Assessment of intestinal barrier function over a time course of porcine epidemic diarrhea virus (PEDV) challenge in growing pigs

In pigs, PEDV causes reduced growth performance, villus atrophy, and impairment of intestinal barrier function and integrity. The objective of this study was to determine the extent to which a PEDV challenge alters jejunum morphology, epithelial apoptosis, crypt cell proliferation, and tight junction proteins over a 14-day infection.

Thirty-two mixed-sex Choice Genetics maternal line approximately 4 weeks-of-age (BW=9.49±1.38 kg) and naïve for PEDV were allotted into 2 treatments: sham (Controls) and PEDV inoculated. Barrows and gilts were distributed equally among 8 pens per treatment and allowed free access to a corn-soybean meal based diet and water. The 16 PEDV pigs were intragastrically inoculated with 10³ TCID₅₀/ml of PEDV isolate on day 0. On 2, 5, 7, and 14 days post-inoculation (dpi), 4 pigs per treatment were euthanized for sample collection. Formalin-fixed samples were paraffin-embedded, sectioned, and stained routinely with H&E as well as immunohistochemically for PEDV antigen, tight junction proteins (claudin 4 and claudin 2), and cellular proliferation (Ki-67). Villus height, crypt depth and villus:crypt ratios were compared for each group. DNA fragmentation (i.e., apoptosis) was assessed by TUNEL assay on sections of fixed jejunum. Caspase 3 and 7 activity of frozen samples was also used to assess apoptosis using a commercial kit. Treatment, dpi, and treatment by dpi interactions were determined with the individual pig as the experimental unit.

There was no statistical difference in PEDV IHC score (P = 0.361) among infected pigs between 2 and 5 dpi and PEDV antigen was not detected after 5 dpi. Claudin 2 IHC score was greater (P < 0.05) in the villus tip of Controls than PEDV pigs; however, there was no difference in claudin 2 along the sides of villi. Claudin 4 staining was numerically lower in PEDV pigs at 2 and 5 dpi, but this was not statistically significant. The PEDV challenge also resulted in time-dependent changes in villus height and crypt depth (P < 0.05). PEDV pigs had more (P < 0.001) Ki67 positive nuclei detected than Controls. There was an interaction (P < 0.05) between treatment and dpi for caspase 3 and 7 activity; Controls had greater activity at 5 dpi, but PEDV pigs had greater activity at 7 dpi. No difference between treatments was observed at 2 and 14 dpi.

In summary, PEDV infected pigs had greater cellular proliferation, time dependent variability in apoptosis signaling, reduced expression of the barrier protein claudin 2, and decreased villus height compared with Controls. This suggests a time dependent impairment of intestinal barrier function through at least 5 dpi in PEDV infected pigs.

Disclosure of Interest: None Declared.