Whole-genome sequencing of veterinary pathogens

Bacterial infections in production animals constitute a considerable burden across the globe and result in impaired animal welfare and production economy. Additionally, veterinary pathogens with zoonotic potential may cause severe infections in humans. Traditional molecular techniques based on gel-electrophoresis and single-locus sequencing has been widely used to characterize such types of veterinary pathogens.

However, DNA sequencing techniques have become fast and cost effective in recent years and whole-genome sequencing data provide a much higher discriminative power and reproducibility than any of the traditional molecular techniques. In this PhD project three important veterinary pathogens (Clostridium perfringens, Escherichia coli and Staphylococcus aureus) were investigated using whole-genome sequencing.

This was done in five different scientific papers which all have been published. Paper I and II In 2014, an increased mortality rate caused by Escherichia coli infections was observed among chickens (Gallus gallus domesticus) from farms in Denmark and other Nordic countries. Therefore, the genetic diversity and relatedness of 114 E. coli isolates primary associated with increased mortality in Nordic countries, was investigated using whole-genome sequencing.

In paper I, the genome assemblies of two avian pathogenic E. coli (APEC) isolates were annotated and the draft genomes deposited in a publicly available database. In paper II, a group of 47 closely related sequence type (ST)117 O78:H4 isolates from both broiler chickens and parent birds collected across the Nordic countries, was observed.

Since most Nordic poultry farmers import birds that are all part of the same breeding pyramid these results indicate that the ST117 O78:H4 isolates were transmitted vertically through this breeding pyramide. Therefore, it was concluded that vertically transmitted ST117 O78:H4 isolates were the main reason for the increased motality rates observed in the Nordic broiler industry.

Paper III Necrotic enteritis (NE) in chickens is primarily caused by pathogenic Clostridium perfringens strains. Studies have shown that three pathogenicity loci (NELoc-1, 2 and 3) and two virulence genes (netB and cnaA) are often carried by C. perfringens isolates collected from chickens with NE. In general, the virulence gene content have not been widely investigated in isolates from turkeys (Maleagris gallopavo) with NE.

However, netB has not been found in high prevalence among isolates from diseased turkeys and prior to this study, no publications using whole-genome sequencing on NE isolates from turkeys, have been published. Here, 30 C. perfringens isolates from both healthy and NE infected chickens and turkeys were analyzed using whole-genome sequencing.

The results showed that NELoc-1 and -3 and the two virulence genes netB and cnaA were significantly more associated with NE isolates from chickens compared to NE isolates from turkeys. Only NELoc-2 was associated with NE isolates from both turkeys and chickens. A putative collagen adhesion gene was discovered among all isolates from diseased turkeys and has been designated cnaD.

Potentially, CnaD could be of importance in regard to the NE pathogenesis in turkeys. In general, these results suggest that the NE pathogenesis in chickens is different from that of turkeys. Paper IV and V Staphylococcus aureus is a pathogen that commonly causes mastitis in dairy cows (Bos taurus).

Many different subtypes, virulence genes and pathogenicity islands have been associated with isolates from bulk tank milk (BTM) and dairy cows with clinical mastitis (CM). Prior to these studies, no Danish S. aureus isolates associated with bovine mastitis have been analyzed using whole-genome sequencing.

In paper V, 157 S. aureus isolates from BTM and dairy cows with CM were whole-genome sequenced and further investigated. In general, the results showed that BTM and CM isolates were of identical genetic background. This indicates that dairy cows can be natural carriers of S. aureus subtypes that in certain cases lead to CM.

A group of isolates that mostly belonged to ST151 carried three pathogenicity islands that were primarily found in this group. The prevalence of resistance genes was generally low but the first ST398 methicillin resistant S. aureus isolate from a Danish dairy cow with CM was observed. In paper IV, the assemblies from this isolate (strain Sa52) were annotated and the draft genome uploaded to a publicly available database