Wild Waterfowl Research Articles

‘The China Virus’: Invasion, Contagion, and the ‘Yellow Peril’

‘The China Virus’: Invasion, Contagion, and the ‘Yellow Peril’

Highly Pathogenic Avian Influenza (H5N1) Landscape Suitability Varies by Wetland Habitats and the Degree of Interface between Wild Waterfowl and Poultry in India.

Highly pathogenic avian influenza (HPAI) virus, subtype H5N1, constitutes one of the world’s most important health and economic concerns given the catastrophic impact of epizootics on the poultry industry, the high mortality attending spillover in humans, and its potential as a source subtype for a future pandemic. Nevertheless, we still lack an adequate understanding of HPAI H5N1 epidemiology and infection ecology. The nature of the wild waterfowl–poultry interface, and the sharing of diverse wetland habitat among these birds, currently underscore important knowledge gaps. India has emerged as a global hotspot for HPAI H5N1, while also providing critical wintering habitat for many species of migratory waterfowl and year-round habitat for several resident waterfowl species. The current study sought to examine the extent to which the wild waterfowl–poultry interface, varied wetland habitat, and climate influence HPAI H5N1 epizootics in poultry in India. Using World Organisation for Animal Health reported outbreaks, this study showed that the wild waterfowl–poultry interface and lacustrine, riparian, and coastal marsh wetland systems were strongly associated with landscape suitability, and these relationships varied by scale. Although increasing poultry density was associated with increasing risk, this was only the case in the absence of wild waterfowl habitat, and only at a local scale. In landscapes increasingly shared between wild waterfowl and poultry, suitability was greater among lower density poultry, again at a local scale only. These findings provide further insight into the occurrence of HPAI H5N1 in India and suggest important landscape targets for blocking the waterfowl–poultry interface to interrupt virus transmission and prevent future outbreaks.

Fine-scale tracking of wild waterfowl and their impact on highly pathogenic avian influenza outbreaks in the Republic of Korea, 2014\u20132015

Wild migratory waterfowl are considered one of the most important reservoirs and long-distance carriers of highly pathogenic avian influenza (HPAI). Our study aimed to explore the spatial and temporal characteristics of wild migratory waterfowl’s wintering habitat in the Republic of Korea (ROK) and to evaluate the impact of these habitats on the risk of HPAI outbreaks in commercial poultry farms. The habitat use of 344 wild migratory waterfowl over four migration cycles was estimated based on tracking records. The association of habitat use with HPAI H5N8 outbreaks in poultry farms was evaluated using a multilevel logistic regression model. We found that a poultry farm within a wild waterfowl habitat had a 3–8 times higher risk of HPAI outbreak than poultry farms located outside of the habitat. The range of wild waterfowl habitats increased during autumn migration, and was associated with the epidemic peak of HPAI outbreaks on domestic poultry farms in the ROK. Our findings provide a better understanding of the dynamics of HPAI infection in the wildlife–domestic poultry interface and may help to establish early detection, and cost-effective preventive measures.

Dynamics and features of manifestation of highly pathogenic avian influenza in the Republic of Tuva

The dynamics and features of the manifestation of highly pathogenic avian influenza A/H5N1 were studied on the territory of the Republic of Tuva, where it was first recorded in 2006 in wild migratory waterfowl of the duck family, mainly great crested grebe ducks. In the epizootic process of highly pathogenic avian flu on the territory of the Republic, causal-temporal relationships with the seasonal migration of wild waterfowl from the countries of South-East Asia have been noted. Epizootics of highly pathogenic avian flu were observed in 2006, 2009, 2010, 2014, 2015, 2016. In 2016, as a result of mutation of the H5N1 virus strain, an outbreak of highly pathogenic avian flu of the H5N8 strain was registered. Outbreaks of highly pathogenic avian flu H5 and H7 subtypes simultaneously with low pathogenic subtypes of avian flu H3 were noted in 2014, H9 – in 2013 and 2014. In the study of blood serum of birds in the years officially free from avian flu, antibodies to hemagglutinins of the virus of different subtypes were detected in 11.2-50.0% of cases. Despite the absence of bird mortality, this indicates a constant circulation of avian influenza agent in the body of waterfowl. On the territory of the Republic, avian flu has not spread among poultry due to natural-geographical, socio-economic conditions. The development of distant pasture farming does not provide for the maintenance and breeding of poultry in the unfavorable area of the lake UvsNuur; the local population does not have a tradition of hunting for wild fowl, which prevents contact between wild and domestic birds; there are no lakes or pond reservoirs near the only poultry farm in the Republic with a small stock, which prevents wild waterfowl from entering and passing through flying passages.

