2. Evaluation of avian influenza vaccines on commercial layer chicks M.M. Amer, Sherein, S.abdelgayed and Abeer, A. Abd El-Baky

3. Introduction Avian influenza (AI) is one of the highly contagious Office of International Epizootics (OIE) list “A” diseases. The disease is also called “fowl plague” due to its high mortality in chicken. Avian influenza (AI) is one of the highly contagious Office of International Epizootics (OIE) list “A” diseases. The disease is also called “fowl plague” due to its high mortality in chicken. On rare occasions, AI viruses have exhibited interspecies transmissibility to human. On rare occasions, AI viruses have exhibited interspecies transmissibility to human.

4. Introduction Different levels of control measures have been implemented to control the outbreaks that included condemnation of infected farms, strict bio-security and vaccination of commercial chickens Different levels of control measures have been implemented to control the outbreaks that included condemnation of infected farms, strict bio-security and vaccination of commercial chickens Vaccination as a supportive tool in AI virus control strategies was implemented to limit spread of H5N1 and to reduce the losses. Vaccination as a supportive tool in AI virus control strategies was implemented to limit spread of H5N1 and to reduce the losses.

5. Introduction Different types of vaccines are already in use that decrease shedding of virus, morbidity, mortality and transmissibility Different types of vaccines are already in use that decrease shedding of virus, morbidity, mortality and transmissibility through increasing resistance to infection and reducing field virus replication. through increasing resistance to infection and reducing field virus replication.

6. Introduction Evaluation of different types of AI vaccines used in Egypt may provide effective vaccination strategy. Evaluation of different types of AI vaccines used in Egypt may provide effective vaccination strategy. Moreover, the combinant vaccine of avian influenza and Newcastle disease (AI&ND) was recently recommended and commercialized for more protection against AI. Moreover, the combinant vaccine of avian influenza and Newcastle disease (AI&ND) was recently recommended and commercialized for more protection against AI.

7. Purpose of the study The purpose of the current study was to Evaluate inactivated monovalent (H5N1) andEvaluate inactivated monovalent (H5N1) and bivalent (H5N1&ND) AI vaccines bivalent (H5N1&ND) AI vaccines through its immune response, clinicopathological and histopathological effects on vaccinated layer chicks.through its immune response, clinicopathological and histopathological effects on vaccinated layer chicks.

8. Materials and Methods Experimental design 90 one day old commercial layer chicks Group A 30 chicks non vaccinated control group Group B 30 chicks vaccinated at 9 th day with inactivated oil adjuvant monovalent (H5N1) AI vaccine Group C 30 chicks vaccinated at 9 th day with inactivated oil adjuvant bivalent (H5N1&ND) AI vaccine

9. Materials and Methods Sampling The Experimental period was 5 weeks collection of samples was performed weekly Body weight (gm) and organ weight (gm) FOR Chicken performance Blood samples from 10 chicks of each group were collected at weekly intervals FOR Clinicopatholo gical and Serological Examinations Tissue specimens including bursa, thymus and spleen were collected weekly FOR Histopathological Examination

10. Materials and Methods Sampling Blood samples The first blood sample was anticoagulated used for evaluating hemogram Total erythrocyte and leukocyte counts, Packed cell volume (PCV%), Hemoglobin concentration, and Differential leukocytic count. The second blood serum sample Used for haemagglutination inhibition (HI) test + biochemical studies Total proteins, Albumin, Globulins, A/G ratio, Aspartate aminotransferase (AST), Alanine amino transferase (ALT), Alkaline phosphatase (ALP), Blood glucose, Uric acid, Creatinine

11. Results and Discussion

14. 3- Clinicopathological Findings  Erythrogram Erythrogram mean values (packed cell volume (PCV %), hemoglobin concentration (Hb), erythrocytes count (RBCs), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC)) of different experimental groups, in comparison to those of control group (A) showed, insignificant changes in vaccinated groups B and C.

15. Results and Discussion 3- Clinicopathological Findings  Leukogram Mean values of leukogram [total leukocyte count (TLC), neutrophil, lymphocyte and monocyte counts] of different experimental groups showed significant leukocytosis due to significant lymphocytosis started from the 2 nd week in all experimental groups.

