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

  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

 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)