Rapid detection and characterisation of infectious bronchitis virus (IBV) from New Zealand using RT-PCR and sequence analysis

Abstract

AIMS: To develop a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect infectious bronchitis virus (IBV) from commercially-raised poultry in New Zealand and compare results with those from virus isolation. To characterise the IBV isolates using sequence analysis.

METHODS: Pooled tissue samples (trachea, kidney, caecal tonsils and cloacal swabs) from 164 broiler and 53 layer flocks located throughout New Zealand were collected in transport medium containing antibiotics. Tissues were homogenised and the resultant supernatant used directly in a RT-PCR assay, and also inoculated into the allantoic cavity of 10-day-old embryonated eggs for virus isolation. Primers for the RT-PCR were selected from an area close to the N-terminus of the S1 (spike) gene and bracketed the hypervariable region 1 (HVR 1). The RT-PCR amplimers were sequenced from both termini, and alignment was constructed and analysed.

RESULTS: From the 217 field samples that were subjected to RT-PCR, 42 (19%) were positive. Twenty-nine (69%) of these RT-PCR-positive, and none of the RT-PCR-negative, samples yielded virus by isolation in chicken embryos. A phylogenetic tree constructed from these amplimers, that spanned the HVR of the S1 gene, revealed the IBV isolates clustered into two demarcated groups which had <60% homology. It is likely that the isolates of one group were derived from the live attenuated vaccine commonly used in New Zealand.

CONCLUSIONS: The RT-PCR assay exhibited higher sensitivity than virus isolation and could be used for rapid diagnosis of IBV in the field. The prevalence of IBV appears to be surprisingly high in New Zealand although the use of pooled samples in the study did not allow accurate calculation of the prevalence in birds. Sequence analysis of a hypervariable region from the S1 gene was informative for the differentiation of closely-related strains.

KEY WORDS:

• Infectious bronchitis
• reverse transcriptase-polymerase chain reaction
• sequence analysis

Acknowledgements

We would like to thank Neil Christensen, and Kent Deitemeyer of Pacificvet Ltd, New Zealand, for supplying samples and funding. Additional funding was from Lincoln University and the Foundation for Research Science and Technology (FRST-Techlink). The first author was the recipient of a MFAT scholarship. IBV strains were supplied by the National Centre for Disease Investigation, Upper Hutt.