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Background: Virulent Newcastle disease virus (NDV, avian Avulavirus-1, APMV-1) induces a highly contagious and lethal systemic disease in gallinaceous poultry. APMV-1 antibody detection is used for surveillance and to control vaccination, but is hampered by cross-reactivity to other subtypes of avian Avulaviruses. Data are lacking concerning the applicability of NDV V proteins as differential diagnostic marker to distinguish vaccinated from virus-infected birds (DIVA strategy). Methods: Full length and C-terminally truncated nucleocapsid (NP) protein, and the unique C-terminal regions of the phospho- (P) and V proteins of the NDV LaSota strain were bacterially expressed as fusion proteins with the multimerization domain of the human C4 binding protein, and used as diagnostic antigens in indirect ELISA. Results: When used as diagnostic antigen in indirect ELISAs, recombinant full-length proved to be a sensitive target to detect seroconversion in chickens after APMV-1 vaccination and infection, but revealed some degree of cross reactivity with sera raised against other APMV subtypes. Cross reactivity was abolished but also sensitivity decreased when employing a C-terminal fragment of the NP of NDV as diagnostic antigen. Antibodies to the NDV V protein were mounted in poultry following NDV infection but also, albeit at lower rates and titers, after vaccination with attenuated NDV vaccines. V-specific seroconversion within the flock was incomplete and titers in individual bird transient. Conclusions: Indirect ELISA based on bacterially expressed recombinant full-length NP compared favorably with a commercial NDV ELISA based on whole virus antigen, but cross reactivity between the NP proteins of different APMV subtypes could compromise specificity. However, specificity increased when using a less conserved C-terminal fragment of NP instead. Moreover, a serological DIVA strategy built on the NDV V protein was not feasible due to reduced immunogenicity of the V protein and frequent use of live-attenuated NDV vaccines. [ABSTRACT FROM AUTHOR]

Background: Swine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important. Results: Several reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1av, H1hu, H1huΔ146-147, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the β-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts. Conclusion: The RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1avN1, H1huN2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1huN2Δ146-147) among H1huN2 viruses, as well as reassortant viruses, such as H1huN1 or H1avN2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies. [ABSTRACT FROM AUTHOR]

Low pathogenic avian influenza viruses (LPAIV) of the subtypes H5 and H7 are known to give rise to highly pathogenic (HP) phenotypes by spontaneous insertional mutations which convert a monobasic trypsin-sensitive endoproteolytical cleavage site (CS) within the hemagglutinin (HA) protein into a polybasic subtilisin-sensitive one. Sporadic outbreaks of notifiable LPAIV H7 infections are continuously recorded in Europe and in Asia, and some lineages showed zoonotic transmission. De novo generation of HPAIV H7 from LPAIV precursors has been reported several times over the past decade. Rapid differentiation between LP and HP H7 virus strains is required as a prerequisite to emplace appropriate control measures. Here, reverse transcription real-time PCR assays (RT-qPCR) were developed and evaluated that allow LP and HP pathotype identification and distinction by probe-assisted detection of the HACS. These new RT-qPCRs allow a sensitive and highly specific pathotype identification of Eurasian subtype H7 AIV in allantoic fluids as well as in diagnostic field samples. RT-qPCR assisted pathotyping presents a rapid and sensitive alternative to pathotyping by animal inoculation or nucleotide sequencing. [ABSTRACT FROM AUTHOR]

Background: Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. Case presentation: Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. Conclusions: Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here 2.2.1.2a related to viruses in poultry in Israel, Gaza and Egypt. Enforcement of biosecurity and constant update of vaccine virus strains may be helpful to protect vaccinated birds and prevent spillover infection to neighbouring countries. [ABSTRACT FROM AUTHOR]

Background: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes. Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations. Methods: The nucleocapsid protein (NP) and HA1 fragments of the hemagglutinin protein (HA) of five different lineages (H1N1av, H1N1pdm, H1pdmN2, H1N2, H3N2) of swine influenza viruses were bacterially expressed and used as diagnostic antigens in indirect ELISA. Results: Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation. Western blotting and indirect ELISA revealed highly subtype-specific antigenic characteristics of the recombinant HA1 proteins although some cross reactivity especially among antigens of the H1 subtype were evident. Discrimination of antibodies directed against four swine influenza virus subtypes co-circulating in Germany was feasible using the indirect ELISA format. Conclusions: Bacterially expressed recombinant NP and HA1 swine influenza virus proteins served as antigens in indirect ELISAs and provided an alternative to commercial blocking NP ELISA and HI assays concerning generic (NP-specific) and HA subtype-specific sero-diagnostics, respectively, on a herd basis. [ABSTRACT FROM AUTHOR]

