Didier Vangeluwe

Experimental infection of Carrion crows (Corvus corone) with two European West Nile virus (WNV) strains

West Nile virus (WNV) has become a wide-spread arbovirus in Europe and the Mediterranean Basin countries. This emerging zoonotic disease disseminated 13 years ago in North America where its impact on animal and public health has been considerable. Although American corvids have been the most reliable avian sentinels for WN surveillance in the United States, there is so far no data available about the susceptibility of their Western European counterparts to WNV. Clinical follow-up and serum, oral swabs and feathers viral RNA load monitoring was herein performed on wild-caught Carrion crows (Corvus corone) experimentally inoculated with two WNV strains, Is98 that was isolated from a stork in Israel where it elicited high rates of avian deaths in 1998, and Fr2000 which was only associated to sporadic equine cases in Camargue, France in 2000. Inoculated crows were sensitive to both WNV infections and, as expected from the available epidemiological data, Is98 induced a higher mortality rate (100% vs. 33%) and a quicker fatal outcome, with higher viral RNA loads detected in the serum, oral swabs and feathers than in the Fr2000 group. Therefore, Carrion crows should also be a target species for WNV surveillance in Western Europe, where reporting for abnormal mortalities could be completed by viral detection in the herein described avian matrices. These experimental findings also emphasize the peculiarity of the European situation where a large spectrum of WNV genetic and pathotypic variants have been so far isolated despite limited WN disease reports in wild birds.

Phylogeographic analysis of avian influenza viruses isolated from Charadriiformes in Belgium confirms intercontinental reassortment in gulls

Nine influenza viruses isolated from gulls and shorebirds in Belgium (2008-2010), including H3N8, H5N2, H6N1, H11N9, H13N6, H13N8, and H16N3 subtypes, were targeted using random amplification and next-generation sequencing. The gene segments of these viruses segregated into three phylogeographic lineage types: (1) segments circulating in waterfowl in Eurasia with sporadic introduction in other species and in the Americas (“Eurasian avian”), (2) segments circulating in American waterfowl with sporadic introduction to other species and regions (“American avian”), and (3) segments circulating exclusively in gulls and shorebirds and having increased connectivity between the two hemispheres (“Charadriiformes specific”). Notably, an H6N1 and an H5N2 isolated from L. argentatus had mainly Eurasian avian genes but shared a matrix segment of American avian origin (first documentation in European gulls of transhemispheric reassortment). These data support the growing evidence of an important role of Charadriiformes birds in the dynamic nature of avian influenza ecology.

Genetic characterization of low pathogenic H5N1 and co-circulating avian influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008

As part of a long-term wild bird monitoring programme, five different low pathogenic (LP) avian influenza viruses (AIVs) were isolated from wild mallards (subtypes H1N1, H4N6, H5N1, H5N3, and H10N7). A LP H5N1 and two co-circulating (same location, same time period) viruses were selected for full genome sequencing. An H1N1 (A/Anas platyrhynchos/Belgium/09-762/2008) and an H5N1 virus (A/Anas platyrhynchos/Belgium/09-762-P1/2008) were isolated on the same day in November 2008, then an H5N3 virus (A/Anas platyrhynchos/09-884/2008) 5 days later in December 2008. All genes of these co-circulating viruses shared common ancestors with recent (2001 to 2007) European wild waterfowl influenza viruses. The H5N1 virus shares genome segments with both the H1N1 (PB1, NA, M) and the H5N3 (PB2, HA) viruses, and all three viruses share the same NS sequence. A double infection with two different PA segments from H5N1 and from H5N3 could be observed for the H1N1 sample. The observed gene constellations resulted from multiple reassortment events between viruses circulating in wild birds in Eurasia. Several internal gene segments from these 2008 viruses and the N3 sequence from the H5N3 show homology with sequences from 2003 H7 outbreaks in Italy (LP) and the Netherlands (highly pathogenic). These data contribute to the growing sequence evidence of the dynamic nature of the avian influenza natural reservoir in Eurasia, and underline the importance of monitoring AIV in wild birds. Genetic information of potential hazard to commercial poultry continues to circulate in this reservoir, including H5 and H7 subtype viruses and genes related to previous AIV outbreaks.

Different replication profiles in specific-pathogen-free chickens of two H7 low pathogenic avian influenza viruses isolated from wild birds.

SUMMARY. During an active wild bird survey conducted in Belgium from 2007 to 2011, two low pathogenic avian influenza (LPAI) H7 viruses were isolated from wild birds: an H7N1 virus from a common shelduck (Tadorna tadorna) and an H7N7 virus from a Canada goose (Branta canadensis). The H7 sequence analyses and intravenous pathogenicity indices indicated that they were both low pathogenic isolates and genetically related to other recent European H7 LPAIs isolated from wild birds. Interestingly, the two isolates showed different replication profiles in specific-pathogen-free (SPF) chickens, but poultry can be at risk from both. Indeed, the H7N1 isolated from the common shelduck had the ability to infect and to replicate efficiently in SPF chickens as indicated by high oropharyngeal and cloacal excretions compatible with efficient transmission as well as strong immune responses. On the other hand, the H7N7 isolated from the Canada goose presented a lower replication profile because the inoculated chickens excreted less virus, mostly via the oropharyngeal route, and only three chickens seroconverted. None of the chickens showed clinical signs during the entire infection. Our study using an SPF chicken model underlines that the mechanisms of adaptation of LPAIs in poultry remain unpredictable and are still poorly understood but it represents a powerful tool to gain a better evaluation of the risks of LPAI circulation in poultry.

