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

Abstract

The development of a real-time polymerase chain reaction (PCR) based on SYBR Green chemistry is described for the quantification of pigeon circovirus (PiCV) DNA in various samples. Plasmid containing a fragment of the PiCV genome was used to create a standard curve and to estimate the viral DNA copies in analysed samples. Both primers were designed in highly conserved regions to avoid false negatives, and amplified a 139-base-pair amplicon. When the amplifications were performed in the presence of cellular DNA extracted from PCR-negative liver, bursa and spleen samples, the detection limits were respectively 20, 20 and 60 copies of genome per milligram of tissue. These limits were 10, 160 and 25 copies/microl for control blood, sera and semen, respectively. For cloacal swab, the detection limit was 200 copies. The assay showed a linear detection over a six-log range (R(2)>0.99) and displayed reliable inter-assay and intra-assay reproducibility. Application of the test to sera samples indicated the presence of the virus in Belgium in 1991, 6 years before PiCV infections were histologically diagnosed. Testing of samples from pigeons with "young pigeon sickness" showed that the viral loads were high in the bursa of Fabricius (up to 2.07 x 10(9) copies/mg), the liver (up to 2.88 x 10(8) copies/mg) and spleen (up to 5.57 x 10(8) copies/mg). For liver, the viral load was significantly higher in sick pigeons than in apparently healthy pigeons. Detection of high quantities of PiCV DNA (up to 1.6 x 10(9) copies/microl) in the sera or blood of some young healthy pigeons indicated that the viral load in this sample type would not be useful as predictive indicator of disease. This work also showed that PiCV DNA can be detected in relatively large amounts in semen (up to 1.0 x 10(7) copies/ejaculate) and cloacal swabs (up to 3.6 x 10(10) copies/swab), confirming that PiCV may be transmitted by vertical and horizontal routes.