Genetic characterization of H9N2 avian influenza viruses isolated from poultry in Poland during 2013/2014.

The study presents molecular characterization of H9N2 avian influenza (AI) isolates from field outbreaks in turkeys that occurred in Poland in 2013-2014. Sequences of all gene segments of one isolate from 2013 (A/turkey/Poland/14/2013(H9N2)) and two isolates from 2014 (A/turkey/Poland/08/2014(H9N2), A/turkey/Poland/09/2014(H9N2)) were obtained and analyzed in search of the phylogenetic relationship and molecular markers of zoonotic potential or increased pathogenicity. All gene segments were shown to originate from the wild bird reservoir and the close relationship of the analyzed isolates proved the link between the outbreaks in 2013 and 2014. However, remarkable molecular differences between isolates from 2013 to 2014 were identified, including mutation in the HA cleavage site (CS) leading to conversion from the PAASNR*GLF to the PAASKR*GLF motif and truncation of the PB1-F2 protein. Additionally, T97I substitution in the PA protein in A/turkey/Poland/08/2014 was detected which can be responsible for enhanced activity of viral polymerase in mammalian cells. However, experimental infection of mice with both isolates from 2014 showed their low pathogenicity, and no statistically significant differences in virus replication were observed between the viruses. Nevertheless, these findings indicate the dynamic evolution of H9N2 in the field emphasizing the need for monitoring of the situation in terms of H9N2 AI in Europe.

Astrovirus-induced "white chicks" condition - field observation, virus detection and preliminary characterization.

Chicken astrovirus (CAstV) was recently indicated as the factor of the "white chicks" condition associated not only with increased embryo/chick mortality but also with weakness and white plumage of hatched chicks. In February 2014, organ samples (livers and kidneys) from dead-in-shell embryos, as well as 1-day-old whitish and normal chicks, were delivered from one hatchery in Poland for disease diagnosis. The samples originated from the same 30-week-old breeder flock in which the only observed abnormal signs were 4-5% decrease in the number of hatched chickens and the presence (about 1%) of weaker chicks with characteristic whitish plumage among normal ones. CAstV was detected in submitted samples and was then isolated in 10-day-old embryonated specific pathogen free (SPF) chicken eggs. We also reproduced an infection model for the "white chicks" condition in SPF layer chickens using the isolated PL/G059/2014 strain as the infectious agent. Results of experimental reproduction of the "white chicks" condition were somewhat more serious than field observation. The administration of the CAstV material into the yolk sac of 8-day-old SPF chicken eggs caused delay and prolongation of hatching, as well as death of embryos/chicks, and also a change of plumage pigmentation. Only two chicks of a total of 10 inoculated SPF eggs survived and were observed for 2 months. A gradual elimination of the CAstV genome was noted in this period. Moreover, a few contact-naive SPF chicks, which had been placed in the same cage, were infected with CAstV. Molecular characterization of detected CAstV was performed by nucleotide sequencing of the full ORF2 region encoding the capsid precursor protein gene. Phylogenetic studies showed that the PL/G059/2014 isolate clustered in the subgroup Aiii of CAstV. In the light of the new classification rules, the Polish PL/G059/2014 CAstV isolate could be assigned to a new species of the Avastrovirus genus.

Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification.

Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.

Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations.

We examined 884 wild birds mainly from the Anseriformes, Charadriiformes and Galliformes orders for infectious bronchitis (IBV)-like coronavirus in Poland between 2008 and 2011. Coronavirus was detected in 31 (3.5%) of the tested birds, with detection rates of 3.5% in Anseriformes and 2.3% in Charadriiformes and as high as 17.6% in Galliformes. From the 31 positive samples, only 10 gave positive results in molecular tests aimed at various IBV genome fragments: five samples were positive for the RdRp gene, four for gene 3, eight for gene N and eight for the 3'-untranslated region fragment. All analysed genome fragments of the coronavirus strains shared different evolutionary branches, resulting in a different phylogenetic tree topology. Most detected fragment genes seem to be IBV-like genes of the most frequently detected lineages of IBV in this geographical region (i.e. Massachusetts, 793B and QX). Two waves of coronavirus infections were identified: one in spring (April and May) and another in late autumn (October to December). To our knowledge this is the first report of the detection of different fragment IBV-like genes in wild bird populations.

Avian influenza H9N2 subtype in Poland--characterization of the isolates and evidence of concomitant infections.

