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In 2011, avian influenza surveillance at the Bangladesh live bird markets (LBMs) showed complete replacement of the highly pathogenic avian influenza (HPAI) H5N1 virus of clade 2.2.2 (Qinghai-like H5N1 lineage) by the HPAI H5N1 clade 2.3.2.1. This clade, which continues to circulate in Bangladesh and neighboring countries, is an intra-and interclade reassortant; its HA, polymerase basic 1 (PB1), polymerase (PA), and nonstructural (NS) genes come from subclade 2.3.2.1a; the polymerase basic 2 (PB2) comes from subclade 2.3.2.1c; and the NA, nucleocapsid protein (NP), and matrix (M) gene from clade 2.3.4.2. The H9N2 influenza viruses cocirculating in the Bangladesh LBMs are also reassortants, possessing five genes (NS, M, NP, PA, and PB1) from an HPAI H7N3 virus previously isolated in Pakistan. Despite frequent coinfection of chickens and ducks, reassortment between these H5N1 and H9N2 viruses has been rare. However, all such reassortants detected in 2011 through 2013 have carried seven genes from the local HPAI H5N1 lineage and the PB1 gene from the Bangladeshi H9N2 clade G1 Mideast, itself derived from HPAI H7N3 virus. Although the live birds we sampled in Bangladesh showed no clinical signs of morbidity, the emergence of this reassortant HPAI H5N1 lineage further complicates endemic circulation of H5N1 viruses in Bangladesh, posing a threat to both poultry and humans.

Since the first H7N9 human case in Shanghai, February 19, 2013, the emerging avian-origin H7N9 influenza A virus has become an epizootic virus in China, posing a potential pandemic threat to public health. From April 2 to April 28, 2013, some 422 oral-pharyngeal and cloacal swabs were collected from birds and environmental surfaces at five live poultry markets (LPMs) and 13 backyard poultry farms (BPFs) across three cities, Wuxi, Suzhou, and Nanjing, in the Yangtze Delta region. In total 22 isolates were recovered, and six were subtyped as H7N9, nine as H9N2, four as H7N9/H9N2, and three unsubtyped influenza A viruses. Genomic sequences showed that the HA and NA genes of the H7N9 viruses were similar to those of the H7N9 human isolates, as well as other avian-origin H7N9 isolates in the region, but the PB1, PA, NP, and MP genes of the sequenced viruses were more diverse. Among the four H7N9/H9N2 mixed infections, three were from LPM, whereas the other one was from the ducks at one BPF, which were H7N9 negative in serologic analyses. A survey of the bird trading records of the LPMs and BPFs indicates that trading was a likely route for virus transmission across these regions. Our results suggested that better biosecurity and more effective vaccination should be implemented in backyard farms, in addition to biosecurity management in LPMs.

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.

Risk management decisions associated with live poultry movement during a highly pathogenic avian influenza (HPAI) outbreak should be carefully considered. Live turkey movements may pose a risk for disease spread. On the other hand, interruptions in scheduled movements can disrupt business continuity. The Secure Turkey Supply (STS) Plan was developed through an industry-government-academic collaboration to address business continuity concerns that might arise during a HPAI outbreak. STS stakeholders proposed outbreak response measure options that were evaluated through risk assessment. The developed approach relies on 1) diagnostic testing of two pooled samples of swabs taken from dead turkeys immediately before movement via the influenza A matrix gene real-time reverse transcriptase polymerase chain reaction (rRT-PCR) test; 2) enhanced biosecurity measures in combination with a premovement isolation period (PMIP), restricting movement onto the premises for a few days before movement to slaughter; and 3) incorporation of a distance factor from known infected flocks such that exposure via local area spread is unlikely. Daily exposure likelihood estimates from spatial kernels from past HPAI outbreaks were coupled with simulation models of disease spread and active surveillance to evaluate active surveillance protocol options that differ with respect to the number of swabs per pooled sample and the timing of the tests in relation to movement. Simulation model results indicate that active surveillance testing, in combination with strict biosecurity, substantially increased HPAI virus detection probability. When distance from a known infected flock was considered, the overall combined likelihood of moving an infected, undetected turkey flock to slaughter was predicted to be lower at 3 and 5 km. The analysis of different active surveillance protocol options is designed to incorporate flexibility into HPAI emergency response plans.

Since 2005, H5N1 highly pathogenic avian influenza virus (HPAIV) has severely impacted the economy and public health in the Middle East (ME) with Egypt as the most affected country. Understanding the high-risk areas and spatiotemporal distribution of the H5N1 HPAIV in poultry is prerequisite for establishing risk-based surveillance activities at a regional level in the ME. Here, we aimed to predict the geographic range of H5N1 HPAIV outbreaks in poultry in the ME using a set of environmental variables and to investigate the spatiotemporal clustering of outbreaks in the region. Data from the ME for the period 2005–14 were analyzed using maximum entropy ecological niche modeling and the permutation model of the scan statistics. The predicted range of high-risk areas (P > 0.60) for H5N1 HPAIV in poultry included parts of the ME northeastern countries, whereas the Egyptian Nile delta and valley were estimated to be the most suitable locations for occurrence of H5N1 HPAIV outbreaks. The most important environmental predictor that contributed to risk for H5N1 HPAIV was the precipitation of the warmest quarter (47.2%), followed by the type of global livestock production system (18.1%). Most significant spatiotemporal clusters (P < 0.001) were detected in Egypt, Turkey, Kuwait, Saudi Arabia, and Sudan. Results suggest that more information related to poultry holding demographics is needed to further improve prediction of risk for H5N1 HPAIV in the ME, whereas the methodology presented here may be useful in guiding the design of surveillance programs and in identifying areas in which underreporting may have occurred.

The largest epidemic of avian influenza (AI) in history attacked poultry and wild birds throughout Taiwan starting January 6, 2015. This study analyzed surveillance results, epidemiologic characteristics, and viral sequences by using government-released information, with the intention to provide recommendations to minimize future pandemic influenza. The H5 clade 2.3.4.4 highly pathogenic AI viruses (HPAIVs) had not been detected in Taiwan before 2015. During this epidemic, four types of etiologic agents were identified: the three novel subtypes H5N2, H5N8, and H5N3 clade 2.3.4.4 HPAIVs and one endemic chicken H5N2 subtype (Mexican-like lineage) of low pathogenic AI viruses. Cocirculation of mixed subtypes also occurred, with H5N2 clade 2.3.4.4 HPAIVs accompanied by the H5N8 and H5N3 subtypes or old H5N2 viruses in the same farm. More than 90% of domestic geese died from this AI epidemic; geese were affected the most at the early outbreaks. The epidemic peaked in mid-January for all three novel H5 subtypes. Spatial epidemiology found that most affected areas were located in southwestern coastal areas. In terrestrial poultry (mostly chickens), different geographic distributions of AI virus subtypes were detected, with hot spots of H5N2 clade 2.3.4.4 vs. past-endemic old H5N2 viruses in Changhwa (P = 0.03) and Yunlin (P = 0.007) counties, respectively, of central Taiwan. Phylogenetic and sequence analyses of all the early 10 Taiwan H5 clade 2.3.4.4 isolates covering the three subtypes showed that they were very different from the HA of the past local H5 viruses from domestic ducks (75%–80%) and chickens (70%–75%). However, they had the highest sequence identity percentages (99.53%–100%), with the HA of A/crane/Kagoshima/KU13/2014(H5N8) isolated on December 7, 2014, in Japan being higher than those of recent American and Korean H5 HPAIVs [A/Northern pintail/Washington/40964/2014 (H5N2) and A/gyrfalcon/Washington/41088-6/2014 (H5N8): 99.02%–99.54% and A/Baikal teal/Korea/Donglim3/2014 (H5N8): 98.61%–99.08%], implying a likely common ancestor of these H5 clade 2.3.4.4 viruses. The multiple subtypes of H5 clade 2.3.4.4 HPAIVs imply high viral reassortment. We recommend establishing an integrated surveillance system, involving clinical, virologic, and serologic surveillance in poultry and wild birds, swine and other mammals prevalent on multiple-animal mixed-type traditional farms, and high-risk human populations, as a crucially important step to minimize future pandemic influenza.