The Single E627K Amino Acid Substitution in PB2 Enhances the Pathogenicity of Wild-Bird-Origin H6N6 Subtype Avian Influenza Virus in Mice

Avian Influenza Viruses (AIVs) are harbored by wild waterfowl as a natural host, and there is a species barrier restricting virus transmission from birds to mammals, including humans. However, it has been reported that, through genetic mutations, AIVs occasionally infect mammals and acquire high pathogenicity. The Amino Acid (aa) substitution of glutamic acid to lysine at position 627 (E627K) in polymerase basic protein 2 (PB2) is one of the wellknown factors underlying mammalian adaptation. Although this substitution was previously observed in mammalian-adapted H5, H7, and H9 AIV subtypes, the impact of this mutation on the mammalian adaptation of other AIV subtypes is not fully verified. Here, we isolated the low pathogenic AIV subtype H6N6 from a wild bird fecal sample in Tokachi Subprefecture, Hokkaido, Japan. We passaged this H6N6 subtype in BALB/c mice four times and acquired the mouse-adapted virus. Whole-genome sequence analysis showed that the adapted virus had only one aa substitution (E627K) in PB2. The adapted virus-inoculated mice tended to show increased weight loss and mortality compared with the original virus-inoculated mice. The viral titer in the lungs of the adapted virus-inoculated mice was significantly higher than that of the original virus-inoculated mice. Additionally, the virus isolated from the lung of the original virus-inoculated mice with serious symptoms harbored the E627K substitution. Our findings indicate the possibility that the PB2 E627K substitution in H6N6 subtype AIV rapidly appears in mammalian hosts and contributes to the enhanced pathogenicity of this virus.

Молекулярно-генетический анализ новых изолятов вируса гриппа птиц субтипа H3N8, выделенных в северных регионах казахстана

Molecular and genetic analysis of viral isolates genes will allow to estimate the spread of influenza A virus in the territory of the Republic of Kazakhstan. Avian influenza control is difficult due to the high variability of the virus genome. The influenza virus circulation among wild waterfowl and the proximity of humans to their habitats is a potential source of threat to public health. Currently, the influenza agent is the subject of detailed study, especially in terms of genetic certification of strains used for the development of diagnostic and prophylactic agents. The aim is studying the molecular genetic properties of new isolates of avian influenza virus subtype H3N8 isolated during the monitoring of avian influenza in the Northern regions of Kazakhstan. Modern methods of molecular biology and genetic engineering were used in the research. In 2018, virological monitoring of wild birds in the Northern regions of Kazakhstan revealed individuals infected with influenza a virus. Pharyngeal swabs of two birds in particular shovelers (Anas clypeata) and teal cracker (Anas querquedula) showed positive reactions for influenza virus type A, subtype H3N8 by PCR. The study obtained isolates of influenza virus A/shoveler/SKO/20/2018 (H3N8) and A/Teal cracker/RMS/45/2018 (H3N8). Results of determining nucleotide sequences and phylogenetic analysis of the new isolates important genes, A/shoveler/SKO/20/2018 (H3N8) and A/Teal cracker/RMS/45/2018 (H3N8) influenza virus in birds are shown. New Kazakhstan isolates of avian influenza virus on the composition of M-gene show the greatest affinity with the strains of avian influenza virus subtype H3N8 isolated in Mongolia in 2018. The molecular genetic analysis results of new isolates of the H3N8 subtype avian influenza virus isolated in the Northern regions of Kazakhstan will be used in the new generation of vaccines production, diagnostics and development of measures to improve the influenza epizootic situation in Kazakhstan. Key words: genetic analysis, avian influenza virus, wild birds, PCR, RNA

Results of scientific expedition to natural biotopes of the Republic of Tyva in 2019 with the purpose of infectious disease monitoring in wild bird populations

