Development and evaluation of real-time TaqMan^®RT-PCR assays for the detection of avian nephritis virus and chicken astrovirus in chickens

Abstract

The development and preliminary evaluations of two TaqMan † -based, real-time reverse transcriptasepolymerase chain reaction (rRT-PCR) assays for the quantitative detection of avian nephritis virus (ANV) and chicken astrovirus (CAstV) RNAs are described. The assays used amplicons generated from the 3' untranslated region of the ANV genome and a conserved region of CAstV open reading frame 1b including its junction with open reading frame 2. High virus RNA levels ( !10 5.99 viral copies) were detected for ANV and CAstV in 81% and 67% gut content samples from growth-retarded broiler flocks. Results from longitudinal surveys of two broiler flocks showed that ANV and CAstV RNAs were detected in most gut content and kidney samples collected at all time points from day 0 to day 35, with RNA levels of both astroviruses being higher in the gut contents than in the kidneys, and with the ANV RNA levels being greater than those of CAstV especially at early (days 7 and 14) time points. When the results obtained for the days 4/5 time-point samples from four broiler flocks with varying growth performances were compared, the two better-performing flocks had 100-fold to 1000-fold less ANV viral copies than the flocks that performed least well. Application of the rRT-PCR tests to samples collected from broiler chicks, which were experimentally infected with a crude gut content inoculum, demonstrated that ANV RNA could be detected in gut content and kidney samples at levels similar to those found at corresponding time points in longitudinal survey samples, whereas CAstV RNA was detected at lower levels than in the longitudinal survey samples, especially in kidney samples.

Chicken astrovirus capsid proteins produced by recombinant baculoviruses: potential use for diagnosis and vaccination

“…The origin and growth in primary chick embryo liver (CEL) cells of the 11672, FP3 and 612 isolates of CAstV were described previously (Todd et al, 2009a). The isolation of the CAstV VF08-29 isolate and its growth in primary CEL and LMH cells was described by Smyth et al (2012).…”

“…CAstV was characterized as a novel astrovirus by Baxendale & Mebatsion (2004), who reported the isolation of three antigenically similar viruses from outbreaks of runtingÁstunting syndrome that occurred in the Netherlands in the 1980s. Since then CAstVs have been detected by reverse transcriptasepolymerase chain reaction (RT-PCR) in intestinal samples from growth-retarded and normal broilers reared in the USA, the UK and other European countries (Day et al, 2007;Smyth et al, 2009Smyth et al, , 2010. The existence of two antigenically different CAstVs, namely isolate 11672 and isolate 612, which share low levels of cross-reactivity as determined by indirect immunofluorescence (IF), has been reported by Todd et al (2009a).…”

“…The existence of two antigenically different CAstVs, namely isolate 11672 and isolate 612, which share low levels of cross-reactivity as determined by indirect immunofluorescence (IF), has been reported by Todd et al (2009a). These isolates are representative of two major CAstV groups, designated A and B, which display very low levels (38 to 40%) of capsid protein identity (Smyth et al, 2012). Seroprevalence investigations involving separate indirect IF tests have shown that infections with CAstVs from both groups are common in UK broiler and broiler breeder flocks, and are widespread in poultry organizations from throughout Europe (Todd et al, 2009b).…”

Development of an indirect enzyme-linked immunosorbent assay test for detecting antibodies to chicken astrovirus in chicken sera

“…They have been detected in a US investigation of gut and faeces samples from healthy broiler chicken flocks and from flocks affected by enteritis and growth problems , and in gut content and kidney samples from UK flocks with growth-retardation problems (Smyth et al, 2009(Smyth et al, , 2010. In addition, CAstVs, originally recognized as ELVs, have also been shown to cause varying degrees of growth retardation following experimental infections of one-day-old specific pathogen free (SPF) or broiler chicks (McNulty et al, 1990;McNeilly et al, 1994).…”

“…CAstV was first characterized as a novel astrovirus by Baxendale and Mebatsion (2004), who reported the isolation of three antigenically similar viruses, exemplified by the P22-18.8.00 isolate, from outbreaks of runting stunting syndrome that occurred in the Netherlands in the 1980s. Since then CAstVs have been detected by reverse transcription-polymerase chain reaction (RT-PCR) in intestinal samples from growth-retarded and normal broilers reared in the USA, India, UK and other European countries Smyth et al, 2009Smyth et al, , 2010Bulbule et al, 2013). The existence of two antigenically different CAstVs, typified by the 612 and 11672 isolates, which share low levels of cross-reactivity as determined by indirect immunofluorescence (IF), has been reported by Todd et al (2009a).…”