Since the first outbreak of low pathogenic avian influenza (LPAI) in 1996, outbreaks of LPAI have become more common in Korea, leading to the development of a nationwide mass vaccination program in 2007. In the case of highly pathogenic avian influenza (HPAI), four outbreaks took place in 2003–04, 2006–07, 2008, and 2010–11; a fifth outbreak began in 2014 and was ongoing at the time of this writing. The length of the four previous outbreaks varied, ranging from 42 days (2008) to 139 days (2010–11). The number of cases reported by farmers that were subsequently confirmed as HPAI also varied, from seven cases in 2006–07 to 53 in 2010–11. The number of farms affected by the outbreaks varied, from a low of 286 (2006–07) with depopulation of 6,473,000 birds, to a high of 1500 farms (2008) with depopulation of 10,200,000 birds. Government compensation for bird depopulation ranged from $253 million to $683 million in the five outbreaks. Despite the damage caused by the five HPAI outbreaks, efficient control strategies have yet to be established. Meanwhile, the situation in the field worsens. Analysis of the five HPAI outbreaks revealed horizontal farm-to-farm transmission as the main factor effecting major economic losses. However, horizontal transmission could not be efficiently prevented because of insufficient transparency within the poultry industry, especially within the duck industry, which is reluctant to report suspicious cases early. Moreover, the experiences and expertise garnered in previous outbreaks has yet to be effectively applied to the management of new outbreaks. Considering the magnitude of the economic damage caused by avian influenza and the increasing likelihood of its endemicity, careful and quantitative analysis of outbreaks and the establishment of control policies are urgently needed.

The risk of highly pathogenic avian influenza (HPAI) virus introduction via import of live poultry results from the probability that infected birds are exported from apparently HPAI-free areas during the silent phase of the epidemic, i.e., the period between an incursion of the virus into a susceptible population and a report on the outbreak by an exporting country. In our study we adapted a stochastic model, previously published in 2010 by Sánchez-Vizcaíno et al., with our own modifications in which the probability of HPAI introduction was assessed as the sum of the probabilities of entry of at least one infected bird from each susceptible species exported from each country into each Polish region (county). The mean annual probability of HPAI introduction into Poland via legal trade of live poultry was very low (3.07 × 10⁻³, which corresponds to 1 outbreak every 326 yr). The highest risk was associated with the import of turkeys (62%) and chickens (33%). The exporting countries that contributed the most to the overall risk were Italy (31%), the Netherlands (24%), and the Czech Republic (17%). The risk was not evenly distributed across the country and it seemed higher in western, north-central, and eastern Poland while several counties of the north-west, central, or south-east parts of the country were at negligible risk. The applied model provides quantitative evidence that the risk of HPAI introduction through legal trade of poultry does not play a major role and that other paths, such as wild birds migrations or illegal trade, should be considered as the most-likely routes along which the virus can be introduced.

To help guide surveillance and control of highly pathogenic avian influenza subtype H5N1 (H5N1-HPAI), the Food and Agriculture Organization of the United Nations in 2004 devised a poultry farm classification system based on a combination of production and biosecurity practices. Four “Sectors” were defined, and this scheme has been widely adopted within Indonesia to guide national surveillance and control strategies. Nevertheless, little detailed research into the robustness of this classification system has been conducted, particularly as it relates to independent, small to medium-sized commercial poultry farms (Sector 3). Through an analysis of questionnaire data collected as part of a survey of layer farms in western and central Java, all of which were classified as Sector 3 by local veterinarians, we provide benchmark data on what defines this sector. A multivariate analysis of the dataset, using hierarchical cluster analysis, identified three groupings of the farms, which were defined by a combination of production-and biosecurity-related variables, particularly those related to farm size and (the lack of) washing and disinfection practices. Nevertheless, the relationship between production-related variables and positive biosecurity practices was poor, and larger farms did not have an overall higher total biosecurity score than small or medium-sized ones. Further research is required to define the properties of poultry farms in Indonesia that are most closely related to effective biosecurity and the prevention of H5N1-HPAI.

Maternally derived antibodies (MDA) are known to provide early protection from disease but also to interfere with vaccination efficacy of young chicks. This interference phenomenon is well described in the literature for viral diseases such as infectious bursal disease, Newcastle disease (ND), and avian influenza (AI). The goal of this work was to investigate the impact of H5 MDA and/or ND virus (NDV) MDA on the vaccine efficacy of a recombinant NDV-H5–vectored vaccine (rNDV-H5) against two antigenically divergent highly pathogenic AI (HPAI) H5N1 challenges. In chickens with both H5 and NDV MDA, a strong interference was observed with reduced clinical protection when compared to vaccinated specific-pathogen-free (SPF) chickens. In contrast, in chickens from commercial suppliers with NDV MDA only, a beneficial impact on the vaccine efficacy was observed with full protection and reduced viral excretion in comparison with rNDV-H5–vaccinated SPF chickens. To distinguish between the respective effects of the H5 and NDV MDA, an SPF model where passive immunity had been artificially induced by inoculations of H5 and NDV hyperimmunized polysera, respectively, was used. In the presence of H5 artificial MDA, a strong interference reflected by a reduction in vaccine protection was demonstrated whereas no interference and even an enhancing protective effect was confirmed in presence of NDV MDA. The present work suggests that H5 and NDV MDA interact differently with the rNDV-H5 vaccine with different consequences on its efficacy, the mechanisms of which require further investigations.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10⁵ and 10⁶ 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Waterfowl play a key role in the epidemiology of the H5N1 subtype of highly pathogenic avian influenza (HPAI) virus; therefore, efficient immunization of domesticated ducks and geese to maximize the impact of other control measures is of great importance. A recombinant (r)HVT-AI, expressing the HA gene of a clade 2.2 H5N1 HPAI strain had been developed and proved to be efficient against different clades of H5N1 HPAI virus in chickens after a single vaccination at 1 day old and could provide long-term immunity. We investigated whether rHVT-AI applied at 1 day old is able to replicate in different species and crossbreeds of ducks and in geese with the aim of collecting data on the possible application of rHVT-AI vaccine in different species of waterfowl for the control of H5N1 HPAI. We tested the possible differences among different waterfowl species, i.e., between geese (Anser anser, domesticated greylag goose), Muscovy ducks (Cairina moschata forma domestica), Pekin ducks (Anas platyrhynchos forma domestica), and mule ducks (Muscovy duck × Pekin duck), in their susceptibility to support the replication of rHVT-AI. Vaccine virus replication was followed by real-time PCR in spleen, bursa, and feather tip samples. Humoral immune response to vaccination was tested using the hemagglutination inhibition (HI) test and H5-specific commercial ELISA. Significant differences among the different waterfowl species regarding the rate of rHVT-AI replication was detected that were not reflected by the same difference in the immune response to vaccination. Replication of the rHVT-AI vaccine was very limited in Pekin ducks, somewhat better in mule ducks, and the vaccine virus was replicating significantly better in Muscovy ducks and geese, reaching 100% detectability at certain time points after administration at 1 day old. Results indicated that the vaccine virus could establish different levels of persistent infection in these species of waterfowl. No humoral immune response could be detected either by HI test or ELISA during the tested postvaccination period (5 wk).

