Highly pathogenic avian influenza (HPAI) is an infectious and highly contagious viral disease of birds (2, 5). The HPAI viruses (HPAIV) emerge as a result of mutation from low-virulence progenitors (low pathogenic avian influenza viruses, LPAIV), for which wild aquatic birds are the natural reservoir (2, 30). Influenza viruses belong to the
HPAI as a global problem is mostly associated with the A/goose/Guangdong/1/1996 (Gs/GD) lineage of HPAIV H5 viruses (H5 Gs/GD), which were first reported in 1996 in China as an H5N1 subtype (8). In subsequent years, the H5 Gs/GD viruses evolved into multiple genetic clades and genotypes through genetic drift (i.e. accumulation of point mutations over time) or reassortment (i.e. switching of viral RNA segments between different influenza viruses co-infecting the same avian host) (10, 18). More than a decade ago, H5 Gs/GD viruses of clade 2.3.4 started to evolve into lower-order sub-clades and it has been shown that the sub-clade 2.3.4.4 viruses (further differentiated into four subgroups unofficially designated 2.3.4.4a to 2.3.4.4d) were unusually prone to reassortment (18, 25). Since 2014, a rapid global expansion of H5 Gs/GD clade 2.3.4.4 has been observed, and frequent reassortment events with LPAIV prevailing in local populations of Eurasian wild birds resulted in the generation of novel genotypes bearing different neuraminidase subtypes, e.g. H5N2, H5N5, H5N6 or H5N8 (15, 18). The viruses spread from Asia to Europe, North America and Africa but the magnitude of the epidemics varied greatly (1, 13, 15).
Poland was among the countries affected by the 2016–17 HPAI epidemic of the H5 Gs/GD clade 2.3.4.4b viruses, which so far has been the largest in Europe (1). On 31 December 2019, a novel HPAIV H5N8 clade 2.3.4.4b genotype was confirmed in Poland (27) and quickly spread across the country. This article provides details on the epidemic with a special focus on the clinical outcome of infections in different poultry species, possible pathways of spread, preventive measures undertaken in response to the situation and molecular characterisation of the representative HPAIV H5N8 isolates.
Between 31 December 2019 and 16 July 2020, a total of 428 poultry flocks were tested, including laying hens, broiler chickens, fattening turkeys, geese, and ducks, breeding geese and duck and guinea fowl (Table 1). Additionally, 84 wild birds mainly comprising swans, ducks and pigeons, were examined in the same period. Descriptive data on the clinical course as well as morbidity and mortality were collected by official veterinarians. Epidemiological investigation was performed to determine the most likely route of virus introduction into the flock.
Number and type of tested flocks per species and production category
Type of poultry holding tested | Laying hens | Broiler chickens | Fattening turkeys | Fattening geese | Breeding geese | Fattening ducks | Breeding ducks | Guinea fowl |
---|---|---|---|---|---|---|---|---|
Flocks suspected of being infected and TTE (passive surveillance) organs from at least 5 birds (dead or sick) as well as oropharyngeal and cloacal swabs from at least 20 birds per flock (dead or sick) | 24 | 25 | 34 | 3 | 2 | 58 | 4 | 3 |
Contact (sick organs from at least 5 birds (dead or sick) as well as oropharyngeal and cloacal swabs from at least 20 birds per flock (dead or sick) oropharyngeal swabs from 60 randomly selected birds per flock | 1 | 0 | 8 | 0 | 0 | 4 | 0 | 0 |
Flocks assumed free of slaughterhouses HPAI before shipment or within to restriction zones oropharyngeal swabs from 60 randomly selected birds per flock | 1 | 146 | 18 | 0 | 0 | 40 | 0 | 1 |
Flocks of HPAI assumed after re-free population organs from dead poultry or swabs taken from their carcasses from up to 10 birds per week during the 21-day period | 3 | 5 | 27 | 5 | 1 | 9 | 6 | 0 |
TOTAL | 29 | 176 | 87 | 8 | 3 | 111 | 10 | 4 |
TTE – testing to exclude
HPAI – highly pathogenic avian influenza
For HTS, all gene segments were amplified using universal primers and a SuperScript III One-Step RT-PCR System with Platinum
A summary of HPAI outbreaks in poultry in Poland reported between 31 December 2019 and 31 March 2020
Production type | Province | Number of outbreaks | Total |
---|---|---|---|
fattening turkeys | Lubelskie | 5 | 12 |
fattening ducks | Wielkopolskie | 3 | 10 |
breeding ducks | Wielkopolskie | 1 | 2 |
breeding geese | Wielkopolskie | 1 | 2 |
laying hens | Wielkopolskie | 1 | 1 |
guinea fowl | Lubelskie | 1 | 1 |
backyard holdings (mostly chickens) | Lubelskie | 2 | 7 |
Total | 35 |
All positive results were obtained for samples collected in the frame of passive surveillance (on grounds of suspicion or as TTE). No virus was detected in clinically healthy birds tested before movement to slaughterhouses or after re-population.
