Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12).

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.

Demonstration of Ornithobacterium rhinotracheale in pheasants (Phasianus colchicus) with pneumonia and airsacculitis.

Outbreaks of respiratory disease were investigated in reared pheasants (Phasianus colchicus) aged approximately 18 to 32 weeks, released into the semi-wild on four shooting estates in southern England. The clinical signs in the affected birds included swelling of the face and eyes, loss of condition, gasping respirations and coughing. The gross pathology findings included sinusitis, airsacculitis, pleural oedema and lung lesions. The histopathological findings in the affected lungs were characterized by a granulomatous pneumonia. Ornithobacterium rhinotracheale (ORT) was isolated from respiratory tract tissues, and 16S rRNA gene sequencing on three isolates revealed two distinct genotypes, one previously associated with some electrophoretic type (ET) 1 strains and the other a novel genotype that clustered among sequences previously associated with ET 3, ET 4, ET 5 and ET 6 isolates. The localization of ORT within the lung tissue was demonstrated by fluorescent in-situ hybridization in the bronchial exudate of three cases, although not within the granulomatous lesions themselves. In each case, ORT was identified as part of a complex of other respiratory agents including avian paramyxovirus type 2, avian coronavirus, Mycoplasma gallisepticum, Mycoplasma synoviae and other Mycoplasma species, Escherichia coli, Pasteurella multocida, other Pasteurellaceae and Syngamus trachea, suggesting synergism with other agents. Exposure to other intercurrent factors, including adverse weather conditions and internal parasitism, may also have exacerbated the severity of disease.

The ecology and age structure of a highly pathogenic avian influenza virus outbreak in wild mute swans.

The first UK epizootic of highly pathogenic (HP) H5N1 influenza in wild birds occurred in 2008, in a population of mute swans that had been the subject of ornithological study for decades. Here we use an innovative combination of ornithological, phylogenetic and immunological approaches to investigate the ecology and age structure of HP H5N1 in nature. We screened samples from swans and waterbirds using PCR and sequenced HP H5N1-positive samples. The outbreak's origin was investigated by linking bird count data with a molecular clock analysis of sampled virus sequences. We used ringing records to reconstruct the age-structure of outbreak mortality, and we estimated the age distribution of prior exposure to avian influenza. Outbreak mortality was low and all HP H5N1-positive mute swans in the affected population were <3 years old. Only the youngest age classes contained an appreciable number of individuals with no detectable antibody responses to viral nucleoprotein. Phylogenetic analysis indicated that the outbreak strain circulated locally for ~1 month before detection and arrived when the immigration rate of migrant waterbirds was highest. Our data are consistent with the hypothesis that HP H5N1 epizootics in wild swans exhibit limited mortality due to immune protection arising from previous exposure. Our study population may represent a valuable resource for investigating the natural ecology and epidemiology of avian influenza.

Outbreak of Newcastle disease due to pigeon paramyxovirus type 1 in grey partridges (Perdix perdix) in Scotland in October 2006.

In October 2006, following an initially non-statutory disease investigation affecting 12-week-old grey partridges (Perdix perdix), an outbreak of Newcastle disease due to infection with the avian paramyxovirus type 1 virus responsible for the current panzootic in pigeons (PPMV-1) was confirmed in Scotland. Two pens of partridges were affected by signs including loss of condition, diarrhoea, progressive neurological signs and mortality totalling approximately 24 per cent, and laboratory evidence of the infection was obtained only in these groups. The premises had approximately 17,000 poultry including a collection of 375 birds of rare breeds, containing endangered breeds of significant conservation value, which were not culled but subjected to a health monitoring and testing programme. Investigations suggested that a population of feral pigeons living above the affected pens of partridges was the likely source of the outbreak. Laboratory and genetic analyses confirmed that the isolate recovered from the clinically affected partridges was PPMV-1, belonging to genetic lineage 4b. However, the virus could not be isolated from or detected in dead pigeons collected from the affected buildings.

Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Recent epidemiology and ecology of influenza A viruses in avian species in Europe and the Middle East.

There have been at least ten distinct outbreaks of LPAI or HPAI in poultry caused by H5 or H7 viruses in the last eight years in Europe and the Middle East. There appears to be an increased occurrence of such episodes consistent with global trends. As a result, surveillance systems have been enhanced to facilitate early detection of infection in poultry, together with active surveillance of wild bird populations. These complementary activities have resulted in the detection of a number of viruses in wild bird populations, including some with high genetic similarity to newly detected viruses in poultry, for example, H7N3 in Italy and H7N7 in the Netherlands. Furthermore, there is evidence for continued circulation of H5 and H7 viruses in wild Anseriformes, thereby presenting a real and current threat for the introduction of viruses to domestic poultry, especially those reared in outdoor production systems. Viruses of H9N2 subtype continue to circulate widely in the Middle East and are associated with significant disease problems in poultry. The epidemiology has the potential to be complicated further by introduction of novel viruses through illegal importation of captive birds, such as was detected with H5N1 in Belgium in 2004. Continual genetic exchange in the avian virus gene pool and independent evolution of all gene segments either within an individual host species or among wild bird hosts suggests that these viruses are not in evolutionary stasis in the natural reservoir.