In April/May 2013, four outbreaks of avian influenza virus (AIV) infections caused by H9N2 subtype were diagnosed in Poland in fattening turkey flocks exhibiting a drop in feed and water intake, depression, respiratory signs and mortality. The subsequent serological survey carried out on samples collected between June 2012 and September 2013 from 92 poultry flocks detected positive sera in two additional meat turkey flocks located in the same province. The analysis of amino acids in the haemagglutinin and neuraminidase glycoproteins revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity, avian-like SAα2,3 receptor specificity and adaptation to domestic poultry. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian clade of wild bird-origin AIVs and had no relationship with H9N2 AIV circulating in poultry in the Middle East and Far East Asia over the past decade. Experimentally infected SPF chickens with the index-case H9N2 virus remained healthy throughout the experiment. On the other hand, ten 3-week-old commercial turkeys infected via the oculonasal route showed respiratory signs and mortality (2/10 birds). Additional diagnostic tests demonstrated the consistent presence of DNA/RNA of Ornithobacterium rhinotracheale, Bordetella avium and, less frequently, of astro-, rota-, reo-, parvo- and adenoviruses in turkeys both from field outbreaks and laboratory experiment. Although no microbiological culture was performed, we speculate that these secondary pathogens could play a role in the pathogenicity of the current H9N2 infections.

Astroviruses in Polish commercial turkey farms in 2009-2012.

Avian astrovirus infections are widespread in many countries, and infections have been connected with enteritis and increased mortality in young birds. In the present study, fecal samples were collected during 2009-2012 from a total of 156 meat turkey flocks. Astrovirus presence and type differentiation was performed with the use of two molecular diagnostic approaches. Out of 156 flocks, 48.7% were found to be TAstV positive. Depending on the method used for type differentiation, TAstV-2 and TAstV-1 prevalence was between 31.4%-41% and 9.6%-15.4%, respectively. No avian nephritis virus was detected. About 30% of astrovirus-positive flocks were infected with both types of TAstV. Phylogenetic analysis based on the partial polymerase gene sequence revealed the genetic variability of isolated TAstV, and most of the detected TAstV-2 belonged to the European lineage of astroviruses. Statistical analysis suggested the positive but weak correlation between the presence of astrovirus and health status (slightly more frequent detection of TAstV in sick, diarrheic birds) and also negative medium correlation between age and astrovirus occurrence.

Experimental infection of different species of birds with pigeon paramyxovirus type 1 virus--evaluation of clinical outcomes, viral shedding, and distribution in tissues.

The virulence of pigeon paramyxovirus type 1 (PPMV-1) for different species of birds was investigated in two independent sets of experiments in which groups of pigeons, chickens, turkeys, quails, and geese (10 birds per group) were inoculated with 10(6) median embryo infectious doses of PPMV-1 isolate: 1) nonpassaged (nPPMV-1, intracerebral pathogenicity [ICPI] value = 1.27) and 2) after six passages in specific-pathogen-free chickens (pPPMV-1, ICPI = 1.46) via the oculonasal route. Naive birds were placed in contact with infected birds (two birds per group) to monitor virus transmission. Clinical observation was performed daily. Additionally, cloacal swabs, oropharyngeal swabs, and selected organ samples were collected on days 2, 4, 7, 10, and 14 postinfection and tested by real-time reverse transcriptase-PCR for estimation of viral shedding and distribution in tissues. Infected pigeons exhibited nervous and digestive tract symptoms, mortality, shedding, and transmission to contact birds. Chickens, turkeys, quails, and geese did not exhibit any clinical signs regardless of the PPMV-1 strain used for inoculation. However, in contrast to quails and geese, chickens and turkeys shed the virus via the oral cavity and cloaca, and transmission to contact birds was also observed. Viral RNA was identified in tissues collected from all pPPMV-1-infected birds, whereas negative results were obtained in the case of tissues taken from nPPMV-1-infected quails and geese. We conclude that the PPMV-1 used in this study was most virulent to pigeons, followed by chickens and turkeys, while quails and geese seem to have the highest level of innate resistance to this strain. However, passaging of PPMV-1 in chickens resulted in the increase of ICPI and noticeable but sometimes contrasting changes in the replication capacities of the virus.

Phylogenetic studies of H3 low pathogenic avian influenza viruses isolated from wild mallards in Poland.

