Sylvie Marché

H5N1 high pathogenicity avian influenza virus survival in different types of water.

Abstract Persistence of H5N1 high pathogenicity avian influenza virus (HPAIV), isolated during the epidemic in wild birds in Poland in 2006, was evaluated in three water samples derived from the sources known to host wild water birds (city pond, Vistula river mouth, and Baltic Sea). The virus was tested at two concentrations (104 and 106 median tissue culture infective dose per milliliter) and at three temperatures (4 C, 10 C, and 20 C), representing average seasonal temperatures in Poland. All tested water samples were filtered before virus inoculation, and one unfiltered sample (Baltic seawater) was also tested. Infectivity was determined twice a week over a 60-day trial period by microtiter endpoint titration. The persistence of the virus varied considerably depending on its concentration and also on physico-chemical parameters of the water, such as temperature and salinity. Avian influenza virus survival was the highest at 4 C and the lowest at 20 C. Prolonged infectivity of the virus in Baltic seawater (brackish, 7.8 ppt) was also seen. In distilled water, the virus retained its infectivity beyond the 60-day study period. Interestingly, a devastating effect of the unfiltered fraction of seawater was seen as the virus disappeared in this fraction the quickest in all studied combinations; thus, biologic factors may also affect infectivity of HPAIV.

Evaluation of Different Strategies for the Use of ELISA Tests as First Screening Tools for Serologic Surveillance of Low Pathogenic Avian Influenza in the Belgian Poultry Sector

Abstract Since the emergence of the highly pathogenic avian influenza H5N1, avian influenza surveillance has been expanded in Europe. The serologic monitoring of domestic poultry is usually accomplished using the reference hemagglutination inhibition (HI) test for the detection of H5 and H7 subtypes. However, as the number of tested sera has been increasing, there is a need for another serologic method that could be used as a preliminary screening test. A comparison of four enzyme-linked immunosorbent assay (ELISA) tests (two indirect and two competitive) was conducted, and they showed good specificity and higher sensitivity than the HI test. The selected ELISA tests were then tested using approximately 800 field sera representative of different poultry species, and a simulation was done to determine the best strategy for screening. The first strategy was testing both gallinaceous and nongallinaceous sera with a competitive ELISA and using the HI test for H5 and H7 as a confirmatory test. The second strategy was testing only gallinaceous bird sera with the indirect ELISA with confirmatory H5 and H7 HI and all nongallinaceous sera by the H5 and H7 HI test. In the Belgian poultry context, the best strategy seems to be the use of a blocking ELISA as the primary screening tool to test all the poultry sera, followed by confirmation by H5 and H7 HI test subtyping.

Redesigning the Serological Surveillance Program for Notifiable Avian Influenza in Belgian Professional Poultry Holdings

Abstract This study was aimed at redesigning the Belgian active surveillance program for domestic birds in professional poultry holdings based on a risk analysis approach. A stochastic quantitative analysis, combining all data sources, was run to obtain sensitivity estimates for the detection of an infected bird in the different risk groups identified. An optimal number of holdings for each risk group was then estimated on the basis of the different sensitivities obtained. This study proved to be a useful tool for decision makers, providing insight on how to reallocate the total amount of samples to be taken in the coming year(s) in Belgium, thus optimizing the field resources and improving efficiency of disease surveillance such as required by the international standards.

The impact of viral tropism and housing conditions on the transmission of three H5/H7 low pathogenic avian influenza viruses in chickens

In this study, shedding and transmission of three H5/H7 low pathogenic avian influenza viruses (LPAIVs) in poultry was characterized and the impact of floor system on transmission was assessed. Transmission experiments were simultaneously conducted with two groups of animals housed on either a grid or a floor covered with litter. Transmission was observed for H5N2 A/Ch/Belgium/150VB/99 LPAIV. This virus was shed almost exclusively via the oropharynx and no impact of floor system was seen. Transmission was also seen for H7N1 A/Ch/Italy/1067/v99 LPAIV, which was shed via both the oropharynx and cloaca. A slight increase in transmission was seen for animals housed on litter. H5N3 A/Anas Platyrhynchos/Belgium/09-884/2008 LPAIV did not spread to susceptible animals, regardless of the floor system. This study shows that environmental factors such as floor systems used in poultry barns may act upon the transmission of LPAIVs. However, the level of influence depends on the virus under consideration and, more specifically, its principal replication sites.

