Search

Garbage management represents a potential pathway of HPAI-virus infection for commercial poultry operations as multiple poultry premises may share a common trash collection service provider, trash collection site (e.g., shared dumpster for multiple premises) or disposal site (e.g., landfill). The types of potentially infectious or contaminated material disposed of in the garbage has not been previously described, but is suspected to vary by poultry industry sector. A survey of representatives from the broiler, turkey, and layer sectors in the United States revealed that many potentially contaminated or infectious items are routinely disposed of in the trash on commercial poultry premises. On-farm garbage management practices, along with trash hauling and disposal practices are thus key components that must be considered in order to evaluate the risk of commercial poultry becoming infected with HPAI-virus.

The response to an agricultural emergency that threatens to destroy crops or animals requires a rapid, coordinated state-level response from the outset. An authority should be established at the local level to initiate and enforce food embargoes, quarantine livestock or poultry premises, depopulate affected or potentially affected animals, and provide indemnity, when appropriate, for those depopulated animals or destroyed products. Depending on the scale of the threat, industry needs, state resources, and response capacity, the authority for these activities currently resides with the state and is supported by federal agencies. However, an all-encompassing all-hazards agricultural emergency response system can be constructed through collaborations with agricultural industry, state responders, and federal agencies. The formed response should include development of permitting guidance for controlled harvest and movement of unaffected crops, animals, and animal products. The ultimate goal is to effectively manage the emergency yet maintain agricultural business continuity.

Enterovirus 71 (EV71) has become an important neurotropic enterovirus in children since the eradication of the poliovirus. A cytokine storm with elevated levels of proinflammatory cytokines is proposed as critical to the pathogenesis of EV71-induced brain stem encephalitis with pulmonary edema, often a fatal complication. Mitogen-activated protein kinases (MAPK) are involved in cellular processes including immune responses and apoptosis. EV71 infection can trigger MAPK activation in human cells, and extracellular signal-regulated kinase (ERK) signaling is essential for EV71 replication. In this study, we report that in EV71-infected human intestinal epithelial cells (HT-29), both ERK1/2 and Jun-N-terminal kinase 1/2 (JNK1/2) were phosphorylated and activated. While MAPK was not involved in the induction of IL-1β, the expressions of IL-6 and IL-8 were positively regulated by both ERK1/2 and JNK1/2, which were also responsible for type I IFN induction. HT-29 cells underwent apoptosis in EV71 infection, but the activation of ERK1/2 and JNK1/2 signaling appeared to suppress induction of TNF-α and FasL and inhibit extrinsic apoptosis in infected cells. Activation of NF-κB was also observed in HT-29 cells in EV71 infection, leading to increased viral replication and proinflammatory cytokine induction. However, the activation of NF-κB was inhibited by the inhibitors of ERK1/2, p38 and JNK1/2, indicating that a cross-talk may exist between MAPK and NF-κB pathways in EV71-infected intestinal epithelial cells. Understanding host responses and their underlying mechanisms may help develop effective therapeutics against EV71 and tools for control of future EV71 outbreaks.

Non-typhoidal Salmonella (NTS) remains a global pathogen that affects a wide range of animal species. We analyzed a large number of NTS isolates of different host origins, including Salmonella Heidelberg (n = 80, avian), S. Dublin (50, bovine), S. Typhimurium var 5- (n = 40, porcine), S. 4,5,12,:i:- (n = 40, porcine), S. Cerro (n = 16, bovine), and S. Montevideo (n = 14, bovine), using virulence profiling of the bcfC, mgtC, ssaC, invE, pefA, stn, sopB, and siiE virulence-associated genes, a biofilm production assay, pulsed field gel electrophoresis, and the full-length sequencing of the fimA (adhesin) and iroN (receptor) genes. We determined a key amino acid substitution, A169 (i.e., threonine changed to alanine at position 169), in the FimA protein that changed ligand affinity of FimA toward N-acetyl-D-glucosamine. This finding clearly indicates the important role of non-synonymous single nucleotide polymorphism (nsSNPs) in adhesin functionality that may impact the host tropism of NTS. This nsSNP was found in S. Heidelberg and S. Cerro isolates. Although this was not the case for the IroN receptor, the phylogeny of this receptor and different host origins of NTS isolates were positively correlated, suggesting existence of specific host immune selective pressures on this unique receptor in S. enterica. We found that pefA, a gene encoding major fimbrial subunit, was the most-segregative virulence factor. It was associated with S. Heidelberg, S. Typhimurium var 5- and S. 4,5,12,:i:- but not with the rest of NTS strains. Further, we observed a significantly higher frequency of non-biofilm producers among NTS strains that do not carry pefA (42.5%) compared to S. Heidelberg (2.5%) and S. Typhimurium var 5- (7.5%) and S. 4,5,12,:i:- (0%). This study provides new insights into the host adaptation of avian and mammalian NTS isolates that are based on the bacterial antigens FimA and IroN as well as the interrelationships between host adaptation, overall genetic relatedness, and virulence potential in these NTS isolates.

Characterizing the health consequences of interactions among animals, humans, and the environment in the face of climatic change, environmental disturbance, and expanding human populations is a critical global challenge in today's world. Exchange of interdisciplinary knowledge in basic and applied sciences and medicine that includes scientists, health professionals, key sponsors, and policy experts revealed that relevant case studies of monkeypox, influenza A, tuberculosis, and HIV can be used to guide strategies for anticipating and responding to new disease threats such as the Ebola and Chickungunya viruses, as well as to improve programs to control existing zoonotic diseases, including tuberculosis. The problem of safely feeding the world while preserving the environment and avoiding issues such as antibiotic resistance in animals and humans requires cooperative scientific problem solving. Food poisoning outbreaks resulting from Salmonella growing in vegetables have demonstrated the need for knowledge of pathogen evolution and adaptation in developing appropriate countermeasures for prevention and policy development. Similarly, pesticide use for efficient crop production must take into consideration bee population declines that threaten the availability of the two-thirds of human foods that are dependent on pollination. This report presents and weighs the objective merits of competing health priorities and identifies gaps in knowledge that threaten health security, to promote discussion of major public policy implications such that they may be decided with at least an underlying platform of facts.

Highlights • We evaluated three processing methods to harvest viral metagenome from intestinal tissue. • Rotaviruses and astroviruses were found in abundance in the gut of a turkey showing no clinical signs of disease. • A customized approach to analyze the data was evaluated. • A true measure of diversity was obtained at much smaller fraction of the total data. • The rarefaction curve plateaued after 90% of the data was sampled at species level., RNA viruses have been associated with enteritis in poultry and have been isolated from diseased birds. The same viral agents have also been detected in healthy flocks bringing into question their role in health and disease. In order to understand better eukaryotic viruses in the gut, this project focused on evaluating alternative methods to purify and concentrate viral particles, which do not involve the use of density gradients, for generating viral metagenome data. In this study, the sequence outcomes of three tissue processing methods have been evaluated and a data analysis pipeline has been established for RNA viruses from the gastrointestinal tract. In addition, with the use of the best method and increased sequencing depth, a glimpse of the RNA viral community in the gastrointestinal tract of a clinically normal 5-week old turkey is presented. The viruses from the Reoviridae and Astroviridae families together accounted for 76.3% of total viruses identified. The rarefaction curve at the species level further indicated that majority of the species diversity was included with the increased sequencing depth, implying that viruses from other viral families were present in very low abundance.

