Background and Aim: Prophylaxis and disease prevention is an essential strategy among biorisk management in poultry farms that stimulate and maintain the birds' immunity. The aim of this study was to investigate the prophylactic, and immune-stimulant influence of Nigella sativa Linn. in broilers under biological stress.

Materials and Methods: A total of 250 1-day-old (ross) chicks were divided into 5 groups; four of which were supplemented with 1.4%, 2.8%, 4.2%, and 5.6% N. sativa Linn., respectively. The four supplemented groups were challenged with Escherichia coli O₁₅₇:H₇ 1.5x10⁸ at a 14th day old. A total of 1050 samples (150 serum, 150 swab, and 750 organ samples) were collected and examined.

Results: A highly significant increase (p<0.01) in 5.6% N. sativa Linn. supplemented group in performance traits (body weight, weight gain, and performance index), biochemical parameters (proteinogram, liver enzymes, and creatinine), immunoglobulins concentration, and immune organs' weight. Meanwile, liver showed improvement of histoarchitecture without fibrosis. Heart showed a mild pericarditis with a mild degree of hydropic degeneration. Bursa, thymus, and spleen showed lymphoid hyperplasia.

Conclusion: A concentration of 5.6% N. sativa Linn. in broiler's feed can improve the immune response and subsequent resistance of broilers against diseases.

Keywords: Broiler, Escherichia coli, histopathology, Nigella sativa Linn., preventive.

1. Attia, Y.A. and Al-Harthi, M.A. (2015) Nigella seed oil as an alternative to antibiotic growth promoters for broiler Chickens. Eur. Poult. Sci., 79: 1-15.

2. Kim, J.D., Nhut, T.M., Hai, T.N. and Ra, C.S. (2011) Effect of dietary essential oils on growth, feed untilization and meat yields of white leg shrimp L. vannamei. Asian Aust. J. Anim. Sci., 24(8): 1136-1141. [Crossref]

3. Hassanpou, H., Zamani-Moghaddam, A.K., Khosravi, M. and Mayahi, M. (2013) Effect of symbiotic on the intestinal morphology and humoral immune response in broiler chickens. Livest. Sci., 153: 116-122. [Crossref]

4. Chen, W., Wang, J.P., Yan, L. and Huang, Y.Q. (2013) Evaluation of probiotics in diets with different nutrient densities on growth performance, blood characteristics, relative organ weight and breast meat characteristics in broilers. Br. Poult. Sci., 54(5): 635-641. [Crossref] [PubMed]

5. Blajman, J.E., Frizzo, L.S., Zbrun, M.V., Astesana, D.M., Fusari, M.L., Soto, L.P., Rosmini, M.R. and Signorini, M.L. (2014) Probiotics and broiler growth performance: A meta-analysis of randomised controlled trials. Br. Poult. Sci., 55(4): 483-494. [Crossref] [PubMed]

6. Wang, X., Farnell, Y.Z., Peebles, E.D., Kiess, A.S., Wamsley, K.G. and Zhai, W. (2016) Effects of prebiotics, probiotics, and their combination on growth performance, small intestine morphology, and resident Lactobacillus of male broilers. Poult. Sci. 95(6): 1332-1340. [Crossref] [PubMed]

7. Al-Bukhari, M.I. (1976) In: Al-Bukhari, S., editor. The Collection of Authentic Sayings of Prophet Mohammad (Peace be Upon Him), Division 71 on Medicine. 2nd ed. Hilal Yayinlari, Ankara.

8. Attia, Y.A., Tag El-Din, A.E., Zeweil, H.S. and Arafat, M.A. (2003) Nutritional evaluation of nigella seed meal and the effect of microbial phytase and amino acids supplementations on its feeding value for Japanese Quai. In: Proceedings 9th Conference on Animal Nutrition. Hurghada, Egypt. p201-217.

