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.
The purpose of this study was to molecularly characterize circoviruses that infect finches and gulls. Circovirus-specific DNAs were isolated using polymerase chain reaction methods from bursa of Fabricius tissues from a Gouldian finch (Chloebia gouldiae) and a herring gull (Larus argentatus) that were known to be circovirus-infected. Nucleotide sequence determination and analysis of cloned genomic DNAs showed that these circoviruses represented novel members of the genus Circovirus of the family Circoviridae, and have been tentatively named Finch circovirus (FiCV) and Gull Circovirus (GuCV). Both new circoviruses shared genome organizational features with previously characterized circoviruses, such that both contained two major, inversely-arranged open reading frames encoding the putative replication-associated and capsid proteins, and both contained a potential stemÁloop and nonanucleotide motif. Phylogenetic analyses based on genome nucleotide sequences and involving the seven additional genus members indicated that FiCV and GuCV were more closely related to canary circovirus, beak and feather disease virus and pigeon circovirus, and that FiCV and canary circovirus were the most closely related avian circoviruses. Pairwise comparisons showed that the capsid proteins of FiCV and GuCV shared highest amino acid identity values with those of canary circovirus (62.0%) and pigeon circovirus (40.6%), respectively. The 5? intergenic region of GuCV was longer (207 nucleotides) and contained more direct and inverse repeated sequences than those of other circoviruses, while the 3? intergenic region of FiCV was notable in being longer (307 nucleotides) than its counterparts in other circoviruses and in containing two long repeats of 77 nucleotides.
Infections with pigeon circovirus (PiCV) occur in young racing pigeons and pigeons raised for meat production and have been reported worldwide, but relatively little is known about the disease induced by PiCV infection. The aim of this study was to investigate how PiCV is transmitted. Using a sensitive polymerase chain reaction (PCR) test, the presence of PiCV was investigated in a wide range of samples from adult pigeons, embryos, breeders and young birds, which were derived from a racing loft that had a clinical history of "young pigeon sickness" and in which PiCV had been previously been diagnosed. Using PCR, PiCV DNA was detected in tissues of 13/20 apparently healthy older birds, aged from 1 to 9 years. Viral DNA was most commonly detected in the respiratory organs, including the trachea, pharynx and lung, followed by tissues such as the spleen, kidney and liver. It was also detected in the ovary and/or testes of some birds. This finding, and the detection of viral DNA in tissues from 8/22 embryos, suggested that PiCV may be vertically transmitted. Testing of pharyngeal and cloacal swabs, and blood samples, collected immediately before the death of the adult pigeons, failed to detect all birds found to be infected at necropsy, suggesting that testing of potential breeding birds would not enable exclusion of infected birds from breeding programmes. Additional PCR testing of cloacal swab samples obtained sequentially from 19 young pigeons showed that while four were excreting virus when 15 days old, only one bird was excreting at the time of weaning (28 days old). The detection of viral DNA in cloacal swab samples from 15.8% of the birds when 37 days old and 100% of birds when 51 days old suggested that most young pigeons probably became infected in the rearing loft.
Nineteen racing pigeons aged from one to five years were examined postmortem. pcr tests showed that the spleens of 16 of them were positive for pigeon circovirus, the livers of six were positive, and blood from one of them was positive for the virus. Five of 44 embryos in embryonated eggs collected from three lofts were positive by pcr, but swabs taken from the crops of 64 adult birds which were feeding one- to 10-day-old squabs in these three lofts were negative for the viral dna.
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