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Development of Chitosan Nanoparticles Targeting Pathogenic Escherichia Coli in Beef and Dairy Cattle

Jeong, Kwang Cheol; Dilorenzo, Nicolas; Galvão, Klibs N; Lamb, G Clifford
University of Florida
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Our long-term goal is to develop solutions for improving animal health in the agricultural setting and enhancing food safety. In this proposal, we will develop applicable chitosan nanoparticles (CN) to reduce pathogens in beef and dairy cattle, targeting Shiga toxin-producing E. coli (STEC) as a model organism. Our preliminary data showed that chitosan microparticles (CM) significantly reduces E. coli O157:H7 in the gastrointestinal tract of cattle and exerts antimicrobial activity by binding to an outer membrane protein, OmpA. Our central hypothesis is that we can increase antimicrobial activity of CN by enhancing the binding activity of CN to pathogens in the GI tract of cattle (Aim #1) and increase specificity to recognize and eliminate target pathogens by conjugation of pathogen specific antibodies to CN (Aim #2). Our rationale for these studies is that the development of CN will contribute a strong impact on reducing the prevalence of zoonotic diseases on farms, enhancing the sustainability of US agriculture.

Specific Aim #1) Enhancement of antimicrobial activity of chitosan nanoparticles. Based on our pilot data, we hypothesize that we can increase antimicrobial activity of chitosan nanoparticles by fabricating properties and sizes of particles that will exert strong antimicrobial activity in the GI tract, allowing us to apply CN as an alternative antimicrobial agent for farm animals. Aim #1, Obj. 1. Development of CN. Aim #1, Obj. 2. Screening of CN candidates. Aim #1, Obj. 3. In vitro risk assessment of CN.

Specific Aim #2) Development of chitosan nanoparticles that selectively bind to E. coli O157:H7 in the GI tract. The working hypothesis is that conjugated CN-anti O157 antibodies (CN-Ab) against E. coli O157:H7 will provide specificity to CN, and thus CN will selectively kill E. coli O157:H7 in the GI tract of cattle. Aim #2, Obj. 1. Chicken antibody (IgY) production. Aim #2, Obj. 2. Evaluation of CN+Ab. Aim #2, Obj. 3 In vitro risk assessment of CN+Ab.

Specific Aim #3) Evaluation of the efficacy of CN and CN-Ab at eliminating STEC and E. coli O157:H7 from dairy and beef cattle at the pre-harvest level. Based on our preliminary data, we expect to observe enhanced antimicrobial activity of CN and CN-Ab when they are administered to cattle orally, providing potential application as a feed additive. Aim #3, Obj. 1. Identification of super-shedders. Aim #3, Obj. 2. Reducing STEC O157 and Non O157 STEC in dairy and beef cattle. Aim #3, Obj. 3. In vivo Risk assessment of CN and CN-Ab administration.

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Pathogenic E. coli strains are significant disease causing organisms that negatively impact not only animal and human health, but also food safety. The clearance of pathogenic E. coli at the pre-harvest level (on farms) is one of the most effective ways to prevent foodborne illness caused by zoonotic pathogens. However, controlling zoonotic pathogens on farms is challenging because multiple layers of risk factors, such as animal and environmental factors, exist in agricultural settings. Currently, several pre-harvest intervention methods have been developed and tested to mitigate pathogens, yet practical solutions that can be applied on farms are not available. In this proposal, we will develop applicable chitosan nanoparticles (CN) to reduce pathogens in beef and dairy cattle, targeting Shiga toxin-producing E. coli (STEC) as a model organism. Moreover, by conjugation of pathogen specific antibodies to CN, we will develop a strategy to target only desired pathogens without disrupting healthy microflora in the gastrointestinal tract. This research is innovative and significant because the outcomes of this project are expected to lead to practical and effective new solutions for mitigating pathogenic E. coli on farms. Furthermore, the solutions can be applied to solve the problems caused by many other foodborne pathogens at the pre-harvest level, greatly enhancing the sustainability of US agricultural systems by improving food safety and animal health.

