Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Background and Aim: Highly pathogenic avian influenza H5N8 virus of clade 2.3.4.4 was newly emerged to Egypt and firstly detected in carcasses of wild birds in November 2016. This study assessed the protection efficacy and virus shedding reduction of three different inactivated avian influenza (AI) H5 (H5N1, H5N2, and H5N3) commercial vaccines against challenge with two newly emerging highly pathogenic AI virus H5N8 Egyptian isolates in specific-pathogen-free (SPF) chicks. Materials and Methods: 10-day-old SPF chicks (n=260) were divided into 20 groups (n=13). Groups 1-5 were vaccinated through the subcutaneous route (S/C) with 0.5 mL of H5N1 vaccine, Groups 6-10 were vaccinated (S/C) with 0.5 mL of H5N2 vaccine, and Groups 11-15 were vaccinated (S/C) with 0.5 mL of H5N3 vaccine. Positive control groups (16-19) were challenged at 25 and 31 days old (2 and 3 weeks post-vaccination [PV]) using H5N8 clade 2.3.4.4 A/duck/Egypt/ F13666A/2017(H5N8) and H5N8 clade 2.3.4.4 A/chicken/Egypt/18FL6/2018(H5N8). Group 20 was left non-vaccinated as a control. All vaccinated groups were divided and challenged with both viruses at 25 and 31 days of age. The viral challenge dose was 0.1 mL of 106 EID50/0.1 mL titer/chick, and it was administered oronasally. All chicks were kept in isolators for 14 days after each challenge. Sera samples were collected weekly and at 2 weeks post-challenge (PC) to detect a humoral immune response. PC mortalities were recorded daily for 10 days to calculate the protection percentages. Tracheal swabs were collected from the challenged chicks in different groups at 3, 5, 7, and 10 days PC. Kidneys and spleens were collected at 3, 5, 7, and 10 days PC and kept in formalin for histopathological examination to assess lesions and severity scores. Tracheal swabs were inoculated in 10-day-old SPF embryonated chicken eggs for virus titration and to calculate shedding levels. Results: All studied vaccines displayed 70-100% protection within 10 days PC. Hemagglutination inhibition results from sera samples revealed antibody titers ranging from 0.6 to 5.4 log2 starting at 1-week PV with the highest titers at 4 weeks PV. Challenged SPF chickens exhibited a notable reduction in virus shedding, with an average of 1.5-2 log10, compared to control birds. Various histopathological lesions with different scores were detected. Conclusion: Our findings suggest that the inadequate virus shedding reduction and protection efficacy of studied vaccines were variable and that the type of vaccine to be used under field conditions should be reconsidered. Study of the variability between the Egyptian old emerged AI (AIV) 2017 H5N8 strains and the new emerging AIV 2018 H5N8 is required to achieve optimal protection and limit the current economic losses.

Background and Aim: Avian influenza (AI), which is one of the major respiratory diseases of poultry, and Escherichia coli (E. coli) have caused major economic losses around the world, including in Egypt. Therefore, in this study, we aimed to produce a vaccine from E. coli O157 and AI H5N1 formulated with Montanide ISA70 for the protection of poultry against both diseases. Materials and Methods: We divided one hundred 3-week-old chicks into four groups: Group 1 was vaccinated with prepared inactivated AI H5N1formulated with Montanide ISA70, Group 2 was vaccinated with inactivated E. coli formulated with Montanide ISA70, Group 3 was vaccinated with combined E. coli and AI H5N1 formulated with Montanide ISA70, and Group 4 was an unvaccinated control group. We measured the immune response using the HI (hemagglutination inhibition) test, enzyme-linked immunosorbent assay (ELISA), and the challenge test. Results: We found the three vaccines to be safe and sterile during all periods of examination and observation. The HI test showed that Group 1 exhibited specific antibody titers of 2.3 log(2), 4.3 log(2), 7.5 log(2), 7.8 log(2), 8 log(2), and 8.1 log(2) from week 2 to week 7, respectively, post-vaccination. Group 3 exhibited antibody titers of 3.3 log(2), 5.8 log(2), 7.8 log(2), 8 log(2), 8.3 log(2), and 8.3 log(2) from week 2 to week 7, respectively, post-vaccination. The immune response in both groups reached a high titer at week 6. The combined inactivated E. coli and AI H5N1 vaccine generated a higher immune response than the inactivated AI H5N1 vaccine, and a significant difference exists between the two groups. For Groups 2 and 3, the ELISA antibody titer exhibited its lowest value, 1996.5 and 2036.7, respectively, at week 1 post-vaccination; whereas, both groups exhibited the highest titers, 2227.7 (for Group 2) and 2287.3 (for Group 3), in week 3 post-booster. The ELISA for the combined inactivated E. coli and AI H5N1 vaccine had a higher titer than did the inactivated E. coli vaccine, and a significant difference exists between the two groups. Moreover, the protection rate was higher in Group 3, with 100% for E. coli and 90% for the AI H5N1 vaccine. Conclusion: Our findings demonstrate that producing a combined vaccine using E. coli and AI H5N1 formulated with Montanide ISA70 is recommended for protection against both diseases.