Human salmonellosis incidence is increasing in the European Union (EU). Salmonellaenterica subsp. enterica serovar Enteriditis, Salmonellaenterica subsp. enterica serovar Typhimurium (including its monophasic variant) and Salmonellaenterica subsp. enterica serovar Infantis represent targets in control programs due to their frequent association with human cases. This study aimed to detect the most prevalent serotypes circulating in Abruzzo and Molise Regions between 2015 and 2020 in the framework of the Italian National Control Program for Salmonellosis in Poultry (PNCS)]. A total of 332 flocks of Abruzzo and Molise Regions were sampled by veterinary services in the period considered, and 2791 samples were taken. Samples were represented by faeces and dust from different categories of poultry flocks: laying hens (n = 284), broilers (n = 998), breeding chickens (n = 1353) and breeding or fattening turkeys (n = 156). Breeding and fattening turkeys had the highest rate of samples positive for Salmonella spp. (52.6%; C.I. 44.8%–60.3%). Faeces recovered through boot socks represented the greatest number of positive samples (18.2%). Salmonellaenterica subsp. enterica serovar Infantis was the prevalent serotype in breeding and fattening turkeys (32.7%; C.I. 25.8%–40.4%) and in broiler flocks (16.5%; C.I. 14.4%–19.0%). Salmonellaenterica subsp. enterica serovar Typhimurium was detected at low levels in laying hens (0.7%; C.I. 0.2%–2.5%) followed by breeding and fattening turkeys (0.6%; C.I. 0.2%–2.5%). Salmonellaenterica subsp. enterica serovar Enteriditis was also detected at low levels in laying hens (2.5%; C.I. 1.2%–5.0%). These findings highlight the role of broilers and breeding/fattening turkeys as reservoirs of Salmonella spp. and, as a consequence, in the diffusion of dangerous serotypes as Salmonellaenterica subsp. enterica serovar Infantis. This information could help veterinary services to analyze local trends and to take decisions not only based on indications from national control programs, but also based on real situations at farms in their own competence areas.

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28The use of whole genome sequencing (WGS) using next generation sequencing (NGS) 29 technology has become a widely accepted method for microbiology laboratories in the 30 application of molecular typing for outbreak tracing and genomic epidemiology. Several 31 studies demonstrated the usefulness of WGS data analysis through Single Nucleotide 32 Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, 33whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing 34 (cgMLST) has not been explored so far. The current study developed an allele-based 35 method cgMLST and compared its performance to the genome-wide SNP approach and 36 the traditional MLVA on a defined sample collection. The dataset comprised of 37 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically 38 unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in 39 this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of 40 the complete genome). We established the potential criteria necessary for inclusion of an 41 isolate into a brucellosis outbreak cluster to be ≤4 loci in the cgMLST and ≤10 in WGS 42 SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance 43 resolution than MLVA, particularly for strains belonging to the same lineage thus allowing 44 diverse and unrelated genotypes to be identified with greater confidence. The application 45 of this cgMLST scheme to the characterization of B. melitensis strains provided insights 46 into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for 47 outbreak investigations in human and animal brucellosis. 48 49 50 51 52 53 3 Introduction 54