The results of the scientific expedition to Tere Khol and Uvs Nuur Lakes in the Republic of Tyva with the purpose of active monitoring of highly dangerous diseases in wild migratory waterfowl and epidemic analysis of these biotope water areas are presented in the paper. The Uvs Nuur Lake is a kind of an indicator for avian influenza introduction to the Russian Federation, because this is the resting and nesting area for many migratory wild birds during the period of mass migrations from Central and South-East Asian countries. In the process of active monitoring the complete autopsy of bird carcasses with description of organs and systems and sampling for laboratory diagnostics were performed. Droppings (pooled samples), parts of internal organs from dead and shot birds, blood (if possible) served as biological and pathological material for testing. While sampling, species were identified using an ornithological guide. The autopsy of dead waterfowl and birds shot for diagnostic purposes demonstrated a high worm burden of nematodes and cestodes. Two samples from European herring gulls were positive for avian influenza type A virus genome and subtype H13N6 was identified in one of them. Avian paramyxovirus serotype 1 (APMV-1), the agent of Newcastle disease, was found in one sample from gulls. The lakes of the Republic of Tyva are the most significant sites for sampling of biological material from wild birds, as the primary detection of highly pathogenic avian influenza virus in this territory is a serious signal of potential further virus spread and a precursor to a probable epizooty. Notwithstanding the absence of AIV very virulent isolate detections in wild bird populations the middle term prognosis for 2020 can be designated as cautious, as the avian influenza epidemic situation is deteriorating globally, especially in the European countries, and the threat of the virus introduction to the Russian territory with migratory birds still exists.

Year‑Round Influenza A Virus Surveillance in Mallards (Anas platyrhynchos) Reveals Genetic Persistence During the Under‑Sampled Spring Season.

Active influenza A virus (IAV) surveillance in wild waterfowl in the United States has revolved around convenience-based sampling methods, resulting in gaps in surveillance during the spring season. We conducted active IAV surveillance in mallards continuously from July 2017 to July 2019 in the coastal marshes of Lake Erie near Port Clinton, Ohio. We aimed to understand ecological and evolutionary dynamics of IAV across multiple seasons, including the under-sampled spring season. We collected 2096 cloacal swabs and estimated a 6.1% (95% confidence interval (CI): 0.050–0.071) prevalence during the study period. Prevalence was lowest during spring (1.0%, 95% CI: 0.004–0.015). Time-stamped phylogenetic analyses revealed local persistence of genetic lineages of multiple gene segments. The PA segment consists of a lineage detected in multiple seasons with a time to most recent common ancestor of 2.48 years (95% highest posterior density: 2.16–2.74). Analysis of the H3 and H6 segments showed close relation between IAVs detected in spring and the following autumn migration. Though the mechanisms behind viral persistence in a single location are not well understood, we provide evidence that viruses can persist across several seasons. Current surveillance methods should be evaluated to ensure they are capturing the breadth of genetic diversity of IAV in waterfowl and prepare for IAV outbreaks in both animals and humans.

Factores ambientales relacionados con la presentación de virus influenza A en aves silvestres

La investigación y el interés por el virus influenza aviar han aumentado considerablemente en las últimas décadas en respuesta a los brotes de influenza aviar de alta patogenicidad en aves de corral y a su potencial zoonótico. Las aves silvestres acuáticas son el principal reservorio del virus en la naturaleza, por lo tanto, la comprensión de la dinámica de infección del virus influenza A (VIA) en estas poblaciones es fundamental para entender su potencial de persistencia en el ambiente y sus posibilidades de transmisión hacia aves domésticas y humanos. Se ha identificado que factores ambientales (como temperatura, precipitaciones, vegetación y características del paisaje, entre otros) pueden tener un importante rol en el mantenimiento y diseminación del virus en las zonas de concentración de aves silvestres. Sin embargo, los estudios que incluyen aspectos ecológicos del virus y que exploran la interacción entre la prevalencia del VIA en aves silvestres y el ambiente, continúan siendo escasos. En esta revisión se resumen los esfuerzos de investigación que se han realizado para identificar a los factores ambientales involucrados en la persistencia y transmisión del VIA en lugares de concentración de aves silvestres y cómo estos factores pueden incidir en la prevalencia del virus en estas poblaciones, generando diferencias en la presentación de la infección entre distintas zonas geográficas.

Pathologic Evaluation of Influenza A H5N8 Infection Outbreaks in Mule Ducks in Bulgaria.

