R. E. Gough

Molecular analysis of the 793/B serotype of infectious bronchitis virus in Great Britain.

Since the winter of 1990/91 respiratory disease of poultry in Great Britain has commonly been associated with the 793/B (or 4/91) serotype of infectious bronchitis virus (IBV). We have sequenced a variable part of the S1 region of the spike protein (5) gene. Comparison of up to 270 nucleotides of 12 British 793/B isolates, obtained in 1991 and 1993, revealed 94 to 100% nucleotide identity with each other. Eleven of them fell into one of two subgroups, A and B, one isolate forming subgroup C. Identity within subgroups A and B was > 98%. The whole S1 gene sequence (1617 nucleotides) was determined for five 793/B isolates, two from each of subgroups A and B and one from subgroup C; nucleotide identity between any two isolates was > 97%. A large proportion of the nucleotide differences corresponded to amino acid changes. The whole S1 amino acid sequence differed by 21 to 25% or more from that of all other published IBV sequences. This extensive difference has probably contributed to the persistence of the 793/B serotype in Britain even though het-erologous vaccines have been used. The finding that the 793/B isolates could be placed into three subgroups suggests that either (a) they had diverged from a common progenitor present, but undetected, in Britain prior to 1990/91 or (b) at least three different strains of the 793/B serotype had entered Britain in or prior to 1990/91.

Variation in the spike protein of the 793/B type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs

The degree of variation exhibited within the 793/B serotype (also known as 4/91 and CR88 serotypes) was investigated with nine French and 10 British isolates, collected between 1985 and 1994. The S1 part (1644 nucleotides) of the spike protein gene of the first known isolate of this serotype, FR/CR85131/85, had 95.9% to 97% nucleotide identity with the other isolates. Partial sequencing of isolates from Iran and Saudi Arabia, isolated in 2000, revealed approximately 95% nucleotide identity with European isolates, including the two live 793/B vaccinal strains, showing that they were not re-isolations of vaccinal virus. The data indicates that strains within the 793/B serotype have ≥96% nucleotide identity within the whole S1 gene and ≥93% nucleotide identity within the first 560 nucleotides, and ≥92% and ≥86% amino acid identities in the corresponding protein regions. This is similar to the identities exhibited within the Massachusetts serotype. Sequence analysis of a 793/B field isolate after passage in embryonated eggs, then in chickens and then again in eggs revealed selection for a serine and alanine at S1 amino acid position 95 in chicken-passaged and egg-passaged virus, respectively. There was no change in pathogenicity. This is the first demonstration at gene sequence level of host-driven selection for infectious bronchitis virus.

Co-circulation of four types of infectious bronchitis virus (793/B, 624/I, B1648 and Massachusetts)

Eighteen isolates of infectious bronchitis virus (IBV) from Italy and Poland in 1997 to 1998 were comprehensively analysed by serum haemagglutination inhibition and virus neutralization tests, and by type-specific polymerase chain reactions and spike protein S1 gene sequencing. Four types of IBV (793/B, 624/I, B1648 and Massachusetts) were detected in Italy, while the presence of 793/B was confirmed in Poland. This showed that not only were four types of IBV co-existing within a single year, but also that several types of IBV have persisted in Europe for many years (at least 13 to 14 years for types B1648 and 793/B). Sequencing of the S1 gene of the 624/I isolate confirmed this as a unique type of IBV.

Chinese QX strain of infectious bronchitis virus isolated in the UK

SIR, — We wish to report the isolation and identification of a strain of infectious bronchitis virus (ibv) which shows a close genetic relationship with the so-called Chinese qx strain of ibv. As far as we are aware, this is the first report of the presence of this virus in the uk. A six- to

Avipoxvirus infection in peregrine falcons (Falco peregrinus) from a reintroduction programme in Germany

Poxvirus infections are common in domestic birds in Germany, but they are rare in birds of prey. Only species of falconidae imported from Arabian or Asian countries have so far tested positive for poxvirus, and, among these, only raptors kept for falconry. As part of a reintroduction programme in the northern county of Mecklenburg-Western Pomerania, which is adjacent to the Baltic Sea, 21 young peregrine falcons were released into the wild; six of them died and one was examined postmortem, its tissues being examined by light and electron microscopy. In addition, an ELISA for fowipox, pigeonpox and canarypox was applied. No virus could be isolated and propagation in culture failed, but virus particles were detected by electron microscopy in lesions from its skin and tongue.

Avian encephalomyelitis virus in reared pheasants: a case study

An outbreak of neurological disease occurred in pheasant chicks on a game farm in 2007. The disease was first seen in the 10th hatching of chicks on the farm. Affected chicks showed trembling and incoordination from the time of hatching, and subsequently blindness and cataract formation was seen in some of the affected chicks at 3 weeks of age. The peak mortality and culling figure was 21.0% in the worst affected hatch, compared with a maximum of 11.7% in the first nine hatches. No further cases were evident by 7.5 weeks of age. Histopathological examination showed a moderate acute encephalomyelitis in some, but not all, of the chicks with neurological signs. The clinical presentation and histopathological findings were typical of vertically transmitted avian encephalomyelitis as seen in chickens, although avian encephalomyelitis virus could not be detected in inoculated embryonated chicken eggs. However, serological testing by enzyme-linked immunosorbent assay for antibodies to the virus was positive in four of five affected 3-week-old birds and in 23 out of 29 adult breeding birds, and reverse transcriptase-polymerase chain reaction testing of RNA extracted from brain and pancreas tissue of affected chicks yielded nucleotide sequences aligned 82% and 83% with three avian encephalomyelitis sequences in a sequence database. The evidence suggested that the neurological disease was attributable to infection with a strain of avian encephalomyelitis virus that appeared to have entered the flock at the start of the breeding season, and was possibly introduced by carrier pheasants brought on to the farm early in the season.