Vaccination is frequently used as a control method for the H9 subtype of low pathogenicity avian influenza virus (AIV), which is widespread in Asia and the Middle East. One of the most important factors for selecting an effective vaccine strain is the antigenic match between the hemagglutinin protein of the vaccine and the strain circulating in the field. To demonstrate the antigenic relationships among H9 AIVs, with a focus on Israeli H9 isolates, antigenic cartography was used to develop a map of H9 AIVs. Based on their antigenic diversity, three isolates from Israel were selected for vaccination-challenge studies: 1) the current vaccine virus, A/chicken/Israel/215/2007 H9N2 (Ck/215); 2) A/chicken/Israel/1163/2011 H9N2 (Ck/1163); and 3) A/ostrich/Israel/1436/2003 (Os/1436). A 50% infective dose (ID₅₀) model was used to determine the effect of the vaccines on susceptibility to infection by using a standardized dose of vaccine. Sera collected immediately prior to challenge showed that Ck/215 was the most immunogenic, followed by Ck/1163 and Os/1436. A significant difference in ID₅₀ was only observed with Ck/215 homologous challenge, where the ID₅₀ was increased by 2 log ₁₀ per bird. The ID₅₀ for Ck/1163 was the same, regardless of vaccine, including sham vaccination. The ID₅₀ for Os/1436 was above the maximum possible dose and therefore could not be established.

Low pathogenic avian influenza H9N2 virus infection has been an important risk to the Egyptian poultry industry since 2011. Economic losses have occurred from early infection and co-infection with other pathogens. Therefore, H9N2 vaccination of broiler chicks as young as 7 days old was recommended. The current inactivated H9N2 vaccines (0.5 ml/bird) administered at a reduced dose (0.25 ml/bird) do not guarantee the delivery of an effective dose for broilers. In this study, the efficacy of the reduced-dose volume (0.3 ml/bird), compared with the regular vaccine dose (0.5 ml/bird) of inactivated H9N2 vaccines using two different commercially available adjuvants, was investigated. The vaccines were prepared from the local H9N2 virus (Ck/EG/114940v/NLQP/11) using the same antigen content: 300 hemagglutinating units. Postvaccination (PV) immune response was monitored using the hemagglutination inhibition test. At 4 wk PV, both vaccinated groups were challenged using the homologous H9N2 strain at a 50% egg infective dose (EID₅₀) of 10⁶ EID₅₀/bird via the intranasal route. Clinical signs, mortality, and virus shedding in oropharyngeal swabs were monitored at 2, 4, 6, and 10 days postchallenge (DPC). The reduced-dose volume of vaccine induced a significantly faster and higher immune response than the regular volume of vaccine at 2 and 3 wk PV. No significant difference in virus shedding between the two vaccine formulas was found (P ≥ 0.05), and both vaccines were able to stop virus shedding by 6 DPC. The reduced-dose volume of vaccine using a suitable oil adjuvant and proper antigen content can be used effectively for early immunization of broiler chicks.

Vaccination against H5N1 highly pathogenic avian influenza (AI) virus (HPAIV) is one of the possible complementary means available for affected countries to control AI when the disease has become, or with a high risk of becoming, endemic. Efficacy of the vaccination against AI relies essentially, but not exclusively, on the capacity of the vaccine to induce immunity against the targeted virus (which is prone to undergo antigenic variations), as well as its capacity to overcome interference with maternal immunity transmitted by immunized breeding hens to their progeny. This property of the vaccine is a prerequisite for its administration at the hatchery, which assures higher and more reliable vaccine coverage of the populations than vaccination at the farm. A recombinant vector vaccine (Vectormune® AI), based on turkey herpesvirus expressing the hemagglutinin gene of an H5N1 HPAIV as an insert, has been used in several experiments conducted in different research laboratories, as well as in controlled field trials. The results have demonstrated a high degree of homologous and cross protection against different genetic clades of the H5N1 HPAIV. Furthermore, vaccine-induced immunity was not impaired by the presence of passive immunity, but on the contrary, cumulated with it for improved early protection. The demonstrated levels of protection against the different challenge viruses exhibited variations in terms of postchallenge mortality, as well as challenge virus shedding. The data presented here highlight the advantages of this vaccine as a useful and reliable tool to complement biosecurity and sanitary policies for better controlling the disease due to HPAIV of H5 subtypes, when the vaccination is applied as a control measure.

An inactivated H5N1 avian influenza (AI) vaccine (Re-6) that bears the HA and NA genes from a clade 2.3.2.1 H5N1 virus, A/duck/Guangdong/S1322/10 (DK/GD/S1322/10), has been used in domestic poultry in China and other Southeast Asian countries to control clade 2.3.2.1 H5N1viruses since 2012. The efficacy of this vaccine against H5N1 viruses isolated in recent years has not been reported. In this study, we evaluated the protection efficacy of the Re-6 vaccine in chickens against challenge with four clade 2.3.2.1 H5N1 viruses, one clade 2.3.4.4 H5N1 virus, and one clade 7.2 H5N1 virus; these viruses were isolated in mainland China, Hong Kong, and the Democratic People's Republic of Korea between 2011 and 2015. The vaccinated chickens were completely protected (no disease signs, virus shedding, or death) from the challenge with the four clade 2.3.2.1 H5N1 viruses. In the clade 7.2 virus–challenged group, all of the vaccinated chickens remained healthy and survived for the entire 2-wk observation period; virus shedding was only detected from 1 of 10 chickens on day 3 postchallenge. In the clade 2.3.4.4 virus–challenged group, 8 of the 10 vaccinated chickens remained healthy and survived the 2-wk observation period; however, virus shedding was detected from 8 of 10 chickens on day 5 postchallenge. These results indicate that the Re-6 vaccine provides solid protection against clade 2.3.2.1, good protection against clade 7.2, and poor protection against clade 2.3.4.4.

Influenza A virus (IAV) surveillance in migratory waterfowl in the United States has primarily occurred during late summer and the autumn southern migration. Data concerning the presence and ecology of IAVs in waterfowl during winter and spring seasons in the U.S. northern latitudes have been limited, mainly due to limited access to waterfowl for sampling. The southwestern Lake Erie Basin is an important stopover site for waterfowl during migration periods, and over the past 28 years, 8.72% of waterfowl sampled in this geographic location have been positive for IAV recovery during summer and autumn (June–December). To gain a better understanding of influenza A viral dynamics in waterfowl populations during winter and spring migration (February through April), cloacal swabs were collected from overwintering and spring-migrating waterfowl in Ohio and Michigan in 2006, 2007, 2013, and 2014. A total of 740 cloacal swabs were collected and tested using virus isolation in embryonating chicken eggs, resulting in the recovery of 33 (4.5%) IAV isolates. The influenza A isolates were recovered from eight waterfowl species in the order Anseriformes. Antigenically, the IAV isolates represent 15 distinct hemagglutinin (HA) and neuraminidase (NA) combinations, with seven (21%) of the isolates reported as mixed infections based on antigenic HA subtyping, NA subtyping, or both. This effort demonstrates the presence of antigenically diverse IAV in waterfowl during overwintering and spring migration at northern latitudes in the United States, thereby contributing to the understanding of the maintenance of diversity among waterfowl-origin IAVs.