The cleavage motif in the hemagglutinin (HA) protein of highly pathogenic H5 and H7 subtypes of avian influenza viruses is characterized by a peptide insertion or a multibasic cleavage site (MBCS). Here, we isolated an H4N2 virus from quails (Quail/CA12) with two additional arginines in the HA cleavage site, PEKRRTR/G, forming an MBCS-like motif. Quail/CA12 is a reassortant virus with the HA and neuraminidase (NA) gene most similar to a duck-isolated H4N2 virus, PD/CA06 with a monobasic HA cleavage site. Quail/CA12 required exogenous trypsin for efficient growth in culture and caused no clinical illness in infected chickens. Quail/CA12 had high binding preference for α2,6-linked sialic acids and showed higher replication and transmission ability in chickens and quails than PD/CA06. Although the H4N2 virus remained low pathogenic, these data suggests that the acquisition of MBCS in the field is not restricted to H5 or H7 subtypes.

Two distinct antigenic clusters were previously identified among the H3N2 swine influenza A viruses (IAVs) and were designated H3N2SIV-alpha and H3N2SIV-beta ( Feng et al., 2013. Journal of Virology 87 (13), 7655–7667 ). A consistent mutation was observed at the position 189 of hemagglutinin (R189K) between H3N2SIV-alpha and H3N2SIV-beta fair isolates. To evaluate the contribution of R189K mutation to the antigenic drift from H3N2SIV-alpha to H3N2SIV-beta, four reassortant viruses with 189R or 189K were generated. The antigenic cartography demonstrated that the R189K mutation in the hemagglutinin of H3N2 IAV contributed to the antigenic drift, separating these viruses into H3N2SIV-alpha to H3N2SIV-beta. This R189K mutation was also found to contribute to the cross-reaction with several ferret sera raised against historical human IAVs with hemagglutinin carrying 189K. This study suggests that the R189K mutation plays a vital role in the antigenicity of swine and human H3N2 IAVs and identification of this antigenic determinant will help us rapidly identify antigenic variants in influenza surveillance.

Background In early 2015, highly pathogenic avian influenza H5N2 caused outbreaks in commercial poultry farms in Minnesota and neighboring states where more than 48 million birds were affected. To date, the origin and transmission pathways of HPAI H5N2 have not been conclusively established. Methods In this study, we analyzed forty-six samples from turkeys and their environment that were collected at different time-points of the outbreak to identify origins and within outbreak evolutionary changes. We performed de-novo whole genome sequencing from primary samples and the most recent common ancestors of the PB2, PA, HA5, M and NS segments were traced back to Japanese HPAI H5N8 isolates. These segments appeared to have diverged from the ancestor around June and November 2014. Results The time to most recent common ancestor analysis for PB1, NP and NA2 segments suggest two likely possibilities of reassortant HPAI H5N2 origin - either a reassortment in Alaska area or multiple reassortments with North American low pathogenic avian influenza strains, before the HPAI H5N2 outbreak strain emerged. Within the outbreak, viruses clustered into two and three subgroups suggesting high substitution rates of 0.702x10-2 - 1.665x10-2 (subs/site/year), over the 5-month outbreak period. Conclusions Data are suggestive of a fast evolving HPAI strain within an outbreak that should be taken into consideration in developing appropriate control strategies in the future. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0605-4) contains supplementary material, which is available to authorized users.

Animal diseases such as foot-and-mouth disease (FMD) have the potential to severely impact food animal production systems. Paradoxically, the collateral damage associated with the outbreak response may create a larger threat to the food supply, social stability, and economic viability of rural communities than the disease itself. When FMD occurs in domestic animals, most developed countries will implement strict movement controls in the area surrounding the infected farm(s). Historically, stopping all animal movements has been considered one of the most effective ways to control FMD and stop disease spread. However, stopping all movements in an area comes at a cost, as there are often uninfected herds and flocks within the control area. The inability to harvest uninfected animals and move their products to processing interrupts the food supply chain and has the potential to result in an enormous waste of safe, nutritious animal products, and create animal welfare situations. In addition, these adverse effects may negatively impact agriculture businesses and the related economy. Effective disease control measures and the security of the food supply thus require a balanced approach based on science and practicality. Evaluating the risks associated with the movement of live animals and products before an outbreak happens provides valuable insights for risk management plans. These plans can optimize animal and product movements while preventing disease spread. Food security benefits from emergency response plans that both control the disease and keep our food system functional. Therefore, emergency response plans must aim to minimize the unintended negative consequence to farmers, food processors, rural communities, and ultimately consumers.

The US National institutes of Health-Centers of Excellence for Influenza Research and Surveillance is a research consortium that funds numerous labs worldwide to conduct influenza A surveillance in diverse animal species. There is no harmonization of testing procedures among these labs; therefore an external quality assurance (EQA) program was implemented to evaluate testing accuracy among labs in the program in 2012. Accurate detection of novel influenza A variants is crucial because of the broad host range and potentially high virulence of the virus in diverse species. Two molecular detection sample sets and 2 serology sample sets (one with avian origin isolates, and one with mammalian origin isolates each) were made available at approximately six month intervals. Participating labs tested the material in accordance with their own protocols. During a five year period a total of 41 labs from 23 countries ordered a total of 132 avian molecular, 121 mammalian molecular and 90 serology sample sets. Testing was completed by 111 individuals. Detection of type A influenza by RT-PCR was reliable with a pass rate (80% or greater agreement with expected results) of 86.6% for avian and 86.2% for mammalian origin isolates. However, identification of subtype by RT-PCR was relatively poor with 54.1% and 75.9% accuracy for avian and mammalian influenza isolates respectively. Serological testing had an overall pass rate of 86.9% and 22/23 labs used commercial ELISA kits. Based on the results of this EQA program six labs modified their procedures to improve accuracy and one lab identified an unknown equipment problem. These data represent the successful implementation of an international EQA program for an infectious disease; insights into the logistics and test design are also discussed.

The perception of risks of exposure to avian influenza and other poultry diseases among adults in Tanzania is influenced by their previous experiences, beliefs, and values, which can stand in the way of learning new approaches. We tested a novel disease risk communication approach centered on elementary school pupils, involving their teachers and parents. Age-appropriate training modules were developed and taught to teachers who then taught their pupils through extracurricular activities. The pupils practiced what they learned through club projects and subsequently transmitted what they learned to their parents. In 2009 we developed a poultry health and production curriculum as part of efforts to prevent and control poultry diseases, including avian influenza, in Tanzania. The curriculum developed for veterinarians and veterinary paraprofessionals was adapted for use with elementary school children and translated into Kiswahili. Twenty teachers from 10 primary schools in Mzumbe ward, Morogoro, were trained by poultry veterinary extension experts on teaching the curriculum to standard 5 pupils (ages 11-12 yr). Pupils and teachers practiced the curriculum in four demonstration chicken coops established on the grounds of the Changarawe, Lubungo, Masanze, and Mzumbe primary schools. By October 2011, at the conclusion of the funded project, a total of 202 girls and 193 boys had been trained. Additionally, 34 adults from surrounding villages made official learning tours to the schools and received training from their children and teachers involved in the projects. With at least 75% of the 395 pupils coming from different households, it can be safely assumed that over 250 households have heard about poultry disease risks and how to manage poultry to prevent those risks.

Studies of pathogen transmission typically overlook that wildlife hosts can include both migrant and resident populations when attempting to model circulation. Through the application of stable isotopes in flight feathers, we estimated the migration strategy of mallards (Anas platyrhynchos) occurring on California wintering grounds. Our study demonstrates that mallards- a principal host of avian influenza virus (AIV) in nature, contribute differently to virus gene flow depending on migration strategy. No difference in AIV prevalence was detected between resident (9.6%), intermediate-distance (9.6%) and long-distance migrants (7.4%). Viral diversity among the three groups was also comparable, possibly owing to viral pool mixing when birds converge at wetlands during winter. However, migrants and residents contributed differently to the virus gene pool at wintering wetlands. Migrants introduced virus from northern breeding grounds (Alaska and the NW Pacific Rim) into the wintering population, facilitating gene flow at continental scales, but circulation of imported virus appeared to be limited. In contrast, resident mallards acted as AIV reservoirs facilitating year-round circulation of limited subtypes (i.e. H5N2) at lower latitudes. This study supports a model of virus exchange in temperate regions driven by the convergence of wild birds with separate geographic origins and exposure histories.

