Detection and isolation of avian paramyxovirus serotype 1 from outbreaks of Newcastle disease in 2002

AVIAN paramyxoviruses (APMVs) comprise nine antigenic types, designated APMV-1 to APMV-9. APMV-1 is a synonym for Newcastle disease virus (NDV) and consists of viruses of both high and low virulence in chickens (Alexander 2000). The EU definition of, and procedures for, diagnosis of Newcastle disease are laid down in Council Directive 92/66/EEC (CEC 1992). The diagnosis of Newcastle disease must be based on virus isolation (VI) and serological distribution of NDV followed by a biological pathogenicity assay, that is, the intracerebral pathogenicity index (ICPI). In contrast, the Office Internationale des Epizooties (OIE 2006) accept the demonstration of multiple basic amino acids at the C terminus of the F2 protein as an alternative to cultivation. Serum antibodies against NDV may be detected by various test methods including the haemagglutination inhibition (HI) test or by ELISA. In the present study, samples from three flocks (A, B and C) of commercial layers or layer pullets (18,000 to 45,000 birds in total), which had experienced outbreaks of Newcastle disease, were examined for the presence of NDV by VI in specific pathogen-free (SPF) chicken embryos and by the use of reverse transcriptase-PCR (RT-PCR). There was no contact between the flocks. Dead or euthanased sick birds were submitted to the national reference laboratory for Newcastle disease (Danish Institute for Food and Veterinary Research). Two submissions of samples were collected from each flock between July 11 and August 21, 2002, designated ‘virology 1’ and ‘virology 2’. The ‘virology 2’ samples were collected when the flocks were killed. Blood samples, collected on one occasion in each flock were tested for the presence of antibodies to APMV. The serum samples were collected up to two days before collection of the ‘virology 1’ samples in the flocks. VI by inoculation in SPF chicken embryos and virus identification conformed to the 92/66/EEC guidelines (CEC 1992). RNA extraction, RT-PCR of the F and HN genes, and sequencing of the cleavage site of the F gene were performed according to Jørgensen and others (1999) and Otim and others (2004). The different methods (virus cultivation v RT-PCR) were compared using Fisher’s exact test to evaluate the null hypothesis of independence of row and columns in a contingency table with fixed marginals (Agresti 2002). Calculations were carried out in R version . 2.0. 1 (R Project 2006). The HI tests for all serum samples from all three flocks were positive. A considerable proportion of the sera, ranging from 50 to 90 per cent within the flocks, had antibody titres of 1:256 or more. The results of the VI and RT-PCR are presented in Table 1. Statistical analysis of the results of the various methods showed a significant difference at 95 per cent significance level between VI and RT-PCR. The P value was 1·4 x 10–2 for VI v RT-PCR for the F gene, 2·4 x 10–3 for VI v RT-PCR for the HN gene, 1·6 x 10–3 for VI v RT-PCR for both F and HN gene positivity, and 5·2 x 10–8 for the F gene v the HN gene. The sequences of the 276 bp fragment of the F gene of virus isolated from tissue samples and of virus detected directly from tissue did not show any differences at the nucleotide level in samples or isolates from any of the three flocks. The amino acid sequence possessed the RRQRR↓F motif at the cleavage site, indicating a APMV-1 virus with high pathogenicity to chickens. Epidemiological circumstances suggest that the outbreaks in flocks A and C were primary outbreaks, while the outbreak in flock B could have been either primary or secondary caused by transmission of NDV from a fourth flock in which isolation of NDV was unsuccessful. Since 1995, the occurrence of Newcastle disease in Denmark has strongly indicated primary introduction of the virus from wild birds (Jørgensen and others 1997, 1999, Jørgensen and Handberg 2001). The source of NDV in the present study may have been feral birds. Compared with earlier outbreaks, it was difficult to isolate the NDV from the flocks in the 2002 outbreaks. A comprehensive programme of inoculation of tissue samples in SPF embryos was carried out. Out of a total of 62 tissue sample pools inoculated, NDV was isolated from six (10 per cent), all of which were brain samples; that is, six of 18 (or 33 per cent) of brain samples were positive for NDV. The RT-PCR procedures detected APMV-1 in a total of 22 (35 per cent) samples. The procedure amplifying the F gene detected APMV-1 in 15 of 18 pools of brain tissue (83 per cent). This procedure (for the F gene) also detected the virus in six of 44 non-brain samples (14 per cent). In total, the procedure amplifying the F gene detected NDV in 21 of 62 pools (34 per cent). The difficulties of VI experienced in the present study may have been due to collection of virological samples at late stages of infection. This assumption is supported by the serological results, which showed a high prevalence of birds with relatively high titres almost at the same time as the initial collection of virological samples (‘virology 1’). Thus, only quantities of virus lower than one egg infective dose or elements of non-infective virus particles may have been present in the samples in which NDV was detected by RT-PCR, but not by VI. In the present study, the detection of NDV by RT-PCR was significantly more sensitive than detection of NDV by inoculation. Furthermore, sequencing of the F protein cleavage site from RT-PCR products derived directly from the tissues of the infected birds revealed information on the virulence Veterinary Record (2006) 159, 561-562