Sean W. Nashold

PRELIMINARY EVALUATION OF A SIMPLE IN VITRO TEST FOR THE DIAGNOSIS OF TYPE C BOTULISM IN WILD BIRDS

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of type C botulinum toxin (Clostridium botulinum) in wild birds. This simple, antigen-capture ELISA utilizes polystyrene immunosticks as the solid substrate, chicken antitoxin (IgY) as the coating antibody, rabbit antitoxin as the primary antibody, and peroxidase-labeled goat-anti-rabbit as the secondary antibody. To evaluate the immunostick ELISA as a diagnostic test for avian botulism, known concentrations of toxin were added to heparinized blood collected from healthy birds and tested by both the ELISA and mouse bioassay. Also, blood samples from 236 bird carcasses submitted to the National Wildlife Health Center (NWHC) for cause of death determinations were tested by both procedures. Using ≤ 0.5 ml as the test volume for both procedures, the ELISA was less sensitive, detecting 0.25 ng/ml of toxin compared to 0.12 ng/ml for the mouse bioassay. Using the same volume of test sample for diagnostic submissions (≤ 0.5 ml), the ELISA was positive for 60% of the 149 clinically-diagnosed cases of botulism, whereas the mouse bioassay was positive for 79%. However, we demonstrated that with larger sample volumes (≥ 1.0 ml), the sensitivity of the ELISA may be equivalent or better than the mouse test due to the concentrating effect of the ELISA procedure. These preliminary results suggest that when adequate sample volumes are available, the immunostick ELISA can replace the mouse test for the diagnosis of botulism in wild birds.

North Atlantic Migratory Bird Flyways Provide Routes for Intercontinental Movement of Avian Influenza Viruses

Avian influenza virus (AIV) in wild birds has been of increasing interest over the last decade due to the emergence of AIVs that cause significant disease and mortality in both poultry and humans. While research clearly demonstrates that AIVs can move across the Pacific or Atlantic Ocean, there has been no data to support the mechanism of how this occurs. In spring and autumn of 2010 and autumn of 2011 we obtained cloacal swab samples from 1078 waterfowl, gulls, and shorebirds of various species in southwest and west Iceland and tested them for AIV. From these, we isolated and fully sequenced the genomes of 29 AIVs from wild caught gulls (Charadriiformes) and waterfowl (Anseriformes) in Iceland. We detected viruses that were entirely (8 of 8 genomic segments) of American lineage, viruses that were entirely of Eurasian lineage, and viruses with mixed American-Eurasian lineage. Prior to this work only 2 AIVs had been reported from wild birds in Iceland and only the sequence from one segment was available in GenBank. This is the first report of finding AIVs of entirely American lineage and Eurasian lineage, as well as reassortant viruses, together in the same geographic location. Our study demonstrates the importance of the North Atlantic as a corridor for the movement of AIVs between Europe and North America.

Diagnosis of Duck Plague in Waterfowl by Polymerase Chain Reaction

A recently developed polymerase chain reaction (PCR) assay was used for diagnosis of duck plague in waterfowl tissues from past and current cases of waterfowl mortality and to identify duck plague virus in combined cloacal/oral-pharyngeal swab samples from healthy mallards (Anas platyrhynchos) after a disease outbreak. The PCR was able to detect viral DNA from all the individual or pooled tissues assayed from 10 waterfowl, including liver and spleen samples from three Muscovy ducks (Cairina moschata domesticus) that did not yield virus isolates. The strong staining intensity of the PCR products from the waterfowl tissues indicated that large amounts of virus were present, even when virus was not isolated. Duck plague DNA was also detected in a cloacal swab sample from a wood duck (Aix sponsa) carcass submitted for diagnosis. The PCR assay identified duck plague DNA in 13 swab samples that produced virus isolates from carrier mallards sampled in 1981 after a duck plague die-off. The duck plague PCR clearly demonstrated the ability to quickly diagnose duck plague in suspect mortality cases and to detect virus shed by carrier waterfowl.

Dispersal of H9N2 influenza A viruses between East Asia and North America by wild birds

Samples were collected from wild birds in western Alaska to assess dispersal of influenza A viruses between East Asia and North America. Two isolates shared nearly identical nucleotide identity at eight genomic segments with H9N2 viruses isolated from China and South Korea providing evidence for intercontinental dispersal by migratory birds.

Experimental Infection of a North American Raptor, American Kestrel (Falco sparverius), with Highly Pathogenic Avian Influenza Virus (H5N1)

Several species of wild raptors have been found in Eurasia infected with highly pathogenic avian influenza virus (HPAIV) subtype H5N1. Should HPAIV (H5N1) reach North America in migratory birds, species of raptors are at risk not only from environmental exposure, but also from consuming infected birds and carcasses. In this study we used American kestrels as a representative species of a North American raptor to examine the effects of HPAIV (H5N1) infection in terms of dose response, viral shedding, pathology, and survival. Our data showed that kestrels are highly susceptible to HPAIV (H5N1). All birds typically died or were euthanized due to severe neurologic disease within 4–5 days of inoculation and shed significant amounts of virus both orally and cloacally, regardless of dose administered. The most consistent microscopic lesions were necrosis in the brain and pancreas. This is the first experimental study of HPAIV infection in a North American raptor and highlights the potential risks to birds of prey if HPAIV (H5N1) is introduced into North America.

