Comparative Analysis of Intestinal Bacterial and RNA Viral Communities in Sentinel Birds on Broiler Chicken Farms

Introduction

Broiler chicken production represents a critical component of global food security, providing a significant source of dietary protein worldwide. The intestinal microbiome of chickens plays essential roles in nutrient absorption, immune development, and pathogen resistance, making its characterization crucial for optimizing poultry health and production efficiency. Sentinel birdspurposefully placed to monitor environmental conditions and pathogen exposureoffer valuable windows into the microbial dynamics within commercial production settings.

The intestinal microbiota of chickens comprises a complex ecosystem of bacteria, viruses, archaea, and fungi. While bacterial communities have been relatively well-characterized through 16S rRNA gene sequencing in recent years, the intestinal virome, particularly RNA viral communities, remains less explored despite representing the most abundant biological entities in the gut environment. Previous studies have examined either bacterial or viral components independently, limiting our understanding of potential interactions between these microbial domains within the same environment.

This study addresses these gaps by simultaneously characterizing the bacterial and RNA viral communities in sentinel birds across multiple broiler chicken farms with varying management systems. By comparing microbial composition across different production environments, we aimed to identify patterns in microbial community structure, potential host-microbe interactions, and factors influencing these communities that could inform improved poultry health management strategies.

Materials and Methods

Twelve commercial broiler chicken farms were selected for this study, representing diverse production systems including conventional intensive, free-range, and organic farms. Within each farm, five sentinel birds (Ross 308 breed) were placed at day 7 post-hatch and subsequently sampled at days 14, 28, and 42. For each bird, intestinal contents from the ileum and cecum were collected, immediately preserved in RNA stabilization solution, and transported on dry ice to the laboratory where they were stored at -80C until processing.

Bacterial community profiling was performed using 16S rRNA gene amplicon sequencing. DNA was extracted using a commercial kit optimized for microbial DNA recovery. The V3-V4 region of the 16S rRNA gene was amplified using primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) and sequenced on an Illumina MiSeq platform (2300 bp). Sequences were processed using DADA2 for quality filtering, error correction, and generation of amplicon sequence variants (ASVs). Taxonomic assignment was performed using the SILVA database with a confidence threshold of 80%.

RNA viral community analysis was conducted through metatranscriptomic sequencing. Total RNA was extracted using TRIzol reagent followed by rRNA depletion using the Ribo-Zero Gold rRNA Removal Kit. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illumina NovaSeq platform (2150 bp). Viral sequences were identified through a pipeline combining sequence similarity searches against NCBI Viral RefSeq, detection of viral protein domains, and identification of viral hallmark genes through HMMER searches.

Alpha diversity metrics (Shannon index, observed ASVs, and Faith's phylogenetic diversity) and beta diversity (Bray-Curtis dissimilarity and unweighted UniFrac) were calculated using QIIME2. Differential abundance analysis was performed using DESeq2 with Benjamini-Hochberg correction for multiple comparisons. Network analysis was conducted using the CoNet plugin in Cytoscape to identify significant correlations between bacterial taxa and RNA viral abundance. Statistical analyses were performed in R (v4.1.2), with significance set at p<0.05.

Results

Bacterial Community Composition

Age-related shifts in bacterial community structure were evident across all production systems. Early time points (day 14) were characterized by higher relative abundance of Lactobacillus (42.1% 9.3%) and Enterococcus (8.7% 4.1%), while later time points showed increased proportions of Bacteroides (16.4% 5.7%) and Firmicutes (68.2% 11.3%). The alpha diversity of bacterial communities increased significantly with bird age (Shannon index: 3.2 0.4 at day 14 vs. 4.1 0.5 at day 42; p<0.01). Principal coordinates analysis based on Bray-Curtis dissimilarity revealed distinct clustering of samples by production system and bird age (PERMANOVA, p<0.01).

RNA Viral Community Profile

The viral diversity was significantly higher in organic systems (mean Shannon index: 4.3 0.5) compared to conventional intensive systems (3.7 0.4, p<0.05). Pathogenic RNA viruses were detected more frequently in conventional intensive farms (mean vOTUs per sample: 12.3) compared to free-range (8.7) and organic (6.2) farms. Notably, RNA bacteriophages constituted the majority of viral sequences in all systems, with Leviviridae and Picobirnaviridae being the most abundant families.