Evaluation of Rapid Antigen Detection Kits for the Diagnosis of Highly Pathogenic Avian Influenza H5N1 Infection

Abstract Early detection of highly pathogenic (HP) strains of avian influenza, especially the HP H5N1, is important in terms of controlling and minimizing the spread of the virus. Several rapid antigen detection kits that are able to detect influenza A viruses in less than 1 hr are commercially available, but only a few of them have been evaluated. In this study, four commercially available rapid tests for veterinary usage and two tests for human usage were evaluated and compared. The evaluation of the detection limits of the different tests established with serial dilution of HP H5N1 indicated that most of them have a detection limit of about 105 to 106 50% tissue culture infectious dose/ml. None of the tests was able to detect virus in oral and cloacal swabs 24 hr post–experimental infection of specific-pathogen-free chickens with HP H5N1. However, 48 hr postinfection, almost all of the rapid tests were able to detect infected birds (dead or alive). Moreover, organs were also successful samples for detection of H5N1 with the rapid tests. Unexpectedly, the specificity was not very high for some tests. However, in general in this study, the tests for veterinary usage showed better sensitivity. To conclude, these tests offer good indicative value in the event of an outbreak, but as a result of their low sensitivity and some aspecific reactions, test results always need to be confirmed by other methods.

An experimental model to analyse the risk of introduction of a duck-originated H5 low-pathogenic avian influenza virus in poultry through close contact and contaminative transmission

Aquatic wild birds are often carriers of low-pathogenic avian influenza viruses (LPAIVs). If H5 and H7 LPAIVs are transmitted to poultry and have the opportunity to circulate, a highly pathogenic AIV may arise. Contact with aquatic wild birds is one of the most important ways in which these LPAIVs can be introduced into poultry flocks. In this study, the transmissibility of a duck-originated H5 LPAIV between ducks and chickens was analysed in a series of animal experiments, using different transmission routes. Results indicate that the outcome of virus intake by chickens exposed to infectious ducks depends on the way the virus is presented. Faecally contaminated drinking water proved to be the most efficient route by which the virus can be transmitted to chickens. The results from this study also suggest that some duck-originated H5 LPAIVs may be introduced to poultry but do not have the potential to become established in poultry populations.

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.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

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

Rapid Detection of Eurasian and American H7 Subtype Influenza A Viruses Using a Single TaqManMGB Real-Time RT-PCR

Abstract A real-time reverse transcription PCR (RRT-PCR) targeting a highly conserved HA2 H7 region was developed for the detection of all H7 subtype avian influenza viruses (PanH7). The wide phylogenetic scope and analytical sensitivity and specificity were validated with the use of a panel of 56 diverse influenza A viruses. The detection limit was determined with the use of serial dilutions of Eurasian isolates A/Ck/BE/06775/2003 and A/Ck/It/1067/v99 and North American isolates A/CK/PA/143586/2001 and A/Quail/PA/20304/1998, to be 1 log10 higher than the detection limit of the generic influenza A matrix RRT-PCR (about 2.5 EID50/reaction compared to 0.25 EID50/reaction for matrix). Diagnostic test properties of PanH7 were determined with the use of 102 swabs from A/Ck/It/1067/v99 experimentally infected chickens, and were not affected by the increased detection limit of PanH7. In comparison to matrix RRT-PCR and virus isolation in embryonated chicken eggs (VI), the PanH7 detected more weakly positive oropharyngeal swabs at the onset of the infection. PanH7 diagnostic sensitivity compared to virus isolation (VI) was 83.3% (compared to 72.2% for matrix RRT-PCR); and diagnostic specificity was 88.1% (94.0% for matrix). The PanH7 test can also be tailored to detect only American (AmH7) or only Eurasian (EurH7) strains by changing the mix of forward and reverse primers used in combination with the unique probe. Overall, this new test is a valuable tool for the detection and identification of H7 subtype influenza A.