Evolution of a reassortant North American gull influenza virus lineage: drift, shift and stability

BackgroundThe role of gulls in the ecology of avian influenza (AI) is different than that of waterfowl. Different constellations of subtypes circulate within the two groups of birds and AI viruses isolated from North American gulls frequently possess reassortant genomes with genetic elements from both North America and Eurasian lineages. A 2008 isolate from a Newfoundland Great Black-backed Gull contained a mix of North American waterfowl, North American gull and Eurasian lineage genes.MethodsWe isolated, sequenced and phylogenetically compared avian influenza viruses from 2009 Canadian wild birds.ResultsWe analyzed six 2009 virus isolates from Canada and found the same phylogenetic lineage had persisted over a larger geographic area, with an expanded host range that included dabbling and diving ducks as well as gulls. All of the 2009 virus isolates contained an internal protein coding set of genes of the same Eurasian lineage genes except PB1 that was from a North American lineage, and these genes continued to evolve by genetic drift. We show evidence that the 2008 Great Black-backed Gull virus was derived from this lineage with a reassortment of a North American PA gene into the more stable core set of internal protein coding genes that has circulated in avian populations for at least 2 years. From this core, the surface glycoprotein genes have switched several times creating H13N6, H13N2, and H16N3 subtypes. These gene segments were from North American lineages except for the H16 and N3 vRNAs.ConclusionsThis process appears similar to genetic shifts seen with swine influenza where a stable “triple reassortant internal gene” core has circulated in swine populations with genetic shifts occurring with hemaggluttinin and neuraminidase proteins getting periodically switched. Thus gulls may serve as genetic mixing vessels for different lineages of avian influenza, similar to the role of swine with regards to human influenza. These findings illustrate the need for continued surveillance in gull and waterfowl populations, both on the Pacific and especially Atlantic coasts of North America, to document virus intercontinental movement and the role of gull species in the evolution and epidemiology of AI.

Evidence for common ancestry among viruses isolated from wild birds in Beringia and highly pathogenic intercontinental reassortant H5N1 and H5N2 influenza A viruses.

Highly pathogenic clade 2.3.4.4 H5N8, H5N2, and H5N1 influenza A viruses were first detected in wild, captive, and domestic birds in North America in November-December 2014. In this study, we used wild waterbird samples collected in Alaska prior to the initial detection of clade 2.3.4.4 H5 influenza A viruses in North America to assess the evidence for: (1) dispersal of highly pathogenic influenza A viruses from East Asia to North America by migratory birds via Alaska and (2) ancestral origins of clade 2.3.4.4 H5 reassortant viruses in Beringia. Although we did not detect highly pathogenic influenza A viruses in our sample collection from western Alaska, we did identify viruses that contained gene segments sharing recent common ancestry with intercontinental reassortant H5N2 and H5N1 viruses. Results of phylogenetic analyses and estimates for times of most recent common ancestry support migratory birds sampled in Beringia as maintaining viral diversity closely related to novel highly pathogenic influenza A virus genotypes detected in North America. Although our results do not elucidate the route by which highly pathogenic influenza A viruses were introduced into North America, genetic evidence is consistent with the hypothesized trans-Beringian route of introduction via migratory birds.

Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin (Calidris alpina), an intercontinental migrant shorebird species.

Please cite this paper as: Hall et al. (2011). Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin (Calidris alpina), an intercontinental migrant shorebird species. Influenza and Other Respiratory Viruses 5(5), 365–372.

Evaluation of Nobuto Filter Paper Strips for the Detection of Avian Influenza Virus Antibody in Waterfowl

SUMMARY. The utility of using Nobuto paper strips for the detection of avian influenza antibodies was examined in mallards (Anas platyrhynchos) experimentally infected with low pathogenic avian influenza viruses. Blood was collected 2 wk after infection and was preserved either as serum or whole blood absorbed onto Nobuto strips. Analysis of samples using a commercially available blocking enzyme-linked immunosorbent assay revealed comparable results (≥96% sensitivity for all methods) between sera stored at −30 C and the Nobuto strip preservation method even when the Nobuto strips were stored up to 3 mo at room temperature (RT). Significant differences were detected in the ratio of sample absorbance to negative control absorbance for Nobuto strips stored at RT compared with sera stored at −30 C, although these differences did not affect the ability of the test to reliably detect positive and negative samples. Nobuto strips are a convenient and sensitive alternative to the collection of serum samples when maintaining appropriate storage temperatures is difficult.

Avian influenza in shorebirds: experimental infection of ruddy turnstones (Arenaria interpres) with avian influenza virus

Please cite this paper as: Hall et al. (2012) Avian influenza in shorebirds: experimental infection of ruddy turnstones (Arenaria interpres) with avian influenza virus. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2012.00358.x.