The immunity profile against H5N1 highly pathogenic avian influenza (HPAI) in the commercial poultry value chain network in Egypt was modeled with the use of different vaccination scenarios. The model estimated the vaccination coverage, the protective seroconversion level, and the duration of immunity for each node of the network and vaccination scenario. Partial budget analysis was used to compare the benefit-cost of the different vaccination scenarios. The model predicted that targeting day-old chick avian influenza (AI) vaccination in industrial and large hatcheries would increase immunity levels in the overall poultry population in Egypt and especially in small commercial poultry farms (from <30% to >60%). This strategy was shown to be more efficient than the current strategy of using inactivated vaccines. Improving HPAI control in the commercial poultry sector in Egypt would have a positive impact to improve disease control.

The Goose/Guangdong-lineage H5 viruses have evolved into diverse clades and subclades based on their hemagglutinin (HA) gene during their circulation in wild birds and poultry. Since late 2013, the clade 2.3.4.4 viruses have become widespread in poultry and wild bird populations around the world. Different subtypes of the clade 2.3.4.4 H5 viruses, including H5N1, H5N2, H5N6, and H5N8, have caused vast disease outbreaks in poultry in Asia, Europe, and North America. In this study, we developed a new H5N1 inactivated vaccine by using a seed virus (designated as Re-8) that contains the HA and NA genes from a clade 2.3.4.4 virus, A/chicken/Guizhou/4/13(H5N1) (CK/GZ/4/13), and its six internal genes from the high-growth A/Puerto Rico/8/1934 (H1N1) virus. We evaluated the protective efficacy of this vaccine in chickens challenged with one H5N1 clade 2.3.2.1b virus and six different subtypes of clade 2.3.4.4 viruses, including H5N1, H5N2, H5N6, and H5N8 strains. In the clade 2.3.2.1b virus DK/GX/S1017/13–challenged groups, half of the vaccinated chickens shed virus through the oropharynx and two birds (20%) died during the observation period. All of the control chickens shed viruses and died within 6 days of infection with challenge virus. All of the vaccinated chickens remained healthy following challenge with the six clade 2.3.4.4 viruses, and virus shedding was not detected from any of these birds; however, all of the control birds shed viruses and died within 4 days of challenge with the clade 2.3.4.4 viruses. Our results indicate that the Re-8 vaccine provides protection against different subtypes of clade 2.3.4.4 H5 viruses.

Since the first report of low pathogenic avian influenza (LPAI) H9N2 virus in Egypt in 2011, the Egyptian poultry industry has suffered from unexpected economic losses as a result of the wide spread of LPAI H9N2. Hence, inactivated H9N2 vaccines have been included in the vaccination programs of different poultry production sectors. The optimal antigen content of avian influenza virus vaccines is essential to reach protective antibody titers. In this study, the correlation between antigen content (based on hemagglutinating units [HAU]) and postvaccination (PV) antibody response of H9N2 inactivated vaccine was studied. Five different vaccine antigen loads (128, 200, 250, 300, and 350 HAU formulas/dose) were investigated in commercial broiler and specific-pathogen-free (SPF) chickens. Vaccine safety and PV antibody responses were monitored. At the fourth week PV only SPF vaccinated groups (128, 200, 250, and 300 HAU/dose) were challenged using LPAI H9N2 (A/Ck/EG/114940v/NLQP/11) virus with 10⁶ EID₅₀/bird. Oropharyngeal swabs were used to monitor virus shedding at 2, 4, 6, and 10 days postchallenge. Results showed that all vaccine formulations were well tolerated, and the highest antibody titers were observed in birds vaccinated with higher HAU. Vaccines containing 128 and 200 HAU/dose did not induce the required protective HI titers by 3 wk PV. Meanwhile, the challenge experiment in SPF chickens showed that 250 and 300 HAU vaccine doses were required to reduce the level and duration of virus shedding. Study results thus suggest that inactivated H9N2 vaccines containing at least 250 HAU/dose will induce the optimal protective titers and minimize virus shedding in SPF chickens.

To evaluate the effect of selection for high laying performance on the capacity to respond to an infection with avian influenza virus (AIV), four different chicken lines were tested: A white layer and a brown layer breed originating from a commercial breeding program, and a white layer and a brown layer line maintained as a conservation flock for decades without any selection. The different chicken breeds were infected with AIV of different pathotypes (low pathogenic to high pathogenic) to evaluate and compare their immunological competence. Morbidity and mortality rates, as well as viral shedding, were investigated as parameters of virus infection. Immune cells in blood samples collected after different time points following inoculation were identified. In general, the chickens of the two phylogenetically related brown layer lines (irrespective of the performance type) were more resistant to infection with the selected AIVs, reflected by a lower mortality rate (low virulent AIV) or a prolonged length of survival before succumbing to the disease (highly virulent AIV). Corresponding to these results, CD8-positive cell counts were reduced in both white layer lines. This observation was also confirmed in an in vivo allogenic transfer experiment, in which brown layers eliminated the transferred cells in a shorter time period. In conclusion, our results do not support the theory of reduced immunological competence of high-performance layer breeds, at least against AIV infection. Instead, brown layer strains had a faster CD8-positive immune cell response after viral or allogenic stimulus than the phylogenetically distant white layers, resulting in better resistance against AIV infection.

Little is known on the interactions between avian influenza virus (AIV) and Newcastle disease virus (NDV) when coinfecting the same poultry host. In a previous study we found that infection of chickens with a mesogenic strain of NDV (mNDV) can reduce highly pathogenic AIV (HPAIV) replication, clinical disease, and mortality. This interaction depended on the titer of the viruses used and the timing of the infections. To further explore the effect of mNDV infectious dose in protecting chickens against HPAIV infection, 2-wk-old birds were inoculated with different doses of mNDV (10⁴, 10⁶, or 10⁷ 50% embryo infective dose [EID₅₀]) 3 days before inoculation with a HPAIV (10⁵ or 10⁶ EID₅₀). Although birds coinfected with the higher mNDV doses (10⁶ or 10⁷) survived for longer than birds inoculated only with HPAIV (10⁵), we did not observe the same protection with the lower dose of mNDV (10⁴) or when given the higher dose of HPAIV (10⁶), indicating that the relation between the titer of the two coinfecting viruses is determinant in the outcome. In a similar experiment, a higher number of 4-wk-old birds survived, and for longer, even when given higher HPAIV doses (10^6.3 and 10^7.3 EID₅₀). In addition, we also examined the duration of protection provided by mNDV (10⁷ EID₅₀) on a HPAIV infection. Five-week-old chickens were inoculated with mNDV followed by inoculation with 10⁶ EID₅₀ of an HPAIV given at 2, 4, 6, or 9 days after the mNDV. HPAIV replication was affected and an increase in survival was found in all coinfected groups when compared to the HPAIV single-inoculated group, but the mortality in coinfected groups was high. In conclusion, previous inoculation with mNDV can affect HPAIV replication in chickens for at least 9 days, but this viral interference is titer dependent.