9. Attia, Y.A., El-Din, A.E.R., Zeweil, H.S., Hussein, A.S. and Arafat, M.A. (2008) The effect of supplementation of enzyme on laying and reproductive performance in Japanese Quail hens fed nigella seed meal. J. Poult. Sci., 45: 110-115. [Crossref]

10. El-Deek, A.A., Hamdy, S.M., Attia, Y.A. and Khalifah, M.M. (2009) Nigella sativa seed oil meal as a source of plant protein in broiler diets. Egypt. Poult. Sci. J., 29: 39-52.

11. Shamsa, M.T., Al-Bayati, M.T.N. and Ibrahim, I. (2013) The effect of garlic and Nigella sativa on some biochemical and production characteristic in broiler chicken. Kufa J. Vet. Med. Sci., 4(1): 75-80.

12. Abbas, T.E.E. and Ahmed, M.E. (2010) Effect of supplementation of Nigella sativa seeds to the broiler chicks diet on the performance and carcass quality. Int. J. Agric. Sci., 2(2): 9-13. [Crossref]

13. National Research Council, NRC. (1994) Nutrient Requirements for Poultry. 9th ed. National Research Council, New York.

14. Kocijancic, D., Felgner, S., Frahm, M., Komoll, R.M., Iljazovic, A., Pawar, V., Rohde, M., Heise, U., Zimmermann, K., Gunzer, F., Hammer, J., Crull, K., Leschner, S. and Weiss, S. (2016) Therapy of solid tumors using probiotic symbioflor-2-restraints and potential. Oncotarget, 7(16): 22605-22622. [Crossref] [PubMed] [PMC]

15. Soliman, E.S. and Hassan, R.A. (2017) Evaluation of superphosphate and meta-bisulfide efficiency in litter treatment on productive performance and immunity of broilers exposed to ammonia stress. Adv. Anim. Vet. Sci., 5(6): 253-259.

16. Coles, E.H. (1986) Veterinary Clinical Pathology. WB Saunders Company, Philadelphia, London.

17. Young, D.S. (2001) Effect of Drugs on Clinical Laboratory Test. 5th ed. AACC Press, Washington, D.C.

18. Wu, Y.N., Yan, F.F., Hu, J.Y., Chen, H., Tucker, C.M., Green, A.R. and Cheng, H.W. (2017) The effect of chronic ammonia exposure on acute-phase proteins, immunoglobulin, and cytokines in laying hens. Poult. Sci., 96(6): 1524-1530. [Crossref] [PubMed]

19. American Public Health Association, American Water Works Association, Water Environment Federation. (2012) Standard Methods for the Examination of Water and Wastewater. 22th ed. American Water Work Association Publications, Washington D.C.

20. Herigstad, B., Hamilton, M. and Heersink, J. (2001) How to optimize the drop plate method for enumerating bacteria. J. Microbiol. Methods, 44(2): 121-129. [Crossref]

21. Cruickshank, R., Duguid, J.P., Marmion, B.P. and Swain, R.H.A. (1980) The Practice of Medical Microbiology. 12th ed., Vol. 2. Churchill Livingstone, Edinburgh, New York. p193, 194, 299.

22. Bancroff, J., Stevenes, A. and Turner, D. (1990) Theory and Practice of Histological Techniques. 3rd ed. Clurechill Livingston, Edinburgh, London.

23. SPSS, Inc. (2001) SPSS for Windows: Computer Software. SPSS, Inc., Chicago.

24. SAS Institute, Inc. (2002) SAS Course Notes on Mixed Model Analysis Using SAS System. SAS Institute, Inc., Cary, NC.

25. Levesque, R. (2007) SPSS Programming and Data Management: A Guide for SPSS and SAS^® Users. 4th ed. SPSS Inc., Chicago IL.

