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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

From: Methodology and application of Escherichia coli F4 and F18 encoding infection models in post-weaning pigs

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)
  1. 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