Location: Food Safety and Enteric Pathogens Research
2019 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), including STEC serotype O157:H7 (O157), are foodborne pathogens that can cause severe disease in humans. Cattle do not get ill from O157 infection, and instead, carry O157 in their intestines which is then shed in their feces. Feces with O157 can contaminate the environment, and cattle hides and carcasses during animal processing, which contributes to food product contamination. Since cattle remain disease-free over the course of O157 colonization, it is difficult to detect colonized animals and apply interventions to eliminate or reduce the bacterium prior to animal processing. Understanding the complex dynamics between animal, bacteria, and intestinal environment is important for developing intervention strategies to effectively reduce O157 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 identified bacterial communities in the RAJ microbiome that distinctly change in the presence/absence of O157. Changing microbiota bacteria could be identified only at the genus level and not at the individual species level preventing the identification of specific bacteria for use as a probiotic to limit O157 colonization at the bovine RAJ. As an alternative, host expressed proteins (cytokines) produced by immune cells at the RAJ are being evaluated for their ability to alter RAJ cells and subsequently limit O157 attachment. Previous data suggest that vaccination with a particular O157 formulation can provide protection, and immune cells in the intestine are producing cytokines that may impact epithelial cells in a manner that limits attachment.
Cattle vaccination is one potential method to limit O157 STEC carriage in cattle. In support of Objective 2: Formulate and assess the efficacy of vaccines for controlling STEC colonization of cattle, an animal study was completed to evaluate the ability of vaccination to induce O157-specific immunity and to reduce O157 STEC in cattle. The vaccine was formulated with a mutant O157 STEC strain which was formulated with different adjuvants and delivered in one or two doses. To assess activation of the immune response following vaccination, researchers isolated blood, blood cells, and feces from cattle at various times following vaccination, and after cattle were inoculated with O157. Analysis of vaccine immunogenicity are ongoing, but preliminary data suggest that it was reduced compared to vaccine studies using a different vaccine strain. In addition, shedding was not limited as observed with other formulations. Collectively, the data suggest that vaccine approaches can limit O157 in cattle, but the vaccine formulation is critical for protection, and specific bacterial compounds are required in the vaccine for efficacy.
Although cattle are a primary reservoir of O157, sheep also harbor O157 and are often used as an economical, alternative ruminant model to study immune activation and STEC colonization. Further refinement of the in-house designed O157 vaccine strain and formulation considerations were warranted prior to a large cattle trial. Thus, sheep were used for rapid and cost-effective evaluation of immune activation after administration of different whole-cell O157 vaccine formulations. Sheep were vaccinated with one of three different vaccine formulations, and blood and feces collected to evaluate O157-specific antibody, and O157-specific cell responses. As expected, vaccination induced O157-specific antibody in the feces and blood. Adjuvant did impact responses, as did the modifications to the O157 vaccine strain. Data analysis is ongoing to further differentiate differences in immune cell activation and identify the O157 proteins recognized by antibody after vaccination with different formulas. The data will be used to design a vaccine trial in cattle, selecting the vaccine expected to provide the greatest protection against challenge.
Rapid and reliable identification of cattle carrying O157 is warranted to reduce carcass contamination. While methods are available to detect O157, they can take days for results. 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, researchers continued analysis of blood metabolites as targets for a rapid, pen-side diagnostic test. Initially, 33 blood metabolites recognized as uniquely present in blood of STEC-colonized cattle were identified in collaboration with researchers at Iowa State University. Additional analysis was performed with different mass spectrometry techniques, and additional blood samples from cattle with and without STEC. Further statistical analysis enabled shortlisting 15 of 33 metabolites for use as potential biomarkers of bovine colonization with O157. In support of the development of a targeted assay, antibodies to 7 of the 15 metabolites were developed and are currently being evaluated in laboratory assays for their ability to accurately identify the specific metabolites.
