Quantification of cell proliferation and alpha-toxin gene expression of Clostridium perfringens in the development of necrotic enteritis in broiler chickens

Abstract

Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b(0) + b(1)X - b(2)X(2). The average C. perfringens count of 5 log(10) CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.

FIG. 1.

Representative standard curve showing the relationship between real-time PCR amplification threshold cycle numbers (Ct) and copy numbers of cpa. PCR amplification efficiencies were 0.95. Log N, log₁₀ copy numbers of alpha-toxin gene mRNA.

FIG. 2.

Progress curves of C. perfringens cell proliferation and alpha-toxin gene expression after challenge. Log CFU/g, log₁₀ CFU per gram of ileal digesta (▪); N, copy number of alpha-toxin gene mRNA (▴). The solid line represents colony counts of C. perfringens, generated from a regression model: y = 1.50694 + 5.41067x − 1.21876x² (r² = 0.3385, n = 60). The dashed line represents mRNA copy numbers of the alpha-toxin gene, generated from a regression model: y = 0.10793 + 6.15743x − 1.32615x² (r² = 0.3709, n = 58). All slopes in the equations are significant (P < 0.0001). Data points are observed mean values with standard deviation bars.

FIG. 3.

Correlation between the cell proliferation and alpha-toxin gene expression of C. perfringens in the ileal digesta of non-bacitracin-treated postchallenge chickens. Panels A through D show data from days 1 through 4 postchallenge, respectively. Log CFU/g, log₁₀ CFU per gram of ileal digesta; N, copy numbers of alpha-toxin gene mRNA; r, Spearman's rank correlation coefficient. Chickens were challenged with C. perfringens on day 18.

FIG. 4.

Relationship between C. perfringens cell proliferation and the incidence of NE.