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Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics

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Abstract

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

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Acknowledgments

The authors thank Moroni Feed Co. (Ephraim, UT, USA) for providing turkeys and feed and Kevin McMahon (Virginia Tech, Blacksburg, VA, USA) for assisting with data analysis. This study was funded by US Department of Agriculture—Agriculture and Food Research Initiative Grants 2005-01326, 2007-35205-17880, and 2009-35205-05302, US Department of Agriculture—National Institute of Food and Agriculture Animal Genome Program Grant 2013-01043, and the Utah and Minnesota Agricultural Experiments Stations.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Author notes

  1. Sumit Rawal

    Present address: Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA

Authors and Affiliations

  1. Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 295 AS/VM, 1988 Fitch Ave., St. Paul, MN, 55108, USA

    Melissa S. Monson, Kristelle M. Mendoza & Kent M. Reed

  2. Data Analysis Core, Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

    Robert E. Settlage

  3. Department of Animal, Dairy and Veterinary Sciences, College of Agriculture, Utah State University, Logan, UT, 84322, USA

    Sumit Rawal & Roger A. Coulombe

  4. School of Biological Sciences, University of Hong Kong, Hong Kong, China

    Hani S. El-Nezami

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Corresponding author

Correspondence to Kent M. Reed.

Electronic supplementary material

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Fig. S1

Relationship between mean expression and log2 FC for each pair-wise comparison. a Aflatoxin B1 (AFB) to control (CNTL). b Probiotic mixture (PB) to CNTL. c Probiotic + aflatoxin B1 (PBAFB) to CNTL. d PBAFB to AFB. e PBAFB to PB. Each plot shows log2 fold change (FC) against mean normalized read counts for predicted transcripts with non-zero expression values in both treatments in the comparison. Transcripts with significant differential expression (q-values ≤ 0.05, determined in DESeq; Anders and Huber 2010) are highlighted in red (GIF 75 kb)

High resolution image (TIFF 509 kb)

Fig. S2

Biological process Gene Ontology (GO) term associations to transcripts with significant differential expression. a Significant transcripts in the aflatoxin B1 (AFB, blue), probiotic mixture (PB, red), and probiotic + aflatoxin B1 (PBAFB, green) groups compared to the control (CNTL) group. b Significant transcripts in the PBAFB group compared to the AFB (blue) or PB (red) groups. Level 2 biological process GO terms were identified using BLAST2GO (Contesa et al. 2005). The distribution of associated GO terms was plotted as the percent of total associations (GIF 59 kb)

High resolution image (TIFF 1041 kb)

Fig. S3

Legend for IPA regulatory and functional pathways. This code is used in Fig. 5 to designate the type of interaction and type of molecule (GIF 13 kb)

High resolution image (TIFF 441 kb)

Fig. S4

Distribution of log2 FC for significant transcripts in the spleen. a Log2 fold change (FC) for significant transcripts in aflatoxin B1 (AFB), probiotic mixture (PB), or probiotic + aflatoxin B1 (PBAFB) compared to control (CNTL). b Log2 FC for significant transcripts in PBAFB compared to AFB or PB. For each pair-wise comparison, a box-plot of log2 FC is shown for predicted transcripts with significant differential expression (q-value ≤ 0.05) and non-zero normalized expression in both treatments. Significantly different means for log2 FC (p-value ≤ 0.05) in each treatment are indicated by an asterisk. Open circles represent outliers in the data (GIF 26 kb)

High resolution image (TIFF 809 kb)

Fig. S5

Volcano plots of log2 FC and significance level for each pair-wise comparison. a Aflatoxin B1 (AFB) to control (CNTL). b Probiotic mixture (PB) to CNTL. c Probiotic + aflatoxin B1 (PBAFB) to CNTL. d PBAFB to AFB. e PBAFB to PB. Each plot shows –log10 p-value against log2 fold change (FC) for predicted transcripts with non-zero expression values in the compared treatments. Transcripts with significant differential expression (q-values ≤ 0.05) are highlighted in red. Highly significant transcripts annotated by BLAST are denoted (GIF 46 kb)

High resolution image (TIFF 1096 kb)

Table S1

Summary of RNA-seq datasets for individual spleen samples (PDF 17 kb)

Table S2

Results of filtering predicted spleen transcripts for depth of coverage and LINE-1 elements (PDF 16 kb)

Table S3

Mapping filtered transcripts to the turkey genome (build UMD 2.01) (PDF 17 kb)

Table S4

Significant DE transcripts identified using DESeq and characterized by BLAST and GO (XLSX 529 kb)

Table S5

Comparative normalized expression of IL2 and GZMA in the spleen (PDF 16 kb)

Table S6

Transcripts with significant DE in both AFB/CNTL and PBAFB/AFB group comparisons (XLSX 17 kb)

Methods S1

RNA-seq and qRT-PCR (DOCX 28 kb)

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Monson, M.S., Settlage, R.E., Mendoza, K.M. et al. Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics. Immunogenetics 67, 163–178 (2015). https://doi.org/10.1007/s00251-014-0825-y

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  • DOI: https://doi.org/10.1007/s00251-014-0825-y

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