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Global gene expression profile of chicken cecal tonsil in response to campylobacter jejuni challenge in broiler lines

ABSTRACT: It is essential to understand host response to Campylobacter jejuni infection in order to genetically improve resistance to its colonization in chickens. A custom Agilent chicken 44K array was used to examine gene expression profiles after Campylobacter jejuni infection of two broiler lines (A and B). Day-old chicks were orally inoculated with C. jejuni. After day 7 post-infection, the cecal tonsil was collected for total RNA isolation and cecal content for bacteria burden quantification. Twenty highest and lowest bacterial burden birds and non-infected birds within each line were used to pool four biological replicates for each group. The pair comparisons among high, low bacterial burden, and non-infected group were used. The signal intensity of each gene was normalized by LOWESS method. A mixed model including the fixed effects of dye, line, treatment and line × treatment interaction, and random effects of slide and array was used to identify differentially expressed genes at P < 0.001 by SAS program. Within line A, there were 61, 163, and 90 genes significantly differentially expressed between high and low bacterial burden, high bacterial burden and non-infected group, and low bacterial burden and non-infected group, respectively; 2637, 1684, 561 genes within line B, respectively. The results suggested that genetics, treatment and genetics × treatment interaction played important role in gene regulation of C. jejuni infection. The findings in the current study will lead the identification of potential candidate genes for genetic resistance to C. jejuni infection in chickens. Keywords: diease state analysis

ORGANISM(S): Gallus gallus

PROVIDER: GSE10257 | GEO | 2010/08/03

SECONDARY ACCESSION(S): PRJNA108449

REPOSITORIES: GEO

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Project description:Campylobacter jejuni (C. jejuni) is a zoonotic pathogen that causes human diarrhea worldwide. Chickens are a natural reservoir of C. jejuni. Understanding the host response to C. jejuni infection at the molecular level will lay the foundation to control human campylobacterosis by reducing food contamination. Two distinct genetic lines, resistant (line A) and susceptible (line B) to C. jejuni colonization, were utilized to profile the host response to C. jejuni infection using an Agilent chicken 44K microarray. Day-old chickens were challenged orally with C. jejuni and spleens collected for total RNA 7 days post-challenge. Twenty infected samples with highest (a) or lowest bacterial number (b) in cecal content and twenty non-infected (c) in each line were randomly pooled into four biological replicates. The pair comparisons among these three groups within each line were analyzed. The signal intensity of each gene was normalized using LOWESS method. A mixed model was used to identify differentially expressed genes by SAS (P < 0.001). This was opposite to previous cecal tonsil microarray result. There were 468, 743, and 939 genes differentially expressed between groups a and c, groups a and b, and groups b and c in line A, respectively, and 201, 37, 126 genes in line B, respectively. More differentially expressed genes in spleen in line A than in line B were found. The results indicated that significantly different response to C. jejuni infection occurred between resistant and susceptible chicken lines, and the effects of interaction between genetics and tissue should be considered. Chickens in two broiler lines were inoculated with 10^5 cfu C. jejuni on one day after hatch. The cecal content and cecal tonsil was collected and bacterial number in cecal content was counted on day 7 after inoculation. Twenty samples were separated into 3 groups (high burden, low burden, and control) based on bacterial burden of cecal content in each line, 5 samples were mixed randomly into one pool. A dual color, balanced design was carried on for all samples. Three comparisons were used in each line, non-infected/susceptible, susceptible/resistant, resistant/non-infected, totally, four biological replicates in each line. A Dye swap was used in each pair of comparisons including AN/AS, AS/AR, AR/AN; BN/BS, BS/BR, and BR/BN. Background subtracted signal intensity were collected from 24 arrays and normalized for data analysis.

Project description:Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, of which Campylobacter jejuni subsp. jejuni (C. jejuni) and C. coli are responsible for approximately 98% of the cases. The ceca of commercial turkeys are the main anatomical site where Campylobacter asymptomatically colonizes. We have previously colonized the ceca of commercial turkey poults with C. jejuni, and demonstrated acute changes in cytokine gene expression in cecal tissue and histologically scored intestinal lesions at 2 days post-inoculation (dpi). The host-response of turkeys to C. coli colonization is unknown. Cecal tonsils (CT) are an important part of the gastrointestinal-associated lymphoid tissue that function to sample material passing in and out of the ceca and generating immune responses against intestinal pathogens. The CT immune response towards Campylobacter is unknown. In this study, we generated a C. coli kanamycin-resistant construct (CcK) for enumeration from cecal contents after experimental challenge. In vitro analysis of CcK demonstrated no changes in motility when compared to the parent isolate, but in vitro growth rates were significantly different than the parent strain. Poults were inoculated by oral gavage with CcK (5x10^7 cfu) or sterile-media (mock-colonized), and euthanized at 1 and 3 dpi. At both time points, CcK was recovered from cecal contents, but not from the mock-colonized group. As a marker of acute inflammation, serum alpha-1 acid glycoprotein was significantly elevated at 3 dpi in CcK inoculated poults compared to mock-infected samples. Significant histological lesions were detected in cecal and CT tissues of CcK colonized poults at 1 and 3 dpi, respectively. RNAseq analysis identified 250 differentially expressed genes (DEG) in CT from CcK colonized poults at 3 dpi, of which 194 were upregulated and 56 were downregulated. From the DEG, 9 significantly enriched biological pathways were identified, including platelet aggregation, response to oxidative stress and negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway. These data suggest that C. coli induced an acute inflammatory response in the intestinal tract of poults, and that platelet aggregation and oxidative stress in the CT may affect the turkey’s ability to resist Campylobacter colonization. Results from this study provide insight into host-response of the turkey CT to Campylobacter colonization. These findings will help to develop and test Campylobacter mitigation strategies to promote food safety in commercial turkeys.

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Global gene expression profile of chicken cecal tonsil in response to campylobacter jejuni challenge in broiler lines

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