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Abstract Background Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade. Findings In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (Log2 3.2± 4.2). The hemagglutinin (HA) and neuraminidase (NA) genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM) and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed. Conclusions Despite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans.

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]

Serologic tests are a valuable tool for retrospective surveillance of avian influenza viruses (AIV) and monitoring of postvaccination host immune response. Yet collection of serum samples, particularly in adult breeder chickens, is laborious, intrusive to birds, and may pose a serious risk to the biosecurity of a flock. In this study we compared the level of AIV-specific antibody titers in eggs and serum samples obtained from broiler breeder chickens vaccinated at 6, 12, and 18 wk of age with H5N2-inactivated vaccine. Nucleocapsid protein-specific ELISA and hemagglutination inhibition test (HI) against homologous as well as heterologous antigens were used. The eggs and sera were collected at 22, 30, 45, and 50 wk of age (i.e., 4, 12, 27, and 32 wk after the third and final immunization, respectively). Using ELISA, the number of positive egg yolk samples decreased over time after vaccination, from 97% to 47%, while the seropositivity rate of serum samples was 97%-100% during the whole investigation period. No antibody titers were detected in egg white. By HI, antibody titers in serum samples were higher than in egg yolk samples. Compared to the homologous H5N2 antigen, significantly lower HI titers were obtained by using a heterologous H5N1 virus of clade 2.2.1.2. In addition, no HI titers were detected in egg yolk and/or serum samples tested against the antigen of an Egyptian H5N1 antigenic drift variant of clade 2.2.1.1. This study indicates that egg yolk may be used to monitor the postvaccination immune status of broiler breeder chickens and retrospective serosurveillance-by HI when a matching antigen is available as well as by ELISA-particularly for up to 12 wk postvaccination. Nota de investigación- Ventajas y limitaciones del uso de yema de huevo en comparación con muestras de suero para la serovigilancia para el virus de la influenza aviar. Las pruebas serológicas son una herramienta valiosa para la vigilancia retrospectiva de los virus de la influenza aviar (AIV) y para dar seguimiento a la respuesta inmune del huésped después de la vacunación. Sin embargo, la colección de muestras de suero, especialmente en reproductoras adultas, es laboriosa, causa estrés para las aves y puede representar un grave riesgo para la bioseguridad de una parvada. En este estudio se comparó el nivel de los títulos de anticuerpos específicos contra el virus de influenza aviar en huevos y en muestras de suero obtenidas de pollos de engorde vacunados a las seis, doce y 18 semanas de edad con una vacuna inactivada con el subtipo H5N2. Se utilizó una prueba de ELISA específica para la nucleocápside viral y una prueba de inhibición de la hemaglutinación (HI) contra antígenos homólogos y heterólogos. Las muestras de huevo y suero se recogieron a las 22, 30, 45 y 50 semanas de edad (es decir, a las cuatro, doce, 27, y 32 semanas después de la tercera y última inmunización, respectivamente). Mediante la prueba de ELISA, el número de muestras de yema de huevo positivas disminuyó con el tiempo después de la vacunación, del 97% al 47%, mientras que la tasa de seropositividad de las muestras de suero fue del 97% −100% durante todo el período de investigación. No se detectaron títulos de anticuerpos en la clara de huevo. Por inhibición de la hemoaglutinación, se observó que los títulos de anticuerpos en las muestras de suero fueron más altas que en las muestras de yema de huevo. En comparación con el antígeno H5N2 homólogo, se obtuvieron títulos de inhibición de la hemoaglutinación significativamente más bajos contra un virus H5N1 heterólogo del clado 2.2.1.2. Además, no se detectaron títulos de inhibición de la hemoaglutinación en la yema de huevo y/o en las muestras de suero analizadas contra el antígeno de una variante por deriva antigénica de un virus H5N1 egipcio del clado 2.2.1.1. Este estudio indica que la yema de huevo puede ser usada para determinar el estado inmune después de la vacunación de pollos de engorde y reproductoras para la vigilancia serológica retrospectiva por inhibición de la hemoaglutinación cuando un antígeno homólogo está disponible, así como por ELISA, particularmente para un máximo de doce semanas después de la vacunación. [ABSTRACT FROM AUTHOR]

The article discusses a study that evaluates the impact of concentration, time of exposure, surface porosity, and organic matter on the ability of four commercial chemical disinfectants to inactivate two H5N1 avian influenza viruses (A/H5N1) in 2006 and 2010 from broiler flocks in Egypt. It uses suspension and carrier tests to examine the virus. It emphasizes the need to use high concentrations of and/or extended time of exposure to disinfectants for efficient inactivation of the virus.