So far, there have been four recorded HPAI epidemics in Poland, all caused by H5 Gs/GD lineage viruses (23, 24, 27, 28). The causative agent of the first two HPAI epidemics (in 2006 and 2007) was H5N1 clade 2.2 virus, but the genetic analysis confirmed that the outbreaks in 2006 and 2007 were caused by separate incursions of genetically distinguishable viruses (23). The highly pathogenic H5 Gs/GD clade 2.3.4.4b viruses were first introduced into Poland in 2016 (28). Between November 2016 and March 2017, 65 outbreaks in poultry and 68 detections in wild birds were caused by two subtypes (mostly H5N8 and H5N5) and at least four different genotypes of the H5 Gs/GD clade 2.3.4.4b virus (27).
The latest 2019/20 HPAIV epidemic in Poland was caused by a novel genotype of H5 Gs/GD clade 2.3.4.4b that has African ancestry and was generated through reassortment between sub-Saharan Africa H5N8 Gs/GD clade 2.3.4.4b viruses (six gene segments) and Eurasian LPAIV (two segments) (27). All the analysed outbreaks in Poland were caused by the same reassortant virus and relatively high homology was noted for all genome segments of Polish strains (>99.3%), suggesting their recent divergence from a common ancestor. This observation was confirmed by phylogenetic analysis employing estimation of divergence times, as the MRCAs for all genome segments of the European viruses from the current epidemic dated back to summer/early autumn 2019.
The role of wild birds in the introduction and spread of the virus was initially questioned, as there were no HPAIV detections in the period preceding the occurrence of the H5N8 virus in Europe and during the epidemic positive cases in wild avifauna were extremely rare (9). However, almost simultaneous detection of genetically similar viruses in Poland, Hungary, Slovakia, Romania, Germany, the Czech Republic and Ukraine (9, 14) pointed indirectly at wild birds as the most probable disseminators of the virus. The possible reasons for the late incursion of the HPAI virus in 2019/20 into Europe compared to the previous epidemics can be explained by the moderate temperatures reported in the moulting areas in Russia in November/December 2019, different routes of virus spread or undetected circulation of the HPAIV H5 virus in wild birds (9). Presently purely speculative reasons for virus detections in wild avian species being infrequent are insufficient surveillance, pre-existing herd immunity elicited by exposures to antigenically similar viruses of H5 Gs/GD clade 2.3.4.4 in previous seasons and/or reduced pathogenicity of the new H5N8 genotype for certain “sentinel” bird species.
