Bovine tuberculosis (bTB) is a zoonotic disease with consequences for public health as well as the economy. In the EU, compulsory eradication programmes have been applied, and most territories in Italy have been reported as disease-free (FTs). However, outbreaks (OBs), i.e., an officially confirmed occurrence of bTB in one or more animals in an establishment, have continued to be reported. In this study we provide an overview of bTB in terms of OB numbers in cattle from Italian FTs. Legislative sources were collected to find the FTs, the relevant declaration of free status year (FSY), and regional control and surveillance plans. Then, descriptive and statistical analyses were applied to the collected OBs. A total of 12 regions and 19 provinces were declared FTs in the 20 years from 2003 to 2023. Differences in regional plans were observed with respect to the percentages of herds that were annually controlled (control frequency). Overall, 370 OBs were recorded. A non-statistically significant decrease in the OB incidence rate after the FSY was declared. However, a notable increase in OBs detected at slaughterhouses after the FSY suggests that control systems (serological tests) at the herd level are not completely effective. Differences in the herds’ control frequencies among FTs seem to not have had a significant influence on the observed OB number. The Tuscany region was the most affected FT based on the OB numbers after the FSY (especially in the last year). Epidemiologically relevant primary determinants seem to be the farming system (semi-extensive and adjacent herds) and the cattle movements from positive incidence areas (trade and animal fairs). The role of wild boars in the disease maintenance cannot be excluded. The results of this study stress the need to revise bTB eradication and surveillance plans based on risk analysis.

The 16S rRNA metabarcoding, based on Next-Generation Sequencing (NGS), is used to assess microbial biodiversity in various matrices, including food. The process involves a "dry-lab" phase where NGS data are processed through bioinformatic pipelines, which finally rely on taxonomic unit assignment against reference databases to assign them at order, genus, and species levels. Today, several public genomic reference databases are available for the taxonomic assignment of the 16S rRNA sequences. In this study, 42 insect-based food products were chosen as food models to find out how reference database choice could affect the microbiome results in food matrices. At the same time, this study aims to evaluate the most suitable reference database to assess the microbial composition of these still poorly investigated products. The V3-V4 region was sequenced by Illumina technology, and the R package “DADA2” used for the bioinformatic analysis. After a bibliographic search, three public databases (SILVA, RDP, NCBI RefSeq) were compared based on amplicon sequence variant (ASV) assignment percentages at different taxonomic levels and diversity indices. SILVA assigned a significantly higher percentage of ASVs to the family and genus levels compared to RefSeq and RDP. However, no significant differences were noted in microbial composition between the databases according to α and β diversity results. A total of 121 genera were identified, with 56.2% detected by all three databases, though some taxa were identified only by one or two. The study highlights the importance of using updated reference databases for accurate microbiome characterization, contributing to the optimization of metabarcoding data analysis in food microbiota studies, including novel foods.