بررسی اثر ادجوانی نانوکیتوزان بر ایمنی زایی واکسن کشته‌ی آئروموناس هیدروفیلا در ماهی کپور معمولی(Cyprinus carpio)

نوع مقاله: میکروبشناسی و ایمنی شناسی

نویسندگان

1 گروه علوم درمانگاهی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز ، ایران

2 دانش آموخته دامپزشکی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران

3 گروه پاتوبیولوژی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران

چکیده

زمینه مطالعه: اخیرا گزارشات متعددی از اثرات تحریک ایمنی و ادجوانی نانوکیتوزان در حیوانات مختلف گزارش شده است. هدف: در این مطالعه، اثر ادجوانی نانوکیتوزان تولید شده از کیتوزان حاصل از کیتوزان پوسته میگو بر ایمنی زایی واکسن کشته آئروموناس هیدروفیلا در ماهی کپور معمولی (Cyprinus carpio) مورد بررسی قرار گرفت. روش کار: تعداد 300 عدد ماهی کپور معمولی (با وزن متوسط 6/5±51 گرم) به چهار گروه (هر گروه 75 ماهی و 25 ماهی در هر تکرار) تقسیم شدند. گروه 1، 2 و 3 با واکسن آئروموناس هیدروفیلا ایمن شدند. در گروه 1 واکسن بدون ادجوان، گروه 2، واکسن همراه ادجوان فروند و گروه 3 واکسن همراه ادجوان نانوکیتوزان استفاده شد. گروه 4 (شاهد) با سرم فیزیولوژی تزریق گردید. ماهی‌های هر گروه در روز صفر و 14 ایمن شدند و به مدت 6 هفته با شرایط مشابه نگهداری و تغذیه شدند. خونگیری در انتهای هفته 2، هفته 4 و هفته 6 تحقیق انجام گرفت. برخی شاخص‌های ایمنی مانند تعداد گلبول‌های سفید خونی، فعالیت لایزوزیم و قدرت ضدباکتریایی سرم، فعالیت کمپلمان، میزان احیای NBT ، پروتئین و گلوبولین تام و عیار آنتی‌بادی ضد آئروموناس هیدروفیلا بررسی شد. در انتهای دوره ماهی‌ها با غلظت LD50 باکتری زنده آئروموناس هیدروفیلا چالش داده شدند و میزان تلفات طی 10 روز ثبت شد. نتایج نشان داد که تعداد گلبول‌های سفید خونی، فعالیت لایزوزیم سرم، میزان گلوبولین، میزان فعالیت NBT و عیار پادتن در دو تیمار فروند و نانوکیتوزان در مراحل نمونه گیری افزایش معنی داری نسبت به تیمار کنترل یافت(P<0.05). در میزان فعالیت ضدباکتریایی و سطح آلبومین سرم در تیمارهای مختلف تفاوت معنی داری مشاهده نگردید(P>0.05). تلفات بعد از چالش نیز در هر دو تیمار نانوکیتوزان و فروند کاهش معنی داری نسبت به تیمار بدون ادجوان و کنترل نشان دادند(P<0.05). نتیجه‌گیری نهایی: بر اساس نتایج این تحقیق نانوکیتوزان دارای خواص ادجوانی مناسب و قابل رقابت با ادجوان فروند بوده و گزینه خوبی برای جایگزینی ادجوان های رایج ماهی می باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Adjuvant effects of nanochitosan on immunogenicity of Aeromonas hydrophila vaccine in Cyprinus carpio

نویسندگان [English]

  • Mojtaba Alishahi 1
  • Ozra Hajipoor 2
  • Masood Ghorbanpoor 3
  • Mehrzad Mesbah 1
1 1Department of Clinical Science, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
2 2Student of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
3 3Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz-Iran
چکیده [English]