Despite the limited sequence variation, the phylogenetic analysis could assist in the investigation of the pathways of virus spread by complementing the findings of classic epidemiological investigations. The first outbreak in Poland was detected on a farm in a region with high turkey density and the abundant water bodies of the Łęczna-Włodawa Lakeland, where wild migratory waterfowl had gathered in large flocks at that time. Indirect contact with wild birds was therefore suggested as the most plausible source of virus introduction onto the farm. The HPAIV-positive holdings detected in the following days on the neighbouring farms were likely caused by human-mediated spread of the virus, owing to the close proximity of the poultry establishments to the location of the index case. However, airborne transmission cannot be ruled out and there are more evidence-based studies to support the role of wind in the dissemination of the virus over short distances (21). The detection of an HPAI H5N8 outbreak in a flock of laying hens on a farm located in Wielkopolskie Province more than 350 km from the index case in Lubelskie Province raised questions about the origin of the pathogen. The phylogenetic studies ruled out the possibility of a direct connection between the first outbreaks in Lubelskie and Wielkopolskie, and so far the only explanation is indirect contact with wild birds. On the other hand, the index case turkey farm was suggested as the source of the outbreak in backyard poultry in Lubelskie, 60 km away. In the days preceding the appearance of signs arousing suspicion of HPAI at the farm where the index case was diagnosed, animal by-products were sold. Epidemiological investigation revealed that they were to feed foxes kept on the same premises where on a small scale, poultry were kept which subsequently tested positive for HPAI. It was later confirmed by whole genome sequencing, which showed that the two viruses were identical. Interestingly, the viruses in question clustered separately from the virus detected at the same time in a nearby (~20 km) backyard flock and thus direct interconnections were excluded. Most of the outbreaks in young fattening ducks detected in February had a common source, i.e. one specific transport company that delivered one-day-old birds to farms in different, sometimes distant locations. It was also corroborated by phylogenetic studies, as the viruses from the outbreaks in ducklings were either identical or differed only at single nucleotide positions. As the risk of vertical transmission of HPAIV is negligible and laboratory examinations excluded the possibility of infection at hatcheries, the young ducklings were most probably infected during transport in a contaminated vehicle.
Control measures applied during the epidemic were in agreement with the Regulation of the Minister of Agriculture and Rural Development of December 18, 2007 on eradication of avian influenza (implementation of Council Directive 2005/94/EC (6)) and included stamping-out, zoning, movement restrictions, cleansing and disinfection, post-outbreak surveillance, information campaigns, housing orders and strengthening other biosecurity measures (9). Although preventive culling can be implemented in a protection zone (i.e. 3 km around the HPAI outbreak), it was not applied during the recent epidemic. Similarly to previous epidemics in Poland (2006-2017), vaccination in poultry and zoo birds was prohibited. In most cases, samples from clinically healthy broiler/meat poultry flocks from restriction zones were tested in a laboratory before shipment to a slaughterhouse. Despite the large number of tested consignments (>200), no positive results were found. Additionally, in spite of the introduction of the virus into regions with intensive poultry production (e.g. Wielkopolskie, Lubuskie, and Lubelskie provinces), secondary spread was rather limited and the outbreaks were quickly contained.
In summary, repeated incursions of HPAI viruses into new countries or territories raise serious concerns for poultry producers. Scientific evidence collected in recent years highlights the predominant role of wild birds in disseminating the HPAI virus to previously disease-free areas (1, 3, 15). However, the 2019/20 HPAI epidemic revealed the ineffectiveness of wild bird surveillance as an early warning tool for disease detection, and the reasons for that failure should be carefully addressed to improve future detectability of the virus in wild avifauna. As the movement of wild birds is beyond human control and most European countries apply a non-vaccination policy, the only means of preventing virus introduction into poultry flocks is strict adherence to biosecurity practices. Furthermore, timely notification of health problems suggestive of HPAI and fast laboratory diagnosis minimise the risk of secondary spread. Based on the experience gathered during the recent epidemic in Poland, passive surveillance in poultry can be very effective. The most common clinical signs predictive of HPAI included higher mortality, drop in food and water consumption, decreased egg production, and neurological signs. However, it is also very important to note that in breeding ducks the mortality was extremely low and neurological symptoms were absent, thus the stereotypical approach to clinical manifestation in the flock engenders the risk of erroneous suspicion and prolonged time to diagnosis. As HPAI will continue to pose a risk in the future, better preparedness including constant vigilance (demonstrably necessary by the late onset of the recent epidemic), increased surveillance activity in wild birds, awareness campaigns among stakeholders, timely diagnosis, and notification of and response to outbreaks in poultry are needed to reduce the risk of occurrence and minimise the losses posed by HPAI.