Guineafowl of different ages were inoculated intravenously with a H6N2 wild waterfowl–origin low pathogenicity avian influenza virus (LPAIV). No clinical disease was observed. The infected birds had atrophy of the spleen, thymus, and cloacal bursa when compared with the noninfected control groups. The central and peripheral lymphoid tissues presented either lymphoproliferative or degenerative lesions that increased in intensity from 14 to 21 days postinoculation (DPI). Lymphoid depletion was present in the bursa, thymic lobes, and spleen T-dependent zone. In contrast, lymphoid proliferation was observed in liver, pancreas, and spleen B-dependent zone. Bronchus associated lymphoid tissue hyperplasia was observed in the lungs of the birds at 14 and 21 DPI. The virus was detected by virus isolation and reverse transcription PCR from both oropharyngeal and cloacal swabs with higher isolation rates from the latter. Most birds from the LPAIV inoculated groups shed virus up to 7 DPI. The virus was infrequently isolated from lung, kidney, liver, bursa, or spleen of infected birds until 14 DPI and from two samples (kidney and spleen, 1-yr-old birds) at 21 DPI. These data indicate that the wild bird–origin LPAIV used in this study caused pantropic infection in guineafowl when inoculated intravenously.

The extensive nature of ostrich farming production systems bears the continual risk of point introductions of avian influenza virus (AIV) from wild birds, but immune status, management, population density, and other causes of stress in ostriches are the ultimate determinants of the severity of the disease in this species. From January 2012 to December 2014, more than 70 incidents of AIV in ostriches were reported in South Africa. These included H5N2 and H7N1 low pathogenicity avian influenza (LPAI) in 2012, H7N7 LPAI in 2013, and H5N2 LPAI in 2014. To resolve the molecular epidemiology in South Africa, the entire South African viral repository from ostriches and wild birds from 1991 to 2013 (n = 42) was resequenced by next-generation sequencing technology to obtain complete genomes for comparison. The phylogenetic results were supplemented with serological data for ostriches from 2012 to 2014, and AIV-detection data from surveillance of 17 762 wild birds sampled over the same period. Phylogenetic evidence pointed to wild birds, e.g., African sacred ibis (Threskiornis aethiopicus), in the dissemination of H7N1 LPAI to ostriches in the Eastern and Western Cape provinces during 2012, in separate incidents that could not be epidemiologically linked. In contrast, the H7N7 LPAI outbreaks in 2013 that were restricted to the Western Cape Province appear to have originated from a single-point introduction from wild birds. Two H5N2 viruses detected in ostriches in 2012 were determined to be LPAI strains that were new introductions, epidemiologically unrelated to the 2011 highly pathogenic avian influenza (HPAI) outbreaks. Seventeen of 27 (63%) ostrich viruses contained the polymerase basic 2 (PB2) E627K marker, and 2 of the ostrich isolates that lacked E627K contained the compensatory Q591K mutation, whereas a third virus had a D701N mutation. Ostriches maintain a low upper- to midtracheal temperature as part of their adaptive physiology for desert survival, which may explain the selection in ratites for E627K or its compensatory mutations—markers that facilitate AIV replication at lower temperatures. An AIV prevalence of 5.6% in wild birds was recorded between 2012 and 2014, considerably higher than AIV prevalence for the southern African region of 2.5%–3.6% reported in the period 2007–2009. Serological prevalence of AI in ostriches was 3.7%, 3.6%, and 6.1% for 2012, 2013, and 2014, respectively. An annual seasonal dip in incidence was evident around March/April (late summer/autumn), with peaks around July/August (mid to late winter). H5, H6, H7, and unidentified serotypes were present at varying levels over the 3-yr period.

Several ecologic factors have been proposed to describe the mechanisms whereby host ecology and the environment influence the transmission of avian influenza viruses (AIVs) in wild birds, including bird’s foraging behavior, migratory pattern, seasonal congregation, the rate of recruitment of juvenile birds, and abiotic factors. However, these ecologic factors are derived from studies that have been conducted in temperate or boreal regions of the Northern Hemisphere. These factors cannot be directly translated to tropical regions, where differences in host ecology and seasonality may produce different ecologic interactions between wild birds and AIV. An extensive dataset of AIV detection in wildfowl and shorebirds sampled across tropical Africa was used to analyze how the distinctive ecologic features of Afrotropical regions may influence the dynamics of AIV transmission in wild birds. The strong seasonality of rainfall and surface area of wetlands allows testing of how the seasonality of wildfowl ecology (reproduction phenology and congregation) is related to AIV seasonal dynamics. The diversity of the African wildfowl community provides the opportunity to investigate the respective influence of migratory behavior, foraging behavior, and phylogeny on species variation in infection rate. Large aggregation sites of shorebirds in Africa allow testing for the existence of AIV infection hot spots. We found that the processes whereby host ecology influence AIV transmission in wild birds in the Afrotropical context operate through ecologic factors (seasonal drying of wetlands and extended and nonsynchronized breeding periods) that are different than the one described in temperate regions, hence, resulting in different patterns of AIV infection dynamics.

In late February 2014, unusually high numbers of wild thick-billed murres (Uria lomvia) were found dead on the coast of South Greenland. To investigate the cause of death, 45 birds were submitted for laboratory examination in Denmark. Avian influenza viruses (AIVs) with subtypes H11N2 and low pathogenic H5N1 were detected in some of the birds. Characterization of the viruses by full genome sequencing revealed that all the gene segments belonged to the North American lineage of AIVs. The seemingly sparse and mixed subtype occurrence of low pathogenic AIVs in these birds, in addition to the emaciated appearance of the birds, suggests that the murre die-off was due to malnutrition as a result of sparse food availability or inclement weather. Here we present the first characterization of AIVs isolated in Greenland, and our results support the idea that wild birds in Greenland may be involved in the movement of AIV between North America and Europe.

In this study, Global Positioning System satellite transmitters were attached to three mallards (Anas platyrhynchos) wintering in South Korea to track their migration routes, stopover sites, breeding sites, and migration patterns. We successfully tracked only one mallard (no. 108917) from November 15, 2011, to November 29, 2013, and determined separate migration routes in two cases of spring migration and one case of fall migration. The mallard repeatedly migrated to the same final destination, even though the travel path varied. We identified six stopover sites: Hunhe River, Liaohe River, Yinma River, Yalu River, Songjeon Bay, and Dahuofang Reservoir in China and South Korea. The wintering sites of two migration cases were discovered to be identical (Gokgyo River in Asan, South Korea). The terminal sites, which were presumed to be breeding grounds, were the same in both cases (Hinggan League in Inner Mongolia Autonomous Region, China). On the basis of the migration routes identified in this study, we suggest that future efforts to control highly pathogenic avian influenza (HPAI) should not only include avian influenza surveillance but also implement flyway-based strategies, with regard to all countries affected by potential HPAI outbreaks.

This work presents the results of studies aimed at assessing the median and maximum distances covered by wild mallards (Anas platyrhynchos; n = 38), hypothetically infected with the high pathogenicity avian influenza virus (HPAIV) during spring migrations, using GPS-GSM tracking and published data on the susceptibility to HPAIV infection and duration of shedding. The model was based on the assumptions that the birds shed virus in the absence of clinical signs during infectious periods (IP) that were assumed to last 1 day (IP₁), 4 days (IP₄), and 8 days (IP₈) and that each day of migration is a hypothetical day of the onset of IP. Using the haversine formula over a sliding timeframe corresponding to each IP, distances were estimated for each duck that undertook migration and then the maximum distance (D_max) was selected. Ten mallards undertook spring migrations but, due to the loss of signal in the GPS-GSM devices, only three ducks were observed during autumn migrations. The following ranges of D_max values were calculated for spring migrations: 124–382 km for IP₁ (median 210 km), 208–632 km for IP₄ (median 342 km), and 213–687 km for IP₈ (median 370 km). The present study provides information that can be used as a data source to perform risk assessment related to the contribution of wild mallards in the dispersal of HPAIV over considerable distances.

