Evaluation of a real time pcr to simultaneously detect and differentiate virulent and non-virulent Newcastle disease virus

Evaluation of a real time PCR to simultaneously detect and differentiate virulent and non-virulent Newcastle disease virus Simone Warner^1, Kim O’Riley^1, Xinlong Wang^1, Ibrahim Diallo^2 & Mark Fegan^1 ^1Department of Primary Industries, Victoria ^2 Biosecurity Sciences Laboratory (Queensland Department of Agriculture, Fisheries and Forestry, DAFF) WEDPP Final Report 31^st July 2012

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PROJECT DETAILS:


Project: Evaluation of a Real time PCR to simultaneously detect and differentiate virulent and non-virulent Newcastle disease virus.
Project Dates: 1st September 2011 to 30th May 2012

Project Leader: Simone Warner

Department of Primary Industries, Victoria

Attwood Centre

475 Mickleham Rd

Attwood, VIC, 3049
Phone: [phone]

Fax: [phone]

Email: [e-mail]

Project Background

Avian paramyxoviruses (APMV) have been grouped into nine distinct serotypes, APMV-1 to APMV-9 (Huovilainen et al., 2001). The most recognized of these is APMV-1, a virus more commonly known as Newcastle disease virus (NDV). The name ‘Newcastle Disease’ is exclusively reserved for the disease that results from infection with strains of APMV-1 that are pathogenic for chickens (Leighton & Heckert, 2007). NDV is a significant avian pathogen with worldwide distribution and is a disease listed by the World Health Organisation. Infection with NDV can cause outbreaks of virulent disease in poultry with devastating economic impact as a result of high mortality and slaughter for disease control. NDV strains are traditionally characterized as one of three pathotypes based on the disease severity in chickens. These include highly virulent (velogenic), intermediate (mesogenic) or non-virulent (lentogenic) (Beard & Hanson, 1984).

APMV and avian influenza viruses (AIV) have been obtained frequently from migratory waterfowl and other wild bird populations including in Australian studies (McKenzie et al., 1984; Peruolis & O’Riley, 2004; Haynes et al., 2009; Hansbro et al., 2010). Waterfowl are important reservoirs of these viruses and are considered as vectors for the transfer to poultry, which can lead to outbreaks of disease (Alexander, 1995). Most of the isolates detected in wild birds have been of low virulence for chickens and would be classified as lentogenic. Australia has been free from outbreaks of virulent Newcastle disease since 2002, when one incident in Victoria in 2002, and another in NSW from 1998-2000 were eradicated. As a consequence of those events, the national ND management Plan was developed, which included the formation of a steering committee to oversee further development and implementation of the plan. The goal of the current ND Management Plan, which is due to finish in 2012, is to reduce the spread of viruses that are precursors to virulent ND virus through the application of biosecurity measures in the poultry industry, as well through a vaccination program using live V4 and inactivated vaccine. The current plan is designed to lead to a risk-based exit strategy that may result in minimal or no vaccination in chicken flocks at the end of the program in 2012.
The most common PCR tests currently used in Australia cannot differentiate between virulent and avirulent strains of APMV. Therefore, whilst the tests are adequate for the detection of APMV, a positive result from a chicken sample causes concern until the virulence can be determined. This is generally delayed by at least a couple of days until after sequencing has been completed. Presently an epidemiological assessment is made, but there is some risk of a wrong decision pending the result of follow-up testing. One type of incorrect decision could result in further spread of virulent ND, with significantly greater control and eradication costs, and the second type, imposition of unnecessary control measures. This project seeks to evaluate a new test capable of differentiating virulent from virulent strains of APMV-1. A PCR test that could quickly and easily confirm that an APMV-1 PCR positive sample was caused by a virulent strain of NDV, as opposed to a V4–like avirulent strain, would be a great advantage in veterinary diagnostics.
Objectives

1. 
   To determine an appropriate NDV PCR to use for the simultaneous detection and differentiation of virulent and avirulent NDV strains
   
2. 
   To screen a selection of NDV positive and negative wild bird samples previously tested in Kim/Wise PCR using new PCR to compare diagnostic sensitivity
   
3. 
   To technology transfer the newly acquired NDV PCR test to Biosecurity Sciences Laboratory (BSL, Queensland Department of Agriculture, Fisheries and Forestry - DAFF)
   
4. 
   Undertake phylogenetic analysis of pathotyping sequencing results to show relationships of clades