H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

Surveillance and investigative diagnosis of a poultry flock in Great Britain co-infected with an influenza A virus and an avirulent avian avulavirus type 1.

A detailed veterinary and laboratory investigation revealed an unusual case of concurrent avian avulavirus type 1 (AAvV-1, formerly called avian paramyxovirus type 1) and low pathogenicity avian influenza (LPAI) virus infections of chickens during March 2010 in a mixed poultry and livestock farm in Great Britain. Respiratory signs and daily mortality of 5-6 birds in a broiler flock 8-weeks of age prompted submission of two carcasses to an Animal and Plant Health Agency (APHA) regional laboratory. Infectious bronchitis virus infection was suspected initially and virus isolation in SPF embryonated fowls' eggs was attempted at APHA-Weybridge. Avirulent AAvV-1 was detected in the first sampling. Both in vitro nucleotide sequencing of the fusion gene and in vivo pathotyping by intracerebral pathogenicity index revealed an avirulent AAvV-1 not definitively ascribed to licensed vaccine. Upon initial detection of the AAvV-1 virus, statutory restrictions were placed on the farm, an official veterinary visit was performed and further samples were submitted to APHA-Weybridge for official statutory disease investigation. An H2N3 LPAI virus was subsequently isolated from tissue samples and swabs submitted from the follow-up statutory investigation. The subtype was confirmed by haemagglutination inhibition test (HAIT) and neuraminidase inhibition (NI) tests on egg-amplified virus. As neither virus was notifiable according to the internationally recognized EU and OIE standards, and/or definitions of disease, statutory farm restrictions were lifted. Veterinary investigations identified the broiler flock to be free-range, next to a river and duck pen, reinforcing the suspicion of wild bird origin for both viruses which may have been co-circulating in ducks. It could not, however, be established as to whether there were separate introductions of the two viruses or whether there had been a single co-introduction of the viruses. The described case highlights the value of integrated surveillance and laboratory approaches, including veterinary field investigations, international standards and definitions of notifiable avian disease, validated RRT-PCR assays, and virus isolation in achieving rapid and accurate diagnostic results.

Detection of non-notifiable H4N6 avian influenza virus in poultry in Great Britain.

A 12-month pilot project for notifiable avian disease (NAD) exclusion testing in chicken and turkey flocks in Great Britain (GB) offered, in partnership with industry, opportunities to carry out differential diagnosis in flocks where NAD was not suspected, and to identify undetected or undiagnosed infections. In May 2014, clinical samples received from a broiler breeder chicken premises that had been experiencing health and production problems for approximately one week tested positive by avian influenza (AI) real-time reverse transcription polymerase chain reaction (RRT-PCR). Following immediate escalation to an official, statutory investigation to rule out the presence of notifiable AI virus (AIV; H5 or H7 subtypes), a non-notifiable H4N6 low pathogenicity (LP) AIV was detected through virus isolation in embryonated specific pathogen free (SPF) fowls' eggs, neuraminidase inhibition test, cleavage site sequencing and AIV subtype H4-specific serology. Premises movement restrictions were lifted, and no further disease control measures were implemented as per the United Kingdom (UK) legislation. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the virus revealed closest relationships to viruses from Mallard ducks in Sweden during 2007 and 2009. In June 2014, clinical suspicion of NAD was reported in a flock of free-range laying chickens elsewhere in GB, due to increasing daily mortality and reduced egg production over a five-day period. An H4N6 LPAIV with an intravenous pathogenicity index of 0.50 was isolated. This virus was genetically highly similar, but not identical, to the virus detected during May 2014. Full viral genome analyses showed characteristics of a strain that had not recently transferred from wild birds, implying spread within the poultry sector had occurred. A stalk deletion in the neuraminidase gene sequence indicated an adaptation of the virus to poultry. Furthermore, there was unexpected evidence of systemic spread of the virus on post-mortem. No other cases were reported. Infection with LPAIVs often result in variable clinical presentation in poultry, making detection of disease more difficult.

The Detection of a Low Pathogenicity Avian Influenza Virus Subtype H9 Infection in a Turkey Breeder Flock in the United Kingdom.

In April 2013, an H9N2 low pathogenicity avian influenza (LPAI) virus was isolated in a turkey breeder farm in Eastern England comprising 4966 birds. Point-of-lay turkey breeding birds had been moved from a rearing site and within 5 days had shown rapid onset of clinical signs of dullness, coughing, and anorexia. Three houses were involved, two contained a total of 4727 turkey hens, and the third housed 239 male turkeys. Around 50% of the hens were affected, whereas the male turkeys demonstrated milder clinical signs. Bird morbidity rose from 10% to 90%, with an increase in mortality in both houses of turkey hens to 17 dead birds in one house and 27 birds in the second house by day 6. The birds were treated with an antibiotic but were not responsive. Postmortem investigation revealed air sacculitis but no infraorbital sinus swellings or sinusitis. Standard samples were collected, and influenza A was detected. H9 virus infection was confirmed in all three houses by detection and subtyping of hemagglutinating agents in embryonated specific-pathogen-free fowls' eggs, which were shown to be viruses of H9N2 subtype using neuraminidase inhibition tests and a suite of real-time reverse transcription PCR assays. LPAI virus pathotype was suggested by cleavage site sequencing, and an intravenous pathogenicity index of 0.00 confirmed that the virus was of low pathogenicity. Therefore, no official disease control measures were required, and despite the high morbidity, birds recovered and were kept in production. Neuraminidase sequence analysis revealed a deletion of 78 nucleotides in the stalk region, suggesting an adaptation of the virus to poultry. Hemagglutinin gene sequences of two of the isolates clustered with a group of H9 viruses containing other contemporary European H9 strains in the Y439/Korean-like group. The closest matches to the two isolates were A/turkey/Netherlands/11015452/11 (H9N2; 97.9-98% nucleotide identity) and A/mallard/Finland/Li13384/10 (H9N2; 97% nucleotide identity). Both PB2 partial sequences were a 100% nucleotide identity with A/mallard/France/090360/09, indicating a European origin of the causative virus. Furthermore, partial sequencing analysis of the remaining genes revealed the virus to be genotypically of European avian origin and therefore of lower risk to public health compared with contemporary viruses in Central and Eastern Asia. Occupational health risks were assessed, and preventative measures were taken.

