• Awrahman S. (2017) Using RNA-Seq to Define Genes and Pathways Affecting Salmonella Vaccine Response in Beef Cattle. Thesis – South Dakota State University

A Salmonella siderophore receptor and porin (SRP) proteins vaccine is an intervention strategy to control Salmonella burden in cattle. The vaccine works by stimulatingimmunity to produce antibodies against bacterial SRPs as siderophore molecules play a major role in transporting iron to bacteria. Blocking iron uptake system by antibodies causes death of bacterial cells. The vaccine can also be useful to protect humans against salmonellosis, which causes high rates of illness and death annually, by reducing shedding of Salmonella in the feces of cattle. Though other researchers have evaluated efficacy and immune response of this Salmonella SRP vaccine, genes and pathways affected by vaccination have not been investigated. Therefore, the aim of this study was to identify differentially expressed genes and pathways in bovine whole blood after vaccination with Salmonella SRPs by RNA sequencing. Five Angus cattle were vaccinated with the Salmonella SRP vaccine, and blood samples were collected at first day of vaccination, day 21 post-vaccination (time of booster vaccination) and finally day 48 post-vaccination. To perform RNA seq, total RNA was extracted from blood samples using the PAXgeneTM Blood RNA kit. Library samples were prepared using the Illumina TruSeq Stranded Total RNA Library PrepKit and sequenced on an Illumina HiSeq sequencer. After processing raw sequencing reads, filtered data was aligned to the bovine viiireference genome (Bta_4.6.1). Cuffdiff was used for pairwise differential expression analysis. The transcript abundance was normalized in Fragments Per Kilobase of transcript per Million mapped reads (FPKM). FDR adjusted p-values (q-value<0.05) were used to identify differentially expressed genes between 1) day 21 and day 0, and 2) day 48 and day 0 of vaccination. Of the 848 differentially expressed (DE) genes at day 21 post-vaccination, one gene was increased in abundance and 26 genes were decreased in abundance with a fold-change >2. Of the 1155 DE genes at day 48 post-vaccination, 20 genes were increased in abundance and 39 genes were decreased in abundance with a fold-change >2. DAVID bioinformatics was used to annotate a list of differentially expressed genes to their correlated GO terms and KEGG pathways using Bos Taurus as a background. Most decreased in abundance genes were annotated to biosynthetic processes of heme and protoporphyrinogen IX. FoxO signaling pathway, AMPK signaling pathway, and porphyrin and chlorophyll metabolism were found significantly decreased in abundance at day 21 post-vaccination. Real-time RT-PCR was used to validate RNA sequencing results for the day 48 to day 0 comparison. All three genes tested (HMB, ALAS2, and ATPIF1) were decreased in abundance at day 48 (P<0.05), confirming our RNA sequencing observations. Our analysis suggests that SalmonellaSRP vaccination decreased in abundance pathways involving heme (iron) metabolism in bovine blood

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