Evaluation of Four Enzyme-Linked Immunosorbent Assays for the Serologic Survey of Avian Influenza in Wild Bird Species

SUMMARY. Wild birds that reside in aquatic environments are the major reservoir of avian influenza viruses (AIVs). Since this reservoir of AIVs forms a constant threat for poultry, many countries have engaged in AIV surveillance. More and more commercial enzyme-linked immunosorbent assays (ELISA) are available for serologic surveillance, but these tests are often developed and validated for use in domestic poultry. However, for a correct interpretation of ELISA test results from wild bird sera, more information is needed. In the present study, four ELISA test kits (ID-Vet IDScreen®, IDEXX FlockChek™ AI MultiS-Screen Ab Test Kit, Synbiotics FluDETECT™BE, and BioChek AIMSp) were compared for the serologic analysis of 172 serum samples from mallard, mute swan, and Canada goose. Samples were selected based on ID-Vet IDScreen results to obtain an approximately equal number of positive and negative samples. In addition, 92 serum samples from experimentally infected specific-pathogen-free (SPF) chickens and Pekin ducks were included in the tests for validation purposes. Cohens kappa statistics and Spearman correlation coefficients were calculated for each combination of two tests and for each bird species. Test agreement for mallard sera varied from poor to moderate, while test results for Canada goose and swan sera agreed from fair to almost perfect. The best agreement was obtained with sera from experimentally infected SPF chickens and Pekin ducks. This study shows that some care must be taken before using nucleoprotein ELISAs for the testing of sera from wild birds and that more reliable validation studies should be considered before their use in the serologic surveillance of wild birds.

Impact of Age, Season, and Flowing vs. Stagnant Water Habitat on Avian Influenza Prevalence in Mute Swan (Cygnus olor) in Belgium

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

Differential Viral Fitness Between H1N1 and H3N8 Avian Influenza Viruses Isolated from Mallards (Anas platyrhynchos)

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

One Decade of Active Avian Influenza Wild Bird Surveillance in Belgium Showed a Higher Viroprevalence in Hunter-Harvested Than in Live-Ringed Birds

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

Mapping critical habitat of waterbirds in the Arctic for risk management in respect of IFC PS6

Economic development and energy exploration are increasing in the Arctic. Important breeding habitats for many waterbird species, which have previously been relatively undisturbed, are now being subjected to these anthropogenic pressures. The conservation of the habitats and the species they support is a significant challenge for sustainable development. Even if governments and corporates operating in this fragile environment are committed to sustainable development, there is little information available to avoid, mitigate and manage environmental risk and impacts. Taking a risk management perspective, we followed the International Finance Corporations’ (IFC) Performance Standard 6 (PS6) criteria on Environmental and Social Sustainability and developed an approach to identify “critical habitat”, as defined in IFC PS6, for waterbird species breeding in the Arctic. While the range of these waterbirds is roughly known, more accuracy is needed for proper risk assessment. We have therefore gone a step further by modelling suitable habitat within these ranges. Depending on the relevance of the species for IFC PS6 and the level of certainty we separated the classes likely and potential critical habitat. We tested the approach for Russian breeding populations of five Anatidae species (White-fronted Goose Anser albifrons, Lesser White-fronted Goose Anser erythropus, Brent Goose Branta bernicla, Redbreasted Goose Branta ruficollis and Bewick’s Swan Cygnus columbianus bewicki). Likely critical habitats were identified through a review of literature and available data for these waterbird species and multi-species congregations. To address the information gap for most of the Russian Arctic a species distribution modelling approach was used. The outputs of this approach were labelled as potential critical habitat, indicating the lower level of certainty than likely critical habitat. Based on existing information the amount of likely critical habitat is estimated to be at least x,xxx,xxx km2. For the five Anatidae species, X,XXX,XXX km2 potential critical habitat was identified; 95% of these areas were outside of the area boundaries of likely critical habitat for the species. Insufficient data in the east of the study area did affect the results, as some areas known to support breeding populations were not identified as suitable. Conversely, species’ distributions may be overpredicted in other areas; It should also be recognized that the analyzed species currently have depressed populations and may therefore only utilize a proportion of suitable habitat available. For risk assessment purposes however, it is better to predict false positives, rather than false negatives. The study indicates that there are large areas in the Arctic that are potentially important for each of the Anatidae species modelled, but are not yet recognised as key important areas. The results confirm that there is still much to learn about waterbird distribution and abundance in the Russian Arctic. Synthesis and applications The critical habitat maps produced do not just provide a new source of information for the economic development sector, but provide it in a way that is relevant to the sector and directly applicable. The maps are useful for initial risk assessments of potential developments, to identify likely impacts and to consider mitigation options, in accordance with IFC PS6. Risk assessors should exercise caution and detailed surveys for any development in areas predicted to be suitable for each species should be carried out.

Complete Coding Sequences of One H9 and Three H7 Low-Pathogenic Influenza Viruses Circulating in Wild Birds in Belgium, 2009 to 2012

ABSTRACT The complete coding sequences of four avian influenza A viruses (two H7N7, one H7N1, and one H9N2) circulating in wild waterfowl in Belgium from 2009 to 2012 were determined using Illumina sequencing. All viral genome segments represent viruses circulating in the Eurasian wild bird population.