Isolation and identification of a reovirus from a lizard, Uromastyx hardwickii, in the United Kingdom

S021 IBX IN December 2000, six carcases of Uromastyx hardwickii lizards were submitted for postmortem examination. These are small insectivorous lizards, originating from Pakistan. The six animals were juveniles, part of a consignment of 50 that had been recently imported into the UK, and had been found dead. Eventually, the whole consignment died. A full postmortem examination carried out on one lizard revealed no abnormalities in the mouth, oesophagus or trachea, but the lungs were dark red in colour. Only a small amount of feed was present in the stomach, and the remainder of the intestinal tract had very scant contents. The liver, kidneys and spleen all appeared normal, but the ureters were prominent and distended by white urate material. Similar findings were noted in all six lizards, with some variation in the degree of red coloration of the lungs. There was a general lack of fat deposits anywhere in the carcases, suggesting a long-term loss of condition of the animals. Examination of intestinal smears showed profuse numbers of, most likely oxyurid, worm eggs and larvae in both the small and large intestine. Routine bacteriological culture techniques found Salmonella Arizonae in the liver, and a similar organism was also detected in samples of heart, blood, lung and small intestine. The significance of this finding to the deaths of the animals is not known. Samples of liver, spleen and small intestine were submitted to the Veterinary Laboratories Agency (VLA) Weybridge for virological examination in January 2001. On receipt, the sample material was processed for examination by electron microscopy by preparing a 20 per cent (w/v) suspension of each tissue in deionised water. Clarified suspensions were concentrated by ultracentrifugation at 32,000 g for one hour at 40C. The pellets were resuspended in a minimal volume of deionised water, stained using 2 per cent potassium phosphotungstate at pH 6-6, and examined by negative contrast electron microscopy. Reovirus-like particles were detected in samples of both pooled spleen/liver and small intestine (Fig 1). Tissues were also processed for attempted virus isolation by inoculation onto confluent monolayers of chick embryo fibroblast (CEF) cells. Cultures were incubated at 28°C for twoand-a-half hours. The culture medium was then replaced with a maintenance medium and the cultures were incubated for up to 12 days at 280C. The cultures were examined daily for cytopathic effect (cpe). Cpe was observed after four days of the initial passage on CEF cultures. The cells became increasingly refractile after the second day, with cell fusion, syncytial formation and detachment from the cell sheet over the following two days. The same effect was reproduced in the second cell culture passage on CEF cells, and also in chick embryo liver cell cultures. This effect is characteristic of reoviruses. The affected cultures were frozen at -80°C, thawed and clarified by centrifugation at 720 g for five minutes. The clarified supernatants were prepared by high-speed centrifugation, as described above, for examination by electron microscopy to confirm the identity of the isolated agent. FIG 1: Reovirus-like particles detected in tissue from the small intestine of a lizard, Uromastyx hardwickii. Bar= 100 nm (a) (b)

Protection in specific pathogen free chickens with live avian metapneumovirus and Newcastle disease virus vaccines applied singly or in combination

This paper describes two experiments. In each experiment, 1-day-old specific pathogen free chicks were divided into three groups. In Experiment 1—[avian metapneumo virus (aMPV) challenge]—one group served as unvaccinated controls; the second group was vaccinated with live aMPV (subtype B) vaccine only, and the third group received the aMPV vaccine in combination with live Newcastle disease virus (NDV) vaccine (VG/GA strain). Oropharyngeal swabs, tissues and blood samples were collected before and after challenge with a virulent subtype aMPV at 21 days post vaccination. Chicks were monitored for post-challenge clinical signs. Swabs and tissues were examined for the detection of challenge aMPV by virus isolation and by reverse-transcriptase polymerase-chain reaction. Sera were assayed for antibodies against aMPV and NDV. The single and combined vaccinated chicks were all protected against clinical signs and no challenge virus was isolated from either of the vaccinated-challenged groups. In Experiment 2 (NDV challenge), as in Experiment 1, chicks were divided into three groups where one group remained as unvaccinated control and the other two groups were vaccinated as above, except that the second group received live NDV vaccine only, instead of aMPV. At 21 days post vaccination, 15 chicks from each of the three groups were removed to a different site and challenged with a virulent NDV (Texas GB strain). Re-isolation of the challenge virus was not attempted. All chicks in both NDV-vaccinated challenged groups were protected against clinical signs and mortality. These results show that, based on parameters monitored for the respective challenge virus, simultaneous application of live aMPV and NDV vaccines did not affect the efficacy of either vaccine.