Evaluation of the immuno-stimulatory effect of aqueous neem (Azadirachta indica) leaf extract against highly pathogenic avian influenza (H5N8) in experimental chickens

The recently detected clade 2.3.4.4 of the highly pathogenic avian influenza (HPAI) H5N8 virus in poultry encouraged us to study the efficacy of the 6 most extensively used saleable H5 poultry vaccinations (bivalent [AI + ND], Re-5 H5N1, H5N1, H5N3, monovalent AI, monovalent ND) with or without aqueous 8% neem (Azadirachta indica) leaf extract as an immunostimulant. One hundred thirty birds were randomly divided into 7 groups. Groups 1, 2, 3, 4, 5, and 6 were divided into 2 subgroups (G1a, G2a, G3a, G4a, G5a, G6a) and (G1b, G2b, G3b, G4b, G5b, G6b) with 10 birds each. Subgroups (G1a, G2a, G3a, G4a, G5a, G6a) received the (bivalent [AI + ND], Re-H5N1, H5N1, H5N3, monovalent AI, monovalent ND) vaccines, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) received the same previous vaccination but treated with neem leaf extract administrated 2 d before and after vaccination, and G7 with 10 birds was kept unvaccinated as positive control group. Clinical signs of the challenged group showed conjunctivitis, closed eyes, cyanosis in comb and wattle, ocular discharge, and greenish diarrhea, while postmortem lesions showed congested trachea and lung, hemorrhage on the shank, proventriculus, and pancreas; gelatinous fluid submandibular, congestion of all organs (septicemia), mottled spleen. The clinical signs and lesions were mild in neem leaf extract treated with bivalent vaccine and Re-H5N1 while moderate in monovalent vaccine and H5N3 with or without neem leaf extract treated and reached severe in the group immunized with H5N1 with or without neem leaf extract treatment. The protection levels in the bivalent vaccine (AI + ND), Re-5 H5N1, and H5N3 treated with neem leaf extract, were 80%, 80%, and 60%, respectively, while bivalent vaccine (AI + ND), Re-5 H5N1 and H5N3 without treatment were 60%, 60%, and 40%, respectively. The virus shedding was prevented in groups vaccinated with bivalent vaccine and Re-H5N1 vaccine treated with neem leaf extract, while decreased in the group vaccinated with H5N3 with neem leaf extract and Re-H5N1 without neem leaf extract compared with H5N3, H5N1, and monovalent vaccine. The immunological response after vaccination was stronger in the bivalent vaccine group than in the other commercial vaccine groups treated with neem leaf extract, with geometric mean titer (GMTs) of 315.2 and 207.9 at the third and fourth weeks, respectively. The use of immunostimulant antiviral medicinal plants, such as neem, completely protected chicken flocks against HPAI (H5N8) and prevented AI virus shedding, leading us to the conclusion that the use of bivalent vaccines induces a higher immune response than other different commercial vaccines.