Development of Methods for Recovery and Quantitation of Viral Nucleic Acids from Broiler Litter

Abstract

We investigated the development of standardised methods of extraction and quantitation of viral nucleic acids from broiler litter. To detect and quantify virus we used fully quantitative Taqman® qPCR assays with plasmid-based standard curves to quantify Marek's disease virus (MDV, dsDNA), infectious laryngotracheitis virus (ILTV, dsDNA), Fowl adenovirus (FadV, dsDNA), chicken anaemia virus (CAV, ssDNA) and infectious bursal disease virus (IBDV, dsRNA). A series of experiments examined the effects of litter washing, blending, bead beating, and removal of inhibitors using polyvinylpolypyrrolidone (PVPP). To evaluate the qPCR assays and the DNA extraction techniques we monitored the recovery of fixed amounts of virus added to litter samples. The three litter types used were hardwood shavings, softwood (pine) shavings and rice hulls. Hardwood shavings were shown to contain high levels of PCR inhibitors but these could be neutralised by PVPP. Detectable virus recovery was good for IBDV and CAV, but low for the dsDNA viruses ILTV and MDV. The third dsDNA virus, FAdv, was unable to be detected. The four detectable viruses were detectable in all fractions of material (retentate after filtration, and in both the pellet and supernatant fractions following centrifugation of the filtrate) with highest concentrations in the pellet. The results indicate that a method based on washing samples with buffer containing 0.15% Tween-80 followed by bead beating and PVPP treatment would enable detection of most DNA and RNA viruses from litter with the greatest concentration of virus found in the pellet fraction after centrifugation. Work is ongoing to resolve the low recovery rate of dsDNA viruses and to simplify the litter processing and DNA extraction further.