First reported detection of influenza A (H1N1)pdm09 in turkeys in the United Kingdom.

We report the first occurrence of pandemic (H1N1) 2009 virus [A(H1N1)pdm09] infection on two epidemiologically linked turkey breeder premises in the United Kingdom during December 2010 and January 2011. Clinically, the birds showed only mild signs of disease, with the major presenting sign being an acute and marked reduction in egg production, leading to the prompt reporting of suspected avian notifiable disease for official investigation. Presence of A(H1N1)pdm09 infection in the United Kingdom turkey breeder flocks was confirmed by detailed laboratory investigations including virus isolation in embryonated specific pathogen-free fowls' eggs, two validated real-time reverse transcription-PCR tests, and nucleotide sequencing of the hemagglutinin and neuraminidase genes. These investigations revealed high nucleotide identity with currently circulating human A(H1N1)pdm09 strains, suggesting that human-to-poultry transmission (reverse zoonosis) was the most likely route of infection. Peak levels of human influenza-like illness community transmission also coincided with the onset of clinical signs in both affected turkey breeder flocks. This case demonstrated the value of the existing passive surveillance framework and associated veterinary and laboratory infrastructure that enables the detection and management of both exotic and new and emerging disease hazards and risks. The case also presents further evidence of the susceptibility of turkeys to infection with influenza A viruses of nonavian origin.

Overview of incursions of Asian H5N1 subtype highly pathogenic avian influenza virus into Great Britain, 2005-2008.

Since 2005 there have been five incursions into Great Britain of highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 related to the ongoing global epizootic. The first incursion occurred in October 2005 in birds held in quarantine after importation from Taiwan. Two incursions related to wild birds: one involved a single dead whooper swan found in March 2006 in the sea off the east coast of Scotland, and the other involved 10 mute swans and a Canada goose found dead over the period extending from late December 2007 to late February 2008 on or close to a swannery on the south coast of England. The other two outbreaks occurred in commercial poultry in January 2007 and November 2007, both in the county of Suffolk. The first of these poultry outbreaks occurred on a large turkey farm, and there was no further spread. The second outbreak occurred on a free-range farm rearing turkeys, ducks, and geese and spread to birds on a second turkey farm that was culled as a dangerous contact. Viruses isolated from these five outbreaks were confirmed to be Asian H5N1 HPAI viruses; the quarantine outbreak was attributed to a clade 2.3 virus and the other four to clade 2.2 viruses. This article describes the outbreaks, their control, and the possible origins of the responsible viruses.

Detection of West Nile virus in the tissues of specific pathogen free chickens and serological response to laboratory infection: a comparative study.

Using an isolate of West Nile virus (WNV) from lineage 1 (Goose/Israel 1998), groups of specific pathogen free chickens were experimentally infected via the subcutaneous or intravenous routes. To evaluate the relative efficiency of detecting the virus in the infected chickens, samples from a range of tissues and organs were examined by virus isolation tests in tissue culture, including Vero, primary chicken embryo liver and fibroblast cells, and polymerase chain reaction (PCR) analyses. Additionally, in order to investigate the serological response of the chickens and produce WNV monospecific antibodies, serum samples were collected from the birds during the trial and analysed for antibodies by virus neutralization (VN) and the plaque-reduction neutralization test (PRNT). No clinical signs or gross pathological changes were seen in any of the inoculated chickens throughout the study. The nested PCR used in the study appeared to be significantly more sensitive at detecting the presence of the virus in both the tissues and the inoculated Vero cell cultures compared with the detection of gross cytopathic changes as observed in infected Vero cell culture. No cytopathic changes were seen in the inoculated avian cell cultures. Following primary inoculation of the chickens there was a weak antibody response 15 days post-inoculation. However, following re-inoculation with inactivated WNV and adjuvant there was a substantial increase in the neutralizing antibody titres when tested 2 weeks later. The results obtained suggested that the PRNT was more sensitive than the conventional VN test. Based on detection of virus and serology there was no evidence of viral transmission to the close contact controls. It can be concluded that the PCR used in this study was more sensitive than virus isolation for the detection of WNV while the PRNT also appeared more sensitive than the conventional VN test.