The poultry industry is faced with numerous challenges associated with infectious diseases and suboptimal performance of flocks. As microbiome research continues to grow, it is becoming clear that poultry health and production performance are partly influenced by nonpathogenic symbionts that occupy different habitats within the bird. This study has defined the baseline composition and overlaps between respiratory and gut bacteria in healthy, optimally performing chicken layers across all stages of the commercial farm sequence. Consequently, the study has set the groundwork for the development of interventions that seek to enhance production performance and to prevent and control infectious diseases through the modulation of gut and respiratory bacteria.

Communities of gut bacteria (microbiota) are known to play roles in resistance to pathogen infection and optimal weight gain in turkey flocks. However, knowledge of turkey respiratory microbiota and its link to gut microbiota is lacking. This study presents a 16S rRNA gene-based census of the turkey respiratory microbiota (nasal cavity and trachea) alongside gut microbiota (cecum and ileum) in two identical commercial Hybrid Converter turkey flocks raised in parallel under typical field commercial conditions. The flocks were housed in adjacent barns during the brood stage and in geographically separated farms during the grow-out stage. Several bacterial taxa that were acquired in the respiratory tract at the beginning of the brood stage persisted throughout the flock cycle, primarily Staphylococcus. Late-emerging predominant taxa in the respiratory tract included Deinococcus and Corynebacterium. Tracheal and nasal microbiota of turkeys were identifiably distinct from one another and from gut microbiota. Nevertheless, gut and respiratory microbiota changed in parallel over time and appeared to share many taxa. During the brood stage, the two flocks generally acquired similar gut and respiratory microbiota, and their average body weights were comparable. However, there were qualitative and quantitative differences in microbial profiles and body weight gain trajectories after the flocks were transferred to geographically separated grow-out farms. Lower weight gain corresponded with emergence of Deinococcus and Ornithobacterium in the respiratory tract, and Fusobacterium and Parasutterella in gut. This study provides an overview of turkey microbiota under field conditions and suggests several hypotheses concerning the respiratory microbiome. IMPORTANCE Turkey meat is an important source of animal protein, and the industry around its production contributes significantly to the agricultural economy. The microorganisms present in the gut of turkeys are known to impact bird health and flock performance. However, the respiratory microbiota in turkeys are entirely unexplored. This study has elucidated the microbiota of respiratory tracts of turkeys from two commercial flocks raised in parallel throughout a normal flock cycle. Further, the study suggests that bacteria originating in the gut or in poultry house environments may influence respiratory communities; and consequently, induce poor performance, either directly or indirectly. Future attempts to develop microbiome-based interventions for turkey health should delimit the contributions of respiratory microbiota and aim to limit disturbances to those communities.

Outbreaks of novel highly pathogenic avian influenza viruses have been reported in poultry species in the United States since 2014. These outbreaks have proven the limitations of biosecurity control programs, and new tools are needed to reinforce the current avian influenza control arsenal. Some enzootic countries have implemented inactivated influenza vaccine (IIV) in their control programs, but there are serious concerns that a long-term use of IIV without eradication may result in the selection of novel antigenically divergent strains. A broadly protective vaccine is needed, such as live-attenuated influenza vaccine (LAIV). We showed in our previous studies that pc4-LAIV (a variant that encodes a C-terminally truncated NS1 protein) can provide significant protection against heterologous challenge virus in chickens vaccinated at 2–4 weeks of age through upregulation of innate and adaptive immune responses. The current study was conducted to compare the performances of pc4-LAIV and IIV in young chickens vaccinated at 1 day of age. A single dose of pc4-LAIV was able to induce stronger innate and mucosal IgA responses and protect young immunologically immature chickens better than a single dose of IIV. Most importantly, when 1-day-old chickens were intranasally primed with pc4-LAIV and subcutaneously boosted with IIV three weeks later, they showed a rapid, robust, and highly cross-reactive serum antibody response and a high level of mucosal IgA antibody response. This vaccination regimen warrants further optimization to increase its range of protection.

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

Influenza A virus (IAV) noncanonical RNAs are bound by retinoic acid–inducible gene I (RIG-I). However, innate immune activation is infrequent and it is not understood why noncanonical IAV RNAs activate RIG-I in a sequence- or RNA structure–dependent manner. We hypothesized that multiple events need to occur before IAV RNA synthesis activates RIG-I and investigated whether RIG-I activation is stimulated by the noncanonical or aberrant transcription of mini viral RNAs (mvRNA), an RNA that is overexpressed in highly pathogenic IAV infections. We find that mvRNAs can cause noncanonical transcription termination through a truncated 5′ polyadenylation signal or a 5′ transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs stimulate the release of an mvRNA and complement RIG-I activation in trans. Overall, our findings indicate that sequential rounds of noncanonical or aberrant viral replication and transcription are needed for innate immune signaling by IAV RNA synthesis.