Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/6/2012
Publication Date: 1/17/2012
Citation: Sharma, V.K., Sacco, R.E., Kunkle, R.A., Bearson, S.M., Palmquist, D.E. 2012. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle. Infection and Immunity. 80(4):1333-1342. Interpretive Summary: Escherichia coli O157:H7 (O157) are Shiga toxin-producing bacteria that infect humans through the consumption of contaminated meats, such as ground beef, unpasteurized dairy products, fresh produce and water. Infections in humans with these bacteria result in symptoms ranging from watery to bloody diarrhea that in young children and elderly individuals could lead to kidney malfunction and even death. Among animals, cattle are the major reservoir for O157. O157 bacteria can colonize and persist in the intestines of cattle without causing disease, but colonized animals can shed O157 in their feces at variable magnitudes and for extended periods of time. Cattle feces are the major risk factor for the contamination of animal hides in feedlots and carcass contamination at slaughter plants leading to the downstream contamination of meat products. Cattle manure is also a major risk factor for the contamination of water resources and environmental spread of O157. According to CDC estimates, O157 causes over 73,000 human illnesses per year with over 2,000 cases of hospitalizations and 61 deaths. Combined economic losses due to human illnesses and food contamination are estimated at almost a billion dollars. Thus, a concerted approach is needed to identify bacterial factors that promote O157 colonization of cattle intestines leading to long-term persistence and fecal shedding of these bacteria by these animals. Bacterial factors identified through the successful execution of these studies could then be tested as potential targets for incorporation into novel vaccines for reducing colonization and persistence of O157 in these animals. In addition, these bacterial targets could potentially be tested in screening procedures designed to identify inhibitors (biological or chemical) of O157 growth by blocking the function of these targets. Thus, combinations of vaccines and biological inhibitors could provide highly effective remedies for reducing colonization and fecal shedding of O157 in cattle, thus reducing the risks of downstream contamination of meats, produce, and water.
Technical Abstract: The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-studied, but the relevance of a functional T3SS and levels of secreted proteins in the colonization process has not been examined. In this study, we compared hha and hha sepB mutants, which were up-regulated in LEE expression but contained either a functional (hha mutant) or a defective T3SS (hha sepB mutant), to the wild-type E. coli O157:H7 in intestinal colonization of cattle. Weaned (n = 4 per group) calves were inoculated orally with 10**10 cfu of the wild-type or hha or hha sepB mutant strains, and feces were collected for 28-days to determine the magnitude and duration of fecal shedding of the inoculated strains. Tissues from the recto-anal junction (RAJ) were collected on day 28 to determine colonization of this site and to assess transcriptional levels of various immune modulators produced by the RAJ cells. The calves inoculated with the hha sepB mutant showed no detectable fecal shedding of this mutant 16-days post inoculation. No significant differences (p less than .05) were observed in the duration nor the magnitude of fecal shedding in calves inoculated with the wild-type strain compared to the hha mutant strain. Microscopic examination of RAJ tissues showed no detectable A/E lesions irrespective of the inoculated strains. The expression of pro-inflammatory cytokines in RAJ was similar between the three groups of calves except for the up-regulation of IL-1 beta in calves inoculated with the hha sepB mutant. The results described herein indicate that T3SS is essential for intestinal colonization, but increased secretion of LEE proteins does not enhance the duration and the magnitude of fecal shedding.