Avian influenza virus (AIV) is an RNA virus that belongs to the
In recent years, large-scale epidemics of HPAI have been mostly associated with the A/goose/Guangdong/ 1/1996 (Gs/GD) lineage of H5 viruses (hereafter referred to as H5 Gs/GD) that can have different NA subtypes (15). Since their first detection in China in the mid-90s, the HA genes of H5 Gs/GD viruses (initially existing as the H5N1 subtype) have evolved into multiple genetic clades, some of which (
The H5 Gs/GD viruses have been introduced to Europe at least 10 times between 2005 and 2020 (21). During this period, Poland has experienced five incursions: in 2006 (clade 2.2 H5N1 subtype), 2007 (clade 2.2 H5N1 subtype), 2016 (clade 2.3.4.4b H5N8 and H5N5 subtypes), 2019 (clade 2.3.4/4.b H5N8 subtype) and 2020 (clade 2.3.4.4b H5N8, H5N5 and H5N1 subtypes) (16, 18, 19, 20). The size of outbreaks varied greatly but the 2020/2021 season was undoubtedly the largest and caused the highest economic losses to the poultry industry.
The paper describes the major characteristics of the 2020/2021 HPAI epidemic in Poland and discusses possible factors that led to the unprecedented spread of the virus.
The surveillance system for HPAI in poultry was composed of passive and active components. Passive surveillance had two elements: suspicion of HPAI (birds showing clinical signs and necropsy findings suggestive of HPAI) and “testing-to-exclude” (TTE) system (
Number and type of flocks per species and production category tested between 24 November 2020 (date of the first outbreak) to 9 August 2021 (date of the last outbreak)
Poultry category | Type of surveillance |
Total | ||
---|---|---|---|---|
Passive surveillance | Contact holdings | Active surveillance | ||
Laying hens | 219 | 60 | 22 | 301 |
Broiler chickens | 88 | 27 | 234 | 349 |
Broiler breeders | 132 | 25 | 5 | 162 |
Dual-chickens purpose | 32 | 15 | 6 | 53 |
Fattening turkeys | 174 | 57 | 157 | 388 |
Turkey breeders | 1 | 0 | 0 | 1 |
Fattening geese | 37 | 4 | 24 | 65 |
Geese breeders | 15 | 1 | 2 | 18 |
Fattening duck | 109 | 11 | 203 | 323 |
Duck breeders | 9 | 1 | 6 | 16 |
Pheasants | 1 | 0 | 0 | 1 |
Guinea fowl | 1 | 2 | 1 | 4 |
Multi-species | 17 | 3 | 1 | 21 |
Total | 835 | 206 | 661 | 1702 |
The virus sequences were obtained by Sanger sequencing of overlapping RT-PCR products or by high-throughput sequencing following amplification of all segments in an RT-PCR. The methods and the analysis pipeline were described previously (18, 20). Whole genome sequences were generated for 27 viruses and for another 50 strains only HA gene sequences were obtained. Additionally, 20 whole genome sequences of Polish strains produced by the European Union Reference Laboratory (IZSVe, Legnaro, Italy) and sequences from other countries available in the GISAID (Global Initiative on Sharing Avian Influenza Data) database were also included in the analysis. Phylogenetic trees were inferred in the MEGA X program (13) by using the maximum likelihood method and Hasegawa–Kishino–Yano model with 100 bootstrap replications.
