Comparative efficacy and immune responses induced by aluminium hydroxide gel (Algel) adjuvanted and non-adjuvanted formalin killed Vibrio alginolyticus vaccines in Asian seabass Lates calcarifer (Bloch, 1790)

Azhar Shahansha T. S.; Pradeep  M. A.; Ramesh Kumar  P; Suresh Babu P. P; Joshy C. G.; Dhanutha N. R.; Nidhiya Kottayi; Nivya A; Sanil N K

doi:10.21077/

Authors

Azhar Shahansha T S ICAR-CMFRI
Pradeep M. A. icar https://orcid.org/0000-0001-9511-1835
Ramesh Kumar P ICAR-CMFRI
Suresh Babu P. P ICAR-CMFRI
Joshy C. G. ICAR-CIFT
Dhanutha N. R ICAR-CMFRI
Nidhiya Kottayi ICAR-CMFRI
Nivya A ICAR-CMFRI
Sanil N. K. ICAR-CMFRI

https://doi.org/10.21077/

Keywords:

formalin-killed vaccine; aluminium hydroxide adjuvant; protective efficacy; vibriosis; immune gene expression

Abstract

Vibriosis caused by Vibrio alginolyticus is an important bacterial disease affecting Asian seabass Lates calcarifer (Bloch, 1790). This study evaluated the protective efficacy and immune responses induced by two intraperitoneally administered formalin killed V. alginolyticus vaccine formulations: a non-adjuvanted bacterin (T1) and an aluminium hydroxide gel (Algel) adjuvanted bacterin (T2). Phosphate buffered saline (PBS) injected fish served as the challenged control (T0), while a non-challenged naive group (T3) was maintained to monitor background mortality. Each vaccine was administered at a dose of 0.2 ml containing 1×10⁸CFU-equivalent per fish. Protection was assessed three weeks after vaccination following intraperitoneal challenge with live V. alginolyticus at 1×10⁸ CFU per fish. Serum agglutination titre, serum bactericidal activity, serum lysozyme activity, and the expression of complement factor H (CFH), Cystatin B, HSP70, and HSP90α in the spleen and head kidney were evaluated at 7, 14 and 21 days post-vaccination (DPV). T2 showed the highest survival (90%) and relative percent survival (RPS; 86.4%), followed by T1 (80% survival; 72.7% RPS), whereas the T0 challenged control showed 26.6% survival and no mortality was observed in the nonchallenged naive group (T3). T2 produced higher serum agglutination titres and lysozyme activity, particularly at 14 DPV. At 21 DPV, bacterial survival percentage was similar between T1 (77.2%) and T2 (77.9%; p>0.05), but both were lower than the T0 control (92.1%). Immune gene expression showed tissue- and time-dependent patterns without a uniform response across the four genes. Overall, both formalin killed vaccine formulations provided substantial protection under experimental challenge conditions, with the Algel adjuvanted formulation demonstrating enhanced survival, RPS, and early humoral and innate immune responses. The practical advantages of Algel over the non-adjuvanted formulation requires further evaluation through analysis of cost-effectiveness, assessment of duration of protective immmunity, booster vaccination studies and field studies.

Keywords: Aluminium hydroxide adjuvant, Formalin killed vaccine, Immune gene expression, Vibriosis

Downloads

Download data is not yet available.

Author Biographies

Azhar Shahansha T S, ICAR-CMFRI

PhD scholar
Pradeep M. A., icar

Senior Scientist, MBFNHD, CMFRI, KOCHI
Ramesh Kumar P, ICAR-CMFRI

Senior Scientist, ICAR-CMFRI
Suresh Babu P. P, ICAR-CMFRI

Principal Scientist
Joshy C. G., ICAR-CIFT

Senior Scientist
Dhanutha N. R, ICAR-CMFRI

Technical Officer
Nidhiya Kottayi , ICAR-CMFRI

PhD Scholar
Nivya A, ICAR-CMFRI

PhD Scholar
Sanil N. K., ICAR-CMFRI

Principal Scientist (ret)

References

Abdy, M. J., Kaur, B., Kalita, P. and Sarma, D. K. (2017). Adjuvant-based enhancement of fish vaccine efficacy against bacterial pathogens: a review. Reviews in Aquaculture, 9: 87-101.

