Abstract
Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.
Highlights
Avian influenza (AI) is an infectious disease in birds caused by Influenza A viruses belonging to the family Orthomyxoviridae
We describe the development of a multiplex serological assay for subtyping of AI antibodies using Luminex xMAP technology
In this study we report the development of a multiplex serological assay that allows the detection of antibodies against all AI viruses (AIV) subtypes in poultry sera using Luminex xMAP technology
Summary
Avian influenza (AI) is an infectious disease in birds caused by Influenza A viruses belonging to the family Orthomyxoviridae. Primarily waterfowl, gulls and shorebirds, are the natural reservoir of the virus and generally show no clinical signs of infection [1]. Wild birds spread AI viruses (AIV) worldwide during migration [2,3,4]. Influenza A viruses are divided into different subtypes based on the antigenicity of their surface glycoproteins haemagglutinin (HA) and neuraminidases (NA) [1,5]. 16 different HA and 9 different NA subtypes are detected in birds, which can be found in different combinations [6]. Some HA subtypes are primarily detected in specific wild bird species, e.g. H13 subtypes are frequently found in gulls but rarely in other birds [7]. The evolution of AI viruses is rapid and unpredictable, which can lead to a sudden appearance of new virus strains with possible new characteristics and risks
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