Diagnostics, epidemiological observations and genomic subtyping in an outbreak of pullorum disease in non-commercial chickens.

Salmonella Gallinarum biovar Pullorum (S. Pullorum) is a poultry pathogen associated with significant economic losses and animal suffering. Strict eradication programmes have eliminated S. Pullorum from the commercial poultry sector in most regions, but occasional outbreaks still occur in the non-commercial population. In 2012, pullorum disease was diagnosed in a non-commercial flock in Sweden. Epidemiological, post-mortem and bacteriological investigations identified three more infected flocks. To study the epidemiological relationships between the flocks and evaluate different subtyping methods for S. Pullorum, 13 isolates from the four infected flocks were investigated by pulsed-field gel electrophoresis (PFGE) and multi-locus variable number tandem repeat analysis (MLVA). Four isolates collected from two non-commercial flocks in 2001 were also included. Six representative isolates from 2012 were further analysed by high-throughput sequencing. To differentiate between biovars Gallinarum and Pullorum, ten regions of difference (RODs) were investigated by in silico PCR. Three different PFGE-types were observed from the four epidemiologically linked farms in 2012 and MLVA further discriminated the isolates. SNP typing based on high-throughput sequencing clustered the four farms from the 2012 outbreak in two pairs. The PFGE, MLVA and high-throughput sequencing results suggested at least two different sources of infection or a common genetically mixed source in 2012. High-throughput sequencing is useful both for subtyping of S. Pullorum and may also be used for differentiating between the two biovars Pullorum and Gallinarum.