In order to study the variation of low pathogenic avian influenza viruses (AIV) of H3 subtype in the natural reservoir, partial genetic characterisation of four AIV isolates of H3 subtype, recovered from wild mallards in Poland in 2006-2010, was performed. Phylogenetic analysis clearly confirms that there is a constant flow of AIV H3 between wild birds in Eurasia and Africa, and, to a limited degree, to North America (Alaska), with an occasional spill-over to poultry. The analysis of the PA gene of one isolate from 2010 suggests that it is closely related to several HPAI H5N1 viruses belonging to clade 2.3.2 and that, therefore, a reassortment event has occurred recently between low pathogenic and H5N1 highly pathogenic AIV.

Genetic characterization of parvoviruses circulating in turkey and chicken flocks in Poland.

Between 2008 and 2011, commercial turkey and chicken flocks in Poland were examined for the presence of turkey parvovirus (TuPV) and chicken parvovirus (ChPV). Clinical samples (10 individual faecal swabs/flock) from 197 turkey flocks (turkeys aged 1 to 19 weeks) and 45 chicken flocks (chickens aged 3 to 17 weeks) were collected in different regions of the country and tested using a PCR assay that targeted the NS1 gene (3'ORF). The prevalence of TuPV was 29.4 % in the flocks tested, while ChPV infections were found in 22.2 % of the studied flocks. Phylogenetic analysis revealed a clear division into three groups: ChPV-like, TuPV-like and a third, previously unrecognized and distinct subgroup, TuPV-LUB, containing exclusively three Polish isolates from turkeys. The isolates from the novel group showed as little as 50.6-64.5 % of nucleotide sequence identity to the prototype chicken and turkey parvovirus strains. Genetic analysis of a ChPV isolate that was classified in the TuPV group strongly suggests a recombination event between chicken and turkey parvoviruses.

One-year molecular survey of astrovirus infection in turkeys in Poland.

The presence of turkey astrovirus (TAstV) was monitored in meat-type turkey flocks in Poland in 2008. Clinical samples (10 individual faecal swabs/flock) from 77 flocks aged 1-19 weeks were collected from different regions of the country. RT-PCR experiments were performed for detection and molecular characterization of TAstV using four sets of primers within the RdRp gene (ORF1b). The prevalence of astrovirus was 34/77 (44.15%) in the flocks tested. TAstV type 2 was associated with 30 of 77 infections (38.9%), either alone or in mixed infections; TAstV type 1 was detected in 9 of 77 flocks (11.6%), either alone or in mixed infections; ANV was detected only in one flock (1.29%) by sequence analysis during this study. Phylogenetic analysis revealed genetic variability in the TAstV strains that were isolated. Some of Polish TAstV-2 strains were genetically related to the North American isolates; however, most of them formed a distinct subgroup of "European" isolates, suggesting their separate origin or evolution. Additionally, due to the high variability of the TAstV sequences, the most suitable method for TAstV typing seems to be sequencing.

Susceptibility of pigeons to clade 1 and 2.2 high pathogenicity avian influenza H5N1 virus.

To assess the susceptibility of pigeons (Columba livia) to infection with H5N1 high pathogenicity avian influenza virus (HPAIV), four groups of 1-yr-old and 4-wk-old racing pigeons (10 birds in each group) were inoculated oculonasally with 106 50% egg infectious dose (EID50) of A/crested eagle/Belgium/01/2004 (clade 1) or A/swan/Poland/305-135V08/2006 (clade 2.2). Contact specific-pathogen-free (SPF) chickens were kept in the same isolators as young pigeons (two chickens per group). At 3, 5, 7, 10, and 14 days postinfection (PI) two pigeons from each infected group were selected randomly, and oropharyngeal and cloacal swabs (pigeons and contact chickens) as well as a number of internal organs (pigeons) were collected for viral RNA detection in real-time reverse transcription PCR (RRT-PCR) and histopathology. At the end of the experiment (14 days PI) blood samples from two pigeons in each group and from contact SPF chickens were also collected, and sera were tested using hemagglutination inhibition (HI) test and blocking enzyme-linked immunosorbent assay (bELISA). During the observation period all pigeons remained clinically healthy, and no gross lesions were observed in any of the infected groups. SPF contact chickens were also healthy and negative in RRT-PCR and HI tests. However, the clade 1 H5N1 virus produced more sustained infection manifested by the presence of histopathologic changes (consisting mainly of mild to moderate hemorrhagic and inflammatory lesions), prolonged persistence of viral RNA (detectable between 3 and 10 days PI) in a variety of tissues of both adult and juvenile birds (with highest RNA load in lungs and brain) as well as slight viral shedding from the trachea and cloaca, but without transmission to SPF contact chickens. Additionally, two clade 1-infected adult pigeons sacrificed at the end of experiment showed seroconversion in bELISA and HI test (using homologous virus as antigen). The viral RNA was found only at day 3 PI in one adult pigeon inoculated with dade 2.2 H5N1 virus, but neither microscopic lesions nor seroconversion were found in any other tested birds inoculated with A/swan/Poland/305-135V08/2006. Our results support the observations that pigeons are resistant to H5N1 HPAIV (no deaths or clinical signs), but there may be clade-dependent differences in the pathogenic potentials of H5N1 HPAIV of Asian origin.