Ahangarzadeh, M., Houshmand, H., Mozanzadeh, M. T., Kakoolaki, S., Nazemroaya, S., Sepahdari, A., Peyghan, R., Ajdari, A., Sadr, A. S. (2023). Effect of killed autogenous polyvalent vaccines against Vibrio harveyi, V. alginolyticus and Streptococcus iniae on survival and immunogenicity of Asian seabass (Lates calcarifer). Fish and Shellfish Immunology, 143: 109226.

Ahn, J. Y., Kim, Y. O., Nam, B. H., Seo, J. K. and Kim, B. S. (2013). Cystatin B expression in tissues of olive flounder following lipopolysaccharide stimulation. Fish and Shellfish Immunology, 35: 1108-1113.

Amir, I., Kaur, A. and Kaur, M. (2022). Inactivated vaccines for combating vibriosis in fish: status and future directions. Fish and Shellfish Immunology, 122: 37-46.

Amir-Danial, Z., Zamri-Saad, M., Amal, M. N. A., Annas, S., Mohamad, A., Jumria, S., Manchanayake, T., Arbania, A. and Ina-Salwany, M. Y. (2023). Field efficacy of a feed-based inactivated vaccine against vibriosis in cage-cultured Asian seabass, Lates calcarifer, in Malaysia. Vaccines, 11(1): 9.

Bastola, R., Noh, G. and Keum, T. (2017). Adjuvants and their roles in immunopotentiation of vaccines. Human Vaccines and Immunotherapeutics, 13: 1461-1476.

Bauer, A. and Young, R. (2000). Vibrio alginolyticus as a human pathogen: a case report and literature review. Clinical Infectious Diseases, 31: 812-815.

Biller, S. A., Nguyen, D. P. and Chen, J. (2021). Serum lysozyme as a biomarker of innate immunity in teleost fish. Fish and Shellfish Immunology, 111: 26-34.

Bomford, R., Stapleton, M., Winsor, S., McKnight, A. and Andronova, T. (1992). The adjuvanticity of alum and its salts: limitations on the use of adjuvant. Vaccine, 10: 1003-1010.

Bromage, E. S. and Owens, L. (2002). Infection of barramundi Lates calcarifer with Streptococcus iniae: effects of different routes of exposure. Diseases of Aquatic Organisms, 52: 199-205.

Bunnoy, A., Na-Nakorn, U., Srisapoome, P. and Kayansamruaj, P. (2022). Characterisation of commercial Asian seabass genetic strains for aquaculture. Aquaculture, 547: 737434.

Byadgi, O., Chen, Y. C., Barnes, A. C., Tsai, M. A., Wang, P. C. and Chen, S. C. (2018). Evaluation of the immune response of hybrid grouper after vaccination with polyvalent formalin-killed Streptococcal bacterin. Fish and Shellfish Immunology, 77: 295-305.

Cao, Y., Ding, X., Zhao, G. and Liu, X. (2018). Immune response and protection elicited by ghost bacterial vaccines against Vibrio harveyi. Fish and Shellfish Immunology, 82: 419-426.

Chang, M. X. (2020). Gene expression analysis of fish immune responses to vaccination: a review. Aquaculture, 523: 735149.

Dadar, M., Dhama, K., Vakharia, V. N., Bhatt, P., Wani, M. Y., Singh, K. P., Karthik, K., Yatoo, M. I., Alagawany, M. and Farag, M. R. (2017). Advances in aquaculture vaccines against fish pathogens: global status and current trends. Reviews in Fisheries Science and Aquaculture, 25: 184-217.

Dhasarathan, P., Ramasamy, S. and Balasundaram, C. (2014). Serum agglutination assay in fish immunology. World Journal of Fish and Marine Sciences, 6: 1-6.

Dong, C. X., Lu, J. F., Wang, Q. and Chen, J. (2013). Formalin-killed cell vaccine protection against infectious spleen and kidney necrosis virus in mandarin fish. Journal of Fish Diseases, 36: 1053-1061.

Du, Y., Han, B., Liu, X., Zhang, Y. and Cao, W. (2024). Inactivated vaccines for aquaculture bacterial diseases: current status and future prospects. Frontiers in Veterinary Science, 11: 1376201.

Du, Y., Hu, X., Miao, L. and Chen, J. (2022). Current status and development prospects of aquatic vaccines. Frontiers in Immunology, 13: 1040336.

Encinas, P., Rodriguez-Milla, M. A., Novoa, B., Estepa, A., Figueras, A. and Coll, J. (2010). Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. BMC Genomics, 11: 1-16.