This article outlines pathomorphologic findings of a study involving commercial mule ducks with confirmed influenza A H5N8 infections after a series of outbreaks in Bulgaria. Examinations were carried out after performing necropsy on dead birds from three different age groups (up to 15, 20 to 30, and 40+ days of age) fattened on different farms. Among birds of all ages, gross lesions were present as lesions affecting the heart. Histologically, the myocardium exhibited severe intermyofibrillar edema, moderate to massive hemorrhages, and degenerative changes. All lesions resulted in single or multiple and small to massive myocardial infarctions. Other affected organs included the brain, lungs, liver, spleen, and pancreas. Nonpurulent lymphocytic encephalitis was found postmortem in ducks that had shown prior clinical nervous signs. Among ducks of all ages, a viral antigen in the cardiomyocytes and the epithelium of air capillaries was found through immunohistochemical detection methods. The results of the present study allowed us to conclude that the highly pathogenic avian influenza A H5N8 viral infection may manifest itself as a systemic illness in commercial mule ducks with septicemic lesions, resulting in high morbidity and mortality rates of up to 100%. Pathomorphologic lesions were somewhat different from those previously reported in wild waterfowl.

Variation in True Metabolizable Energy Among Aquatic Vegetation and Ducks

ABSTRACTAvian diet quality is typically measured using true metabolizable energy (TMEN), which is a measure of assimilable energy of food items accounting for innate endogenous losses. Originally developed for use in the poultry industry, TMEN methods have been adapted to determine the value of natural foods consumed by waterfowl to parameterize bioenergetics models for conservation planning. Because there is little knowledge of the variation in TMEN estimates among food items and waterfowl species, we investigated TMEN of 6 common species of submersed aquatic vegetation for mallards (Anas platyrhynchos; i.e., a diet generalist) and gadwall (Mareca strepera; i.e., a diet specialist) in the midwestern United States during autumn 2015–2017. We precision fed and collected excreta from ducks using standard bioassays to estimate TMEN. Mallards had slightly greater TMEN than gadwall, but there was considerable variation in TMEN among vegetation species, duck species, and individuals within each species. True metabolizable energy (±SE; kcal/g[dry]) for mallards was greatest for Canadian waterweed (Elodea canadensis; 1.66 ± 0.26), followed by coontail (Ceratophyllum demersum; 1.51 ± 0.28), southern naiad (Najas guadalupensis; 1.37 ± 0.39), sago pondweed (Stuckenia pectinata; 0.50 ± 0.22), wild celery (Vallisneria americana; 0.05 ± 0.42), and Eurasian watermilfoil (Myriophyllum spicatum; –0.13 ± 0.42). Mean TMEN for gadwall was greatest for Eurasian watermilfoil (0.77 ± 0.32), followed by Canadian waterweed (0.70 ± 0.31), coontail (0.55 ± 0.28), southern naiad (–0.61 ± 0.34), wild celery (–0.98 ± 0.39), and sago pondweed (–1.07 ± 0.33). Generally, TMEN for most vegetation species was less than agricultural grains, but it was similar to ranges reported for seeds of naturally occurring hydrophytic vegetation and aquatic macroinvertebrates. We recommend that conservation planners incorporate species‐specific TMEN estimates in bioenergetics models and that future researchers improve TMEN assays for wild waterfowl following our recommendations. © 2020 The Wildlife Society.

Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines

BackgroundAquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation.MethodsThe virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID50) assay, respectively.ResultsThe isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 105 to 106 FFU / 106 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 104 to 105 FFU / 106 cells. DF-1 produced minimal infectious titre.ConclusionsThis study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.

Combination of multiplex reverse transcription recombinase polymerase amplification assay and capillary electrophoresis provides high sensitive and high-throughput simultaneous detection of avian influenza virus subtypes.

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.

First record of mass wild waterfowl mortality due to Clostridium botulinum in Brazilian semiarid.

In 2008, 270 wild birds from aquatic environments were found dead or debilitated on the banks of smaller lakes that had been formed due to the decrease in the level of the holding lake of the Sobradinho Dam located on the São Francisco River in the Caatinga of Bahia, Brazil. The outbreak occurred months after the dam's partial drainage, with the formation of puddles that accumulated decomposing organic material. Amongst the 270 individuals examined and/or found dead, the majority (50%) of the birds found belonged to the Anatidae family. The debilitated birds presented neurological clinical signs including lack of motor coordination, weakness, grave flaccid paralysis in the legs, wings, neck and eyelids, diarrhea, and dyspnea. Tissue samples of the birds were collected, as were water samples and samples of the substrate of the lakes. Zoonotic arboviroses or heavy metals were not detected. Analyses of liver and digestive tract content samples through bioassay and serum neutralization in mice revealed the presence of type C botulinic toxin in the viscerae samples, and type D in sediment samples. According to our knowledge, this is the first record of an outbreak of botulism in wild birds in natural conditions in Brazil.

