ITS-based metabarcoding of broiler and layer chicken litter: Linking fungal diversity to environmental and One Health risks.

The One Health concept highlights the interconnectedness of environmental, animal, and human health, particularly in agricultural environments that can serve as microbial reservoirs. This context positions poultry farming as a potential source of environmental fungal pathogens. This study aimed to characterize the structure and taxonomic composition of the fungal communities across diverse commercial poultry farms and inventory genera with known clinical significance to humans. We analyzed samples collected from three distinct Poultry houses using high-throughput sequencing of the fungal Internal Transcribed Spacer (ITS) regions. Specifically, both ITS1 (using primers ITS1F/ITS2) and ITS2 (using primers ITS3/ITS4) ribosomal RNA gene regions were sequenced. This dual-marker strategy was employed as a methodological approach to mitigate primer-mediated biases and ensure the capture of greater fungal richness across complex environmental matrices. Data analysis included alpha and beta diversity metrics, taxonomic assignment, and functional profiling via FUNGuild. We observed that the selection of the marker gene significantly impacted diversity estimates, with the ITS1 region consistently revealing higher fungal richness than ITS2. Each Poultry house maintained a distinct and unique fungal community structure. Crucially, the analysis identified several clinically relevant genera, including Aspergillus, Trichosporon, and Microascus, confirming the role of agricultural settings as reservoirs for potential human pathogens. Although harmless saprotrophic fungi were predominant, this study provides clear evidence for the presence of pathogenic taxa in the poultry environment. The findings underscore that a multi-marker sequencing approach (ITS1 and ITS2) is essential to accurately capture true fungal diversity and effectively identify public health risks, thereby strongly reinforcing the need for One Health biosecurity perspectives in poultry farming.