H5N1 high pathogenicity avian influenza virus survival in different types of water.

Persistence of H5N1 high pathogenicity avian influenza virus (HPAIV), isolated during the epidemic in wild birds in Poland in 2006, was evaluated in three water samples derived from the sources known to host wild water birds (city pond, Vistula river mouth, and Baltic Sea). The virus was tested at two concentrations (10(4) and 10(6) median tissue culture infective dose per milliliter) and at three temperatures (4 C, 10 C, and 20 C), representing average seasonal temperatures in Poland. All tested water samples were filtered before virus inoculation, and one unfiltered sample (Baltic seawater) was also tested. Infectivity was determined twice a week over a 60-day trial period by microtiter endpoint titration. The persistence of the virus varied considerably depending on its concentration and also on physico-chemical parameters of the water, such as temperature and salinity. Avian influenza virus survival was the highest at 4 C and the lowest at 20 C. Prolonged infectivity of the virus in Baltic seawater (brackish, 7.8 ppt) was also seen. In distilled water, the virus retained its infectivity beyond the 60-day study period. Interestingly, a devastating effect of the unfiltered fraction of seawater was seen as the virus disappeared in this fraction the quickest in all studied combinations; thus, biologic factors may also affect infectivity of HPAIV.

Full-length genome sequencing of the Polish HPAI H5N1 viruses suggests separate introductions in 2006 and 2007.

This paper describes the results of the molecular and phylogenetic analysis of seven highly pathogenic avian influenza (HPAI) H5N1 strains isolated in 2006 (n = 5) and 2007 (n = 2) from wild birds and poultry in Poland. The whole genome sequence of these isolates was determined. All of the isolates possessed the hemagglutinin (HA) cleavage site sequence PQGERRRKKR*GLF typical of HPAI. Molecular markers associated with increased adaptation and virulence in mammals, as well as susceptibility to neuraminidase inhibitors, were revealed in the HA, neuraminidase (NA), and PB2 proteins. Based on the sequencing results related to the HA and NA genomic segments, H5N1 viruses circulating in Poland all belong to lineage 2.2. However, isolates isolated in 2006 were genetically distinct from those isolated in 2007 and grouped in different sublineages. H5N1 viruses isolated from wild birds in 2006 are almost identical to each other (99.9% HA; 99.6%-100% NA), and they are grouped within a cluster of viruses isolated in Germany from wild and domestic birds and mammals in 2006. Isolates from 2007 are also closely related to each other (nucleotide homologies 99.9% and 100% for HA and NA, respectively), and they are grouped together with isolates from wild and domestic birds collected in Eastern and Central Europe (Romania, Germany), and the Middle East (Kuwait, Saudi Arabia). Phylogenetic analysis of the sequences related to the internal proteins confirmed the results obtained for the HA and NA genes. Overall, the results indicate that HPAI H5N1 in Poland in 2006-07 was caused by at least two separate incursions of genetically distinct viruses.

Highly pathogenic avian influenza virus (H5N1) in frozen duck carcasses, Germany, 2007.