In a previous study, we found clear differences in pathogenicity and response to vaccination against H5N1 highly pathogenic avian influenza (HPAI; HA dade 2.3.4) between Pekin (Anas platyrhynchos var. domestica) and Muscovy (Cairina moschata) ducks vaccinated using a commercial inactivated vaccine (Re-1). The objective of the present study was to further investigate the pathogenicity of H5N1 HPAI viruses in different species of ducks by examining clinical signs and innate immune responses to infection with a different strain of H5N1 HPAI virus (HA clade 1) in two domestic ducks, Pekin and Muscovy, and one wild-type duck, mallard (Anas platyrhynchos). Protection conferred by vaccination using the Re-1 vaccine against infection with this virus was also compared between Pekin and Muscovy ducks. Differences in pathogenicity were observed among the virus-infected ducks, as the Muscovy ducks died 2 days earlier than did the Pekin and mallard ducks, and they presented more-severe neurologic signs. Conversely, the Pekin and mallard ducks had significantly higher body temperatures at 2 days postinfection (dpi) than did the Muscovy ducks, indicating possible differences in innate immune responses. However, similar expression of innate immune-related genes was found in the spleens of virus-infected ducks at this time point. In all three duck species, there was up-regulation of IFN-alpha, IFN-gamma, IL-6, CCL19, RIG-I, and MHC class I and down-regulation of MHC class II, but variable expression of IL-18 and TLR7. As in our previous study, vaccinated Muscovy ducks showed less protection against virus infection than did Pekin ducks, as evidenced by the higher mortality and higher number of Muscovy ducks shedding virus when compared to Pekin ducks. In conclusion, infection with an H5N1 HPAI virus produced a systemic infection with high mortality in all three duck species; however, the disease was more severe in Muscovy ducks, which also had a poor response to vaccination. The differences in response to virus infection could not be explained by differences in the innate immune responses between the different types of ducks when examined at 2 days dpi, and earlier time points need to be evaluated.

Evidence accumulating in 2011–2012 indicates that there is significant intra- and inter-species transmission of influenza A viruses at agricultural fairs, which has renewed interest in this unique human/swine interface. Six human cases of influenza A (H3N2) variant (H3N2v) virus infections were epidemiologically linked to swine exposure at fairs in the United States in 2011. In 2012, the number of H3N2v cases in the Midwest had exceeded 300 from early July to September, 2012. Prospective influenza A virus surveillance among pigs at Ohio fairs resulted in the detection of H3N2pM (H3N2 influenza A viruses containing the matrix (M) gene from the influenza A (H1N1) pdm09 virus). These H3N2pM viruses were temporally and spatially linked to several human H3N2v cases. Complete genomic analyses of these H3N2pM isolates demonstrated >99% nucleotide similarity to the H3N2v isolates recovered from human cases. Actions to mitigate the bidirectional interspecies transmission of influenza A virus between people and animals at agricultural fairs may be warranted.

Domestic ducks are key intermediates in the transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses, and therefore are included in vaccination programs to control H5N1 HPAI. Although vaccination has proven effective in protecting ducks against disease, different species of domestic ducks appear to respond differently to vaccination, and shedding of the virus may still occur in clinically healthy vaccinated populations. In this study we compared the response to vaccination between two common domestic duck species, Pekin (Anas platyrhynchos domesticus) and Muscovy (Cairina moschata), which were vaccinated with a commercial inactivated vaccine using one of three different schedules in order to elicit protection to H5N1 HPAI before one month of age. Clear differences in responses to vaccination were observed; the Muscovy ducks developed lower viral antibody titers induced by the same vaccination as Pekin ducks and presented with higher morbidity and mortality after challenge with an H5N1 HPAI virus. When comparing the response to infection in non-vaccinated ducks, differences were also observed, with infected Muscovy ducks presenting a lower mean death time and more severe neurological signs than Pekin ducks. However Pekin ducks had significantly higher body temperatures and higher levels of nitric oxide in the blood at 2 days post challenge than Muscovy ducks, indicating possible differences in innate immune responses. Comparison of the expression of innate immune related genes in spleens of the non-vaccinated infected ducks showed differences including significantly higher levels of expression of RIG-I in Pekin ducks and of IL-6 in Muscovy ducks. Both duck species showed an up-regulation of IFNα and MHC-I expression, and a down-regulation of MHC-II. In conclusion, differences in response to infection and vaccination were observed between the two domestic duck species. This information should be taken into account when developing effective vaccination programs for controlling H5N1 HPAI in different species of ducks.

Oropharyngeal and cloacal swabs were collected from poultry sold in two live bird market (LBM) systems to estimate the prevalence of low pathogenicity avian influenza virus (LPAIV) shedding during the summer and fall of 2005. Random sampling was conducted in three LBMs in Minnesota where 50 birds were sampled twice weekly for 4 wk, and in three LBMs in a California marketing system. A stratified systematic sampling method was used to collect samples from Southern California LBMs, where LPAIV was detected during routine surveillance. No LPAIV was detected in the LBM system in Minnesota where realtime reverse transcription-PCR (RT-PCR) was conducted on oropharyngeal samples. RT-PCR was performed on swabs taken from 290 of 14,000, 65 of 252, and 60 of 211 birds at the three Southern California LBMs. The number of samples collected was based on the number of birds, age of the birds, and number of species present in the LBM. Virus isolation, subtyping, and sequencing of the hemagglutinin, neuraminidase, and other internal protein genes was performed on AIV-positive samples. The estimated prevalence of LPAIV in California was 0.345% in an LBM/supply farm with multiple ages of Japanese quail, 3% in an LBM with multiple ages and strains of chickens present, and 49.8% in an LBM with multiple species, multiple strains, and multiple ages. The positive virus samples were all LPAIV H6N2 and closely related to viruses isolated from Southern California in 2001 and 2004. Little or no comingling of poultry may contribute to little or no LPAIV detection in the LBMs.

The avian influenza virus H9N2 subtype has circulated in wild birds, is prevalent in domestic poultry, and has successfully crossed the species boundary to infect humans. Phylogenetic analyses showed that viruses of this subtype appear to have contributed to the generation of highly pathogenic H5N1 viruses. Little is known about the host responses to H9N2 viruses in human airway respiratory epithelium, the primary portal for viral infection. Using an apically differentiated primary human tracheobronchial epithelial (TBE) culture, we examined host immune responses to infection by an avian H9N2 virus, in comparison with a human H9N2 isolate. We found that IFN-β was the prominent antiviral component, whereas interferon gamma-induced protein 10 kDa (IP-10), chemokine (C-C motif) ligand (CCL)-5 and TNF-α may be critical in proinflammatory responses to H9N2 viruses. In contrast, proinflammatory IL-1β, IL-8, and even IL-6 may only play a minor role in pathogenicity. Apparently Toll-like receptor (TLR)-3, TLR-7, and melanoma differentiation- associated gene 5 (MDA-5) contributed to the innate immunity against theH9N2viruses, andMDA-5was important in the induction of IFN-β. We showed that the avian H9N2 virus induced apoptosis through the mitochondria/cytochrome c-mediated intrinsic pathway, in addition to the caspase 8-mediated extrinsic pathway, as evidenced by the cytosolic presence of active caspase 9 and cytochrome c, independent of truncated BH3 interacting domain death agonist (Bid) activation. Further, we demonstrated that FLICE-like inhibitory protein (FLIP), an apoptotic dual regulator, and the p53- dependent Bcl-2 family members, Bax and Bcl-x s, appeared to be involved in the regulation of extrinsic and intrinsic apoptotic pathways, respectively. The findings in this study will further our understanding of host defense mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium., link_to_subscribed_fulltext