Background: Systematic pathological investigations to assess the health status of seabirds and coastal birds in Germany were performed. The investigation was conducted to obtain data on possible causes of decline in seabird and coastal bird populations. Methods: 48 individuals of 11 different species of seabirds and coastal birds were collected by the stranding network along the entire German North Sea coast from 1997 to 2008, including mainly waders such as Eurasian oystercatchers (Haematopus ostralegus) and red knots (Calidris canutus) as well as seabirds such as northern fulmars (Fulmaris glacialis) and common scoters (Melanitta nigra). For most birds (n = 31) found dead along the shore no obvious cause of death was evident, while 17 individuals were killed by collisions with lighthouses. Results: Overall, the nutritional status of the investigated birds was very poor, and the body mass in most cases was significantly lower compared to masses of living birds caught during the same periods of the year. This is partly linked to chronic parasitic or bacterial infections in different organs or to septicaemia. In some cases infections with zoonotic tuberculosis caused by Mycobacterium spp. were found. Avian influenza was not found in any of the collected birds. Conclusion: The presented data contribute to the evaluation of the health status of birds in the German North Sea. Moreover, they present an important tool for the assessment of potential pathogens with an impact on the health status of seabirds and coastal birds. [ABSTRACT FROM AUTHOR]

Background: There is a continuing threat of human infections with avian influenza viruses (AIV). In this regard falconers might be a potential risk group because they have close contact to their hunting birds (raptors such as falcons and hawks) as well as their avian prey such as gulls and ducks. Both (hunting birds and prey birds) seem to be highly susceptible to some AIV strains, especially H5N1. We therefore conducted a field study to investigate AIV infections in falconers, their falconry birds as well as prey birds. Findings: During 2 hunting seasons (2006/2007 and 2007/2008) falconers took tracheal and cloacal swabs from 1080 prey birds that were captured by their falconry birds (n = 54) in Germany. AIV-RNA of subtypes H6, H9, or H13 was detected in swabs of 4.1% of gulls (n = 74) and 3.8% of ducks (n = 53) using RT-PCR. The remaining 953 sampled prey birds and all falconry birds were negative. Blood samples of the falconry birds tested negative for AIV specific antibodies. Serum samples from all 43 falconers reacted positive in influenza A virus-specific ELISA, but remained negative using microneutralisation test against subtypes H5 and H7 and haemagglutination inhibition test against subtypes H6, H9 and H13. Conclusion: Although we were able to detect AIV-RNA in samples from prey birds, the corresponding falconry birds and falconers did not become infected. Currently falconers do not seem to carry a high risk for getting infected with AIV through handling their falconry birds and their prey. [ABSTRACT FROM AUTHOR]

Background: The endemic status of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR) protocols due to mismatches in the primers/probe binding sites. Results: We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV). A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt. Conclusions: The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt. [ABSTRACT FROM AUTHOR]

Background: Virological, molecular and serological studies were carried out to determine the status of infections with avian influenza viruses (AIV) in different species of wild waterbirds in Iran during 2003-2007. Samples were collected from 1146 birds representing 45 different species with the majority of samples originating from ducks, coots and shorebirds. Samples originated from 6 different provinces representative for the 15 most important wintering sites of migratory waterbirds in Iran. Results: Overall, AIV were detected in approximately 3.4% of the samples. However, prevalence was higher (up to 8.3%) at selected locations and for certain species. No highly pathogenic avian influenza, including H5N1 was detected. A total of 35 AIVs were detected from cloacal or oropharyngeal swab samples. These positive samples originated mainly from Mallards and Common Teals. Of 711 serum samples tested for AIV antibodies, 345 (48.5%) were positive by using a nucleoprotein-specific competitive ELISA (NP-C-ELISA). Ducks including Mallard, Common Teal, Common Pochard, Northern Shoveler and Eurasian Wigeon revealed the highest antibody prevalence ranging from 44 to 75%. Conclusion: Results of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. Mallard and Common Teal exhibited the highest number of positives in virological and serological investigations: 43% and 26% virological positive cases and 24% and 46% serological positive reactions, respectively. These two species may play an important role in the ecology and perpetuation of influenza viruses in this region. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected. [ABSTRACT FROM AUTHOR]

Background: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes. Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations., Methods: The nucleocapsid protein (NP) and HA1 fragments of the hemagglutinin protein (HA) of five different lineages (H1N1av, H1N1pdm, H1pdmN2, H1N2, H3N2) of swine influenza viruses were bacterially expressed and used as diagnostic antigens in indirect ELISA., Results: Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation. Western blotting and indirect ELISA revealed highly subtype-specific antigenic characteristics of the recombinant HA1 proteins although some cross reactivity especially among antigens of the H1 subtype were evident. Discrimination of antibodies directed against four swine influenza virus subtypes co-circulating in Germany was feasible using the indirect ELISA format., Conclusions: Bacterially expressed recombinant NP and HA1 swine influenza virus proteins served as antigens in indirect ELISAs and provided an alternative to commercial blocking NP ELISA and HI assays concerning generic (NP-specific) and HA subtype-specific sero-diagnostics, respectively, on a herd basis.