Due to their probable role in the spread of Asian highly pathogenic avian influenza (HPAI) H5N1 virus, and in order to explore its implication in the low pathogenic avian influenza (LPAI) virus epidemiology, mute swans represent one particular wild bird species specifically targeted in the avian influenza (AI) surveillance elaborated in Belgium. A total of 640 individual mute swans have been sampled during a 4-yr AI surveillance program (2007–2010) to determine the AI seroprevalence and viroprevalence in this species; all were analyzed through age, temporal, and habitat (flowing and stagnant water) factors. Using a nucleoprotein (NP)-based ELISA, a global antibody prevalence of 35% has been found and was characterized by two peaks in the winter and the summer that might be indicative of a greater LPAI virus circulation in the autumn than in the spring. A significantly higher antibody prevalence was detected in adult swans (53.8%) as compared to juveniles (15.5%). In contrast, a low prevalence of infection (2.7%) was found, mainly in juvenile mute swans and only during the autumn migration period. Interestingly, an impact of water habitat was observed based on the comparison of the antibody prevalence and prevalence of infection from swan populations living on stagnant water vs. flowing water, suggesting that stagnant water provides a more-favorable environment for LPAI persistence and transmission.

One of the longest-persisting avian influenza viruses in history, highly pathogenic avian influenza virus (HPAIV) A(H5N1), continues to evolve after 18 yr, advancing the threat of a global pandemic. Wild waterfowl (family Anatidae) are reported as secondary transmitters of HPAIV and primary reservoirs for low-pathogenic avian influenza viruses, yet spatial inputs for disease risk modeling for this group have been lacking. Using geographic information software and Monte Carlo simulations, we developed geospatial indices of waterfowl abundance at 1 and 30 km resolutions and for the breeding and wintering seasons for China, the epicenter of H5N1. Two spatial layers were developed: cumulative waterfowl abundance (W_AB), a measure of predicted abundance across species, and cumulative abundance weighted by H5N1 prevalence (W_PR), whereby abundance for each species was adjusted based on prevalence values and then totaled across species. Spatial patterns of the model output differed between seasons, with higher W_AB and W_PR in the northern and western regions of China for the breeding season and in the southeast for the wintering season. Uncertainty measures indicated highest error in southeastern China for both W_AB and W_PR. We also explored the effect of resampling waterfowl layers from 1 to 30 km resolution for multiscale risk modeling. Results indicated low average difference (less than 0.16 and 0.01 standard deviations for W_AB and W_PR, respectively), with greatest differences in the north for the breeding season and southeast for the wintering season. This work provides the first geospatial models of waterfowl abundance available for China. The indices provide important inputs for modeling disease transmission risk at the interface of poultry and wild birds. These models are easily adaptable, have broad utility to both disease and conservation needs, and will be available to the scientific community for advanced modeling applications.

Wild waterfowl in the order Anseriformes are recognized reservoirs for influenza A viruses (IAVs); however, prevalence of infection can vary greatly by species. Few isolates of IAVs have been reported from snow geese (Chen caerulescens), and generally they have not been regarded as an important component of this reservoir. In February 2013, 151 combined cloacal and oropharangeal swabs and 147 serum samples were collected from snow geese wintering on the Gulf coast of Texas. None of the swab samples tested positive by virus isolation, but antibodies to IAVs were detected in 87 (59%) birds tested by competitive blocking ELISA (bELISA). To further characterize these detected antibodies, positive samples were tested by virus microneutralization (MN) for antibodies to viruses representing 14 hemagglutinin subtypes (HA1–HA12, H14, and H15). By MN, antibodies to H1 (n = 41; 47%), H5 (n = 32; 37%), H6 (n = 49; 56%), H9 (n = 50; 57%), and H12 (n = 24; 28%) were detected. Snow goose populations have increased in North America since the 1960s, and their association with agricultural lands provides a potential indirect source of IAV infection for domestic poultry. This potential, as well as the detection of antibodies to HA subtypes H5, H9, and H12 that are not well represented in other waterfowl species, suggests that further snow geese surveillance is indicated.

Gulls are the known reservoir for H13 and H16 influenza A viruses (IAV) but also host a diversity of other IAV subtypes. Gulls also share habitats with both ducks and shorebirds, increasing the potential for cross-species IAV transmission. We serologically tested laughing gulls (Leucophaeus atricilla) collected at Delaware Bay during May when they were in direct contact with IAV-infected shorebirds; both species feed on horseshoe crab (Limulus polyphemus) eggs on beaches during this month. From 2010 to 2014, antibody prevalence as determined by competitive blocking enzyme-linked immunosorbent assay ranged from 25%–72%. Antibodies to H13 and H16 were detected by hemagglutination inhibition (HI) tests in 12% and 24% of tested gulls, respectively. Results from virus microneutralization (MN) tests for antibodies to H1–H12, H14, and H15 varied among years but the highest prevalence of neutralizing antibodies was detected against H1 (24%), H5 (25%), H6 (35%), H9 (33%), and H11 (42%) IAV. The subtype diversity identified by serology in gulls was dominated by Group 1 HA subtypes and only partially reflected the diversity of IAV subtypes isolated from shorebirds.

Besides humans, H3 subtypes of influenza A viruses (IAVs) can infect various animal hosts, including avian, swine, equine, canine, and sea mammal species. These H3 viruses are both antigenically and genetically diverse. Here, we characterized the antigenic diversity of contemporary H3 avian IAVs recovered from migratory birds in North America. Hemagglutination inhibition (HI) assays were performed on 37 H3 isolates of avian IAVs recovered from 2007 to 2011 using generated reference chicken sera. These isolates were recovered from samples taken in the Atlantic, Mississippi, Central, and Pacific waterfowl migration flyways. Antisera to all the tested H3 isolates cross-reacted with each other and, to a lesser extent, with those to H3 canine and H3 equine IAVs. Antigenic cartography showed that the largest antigenic distance among the 37 avian IAVs is about four units, and each unit corresponds to a 2 log&thinsp;₂ difference in the HI titer. However, none of the tested H3 IAVs cross-reacted with ferret sera derived from contemporary swine and human IAVs. Our results showed that the H3 avian IAVs we tested lacked significant antigenic diversity, and these viruses were antigenically different from those circulating in swine and human populations. This suggests that H3 avian IAVs in North American waterfowl are antigenically relatively stable.