Isolation of a highly pathogenic influenza A virus of subtype H7N3 from a peregrine falcon ( Falco peregrinus ).

A peregrine falcon ( Falco peregrinus ) was presented to the Al Safa Falcon Clinic in Dubai, UnitedArab Emirates unable to stand. Four hours after hospitalization, the bird died despite supportive care and calcium disodiumedetate treatment. The falcon had been on a hunting trip to Syria with its owner of 2 years, prior to its death. The carcass was submitted to the Central Veterinary Research Laboratory in Dubai where it was subjected to postmortem examination. Investigations resulted in the isolation of an influenza A virus subtype H7N3, which proved to be highly pathogenic for chickens.

Characterization of Newcastle disease viruses isolated in Italy in 2000.

Thirty-two Newcastle disease virus isolates from the 2000 Italian epidemic were characterized by monoclonal antibody binding pattern and nucleotide sequencing of approximately 400 base pairs of the fusion gene. In addition, the pathogenicity of six of these isolates was assessed by means of the intracerebral pathogenicity test (ICPI). The strains tested exhibited an ICPI ranging from 1.6 to 2.0. On the basis of the monoclonal antibody binding pattern, all isolates could be classified as belonging to group C1. Both monoclonal antibody and genomic analysis revealed a very high degree of homology, indicating a common source of infection. On the basis of the phylogenetic analysis, it appears that the Italian isolates are closely related to the recent isolates from the UK, Scandinavia and South East Europe, thus suggesting the circulation of this viral strain in Europe during the past 5 years.

Isolation of an influenza A virus of unusual subtype (H1N7) from pigs in England, and the subsequent experimental transmission from pig to pig.

A novel H1N7 influenza virus (A/swine/Eng/191973/92) was isolated from nasal swabs collected from two pigs on a farm where there had been recent clinical disease due to infection with an H1N1 virus (A/swine/Eng/195852/92). Antigenically, the haemagglutinin (HA) of the H1N7 virus was related most closely to the HA of A/USSR/90/77, whilst the neuraminidase (NA) appeared to be related most closely to the NA of A/equine/Prague/1/56 (H7N7). Pigs infected experimentally with A/swine/Eng/191973/92 developed mild clinical signs, excreted virus into the nasal passages for up to nine days after infection, appeared normal at necropsy, transmitted the virus to sentinel pigs, but seven out of eight pigs failed to seroconvert. These findings suggest that the H1N7 virus has a low pathogenicity for pigs, resulting in limited virus multiplication which is insufficient to stimulate a detectable primary humoral immune response.

Pathogenesis of H7 influenza A viruses isolated from ostriches in the homologous host infected experimentally.

Infections of ostriches with avian influenza A viruses are generally associated with clinical disease, but the occasional high mortality in young birds does not appear to be related directly to virus pathotype. In this study we investigated the pathogenesis of two H7 viruses for 11-wk-old ostriches inoculated intranasally, and clinical symptoms, virus excretion, and immune response were studied. One of the viruses (A/Ostrich/Italy/1038/00) was highly pathogenic for chickens, whereas the other (A/Ostrich/South Africa/1609/91) was of low pathogenicity for chickens. Clinical signs in ostriches receiving virulent virus were slight depression and hemorrhagic diarrhea, while the group receiving avirulent virus was clinically normal except for green diarrhea. Both viruses were transmitted to in-contact sentinel birds housed with the infected groups 3 days postinfection. Postmortem examination of the birds infected (including the sentinel bird) with virus highly pathogenic for chickens were grossly normal except for localized pneumonic lesions. The results of the study are presented and discussed.

Newcastle disease and avian influenza A virus in wild waterfowl in South Africa.

In an intensive ostrich farming area in South Africa with a history of ostrich influenza outbreaks, we conducted a survey of avian influenza virus (AIV) and Newcastle disease virus (NDV) in wild aquatic birds. During late autumn and winter 1998, the time of year when outbreaks in ostriches typically start to occur, 262 aquatic birds comprising 14 species were sampled and tested for both virus infections. From eight samples, AIV, serotype H10N9, could be isolated. All isolates were apathogenic as determined by the intravenous pathogenicity index (0.00). Conversely, none of 33 sera of these wild birds showed antibodies against H10. However, one bird was found serologically positive for H6 AIV. This AIV serotype was later isolated from ostriches during an avian influenza outbreak in this area. No NDV was isolated although 34 of 46 serum samples contained NDV-specific antibodies. This is the first H10N9 isolate to be reported from Africa. In addition, our data support the notion that wild aquatic birds may function as a reservoir for AIV and NDV in South Africa.

Comparison of Newcastle disease viruses isolated from cormorants in Canada and the USA in 1975, 1990 and 1992.

