Location: Food Safety and Enteric Pathogens Research
2018 Annual Report
Objectives
Since antibiotics are neither recommended nor used for STEC treatment in humans or carrier-animals, strategies as described in the National Strategy for Combating Antibiotic Resistant Bacteria (CARB; 1) are needed and will be investigated for STEC control in cattle as exemplified in the following objectives:
Objective 1: Understand the impact of the bovine intestinal environment, especially at the rectoanal junction, and the molecular mechanisms that promote or inhibit colonization, adherence, and persistence of STEC in cattle and develop intervention strategies to control STEC colonization.
Subobjective 1A: Identify bacteria in the rectoanal junction (RAJ) microbiome that could effectively interfere with STEC colonization for possible use in probiotic applications.
Subobjective 1B: Identify bacterial ligands and tissue receptors involved in STEC adherence to the RAJ.
Objective 2: Formulate and assess the efficacy of vaccines for controlling STEC colonization of cattle based on whole-cell and subunit vaccines and identify proteins and epitopes conserved in STEC.
Objective 3: Define potential biomarkers using systems-based approaches that will allow the development of rapid diagnostic tests to identify STEC-colonized cattle.
Approach
Experimental animal and animal organ/tissue culture models will be used for determining qualitative and quantitative changes in bacterial communities constituting the rectoanal junction (RAJ) and fecal microbiomes, in response to colonization of cattle intestines by STEC serotype O157:H7 (O157). These microbiome changes will be correlated with the duration and magnitude of fecal O157 shedding by colonized cattle and compared to fecal/RAJ microbiome of non-colonized cattle. The results from these comparative studies will be used for identifying bacterial species that are part of the cattle intestinal microbiome and can compete effectively with O157 for bovine intestinal colonization. In vitro organ and tissue culture systems will be used for evaluating effects of these bacteria on O157 colonization at the RAJ. Proteomics-based techniques will be used for identifying proteins that are used by O157 to adhere to RAJ epithelial cells. Analogs mimicking these proteins will be designed and evaluated for their ability to interfere with O157 adherence to the RAJ epithelial cells. The existing O157 mutant vaccine strain will be modified and will be used as a whole-cell killed vaccine for determining if vaccination of cattle with the modified vaccine would show increased efficacy in controlling O157 colonization of cattle compared to the unmodified mutant strain-based vaccine. Metabolomics-based techniques will be used to identify metabolites present in blood samples of O157-colonized and non-colonized cattle. Few metabolites that are uniquely present in O157-colonized cattle will be evaluated for use as biomarkers to differentiate such cattle from the non-colonized animals.
Progress Report
Shiga toxin-producing Escherichia coli (STEC), often referred to as O157, are foodborne pathogens that can cause severe disease in humans. Cattle carry STEC in their intestines without any disease and shed STEC in their feces, which can result in contamination of cattle hides and carcasses during animal processing, contaminating food products. Since cattle remain disease-free over the course of STEC colonization, it is difficult to detect colonized animals and apply interventions to eliminate or reduce the bacterium prior to slaughter. Understanding the complex dynamics between animal, bacteria, and intestinal environment is important for developing intervention strategies to effectively reduce STEC in cattle prior to and/or at slaughter.
Under Objective 1: Subobjective 1A: Identify bacteria in the bovine rectoanal junction (RAJ) microbiome that can interfere with STEC colonization, researchers evaluated if O157 colonization altered bacterial communities (microbiome/microbiota) normally found at the RAJ of cattle experimentally challenged with O157. Nucleic acid was extracted from RAJ swab samples collected at various times post-challenge and subjected to 16S ribosomal ribonucleic acid (rRNA) gene sequencing. Preliminary results indicate small shifts in bacterial populations, and data analysis is ongoing. These data will provide important information about the impact of experimental O157 infection on the cattle RAJ microbiota and may aid in development of strategies to limit STEC colonization.
Also under the same Subobjective 1A, an in vitro cellular model of the RAJ was developed. An in vitro organ culture (IVOC) of the RAJ was optimized after several trials exploring different tissue scaffold and media conditions. The final protocol resulted in IVOCs that were viable for five days in culture without inclusion of anti-bacterial agents and environmental conditions mimicking the intestinal cell level (temperature, oxygen levels). Studies are ongoing to evaluate O157 interactions with the RAJ-IVOC, and for use as a model to evaluate strategies for limiting attachment and colonization at the epithelial layer. In addition, the RAJ-IVOC could provide a suitable alternative to a live animal to study O157 colonization at the intestinal tissue level in some instances.