We have examined a variety of sampling strategies for detecting pathogens in turkey flocks undergoing infections with low pathogenicity avian influenza virus (LPAIV). We found that viral RNA was widely distributed in the barn environment of turkey flocks undergoing an active LPAIV infection and was in both water and drinker biofilm samples. Viral RNA was concentrated in drinker biofilm and sediment and was detectable using real-time reverse-transcription polymerase chain reaction (RRT-PCR) and by virus isolation. Drinker biofilm sample results correlated with concurrently collected oropharyngeal (OP) sample results from flocks on a farm with LPAI in which the two sampling strategies were directly compared. To evaluate the utility of biofilm sampling for the detection of highly pathogenic avian influenza virus (HPAIV), biofilm and OP swabs from mortality pools were collected daily from negative turkey flocks on an HPAI-positive premise. The biofilm swabs were positive 1-2 days prior to positives appearing in the OP sample pools. The drinker biofilm sampling strategy overcame the difficulty of finding a subclinical infectious bird in a population by collecting material from a large number of individuals and testing a sample in which a positive signal persists for several days to weeks. The sampling method is convenient for use in turkey barns and has been reliably used in both active and passive surveillance programs for LPAIV and HPAIV using RRT-PCR.Muestreo ambiental para el virus de influenza A en casetas de pavos. Se han examinado una variedad de estrategias de muestreo para detectar patógenos en parvadas de pavos que sufren infecciones con el virus de la influenza aviar de baja patogenicidad (con las siglas en inglés LPAIV). Se encontró que el ARN viral se distribuyó ampliamente en el ambiente de las casetas con parvadas de pavos con infección activa por el virus de la influenza aviar de baja patogenicidad y se determinó tanto en muestras de agua como en muestras de la biopelícula de bebederos. El ARN viral se concentró en la biopelícula y en el sedimento de bebederos y se detectó mediante transcripción reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR) y mediante el aislamiento del virus. Los resultados de la muestra de la biopelícula del bebedero se correlacionaron con los resultados de la muestra orofaríngea (OP) colectada de forma simultánea de parvadas en una granja con influenza aviar de baja patogenicidad en las que se compararon directamente las dos estrategias de muestreo. Para evaluar la utilidad del muestreo de la biopelícula para la detección del virus de la influenza aviar altamente patógena (HPAIV), se recolectaron diariamente biopelículas e hisopos orofaríngeos de grupos de mortalidad de parvadas de pavos negativas en una granja positiva para la influenza aviar de alta patogenicidad. Los hisopos de biopelículas fueron positivos de uno a dos días antes de que aparecieran resultados positivos en las muestras orofaríngeas agrupadas. La estrategia de muestreo de la biopelícula del bebedero eliminó la dificultad de encontrar un ave infectada subclínicamente en una población al recolectar material de un gran número de individuos y analizar una muestra en la que persiste una señal positiva durante varios días o semanas. El método de muestreo es adecuado para su uso en casetas de pavos y se ha utilizado de manera confiable en los programas de vigilancia activa y pasiva para el virus de influenza aviar tanto de baja como de alta patogenicidad utilizando transcripción reversa y reacción en cadena de la polimerasa en tiempo real.

Avian influenza viruses (AIVs) pose a significant threat to public health, and viral subtypes circulating in natural avian reservoirs can contribute to the emergence of pathogenic influenza viruses in humans. We investigated the prevalence and distribution of AIVs in 8826 migratory and resident wild birds in North America along the Pacific flyway, which is a major north-south migration pathway that overlaps with four other flyways in Alaska providing opportunities for mixing of Eurasian and American origin influenza viruses. Overall, the prevalence of AIVs was low (1%) among the wide range of avian species tested, but we detected AIVs in 69 hunter-harvested waterfowl (Anseriformes) sampled at a national wildlife refuge in California from October 2007 to January 2008. A wide range of subtypes were detected in waterfowl with H6N1, H10N7, H7N3, and H3N5 being the most common. We suspect H6N1 was introduced or remerged in 2007 at this key wintering site for waterfowl along the Pacific Flyway. Over a 3-week period, 13 H6N1 AIVs were isolated from two northern pintails (Anas acuta), three northern shovelers (Anas clypeata), three ring-necked ducks (Aythya collaris), four American widgeon (Anas americana), and one gadwall (Anas strepera). We conclude that a diverse array of AIVs was present and that cross-species transmission was occurring among waterfowl in the central valley wetlands of California.

Birds within the orders Charadriiformes (shorebirds, gulls) and Anseriformes (waterfowl) are reservoir hosts for avian influenza (AI) viruses, but their role in the transmission dynamics of AI viruses is unclear. To date, waterfowl have been the predominant focal species for most surveillance and epidemiological studies, yet gulls, in particular, have been shown to harbor reassortant AI viruses of both North American and Eurasian lineages and are underrepresented in North American surveillance efforts. To address this gap in surveillance, 1346 ring-billed gulls (Larus delawarensis) were sampled during spring and fall migrations and at three breeding sites in 2017 across Minnesota. Results indicate noticeable age-cohort dynamics in AI virus prevalence within ring-billed gulls in Minnesota. Immunologically naive juveniles represented the cohort with the highest prevalence rate (57.8%). Regardless of age, more gulls had AI virus detected in oropharyngeal (OP) than in cloacal (CL) swabs. The high AI virus prevalence within ring-billed gulls, particularly in immunologically naive birds, warrants further targeted surveillance efforts of ring-billed gulls and other closely related species.

Mutations in a wild duck isolate of avian influenza virus were detected in isolates shed by chickens within 1 day after inoculation. The newly adapted virus was transmitted to naive chickens in direct contact and sharing food and water. Two consistent amino acid substitutions in the hemagglutinin have been identified, A198V and S274F, and may be important in transmissibility. Mutants with a 30–amino acid deletion in the neuraminidase stalk region 43–72 (N9 numbering) were recovered from inoculated chickens, but not from naive chickens in contact. The NA stalk mutant virus did not replicate well in Pekin ducks. In vivo viral replication was at low titers and a change in tropism from the respiratory to the digestive tract was observed. Our results indicated that there is a rapid genetic adaptation of wild bird isolates in poultry species, but that resultant viruses may have phenotypes that are intermediate and not fully adapted to the new host.

Public gene sequence databases have become important research tools to understand viruses and other organisms. Evidence suggests that the identifying information for some of the sequences in these databases might not belong to the sequences they are associated with. We developed two tests to conduct a comprehensive analysis of all published sequences of the hemaglutinin and neuramidase genes of avian influenza viruses (AIVs) to identify sequences that may have been misclassified. One test identified sequence pairs with highly similar nucleotide sequences despite a difference of several years between their sampling dates. Another test, which was applied to samples sequenced and deposited more than once, detected sequences with more nucleotide differences to their own than to their closest relatives. All sequences identified as misclassified were further traced to relevant publications to assess the likelihood of contamination and determine if any conclusions were associated with the use of these sequences. Our results suggested that among 4040 published gene sequences examined, approximately 0.8% might be misclassified and that publications using these sequences may include inaccurate statements. Findings from this report suggest that using laboratory-adapted strains and handling multiple samples simultaneously increases the risk of contamination. The tests reported here may be useful for screening new submissions to public sequence databases.

The long-standing evolutionary and ecological relationships between wild birds and influenza A viruses has created a broad pool of viral genetic diversity and a reservoir of potentially transmissible viruses. An understanding of these relationships can help us identify and modify critical control points to reduce transmission of avian influenza viruses into animal and human populations.

