Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2019

Investigating the use of Nanopore sequencing for pathogen surveillance in aquatic birds (#117)

Alistair R Legione 1 , Glenn F Browning 2 , Marc S Marenda 1 , Paola K Vaz 1 , Omid Fakhri 1 , Marjolein van Polanen Petel 3 , Peter Dann 3 , Nicholas D Crosbie 4 , Joanne M Devlin 1
  1. Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia
  2. Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia
  3. Philip Island Nature Parks, Cowes, Victoria, Australia
  4. Melbourne Water, Melbourne, Victoria, Australia

Victoria has diverse avifauna and supports a range of sites where large numbers congregate to breed, migrate, or escape drought. Two important sites for Victorian birds are Melbourne Water’s Western Treatment Plant (WTP), which supports tens of thousands of waterfowl, and Phillip Island Nature Parks (PINP), which supports the largest little penguin colony in the world. Due to the ecological and economic importance of these populations, pathogen surveillance is a desirable management tool to identify potential threats and the risk of disease in advance of, and to reduce the incidence of, mass mortality events. With the advent of third-generation sequencing technology, such as Nanopore sequencing through the MinION device, it has become possible for in-field (or near-field) sequencing of samples for rapid simultaneous detection of organisms of interest, and previously unknown potential pathogens. We collected field faecal samples from 46 penguins (PINP) and 121 waterfowl (WTP) to determine optimal extraction methodologies for Nanopore sequencing and investigate the potential of this tool for pathogen surveillance. We obtained the best results for quality and concentration of nucleic acids using traditional phenol-chloroform extraction methods, followed by a kit-based post-extraction ‘clean up’. We found that kit-based extraction could be used on samples after concentration by ultracentrifugation. Sequencing was undertaken on a subset of samples to determine which library preparation kit provided the best results. Optimal results were obtained by using Nanopore’s low input sequencing kits. The viruses most commonly identified by k-mer matching algorithms were bacteriophage, and invertebrate and plant viruses. DNA from Clostridium botulinum and Clostridium perfringens was detected in 6/18 and 8/18 samples, respectively, of the sequenced waterfowl species. These organisms are known to cause significant morbidity and mortality to avian specieswhen infections result in disease outbreaks. Further advances in automation of extraction of nucleic acids and subsequent library preparation, Nanopore sequencing technology itself, and rapid dataanalysis, may allow in-field pathogen surveillance of important wildlife populations in the near future.