Characterization and Phylodynamics of Reassortant H12Nx Viruses in Northern Eurasia.

Wild waterfowl birds are known to be the main reservoir for a variety of avian influenza viruses of different subtypes. Some subtypes, such as H2Nx, H8Nx, H12Nx, and H14Nx, occur relatively rarely in nature. During 10-year long-term surveillance, we isolated five rare H12N5 and one H12N2 viruses in three different distinct geographic regions of Northern Eurasia and studied their characteristics. H12N2 from the Far East region was a double reassortant containing hemagglutinin (HA), non-structural (NS) and nucleoprotein (NP) segments of the American lineage and others from the classical Eurasian avian-like lineage. H12N5 viruses contain Eurasian lineage segments. We suggest a phylogeographical scheme for reassortment events associated with geographical groups of aquatic birds and their migration flyways. The H12N2 virus is of particular interest as this subtype has been found in common teal in the Russian Far East region, and it has a strong relation to North American avian influenza virus lineages, clearly showing that viral exchange of segments between the two continents does occur. Our results emphasize the importance of Avian Influenza Virus (AIV) surveillance in Northern Eurasia for the annual screening of virus characteristics, including the genetic constellation of rare virus subtypes, to understand the evolutionary ecology of AIV.

Domestic ducks play a major role in the maintenance and spread of H5N8 highly pathogenic avian influenza viruses in South Korea.

The H5N8 highly pathogenic avian influenza viruses (HPAIVs) belonging to clade 2.3.4.4 spread from Eastern China to Korea in 2014 and caused outbreaks in domestic poultry until 2016. To understand how H5N8 HPAIVs spread at host species level in Korea during 2014-2016, a Bayesian phylogenetic analysis was used for ancestral state reconstruction and estimation of the host transition dynamics between wild waterfowl, domestic ducks and chickens. Our data support that H5N8 HPAIV most likely transmitted from wild waterfowl to domestic ducks, and then maintained in domestic ducks followed by dispersal of HPAIV from domestic ducks to chickens, suggesting domestic duck population plays a central role in the maintenance, amplification and spread of wild HPAIV to terrestrial poultry in Korea.

A novel reassortant clade 2.3.4.4 highly pathogenic avian influenza H5N6 virus identified in South Korea in 2018

Since 2017, clade 2.3.4.4b H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been detected over a broad geographic region, including Eurasia. These viruses have evolved through reassortment with Eurasian low pathogenic avian influenza viruses (LPAIVs), resulting in multiple genotypes. Here, we sequenced the full-length genome of 15 H5N6 HPAIVs collected from wild birds and poultry farms in South Korea from January to March 2018. A comparative phylogenetic analysis was then conducted. Three distinct genotypes were identified in South Korea during 2017/2018, including a novel reassortant genotype, H214. The novel reassortant H5N6 viruses isolated in this study possessed PB2, PA, and NP gene segments of Eurasian LPAIV on a genetic backbone of the H35-like genotype, which was identified in Korea and the Netherlands during 2017. Bayesian molecular clock analysis suggested that the novel reassortant viruses were generated most likely during the fall migration/wintering season of migratory waterfowl in 2017. Considering the continued emergence and spread of clade 2.3.4.4 HPAIV, enhanced surveillance of wild waterfowl is needed for early detection of HPAIV incursions.

Pan-genome analysis of Riemerella anatipestifer reveals its genomic diversity and acquired antibiotic resistance associated with genomic islands.

