Combined rotavirus and K99 Escherichia coli infection in gnotobiotic pigs
Authors: Benfield David A. , Francis David H. , McAdaragh John P. , Johnson Darrell D. , Bergeland Martin E. , Rossow Kurt , Moore Ron 
Published: February 2025

SUMMARY
Fifty nine 3-day-old gnotobiotic pigs were randomly assigned to 4 experimental groups: 14 pigs were orally inoculated with rotavirus (rv), 14 were orally inoculated with enterotoxigenic Escherichia coli (etec), 18 were orally inoculated with both agents, and 13 were controls. Pigs inoculated with rv plus etec were given the rv inoculum at 3 days of age and then, 24 hours later, were given the etec inoculum. Three pigs inoculated only with rv, 3 pigs inoculated only with etec, 4 pigs inoculated with rv plus etec, and 3 pigs in the control group were euthanatized at 5 and 7 days of age. Two pigs in each of the 4 experimental groups also were euthanatized at 9 days of age. Intestinal segments from 6 sites in the small intestine were examined by virologic, bacteriologic, and histologic procedures. For 10 days after inoculation, the remaining pigs in each group were observed clinically to monitor severity and duration of diarrhea, mortality, and shedding of rv or etec.
Pigs inoculated with the combined rv plus etec inoculum developed more severe diarrhea, compared with pigs inoculated with the single agents; all dually inoculated pigs died between 3 and 6 days after inoculation. There was no mortality in pigs inoculated with either rv or etec.
Lesions were restricted to the small intestine in pigs inoculated with rv plus etec and in pigs inoculated with rv or etec. There was no difference in the severity of the villus atrophy between the dually inoculated pigs and pigs inoculated only with rv. The pigs inoculated only with etec at 5 days of age had villus atrophy in the distal half of the small intestine, which progressed proximally to involve all segments except the duodenum in pigs euthanatized at 9 days of age. Escherichia coli organisms were observed in all segments of the small intestine by light microscopy and in Gram-stained intestinal smears from pigs inoculated only with etec or dually inoculated with rv plus etec. The etec organisms in the proximal half of the small intestine were not adherent to intestinal villi, but were prevalent in the lumen of the small intestine, and a few expressed K99 antigen. In the distal half of the small intestine of the dually inoculated pigs or of the pigs inoculated only with etec, E coli organisms were adherent to villous epithelial cells of the small intestine, expressed K99 antigen, and were most numerous in the terminal aspect of the ileum. The concentration of bacteria (colony-forming units per centimeter of intestine) in the jejunum and ileum was significantly (P ≤ 0.01) higher in the dually inoculated pigs euthanatized at 7 and 9 days, compared with bacterial counts from similar specimens from pigs inoculated only with etec. The more severe clinical disease and mortality in the pigs inoculated with rv plus etec were attributed to a combined malabsorption and hypersecretory diarrhea, which resulted in excessive dehydration and loss of flesh not found in pigs inoculated with only one agent. We concluded that the combined inoculum induced an additive, but not a synergistic effect, because lesions were no more severe in the dually inoculated pigs, compared with those in the single agent-inoculated pigs. Rotavirus did not consistently enhance the growth of E coli in the small intestine of the dually inoculated pigs.