Erfanmanesh, A., Beikzadeh, B., Khanzadeh, M. and Alishahi, M. (2024). Immuno-protective response of Asian seabass (Lates calcarifer) to inactivated vaccines against Streptococcus iniae and Vibrio harveyi. BMC Veterinary Research, 20: 89.

Firdaus, N., Arwin, M. and Samsul, H. (2013). Immune responses of grouper Epinephelus spp. to oil-adjuvanted inactivated vaccines. Aquaculture, 408: 184-189.

Ferraboschi, P., Ciceri, S., & Grisenti, P. (2021). Applications of Lysozyme, an Innate Immune Defense Factor, as an Alternative Antibiotic. Antibiotics (Basel, Switzerland), 10(12), 1534. https://doi.org/10.3390/antibiotics10121534.

Gu, Q. Q., Wang, G. H., Li, N. Q., Hao, D. F., Liu, H. M., Wang, C. B., Hu, Y. H., & Zhang, M. (2021). Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus. Fish & shellfish immunology, 118, 197–204. https://doi.org/10.1016/j.fsi.2021.09.008.

Huang, H.-Y., Chen, Y.-C., Wang, P.-C., Tsai, M.-A., Yeh, S.-C., Liang, H.-J., & Chen, S.-C. (2014). Efficacy of a formalin-inactivated vaccine against Streptococcus iniae infection in the farmed grouper Epinephelus coioides by intraperitoneal immunization. Vaccine, 32(51), 7014–7020. https://doi.org/10.1016/j.vaccine.2014.08.039.

Ji, W., Sun, B., Liu, J., Sun, X. and Shang, N. (2020). Development and application of formalin-inactivated and recombinant vaccines against fish Vibrio infections. Reviews in Aquaculture, 12: 1831-1846.

Jiao, X. D., Cheng, S., Hu, Y. H. and Sun, L. (2010). Comparative study of the effects of aluminium adjuvants and Freund's incomplete adjuvant on the immune response to an Edwardsiella tarda major antigen. Vaccine, 28: 1832-1837.

Khorshidian, A., Yousefi, M., Parsaei, M., Hosseini, S. M. and Mortazavian, A. M. (2022). Lysozyme: a review on mechanisms of antibacterial activity, its role in immune system, and application in food industry. Food and Bioprocess Technology, 15: 1447-1468.

Krupesha, S. R., Sharma, S. R. K., Hari, P. and Bindulekha, K. J. (2012). Vibrio alginolyticus infection in Asian seabass, Lates calcarifer, in open sea cage culture. Aquaculture Research, 44: 314-319.

Kumareswaran, K. and Jayasinghe, S. (2022). Aquaculture disease management: current practices and future directions. Reviews in Aquaculture, 14: 1849-1873.

Lan, N. G. T., Dong, H. T., Vinh, N. T., Senapin, S., Shinn, A. P., Salin, K. R. and Rodkhum, C. (2024). Immersion prime and oral boost vaccination with an inactivated Vibrio harveyi vaccine confers a specific immune response and protection in Asian seabass (Lates calcarifer). Fish and Shellfish Immunology, 144: 109293.

Lan, N. G. T., Dong, H. T., Vinh, N. T., Senapin, S., Shinn, A. P., Salin, K. R. and Rodkhum, C. (2025). Non-invasive vaccination enhances immune response and protects Asian seabass (Lates calcarifer) against Vibrio vulnificus infection. Fish and Shellfish Immunology Reports, 7: 100225.

Lindblad, E. B. (2004). Aluminium compounds for use in vaccines. Immunology and Cell Biology, 82: 497-505.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8. doi: 10.1006/meth.2001.1262. PMID: 11846609.

Ma, Y., Zhang, L., Zhao, Z. and Zhou, H. (2019). Vaccine development strategies for controlling Vibrio diseases in marine fish aquaculture. Reviews in Aquaculture, 11: 663-680.

Magister, Š. and Kos, J. (2013). Cystatins in immune system. Journal of Cancer, 4: 45-56.

Magnadóttir, B. (2006). Innate immunity of fish (overview). Fish and Shellfish Immunology, 20: 137-151.

Manchanayake, T., Hossain, S., Deczkowska, R. M. and De Silva, B. C. J. (2023). Vibrio alginolyticus in aquaculture: emerging challenges and management. Aquaculture International, 31: 1-22.

Meza, K., Espinoza, J. C., Ramos-Quispe, T. and Merino, C. (2019). Assessment of fish vaccination routes against Streptococcus agalactiae in Nile tilapia: comparison of immunisation outcomes. Aquaculture, 505: 313-317.

