Investigation of host mechanisms associated with Escherichia coli O157:H7 super-shedding in beef steers

• Author / Creator

  Wang, Ou

• Cattle shedding > 104 CFU E. coli O157/gram of feces are defined as super-shedder (SS) which are a major disseminator of this foodborne pathogen into the environment. The recto-anal junction (RAJ) has been identified as the primary colonization site of E. coli O157within the gastrointestinal tract. To date, the mechanisms of E. coli O157 super-shedding in beef cattle remain unidentified. This thesis aimed to identify host mechanisms that are potentially associated with super-shedding through performing four studies on host gene expression throughout the intestinal tract and associated regulation mechanisms. Study 1 performed comparative transcriptomic analysis of rectal tissue collected from 5 SS and 4 cattle free from fecal E. coli O157 (non-shedders, NS) using RNA-Seq, and identified 58 differentially expressed (DE) genes including 11 that were up-regulated and 47 that were down-regulated in SS compared to NS. Functional analysis of these DE genes revealed that 31 down-regulated genes were potentially associated with reduced innate and adaptive immunity including decreased quantity and migration of immune cells such as lymphocytes, neutrophils and dendritic cells in SS. This suggests that the RAJ of SS may be less effective in terms of immune protection. Study 2 performed RNA-Seq to characterize and compare the transcriptomes of the whole intestinal tissues of SS and NS to understand whether other gut regions could also be associated with super shedding. In total, 351 DE genes were identified throughout the gastrointestinal tract between SS and NS, with 101 being up-regulated and 250 down-regulated in SS. Functional analysis of these DE genes suggested increased T-cell responses and cholesterol absorption in the distal jejunum and descending colon, and decreased B-cell maturation in the distal jejunum of SS. Further, single nucleotide polymorphism analysis of these DE genes identified association between 33 SNPs in 7 DE genes and E. coli O157 shedding. The functions of these 7 genes were associated with leukocyte activation and cholesterol transportation, suggesting that host genetic variation may influence gene expression leading to altered immune functions and cholesterol transportation in SS. Study 3 analyzed miRNAomes of the whole intestinal tract of SS and NS, aiming to identify the possible post transcriptional regulatory mechanisms that altered gene expression observed in studies 1 and 2. In total, the number of DE miRNAs ranged from 1 (in descending colon) to 8 (in distal jejunum) and 7 miRNAs were up-regulated and 7 were down-regulated throughout the gut of SS. Functional analysis indicated that the DE miRNAs potentially regulate genes involved in host immune function including hematological system development and immune cell trafficking. These findings suggest that the alternation of miRNA expression may be one of the regulatory mechanisms responsible for the altered expression of genes associated with immune function in the gut of SS. In study 4, bacterial 16S rRNA gene amplicon profiling was performed to characterize the RAJ mucosa associated microbiota and their functions (using PICRUSt). Differential abundance analysis of operational taxonomic units (OTUs) identified 2 OTUs unique to SS which potentially represented members Bacteroides and Clostridium, and 7 OTUs unique to NS which potentially represented members of Coprococcus, Prevotella, Clostridium and Paludibacter. The relative abundance of microbial taxa and their function were correlated with the DE genes of RAJ reported in study 2, with results suggesting that Clostridium, Coprococcus and Paludibacter may alter the host host epithelium in a manner that deters the colonization of the RAJ by E. coli O157. In summary, this thesis provides knowledge of the host gut gene expression, miRNA regulation, and host-microbial interaction in beef cattle that differ in their E. coli O157 shedding capacity. Our findings suggest that E. coli O157 shedding in cattle could be systematically attributed to differences in host immune responses, genetic variation, miRNA regulation, and the nature of the gut microbiota.

• Subjects / Keywords

  • Gut microbiome
  • Super-shedder
  • Transcriptome profiling
  • miRNA profiling
  • E. coli O157

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3HM5320R

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.