Accomplishments
1. O157-specific fecal antibodies limit attachment of O157 to cattle intestinal cells. O157 attachment to intestinal cells leads to intestinal colonization and fecal shedding of O157 in cattle. Reducing shedding of O157 by cattle through vaccination is a practical approach for lowering both the risk of O157 contamination of foods and incidence of O157-related human illnesses. ARS researchers in Ames, Iowa, demonstrated that vaccinating cattle with a specific vaccine formulation (a mutant O157 with immune stimulant) induced an immune response that reduced O157 colonization in cattle. One of the immune response factors (IgA) detected in the feces of vaccinated cattle was able to block attachment of O157 to epithelial cells. Vaccination also induced specific immune cells and cytokines known to limit bacteria attachment to cattle intestinal cells. These results provide a platform for developing improved vaccines for controlling O157 colonization and fecal shedding in cattle; outcomes essential for preventing transmission of these bacteria to humans.
2. Stress alters gastrointestinal microbiome in light-weight dairy calves. Stress can lead to changes in normal behavior, growth, immunity, and impact gastrointestinal tract (GIT) microbiome which in turn can negatively affect cattle health. Administration of analgesics may reduce pain, but it’s unclear if the biological impact of stress is also mitigated with analgesics. ARS researchers in Ames, Iowa, and colleagues at Iowa State University and Kansas State University discovered that dehorning and castration, two common stressors, caused changes to the GIT microbiome regardless of analgesic administration, and these changes were most pronounced in lightweight calves. The work highlights that methods to reduce pain may not alter the biological impact of a stressful procedure, and different approaches may be warranted in animals of different sizes. Reducing stress is important for overall animal health which in turn may influence efficacious immune responses to vaccines and other therapies.
3. Identified O157 protein involved in initial attachment to human intestinal cells. O157 uses a well-characterized mechanism for intimately attaching to human intestinal cells and causing disease, and mechanisms of attachment to human versus cattle cells are different. Prior to intimate cell attachment there is a process of initial attachment, which is not well understood. ARS researchers in Ames, Iowa, along with collaborators at Pennsylvania State University identified the O157 protein carbon starvation-inducible lipoprotein, or Slp, which is involved in initial attachment. Slp interacts with a protein on the human intestinal cells called the polymeric immunoglobulin receptor or pIgR. O157 protein such as Slp, involved in initial attachment, may be targeted for human therapeutic interventions, such as vaccines that could interfere with O157 attachment to human intestinal cells and hence prevent disease.
Review Publications
Mir, R.A., Kudva, I.T. 2019. Antibiotic resistant Shiga toxin-producing Escherichia coli: An overview of prevalence and intervention strategies. Zoonoses and Public Health. 66:1-13. https://doi.org/10.1111/zph.12533.
Mir, R.A., Kleinhenz, M.D., Coetzee, J.F., Allen, H.K., Kudva, I.T. 2019. Fecal microbiota changes associated with dehorning and castration stress primarily affects light-weight dairy calves. PLoS One. 14(1):e0210203. https://doi.org/10.1371/journal.pone.0210203.
Sharma, V.K., Akavaram, S.N., Schaut, R.G., Bayles, D.O. 2019. Comparative genomics reveals structural and functional features specific to the genome of a foodborne Escherichia coli O157:H7. BMC Genomics. 20:196. https://doi.org/10.1186/s12864-019-5568-6.
Schaut, R.G., Boggiatto, P.M., Loving, C.L., Sharma, V.K. 2019. Cellular and mucosal immune responses following vaccination with inactivated mutant of Escherichia coli O157:H7. Scientific Reports. 9:6401. https://doi.org/10.1038/s41598-019-42861-z.
Fedorchuk, C., Kudva, I.T., Kariyawasam, S. 2019. The Escherichia coli O157:H7 carbon starvation-inducible lipoprotein Slp contributes to initial adherence in vitro via the human polymeric immunoglobulin receptor. PLoS One. 14(6):e0216791. https://doi.org/10.1371/journal.pone.0216791.