Specific Aim #1) Enhancement of antimicrobial activity of chitosan nanoparticles. In specific Aim #1, we will enhance CN activity by optimizing CN preparation conditions and reducing particle sizes. We found that current CN (size = 600 nm ± 10 nm) binds to OmpA, resulting in disruption of membrane integrity. Our working hypothesis is that if we reduce the size of CN (<100 nm), we can increase the binding specificity of CN to OmpA (diameter of OmpA is about 2 nm) through reducing non-specific binding of CN to other surface molecules. Our hypothesis is supported by our own data that the ompA mutant showed decreased CN binding compared to the WT strain. Aim #1, Obj. 1. Development of CN. 1.1.2. Preparation of nanoparticles. Chitosan nanoparticles will be prepared as follows with modifications to test the variables mentioned above. A 1% (wt/vol) chitosan solution will be prepared from chitosan (Sigma Chemical Co., St. Louis, MO) in 2% acetic acid and 1% Tween 80. To facilitate cross-linking, the chitosan solution will be stirred and sonicated with the addition of 2 ml of sodium sulfate (10% [wt/vol]) into 100 ml of chitosan solution. Mixing and sonication will be continued. Total length of sonication time and sonication power will be varied to obtain desired chitosan nanoparticle size (<100 nm). The CN will be collected by centrifugation (12,000 x g) for 10 minutes, washed three times with sterile water, and freeze-dried. 1.1.3. Characterization of CN. Particle size and zeta potential will be measured using a Zetasizer Nano-ZS (Malvern instruments). The zeta potential will be measured in demineralized water at neutral pH. Morphology of CN will be analyzed by scanning electron microscopy (SEM) to evaluate approximate size, form, uniformity, and the formation of aggregates. Aim #1, Obj. 3. In vitro risk assessment of CN. In vitro risk assessment will be conducted to evaluate potential negative impact of developed CN. Batch culture studies will be conducted to determine the effects of CN on in vitro ruminal fermentation and in vitro true dry matter digestibility (IVTDMD) using 10 fold higher doses than that will be used for the animal studies. In vitro fermentation. In vitro incubations will be conducted in 250-mL bottles and gas production kinetics will be recorded using the Ankom Gas Monitoring System (Ankom Technologies, Fairport, NY). Aim #2, Obj. 1. Chicken antibody (IgY) production. 2.1.2. Preparation of antigens. Target genes (i.e. the eae gene, specific and necessary for EHEC Type III secretion system) for antigens will be cloned and expressed in an avirulent E. coli strain under Ptac promoter in pQE30. After IPTG induction, cells will be collected and lysed by sonication. The cell lysate will be passed through a Ni-NTA column to purify and then His-tagged proteins will be eluted with elution buffer. The purified proteins will be separated using a preparative SDS-PAGE gel to elute antigens without contamination with other proteins. Gel-purified proteins will be used for the vaccination of hens. In addition, we will produce IgY against lipopolysaccharide (LPS) of STEC strains. The Big 6 non-O157 STEC serotypes (O26, O45, O103, O111, O121, and O145) have different O-antigens. Therefore, we will isolate O-antigens from the Big 6 serotype strains (purchased from the ATCC) and E. coli O157:H7 strains using the phenol-water extraction method to vaccinate the hens (Morrison and Leive 1975). 2.2.2. Specificity against E. coli O157:H7 and antimicrobial activity. Specificity of CN+Ab conjugant will be assessed by in vitro antimicrobial assay (Aim #1, Obj. 2) with minor modifications. Bacterial cultures containing E. coli O157 (eae +) and E. coli K12 (eae -) will be co-incubated with CN+Ab for 6 hours, and enumeration of viable cells will be completed by the plating count method on E. coli O157 specific agar (MacConkey + cefixime + tellurite) and non-selective media. If CN+Ab is specific to E. coli O157, E. coli K12 strain will not be affected by CN-Ab. Moreover, IgY produced against O-antigen, will be evaluated if it can recognize E. coli O157 specifically in a mixture of other serotypes (i.e. the Big 6 non-O157 STEC). To verify the specificity, we will conduct multiplex PCR to identify the surviving colonies. Aim #3, Obj. 1. Identification of super-shedders. 3.1.3. Identification of super-shedding cattle to evaluate CN or CN+Ab efficacy. Rectal anal junction (RAJ) swab samples will be randomly collected from approximately 300 cattle from each farm at NFREC and DRU and be transported to the lab in Cary-Blair transport medium on ice within 6 hours of collection to minimize bacterial growth. Our preliminary data indicate that about 10% of cattle in the herd carry more than 104 STEC/g of feces, thus we expect to isolate about 30 super-shedders from each farm. Swab samples will be resuspended in 2 ml of Tryptic soy broth (Difco) and one milliliter of resuspended samples will be spun briefly to collect bacterial cells, then plated on MacConkey agar (Difco) to determine the number of Gram-negative bacteria. The other 1 ml of samples will be frozen in 15% glycerol at -80°C for further studies. Aim #3, Obj. 2. Reducing STEC O157 and Non O157 STEC in dairy and beef cattle. 3.2.1. CN and CN-Ab treatment against super-shedders. Once animals are determined as super-shedders (shedding >104 STEC/g of feces), they will be housed in Feed Efficiency Facility (FEF) located at NFREC and DRU. The FEF is equipped with a Grow Safe system that allows the measurement of individual feed intake via radio frequency identification. Super-shedders will be grouped into three pens of 10 cows and the two treatment groups (CN or CN-Ab) will be compared with a non-treatment group (control group). Eight grams of CN or CN-Ab will be mixed with 3.6 kg of feed and administered daily for 6 days to the treatment group. We tentatively decided to treat animals with 8 grams of CN or CN-Ab for 6 days according to the previous data (Jeong, et al. 2011) where E. coli O157:H7 was effectively removed from the GI tract of cattle with CM treatment in 60% of treated animals. However the dose of CN and CN+Ab will be adjusted according to the results of antimicrobial assay of Aim #1 and Aim #2. STEC and STEC-O157 will be enumerated daily for 6 days to determine the antimicrobial activity of treatment. Aim #3, Obj. 3. In vivo Risk assessment of CN and CN-antibody treatment. 3.3.1 Heath evaluations. A complete physical examination will be performed on every cow during the treatment and monitoring period (total of 8 days) to evaluate if CN or CN-antibody treatment will have any negative effect on cow's health. Physical examination will include evaluation of attitude, respiration and heart rate, rumen contraction rate, rectal temperature, and fecal score. Sick cattle usually present with one of the following: decreased attitude (depressed), decreased or increased respiration, heart, or rumen contraction rate, increased rectal temperature, or change in fecal consistency (looser or firmer) (Galvao, et al. 2010).

Funding Source
Nat'l. Inst. of Food and Agriculture
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Natural Toxins
Bacterial Pathogens
Escherichia coli
Sanitation and Quality Standards
Meat, Poultry, Game