The influence of Spirulina extract on pathogenicity, immune response, and vaccine efficacy against H9N2 avian influenza virus in specific pathogen free chickens.

ABSTRACT

Avian influenza (AI) viruses pose a risk to the worldwide poultry industry. Ultimately, improving the efficiency of the H9N2 vaccine is necessary to better control low-pathogenic avian influenza-H9N2 by using natural immunostimulant. Therefore, the goal of the present study was to examine varying doses of the cyanobacterium Spirulina extract on the effectiveness of H9N2 vaccine. Thus, a total of 150 specific pathogen-free (SPF) chickens were allocated into 6 groups, 25 birds each, as follow G1, G2, and G6 were supplemented with 200, 400, and 400 mg Spirulina extract/kg feed, respectively, whilst the feed in G3, G4, and G5 were not supplemented with Spirulina extract. At 21-days-old, only the chickens in G1, G2, and G3 were vaccinated with the H9N2 AI vaccine. After 4 wk postvaccination, the chickens in G1, G2, G3, G4, and G6 were challenged with H9N2 AI Egyptian strain. The challenged virus was selected from a recent circulating Egyptian strain during 2022, and it was related to A/quail/Hong Kong/G1/97-like virus lineage and clustered with G1-B sub-lineage EGY-2 group. It had a high amino acids identity percentage of 92.6% with the A/chicken/Iran/av1221/1998 (Boehringer Ingelheim) vaccine. The results of real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) revealed that no shedding of the virus was reported in G1, G2, G3, and G5. The supplementation of Spirulina extract in low (200 mg/kg of feed) and high (400 mg/kg of feed) concentration with the birds vaccinated with H9N2 AI vaccine (G1 and G2) induced prominent immuno-stimulatory effect in a dose dependent manner where it strongly enhanced the phagocytic activities of broilers' peripheral blood monocytes, and lysozyme at all days postvaccination (dpv) and days postchallenge (dpc) compared to other groups with significant differences at all day of experiment and 21st dpv, 28th dpv, 7th dpc, and 14th dpc, respectively. The supplementation with Spirulina extract in G1 and G2 induced the highest hemagglutination inhibition antibody titer in a dose-dependent manner at all-time intervals. The antibody titer postvaccination was significantly increased in G1 and G2 at 14th, and 21st dpv, in comparison with G3. Furthermore, G1 and G2 showed higher significant antibody titers at 7th and 14th dpc, compared to other groups. Furthermore, Spirulina extract (200 and 400 mg/kg feed) in G1 and G2 showed anti-inflammatory effect in a dose dependant manner by downregulating nitric oxide levels at all times postchallenge with a significant difference at 3 to 7 dpc compared to G3, G4, and G6, with improved histopathological alterations in the trachea, lung, kidney, spleen, and bursa of Fabricius.  G6 supplied with 400 mg/kg Spirulina extract feed only without vaccination had a similar effect as vaccinated groups on innate immunity. However, it delayed the production of antibodies and did not prevent viral shedding as in vaccinated groups. In conclusion, vaccination in conjunction with either dose of Spirulina extract (G1, and G2) prevents viral shedding, increases the immune response, and reduces inflammation and histopathological change caused by H9N2 AI infection in a dose dependent manner. We recommend the use of 400 mg Spirulina extract/kg feed as a natural immunostimulant in conjunction with the H9N2 vaccine to achieve the highest possible level of protection against H9N2 AI infection.