We conducted phylogenetic and epidemiologic analyses to determine sources of outbreaks of highly pathogenic avian influenza virus (HPAIV), subtype H5N1, in poultry holdings in 2007 in Germany, and a suspected incursion of HPAIV into the food chain through contaminated deep-frozen duck carcasses. In summer 2007, HPAIV (H5N1) outbreaks in 3 poultry holdings in Germany were temporally, spatially, and phylogenetically linked to outbreaks in wild aquatic birds. Detection of HPAIV (H5N1) in frozen duck carcass samples of retained slaughter batches of 1 farm indicated that silent infection had occurred for some time before the incidental detection. Phylogenetic analysis established a direct epidemiologic link between HPAIV isolated from duck meat and strains isolated from 3 further outbreaks in December 2007 in backyard chickens that had access to uncooked offal from commercial deep-frozen duck carcasses. Measures that will prevent such undetected introduction of HPAIV (H5N1) into the food chain are urgently required.

A Recombinant Turkey Herpesvirus Expressing F and HN Genes of Avian Avulavirus-1 (AAvV-1) Genotype VI Confers Cross-Protection against Challenge with Virulent AAvV-1 Genotypes IV and VII in Chickens.

Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI ("pigeon variant" of AAvV-1). This recombinant viral vaccine candidate was used to subcutaneously immunize one group of specific pathogen-free (SPF) chickens and two groups of broiler chickens (20 one-day-old birds/group). Humoral immune response was evaluated by hemagglutination-inhibition test and enzyme-linked immunosorbent assay (ELISA). The efficacy of the immunization was assessed in two separate challenge studies performed at 6 weeks of age with the use of virulent AAvV-1 strains representing heterologous genotypes IV and VII. The developed vaccine candidate elicited complete protection in SPF chickens since none of the birds became sick or died during the 2-week observation period. In the broiler groups, 90% and 100% clinical protection were achieved after challenges with AAvV-1 of IV and VII genotypes, respectively. We found no obvious relationship between antibody levels and protection assessed in broilers in the challenge study. The developed recombinant HVT-NDV-F-HN construct containing genes from a genotype VI AAvV-1 offers promising results as a potential vaccine candidate against ND in chickens.

The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen.

Hemagglutinin glycoprotein (HA) is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm) and with low-mannose glycosylation (H5Man5) were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies.

The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

Correction: A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.pone.0172008.].

A prime/boost vaccination with HA DNA and Pichia-produced HA protein elicits a strong humoral response in chickens against H5N1.

Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA-specific humoral immune responses in chickens. In two independent experiments, anti-HA antibodies were detected in sera collected two weeks after the first dose and the response was enhanced by the second dose of a vaccine, regardless of the type of subunit vaccine (DNA or recombinant protein) administered. The serum collected from chickens two weeks after the second dose was characterized by three types of assays: indirect ELISA, hemagglutination inhibition (HI) and a diagnostic test based on H5 antibody competition. Although the indirect ELISA failed to detect superiority of any of the three vaccine regimens, the other two tests clearly indicated that priming of chickens with the DNA vaccine significantly enhanced the protective potential of the recombinant protein vaccine produced in P. pastoris.

SURVEILLANCE FOR AVIAN INFLUENZA VIRUS IN WILD BIRDS IN POLAND, 2008-15.

We tested wild birds in Poland during 2008-15 for avian influenza virus (AIV). We took 10,312 swabs and feces samples from 6,314 live birds representing 12 orders and 84 bird species, mostly from orders Anseriformes and Charadriiformes, for testing and characterization by various PCR methods. From PCR-positive samples, we attempted to isolate and subtype the virus. The RNA of AIV was detected in 1.8% (95% confidence interval [CI], 1.5-2.1%) of birds represented by 48 Mallards ( Anas platyrhynchos ), 11 Mute Swans ( Cygnus olor ), 48 Common Teals ( Anas crecca ), three Black-headed Gulls (Chroicocephalus ridibundus), one Common Coot ( Fulica atra ), one Garganey (Spatula querquedula), and one unidentified bird species. Overall, the prevalence of AIV detection in Mallards and Mute Swans (the most frequently sampled species) was 2.0% (95% CI, 1.4-2.5%) and 0.5% (95% CI, 0.2-0.8%), respectively; the difference was statistically significant (P=0.000). Hemagglutinin subtypes from H1 to H13 were identified, including H5 and H7 low pathogenic AIV subtypes. Mallards and Common Teals harbored the greatest diversity of subtypes. We observed seasonality of viral detection in Mallards, with higher AIV prevalence in late summer and autumn than in winter and spring. In addition, two peaks in AIV prevalence in summer (August) and autumn (November) were demonstrated for Mallards. The prevalence of AIV in Mute Swans did not show any statistically significant seasonal patterns.