The HPAI epidemic in the 2020/2021 season had three discernible but sometimes overlapping phases with different epidemiological dynamics (Fig. 1). The landmarks of the phases were determined by the first detection of the virus at the end of November 2020 with subsequent outbreaks that followed in the next weeks until the beginning of February 2021 (phase I, Fig. 2a), severe weather conditions in February that triggered an epidemic in wild birds in the following weeks (phase II, Fig. 2b) and further escalation of the epidemic (from March 2021 onwards with the peak in April), mostly associated with the introduction of viruses into high-density poultry regions (phase III, Fig. 2c). In total, between 24 November 2020 and 9 August 2021, 357 HPAIV H5Nx outbreaks in poultry, 2 outbreaks in captive birds and 92 events in wild birds involving >150 birds were confirmed, with most of the poultry outbreaks localised in Mazowieckie (n = 132) and Wielkopolskie (n = 100) provinces (Fig. 2d). The highest number of outbreaks was found in chicken layers (both breeders and hens laying eggs for consumption, n = 118) followed by meat turkeys (n = 75), fattening ducks (n = 62), breeder geese (n = 9), fattening geese (n = 8), broiler chickens (n = 6) and breeder ducks (n = 5). Twenty-four positive commercial holdings were multispecies farms. The virus was also detected in 49 non-commercial, mostly backyard poultry holdings, where chickens were the predominant species. All outbreaks in poultry were caused by the HPAIV H5N8 subtype. The clinical signs observed in infected poultry did not substantially differ from those typically described for HPAI. The disease usually began with the drop in feed and/or water consumption that was accompanied (or quickly followed) by increased mortality and a drop in egg production (the latter not always observed in hens at the onset of clinical problems), depression, on some occasions shortness of breath, other respiratory signs (conjunctivitis, nasal discharge, sneezing) and diarrhoea. The neurological signs were very clearly seen in fattening ducks and entailed a broad spectrum of symptoms including: ataxia, tremors, lying on the back and pedalling with the legs, torticollis and paralysis. Breeder ducks usually did not display obvious clinical signs, and the drop in feed/water intake and decreased production of eggs (by approximately 50%) were the only observed abnormalities. As for wild birds, the vast majority of HPAIV detections were made in swans (mostly mute swans,
A total of 97 HA sequences of Polish strains were analysed. All tested viruses belonged to the 2.3.4.4b clade and they grouped together with other HPAIV H5Nx which have circulated in Europe since autumn 2020. A significant intra-clade variation was found with spatio-temporal clusters that were identified for viruses detected in high-density poultry areas (Fig. 3). The analysis of fully-sequenced HPAIV H5Nx strains from Poland (n = 47) revealed the existence of one genotype of H5N8 subtype (genotype A). Conformations of genome segments of Polish H5N8 and H5N1 viruses were similar to those found in other European countries. In contrast, the H5N5 isolate showed a unique gene constellation as its PB2, PB1, NP and NA clustered with Eurasian LPAIV strains, whereas the remaining segments were highly similar to the 2020/21 H5N8 viruses. In two HPAIV H5N8 strains found in domestic ducks and geese at the beginning of May 2021, single mutations (PB2 D701N) associated with increased polymerase activity and replication in mammalian cell lines were detected (9).
The first detection of H5 Gs/GD viruses in the 2020/2021 season in Europe was confirmed in mid-October 2020 in the Netherlands and was followed by notifications from the United Kingdom, Germany, Denmark, Ireland, Belgium, France and Sweden (1). The detections were made in poultry, wild and captive birds. Early in the season, the barnacle goose (
The first confirmation of HPAI H5Nx in Poland was achieved quite late (24th November) in comparison to other European countries and the 2020/2021 HPAI season in Poland with its three major phases began with a rather gradual escalation. The first phase (between November and the start of February) was characterised by a small number of outbreaks in poultry (weekly median value = 3) and a very small number of wild bird cases, which on several occasions were detected only in active searches for carcasses in close proximity to virus-positive poultry holdings. With the exception of a small region in western Poland (Wolsztyn and adjacent counties in Wielkopolskie province), where to some extent a secondary spread was observed, the existing evidence coming from epizootic investigations and phylogenetic studies suggests that most of the HPAIV-positive poultry holdings were primary outbreaks that arose as a result of direct or indirect contact with wild birds. Interestingly, a lot of positive holdings were big commercial farms. In total, by the end of January 2021, 32 outbreaks in poultry and 13 detections in wild birds (in 7 locations) were confirmed. The situation was not advantageous but was stable.
In February 2021, a significant drop in temperatures (below −15℃) coincided with the field observation of moderate and mass mortalities among wild birds across a large area of Poland and marked the second phase of the HPAI season. It was particularly noticeable around the Gdańsk Bay in northern Poland. Samples collected from wild birds, mostly mute swans but also other waterfowl and birds of prey, on many occasions tested positive for an H5 virus that in the overwhelming majority of cases was subtyped as H5N8 virus. The sudden aggravation of the epidemiological situation resembled that in February 2006, when the peak of HPAIV H5N1 detections in wild birds in Europe occurred during harsh winter conditions (11). Possibly, gatherings of wild birds coming from different regions to places where the chances of survival were higher (
In March, as temperatures started to rise, wild birds dispersed and an increasing number of outbreaks in poultry was noted shortly afterwards. Disturbingly, the H5N8 virus spread into regions with high concentrations of poultry production. First it struck in the Wielkopolskie province, where between March and May, 81 outbreaks in poultry were confirmed. The region is abundant with domestic waterfowl production and indeed, most of the HPAI H5N8 outbreaks were detected in ducks and geese. It is noteworthy that approximately 20% of positive holdings (mostly fattening Pekin ducks) were detected through testing of healthy meat flocks before shipment to the slaughterhouse. Infection with H5 clade 2.3.4.4 (especially early viruses – or “group A” – present in Europe in 2014/15) can be asymptomatic in Pekin ducks, but the representatives of the more recent H5N8 of this clade (“group B”) are usually virulent in this species (6, 12). Additionally, based on the feedback received from veterinary inspection, re-visits of the farms following positive laboratory results led to the observation that at least some of the birds in the flock had already started to sicken. Thus, the positive results in asymptomatic flocks of waterfowl were more likely associated with the detection of infection during the incubation period rather than the possibility of subclinical infection. Field observations in the same region confirmed that in non-adult domestic waterfowl, especially Pekin ducks, the disease was severe with a range of neurological signs and high mortality. Nonetheless, the obtained results highlight the indispensability of laboratory testing of apparently healthy meat duck and geese flocks from areas under restriction before movement to slaughterhouses.