BACKGROUND: Recently chitosan nanoparticles have been applied as an immunostimulant and adjuvant in biomedical fields. OBJECTIVES: In this study the adjuvant effects of nanochitosan, derived from shrimp shell's chithin, on immunogenicity of Aeromonas hydrophila vaccine in common carp were evaluated. METHODS: Totally 300 juvenile common carp (51±5.6 g, Mean ±SD) were randomly divided into four groups each contained three subgroups with 25 fishes. Groups 1, 2 and 3 were immunized intraperitoneally with A. hydrophila vaccine. No adjuvant used in group 1, but nanochitosan and freund adjuvant were used with vaccine in groups 2 and 3 respectively. Group 4 (Control) injected with normal saline instead of vaccine. Fish were immunised at days zero and 14 and fishes were reared in the same situation for 6 weeks. Serum samples were taken every other weeks and immunological parameters including WBC, serum lysozyme and bactericidal activity, serum complement activity, NBT reduction activity, total protein and globulin levels and anti A.hydrophila antibody titer were compared among the groups. At the end of experiment all groups were challenged with LD50 concentration of A. hydrophila and mortality recorded for ten days. RESULTS: WBC, serum lysozyme and complement activity, NBT activity, globulin level and anti A.hydrophila antibody titer were increased in nanochitosan and freund groups compare to control group (P<0.05). Serum bactericidal activity and serum albumin level showed no significant change among the groups (P>0.05). Challenge mortality decreased in immunised groups compare to control. CONCLUSIONS: Nonochitosan posses proper adjuvant effects which is comparable with freund adjuvant and can be a good candidate as an alternative to current adjuvant in fish vaccines.

کلیدواژه‌ها [English]

  • Nanochitosan
  • Adjuvant
  • Cyprinus carpio
  • Aeromonas hydrophila
Aathi, K., Ramasubramanian, V., Uthayakumar, V., Munirasu, S. (2013) Effect of supplemented diet on survival, growth, hematological, biochemical and immunological responses of Indian major carp Labeo rohita. Int Res J Pharm. 4:141-147.##

Abdou E.S., Nagy, S.A., Elsabee, Z.M. (2008) Extraction and characterization of chitin and chitosan from local sources, Bioresour Technol. 99: 1359-1367.##

Aguilar, J.C., Rodriguez, E.G. (2007) Vaccine adjuvants revisited. Vaccine. (25-19): 3752-3762.##

Alishahi A. and Auder, M. (2012) Applications of chitosan in the seafood industry and aquaculture: A review. Food Bioprocess Tech. 5:817-830.##

Altun S., Kubilay, A.C., Ekici, S., Işıl, B., Diler, O. (2010) Oral Vaccination Against Lactococcosis in Rainbow Trout (Oncorhynchusmykiss) Using Sodium Alginate and Poly (lactide-co-glycolide) Carrier.Kafkas University, Veterinary Faculty Letter. 16 (Suppl-B): S211-S217. ##

Anderson, D.P., Siwicki, A.K. (1994) Duration of protection against Aeromonas salmonicida in brook trout immunostimulated with glucan or chitosan by injection or immersion. Prog Fish Cult. 56: 258-261.##

Aravena, A.R., Sandino, A.M., Spencer, E. (2013) Nanoparticles and microparticles of polymers and polysaccharides to administer fish vaccines. Biol Res. 46: 407-419.##

Audran, R., Peter, K., Dannull, J., Men, Y., Scandella, E., Groettru, M., Gander, B., Corradin, G. (2003) Encapsulation of peptides in biodegradable microspheres prolongs their MHC class-I presentation by dendritic cells and macrophages in vitro. Vaccine. 21(11-12):1250-1255.##

Bar-Ilan, O., Albrecht, R.M., Fako, V.E.,  Furgeson, D.Y. (2009) Toxicity assessments of multisized gold and silver nanoparticles in zebrafish embryos, Small. 5: 1897-1910.##

Baulny, M.O.D., Quentell, C., Fournierl, V., Amour, F., Le gouvello, R. (1996) Effect of long-term oral administration of β-glucan as an immunostimulant or an adjuvant on some non-specific parameters of the immune response of turbot Scophthalmus maximus. Dis Aquat Org. 26:139-147.##

Behera, T., Nanda, P.K., Mohanty, C., Mohapatra, D., Swain, P., DAS, B.K., Routray, P., Mishra, B.K., Sahoo, S.K. (2010) Parenteral immunization of fish, Labeo rohita with Poly D, L-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate andadaptive immune responses. Fish Shellfish Immunol. 28:320-325.##

Diwan, M., Elamanchili, P., Lane, H., Gainer, A., Samuel, J. (2003) Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses. J Drug Target. 11(8-10):495-507.##

Du, Y., Zhao, Y., Dai, S., Yang, B. (2009) Preparation of water-soluble chitosan from shrimp shell and its antibacterial activity. Innov Food Sci. Emerg. 10: 103-107.##

Dzung, H.N., Hong, V.D.T., Phuong, N.T., Quynh, N.T., Hiep, D.M. (2011)  Chitosan Nanoparticle as a Novel Delivery System for A/H1n1 Influenza Vaccine: Safe Property and Immunogenicity in Mice. World Academy of Science,J. Engine Technol. 60:1839-1847.##

Ellis, A.E. (1990) Lysozyme assays. In: Techniques in fish immunology, Fair Haven NJ: SOS Publications. Stolen, J.S, Fletcher, T.C., Anderson, D.P., Roberson, B.S., van Muiswinkel, W.B. (eds.) p. 101-103.##

Engstad, R.E., Robertsen, B., Frivold, E. (1992) Yeast glucan induces increase in lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish Shellfish Immunol. 2: 287-297.

