of Campylobacter in the ceca of chickens by at least one-million fold via oral treatment of broilers with specific bacteriocins. By reducing the levels of pathogens in the broiler intestinal tract, corresponding cross contamination of poultry meat will be proportionately diminished and consumer exposure to these pathogens will be decreased. We have conducted experiments to scale-up production of bacteriocin E-760 to conduct our planned field trial during this next FY.
Antagonists against Clostridium perfringens. We continued research with the objective of creating and characterizing a collection of C. perfringens cultures isolated from Russian chickens. Of all samples derived from 48-day-old broilers, C. perfringens was found only in one third of the samples. Genetic and cultural properties of 20 newly isolated strains of C. perfringens have been initiated.
Anti-C. perfringens activities of 362 lactic acid bacterial isolates and enterococci isolated from broilers were examined and 104 isolates of microbial antagonists were selected for analysis. A bacteriocin is being produced by Lactobacillus salivarius that possesses anti-C. perfringens activity.
Seventeen new bacteriophages with specific anti- C. perfringens activity were isolated from chickens and environmental sources. The bacterial viruses possessed either narrow or rather narrow host specificity in most cases.
Experiments on modeling C. perfringens infections in chickens were initiated and colonizing activities of some strains of C.perfringens in broilers were assessed. It was shown that it is possible to induce intestinal C. perfringens infection in broilers by feeding them either with rye or fish meals.
Effect of coprophagia on colonization of broiler chicks with Campylobacter. Coprophagia (oral ingestion of fecal matter) is a normal activity of commercial broiler chickens. Studies to determine the population of a specific pathogen necessary to colonize 50% of experimental animals inoculated (colonization dose 50% or CD50) can be misled by coprophagous activity among animals. Experiments were conducted to determine the effect of coprophagia by orally administering measured amounts of bacteria to newly hatched chicks. The chicks were housed either individually in cages preventing this activity, or in isolation units containing groups of birds where coprophagia was allowed. The CD50 was calculated and results from the two models were compared. Elimination of coprophagia as an uncontrolled variable led to a more clear determination of the estimated CD50 as demonstrated in the individually-housed chick model. Coprophagous activity obscured results in the chicken experimental model where it was not prevented. The individual bird challenge model is therefore recommended to researchers rather than the group challenge model to determine colonization dosage.
Isolation and purification of a novel bacteriocin from Enterococcus spp. with broad spectrum inhibitory activity.
Campylobacter jejuni is a Gram-negative human food-borne pathogen of primary importance. Poultry are frequently contaminated with C. jejuni during production with the majority of commercial U.S. flocks are positive for the organism by the time birds reach market age of 6 weeks. There is a need for effective interventions that may be practically applied in the poultry industry to reduce colonization of poultry with C. jejuni and subsequently reduce consumer exposure to this pathogen. We reported the isolation and purification of a peptide bacteriocin produced by an Entercoccus species isolated from chicken ceca with broad spectrum activity against both Gram-positive and Gram-negative bacteria and demonstrated the ability of the purified bacteriocin to reduce colonization of poultry by Campylobacter spp. Administration of bacteriocin E-760 treated feed significantly (P< 0.05) reduced colonization of young broiler chicks experimentally challenged with two strains of C. jejuni by more than a million fold. Bacteriocin E-760 also significantly (P<0.05) reduced colonization of naturally acquired Campylobacter spp. in market age broilers when administered in treated feed for four days prior to analysis. This information will be useful to researchers in government, academia and the poultry industry. Bacteriocin E-760 could be developed as a practical on-farm intervention for reducing consumer exposure to pathogenic campylobacter.
The accomplishment addresses Problem 1.14 Intervention Strategies in the National Program 108 Food Safety 2006-2010 Action Plan. Intervention strategies, including both products and practices, are needed to reduce colonization and shedding of epizootic pathogens by food producing animals. This refers to Component 1.1 (Pathogens, Toxins, and Chemical Contaminants Pre-harvest) of the 2005-2009 National Program 108 (Food Safety-animal and plant products) Vision-Strategy Document/Action Plan. Emphasis is placed specifically on subcomponents 1.1.3 (Ecology, Host Pathogen and Chemical Contaminants Relationships), and 1.1.4 (Intervention Strategies).
5.Significant Activities that Support Special Target Populations
|Number of new CRADAs and MTAs||1|
|Number of active CRADAs and MTAs||2|
|Number of invention disclosures submitted||1|
|Number of patent applications filed||3|
|Number of U.S. patents granted||1|
|Number of non-peer reviewed presentations and proceedings||6|
Cole, K., Farnell, M., Donoghue, A.M., Stern, N.J., Svetoch, E.A., Eruslanov, B.N., Volodina, L.I., Kovalev, Y.N., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Levchuk, V.P., Pokhilenko, V.D., Borzenkov, V.N., Svetoch, O.E., Kudryavtseva, T.Y., Reyes-Herrera, I., Blore, P.J., Solis De Los Santos, F., Donoghue, D.J. 2006. Bacteriocins reduce campylobacter colonization and alter gut morphology in turkey poults. Poultry Science. 85:1570-1575.
Georgsson, F., Porkelsson, A.E., Geirsdottir, M., Stern, N.J. 2006. The influence of freezing and duration of storage on campylobacter and indicator bacteria in broiler carcasses. International Journal of Food Microbiology. 23(7):677-683.
Sosunov, V., Mischenko, V., Eruslanov, B., Svetoch, E., Shakina, Y., Stern, N.J., Majorov, K., Sorokoumova, G., Selishcheva, A., Apt, A. 2007. Antimycobacterial activity of bacteriocins and their complexes with liposomes. Antimicrobial Chemotherapy. Vol 59. pg 919-925.