High pathogenic (HP) H5N1 avian influenza (AI) infection has been reported in domestic poultry, wildlife, and human populations since 1996. Risk of infection is associated with direct contact with infected birds. The mode of H5N1 spread from Asia to Europe, Africa and the Far East is unclear; risk factors such as legal and illegal domestic poultry and exotic bird trade, and migratory bird movements have been documented. Measures used to control disease such as culling, stamping out, cleaning and disinfection, and vaccination have not been successful in eradicating H5N1 in Asia, but have been effective in Europe.

Vertebrate immune responses to ectoparasites influence pathogen transmission and host fitness costs. Few studies have characterized natural immune responses to ectoparasites and resultant fitness effects on the ectoparasite. These are critical gaps in understanding vertebrate–ectoparasite interaction, disease ecology and host-parasite co-adaptation. This study focused on an ectoparasite of birds—the northern fowl mite (NFM) (Ornithonyssus sylviarum). Based on prior evidence that chickens develop resistance to NFM, these experiments tested two hypotheses: (i) skin inflammation blocks mite access to blood, impairing development, reproduction and survival; and (ii) host immunogenetic variation influences the inflammatory response and subsequent effects on the ectoparasite. On infested hosts, histology of skin inflammation revealed increased epidermal cell number and size, immigration of leukocytes and deposition of serous exudates on the skin surface. Survival of adult mites and their offspring decreased as the area of skin inflammation increased during an infestation. Inflammation increased the distance to blood vessels beyond the length of mite mouthparts (100–160 μm) and prevented protonymphs and adults from reaching a blood source. Consequently, protonymphs could not complete development, evidenced by a significant inverse relationship between inflammation and protonymph feeding success, as well as an increasing protonymph/adult ratio. Adult females were unable to feed and reproduce, indicated by an inverse relationship between inflammation and egg production, and decreasing female/juvenile ratio. These combined impacts of host inflammation reversed NFM population growth. Intensity of inflammation was influenced by the genotype of the major histocompatibility complex (MHC), supporting previous research that linked these immunological loci with NFM resistance. Overall, these data provide a model for a mechanism of avian resistance to an ectoparasitic arthropod and the fitness costs to the parasite of that host defense.

The NS1 protein is known to suppress immune responses in influenza virus-infected hosts. However, the role of NS1 in apoptosis in infected cells is disputed. In this study, through the use of a mutant A/pheasant/California/2373/1998 (H9N2) avian influenza virus (AIV) with a truncated NS1, we have demonstrated that a functional NS1 protein suppresses the induction of interferons in chicken macrophages. However, NS1 appeared to be irrelevant to the regulation of cytokines interleukin (IL)-1βand IL-6, indicating that distinct mechanisms may be employed in the regulation of antiviral and proinflammatory cytokines in chicken immune cells. Our study also showed that this H9N2 AIV induced apoptosis extrinsically through the Fas/Fas ligand (FasL)-mediated pathway. We found that NS1 suppressed the apoptotic process through suppression of the induction of FasL, but not tumour necrosis factor-αor TNF-related apoptosis-inducing ligand. Furthermore, our data indicated that the disruption of a potential binding site for the p85βsubunit of phosphoinositide 3-kinase in the carboxyl terminus of NS1, while having no effect on the regulation of IFN induction, may contribute to the suppression of Fas/FasL-mediated apoptosis. Therefore, suppression of Fas/FasL-mediated apoptosis by NS1 is one of the critical mechanisms necessary to increase infectivity in AIV-infected chicken macrophages.

The duck and chicken are important hosts of avian influenza virus (AIV) with distinctive responses to infection. Frequently, AIV infections in ducks are asymptomatic and long-lasting in contrast to the clinically apparent and transient infections observed in chickens. These differences may be due in part to the host response to AIV infection. Using real-time quantitative PCR, we examined the expression of immune-related genes in response to low pathogenic AIV H11N9 infection in peripheral blood mononuclear cells (PBMC) isolated from the blood of chickens and Pekin ducks. While chicken PBMC expressed IL-1beta and IL-6 at high levels similar to mammalian species, duck PBMC expression levels were minimal or unchanged. Similarly, duck IFN-beta expression was nearly unaffected, whereas chicken expression was highly upregulated. Chicken IFN-gamma was expressed to higher levels than duck IFN-gamma, while IFN-alpha was expressed similarly by both species. IL-2 was elevated early in infection in duck PBMC, but returned to baseline levels by the end of the experiment; in contrast, IL-2 was weakly induced in chicken PBMC at late time points. TLR-7 and MHC class I molecule expressions were conserved between species, whereas duck MHC class II expression was downregulated and chicken expression was unchanged. These results show distinct PBMC expression patterns of pro-inflammatory cytokines and IFNs between species. The differences in pro-inflammatory cytokine and IFN expression reflect the asymptomatic and lasting infection observed in ducks and the tendency towards clinical signs and rapid clearance seen in chickens. These results highlight important differences in the host response to AIV of two species thought to be critical in the genesis and maintenance of epidemic strains of AIV.

The Avian Influenza Coordinated Agriculture Project (AICAP) entitled “Prevention and Control of Avian Influenza in the US” strives to be a significant point of reference for the poultry industry and the general public in matters related to the biology, risks associated with, and the methods used to prevent and control avian influenza. To this end, AICAP has been remarkably successful in generating research data, publications through an extensive network of university- and agency-based researchers, and extending findings to stakeholders. An overview of the highlights of AICAP research is presented.

Live bird markets (LBM) are essential for marketing poultry in many developing countries, and they are a preferred place for many people to purchase poultry for consumption throughout the world. Live bird markets are typically urban and have a permanent structure in which birds can be housed until they are sold. Live bird markets bring together a mixture of bird species that meet the preferences of their customers and that are commonly produced by multiple suppliers. The mixture of species, the lack of all-in-all-out management, and multiple suppliers are all features that make LBM potential sources of avian influenza viruses (AIV), especially for their supply flocks. Live bird markets have been linked to many outbreaks of avian influenza internationally and in the United States. Avian influenza virus is endemic in many, but not all, LBM systems. For instance, AIV has not been isolated from the Southern California LBM system since December 2005, although the risk of new introductions remains. The California LBM system is much smaller than the New York system, handles fewer birds, and has fewer bird suppliers, which, combined with recent avian influenza prevention and control plans, have enabled it to be AIV free for nearly 3 yr.

In this experiment we evaluated the transmission characteristics of a chicken-adapted low-pathogenicity avian influenza virus (LPAIV) of subtype H6N2, from infected chickens to Japanese quail and Pekin ducks, which are commonly sold in live bird markets located in Southern California. The layout of the cages and bird-handling practices were similar to those found in Southern California live bird markets. Five out of 20 chickens were inoculated with LPAIV H6N2, and placed in direct contact with five chickens and in indirect contact with 10 chickens, 10 Japanese quail and 10 Pekin ducks. Transmission of LPAIV was followed in each bird daily for 15 days post inoculation by real-time reverse transcriptase-polymerase chain reaction testing of oropharyngeal and cloacal swab samples. This strain of H6N2 LPAIV, isolated from commercial poultry in California, was transmitted to chickens, quail, and ducks from chickens. An antibody response was detected in ducks by haemagglutination inhibition tests, but avian influenza virus was only detected by reverse transcriptase-polymerase chain reaction in one duck. Avian influenza virus was detected in quail (5 and 7 days post inoculation) before chickens (8 and 9 days post inoculation), all of which were in indirect contact with infected chickens; however, this difference was not statistically significant.

