Acquired thermotolerance (AT) is the ability of cells to survive a normally lethal temperature treatment as a consequence of pretreatment at an elevated but sublethal temperature. In yeast and cyanobacteria, the expression of the HSP100/ClpB protein is required for the AT response. To determine whether the HSP100/ClpB protein is associated with this response in lima bean (Phaseolus lunatus), we have cloned an HSP100/ClpB homolog and assessed expression of the two gene copies under heat stress conditions, which induce AT. Transcription of the cytoplasmically localized HSP100/ClpB protein genes is stringently controlled by heat stress in both of the laboratory and field heat stress conditions. From a heat-induced cDNA library, we identified a clone of a putative chloroplast-targeted (cp) HSP100/ClpB protein gene sequence. The cp HSP100/ ClpB protein genes are constitutively expressed, but transcript levels increase post-heat stress in laboratory heat stress experiments. In field conditions the genes for the cp HSP100/ClpB are constitutively expressed. Although we were unable to correlate differences in the timing of AT response with the expression or genetic structure of the HSP100/ClpB genes in heat-tolerant or-sensitive varieties of lima bean, we clearly demonstrate the association of expression of HSP100/ClpB proteins with heat response in this species.

Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.

Mycoplasma gallisepticum (MG) has repeatedly emerged as a serious problem in U.S. broiler, layer, and turkey industries. Tracing the source of an outbreak is essential if MG control is to be accomplished. Amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP) are valuable tools used to study MG epidemiology, allowing diagnosticians to determine the source of MG infections. In some past outbreaks, AFLP, RAPD, and RFLP fingerprinting, which require pure MG cultures, were not successful because of contaminating nonpathogenic mycoplasmas from field samples. The objective of this research was to develop a method to separate rapidly growing nonpathogenic avian mycoplasma species from slower-growing MG field strains. Mixtures of MG and three separate nonpathogenic avian mycoplasmas were inoculated onto chick embryo fibroblasts cells (CEF) allowing MG to penetrate the CEF cells. Later, gentamicin sulphate was added to the culture, eliminating the nonpathogenic mycoplasmas and allowing MG to be isolated in pure culture. Mixtures of Mycoplasma synoviae (MS) and MG could not be separated in this assay. However, removal of nicotinamide adenine dinucleotide and cysteine hydrochloride during serial passage in Frey broth medium successfully eliminated growth of MS.

Discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. Macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. IFNγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. This study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages.A large-scale RNA sequencing approach was employed to sequence the RNA of purified macrophages from each haplotype group (B2 vs. B19) during differentiation and after stimulation. Our results demonstrate that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation.

Disease resistance and susceptibility has been associated with the major histocompatibility complex (MHC) in several species. This study aimed to better characterize the molecular basis for dramatically different nitric oxide production and associated immune function between the B2 and the B19 haplotype chicken macrophages in response to IFNγ stimulation, previously shown by our laboratory. A next generation RNA sequencing approach was employed to analyze gene expression from purified macrophages from each haplotype group (B2 vs. B19) across 9 time points spanning seven days. Ultimately, our data shows global dysregulation of gene expression in B19 haplotypes correlating with increased disease susceptibility of this haplotype. Our results demonstrate that transcription factors (TF) exhibit divergent expression between B2 and B19 haplotype cells both prior to and after stimulation. Our results also reveal that miRNAs exhibit divergent expression between B2 and B19 haplotype cells both, prior to, and after stimulation with IFNγ. In contrast to TFs, miRNAs can modulate gene product levels post-transcriptionally. We also identify ubiquitin-related enzymes and factors that exhibit divergent expression between B2 and B19 haplotype cells. Some of our results indicate that snoRNAs exhibit divergent expression between B2 and B19 haplotype cells, which is noteworthy because snoRNAs can modulate genes epigenetically. Taken together our results suggest that the global dysregulation of gene expression observed in B19 haplotypes can at least in part be attributed to differential epigenetic regulation.