By Rodrigo C. Paiva and Marcelo N. Almeida, Iowa State University; and Lucina Galina Pantoja, PIC

Escherichia coli is a type of gram-negative bacteria that can cause disease in pigs, such as neonatal and post-weaning diarrhea, septicemia, polyserositis, arthritis, mastitis, urogenital infections and edema disease. The Iowa State University Veterinary Diagnostic Laboratory has observed an increase in cases of post-weaning diarrhea caused by E. coli from 2019 through 2023.

Genotyping conducted on a subset of post-weaning colibacillosis cases from previous years (2010-2018) indicated a balance between F18 and K88 fimbrial types, with 56.9% (range: 48.8% to 63.8%) of isolates positive for F18 and 43.1% (range: 36.2% to 51.2%) for K88. However, during the period of increased post-weaning colibacillosis diagnoses, F18 E. coli has become the predominant fimbrial type in 77.9% (range: 63.8% to 88.8%) of cases.

Various reasons have been speculated for the heightened activity of F18 E. coli, including changes in antimicrobial practices due to market restrictions and stricter regulations, increased antimicrobial resistance, higher virulence in contemporary isolates, and the failure of immunological interventions (avirulent vaccines or competitive exclusion products) to prevent disease expression. Another hypothesis was the genetic susceptibility of specific breeds to F18 E. coli.

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The expression of specific receptors on enterocytes for the attachment of F18 E. coli is regulated by the FUT1 gene. A point mutation G>A at base-pair 307 in the FUT1 gene can influence the expression of the receptor for F18 fimbrial attachment. Pigs with the FUT1AA genotype are resistant, while pigs with the FUT1GA or FUTGG genotype are susceptible to colonization by F18 E. coli. Therefore, this study aimed to investigate the effect of challenge with a contemporary F18 E. coli isolate of high virulence in susceptible and resistant pigs.

Materials and methods

Twenty 21-day-old pigs (10 susceptible and 10 resistant) were sourced from PIC genetics with specific FUT1. PIC performed FUT1 genotyping to select pigs before shipment to ISU. At arrival, pigs were placed in two rooms according to the resistance and susceptibility, weighed, and acclimated for three days before the challenge. Pigs with resistant and susceptible genotypes were housed in the same pens (Figure 1).

Figure 1: Schematic figure of the experimental design of the rooms.

After three days of acclimation (0 days post-infection, [dpi]), all pigs were inoculated with 10 mL (1.08 x 1010 CFU/ml) of an F18:LT:STa:STb:Stx2e wild-type isolates by oral gavage. Pigs were weighed individually on dpi -3, 0 and 7. Rectal swabs were collected on dpi 0, 1, 2, 3, 5 and 7. The severity of diarrhea was evaluated daily with fecal score visually assessed using the following scale: 0 = solid, 1 = semi-solid, 2 = semiliquid and 3 = liquid. A fecal score ≥ 2 was considered diarrhea. On dpi 7, all pigs were euthanized, and sections of the duodenum, proximal, mid and distal jejunum, and ileum were collected and fixed in 10% neutral buffered formalin and refrigerated for histopathological evaluation and bacteriology culture.

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The fecal swabs were plated onto TSA agar with 5% sheep blood and incubated overnight at 37°C. The hemolytic E. coli shedding was assessed based on a semiquantitative method measured using a 5-point scale ranging from 0 to 4 according to the number of streaked sections that had viable E. coli, where 0 corresponded to no growth, 1 corresponded to growth in the primary streak, 2 corresponded to growth extending into the secondary streak, 3 corresponded to growth into the tertiary streak, and 4 corresponded to growth into the quaternary section of the agar plate.

The presence of hemolytic E. coli on individual colonies isolated was confirmed using the Matrix-Assisted Laser Desorption/Ionization time-of-flight mass spectrometry technique. One section of the duodenum, proximal, mid and distal jejunum, ileum and colon were evaluated histologically. Sections were evaluated for bacterial attachment consistent with E. coli morphology.

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Results

All groups of pigs showed similar E. coli fecal shedding scores during the study period. However, on average, susceptible group had a numerical higher fecal score than the resistant group (Figure 2).

Figure 2: Fecal shedding of the groups during the study period.

The average daily fecal score was statistically higher for pigs in the susceptible group compared to the resistant pigs (Figure 3).

Figure 3:  Fecal score of the groups during the study period.

Although no statistical difference was observed between groups during the study period, a higher mortality rate was observed in the susceptible groups, while no mortality was observed for pigs in the resistant groups (Figure 4).

Figure 4: Survival curve of the groups during the study period.

The weight at day 7, adjusted by the weight at day 0, was statistically higher for resistant pigs compared to susceptible (Figure 5).

Figure 5: Weight at the end of the study period.

A statistical difference was also noted in assessing E. coli colonization of the small intestines. Notably, none of the pigs in the resistant group exhibited E. coli colonization in any of the microscopically assessed sections of the small intestine. In contrast, 90% of the pigs in the susceptible group showed colonization in at least one section of the small intestine.

Summary