House flies (Musca domestica) and little house flies (Fannia canicularis) were examined for their ability to take up and harbor a velogenic strain of exotic Newcastle disease virus (ENDV) (family Paramyxoviridae, genus Avulavirus). Laboratory-reared flies were allowed to feed on evaporated milk containing ENDV at a virus concentration of 10(8.3) egg infectious dose (EID)50/0.1 ml or on poultry feces containing an ENDV titer of 10(5.8) EID50/0.1 g. Flies exposed to either infectious food source for 24 hr became transiently infected with virus. Virus persisted predominantly in the mid- and hindgut, with relatively little virus isolated from the remainder of the fly body. Virus persisted similarly in both fly species that were fed evaporated milk containing ENDV, with a maximum ENDV titer of 10(5.98) EID50/fly for the house fly and 10(4.78) EID50/fly for the little house fly at 1 day postexposure; titers decreased on subsequent days to 10(2.38) EID50/fly for house fly andor = 1 EID50/fly for little house fly at 5 days postexposure. Both fly species acquired viral titers greater than the infective dose for a susceptible chicken (10(3.0) EID50-10(4.0) EID50). In addition, flies fed evaporated milk containing a high titer of ENDV maintained viral titers above the infective dose for up to 4 days postexposure to the infectious food source. Flies fed on infective feces retained a chicken infective dose for only one day. The decrease in viral titer over time was significantly explained by logistic regression for both fly species (P0.05). The slope of the regression line was not different for the two fly species (P0.05), indicating a similar rate of virus loss.

Although live bird markets (LBMs) have been associated with outbreaks of avian influenza (AI), there are some LBM systems where AI outbreaks are extremely rare events. The California LBMs have not had any detected avian influenza viruses (AIVs) since December 2005. Responses to a detailed questionnaire on the practices and characteristics of the participants in the California low-pathogenic (LP) AI control program have been described to characterize possible reasons for the lack of AI outbreaks in LBMs. Compliance with an LPAI control program that contains active surveillance, prevention, and rapid response measures by those involved in the LBM system, rendering services to dispose of carcasses, no wholesalers, and few third-party bird deliveries was associated with the lack of LPAIV circulating in the Southern California LBM system.

Several RNA viruses such as astrovirus, rotavirus, reovirus and parvovirus have been detected in both healthy and diseased commercial poultry flocks. The aim of this study was to characterize (a) the development of the RNA viral community in the small intestines of healthy broiler chickens from hatch through 6 weeks of age (market age) and (b) the contribution of the breeder source vs. bird age in development of the community structure. Intestinal tissue samples were harvested from breeders and their progeny, processed for viral RNA extraction and sequenced using Illumina Hiseq sequencing technology resulting in 100 bp PE reads. The results from this study indicated that the breeder source influenced the RNA viral community only at hatch but later environment i.e. bird age had the more significant effect. The most abundant RNA viral family detected at 2, 4 and 6 weeks of age was Astroviridae, which decreased in abundance with age while the abundance of Picornaviridae increased with age.

The California 99 (Cal99) variant of infectious bronchitis virus (IBV) was first recovered in 1999 from vaccinated broiler chicken flocks in Central California. The S1 hypervariable region of Cal99 genome was most closely related to Arkansas (Ark) serotype viruses. In this study, the complete genome of Cal99 was sequenced, and the structural protein genes were compared with those of commonly used IBV vaccines as well as those of isolates from naturally occurring outbreaks in different parts of the world, to elucidate potential sources of genetic material. Based on sequence comparison, the prototype Cal99 virus is similar to the apathogenic ArkDPI virus, except in the S1 gene and stretches of sequence in the S2 and M structural protein genes, which are more related to Connecticut (Conn) and Massachusetts (Mass) strain viruses, respectively. We speculate that these two fragments came from a Conn and a Mass virus, respectively, and were incorporated into a virus largely derived from ArkDPI. Since Ark, Conn and Mass strains have been simultaneously used as live vaccines in California, both point mutations and recombination among vaccine strains may have contributed to the emergence of the Cal99 variant virus. Analysis of the structural protein genes of six Cal99 isolates demonstrated that viruses of this serotype may differ substantially in the non-S1 structural genes. Finally, we performed a challenge study with Cal99 and demonstrated that the virus causes late-onset respiratory disease, with a severity comparable to that of the M41 IBV challenge strain.

The mycotoxin, aflatoxin B1 (AFB1) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB1 can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo) are especially sensitive to aflatoxicosis, while Eastern wild turkeys (M. g. silvestris) are likely more resistant. In ovo exposure provided a controlled AFB1 challenge and comparison of domesticated and wild turkeys. Gene expression responses to AFB1 in the embryonic hepatic transcriptome were examined using RNA-sequencing (RNA-seq). Eggs were injected with AFB1 (1 μg) or sham control and dissected for liver tissue after 1 day or 5 days of exposure. Libraries from domesticated turkey (n = 24) and wild turkey (n = 15) produced 89.2 Gb of sequence. Approximately 670 M reads were mapped to a turkey gene set. Differential expression analysis identified 1535 significant genes with |log2 fold change| ≥ 1.0 in at least one pair-wise comparison. AFB1 effects were dependent on exposure time and turkey type, occurred more rapidly in domesticated turkeys, and led to notable up-regulation in cell cycle regulators, NRF2-mediated response genes and coagulation factors. Further investigation of NRF2-response genes may identify targets to improve poultry resistance.

Numerous interspecies disease transmission events, Ebola virus being a recent and cogent example, highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a comprehensive scientific manner. The diversity of information gained from the natural, social, behavioral, and systems sciences is critical to developing and sustainably promoting integrated health approaches that can be implemented at the local, national, and international levels to meet grand challenges. The Concept of One Medicine One Science (COMOS) as outlined herein describes the interplay between scientific knowledge that underpins health and medicine and efforts toward stabilizing local systems using 2 linked case studies: the food system and emerging infectious disease. Forums such as the International Conference of One Medicine One Science (iCOMOS), where science and policy can be debated together, missing pieces identified, and science-based collaborations formed among industry, governmental, and nongovernmental policy makers and funders, is an essential step in addressing global health. The expertise of multiple disciplines and research foci to support policy development is critical to the implementation of one health and the successful achievement of global health security goals.大量跨物种疾病传播事件强调了 人、动物和环境卫生之间的复杂 相互作用，以及用综合性科学方 式解决医疗和健康问题的重要 性，埃博拉病毒的大量跨物种疾 病传播事件就是近期一种有说服 力的示例。从自然、社会、行为 学和系统科学获得的各种信息对 于开发和可持续地促进实施能够 迎接本地、国内和国际水平挑战 的综合医疗方法至关重要。此处 概述的“ 一种疾病一门科学 (COMOS)”理念描述了支撑健康 与医学的科学知识的相互影响， 以及使用两项关联的病例研究（ 食物系统和新发传染病）为稳定 本地医疗系统所做的努力。论坛 （如一种疾病一门科学国际会议 (iCOMOS) 中），参与者可以一 起讨论相关科学和政策，鉴别缺 失的信息，并在行业、政府和非 政府政策制定者和投资者间形成 以科学为基础的合作，因而借由 论坛平台是解决全球医疗问题必 不可少的步骤。支持政策制定的 多学科专门知识和研究焦点对于 实施一体化健康和成功实现全球 医疗安全目标至关重要。.Los numerosos acontecimientos de transmisión de enfermedades entre especies, de los cuales el virus del Ébola es un ejemplo claro y reciente, ponen de manifiesto las complejas interacciones que existen entre la salud humana, animal y medioambiental, así como la importancia de abordar la medicina y la salud de una manera científica e integral. La diversidad de la información obtenida de las ciencias naturales, sociales, conductuales y de los sistemas es fundamental para el desarrollo y fomento sostenibles de enfoques integrados de salud que puedan implementarse a nivel local, nacional e internacional para atender los grandes retos. El concepto de Una medicina una ciencia (Concept of One Medicine One Science, COMOS) esbozado aquí describe la interrelación entre el conocimiento científico que sustenta la salud y la medicina, y los esfuerzos hacia la estabilización de los sistemas locales por medio de dos estudios de casos relacionados: el sistema alimentario y las enfermedades infecciosas emergentes. Los foros como la Conferencia internacional de una medicina una ciencia (International Conference of One Medicine One Science, iCOMOS), donde se hace posible el debate conjunto de la ciencia y la política, la identificación de eslabones perdidos, y la formación de colaboraciones basadas en la ciencia entre los formuladores y fundadores de políticas industriales, gubernamentales y no gubernamentales, representan un paso decisivo para abordar el tema de la salud mundial. La experiencia de múltiples disciplinas y enfoques de investigación para apoyar el desarrollo de políticas es fundamental para la implementación de una salud y el logro de los objetivos relativos a la seguridad de la salud mundial.