26. Benhelima, A., Omar, Z.K., Hemida, H., Benmahdi, T. and Addou, A. (2016) Nephroprotective and diuretic effect of Nigella sativa L. seeds oil on lithiasic Wistar rats. Afr. J. Tradit. Complement Altern. Med., 13(6): 204-214. [Crossref] [PubMed] [PMC]

27. EL-Shoukary, R.D.M., Darwish, M.H.A. and Abdel-Rahman, M.A.M. (2017) Behavioral, performance, carcass traits and hormonal changes of heat stressed broilers feeding black and coriander seeds. J. Adv. Vet. Res., 4(3): 97-101.

28. Boka, J., Mahdavi, A.H., Samie, A.H. and Jahanian, R. (2014). Effect of different levels of black cumin (Nigella sativa L.) on performance, intestinal Escherichia coli colonization and jejunal morphology in laying hens. Anim. Physiol. Anim. Nutr., 98(2): 373-383. [Crossref] [PubMed]

29. Jahan, M.S., Khairunnesa, M., Afrin, S. and Ali, M.S. (2015) Dietary black cumin (Nizella sativa) seed meal on growth and meat yield performance of broilers. SAARC J. Agric., 13(2): 151-160. [Crossref]

30. Majeed, L.H.A., Abdelati, K.A., Al-Bagir, N.M., Alhaidary, A., Mohamed, H.E. and Beynen, A.C. (2010) Performance of broiler chickens fed diets containing low inclusion levels of black cumin seed. J. Anim. Vet. Adv., 9(21): 2725-2728.

31. Nasir, Z. and Grashorn, M.A. (2010) Effect of Echinacea Purpurea and Nigella Sativa supplementation on broiler performance, carcass and meat quality. J. Anim. Feed Sci., 19: 94-104. [Crossref]

32. Marian H. Ghaly, Ashraf A. Elghoneimy, Hussein K. Mohamed and Marwa F. Ali (2017) Biochemical and histopathological effects of dietary supplementation of Nigella sativa and Mentha piperita oils to Broilers. J. Adv. Vet. Res., 7(1): 7-15.

33. Al-Beitawi, N. and El-Ghousein, S.S. (2008) Effect of feeding different levels of Nigella sativa seeds (black cumin) on performance, blood constituents and carcass characteristics of broiler chicks. Int. J. Poult. Sci., 7: 715-721. [Crossref]

34. Salam, S., Sunarti, D. and Isroli, I. (2013) Physiological responses of blood and immune organs of broiler chicken fed dietary black cumin powder (Nigella sativa) during dry seasons. J. Indones. Trop. Anim. Agric., 38(3): 185-191. [Crossref]

35. Azeem, T., Rehmanb, Z.U., Umar, S., Asifa, M., Arifc, M. and Rahmanc, A. (2014) Effect of Nigella sativa on poultry health and production: A review. Sci. Lett., 2(2): 76-82.

36. Hermes, I.H., Attia, F.M., Ibrahim, K.A. and El-Nesr, S.S. (2010) Physiological responses of broiler chickens to different dietary forms and levels of Nigella sativa L., Egyptian during summer season. J. Agric. Vet. Sci., 4(1): 17-33.

37. Sogut, B., Celik, I. and Tuluce, Y. (2008) The effects of diet supplemented with black cumin (Nigella sativa L.) upon immune potential and antioxidant marker enzymes and lipid peroxidation in broiler chicks. J. Anim. Vet. Adv., 7: 1196-1199.

38. Al-Mufarrej, S.I. (2014) Immune-responsiveness and performance of broiler chickens fed black cumin (Nigella Sativa L.) powder. J. Saudi Soc. Agric. Sci., 13(1): 75-80. [Crossref]

39. Umar, S., Rehman, A., Younus, M., Nisa, Q., Ali, A., Shahzad, M., Abdullah-Shah, M.A., Munir, M.T., Aslam, H.B. and Yaqoob, M. (2016) Effects of Nigella sativa on immune responses and pathogenesis of avian influenza (H9N2) virus in turkeys. J. Appl. Poult. Res., 25(1): 95-103. [Crossref]

