Diagnosis of duck circovirus infections by conventional and real-time polymerase chain reaction tests

Abstract

Development of the first conventional and real-time polymerase chain reaction (PCR) tests for the diagnoses of duck circovirus (DuCV) infections is described. Both tests amplified a 230 bp fragment specific to the DuCV Rep gene. Although both tests had the same detection limit (13 )/10 3 target DNA/ml) when the target DNA was diluted in water, the detection limit of the real-time test (13 )/10 4 target DNA/ml) was 10-fold less than the conventional test (13)/10 5 target DNA/ml) when the amplifications were performed in the presence of cellular DNA. Using the conventional PCR test, DuCV DNA was detected in 85 (84%) of 101 bursa of Fabricius samples from dead or sick ducks, aged between 1 and 12 weeks, and in samples from 35 (94%) of 37 flocks. Application of the SYBR Green-based real-time PCR test to 54 selected bursa of Fabricius samples indicated that more samples were positive by real-time PCR than by conventional PCR, allowed the numbers of genome copies to be estimated and showed that some bursa of Fabricius samples contained over 10 13 genome copies/g tissue. Although DuCV infections were detected in birds aged from 1 to 12 weeks, higher virus DNA levels were detected in ducks aged older than 5 weeks than in ducks younger than 5 weeks. An in situ hybridization method for the detection of DuCV in histological samples was also developed. Additional work is required to determine the clinicopathological significance of DuCV infections.

Quantification of pigeon circovirus in serum, blood, semen and different tissues of naturally infected pigeons using a real-time polymerase chain reaction

“…The data indicated that PiCV load in sick young pigeons is particularly high in tissues such as the liver (ranging from 2.65)10 5 to 2.88)10 8 copies genome/mg), the spleen (6.88)10 5 to 5.57)10 8 copies genome/mg) and the BF (1.25 )10 6 to 2.07)10 9 copies genome/mg). High virus loads have also been detected by semiquantitative dot blot hybridization in BF tissues of pigeons (Todd et al, 2002), and similar results were recorded for duck circovirus infection by real-time PCR (Fringuelli et al, 2005). Although the numbers of birds investigated were small, a significant difference in the viral loads of liver was detected between YPS and apparently healthy infected pigeons.…”

Evidence of possible vertical transmission of duck circovirus

“…Because of the absence of available cell culture for the isolation of DuCV, the PCR assays were often used for detecting DuCVs (Hattermann et al, 2003;Fringuelli et al, 2005;Jiang et al, 2008;Zhang et al, 2009;Wan et al, 2011;Wang et al, 2011). However, these methods cannot be used to distinguish mixed infection of DuCV-1 and DuCV-2.…”

“…There were multiple local lesions in the spleen, thymus and BF of ducks infected with DuCV (Liu et al, 2010b). Surveys would suggest a high prevalence of the virus in flocks experiencing morbidity and mortality as well as those that appear clinically normal (Fringuelli et al, 2005;Chen et al, 2006;Banda et al, 2007;Jiang et al, 2008;Zhang et al, 2009;Liu et al, 2010a).…”

Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos

“…PCR assays to detect duck circovirus (DuCV) in duck embryos and goose circovirus (GoCV) and goose haemorrhagic polyomavirus (GHPV) in samples from goose embryos were also conducted. The primers for detection of those viruses and PCR reaction parameters were described by Sellers et al (2004) for ARV, by Wozniakowski et al (2009) for DDV, by Plummer et al (1998) for DEV, by Fringuelli et al (2005) for DuCV, by Ball et al (2004) for GoCV and by Guerin et al (2000) for GHPV.…”