Thermoresistant Newcastle disease vaccine effectively protects SPF, native, and commercial chickens in challenge with virulent virus.

BACKGROUND: Due to the more stability and a better homogenecity in immune response, the use of thermoresistant vaccines in different chicken types has been increased. OBJECTIVE: This study aimed to evaluate the efficacy of a newly developed Newcastle disease vaccine (ND.TR.IR) originating from I-2 strain in specific pathogen-free (SPF) and native and broiler chickens. METHODS: Following determination of pathogenicity indices on the candidate seed, three efficacy examinations were conducted. In the first experiment, 120 1-day-old SPF chickens were randomly allocated to six groups and either vaccinated with ND.TR.IR via eye drop at 1, 7, and 21 days of age (V1 , V7 , and V21 ), or considered as non-vaccinated control groups (C1 , C7 , and C21 ). At 20th post-vaccination day, sera hemagglutination inhibition (HI) antibody titres against ND virus (NDV) were measured and then the chickens were challenged by virulent NDV (vNDV). In the second and third experiments, the efficacy of ND.TR.IR vaccine was compared to routine vaccination program (B1 and LaSota) in native and broiler chickens that were vaccinated at 10 and 20 days of age, respectively. The HI antibody titres were measured on 10, 20, 30, and 40 days of age, and also challenge efficacy test with vNDV was conducted on 30 days of age. RESULTS: The studied virus, as a vaccinal seed, complied with the pathogenicity indices of avirulent NDV and molecular identity of I-2 strain. In the efficacy evaluation trials, the vaccinated chickens had higher HI antibody titres against NDV compared with their corresponding control chickens (p < 0.05). Results of the challenge tests indicated 95% and 100% protection against vNDV in native, SPF, and broiler-vaccinated chickens, respectively. CONCLUSIONS: The present findings indicated that administration of ND.TR.IR induced appropriate HI antibody titres against NDV in SPF, native, and broiler chickens associated with good protection in efficacy test.

Comparison of the Impact of Different Administration Routes on the Efficacy of a Thermoresistant Newcastle Disease Vaccine in Chickens.

Route of vaccine administration has a great impact on immunization and protection outcomes in chickens. This study was conducted to compare the effect of different administration routes on the efficacy of a thermoresistant Newcastle disease (ND) vaccine (ND.TR.IR) in chickens. A total of 100 one-day-old specific pathogen-free chicks were divided into five groups (n = 20 chicks per group) and vaccinated through different routes at 10 and 20 days of age. Treatments included no vaccination (control [C]), 1 dose inoculation through eye drop (ED), 1 dose inoculation through drinking water (DW), 1 dose inoculation through feed (FV1), and 10 doses inoculation through feed (FV10). At 20 and 34 days of age, antibody titers were measured against ND virus (NDV) in all the chickens by hemagglutination inhibition (HI) test. Chicks immunized with ND.TR.IR vaccine through different routes of administration also were intramuscularly challenged with a local virulent NDV (vNDV) (Ck/ir/Beh/2011) 14 days after booster vaccination (at 34 days of age). Our results showed that in comparison with the FVs groups, the immunized chicks through ED induced a higher HI antibody titers at 20 days of age (p < 0.05). Meanwhile, vaccination through ED induced higher HI antibody titers at day 34 of age compared with all other groups (p > 0.05). The percentages of the protective HI antibody titers (≥log23) detected in ED and DW groups at 20 days of age were higher than those detected in the FV1 group (p < 0.05). However, routes of vaccination had no significant effect on the rate of protective titers at day 34 of age (100%, 90%, 75%, and 85% for ED, DW, FV1, and FV10, respectively). The percentage of post-NDV challenge survived chickens was not affected by the route of vaccination (p > 0.05), but immunization of chicks with ND.TR.IR in FV1 group provided relatively poorer protection when compared with the other groups (90% vs. 100%, respectively). Altogether, immunization of chicks with ND.TR.IR vaccine through different routes of administration induced protective NDV antibody HI titers, and provided protection against vNDV. However, when the vaccine was administrated through feed, a higher dose of vaccine is recommended.

Comparative assessment of humoral immune responses of aluminum hydroxide and oil-emulsion adjuvants in Influenza (H9N2) and Newcastle inactive vaccines to chickens.

Context Adjuvants are compounds used in the preparation of inactive vaccines to enhance the immune response. Aluminum hydroxide (alum) is one of the first compounds approved by the Food and Drug Administration, which is used as adjuvants in vaccine products for humans. Montanide ISA 70 is an oil-emulsion adjuvant and is used in poultry inactive vaccines. Objective In this study, the effects of alum adjuvant on the efficiency and induction of immune response in inactive vaccines of Influenza and Newcastle are compared with those of ISA 70. Materials and methods Six groups of 7-d-old specific-pathogen-free chickens were inoculated with 0.3 ml of the prepared vaccines via the subcutaneous route in the neck. Immune response in each group after 7, 14, 21, 31, 41, and 45 d was evaluated using the technique of hemagglutination inhibition. Results The results were compared using SPSS software. Results showed that vaccines containing adjuvant ISA 70 depicted a higher increase in the immune response and adjuvant of 20% alum is similar to adjuvant of ISA 70 in boosting the immune system. There was no statistically significant difference between 10% and 20% alum, but these adjuvants are visibly different from ISA 70. Conclusion In conclusion, alum can be used as an easily accessible, harmless, and effective adjuvant; however, to increase the immune period using the inactive vaccines for poultry, more research would be necessary.

Expression of Factor X in BHK-21 Cells Promotes Low Pathogenic Influenza Viruses Replication.

A cDNA clone for factor 10 (FX) isolated from chicken embryo inserted into the mammalian cell expression vector pCDNA3.1 was transfected into the baby hamster kidney (BHK-21) cell line. The generated BHK-21 cells with inducible expression of FX were used to investigate the efficacy of the serine transmembrane protease to proteolytic activation of influenza virus hemagglutinin (HA) with monobasic cleavage site. Data showed that the BHK-21/FX stably expressed FX after ten serial passages. The cells could proteolytically cleave the HA of low pathogenic avian influenza virus at multiplicity of infection 0.01. Growth kinetics of the virus on BHK-21/FX, BHK-21, and MDCK cells were evaluated by titrations of virus particles in each culture supernatant. Efficient multicycle viral replication was markedly detected in the cell at subsequent passages. Virus titration demonstrated that BHK-21/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in BHK-21 or MDCK cells with TPCK-trypsin. The results indicate potential application for the BHK-21/FX in influenza virus replication procedure and related studies.

Evolutionary characterization of hemagglutinin gene of H9N2 influenza viruses isolated from Asia.

The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994-2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection.

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus.

Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA(1) genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA(1) had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.

Development of rHA1-ELISA for specific and sensitive detection of H9 subtype influenza virus.

A recombinant antigen-based single serum dilution ELISA was developed for simultaneous detection and subtyping of influenza viruses. Recombinant baculovirus encoding the hemagglutinin (HA(1) subunit) of H9N2 virus was generated. To evaluate the rHA1-ELISA, microplates were coated with purified HA1 protein and tested with reference control sera. Subsequently, 92 field sera collected from chickens suspected to be infected with H9N2 AIV were employed to test the efficacy of the rHA1-ELISA. The sera were tested simultaneously by HI and a commercial AIV ELISA kit. The rHA1-ELISA appeared to be highly specific and sensitive for direct detection of H9N2 antibodies in serum samples.