Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
VETERINARY MEDICINE
Non Technical Summary
Post-weaning diarrhea caused by enterotoxigenic E. coli (ETEC) strains continues to be a problem for the swine industry. The completion of this study will determine whether or not E. coli strains recovered from sick piglets that do not produce either F4 or F18 fimbriae are porcine pathogens. This will allow veterinarians and producers to make informed decisions regarding herd management when these E. coli strains are recovered from their animals.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Objective 1. Determine if F18 and K88 negative E. coli bind to epithelial cells in vitro. E. coli strains from sick piglets that do not produce any known fimbriae associated with post-weaning diarrhea/edema disease will be tested to their ability to adhere to epithelial cell lines using a standard adherence assay. Strains will also be screened for the production of enterotoxins and/or Shiga toxin 2e. Several strains that adhere in vitro to epithelial cell lines and produce various toxin combinations will tested in vivo in objective 2. Objective 2. Determine if F18 and K88 negative E. coli cause disease in experimentally infected piglets. Strains screened in objective 1 that adhere to epithelial cell lines well in vitro and produce high levels of enterotoxins(s) and/or Shiga toxin 2e will be tested in 3-week old piglets for their ability to cause disease in an experimental setting. Groups of 10 piglets each will be tested with each strain. Feces will be collected and
levels of enterotoxins and/or Shiga toxin 2e will be measured.
Project Methods
Objective 1. Determine if F18 and K88 negative E. coli bind to epithelial cells in vitro. A collection of E. coli strains from the ISU diagnostic laboratory recovered from pigs with diarrhea will be analyzed by multiplex PCR for toxin and fimbriae genes (sta, stb, lt, stx2e, f18, f4, eae) that are commonly associated with post-weaning diarrhea and/or edema disease. Strains that are negative for F18, F4 (K88) and eae (intimin) will be assessed for their ability to adhere to the Hep-2 cell line (human intestinal) and the IPEC-J2 (porcine jejunal) cell line using standard methods. The addition of mannose to the medium will ensure that adherence by type 1 fimbriae is negated. Strains that adhere to the cell lines will be tested for in vitro toxin production by ELISA (LT and Sta) or by Vero cell assay. Strains that produce the highest levels of enterotoxins(s) and/or Shiga toxin and adhere to both cell lines will be selected to use in objective 2. If the strains are
demonstrated to cause disease experimentally (objective 2), additional studies using scanning electron microscopy (SEM with negative staining) will be performed to determine if the strain(s) produce novel fimbriae or type IV pili that mediate adhesion. Transmission electron microscopy will also be used to examine the nature of the bacteria-epithelial cell relationship using the cell lines (tight adherence, invasion, capsule, ect). Objective 2. Determine if F18 and K88 negative E. coli cause disease in experimentally infected piglets. Young piglets (3 weeks old) will be randomly divided into groups and fed a high soy diet. One week after weaning the piglets will be challenged with 1010 cfu of each inoculum strain. Pigs will be monitored for clinical signs of edema disease and/or diarrhea. Individual fecal samples will be collected daily on days 1-10 for measurement of Stx2e and/or LT, and for quantitative bacterial counts of the inoculum strain on days 3, 7, and 10 post-inoculation
(pi. Weights of individual piglets will be taken on days -1, 4, 7 and 13 pi. Moribund piglets (excessive weight loss, severe dehydration, severe depression, neurological signs) will be euthanized and necropsied. At 2 weeks pi the remaining piglets will be necropsied. Gross lesions will be noted; terminal ileum, mid-ileum, jejunum and brain stem will be collected for histopathology (H & E staining). Sections will be examined for vascular necrosis indicative of edema disease and for bacterial layers. If layers are visible on histopathology sections, laser capture microscopy will be used to recover bacterial DNA for PCR evaluation to confirm that the layers are composed of the inoculum strain.