The article discusses a study which examined the efficacy of using modified H5 real time-reverse transcriptase-polymerase chain reaction (RRT-PCR) oligonucleotides for detection of divergent avian influenza H5N1 viruses in Egypt. Information is provided on the methods used in conducting the study. Data show the detection limit of modified and original RRT-PCR assays, the avian influenza viruses for comparison between modified and original RRT-PCR, and results of RRT-PCR assays of swab samples.

Highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 causes a devastating disease in poultry but when it accidentally infects humans it can cause death. Therefore, decrease the incidence of H5N1 in humans needs to focus on prevention and control of poultry infections. Conventional control strategies in poultry based on surveillance, stamping out, movement restriction and enforcement of biosecurity measures did not prevent the virus spreading, particularly in developing countries. Several challenges limit efficiency of the vaccines to prevent outbreaks of HPAIV H5N1 in endemic countries. Alternative and complementary approaches to reduce the current burden of H5N1 epidemics in poultry should be encouraged. The use of antiviral chemotherapy and natural compounds, avian-cytokines, RNA interference, genetic breeding and/or development of transgenic poultry warrant further evaluation as integrated intervention strategies for control of HPAIV H5N1 in poultry [ABSTRACT FROM AUTHOR]

The article discusses a research study on detecting divergent avian influenza H5N1 viruses in Egypt through the modified H5 real-time reverse transcriptase (RRT)-polymerase chain reaction (PCR) oligonucleotides. With 104 percent amplification efficiency, the modified RRT-PCR assay was seen to be more sensitive in detecting Egyptian isolates than the original procedure. Results showed 61 field samples registered positive in the modified assay and 51 samples registered positive in the original protocol.

The present study was performed to evaluate the findings in chickens vaccinated with various regimes and challenged with highly pathogenic avian influenza virus (H5). Forty SPF chicks were divided into four groups of 10 birds each, in which group I served as negative control. Group II was challenged with H5N1 avian influenza virus (positive control) at day 31. Group III was given inactivated vaccine at day 10, then challenged with H5N1 virus at day 31. Group IV was vaccinated with recombinant fowlpox vaccine at day old and boosted with inactivated vaccine at day 10, then challenged with HPAIV at day 31. Quantitative RRT-PCR was carried out on tracheal swabs of living birds and organs of dead birds to evaluate viral load. In addition, specimens from trachea, lungs, bursa of Fabricius, spleen and brain were collected from all birds for histopathologic, immunohistologic and electron microscopic examination. Viral RNA and antigen were demonstrated in examined organs in group II only using indirect immunoperoxidase and quantitative RRT-PCR. The pathological lesions detected were severe in group II, far less in group III and mild in groups I and IV. In conclusion, vaccination regime involving the use of two different vaccines resulted in much alleviation of the pathological alterations and conferred a better protection of chicken against highly pathogenic avian influenza than the use of one vaccine. [ABSTRACT FROM AUTHOR]

Abstract: In Egypt, continuous circulation of highly pathogenic avian influenza (HPAI) H5N1 viruses of clade 2.2.1 in vaccinated commercial poultry challenges strenuous control efforts. Here, vaccine-derived maternal AIV H5 specific immunity in one-day old chicks was investigated as a factor of vaccine failure in long-term blanket vaccination campaigns in broiler chickens. H5 seropositive one-day old chicks were derived from breeders repeatedly immunized with a commercial inactivated vaccine based on the Potsdam/H5N2 strain. When challenged using the antigenically related HPAIV strain Italy/98 (H5N2) clinical protection was achieved until at least 10 days post-hatch although virus replication was not fully suppressed. No protection at all was observed against the Egyptian HPAIV strain EGYvar/H5N1 representing a vaccine escape lineage. Other groups of chicks with maternal immunity were vaccinated once at 3 or 14 days of age using either the Potsdam/H5N2 vaccine or a vaccine based on EGYvar/H5N1. At day 35 of age these chicks were challenged with the Egyptian HPAIV strain EGYcls/H5N1 which co-circulates with EGYvar/H5N1 but does not represent an antigenic drift variant. The Potsdam/H5N2 vaccinated groups were not protected against EGYcls/H5N1 infection while, in contrast, the EGYvar/H5N1 vaccinated chicks withstand challenge with EGYvar/H5N1 infection. In addition, the results showed that maternal antibodies could interfere with the immune response when a homologous vaccine strain was used. [Copyright &y& Elsevier]

