Table 4.
Main features for assessing an ETEC challenge trial, including the setting of the model and the criteria for evaluation of the challenge effectiveness
| Setting of the challenge model | ||
| Animal selection | ||
| Genetic markers which assist in ETEC susceptibility | ETEC F4: non coding region [54]; MUC4 [48]; MUC13 [49, 50] | ETEC F18: FUT1 [60]; BPI [62] |
| Sanitary status of the farm | Previous ETEC infection on the farm; Vaccine prophylaxis | |
| Suggested animal preconditioning | Treatment with narrow-spectrum antibiotics | |
| Fasting for 3 h + 62 mL of a 1.4% NaHCO3-solution | ||
| Control group | Negative control group -- > recommended if a feed intervention is predicted to influence the PWD via immunological mechanisms | |
| Timing of the inoculum | 4–7 dpw -- > multifactorial stress of weaning followed by a drop in passive immunity | |
| Inoculation method and dosage | Oral administration: to comply with the Refinement approach | |
| ETEC F4: 1×108 CFU/mL - 1.5 mL of 1010 CFU/mL | ETEC F18: 5 mL of 108 CFU/mL - 10 mL of a 1011 CFU/mL | |
| Evaluation of the challenge effectiveness | ||
| Diarrhea and related indicators | Fecal score: daily from a day before ETEC inoculation until the pigs recovered | |
| Diarrhea incidence: peak during the first week post-ETEC inoculation | ||
| Days with diarrhea: daily from a day before ETEC inoculation until the pigs recovered | ||
| Fecal DM: daily from a day before ETEC inoculation until the pigs recovered: 12.9–20.4% during dpi 1 to 3 | ||
| Rectal temperature | 39.0–39.5 °C pre-challenge to > 40.0 °C 6 h’ post-inoculation, then decreasing gradually | |
| Bacterial fecal shedding | Evaluation of ETEC F4 and F18 fecal shedding in the period from before inoculation and 3–4 dpi | |
| ETEC F4: 2 dpi (5.97 × 108 F4 per gram of feces), then decreasing suddenly | ETEC F18: 3–5 dpi (9.9 × 107 F18 per gram of feces), then decreasing gradually | |
| Immunoglobulins | Secretory IgA: mucosal and/or bile samples | |
| F4-specific and F18-specific IgA from blood plasma at 5 dpi | ||
| Expression of ETEC specific receptors in the intestinal mucosa | Post-mortem in vitro adhesion test | |
| Intestinal organ culture (PIOC) | ||
Abbreviations: ETEC Enterotoxigenic Escherichia coli, MUC4 Mucine 4, MUC13 Mucine 13, FUT1 Fucosyltransferase 1, BPI Bactericidal/permeability increasing protein, PWD Post-weaning diarrhea, dpw Days post-weaning, DM Dry matter, dpi Days post-inoculum, IgA Immunoglobulin A, PIOC Intestinal organ culture