40. Erener, G., Altop, N., Ocak, H., Aksoy, S. and Ozturk, E. (2010) Influence of black cumin seed (Nigella sativa L) and seed extract on broilers performance and total coliform bacteria count. Asian J. Anim. Vet. Adv., 5: 128-135. [Crossref]

41. Altunoglu, Y.C., Bilen, S., Ulu, F. and Biswas, G. (2017). Immune responses to methanolic extract of black cumin (Nigella sativa) in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol., 67: 103-109. [Crossref] [PubMed]

42. Shewita, R.S. and Taha, A.E. (2011) Effect of dietary supplementation of different levels of black seed (Nigella Sativa L.) on growth, performance, immunological, hematological and carcass parameters of broiler chicks. World Acad. Sci. Eng. Technol., 77: 788-794.

43. Shettima, Y.M., El-Yuguda, A.D., Zanna, M.Y., Abubakar, M.B., Hamisu, T.M., Maina, M.M., Andrew, A., Hambali, I.U. and Baba, S.S. (2016) Serological evidence of infectious bronchitis virus among some poultry species in Maiduguri, Nigeria. Alex. J. Vet. Sci., 51(1): 135-139. [Crossref]

44. Hassanin, O., Abdallah, F. and Awad, A. (2014) Effects of florfenicol on the immune responses and the interferon-inducible genes in broiler chickens under the impact of E. coli infection. Vet. Res. Commun., 38(1): 51-58. [Crossref] [PubMed]

45. Jasim, W.K., Hassan, M.S. and Keam, G.G. (2016) Study the effect of Nigella sativa on thyroid function and reproductive hormone of female rat. J. Contemp. Med. Sci., 2(6): 67-69.

46. Mehta, B.K. and Gupta, M. (2016) A rapid and pollutionless microwave assisted efficient extraction from seeds of Nigella sativa. Nat. Prod., 12(2): 62-67.

47. Chahal, K.K., Dhaiwal, K., Kumar, A., Kataria, D. and Singla, N. (2017) Chemical composition of Trachyspermum ammi L. and its biological properties: A review. J. Pharmacogn. Phytochem., 6(3): 131-140.

48. Kooti, W., Hassanzadeh-Noohi, Z., Sharafi-Ahvazi, N., Asadi-Samani, M. and Ashtary-Larky, D. (2016) Phytochemistry, pharmacology, and therapeutic uses of black seed (Nigella sativa). Chin. J. Nat. Med., 14(10): 732-745. [Crossref]

49. Bopp, C.A., Brenner, F.W., Wells, J.G. and Strockbine, N.A. (2005) Escherichia, Shigella and Salmonella. In: Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., editors. Manual of Clinical Microbiology. American Society for Microbiology, Washington, DC.

50. Nakamura, K., Yuasa, N., Abe, H. and Narita, M. (1990) Effect of infectious bursal disease virus on infections produced by Escherichia coli of high and low virulence in chickens. Avian Pathol., 19: 713-721. [Crossref] [PubMed]

51. Mutabagani, A. and El-Mehdy, S.A.M. (1997) A study of the anti-inflammatory activity of Nigella sativa Linn and thymoquinone in rats. Saudi Pharm. J., 5: 110-113.

52. El-Daly, E.S. (1998) Protective effect of cysteine and Vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin induced toxicity in rats. J. Pharm. Belg, 53: 87-95. [PubMed]

53. Daba, M.H. and Abdel-Rehman, M.S. (1998) Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicol. Lett., 16: 23-29. [Crossref]

54. Swamy, S.W. and Tan, B.K.H. (2000) Cytotoxic and immune-potentiating effect of ethanoic extract of N. sativa L. seeds. J. Ethnopharmacol., 70: 1-7. [Crossref]

55. Sokmen, A., Jones, B.M. and Erturk, M. (1999) Antimicrobial activity of extracts from the cell cultures of some Turkish plants. Phytother. Res., 13: 355-357. [Crossref]