Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Immunological and pathological effects of vitamin E with Fetomune Plus ® on chickens experimentally infected with avian influenza virus H9N2 Walaa F. Awadin a , Abdelfattah H. Eladl b, ⁎ , Reham A. El-Shafei c , Mohamed A. El-Adl d , Hanaa S. Ali e a Department of Pathology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt b Department of Poultry Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt c Department of Pharmacology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt d Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Mansoura University, Egypt e Department of Pathology, Animal Health Research Institute, Mansoura branch, Egypt ARTICLE INFO Keywords: AIV H9N2 Vitamin E Fetomune Plus ® Gene expression Histopathology Immunohistochemistry Chickens ABSTRACT Avian influenza virus (AIV) H9N2 infection causes economic losses on poultry farms, and immunostimulants are essential for improving chicken immunity. This study evaluated the immunological and pathological effects of vitamin E with Fetomune Plus ® (a commercial product based on a yeast extract and vitamins) on chickens experimentally infected with AIV H9N2. Three groups of white Hy-Line chicks were included. The G1 group was kept as an uninfected untreated control, the G2 group was intranasally infected with the AIV H9N2 strain (0.5 ml of 10 6 50% egg infectious dose (EID 50 )), and the G3 group was infected and treated with vitamin E (200 mg/kg of diet) and Fetomune Plus ® (1 ml/liter of drinking water) for four weeks. The gene expression of interferon- gamma (IFN-γ), interleukin (IL)-6, and IL-2 was determined at 3, 5 and 7 days post-infection (PI). Virus shedding titers and rates and haemagglutination inhibition (HI) antibody titers were detected. Clinical signs, mortalities and post-mortem lesions were recorded. The birds were weighed, and relative organ weights were calculated. Tissue specimens were taken for histopathological examination and immunohistochemistry (IHC). The expres- sion of IFN-γ in the duodenum revealed a significant increase in G2 compared to G3 at 3 days PI, while the duodenal and splenic expression of IL-6 was significantly increased in G2 compared to G3 at 5 days PI. IL-2 was overexpressed in the duodenum in G3 compared to G2 at 3 and 5 days PI. A significant decrease (P ≤ 0.05) in the virus shedding titer and an increase in the HI titers were detected in G3 compared to G2. The clinical signs and the mortality rate were clearly appeared in G2 than in G3. By IHC, lower H9N2 staining intensity was observed in the examined organs from G3 than in those from G2. In conclusion, as a first report, vitamin E with Fetomune Plus ® supplementation for four weeks could improve the immunological and pathological effects of H9N2 in- fection on chickens. 1. Introduction Avian influenza virus (AIV) H9N2 circulates widely in domestic poultry around the world (Karimi-Madab et al., 2010). The patho- genicity of AIV H9N2 is variable and depends on the presence of other pathogens. Although AIV H9N2 is classified as a low pathogenic strain, it can cause serious disease and high mortality in broilers due to mixed infections (Seifi et al., 2010). The genome sequence of the first AIV H9N2 isolate from chickens in Egypt was reported in 2012 by Abdel- Moneim et al. (2012). Commonly, the immune response in poultry can be enhanced by preventing deficiency syndromes via the administration of vitamins at https://doi.org/10.1016/j.vetmic.2019.02.028 Received 2 January 2019; Received in revised form 19 February 2019; Accepted 19 February 2019 Abbreviations: AIV, avian influenza virus; LPAI, low pathogenic avian influenza; IHC, immunohistochemistry; IL, interleukin; IFN-γ, interferon-gamma; HI, hae- magglutination inhibition; GMTs, geometric mean titers; BW, body weight; PI, post-infection; RNA, ribonucleic acid; PCR, polymerase chain reaction; cDNA, complementary deoxynucleic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPF, high power field; PBS, phosphate buffered saline; NDV, Newcastle disease virus; MEVAC, Middle East for veterinary vaccines; EID 50 , 50% embryo infective dose; DAB, diaminobenzidine; Ct, threshold cycle; NLQP, National la- boratory for veterinary quality control on poultry production; HA, haemagglutinin; ANOVA, analysis of variance; IKKγ,IκBα kinase gamma; NF-κB, nuclear factor- κappaB ⁎ Corresponding author. E-mail address: abdelfatahpo@yahoo.com (A.H. Eladl). Veterinary Microbiology 231 (2019) 24–32 0378-1135/ © 2019 Elsevier B.V. All rights reserved. T