Riemerella anatipestifer is a gram-negative bacterium that leads to severe contagious septicemia in ducks, turkeys, chickens, and wild waterfowl. Here, a pan-genome with 32 R. anatipestifer genomes is re-established, and the mathematical model is calculated to evaluate the expansion of R. anatipestifer genomes, which were determined to be open. Average nucleotide identity (ANI) and phylogenetic analysis preliminarily clarify intraspecies variation and distance. Comparative genomic analysis of R. anatipestifer found that horizontal gene transfer events, which provide an expressway for the recruitment of novel functionalities and facilitate genetic diversity in microbial genomes, play a key role in the process of acquiring and transmitting antibiotic-resistance genes in R. anatipestifer. Furthermore, a new antibiotic-resistance gene cluster was identified in the same loci in 14 genomes. The uneven distribution of virulence factors was also confirmed by our results. Our study suggests that the ability to acquire foreign genes (such as antibiotic-resistance genes) increases the adaptability of R. anatipestifer, and the virulence genes with little mobility are highly conserved in R. anatipestifer.

Outbreaks of highly pathogenic avian influenza in zoo birds caused by HA clade 2.3.4.4 H5N6 subtype viruses in Japan in winter 2016.

In late 2016, two zoos, one in northern Japan and the other in central Japan, experienced highly pathogenic avian influenza (HPAI) outbreaks, in which multiple zoo birds were infected with H5N6 subtype HPAI virus (HPAIV). Here, we report an overview of these HPAI outbreaks. HPAIV infections were confirmed by virus isolation in three black swans (Cygnus atratus) and three snowy owls (Bubo scandiacus) kept in the Omoriyama Zoo hospital. At Higashiyama Zoo and Botanical Gardens, following the death of a black swan at a zoo pond, nine waterfowl, including two black swans, four cackling geese (Branta hutchinsii leucopareia), two mallards (Anas platyrhynchos), and a wigeon (Anas penelope), died after HPAIV infection in isolation facilities. Based on the presence of H5-specific antibodies in their sera, two surviving black swans and a surviving mallard at Higashiyama Zoo appeared to have HPAIV infection, although the virus was not isolated. The detectable levels of antibodies (≥10 HI) were maintained for at least 5-9months, as determined by haemagglutinin inhibition test. Isolation of two H5N6 subtype HPAIVs from an open-air pond where affected zoo birds were previously housed at Higashiyama Zoo strongly indicates that wild waterfowl associated with aquatic environments brought the virus to the zoo. The phylogenetic relationships of the 18 isolates indicated direct viral transmission among birds within each zoo. In both zoos, containment of suspected birds in isolation facilities might have allowed the virus spread among birds inside the facility. However, maintaining containment measures and strict sanitation procedures could facilitate successful physical containment and clearance of HPAIV in both zoos.

LIMITED DETECTION OF ANTIBODIES TO CLADE 2.3.4.4 A/GOOSE/GUANGDONG/1/1996 LINEAGE HIGHLY PATHOGENIC H5 AVIAN INFLUENZA VIRUS IN NORTH AMERICAN WATERFOWL

During 2014, highly pathogenic (HP) influenza A viruses (IAVs) of the A/Goose/Guangdong/1/1996 lineage (GsGD-HP-H5), originating from Asia, were detected in domestic poultry and wild birds in Canada and the US. These clade 2.3.4.4 GsGD-HP-H5 viruses included reassortants possessing North American lineage gene segments; were detected in wild birds in the Pacific, Central, and Mississippi flyways; and caused the largest HP IAV outbreak in poultry in US history. To determine if an antibody response indicative of previous infection with clade 2.3.4.4 GsGD-HP-H5 IAV could be detected in North American wild waterfowl sampled before, during, and after the 2014-15 outbreak, sera from 2,793 geese and 3,715 ducks were tested by blocking enzyme-linked immunosorbent assay and hemagglutination inhibition (HI) tests using both clade 2.3.4.4 GsGD-HPH5 and North American lineage low pathogenic (LP) H5 IAV antigens. We detected an antibody response meeting a comparative titer-based criteria (HI titer observed with 2.3.4.4 GsGD-HP-H5 antigens exceeded the titer observed for LP H5 antigen by two or more dilutions) for previous infection with clade 2.3.4.4 GsGD-HP-H5 IAV in only five birds, one Blue-winged Teal (Spatula discors) sampled during the outbreak and three Mallards (Anas platyrhynchos) and one Canada Goose (Branta canadensis) sampled during the post-outbreak period. These serologic results are consistent with the spatiotemporal extent of the outbreak in wild birds in North America during 2014 and 2015 and limited exposure of waterfowl to GsGD-HP-H5 IAV, particularly in the central and eastern US.