Benjamin R. Wilcox

Influenza-A Viruses in Ducks in Northwestern Minnesota: Fine Scale Spatial and Temporal Variation in Prevalence and Subtype Diversity

Waterfowl from northwestern Minnesota were sampled by cloacal swabbing for Avian Influenza Virus (AIV) from July – October in 2007 and 2008. AIV was detected in 222 (9.1%) of 2,441 ducks in 2007 and in 438 (17.9%) of 2,452 ducks in 2008. Prevalence of AIV peaked in late summer. We detected 27 AIV subtypes during 2007 and 31 during 2008. Ten hemagglutinin (HA) subtypes were detected each year (i.e., H1, 3–8, and 10–12 during 2007; H1-8, 10 and 11 during 2008). All neuraminidase (NA) subtypes were detected during each year of the study. Subtype diversity varied between years and increased with prevalence into September. Predominant subtypes during 2007 (comprising ≥5% of subtype diversity) included H1N1, H3N6, H3N8, H4N6, H7N3, H10N7, and H11N9. Predominant subtypes during 2008 included H3N6, H3N8, H4N6, H4N8, H6N1, and H10N7. Additionally, within each HA subtype, the same predominant HA/NA subtype combinations were detected each year and included H1N1, H3N8, H4N6, H5N2, H6N1, H7N3, H8N4, H10N7, and H11N9. The H2N3 and H12N5 viruses also predominated within the H2 and H12 subtypes, respectively, but only were detected during a single year (H2 and H12 viruses were not detected during 2007 and 2008, respectively). Mallards were the predominant species sampled (63.7% of the total), and 531 AIV were isolated from this species (80.5% of the total isolates). Mallard data collected during both years adequately described the observed temporal and spatial prevalence from the total sample and also adequately represented subtype diversity. Juvenile mallards also were adequate in describing the temporal and spatial prevalence of AIV as well as subtype diversity.

Avian Influenza Virus in Aquatic Habitats: What Do We Need to Learn?

Abstract Although aquatic habitats utilized by wild and domestic birds potentially can provide a bridge for avian influenza virus (AIV) transmission among many diverse hosts, the factors controlling environmental persistence and transmission via these habitats are poorly understood. AIV has been detected in water samples collected in the field, and under experimental laboratory conditions, these viruses can remain infective in water for periods of time that would be consistent with an environmental reservoir. However, the application of laboratory results to field realities is complicated by the complexity and scale of these systems. In this brief review, we present a summary of existing research on the environmental tenacity of AIV, provide an example of the challenges associated with the application of laboratory results to the field realities associated with detection of AIV from environmental sources, and identify gaps in our current understanding of the factors potentially affecting AIV infectivity in the environment, specifically from aquatic habitats utilized by wild birds.

Prevalence of antibodies to type A influenza virus in wild avian species using two serologic assays

Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

West Nile Virus Antibody Prevalence in American Crows (Corvus brachyrhynchos) and Fish Crows (Corvus ossifragus) in Georgia, U.S.A

Abstract Crows have been the centerpiece of avian West Nile virus (WNV) surveillance and research in North America. This work has demonstrated variation in susceptibility to WNV infection between American (Corvus brachyrhynchos) and Fish Crows (Corvus ossifragus). The higher WNV-associated mortality rate in American Crows compared with Fish Crows suggests that WNV antibody prevalence would be greater in the Fish Crow population. The objectives of this study were to 1) determine whether Fish Crows had higher WNV antibody prevalence than American Crows, 2) determine the persistence of antibodies to WNV in naturally infected Fish Crows, and 3) develop a technique to distinguish Fish Crows from American Crows on the basis of sequence analysis and restriction enzyme digestion of a mitochondrial DNA fragment. West Nile virus antibody prevalence was 16.5% (n = 97) in Fish Crows and 5.7% in American Crows (n = 53) collected from Georgia between 2004 and 2006. Antibodies persisted at high titers for 12 mo in Fish Crows. This is the first report of WNV antibody persistence in a crow species. A polymerase chain reaction technique paired with restriction enzyme digestion easily distinguished American Crows from Fish Crows on the basis of a mitochondrial DNA fragment.

Evidence for seasonal patterns in the relative abundance of avian influenza virus subtypes in blue-winged teal ( Anas discors )

Abstract Seasonal dynamics of influenza A viruses (IAVs) are driven by host density and population immunity. Through an analysis of subtypic data for IAVs isolated from Blue-winged Teal (Anas discors), we present evidence for seasonal patterns in the relative abundance of viral subtypes in spring and summer/autumn.

ARE PASSERINE BIRDS RESERVOIRS FOR INFLUENZA A VIRUSES

Abstract Although peridomestic passerine species have been involved in influenza A virus (IAV) outbreaks in poultry, there is little evidence to indicate they serve as reservoirs for these viruses under natural conditions. Recent molecular-based detections of IAV in terrestrial wild birds have challenged this paradigm, and it has been suggested that additional research is warranted to better define the role of these birds as IAV hosts. To address this need, we reviewed the published literature reporting results from IAV surveillance of passerines. We also conducted prospective virologic and serologic surveillance of North American passerines for IAVs. The literature review included 60 publications from 1975–2013 that reported results from 829 species of passerines and other terrestrial birds. In our prospective study during 2010 and 2011, 3,868 serum samples and 900 swab samples were collected and tested from 102 terrestrial wild bird species from Georgia, New Jersey, Delaware, and Minnesota, USA. Antibodies to the nucleoprotein of IAV were detected with a commercial blocking enzyme-linked immunosorbent assay in 4/3,868 serum samples (0.1%); all positive samples were from Minnesota. No virus was detected in 900 swab samples by virus isolation in embryonated chicken eggs or matrix real-time reverse transcriptase PCR. Our results are consistent with historic literature; although passerines and terrestrial wild birds may have a limited role in the epidemiology of IAV when associated with infected domestic poultry or other aberrant hosts, there is no evidence supporting their involvement as natural reservoirs for IAV.

