Adhesin-Specific Nanoparticles for Removal of Campylobacter jejuni from Poultry

Campylobacter jejuni are bacteria which cause abdominal cramps and profuse bloody diarrhea in humans. Large case-control studies have shown that 50-70% of campylobacter infections can be traced to poultry meat products which have been contaminated with intestinal contents during processing. There are currently no practical and effective methods for addressing this food-safety problem. The overall goal of this program is to develop and evaluate a novel, farm-based strategy for the removal of campylobacters from poultry intestinal tracts prior to processing. This strategy will employ bioactive nanoparticles specifically designed to bind to the biomolecular structures on the surfaces of campylobacters. A broad variety of potentially bioactive nanoparticles will first be synthesized. They will then be tested in vitro for bioactivity assessment and human handling and exposure safety evaluation in order to identify the most effective and safe nanoparticles designs. These will then be orally administered to both chickens and turkeys under actual farm conditions to dislodge colonized campylobacters from the intestinal lining and facilitate their fecal expulsion. The birds' intestinal contents will be tested for reductions of campylobacter levels to assess the effectiveness of this treatment for removal of campylobacters and other food-borne microbial pathogens in poultry products. This overall program will be accomplished through the integration of research, education, and extension for the control of food-borne microbial pathogens in poultry meat products.
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The proposed program will be undertaken in four inter-related components. In the first part, nanoparticles will be synthesized in various controlled size ranges and functionalized with selected surface functional groups. Two kinds of nanoparticle systems will be employed. One is based on the self-assembly of organic polymers structured with intrapolymer binary pseudo-phase separation character. Here, self-assembly is driven by the experimental conditions for micellar formation. Average sizes of the nanoparticles will be adjusted by controlling the micellar formation conditions. Upon the formation of the nanoparticles, a photochemical curing process will be applied in order to produce intra-particle cross-links, and thus "lock" in the nanoparticle structure to the formed shapes, sizes, and morphology. The other nanoparticle system will be based on the organic chemical functionalization of inorganic nanoparticles produced via applications of supercritical fluid processing techniques. The nanoparticles will be functionalized with various polysaccharide and polypeptide moieties selected to promote adhesion with the targeted bacterial systems. In the second part of the program, the bioactivity of the synthesized nanoparticles will be assessed using in vitro cell culture studies with the selected target microbial pathogens (Campylobacter jejuni , Campylobacter coli, Salmonella enteritidis, Escherichia coli, and Lactobacillus acidophilus.). The nanoparticles demonstrating the greatest ability to agglutinate the targeted bacterial cells will be evaluated in terms of human handling and exposure safety as the third component of the program. Finally, the nanoparticles demonstrating the greatest bioactivity and which have passed the handling and exposure safety protocols, will be used in actual farm-based extension service studies. In this fourth component of the overall program, bioactivity studies will be conducted to assess the ability of the designed nanoparticles to agglutinate the targeted bacteria strains in vivo and purge them from the intestinal tracts of chickens and poultry prior to food-processing. If successful, this research has the potential to provide a very effective and cost efficient method to control food-borne microbial pathogens at their source.
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The objective of this project was to develop and evaluate bioactive nanoparticles (NPs) that exhibit specific binding capabilities to targeted bacterial agents that have the potential to serve as non-antibiotic based anti-bacterial agents for use in poultry production. The program was divided up into 4 components: A) Synthesis & characterization, B) In vitro bioactivity assessment, C) Assessment of the use of the bioactive NPs in poultry, and D) In vitro and in vivo exposure sensitivity evaluation. Under part A, polystyrene-based NPs were developed as the NP system. Different types of bioactive agents (carbohydrates (mannose, galactose), peptides, proteins, and protein-coupled carbohydrates) were attached to the NPs by polyethylene glycol linkers. Under part B, we demonstrated that mannose-NPs specifically adhere to and agglutinate E. coli strain ORN178 (fimH+). Pre-exposure of ORN178 to free mannose was shown to block NP-ORN178 binding, NP-ORN178 binding and bacterial agglutination was directly visualized by transmission electron microscopy and fluorescence microscopy, and NP exposure caused a 99% reduction in colony forming units of ORN 178. In addition, NPs did not cause ORN208, which express abnormal pili, to aggregate, thus indicating that binding was occurring via the bacterial pili adhesins. Galactose-NPs did not bind to either type of E. coli and neither mannose nor galactose-NPs bound C. jejuni. This finding further demonstrates that bioactive NPs can be developed that are bacteria-specific. Although we were seeking to find NPs that would bind to C. jejuni, the fact that NPs with specific bioactivity were developed provides evidence that it is possible that C. jejuni could also be targeted with other types of bioactive agents. Under part C (in vivo evaluation in poultry), studies determined that the administration of the mannose-NPs to poults by oral gavage did not have any apparent physical effect on weight gain and behavior. No effects were found, however, in regards to the ability of the synthesize NPs inhibit C. jejuni infection. This result is consistent with the in vitro bioactivity studies and indicates that the types of bioactive agents used in these studies (e.g., mannose, galactose) are not bioactive with respect to C. jejuni. Finally, in part D, cell culture studies, which were conducted with human fibroblasts (lung, colon, and dermal) and rat lung macrophages, showed no significant levels of NP toxicity. In addition, in vivo studies were conducted using IRB-approved protocols. NZW rabbits were used for dermal and ocular studies and Sprague Dawley rats were used for inhalation and ingestion studies. The results showed no significant levels of edema or erythema from dermal tests and no erythema, chemosis, or iritis from ocular tests. Histological slides from organ tissues from the ingestion and inhalation studies did not show any significant levels of inflammation. Therefore there is no indication of any significant level of sensitivity reaction cause by nanoparticle exposure.
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This program has shown that it is feasible to synthesize nanoparticles (NPs) that are able to directly bind to and agglutinate targeted bacteria. Furthermore, the developed anti-bacterial agents have not been found to exhibit any significant levels of toxicity effects at either the cellular or tissue levels. These results are of importance in order to provide indication that this type of bioactive NP design can be safely used in poultry without interfering with normal growth and development, and that they could be safely handled by those administrating the NPs in an agricultural setting for the control of bacterial infection. This was the expected result because the NPs function as antibacterial agents by physically binding to the bacteria with the concept of then inhibiting them from being able to infect the host, as opposed to actual killing the bacteria through intracellular biochemical processes. Most importantly, although further research is needed, these antibacterial agents have the potential to provide alternatives to the use of antibiotics for the control of bacterial infection in the poultry industry. Also, because the bioactive NPs function by binding to the same functional units of the bacteria (i.e., bacterial adhesins) that the bacteria use to infect their host, mutations that may occur that would inhibit NP binding would also likely interfere with the ability of the bacteria to bind to the host. This potentially provides a system that will be inherently resistant to the development of infectious strains of bacterial that are resistant to these agents.