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

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.

Sequence comparison of pigeon circoviruses.

The genome sequences of eight pigeon circoviruses (PiCV) were determined and compared with four previously published sequences. The viruses compared were from the USA, five European countries, China and Australia and included PiCVs from racing, feral, ornamental and meat pigeons and a Senegal dove (Streptopelia senegalensis). The 12 PiCV genomes, ranging from 2032 to 2040 nucleotides in length, displayed similar organizations. Pairwise comparisons showed that the genome nucleotide sequence identities ranged from 85.1% to 97.8% and that the amino acid identities of the putative replication associated (Rep) and putative capsid (Cap) proteins displayed ranges of 91.5-99.1% and 73.0-99.3%, respectively. Comparative analyses identified conserved nucleotide sequences within the Rep gene and 3' intergenic regions, which would be suitable for diagnostic PCR primers, and variable amino acid sequences within the capsid proteins, which should be considered when selecting virus isolates for vaccine development.

Detection of pigeon circovirus in cloacal swabs: implications for diagnosis, epidemiology and control.

Pigeon circovirus (picv) was detected in cloacal swab samples by means of a newly-developed, sensitive pcr. An initial investigation of 17 Belgian racing pigeons aged up to eight months showed that rates of detection of 88 per cent and above were achieved using samples of cloacal swab, blood and bursa of Fabricius. The sampling of 15 caged pigeons six times when they were from three to 31 weeks of age indicated that picv infections were more readily detected in cloacal swabs than in blood, and that the virus could be detected in cloacal swabs for longer periods after infection than in blood. picv infections were detected in cloacal swabs from 38 of 47 young pigeons aged from two to 31 weeks, from 12 racing lofts, which had clinical signs including diarrhoea and weight loss, regurgitation and respiratory signs. Samples from birds from two infected lofts indicated that picv could be detected in some birds for at least 27 weeks. Although nine of 14 pigeons aged from 32 to 45 weeks were virus-positive, picv was detected in only one of 18 adult pigeons that originated from four infected lofts.

Observations on detection, excretion and transmission of pigeon circovirus in adult, young and embryonic pigeons.

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.

New data on the transmission of pigeon circovirus.

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.

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections.

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

Efficacy of an inactivated aqueous-suspension Newcastle disease virus vaccine against paramyxovirus type 1 infection in young pigeons with varying amounts of maternal antibody.

Pigeons aged 3 weeks were vaccinated, subcutaneously, with an inactivated aqueous-suspension LaSota vaccine. Irrespective of the level of maternally-derived antibodies the single vaccination gave protection lasting 1 year as shown by resistance against an intramuscular challenge with a virulent 'pigeon' PMV-1 strain.

Assessment of vaccination of pigeons against paramyxovirus type 1 infection with inactivated aqueous-suspension or oil-emulsion vaccines.

The immune response and resistance to PMV1 challenge of pigeons vaccinated with inactivated oil-emulsion (OE) or aqueous-suspension (AS) vaccines were compared. ASa was based on NDV LaSota strain, ASb on NDV Terumo strain, OEa on NDV Poletti strain and OEb on NDV Ulster 2C strain. Groups were each vaccinated subcutaneously with one of ASa (0.2 ml), ASb (0.2 ml), OEa (0.5 ml), OEa (0.2 ml) and OEb (0.5 ml). An unvaccinated group was kept as control. The immune response measured by HI tests was the highest with the single 0.2 ml dose of ASa vaccine. The single 0.2 ml dose of OEa vaccine produced a poor HI response. Challenge with 'pigeon' PMV1 (intramuscular + intranasal/ocular) one month after vaccination resulted in the vast majority of unvaccinated pigeons excreting virus in the laryngeal secretions and the faeces and all . birds died. In comparison, virus shedding and morbidity-mortality were significantly reduced in the five challenged vaccinated groups. However, morbidity-mortality was higher in the OE groups than in the AS groups. No ASa vaccinated pigeon developed clinical signs and only one ASb vaccinated pigeon presented nervous signs. In contrast, morbidity-mortality reached 30 and 35% in the OE vaccinated groups. Only the inactivated aqueous-suspension vaccines, especially that prepared from NDV LaSota strain, gave, after one dose injection (0.2 ml), high resistance to a severe challenge with 'pigeon' PMV1.

Free and conjugated zeranol residues determined by radio-immunoassay in urine and plasma of calves treated with Forplix.

Anti-zeranol 7-hemisuccinate-bovine serum albumin was raised in rabbits. This antiserum was used in a radio-immunoassay of zeranol residues in urine and plasma of calves implanted with Forplix. The antibody was specific for zeranol but cross-reacted with its metabolite zearalenone and the mycotoxin zearalenone. Detection limits in plasma and in urine were 100 pg/ml and 1 ng/ml respectively. In veal calves treated with zeranol containing implant, no residues were detected in plasma even if plasma proteins were hydrolysed with pronase before the radio-immunoassay. Free and conjugated residues in urine were easily measured. The urine concentration of conjugated residues increased markedly after the 20th day of treatment and was still high (19 ppb) at day 40.

[Infectious bursal disease: distribution and persistence of the virus in inoculated chickens. Study on the transmission of the disease]. / Maladie de gumboro: distribution et persistance du virus chez le poussin inocule. Etudes sur la transmission de la maladie.

The distribution, concentration and persistence of infectious bursal disease virus (IBDV) in the lymphoid organs of inoculated chickens, and its persistence in contaminated premises was examined. The virus only multiplied in the bursa of Fabricius where it induced degeneration and necrosis of the lymphoid cells. It persisted for 10 days in this organ and the highest viral concentrations were observed between the 4th and 8th day following inoculation. The virus was found at a low concentration in the spleen and thymus only during the viraemia phase. The inoculated chickens shed virus in the excreta during the first days of infection. The disease was transmitted to other chickens by direct contact with birds which had been inoculated 4, 10 and 14 days previously with IBDV. Litter on which infected chickens had been reared had a high level of infectivity for 30 days after removal from the chickens and still had some infectivity after 60 days. The long life of the virus in an infected house explains its persistence on infected farms and its transmission to successive flocks.

[Herpes virus isolated from pigeons (author's transl)]. / Isolement d'un virus herpès dans un élevage de pigeons de chair

A virus has been isolated from pigeons with the clinical symptoms of "infectious coryza". The virus produces numerous pocks on the chorio-allantoic membranes; livers from infected embryos have areas of necrosis. Microscopic examinations reveals basophilic and eosinophilic intranuclear inclusions. The virus produces a cytopathic effect of rounded refractile cells after 24 h with rapidly spreads throughout the monolayer. The virus is sensitive to chloroform. Electron microscopy of negatively stained lysed cell preparations shows that the virus is a herpesvirus. The strain is pathogenic for pigeons. Lesions produced by intralaryngeal inoculation consists of small necrotic foci in the laryngeal epithelium and in the liver.