Transcriptional profiles of host-pathogen responses to necrotic enteritis and differential regulation of immune genes in two inbreed chicken lines showing disparate disease susceptibility

Abstract

Necrotic enteritis (NE) is an important intestinal infectious disease of commercial poultry flocks caused by Clostridium perfringens. Using an experimental model of NE involving co-infection with C. perfringens and Eimeria maxima, transcriptome profiling and functional genomics approaches were applied to identify the genetic mechanisms that might regulate the host response to this disease. Microarray hybridization identified 1,049 transcripts whose levels were altered (601 increased, 448 decreased) in intestinal lymphocytes from C. perfringens/E. maxima co-infected Ross chickens compared with uninfected controls. Five biological functions, all related to host immunity and inflammation, and 11 pathways were identified from this dataset. To further elucidate the role of host genetics in NE susceptibility, two inbred chicken lines, ADOL line 6 and line 7 which share an identical B2 major histocompatibility complex haplotype but differ in their susceptibility to virus infection, were compared for clinical symptoms and the expression levels of a panel of immune-related genes during experimental NE. Line 6 chickens were more susceptible to development of experimental NE compared with line 7, as revealed by decreased body weight gain and increased E. maxima oocyst shedding. Of 21 immune-related genes examined, 15 were increased in C. perfringens/E. maxima co-infected line 6 vs. line 7 chickens. These results suggest that immune pathways are activated in response to experimental NE infection and that genetic determinants outside of the chicken B complex influence resistance to this disease.

Figure 1. Effect of C. perfringens/E. maxima co-infection of Ross chickens on body weight gains and intestinal lesion scores.

(A) Chickens were uninfected (Control) or co-infected with C. perfringens and E. maxima (NE) and body weight gains were measured between days 0–9 post-infection with E. maxima. (B) Gut lesion scores were determined at day 2 post-infection with C. perfringens on a scale from 0 (none) to 4 (high) in a blinded fashion by three independent observers. Each bar represents the mean ± SD value (n = 8). *, P<0.05; ***, P<0.001.

Figure 2. The first (A) and seventh (B) networks of genes most significantly associated with the differentially expressed transcripts in C. perfringens/E. maxima co-infected Ross chickens compared with uninfected controls.

Up- and down-regulated genes are illustrated by red and green, respectively. The color intensity directly correlates with the difference in the expression level of the corresponding gene compared between line 6 and line 7 chickens.

Figure 3. Comparison of the fold changes in the expression levels of 15 immune-related gene transcripts in intestinal IELs of C. perfringens/E. maxima co-infected Ross chickens (NE) compared with uninfected controls (Control).

Transcript levels were analyzed by microarray hybridization and qRT-PCR in both groups. Each bar represents the mean ± SD value of the fold change in the transcript level (n = 3). ANXA1, Annexin A1; APP, Amyloid precursor protein; ARHGEF6, Rho guanine nucleotide exchange factor 6; CALB1, Calbindin 1; COL1A2, Collagen, type 1, alpha 2; CXCL14, Chemokine (C-X-C motif) ligand 14; GJA1, Gap junction alpha 1; HSP90B1, Heat shock protein 90kDa beta 1; VEGFA, Vascular endothelial growth factor A; ALCAM, Activated leukocyte cell adhesion molecule; MTTP, Microsomal triglyceride transfer protein; LCP1, Lymphocyte cytosolic protein 1; TNFSF11B, Tumor necrosis factor superfamily 11; SERPINE1, Serpin peptidase inhibitor, clade F1.

Figure 4. Effect of C. perfringens/E. maxima co-infection of ADOL line 6 and line 7 chickens on body weight gains and oocyst shedding.

(A) Chickens were uninfected (Control) or co-infected with C. perfringens and E. maxima (NE) and body weight gains were measured between days 0–9 post-infection with E. maxima. Bars with different letters are significantly different according to the Duncan's multiple range test (P<0.05). (B) Fecal oocyst shedding was measured between days 0–9 post-infection with E. maxima in C. perfringens/E. maxima co-infected chickens. Each bar represents the mean ± SD value (n = 8). *, P<0.05.