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ABSTRACTAs more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturatingSalmonella entericaserotype Typhimurium mutant library recovered from selection under 3 differentin vitrogrowth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes inS. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD,glnL,recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes inSalmonellaand the other approximately 70 bacterial species for which EZ:Tn5mutagenesis has been established.

Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers.

Attenuated Salmonella Enteriditis (ΔSE) recombinant vaccine vectors incorporating a Salmonella flagellar filament protein (fliC) subunit, a putative cell-mediated epitope, for expression of the lamB gene (encoding a maltose outer membrane porin), with or without co-expression of a putative immune-enhancing CD154 oligopeptide, were developed and compared with wild-type Salmonella Enteriditis (experiments 1 and 2) or the attenuated ΔSE empty vector (experiment 3) as initial vaccine candidates against Salmonella infection. A total of 3 experiments were performed to assess the infection and clearance rate of each of these constructs. Each construct or Salmonella Enteriditis was orally administered to broiler chicks at day of hatch by oral gavage (~10(8) cfu/chick). In experiments 1 to 3, liver-spleen and cecal tonsils were removed aseptically for recovery of wild-type Salmonella Enteriditis or ΔSE mutants. These experiments suggested that cell surface expression of fliC alone markedly increased the clearance rate of the vector at or before 21d postvaccination in all 3 experiments. In a fourth experiment, broilers were vaccinated with one of the vaccine constructs or the ΔSE empty vector and then challenged with wild-type Salmonella Typhimurium. At 19 d posthatch, 16 d postinfection, neither candidate protected against challenge significantly better than the ΔSE empty vector, although there was significantly less Salmonella recovered from vaccinated chickens as compared with nonvaccinated controls. These experiments indicate that these experimental vaccines did not protect against heterologous challenge or enhance clearance after Salmonella Typhimurium challenge; as such, their value as vaccines is limited. The increased clearance of the candidate vaccines, particularly the vector expressing fliC alone, may have value in that the fliC epitope may decrease the clearance time of other recombinant vectored Salmonella vaccines.

Salmonellosis in the United States is one of the most costly foodborne diseases. Given that Salmonella can originate from a wide variety of environments, reduction of this organism at all stages of poultry production is critical. Salmonella species can encounter various environmental stress conditions that can dramatically influence their survival and virulence. Previous knowledge of Salmonella species genomic regulation of metabolism and physiology in relation to poultry is based on limited information of a few well-characterized genes. Consequently, although there is some information about environmental signals that control Salmonella growth and pathogenesis, much still remains unknown. Advancements in DNA sequencing technologies revolutionized the way bacteria were studied and molecular tools such as microarrays have subsequently been used for comprehensive transcriptomic analysis of Salmonella. With microarray analysis, the expression levels of each single gene in the Salmonella genome can be directly assessed and previously unknown genetic systems that are required for Salmonella growth and survival in the poultry production cycle can be elucidated. This represents an opportunity for development of novel approaches for limiting Salmonella establishment in all phases of poultry production. In this review, recent advances in transcriptome-microarray technologies that are facilitating a better understanding of Salmonella biology in poultry production and processing are discussed.