The California poultry industry experienced an outbreak of H6N2 avian influenza beginning in February 2000. The initial infections were detected in three commercial egg-laying flocks and a single noncommercial backyard flock but later spread to new premises. The vaccination of pullet flocks with a commercially prepared, killed autogenous vaccine prior to their placements on farms with infected or previously infected flocks was used as a part of the eradication programs for some multiage, commercial egg production farms. The purpose of this study was to follow three vaccinated flocks on two commercial farms to track the immune responses to vaccination. The antibody-mediated responses of the three flocks followed in this study were markedly different. One flock achieved 100% seroconversion at 12.5 wk of age, but by 32 wk of age, all of the hens were seronegative by agar gel immunodiffusion (AGID). In contrast, at 32 wk of age, flocks from the other farm (flocks 2A and 2B) were 95% and 72% seropositive by AGID, respectively. Of the differences that were identified between the vaccination protocols on the two farms, the distinction that could explain the level of disparity between responses is the delivery of the second dose of vaccine with a bacterin on the first farm, which may have interfered with the persistence of immunity in this flock. Hens from flocks 2A and 2B were experimentally challenged at 25 wk of age with H6N2 avian influenza virus. Hens from flock 2A did not transmit virus to naive contact-exposed hens, but hens from flock 2B did. At 34 wk of age, hens from flock 2A were again challenged and naive contact-exposed hens were infected in this second trial. These challenge experiments served to demonstrate that despite detectable antibody responses in flocks 2A and 2B, the birds were protected from infection for less than 21 wk after the second vaccination.

Induced molting in egg-laying hens is an important method for maximizing hen egg production and quality as well as hen health in commercial settings; however, there is growing societal concern over its effects on hen well-being. Using individual hens as their own controls, this research examined the behavior of hens subjected to different treatments of induced molting under premolt, molt, and postmolt conditions. Cage pecking increased in fast-induced subjects and aggression increased in fast-induced and nonfast-induced subjects during the molt. Gakel calling and several aspects of its acoustic structure were much higher during the molt condition in fast-induced subjects only. These data suggest that nonfast-induced molting treatments provide an effective method for inducing molting in hens and improving their well-being by minimizing discomfort due to food deprivation. In addition, these data further support that gakel vocalizations in hens may serve as an effective indicator for assessing well-being in a species otherwise behaviorally stoic in expressing stress or discomfort.

Five antigen capture immunoassay test kits, Directigen Flu A (Becton Dickinson), QuickVue Influenza test kit (Quidel), FLU OIA (ThermoBiostar), Zstat Flu (ZymeTx, Inc.) and NOW FLU A Test (Binax) were used to detect avian influenza virus (AIV) in clinical specimens as per manufacturers' protocols. Each kit was shown to be specific for AIV propagated in embryonating chicken eggs (ECE); other respiratory viruses of poultry tested gave negative results. The Directigen Flu A kit proved to be 10-fold more sensitive than the other kits, capable of detecting 10(4.7) mean embryo lethal dose (ELD50)/ml in allantoic fluid; this is more sensitive than the hemagglutination test using chicken erythrocytes. None of the kits proved to be sufficiently sensitive to reliably detect AIV in oropharyngeal and cloacal swabs collected from chickens experimentally infected with AIV subtype H6N2. In two different experiments, individual swabs and pools of five or six swabs were tested. By virus isolation, 39 individual oropharyngeal swabs tested positive for AIV, but Directigen and Flu OIA only detected 2/39 and NOW FLU A 1/39. Zstat and QuickVue did not detect any. Five individual cloacal swabs positive by virus isolation were negative with all five kits. In a second experiment using pools of five swabs, 26 swab pools were positive by virus isolation and 5/26 were positive by Directigen, the only kit to provide any positive results. Five cloacal swab pools were also positive by virus isolation and 1/5 was positive by Directigen; all other test kits were negative. All of these experiments were performed using the H6N2 subtype of AIV. The results are disappointing, as the kits have proven to be insensitive for detecting AIV when compared with the gold standard, virus isolation. This limits their use in diagnostic field investigations. Individual or groups of chickens could be assumed to be positive for AIV if positive by any of the kits, but a negative result with any of the kits would not prove that birds were AIV free.

We tested blood samples from domestic and captive farmed animals in Minnesota, USA, to determine exposure to severe fever with thrombocytopenia syndrome virus and Heartland-like virus. We found antibodies against virus nucleoproteins in 10%–18% of samples from cattle, sheep, goats, deer, and elk in 24 Minnesota counties.

SUMMARY Hemorrhagic enteritis virus (HEV) is a type II avian adenovirus that causes intestinal hemorrhages accompanied with immunosuppression in 4-to-12-wk-old turkeys. In the present study, a hexon gene-based, quantitative real-time PCR with TaqMan probe was developed and applied to tissue samples from poultry farms to detect and quantify HEV genome copy numbers. The method was confirmed to be rapid, specific, and sensitive for the detection of HEV. This method is an excellent research and diagnostic tool that can be used to study pathogenesis and to gain insights into different phases of infection on poultry farms and for high-throughput epidemiologic investigations.

Chicken anemia virus (CAV) can cause a disease syndrome characterized by severe anemia, bone marrow atrophy, and severe immunosuppression in young chicks. Maternal antibodies prevent these clinical signs but do not prevent infection, transmission of the virus, or immunosuppression. The clinical disease is rare today because of the widespread practice of vaccinating breeders, but the subclinical form of the disease is ubiquitous. The dynamics of CAV infection, CAV antibody responses, relative lymphoid organ weights, and associated lesions were studied in two broiler flocks from a commercial producer. Both groups had detectable CAV antibodies at hatch, which waned over the first 3 wk of life. Both groups had detectable CAV DNA in both thymi and bursae over the same period. At 35 days of age, virus was detectable by polymerase chain reaction in 16 of 20 chickens, and 7 of 20 had detectable antibodies. By 42 days of age, virus was detectable in 18 of 20 chickens, and 18 of 20 had antibodies to CAV. We observed a decrease in relative thymic weights beginning at 35 days of age, coincidental withthe detection of CAV in the thymus. Bursal sizes began to decrease at 28 days of age, coincidental with a rise in antibody titers to infectious bursal disease virus. In this study, we demonstrated that under typical field conditions CAV infections in broilers have unique dynamics unlike those reported in egg laying strains of chickens managed under specific-pathogen-free conditions.