Evensen, O., Brudeseth, B., Mutoloki, S. (2005) The vaccine formulation and its role in inflammatory processes in fish- effects and adverse effects. Progress in Fish Vaccinology, 16: 117-125.##

Fredriksen, B.N., Grip, J. (2012) PLGA/PLA micro- and nanoparticle formulations serve as antigen depots and induce elevated humoral responses after immunization of Atlantic salmon (Salmo salar). Vaccine. 30:656-67.##

Fujimura, Y., Akisada, T., Harada, T., Haruma, K. (2006) Uptake of microparticles into the epithelium of human nasopharyngeal lymphoid tissue. Med Mol Morphol. 39: 181-6.##

Gopalakannan, A., Arul, V. (2006) Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture. 255: 179-187.##

Habeeb, F., Stables, G., Bradbury, F., Nong, S. (2007) The inner gel component of Aloe vera suppresses bacterial-induced pro-inflammatory cytokines from human immune cells. Methods, 42: 388-393.##

Harikrishnan R., Kim M.C., Kim J.S., Han Y.J., Jang I.S., Balasundaram, C., SooJanga, I.,  Balasundaramb, C., Heoa, M. S. (2010) Immune response and expression analysis of cathepsin K in goldfish during Aeromonas hydrophila infection. Fish and Shellfish Immunol. 28: 511-516.##

Harikrishnan, R., Balasundaram, C., Heo, M.S. (2012) Inonotus obliquus containing diet enhances the innate immune mechanism and disease resistance in olive flounder Paralichythys olivaceus against Uronema marinum. Fish Shellfish Immunol. 32: 1148-1154.##

Harikrishnan, R., Nisha, M.R., Balasundaram, C. (2003) Hematological and biochemical parameters in common carp, Cyprinus carpio, following herbal treatment for Aeromonas hydrophila infection. Aquaculture. 221: 41-50.##

Illum, L., Jabbal-Gill, I., Hinchcliffe, M., Fisher, A.N., Davis, S.S. (2001) Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliv Rev. 51: 81-96.##

Ispir, U., Dorucu, M. (2005) A study on the effects of  Levamisole on the Immune System of Rainbow Trout (Oncorhynchus mykiss, Walbaum). Turk J Vet Anim Sci. 29:1169-1176.##

Iwama, G., Nakanishi, T. (1996) The fish immune system: organism, pathogen and environment. Fish physiology, vol. 15, (1st ed.) Academic Press, San Diego. p. 12-42, 64-67, 105-139.##

Kajita, Y., Sakai, M., Atsuta, S, Kobayash, M. (1990) The immunonodulatory effects of levamisole on rainbow trout, Oncorhynchus mykiss. Fish Pathol. 25:93-98.##

Khushiramani, R., Girisha, S.K., Karunasagar, I., Karunasagar, I. (2007) Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish. Protein Expr Purif. 51: 303-307.##

Li, X., Kong, X., Shi, S., Zheng, X., Guo, G., Wei, Y., Qian, Z.H. (2008) Preparation of alginate coated chitosan microparticles for vaccine delivery. BMC Biotechnol. 2008. 8: 89.##

Lin, S., Mao, S., Guan, Y., Luo, L., Luo, L., Pan, Y. (2012) Effects of dietary chitosan oligosaccharides and Bacillus coagulans on the growth, innate immunity and resistance of koi (Cyprinus carpio koi). Aquaculture. 342-343: 36-41.##

Lin, S., Pan, Y., Luo, L., Li, L. (2011) Effects of dietary β-1, 3-glucan, chitosan or raffinose on the growth, innate immunity and resistance of koi (Cyprinus carpio koi). Fish & Shellfish Immunol. 31: 788-794.##

Maqsood, S., Singh, P., Samoon, M.H., Balange, A.K. (2010) Effect of dietary chitosan on nonspecific immune response and growth of Cyprinus carpio challenged with Aeromonas hydrophila. Int Aquat Res. 2: 77-85.##

