Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of lameness in commercial broiler chickens worldwide. BCO represents substantial production loss and welfare issues of chickens. The bacterial species or communities underlying BCO pathogenesis still remain to be fully characterized. To gain insights on blood microbiota in broilers and its potential association with BCO, blood samples collected from healthy (n = 240) and lame (n = 12) chickens were analyzed by deep sequencing of 16S RNA genes. The chicken blood microbiota were dominated by Proteobacteria (60.58% ± 0.65) followed by Bactroidetes (13.99% ± 0.29), Firmicutes (11.45% ± 0.51), Actinobacteria (10.21% ± 0.37) and Cyanobacteria (1.96% ± 0.21) that constituted 98.18% (± 0.22) of the whole phyla. The bacterial communities consist of 30–40 OTUs in the blood of broiler chickens, regardless of ages and other environmental or host conditions, and the blood microbiomes of BCO chickens were largely distinct from those of healthy chickens. In addition, Linear discriminant analysis (LDA) effect size (LEfSe) method revealed that Staphylococcus, Granulicatella, and Microbacterium were significantly enriched in BCO chickens as compared to healthy chickens. The results from this study have significant implications in understanding blood microbiota present in broiler chickens and its potential role in BCO pathogenesis.

Highlights d Human gut bacteria composition correlates with severity of malaria d Gut bacteria dynamically modulate spleen GC reactions to systemic infection d Modulating gut bacteria can halt escalating parasitemia d Antibiotic-induced changes in gut bacteria can boost anti-Plasmodium host immunity

A comprehensive understanding of genotype-phenotype links in bacteria is the primary theme of bacterial functional genomics. Transposon sequencing (Tn-seq) or its equivalent methods that combine random transposon mutagenesis and next-generation sequencing (NGS) represent a powerful approach to understand gene functions in bacteria on a genome-wide scale. This approach has been utilized in a variety of bacterial species to provide comprehensive information on gene functions related to various phenotypes or biological processes of significance. With further improvements in the molecular protocol for specific amplification of transposon junction sequences and increasing capacity of next generation sequencing technologies, the applications of Tn-seq have been expanding to tackle questions that are important yet difficult to address in the past. In this review, we will discuss the technical aspects of different Tn-seq methods along with their pros and cons to provide a helpful guidance for those who want to implement or improve Tn-seq for their own research projects. In addition, we also provide a comprehensive summary of recent published studies based on Tn-seq methods to give an updated perspective on the current and emerging applications of Tn-seq.

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.