Seventeen Newcastle disease virus (NDV) isolates obtained from cormorants, turkeys, a pelican, and a gull in Canada and the USA collected in 1975, 1990 and 1992 were analyzed for relatedness by monoclonal antibody profiling. In addition, nucleotide sequence analysis was performed in two areas of the fusion (F) gene for 5 of the isolates. No difference in the antigenicity of these 17 viruses, as determined by monoclonal antibody binding patterns, was seen. The amino acid sequences obtained via nucleotide sequencing at the cleavage site of the F protein showed that all the isolates tested had two pairs of basic amino acids immediately upstream of the cleavage site, and a phenylalanine residue at the N-terminus of the F1 protein, which is consistent with velogenic NDV. The deduced amino acid sequence obtained at the cleavage site of the F protein from 6 of the isolates was virtually identical regardless of the species, year of isolation, or location. However, the 1975 cormorant isolate showed marked differences from the 1990-1992 isolates in the nucleotide and deduced amino acid sequence of the F gene signal region. These data indicate that the 1990 and 1992 outbreaks were caused by the same epizootic virus and further suggest that the population of NDV in these wild birds may be very stable. The belief that the velogenic NDV circulating in cormorants in 1992 was transmitted into the free-ranging turkey flocks located near the cormorants in North Dakota is supported by the present study in which no distinction could be made between the viruses isolated from turkeys or wild birds.

Characterisation of an antigenically unusual virus responsible for two outbreaks of Newcastle disease in the Republic of Ireland in 1990.

Antigenic characterisation of two highly virulent virus isolates from outbreaks of Newcastle disease on two closely connected farms in County Monaghan, Republic of Ireland, in 1990 showed the viruses to be indistinguishable but unlike other Newcastle disease viruses so far tested. However, they appeared to be antigenically closest to avirulent viruses isolated from waterfowl from several countries and from chickens in Northern Ireland in 1986. Despite the antigenic differences, chickens vaccinated with a live commercial Hitchner B1 vaccine were protected against intramuscular challenge with one of the virulent isolates.

Similarity of avian paramyxovirus serotype 1 isolates of low virulence for chickens obtained from contaminated poultry vaccines and from poultry flocks.

At present Denmark has the status of a 'non-vaccinating' country for Newcastle disease and its poultry population should therefore be free of antibodies to avian paramyxovirus 1 (APMV-1). Three live avian vaccines against infectious bronchitis, avian encephalomyelitis, and chick anaemia which had been found to be contaminated with APMV-1 viruses of low virulence for chickens were examined. The vaccines were produced by the same company and the affected batches had been used in Denmark in 1996/97. Furthermore, APMV-1 isolates of low virulence were obtained from three commercial broiler breeder flocks, one of which had been vaccinated with two of the contaminated vaccines. The flocks belonged to the same hatchery organisation. A comparison of viral F0 gene sequences and typing of virus isolates with a panel of monoclonal antibodies showed that the vaccine and field isolates were identical.

Strains of avian paramyxovirus type 1 of low pathogenicity for chickens isolated from poultry and wild birds in Denmark.

Twenty-one strains of avian paramyxovirus type 1 of low virulence for chickens were isolated in Denmark between 1996 and the beginning of 2003. The low virulence of the strains was demonstrated by sequencing the fusion (F) gene at the cleavage site motif and in some cases by determining the intracerebral pathogenicity index in day-old chicks. By using a panel of monoclonal antibodies it was shown that the isolates belonged to four different antigenic groups (five C2 isolates, six E isolates, six H isolates and four G/Q isolates). They were placed in three distinguishable genetic groups by phylogenetic analysis of a partial sequence of the F gene. The origin of the six E isolates was probably contaminated vaccines; the other viruses were isolated from wild birds and from poultry which probably came into contact with wild birds.

Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997.

Antigenic and genetic analyses of viruses from the 11 outbreaks of Newcastle disease in Great Britain, 12 of the outbreaks in Northern Ireland and the single outbreak in the Republic of Ireland which occurred in 1997, indicated that they were all essentially similar. In addition, the viruses from the British Isles were very similar to viruses isolated from three outbreaks in pheasants in Denmark between August and November 1996, from a goosander in Finland in September 1996, from an outbreak in chickens in Norway in February 1997, and from an outbreak in chickens in Sweden in November 1997. Viruses from outbreaks in other countries during 1995 to 1997 could be distinguished antigenically and/or genetically from the 1996 to 1997 Scandinavian/British Isles isolates, as could viruses responsible for two separate outbreaks in caged birds in quarantine premises in Great Britain in March 1997. Minor nucleotide differences in the 413-base region of the fusion gene and the 187-base region of the haemagglutinin-neuraminidase gene sequenced in this study allowed the 1996 to 1997 Scandinavian/British Isles isolates to be divided into groups. These groups broadly corresponded to the clusters of disease outbreaks, but suggested that the discrete outbreak in Scotland was probably the result of virus spread from Northern Ireland. Overall, the antigenic and genetic analyses of these viruses were consistent with the theory that the virus was introduced into the British Isles by migratory birds moving from north-east Europe. However, it was not possible to rule out other sources, such as the movement of pheasants from Denmark.

Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests.

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.