Today, humans, animals, and the environment are remarkably interconnected and interdependent at a global level through international commerce and movement. Thus, we have access to safe and nutritious food that fuels health, medicines and vaccines that protect us and our animals, and natural resources that support good living standards. However, conflicts arise as exponentially growing populations require more food, demand better living standards, and act to preserve the environment. How do we simultaneously produce more food, reduce disease, afford equitable living standards, and create an environment fit for humans, our animals, and wildlife? Science has played a critical role in finding solutions to many of these challenges, but difficult conflicts continue to emerge. For example, strategies that promote efficient production of food—such as concentrated farming systems, monoculture cropping, and chemical inputs of fertilizer, pesticides, and herbicides—have unintended consequences that threaten human, animal, and environmental health ([ 1 ][1]). A more integrated, holistic problem-solving approach informed by science is needed for development of public policies that address these complex problems. A growing “one health” dialogue has focused on emerging disease surveillance, public health preparedness, and policy issues, with less attention being given to connecting these issues to the scientific foundations that underpin pathogen emergence, global health threats, food security, and environmental health. The imbalance has resulted in a compartmentalization of research and policy, sometimes diluting or compromising the efficacy of the one health movement. For example, lack of balanced scientific input imperils policies affecting antibiotic use for efficient food production and, more seriously, modification of plants for improved production of foods under adverse conditions in impoverished countries. There is value in looking back at instances of scientifically informed decision-making that have broadly benefited human, animal, and environmental health, such as the U.S. Clean Air Act of 1970 and the U.S. Clean Water Act of 1972. Human health security today relies on finding common ground at the intersection of humans, animals, and the environment among diverse opinions and values. The concept of One Medicine One Science–based forums that bring together a diversity of scientists, policy professionals, medical experts, food producers, and other relevant stakeholders provides an important opportunity to present scientific knowledge that objectively informs public policy development ([ 2 ][2]). In the absence of scientific information, for instance, concerns about foods genetically modified for enhanced traits have resulted in European markets being closed to African farmers trying to better their standard of living. It has also led to at-risk pregnant women and children lacking access to golden rice, whose high vitamin A content could alleviate as many as 500,000 cases of irreversible blindness in pregnant women and children every year ([ 3 ][3]). Scientifically informed public policies are also needed to find solutions to foreseeable food supply limitations, prepare for existing foreign animal and human disease pathogens (e.g., African swine fever virus and Chikungunya virus), and deal with environmental implications of extreme energy production (e.g., fracking effects on water quality) or industrial pollution of agricultural land in China (4). Overall, balancing competing priorities is a major challenge as societies seek to maximize human health, animal health and welfare, and environmental integrity. Reductionism may be a natural reaction to complexity, but we are in need of evolving ways to discuss, understand, and address these complex challenges. ![Figure][4] Strategies to increase food production, such as pesticide use, have unintended consequences. PHOTO: LENS/GETTY IMAGES 1. [↵][5] 1. R. S. DeFries, 2. J. A. Foley, 3. G. P. Asner , Front. Ecol. Environ. 2, 249 (2004). [OpenUrl][6][CrossRef][7][Web of Science][8] 2. [↵][9] 1. D. A. Travis 2. et al ., Ann. N.Y. Acad. Sci. 1334, 26 (2014). [OpenUrl][10][CrossRef][11][PubMed][12] 3. [↵][13] Golden Rice Project ([www.goldenrice.org][14]). 4. 1. C. Larson , Science 343, 1415 (2014). [OpenUrl][15][Abstract/FREE Full Text][16] [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: pending:yes [5]: #xref-ref-1-1 "View reference 1 in text" [6]: {openurl}?query=rft.jtitle%253DFront.%2BEcol.%2BEnviron.%26rft.volume%253D2%26rft.spage%253D249%26rft_id%253Dinfo%253Adoi%252F10.1890%252F1540-9295%25282004%2529002%255B0249%253ALCBHNA%255D2.0.CO%253B2%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [7]: /lookup/external-ref?access_num=10.1890/1540-9295(2004)002[0249:LCBHNA]2.0.CO;2&link_type=DOI [8]: /lookup/external-ref?access_num=000223960800016&link_type=ISI [9]: #xref-ref-2-1 "View reference 2 in text" [10]: {openurl}?query=rft.jtitle%253DAnn.%2BN.Y.%2BAcad.%2BSci.%26rft.volume%253D1334%26rft.spage%253D26%26rft_id%253Dinfo%253Adoi%252F10.1111%252Fnyas.12601%26rft_id%253Dinfo%253Apmid%252F25476836%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [11]: /lookup/external-ref?access_num=10.1111/nyas.12601&link_type=DOI [12]: /lookup/external-ref?access_num=25476836&link_type=MED&atom=%2Fsci%2F347%2F6219%2F242.1.atom [13]: #xref-ref-3-1 "View reference 3 in text" [14]: http://www.goldenrice.org [15]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.issn%253D0036-8075%26rft.aulast%253DLarson%26rft.auinit1%253DC.%26rft.volume%253D343%26rft.issue%253D6178%26rft.spage%253D1415%26rft.epage%253D1416%26rft.atitle%253DChina%2BGets%2BSerious%2BAbout%2BIts%2BPollutant-Laden%2BSoil%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.343.6178.1415%26rft_id%253Dinfo%253Apmid%252F24675928%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [16]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEzOiIzNDMvNjE3OC8xNDE1IjtzOjQ6ImF0b20iO3M6MjQ6Ii9zY2kvMzQ3LzYyMTkvMjQyLjEuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA) and nonstructural proteins (NS) among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF) in the cleavage site in HA and glutamate at position 92 (D92E) in NS1. This is the first report of the pathogenicity of the HPAIVH5N1 strain currently circulating in naturally infected poultry in Egypt, which may provide unique insights into the viral pathogenesis in HPAIV-infected chickens and ducks.

Six type-A low pathogenic influenza viruses from 14 Red-necked Grebes (Podiceps grisegena) from Agassiz National Wildlife Refuge were sequenced. The grebe viruses were closely related to North American duck viruses. The genetic and temporal subtype consistency between the duck and grebe isolates suggest spillover events, potentially enhanced by feather eating.

Introductions of H7 influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAVs in Minnesota (MN) turkey farms during 2009 and 2011. The full genome was sequenced from eight isolates as well as the haemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum-likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAVs, estimated the time and geographical origin of the ancestral viruses, and determined the relatedness between the 2009 and 2011 turkey-origin H7N9 IAVs. Analyses supported that the 2009 and 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further supported that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAVs isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAVs could not be determined, our findings suggested that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi Flyway.

Avian infection studies with influenza A virus are an important means of assessing host susceptibility, viral pathogenesis, host responses to infection, mechanisms of transmission, and viral pathotype. Complex systems and natural settings may also be explored with carefully designed infection studies. In this chapter, we explore the elements of infection studies, general guidelines for choosing a virus to use, host selection, and many aspects of study design.

The structure of the icosahedral adenovirus capsid is highly conserved among Adenoviridae. In its native form, the hexon is the major capsid protein. The nascent hexon requires the 100 kDa folding protein to fold into its native, trimeric form. The hexon and 100 kDa folding protein were co-expressed in a fowlpox virus (FPV) vector and in the recombinant FPVs (rFPVs) in which the hexon and 100 kDa folding protein genes are cloned head to tail, the native hexon could be detected with indirect immunofluorescence and immunoprecipitation using a native hexon monoclonal antibody. The FPV-@X100 construct, in which the 100 kDa folding protein gene follows the hexon gene in a head to tail fashion, elicited the best humoral response in chickens. An attenuated HEV commercial vaccine elicited higher and longer lasting anti-HEV titers than FPV-@X100. Humoral immunity was also compared in turkeys inoculated with rFPVs expressing the hexon alone, the 100 kDa folding protein alone, or expressing both genes in different configurations. No anti-HEV humoral immune response was detected in turkeys inoculated with the rFPVs expressing the hexon alone or the 100 kDa folding protein alone.