In support of Objective 1: Subobjective 1B: Identify bacterial ligands and tissue receptors involved in STEC adherence, a unique protocol was standardized for identifying proteins on animal cells that interact with bacteria. This protocol is presently being used to isolate proteins present on different cells at the RAJ (tissue receptors) that directly interact with the STEC bacteria. Using a similar protocol, researchers also identified O157 proteins that interact with the two different RAJ cell types. After proteomic analysis, 84 O157 proteins that interact with the RAJ squamous epithelial (RSE) cells, and 89 O157 proteins that interact with the follicle associate epithelial (FAE) cells were identified. The isolated O157 proteins were used in an assay to evaluate ability to block adherence, and results indicate that the pool of O157 proteins were able to block adherence of O157 to RSE and FAE cells.
Vaccines serve as a potential intervention strategy to reduce O157 colonization and fecal shedding in cattle. Under Objective 2: Formulate and assess the efficacy of vaccines for controlling STEC colonization of cattle, a study was completed to further optimize the vaccine formulation. Specifically, an inactivated, modified strain of 0157 was mixed with one of the three different commercially available adjuvants and used in an immunogenicity and efficacy study in cattle. Results indicate that adjuvant type directly impacts the immune response to vaccine antigen, and degree of efficacy. Overall, these data are important for future testing, and suggesting that future evaluation will require formulation with specific types of adjuvant for significantly limiting shedding of O157 from cattle. In addition, research included evaluation of the intestinal microbiota, and results indicate that vaccination alone can impact bacterial community structure in the feces. Thus, it will be important to understand if this alteration in microbiota has any negative (or positive) impact on animal productivity.
In support of Objective 3: Define potential biomarkers using systems-based approaches that will allow for the development of rapid diagnostic tests to identify STEC-colonized cattle, a collaboration was established with Iowa State University, which led to the identification of 33 blood metabolites that are uniquely present in blood of STEC colonized cattle, but not non-infected controls. Work is ongoing to validate these findings using blood from cattle colonized with other O157 strains.
Accomplishments
1. Identified genetic features unique to super-shed Escherichia coli O157 (O157). Shiga-toxin producing Esherichia coli serotype 0157 causes intestinal disease in humans, but colonizes cattle intestines without causing disease. Cattle that shed O157 in numbers greater than or equal to 10(4) colony forming units per gram of feces are referred to as super-shedders. Super-shedding cattle can easily disseminate O157 within large herds, which may increase transmission to meat at slaughter. The super-shed O157 bacteria may possess unique genetic features contributing to their increased adherence and persistence in the bovine intestinal tract. In a recently completed study by ARS researchers in Ames, Iowa, the genomes of two super-shed O157 strains were sequenced, analyzed and compared to other previously characterized O157 strains. Super-shed O157 have unique, recently acquired, genetic features especially in genes associated with motility, adherence, and metabolism that could influence their persistent colonization of bovine intestines. The proteins encoded by these genes could be developed into blocking therapies aimed at preventing super-shed O157 colonization of cattle. Limiting shedding from cattle is important for maintaining a safe and secure global food supply.
2. Improved a killed whole-cell O157 vaccine formulation to enhance immune responses in cattle. Shiga-toxin producing Esherichia coli serotype 0157 results in foodborne disease in humans, but colonizes cattle intestines without causing disease. Vaccination of cattle is considered an important option for reducing O157 carriage and thereby reducing human illnesses. However most vaccines, especially killed or chemically-inactivated whole-cell bacterial vaccines, require adjuvants (immune response enhancer) for optimal response against vaccine antigens. ARS researchers in Ames, Iowa, performed a study in which cattle were vaccinated with two doses of a vaccine formulation prepared by combining a mutant strain of O157 and a commercially available adjuvant. The vaccine-adjuvant combination not only enhanced immunity against O157 but also efficacy. Specifically, fecal shedding of O157 was significantly reduced after experimental challenge in animals that received the adjuvant formulation. These findings show that vaccine formulations for STEC must be tailored for optimal immune activation and memory response to reduce O157 colonization and fecal shedding in cattle after exposure. Successful vaccination protocols can provide a mechanism for limiting food and environmental contamination, which are critical for reducing transmission of O157 bacteria to humans.
