Detection of chicken astrovirus by reverse transcriptase-polymerase chain reaction

Abstract

The development of a reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting chicken astroviruses (CAstV) is described. Primers, which amplified a fragment of 510 base pairs, were located in conserved regions within the ORF 1b (RNA polymerase) gene. The limit of detection of the test was estimated to be approximately 60 viral copies using a 10-fold dilution series of in vitro transcribed RNA. Positive signals were produced with representative CAstV samples, some of which were not detected by a previously described RT-PCR test for detecting CAstV, but other avian astroviruses including avian nephritis virus and duck hepatitis virus types 2 and 3 tested negative. When applied to gut content samples and swabs from UK and German broiler flocks with growth problems, CAstVs were detected by RT-PCR in 50/52 (96%) samples. CAstVs were detected in between 30% and 72.5% pooled gut content samples from longitudinal surveys of four broiler flocks displaying below-average performances. Whereas all day 0 samples were CAstV-negative, high detection rates were observed when the surveyed birds were aged 4, 5 and 7 days. Based on partial ORF 1b sequences, a phylogenetic analysis of 20 CAstVs indicated the existence of two groups. One group comprised four CAstV isolates, including FP3 and 11672, and two field CAstVs, which shared >94% nucleotide identity. The remaining 14 CAstVs, comprising the first characterized CAstV and 612 isolates and 12 field CAstVs, shared 85% to 99% nucleotide identity and displayed 76% to 79% nucleotide identity with the 11672-like and FP3-like CAstVs. © 2009 Houghton Trust Ltd.