SUMMARY
Necrotic enteritis (NE) is a significant intestinal disease that negatively impacts producer economics and animal welfare. Chickens are predisposed to this disease by infection with the parasite Eimeria, resulting in coccidiosis and subsequent Clostridium perfringens overgrowth. Pathology is caused by damage to enterocytes of the small intestine and blunting of the villus. The hypothesis of this study was that NE results in disruption of cell–cell communication crucial for intestinal stem cell proliferation, differentiation, and restoration of absorptive function. Developmental Wnt and Notch signaling pathways fate stem cells towards formation of specialized secretory or absorptive cell types, shaping intestinal epithelium composition and morphology. The objective of this study was to use a subclinical NE challenge model to analyze effects of C. perfringens and Eimeria maxima on intestinal morphology and developmental signaling pathways. Chicks were placed, at hatch, in battery cages, and they were orally administered Eimeria maxima alone (EM) at 14 days of age, C. perfringens alone (CP) at 19 days of age, or E. maxima at 14 days of age, followed 5 days later with C. perfringens (EM/CP). Uninfected chickens served as the nonchallenged control (NC) group. Gene expression was measured using in situ hybridization and quantitative PCR in the small intestine of chickens at 14, 17, 19, 21, and 23 days of age. Intestinal tissues were histologically scored for lesions, inflammation and coccidia. Statistical analysis was performed using Tukey’s Honestly Significant Difference test, Steel-Dwass All Pairs, Kruskal-Wallis, or Dunn’s multiple-comparisons tests. EM and EM/CP infections considerably impacted jejunum morphology by increasing crypt depth and shortening villi (P < 0.05). Crypt depth elongation was observed exclusively in chickens inoculated with EM or EM/CP, but villus blunting was apparent in EM, CP, and EM/CP chickens compared to NC. Histological observations of the chicken’s small intestine revealed an increase in the cumulative pathology scores in chickens with EM or EM/CP (P < 0.05). Expression of hairy and enhancer of split-1 (Hes1) messenger RNA (mRNA) that regulates stem cell differentiation and inflammation decreased in EM-, CP-, and EM/CP-infected chickens. Hes1 mRNA was localized to intestinal crypts and lymph within the villi using in situ hybridization, indicating that the decrease in expression could be related to intestinal stem cells or macrophages in the lymph that contribute to the immune response. Increased goblet cell density and relative expression of mucin 2 mRNA was observed in CP-infected chickens compared to co-infection with EM/CP. The contribution of each pathogen to altered villus height, crypt depth, stem cell proliferation and differentiation, and developmental signaling in the jejunum of broiler chickens was revealed by single infection. Further research into mechanisms that influence Notch and Wnt signaling in broilers could aid in restoring intestinal homeostasis after infection and lowering economic and animal welfare costs.