Matsuyama T., Fujiwara A., Nakayasu C., Kamaishi T., Oseko N., Ikuo Hirono, I., Aoki, T. (2007) Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda. Fish and Shellfish Immunol. 22: 598-607.##

Nakhla, A.N., Szalai, A.J., Banoub, J.H., Keough, K.M.W. (1997) Serum anti- LPS antibody production by rainbow trout (Oncorhynchus mykiss) in response to the administration of free and liposomally-incorporated LPS from Aeromonas salmonicida. Fish & Shellfish Immunol. 7:387-401.##

Nayak, D.K., Asha, A., Shankar, K.M., Mohan, C.V. (2004) Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus- a carnivore model. Fish and Shellfish Immunol. 16: 613-619.##

Olivier, G., Evelyn, T.P.T., Lallier, R. (1985) Immunity to Aeromonas salmonicida in coho salmon (Oncorhynchus kisutch). induced by modified Freund’s complete adjuvant: its non-specific nature and the probable role of macrophages in the phenomenon. Dev Comp Immunol. 9: 419-432.##

Paterson, WD., Fryer, J.L. (1974) Immune responses of juvenile coho salmon (Oncorhynchus kisutch) to Aeromonas salmonicida cells administered intraperitoneally in Freund’s complete adjuvant. J Fish Res. 31: 1751-1755.##

Rohovec, J.S., Winton, J.R., Fryer, J.L. (1981) Bacterins and vaccines for control of infectious diseases in fish. National science councils Taipei, taiwas, Republic of china. 2: 115-121.##

Sahoo, P.K., Kumarai, J., Mishra, B.K. (2005) Nonspecific immune responses in juveniles of Indian major carps. J Appl Ichthyol. 21: 151-155.##

Schaperclaus, W., Kulow, H., Schreckenbach, K. (1991) Hematological and serological technique. In: Fish Disease. Kothekar, VS. (ed.) (2nd ed.). Connaught circus, Gulab primlani, Oxonian press Pvt. Ltd. New Delhi, India. p. 71-108.##

Selvaraj, V., Sampath. K. and Sekar. V. (2005) Administration of yeast glucan enhances survival and some non-specific and specific immune parameters in carp (Cyprinus carpio) infected with Aeromonas hydrophila. Fish and Shellfish Immunol. 19: 293-306.##

Shen, P., Fan, X., Li, G. (1999) Microbiology experiment. (3rd ed.) Beijing: Higher Education Press; [in Chinese] 165e167.##

Siwicki, A.K., Anderson, D.P., Rumsey, G.L. (1994) Dietary intake of Immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Vet Immunol Immunopathol.41: 125-139.##

Soltani, M., Pourgholam, R. (2007) Lysozyme activity of grass carp (Ctenopharingodon idella) following exposure to sublethal concentrations of organophosphate, diazinon. J Vet Res. 62:49-52.

Sonia, T.A., Sharma, P. (2011) Chitosan and Its derivatives for drug delivery perspective, Adv Polym Sci. 243: 23-54##

Sun, Y., Liu, C.S., Sun, L. (2011) A multivalent killed whole-cell vaccine induces effective protection against Edwardsiella tarda and Vibrio anguillarum. Fish and Shellfish Immunol. 31: 595-599.##

Swain, P.S., Dash, P.K., Sahoo, P., Routray, S.K., Sahoo S.D., Gupta P.K., Meher, N. (2007) Non-specific immune parameters of brood Indian major carp Labeo rohita and their seasonal variations. Fish and Shellfish Immunol. 22: 38-43.##

Tafi, A.A., Meshkini S., Tukmechi, A. (1392) Effects of Chitosan on some immune response of rainbow trout (Oncorhynchus mykiss) and enhance resistance against a pathogenic Aeromonas hydrophila fallowing experimental infection. J Animal Res. 26:468-477.##

Tian, J.Y., SUN, X.Q., CHEN, X.G. (2008) Formation and oral administration of alginate microspheres loaded with pDNA coding for lymphocystis disease virus (LCDV) to Japanese flounder. Fish Shell fish Immunol. 24:592-599.##

Zheng, Z., Yingeng, W., Qingyin, W., Nannan, D., Meijie, L., Jiangbo, Q., Bin, L., Lan, W.l. (2012) Study on the immune enhancement of different immunoadjuvants used in the pentavalent vaccine for turbots. Fish and Shellfish Immunol. 32: 391-395.##

Zheng, F.R., Sun, X.Q., Liu, H.Z., Zhang, J.X. (2006) Study on the distribution and expression of a DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus). Aquaculture. 261:1128-1134.##