16. Results and Discussion 3- Clinicopathological Findings  Leukogram  This lymphocytosis was a result of immunostimulatory effect of vaccination.  Lymphocytosis which observed in group C (vaccinated with bivalent AI vaccine) was higher than those in group B (vaccinated with monovalent AI vaccine)  The avian influenza vaccines have been shown to induce antigen specific lymphocyte responses that may explain the observed lymphocytosis

17. Results and Discussion 3- Clinicopathological Findings  Serum Biochemical Evaluation  Compared to control group, protein profile results showed no significant changes were observed in albumin concentration.  while, significant hyperproteinemia due to hyperglobulinemia with significant decrease in A/G ratio started from 2 nd week in all experimental groups was recorded.

18. Results and Discussion 3- Clinicopathological Findings  Serum Biochemical Evaluation  This hyperglobulinemia may be attributed to the high levels of gamma globulins (immunoglobulin especially IgG and IgA) associated with chicken vaccination by AI vaccine  Hyperglobulinemia which observed in group C (vaccinated with bivalent AI vaccine) was higher than those in group B (vaccinated with monovalent AI vaccine).

19. Results and Discussion 3- Clinicopathological Findings  Serum Biochemical Evaluation  Activities of serum liver enzymes (AST, ALT and ALP) and concentrations of blood glucose, serum creatinine and uric acid showed; insignificant changes throughout the experiment in all groups.  This revealed that the vaccination with both inactivated monovalent and bivalent AI vaccines did not affect liver and kidney functions.

20. Results and Discussion 4- Histopathological Findings  Bursa of Fabricious 1 st week  All groups were normal

21. Bursa of fabricious from all groups at 1week showing apparently normal lymphoid Follicles (H&E X 200).

22. Results and Discussion 4- Histopathological Findings  Bursa of Fabricious 2 nd & 3 rd weeks Groups A & C were normal. Group B revealed interfollicular congestion

23. Bursa of fabricious from group B at 2 nd & 3 rd weeks showing interfollicular congestion (H&E X 200).

24. Results and Discussion 4- Histopathological Findings  Bursa of Fabricious 4 th & 5th weeks Group A was normal. Group B revealed interfollicular edema at 4 th week and normal follicles at 5 th week. Group C revealed lymphocytic hyperplasia and lymphoblastic activation at4 th &5 th weeks

25. Bursa of fabricious from group B at 4 th week showing interfollicular edema (H&E X 200).

26. Bursa of fabricious from group C at 4 th & 5 th week showing lymphocytic hyperplasia and lymphoblastic activation (H&E X 200).

27. Results and Discussion 4- Histopathological Findings  Thymus 1 st & 2 nd weeks All groups were normal

28. Thymus from all groups at 1 st & 2 nd weeks showing apparently normal cortex and medulla (H&E X 200).

29. Results and Discussion 4- Histopathological Findings  Thymus 3 rd week Groups A & C were normal Group B showed congested medulla

30. Thymus from group B at 3 rd week showing congested medulla (H&E X 200).

31. Results and Discussion 4- Histopathological Findings  Thymus 4 th & 5 th weeks Groups A & B were normal Group C showed slight lymphocytic hyperplasia

32. Thymus from group C at 4 th & 5 th weeks showing revealed slight lymphocytic hyperplasia (H&E X 200).

33. Results and Discussion 4- Histopathological Findings  Spleen 1 st & 2 nd weeks All groups were normal

34. Spleen from all groups at 1 st & 2 nd weeks showing apparently normal lymphoid follicles (H&E X 200).

35. Results and Discussion 4- Histopathological Findings  Spleen 3 rd & 4 th weeks groups A & C were normal Group B showed slight lymphocytic depletion

36. Spleen from group B at 3 rd & 4 th weeks showing slight lymphocytic depletion (H&E X 200).

37. Results and Discussion 4- Histopathological Findings  Spleen 5 th week Group A & B were normal Group C showed lymphoblastic activation

38. Spleen from group C at 5 th week showing lymphoblastic activation (H&E X 200).

39.  From the present study, it is concluded that, Bivalent (H5N1&ND)AI vaccine has immunostimulatory effect higher than monovalent (H5N1)AI vaccine.  which reflected on increasing the immune response of its vaccinated chicken against AI virus.  which manifested by its higher globulins concentration and higher mean (HI) antibody titers of chicken against avian influenza virus (AIV) and confirmed histopathologically by the observed hyperplasia of the lymphoid organs. Conclusion