In 2014, clade 2.3.4.4 H5N8 highly pathogenic avian influenza (HPAI) viruses spread across the Republic of Korea and ultimately were reported in China, Japan, Russia, and Europe. Mortality associated with a reassortant HPAI H5N2 virus was detected in poultry farms in western Canada at the end of November. The same strain (with identical genetic structure) was then detected in free-living wild birds that had died prior to December 8, 2014, of unrelated causes in Whatcom County, Washington, U. S. A., in an area contiguous with the index Canadian location. A gyrfalcon (Falco rusticolus) that had hunted and fed on an American wigeon (Anas americana) on December 6, 2014, in the same area, and died 2 days later, tested positive for the Eurasian-origin HPAI H5N8. Subsequently, an active surveillance program using hunter-harvested waterfowl in Washington and Oregon detected 10 HPAI H5 viruses, of three different subtypes (four H5N2, three H5N8, and three H5N1) with four segments in common (HA, PB2, NP, and MA). In addition, a mortality-based passive surveillance program detected 18 HPAI (14 H5N2 and four H5N8) cases from Idaho, Kansas, Oregon, Minnesota, Montana, Washington, and Wisconsin. Comparatively, mortality-based passive surveillance appears to have detected these HPAI infections at a higher rate than active surveillance during the period following initial introduction into the United States.

Waterfowl species are known to harbor the greatest diversity of low pathogenicity influenza A virus (LPAIV) subtypes and are recognized as their main natural reservoir. In Guatemala there is evidence of circulation of LPAIV in wild ducks; however, the bird species contributing to viral diversity during the winter migration in Central America are unknown. In this study, samples obtained from 1250 hunter-killed birds from 22 different species were collected on the Pacific coast of Guatemala during three winter migration seasons between 2010 and 2013. Prevalence of LPAIV detected by real-time reverse-transcriptase polymerase chain reaction was 38.2%, 23.5%, and 24.7% in the 2010–11, 2011–12, and 2012–13 seasons, respectively. The highest virus prevalence was detected in the northern shoveler (Anas clypeata), followed by the blue-winged teal (Anas discors). The majority of positive samples and viral isolates were obtained from the blue-winged teal. Analysis of LPAIV prevalence over time in this species indicated a decreasing trend in monthly prevalence within a migration season. Sixty-eight viruses were isolated, and nine HA and seven NA subtypes were identified in 19 subtype combinations. In 2012–13 the most prevalent subtype was H14, a subtype identified for the first time in the Western Hemisphere in 2010. The results from this study represent the most detailed description available to date of LPAIV circulation in Central America.

Wild bird surveillance for avian influenza virus (AIV) was conducted from 2001 to 2012 in the Azov - Black Sea region of the Ukraine, considered part of the transcontinental wild bird migration routes from northern Asia and Europe to the Mediterranean, Africa, and southwest Asia. A total of 6281 samples were collected from wild birds representing 27 families and eight orders for virus isolation. From these samples, 69 AIVs belonging to 15 of the 16 known hemagglutinin (HA) subtypes and seven of nine known neuraminidase (NA) subtypes were isolated. No H14, N5, or N9 subtypes were identified. In total, nine H6, eight H1, nine H5, seven H7, six H11, six H4, five H3, five H10, four H8, three H2, three H9, one H12, one H13, one H15, and one H16 HA subtypes were isolated. As for the NA subtypes, twelve N2, nine N6, eight N8, seven N7, six N3, four N4, and one undetermined were isolated. There were 27 HA and NA antigen combinations. All isolates were low pathogenic AIV except for eight highly pathogenic (HP) AIVs that were isolated during the H5N1 HPAI outbreaks of 2006–08. Sequencing and phylogenetic analysis of the HA genes revealed epidemiological connections between the Azov-Black Sea regions and Europe, Russia, Mongolia, and Southeast Asia. H1, H2, H3, H7, H8, H6, H9, and H13 AIV subtypes were closely related to European, Russian, Mongolian, and Georgian AIV isolates. H10, H11, and H12 AIV subtypes were epidemiologically linked to viruses from Europe and Southeast Asia. Serology conducted on serum and egg yolk samples also demonstrated previous exposure of many wild bird species to different AIVs. Our results demonstrate the great genetic diversity of AIVs in wild birds in the Azov-Black Sea region as well as the importance of this region for monitoring and studying the ecology of influenza viruses. This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.

Wild waterbirds, specifically waterfowl, gulls, and shorebirds, are recognized as the primordial reservoir of influenza A viruses (IAVs). However, the role of seabirds, an abundant, diverse, and globally distributed group of birds, in the perpetuation and transmission of IAVs is less clear. Here we summarize published and publicly available data for influenza viruses in seabirds, which for the purposes of this study are defined as birds that exhibit a largely or exclusively pelagic lifestyle and exclude waterfowl, gulls, and shorebirds, and we review this collective dataset to assess the role of seabirds in the influenza A ecology. Since 1961, more than 40,000 samples have been collected worldwide from the seabirds considered here and screened, using a variety of techniques, for evidence of active or past IAV infection. From these data, the overall prevalence of active infection has been estimated to be very low; however, serological data provide evidence that some seabird species are more frequently exposed to IAVs. Sequence data for viruses from seabirds are limited, except for murres (common murre, Uria aalge, and thick-billed murre, Uria lomvia; family Alcidae) for which there are full or partial genome sequences available for more than 80 viruses. Characterization of these viruses suggests that murres are infected with Group 1 hemagglutinin subtype viruses more frequently as compared to Group 2 and also indicates that these northern, circumpolar birds are frequently infected by intercontinental reassortant viruses. Greater temporal and spatial sampling and characterization of additional viruses are required to better understand the role of seabirds in global IAV dynamics.

Active monitoring of avian influenza (AI) viruses in wild birds was initiated in Belgium in 2005 in response to the first highly pathogenic avian influenza (HPAI) H5N1 outbreaks occurring in Europe. In Belgium, active wild bird surveillance that targeted live-ringed and hunter-harvested wild birds was developed and maintained from 2005 onward. After one decade, this program assimilated, analyzed, and reported on over 35,000 swabs. The 2009–2014 datasets were used for the current analysis because detailed information was available for this period. The overall prevalence of avian influenza (AI) in samples from live-ringed birds during this period was 0.48% whereas it was 6.12% in hunter-harvested samples. While the ringing sampling targeted a large number of bird species and was realized over the years, the hunting sampling was mainly concentrated on mallard (Anas platyrhynchos) during the hunting season, from mid-August to late January. Even when using just AI prevalence for live-ringed A. platyrhynchos during the hunting season, the value remained significantly lower (2.10%) compared to that detected for hunter-harvested mallards. One explanation for this significant difference in viroprevalence in hunter-harvested mallards was the game restocking practice, which released captive-bred birds in the wild before the hunting period. Indeed, the released game restocking birds, having an AI-naïve immune status, could act as local amplifiers of AI viruses already circulating in the wild, and this could affect AI epidemiology. Also, the release into the wild of noncontrolled restocking birds might lead to the introduction of new strains in the natural environment, leading to increased AI presence in the environment. Consequently, the release of naïve or infected restocking birds may affect AI dynamics.

Although low pathogenicity avian influenza viruses (LPAIV) are detected in shorebirds at Delaware Bay annually, little is known about affected species habitat preferences or the movement patterns that might influence virus transmission and spread. During the 5-wk spring migration stopover period during 2007–2008, we conducted a radiotelemetry study of often-infected ruddy turnstones (Arenaria interpres morinella; n = 60) and rarely infected sanderlings (Calidris alba; n = 20) to identify locations and habitats important to these species (during daytime and nighttime), determine the extent of overlap with other AIV reservoir species or poultry production areas, reveal possible movements of AIV around the Bay, and assess whether long-distance movement of AIV is likely after shorebird departure. Ruddy turnstones and sanderlings both fed on Bay beaches during the daytime. However, sanderlings used remote sandy points and islands during the nighttime while ruddy turnstones primarily used salt marsh harboring waterfowl and gull breeding colonies, suggesting that this environment supports AIV circulation. Shorebird locations were farther from agricultural land and poultry operations than were random locations, suggesting selection away from poultry. Further, there was no areal overlap between shorebird home ranges and poultry production areas. Only 37% (22/60) of ruddy turnstones crossed into Delaware from capture sites in New Jersey, suggesting partial site fidelity and AIV gene pool separation between the states. Ruddy turnstones departed en masse around June 1 when AIV prevalence was low or declining, suggesting that a limited number of birds could disperse AIV onto the breeding grounds. This study provides needed insight into AIV and migratory host ecology, and results can inform both domestic animal AIV prevention and shorebird conservation efforts.

