Since 2007, a national Salmonella control program including obligatory vaccination has been ongoing in Belgium. In this context, the aim of the present study was to investigate the diversity of Salmonella enterica serovar Enteritidis isolates on 5 persistently contaminated Belgian layer farms and to examine the potential sources and transmission routes of Salmonella Enteritidis contamination on the farms during successive laying rounds. A collection of 346 Salmonella isolates originating from the sampled farms were characterized using a combination of multilocus variable number of tandem repeat analysis (MLVA) and phage typing (PT). On each farm, one or 2 dominant MLVA-PT types were found during successive laying cycles. The dominant MLVA type was different for each of the individual farms, but some farms shared the same dominant phage type. Isolates recovered from hens' feces and ceca, egg contents, eggshells, vermin (mice, rats, red mites, and flies), and pets (dog and cat feces) had the same MLVA-PT type also found in the inside henhouse environment of the respective layer farm. Persistent types were identified in the layer farm inside environment (henhouse and egg collecting area). Furthermore, this study demonstrated cross-contamination of Salmonella between henhouses and between the henhouse and the egg collecting area. Additional isolates with a different MLVA-PT type were also recovered, mainly from the egg collecting area. A potential risk for cross-contamination of Salmonella between the individual layer farms and their egg trader was identified