Abstract: Circulation of an antigenically variant lineage of highly pathogenic avian influenza (HPAI) H5N1 virus in chicken breeder flocks in Egypt is a continuing problem. The protective efficacy of multiple repeated vaccinations using the currently available H5N2 vaccines is unclear. Here, broiler breeder chickens were vaccinated at weeks 6, 12 and 18 with an inactivated H5N2 commercial vaccine. HI-titer against an Egyptian H5N1 field isolate of classic clade 2.2.1 (EGYcls/H5N1) were significantly lower after the first immunization but increased after booster vaccinations. In contrast, no HI titers were induced against an antigenically distinct field virus of the variant lineage of clade 2.2.1 (EGYvar/H5N1). Upon challenge at week 50 mild, if any, clinical signs were observed in the group infected with EGYcls/H5N1 although one of eight (12.5%) birds died. Mortality reached 6/8 (75%) in the EGYvar/H5N1 challenge group. Virus excretion in all vaccinated groups was reduced in amplitude, but in vaccinated surviving birds, time of virus excretion was extended to up to ten days. Strikingly, challenged vaccinated birds kept laying eggs almost throughout the observation period. Virus was detected on the outer egg-shell of 17 of 40 eggs. The majority of the infected eggs were derived from the EGYcls/H5N1 challenged animals; here the virus was detected also in the yolk and albumin. Repeated vaccination using a commercial H5N2-based vaccine broadened the antigen profile of induced antibodies but did not provide adequate protection against heterologous virus variant. In addition, the observation of AIV contaminated eggs from infected flocks highlights the risk of silent virus spread by vaccinated animals and point to eggs as a possible vector. [Copyright &y& Elsevier]

Abstract: The poultry populations of Egypt are endemically infected by highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1. Vaccination was chosen as an auxiliary tool to control HPAIV in poultry. Potency of commercial vaccines regarding emerging variants is under discussion. In the current study efficacy of four different inactivated whole H5 virus vaccines representing different sublineages of HPAIV H5N1 were tested in chickens against challenge viruses currently co-circulating in Egypt and representing two antigenically widely distinct HPAIV H5N1 lineages, i.e., “variant” (clade 2.2.1var) and “proper” (clade 2.2.1pro) viruses. All vaccines induced clinical protection against challenge with 2.2.1pro Egyptian strains. In contrast, when challenged with a variant strain, only chickens vaccinated with the homologous Egyptian clade 2.2.1var virus or an inactivated re-assorted H5N1 strain (Re-5, clade 2.3) were protected. However, only the homologous virus induced sterile immunity whereas chickens clinically protected after Re-5 vaccination shed virus at day two after infection indistinguishable to H5N2 vaccines. In conclusion, monitoring vaccine-driven evolution of HPAIV H5N1 by surveillance, antigenic characterization, and challenge studies is essential to assess efficacy of AIV vaccination campaigns. [Copyright &y& Elsevier]

Abstract: In contrast to chickens, there is a paucity of information on the potency of H5 vaccines to protect turkeys against the highly pathogenic avian influenza (HPAI) H5N1 virus infections. In this study, 4 groups, 10 turkey poults each, were vaccinated at seven days old with one of H5N2 or H5N1 commercial vaccines or one of two prepared H5N1 vaccines from a local Egyptian variant HPAI H5N1 (EGYvar/H5N1) strain. At 35 days age, all vaccinated and 10 non vaccinated birds were challenged intranasal with 106 EID50/0.1ml of EGYvar/H5N1. All vaccines used in this study were immunogenic in turkeys. There was no cross reaction between the commercial vaccines and the Egyptian variant H5N1 antigen as obtained by the hemagglutination inhibition test. Birds vaccinated with H5N2 vaccine were died, while other H5N1 vaccinated groups have had 20–40% mortality. The highest virus excretion was found in non-vaccinated infected and H5N2 vaccinated birds. Eleven peculiar amino acid substitutions in H5 protein of the variant strain were existed neither in the vaccine strains nor in the earliest H5N1 virus introduced into Egypt in 2006. In conclusion, single vaccination at seven days old is inadequate for protection of meat turkeys against variant HPAI H5N1 challenge and multi-dose vaccination at older age is recommended. For the foreseeable future, continuous evaluation of the current vaccines in H5N1 endemic countries in the face of virus evolution is a paramount challenge to mitigate the socio-economic impact of the virus. [Copyright &y& Elsevier]