Homosubtypic and heterosubtypic immunity in mallards (Anas platyrhynchos) play an important role in the avian influenza virus (AIV) diversity. The mechanisms of AIV replication among wild birds and the role of immunity in AIV diversity have thus not been completely clarified. During the monitoring of AI circulation among wild waterfowl in 2007–2008, two viruses (H3N8 and H1N1) were isolated from ducks caught in a funnel trap located in La Hulpe wetland in Belgium. H3N8 viruses were revealed to be more prevalent in the mallard population than was H1N1, which might suggest a better adaptation to this species. In order to investigate this hypothesis, we characterized both isolated viruses biologically by experimental inoculation. Virus excretion and humoral response induced by both isolated viruses were evaluated in mallards after a first infection followed by a homo-or heterosubtypic reinfection under controlled experimental conditions. The H1N1 virus had a delayed peak of excretion of 4 days compared to the H3N8, but the virus shedding was more limited, earlier, and shorter after each reinfection. Moreover, the H3N8 virus could spread to all ducks after homo- or heterosubtypic reinfections and during a longer period. Although the humoral response induced by both viruses after infection and reinfection could be detected efficiently by competitive ELISA, only a minimal H1 antibody response and almost no H3-specific antibodies could be detected by the HI test. Our results suggest that the H3N8 isolate replicates better in mallards under experimental controlled conditions.

Avian influenza subtype H9N2 is endemic in many countries in the Middle East. The reported prevalence of infection was variable between countries and ranged from 28.7% in Tunisia to 71% in Jordan. Several commercial killed whole-virus vaccine products are used as monovalent or bivalent mixed with Newcastle disease virus. Recently, we have noticed that many of the vaccinated broiler flocks did not show a production advantage over nonvaccinated flocks in the field. A new avian influenza field virus (H9N2) was isolated from these vaccinated and infected broiler flocks in 2013. This virus had 89.1% similarity of its hemagglutinin (HA) gene to the classical virus used for manufacturing the classical vaccine. Inactivated autogenous vaccine was manufactured from this new field isolate to investigate its serological response and protection in specific-pathogen-free (SPF) and breeder-male chickens compared to the classical vaccine. Oropharyngeal virus shedding of vaccinated breeder-male chickens was evaluated at 3, 9, 10, and 14 days postchallenge (DPC). Percentage of chickens shedding the virus at 3 DPC was 64%, 50%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 7 DPC percentage of virus shedding was 42%, 7%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 10 DPC only 9% of classical vaccine group was shedding the virus and there was no virus shedding in any of the groups at 14 DPC. There was statistical significance difference (P < 0.05) in shedding only at 7 DPC between the autogenous vaccine group and the other two groups. At 42 days of age (14 DPC), average body weight was 2.720, 2.745, 2.290, and 2.760 kg for the classical vaccine group, autogenous vaccine group, control challenged group, and control unchallenged group, respectively. Only the control challenged group had significantly (P < 0.05) lower average body weight. In another experiment, vaccinated SPF chicks had hemagglutination inhibition (HI) geometric mean titers (GMTs), with classical antigen, of 8.7 and 3.1 log ₂ for classical and autogenous vaccine groups, respectively. When the autogenous antigen was used for HI, GMTs were 6.0 and 8.1 log ₂, respectively. Both vaccines protected against body weight suppression after challenge. However, autogenous vaccine elicited significantly higher HI titer and reduced viral shedding at 7 DPC. In conclusion, it is important to revise the vaccine virus strains used in each region to protect against and control infection from new field strains. Further field experiments are needed to demonstrate the efficacy of new vaccines under field conditions.

Subtype H3 influenza A viruses (IAVs) are abundant in wild waterfowl and also infect humans, pigs, horses, dogs, and seals. In Minnesota, turkeys are important and frequent hosts of IAV from wild waterfowl and from pigs. Over 48 yr of surveillance history, 11 hemagglutinin (HA) subtypes of IAV from waterfowl, as well as two HA subtypes from swine, H1 and H3, have infected turkeys in Minnesota. However, there have only been two cases of avian-origin H3 IAV infections in turkeys during this 48-yr period. The first avian-origin IAV infection was detected in seven breeder and commercial flocks in 1982 and was caused by a mixed H3H4/N2 infection. In 2013, an avian-origin H3H9/N2 outbreak occurred in five flocks of turkeys between 15 and 56 wk of age. Phylogenetic analysis of the HA gene segment from the 2013 isolate indicated that the virus was related to a wild bird lineage H3 IAV. A meta-analysis of historical H3 infections in domesticated poultry demonstrated that avian-origin H3 infections have occurred in chickens and ducks but were rare in turkeys. H9N2 virus was subsequently selected during the egg cultivation of the 2013 H3H9/N2 mixed virus. A growth curve analysis suggested that passage 3 of A/Turkey/Minnesota/13-20710-2/2013(mixed) had a slightly lower replication rate than a similar avian-origin H3N2. The challenge studies indicated that the infectious dose of avian-origin H3N2 for turkey poults was greater than 10⁶ 50% egg infective dose. Considered together, these data suggest that avian-origin H3 introductions to turkeys are rare events.

Surveillance of notifiable avian influenza (NAI) virus is mandatory in European member states, and each year a serological survey is performed to detect H5 and H7 circulation in poultry holdings. In Belgium, this serological monitoring is a combination of a stratified and a risk-based approach and is applied to commercial holdings with more than 200 birds. Moreover, a competitive nucleoprotein (NP) ELISA has been used as first screening method since 2010. A retrospective analysis of the serological monitoring performed from 2007 through 2013 showed sporadic circulation of notifiable low-pathogenicity avian influenza (LPAI) viruses in Belgian holdings with a fluctuating apparent flock seroprevalence according to years and species. Overall, the highest apparent flock seroprevalence was detected for the H5 subtype in domestic Anatidae, with 20%–50% for breeding geese and 4%–9% for fattening ducks. Positive serology against non-H5/H7 viruses was also observed in the same species with the use of the IDScreen influenza A antibody competition ELISA kit (ID-vet NP ELISA), and confirmed by isolation of H2, H3, H6, and H9 LPAI viruses. Among Galliformes, the apparent flock seroprevalence was lower, ranging between 0.3% and 1.3%. Circulation of notifiable LPAI viruses was only observed in laying hens with a similar seroprevalence for H5 and H7. Based on ID-vet NP ELISA results, no circulation of LPAI viruses, regardless the subtype, was observed in breeding chickens and fattening turkeys. Retrospectively, the use of an ELISA as first-line test not only reduced the number of hemagglutination inhibition tests to be performed, but also gave a broader evaluation of the prevalence of LPAI viruses in general, and might help to identify the most at-risk farms.