The virus caused the most devastating impact in the northern part of Mazowieckie province, particularly in Żuromin and Mława counties. For many years, this region has been associated with intensive poultry farming (mostly laying hens, broiler breeders and broilers) but in the past 15 years the total production has almost tripled. Before the 2020/21 season, the presence of HPAI (caused by H5N1 clade 2.2.2 virus) was detected in the region once in 2007 (17) but the outbreaks were confined to a few commercial layer holdings and the situation was quickly contained. The first outbreak in the current season was confirmed on 23 March 2021, but this time the attempts to stop the spread of the virus were unsuccessful and altogether >100 outbreaks in the two named counties were confirmed, most of them on large industrial farms. In order to stop the fast spread of the virus in some high-density areas, preventive culling in a 1-km radius was applied to supplement conventional control measures foreseen by the legislation. The total number of birds that died or were destroyed in outbreaks, contact holdings and other holdings located in the 1-km buffer zone exceeded 11 million. Undoubtedly, the high concentration of poultry farms located in close proximity to one another was the major drivers that favoured the rapid spread of the virus. The pathogen was dispersed by as yet not specifically identified human activities and possibly by the wind (this only over short distances). Interestingly, the statistical analysis using linear and non-linear models on data from the 2007, 2016/2017 and 2019/2020 epidemics in Poland showed that greater chicken population density did not increase the probability of HPAI outbreak occurrence (logistic regression showed even a negative impact) (10). Indeed, before the 2020/2021 season, HPAI epidemics rarely affected chickens and had the highest impact on turkeys, geese and ducks. Also in other European countries (
Although the Mazowieckie province was hardest hit during the spring of the 2020/2021 epidemic, positive results were being obtained from other regions of Poland. The virus was detected in poultry in 15 out of the 16 provinces of the country. In addition to primary outbreaks originating from wild birds, at least two human-mediated modes of virus spread were identified: intra-EU trade with infected ducklings (healthy at the time of transport) and illegal trade of infected pullets to non-commercial holdings (5). The overall direct losses of the HPAI in the 2020/2021 season exceeded 14 million birds, but as pointed out above, about 80% of the losses were concentrated in Mazowieckie province (5).
Phylogenetic studies confirmed the extensive evolution of the viruses expressed by significant variation of the HA gene. The analysis of the tree topology supports the hypothesis about wild birds as the source of primary outbreaks both in terrestrial and aquatic poultry, but on the other hand, the clusters revealed in the Wielkopolskie and Mazowieckie provinces demonstrate that secondary spread also played a role in high-density regions. Investigation of the genome segment constellation revealed the existence of only one genotype of H5N8 (genotype A) in Poland out of seven that have been identified in Europe and Central Asia (2). However, it should be stressed that only a small fraction of viruses detected in Poland have been sequenced and the circulation of other genotypes cannot be ruled out. Interestingly, the Polish H5N5 isolate from a tufted duck was the only representative of a unique genotype not present in other European countries. This virus probably emerged by reassortment of H5N8 HPAIV and low-pathogenic strains circulating in wild birds. In some cases, the observation of genome changes over time provided evidence of a slow evolution towards the selection of molecular markers characteristic of increased adaptation to mammals (
To summarise, the HPAI H5Nx epidemic in 2020/2021 was unquestionably the largest ever recorded in Poland in terms of duration, number of affected counties, number of positive holdings and affected birds. The principal control measures foreseen in the legislation such as culling and movement restrictions, although effective in areas with low- and medium poultry concentrations, seemed to have minimal impact on the virus spread in regions with very high poultry density. Additional measures should be considered in such areas to minimise the huge costs of an epidemic,