Detection and characterisation of canine astrovirus, canine parvovirus and canine papillomavirus in puppies using next generation sequencing

Abstract

Gastroenteritis in young animals is a clinical presentation with many infectious and non- infectious aetiologies. We used next generation sequencing (NGS) to investigate the possible infectious causes of gastroenteritis in puppies from a dog kennel in Victoria, Australia. The near complete genome of a canine astrovirus was obtained from pooled faecal samples, and was found to be 94.7% identical with a canine astrovirus detected in the United Kingdom in 2012. The phylogenetic analysis of the capsid gene found similarities to those of canine astroviruses identified in Italy in 2005 and in UK and Hungary in 2012, but distant from that of a canine astrovirus previously identified in Australia in 2012. Thus, different serotypes of canine astrovirus are likely circulating in Australia. The close relationship to European astroviruses also suggested that there had been recent movements of ancestor canine astroviruses between Australia and Europe. NGS also detected other infections in the puppies including several canine papillomaviruses and a canine parvovirus (vaccine strain) as well as a very low level of campylobacter. Canine astrovirus was the probable cause of diarrhoea in these puppies, with the possible involvement of campylobacter bacteria. NGS was effective as a non-targeted method to determine the likely infectious cause of gastroenteritis.

Detection of a Reassortant H9N2 Avian Influenza Virus with Intercontinental Gene Segments in a Resident Australian Chestnut Teal

“…All samples, both pooled and individual, were processed, and virus particles were enriched using a protocol described previously [36,37]. Briefly, the samples were homogenised at 25 Hz for 2 min, followed by centrifugation at 17,000× g for 3 min, and the supernatant was filtered using a 0.8 µm polyethersulfone (PES) spin-filter.…”

“…The samples were then divided into two, one aliquot ultracentrifuged at 178,000× g for 1 h and the other not ultracentrifuged. Both of the aliquots were Viruses 2020, 12, 88 3 of 13 then treated with benzonase and micrococcal nuclease for 2 h [36,37]. The ultracentrifuged samples were then treated with 200 µM PMAxx for 30 min in a PMA-Lit LED Photolysis Device, as indicated by the manufacturer (Biotium, Fremont, CA, USA).…”

“…Finally, nucleic acids were extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The cDNA synthesis and DNA amplification were carried out using the SeqPlex RNA Amplification Kit (Sigma, St. Louis, MO, USA), as per the manufacturer's protocol [36,37].…”

“…Synthesized cDNA was then amplified using 25× PathAmp FluA PCR Primer Mix and 5 U/µL SuperTaq plus PCR Enzyme (Applied Biosystems) at 95 • C for 4 min, with 40 cycles of 95 • C for 15 s, 55 • C for 30 s, and 68 • C for 2 min, with a final 68 • C step for 7 min using an Applied Biosystems ProFlex PCR system (Thermofisher Scientific, Waltham, MA, USA). The amplified products were purified using 1.5× Agencourt AMPure XP kit (Beckman Coulter, Brea, CA, USA), and 5 µL of the purified products were then fragmented and amplified using the same SeqPlex Kit (Sigma), as described above, following the manufacturer's protocol [36,37].…”

A preliminary investigation of major bacterial antibiotic resistance genes present in Australian fecal samples using shotgun metagenomics and targeted sequencing

“…A pooled dog fecal sample (DFS) originating from two pups (7 weeks of age) was collected from the ground immediately after being deposited, with the consent of the owner 31 . These puppies were healthy at the time of collection, but they had been sick just 1-2 weeks before and an astrovirus was detected in the 7-week sample 31 . However, they were originally thought to have a Campylobacter infection and were likely treated with antibiotics at 5 weeks of age.…”