Canada Geese and the Epidemiology of Avian Influenza Viruses

Canada geese (Branta canadensis) are numerous, highly visible, and widely distributed in both migratory and resident populations in North America; as a member of the order Anseriformes, they are often suggested as a potential reservoir and source for avian influenza (AI) viruses. To further examine the role of Canada Geese in the ecology of AI, we re-evaluated existing literature related to AI virus in this species and tested breeding populations of Canada Geese from three states (Georgia, West Virginia, and Minnesota, USA) by virus isolation and serology. The ability of AI virus to persist in goose feces under experimental conditions also was evaluated as an additional measure of the potential for this species to serve as an AI virus reservoir. Virus was not isolated from 1,668 cloacal swabs and type-specific antibody prevalence was low (4/335, 1.2%). Finally, under experimental conditions, AI virus persistence in goose feces and in water contaminated with goose feces was limited as compared to published estimates from duck feces and water. Our results are consistent with historic reports of a low prevalence of AI virus infection in this species, and we suggest that Canada Geese play a minor, if any, role as a reservoir for low pathogenic AI viruses that naturally circulate in wild bird populations.

Isolation of Influenza A Viruses from Wild Ducks and Feathers in Minnesota (2010–2011)

SUMMARY We investigated the feasibility of testing feathers as a complementary approach to detect low pathogenic influenza A viruses (IAVs) in wild duck populations. Feathers on the ground were collected at four duck capture sites during 2010 and 2011, in Minnesota, U. S. A. IAVs were isolated from both feathers and cloacal swabs sampled from ducks at the time of capture. Although virus isolation rates from feather and cloacal swabs were inconsistent between collections, the overall rate of isolation was greatest from the feather samples. Viruses isolated from feathers also reflected the subtype diversity observed in cloacal swab isolates but resulted in many more isolates that contained more than one virus. Our study suggests that testing feathers may represent an alternative noninvasive approach to recover viruses and estimate subtype abundance and diversity. RESUMEN Nota de Investigación—Aislamiento del virus de la influenza aviar de patos silvestres y de plumas en Minnesota (2010–2011). Se investigó la viabilidad de analizar las plumas como un enfoque complementario para detectar al virus de influenza aviar tipo A de baja patogenicidad (IAV) en poblaciones de patos silvestres. Se recolectaron plumas del suelo de cuatro sitios de captura de patos durante los años 2010 y 2011, en Minnesota, en los Estados Unidos. Los virus de influenza aviar de baja patogenicidad fueron aislados de las plumas y de los hisopos cloacales recolectados de los patos en el momento de la captura. Aunque los porcentajes de aislamiento del virus de las plumas y de los hisopos cloacales fueron inconsistentes entre las recolecciones, la tasa global de aislamiento era mayor en las muestras de plumas. Los virus aislados a partir de las plumas también reflejaron la diversidad de subtipos observados en los aislados de hisopos cloacales pero resultaron en muchos más aislamientos que contenían más de un virus. Nuestro estudio sugiere que el análisis de las plumas puede representar un enfoque alternativo no invasivo para recuperar los virus y estimar la abundancia de subtipos y su diversidad.

Optimizing Surveillance for South American Origin Influenza A Viruses Along the United States Gulf Coast Through Genomic Characterization of Isolates from Blue‐winged Teal (Anas discors)

Relative to research focused on inter-continental viral exchange between Eurasia and North America, less attention has been directed towards understanding the redistribution of influenza A viruses (IAVs) by wild birds between North America and South America. In this study, we genomically characterized 45 viruses isolated from blue-winged teal (Anas discors) along the Texas and Louisiana Gulf Coast during March of 2012 and 2013, coincident with northward migration of this species from Neotropical wintering areas to breeding grounds in the United States and Canada. No evidence of South American lineage genes was detected in IAVs isolated from blue-winged teal supporting restricted viral gene flow between the United States and southern South America. However, it is plausible that blue-winged teal redistribute IAVs between North American breeding grounds and wintering areas throughout the Neotropics, including northern South America, and that viral gene flow is limited by geographical barriers further south (e.g., the Amazon Basin). Surveillance for the introduction of IAVs from Central America and northern South America into the United States may be further optimized through genomic characterization of viruses resulting from coordinated, concurrent sampling efforts targeting blue-winged teal and sympatric species throughout the Neotropics and along the United States Gulf Coast.

Influenza A Viruses in American White Pelican (Pelecanus erythrorhynchos)

The role of many wild waterbird species in the ecology and epidemiology of avian influenza viruses (AIV) remains unclear. We report the first isolation of AIV from American White Pelicans (Pelecanus erythror-hynchos; Pelecaniformes) in North America. Two H13N9 AIVs were isolated from hatchling birds in breeding colonies in Minnesota, USA, during 2007 and 2008. Based on molecular sequencing of the hemagglutinin and neur-aminidase genes, the 2008 virus was genetically related to AIVs previously isolated from gulls and shorebirds in North America. The 2007 isolate was most related to AIVs from Eurasian gulls and North American ducks, reflecting both global movement of these viruses and reassortment between viruses associated with duck and gull reservoirs.