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Substantial variation in the virulence of Newcastle disease virus (NDV) isolates means that the detection of NDV or evidence of infection is insufficient for an adequate diagnosis, as control measures for avirulent viruses are very different to those for virulent viruses. Diagnosis therefore requires further characterization, at least as to whether an isolate is virulent or avirulent. Conventional detection and differentiation of ND viruses is perceived as slow, laborious and requiring an undesirable use of in vivo techniques. In addition, further characterization is needed to give greater information on origin and spread. This review concentrates on the application of monoclonal antibody and molecular biological approaches. Panels of monoclonal antibodies were a major advance for the characterization of NDV isolates, although confirmation of virulence for poultry still required in vivo testing. As molecular-based techniques become easier and more reliable, they are likely to supersede the use of monoclonal antibodies, especially for characterizing viruses for epidemiological purposes. The attraction of molecular-based techniques is that they may be able to cover all three aspects of Newcastle disease diagnosis (detection of virus, characterization, including inference of virulence, and epidemiology) quickly, accurately and definitively in a single test. A number of approaches based on the reverse transcriptase polymerase chain reaction have been developed, with subsequent analysis of the product by restriction enzyme analysis, probe hybridization and nucleotide sequencing. Although extensive variation among NDVs still poses technical problems, the real and potential advantages of a molecular biological approach to Newcastle disease diagnosis appear to be overwhelming.

A real-time reverse-transcription PCR (rRT-PCR) that targets a region of the polymerase (L) gene was developed to detect all known lineages of avian paramyxovirus type 1 (APMV-1), also known as Newcastle disease virus (NDV). A panel of 23 viruses representing the current known phylogenetic diversity of the APMV-1 population with a bias towards the more recent European strains, which had been grown in embryonated fowls' eggs, were tested. A range of positive and negative clinical samples (n = 350) provided by the National Reference Laboratory and International Reference Laboratory at VLA Weybridge were also tested. Positive clinical material included samples considered representative of lineages 3, 4 and 5 obtained from chickens, ducks, pigeons and partridges. The negative sample population was obtained from chickens, turkeys and ducks. The APMV-1 L gene rRT-PCR gave high relative sensitivity (96.05%) and specificity (98.18%) when compared with virus isolation in embryonated fowls' eggs. It is proposed that this assay could provide a first-line screening tool for the detection of APMV-1 in clinical samples.

Antigenic and genetic analyses of viruses from the 11 outbreaks of Newcastle disease in Great Britain, 12 of the outbreaks in Northern Ireland and the single outbreak in the Republic of Ireland which occurred in 1997, indicated that they were all essentially similar. In addition, the viruses from the British Isles were very similar to viruses isolated from three outbreaks in pheasants in Denmark between August and November 1996, from a goosander in Finland in September 1996, from an outbreak in chickens in Norway in February 1997, and from an outbreak in chickens in Sweden in November 1997. Viruses from outbreaks in other countries during 1995 to 1997 could be distinguished antigenically and/or genetically from the 1996 to 1997 Scandinavian/British Isles isolates, as could viruses responsible for two separate outbreaks in caged birds in quarantine premises in Great Britain in March 1997. Minor nucleotide differences in the 413-base region of the fusion gene and the 187-base region of the haemagglutinin-neuraminidase gene sequenced in this study allowed the 1996 to 1997 Scandinavian/British Isles isolates to be divided into groups. These groups broadly corresponded to the clusters of disease outbreaks, but suggested that the discrete outbreak in Scotland was probably the result of virus spread from Northern Ireland. Overall, the antigenic and genetic analyses of these viruses were consistent with the theory that the virus was introduced into the British Isles by migratory birds moving from north-east Europe. However, it was not possible to rule out other sources, such as the movement of pheasants from Denmark.

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A range of virus doses were used to infect 3-week-old chickens, turkeys and ducks intranasally/intraocularly, and infection was confirmed by the detection of virus shedding from the buccal or cloacal route by analysis of swabs collected using real-time reverse transcriptase-polymerase chain reaction assays. The median infectious dose (ID 50 ) and the median lethal dose (LD 50 ) values for two highly pathogenic avian influenza (HPAI) viruses of H5N1 and H7N1 subtypes and one virulent Newcastle disease virus (NDV) were determined for each virus and host combination. For both HPAI viruses, turkeys were 100-fold more susceptible to infection than chickens, while both these hosts were 10-fold more susceptible to H5N1 virus than the H7N1 virus. All infected chickens and turkeys died. Ducks were also much more readily infected with the H5N1 virus (ID 50 510 1 median embryo infective dose [EID 50 ]) than the H7N1 virus (ID 50 0 10 4.2 EID 50 ). However, the most notable difference between the two viruses was their virulence for ducks, with a LD 50 of 10 3 EID 50 for the H5N1 virus, but no deaths in ducks being attributed to infection with H7N1 virus even at the highest dose (10 6 EID 50 ). For both HPAI virus infections of ducks, the ID 50 was lower than the LD 50 , indicating that infected birds were able to survive and thus excrete virus over a longer period than chickens and turkeys. The NDV strain used did not appear to establish infection in ducks even at the highest dose used (10 6 EID 50 ). Some turkeys challenged with 10 6 EID 50 , but not other doses, of NDV excreted virus for a number of days (ID 50 010 4.6 EID 50 ), but none died. In marked contrast, chickens were shown to be extremely susceptible to infection and all infected chickens died (ID 50 /LD 50 010 1.9 EID 50 ).

Newcastle disease (ND) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious disease and high flock mortality that may result from some ND virus (NDV) infections, but also through the economic impact that may ensue due to trading restrictions and embargoes placed on areas and countries where outbreaks have occurred. All ages of pheasants are purported to be highly susceptible to infection with NDV, but clinical signs and mortality levels in infected birds vary considerably. This article reviews the available literature relating to infections in pheasants, describing the clinical presentation of the disease and the epidemiological role these hosts may have in the spread of ND.