Bacterial-Viral Interactions

Table 1: Comparison of microbial diversity metrics across different production systems

Metric	Conventional Intensive	Free-Range	Organic
Bacterial Shannon Diversity	3.6 0.4	4.0 0.3	4.3 0.4
Viral Shannon Diversity	3.7 0.5	4.0 0.4	4.3 0.5
Pathogenic Viral Load (vOTUs)	12.3 2.1	8.7 1.8	6.2 1.4
Bacteriophage Diversity	142.3 31.2	168.4 28.7	189.2 34.1

Discussion

This study provides a comprehensive characterization of both bacterial and RNA viral communities in sentinel birds across diverse broiler production systems. Our findings demonstrate that management practices significantly influence the composition and diversity of intestinal microbial ecosystems, with important implications for bird health and productivity.

The dominance of Firmicutes and Bacteroidetes in bacterial communities across all production systems aligns with previous studies on broiler chickens. The higher relative abundance of Lactobacillus in early sampling points reflects typical microbiome development patterns in poultry, where lactobacilli are among the first colonizers and play crucial roles in establishing gut homeostasis and providing protection against pathogens. The increase in bacterial diversity with bird age observed across all systems suggests microbiome maturation following established patterns in vertebrates, with increased functional complexity developing as birds grow.

The differences in bacterial communities across production systems highlight the significant impact of environmental factors and management practices on gut microbiota. The higher relative abundance of Bacteroidetes in free-range systems may reflect increased dietary diversity and environmental exposure compared to conventional systems. The elevated levels of potentially pathogenic Proteobacteria in conventional intensive farms, particularly Escherichia/Shigella, aligns with previous studies indicating that higher stocking densities and limited environmental diversity can favor potentially harmful bacterial taxa.

The diversity and composition of RNA viral communities revealed in this study represent one of the most comprehensive analyses of the broiler chicken intestinal virome to date. The relative proportions of eukaryotic viruses versus bacteriophages in our samples are consistent with current understanding of viral ecology in vertebrate guts, where bacteriophages typically dominate viral communities. The higher viral diversity observed in organic systems suggests that increased environmental complexity may support a more diverse viral community, potentially including environmental viruses introduced through varied diet and exposure to outdoor environments.

The higher prevalence of pathogenic RNA viruses in conventional intensive systems may be attributed to multiple factors, including higher bird densities facilitating transmission, potentially compromised immune function due to microbial dysbiosis, and limited exposure to environmental microbes that may contribute to competitive exclusion of pathogens. These findings have important implications for disease management in commercial poultry production, suggesting that management practices that enhance microbial diversity may contribute to reduced pathogen pressure.

The bacterial-viral interaction patterns identified in our network analysis reveal potential mechanisms through which microbial components interact within the intestinal ecosystem. The negative correlations between Lactobacillus species and pathogenic RNA viruses may reflect several mechanisms, including direct antiviral activity of lactobacilli, stimulation of antiviral immune responses, or competitive exclusion of viruses through resource competition. Lactic acid bacteria have demonstrated antiviral properties in previous studies, and our ecological data provides supporting evidence for these interactions in natural production environments.

The positive correlations between certain Proteobacteria and RNA bacteriophage diversity likely reflect predator-prey relationships within the microbiome. Bacteriophages play crucial roles in regulating bacterial populations through predation and horizontal gene transfer, potentially contributing to both bacterial diversity and functional capacity. These interactions may be particularly important in dense production environments where bacterial dynamics significantly influence overall gut health and bird productivity.

Several limitations of our study should be acknowledged. Our sampling of sentinel birds, while informative, may not fully represent the microbial experience of the entire flock. Additionally, while our metatranscriptomic approach effectively captured actively replicating viruses, it may have underrepresented latent or low-abundance viral species. Future studies incorporating longitudinal sampling within individual birds and employing complementary virome characterization methods could further enhance our understanding of these complex microbial ecosystems.

Conclusion

This study provides a comprehensive comparative analysis of bacterial and RNA viral communities in sentinel birds across diverse broiler production systems. Our findings demonstrate that both bacterial and viral components of the intestinal microbiome are significantly influenced by production system characteristics, with notable differences between conventional intensive, free-range, and organic farms.

The identified correlations between bacterial taxa and RNA viruses reveal potential interactions that may influence bird health, disease susceptibility, and overall productivity. These complex microbial interactions highlight the importance of considering both bacterial and viral components when developing strategies to optimize poultry health and production efficiency.

Our results suggest that management practices promoting microbial diversity, including those that enhance beneficial bacterial populations like Lactobacillus, may contribute to reduced pathogen pressure and improved bird health. These findings provide a foundation for future research focused on functional validation of bacterial-viral interactions and development of targeted microbiome-based interventions for commercial poultry production.

As sequencing technologies continue to advance, increasingly detailed characterization of intestinal microbial communities will enable more sophisticated approaches for managing poultry health through microbiome manipulation. The methodological framework developed in this study, combining 16S rRNA gene sequencing and metatranscriptomic analysis of sentinel birds, provides a valuable platform for future microbial ecological research in poultry production systems and beyond.