Miccoli, A., Buonocore, F. and Scapigliati, G. (2021). Immune parameters and vaccine efficacy assessment in teleost fish. Fish and Shellfish Immunology, 112: 12-25.

Mohd, J. A., Ahmad, A., Azmai, M. N. A., Ina-Salwany, M. Y., Mazelan, S. and Nor, A. M. (2013). Expression of HSP70 and HSP90 in Asian seabass during early Cryptocaryon irritans infection. Fish and Shellfish Immunology, 35: 1163-1170.

Mokhtar, D. M., Sayed, R. K. A. and Abd El-Atti, M. S. (2023). Serum bactericidal assay and adaptive immunity in vaccinated fish: a current review. Frontiers in Immunology, 14: 1116025.

Mondal, M. and Thomas, J. (2022). Adjuvants in veterinary vaccines: key players in immunomodulation. Veterinary World, 15: 1-11.

Murray, C. K. and Fletcher, T. C. (1976). The immunohistochemical localisation of lysozyme in plaice (Pleuronectes platessa L.) tissues. Journal of Fish Biology, 9: 329-334.

Mutoloki, S. and Evensen, Ø. (2015). Intraperitoneal injection of fish vaccine. Methods in Molecular Biology, 1260: 185-192.

Najafpour, B., Hahlbeck, E. and Franzreb, M. (2020). Complement factor H as an immune regulator in teleost fish: roles in inflammation control and tissue repair. Fish and Shellfish Immunology, 104: 617-625.

Nakao, M., Tsujikura, M., Ichiki, S., Vo, T. K. and Somamoto, T. (2011). The complement system in teleost fish: progress of post-homolog-searching studies. Developmental and Comparative Immunology, 35: 1296-1308.

Natnan, M. E., Baharum, S. N., Ludin, N. A. and Aizat, W. M. (2021). Integration of omics approaches in understanding disease in fish. Aquaculture, 545: 737249.

Nguyen, H. T., Thu Nguyen, T. T., Tsai, M. A., Ya-Zhen, E., Wang, P. C., & Chen, S. C. (2017). A formalin-inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides). Fish & shellfish immunology, 65, 118–126. https://doi.org/10.1016/j.fsi.2017.04.008

Ohtani, M., Villumsen, K. R., Strøm, H. K. and Raida, M. K. (2019). 3D visualisation of the innate immune response against Yersinia ruckeri in rainbow trout. PLoS ONE, 14: e0221863.

Pellegrino, P., Perrone, V., Radice, S. and Clementi, E. (2015). Vaccine adjuvants and their immunological mechanisms of action. Journal of Immunology Research, 2015: 1-7.

Pepi, M. and Focardi, S. (2021). Antibiotic-resistant bacteria in aquaculture and climate change: a challenge for health in the Mediterranean area. International Journal of Environmental Research and Public Health, 18: 5723.

Percie du Sert, N., Hurst, V., Ahluwalia, A., Alam, S., Avey, M. T., Baker, M. and Würbel, H. (2020). The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLOS Biology, 18: e3000410.

Rameshkumar, G., Rajan, K. E., Ravichandran, S. and Ajith Kumar, T. T. (2014). Bacterial pathogens of hatchery-reared Asian seabass. Indian Journal of Fisheries, 61: 1-6.

Rameshkumar, G., Ravichandran, S. and Kumaravel, K. (2017). Incidence of Vibrio alginolyticus infection in Asian seabass culture systems in India. Aquaculture Research, 48: 3017-3024.

Rao, S. R., Bhatt, P. and Jain, R. (2013). Modified delta-delta CT method for gene expression calculation in RT-qPCR. International Journal of Molecular Sciences, 14: 24484-24498.

Rebl, A. and Goldammer, T. (2018). Under control: the innate immunity of fish from the inhibitors' perspective. Fish and Shellfish Immunology, 77: 328-349.

Reed, L. J., & Muench, H. (1938). A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology, 27(3), 493–497.

Rosini, R., Nicchi, S., Pizza, M. and Rappuoli, R. (2020). Vaccines against antimicrobial resistance. Frontiers in Immunology, 11: 1048.

Serwecińska, L. (2020). Antimicrobials and antibiotic resistant bacteria: a risk to the environment and to public health. Water, 12: 3313.

Shah, S. Q. A., Colquhoun, D. J. and Nikuli, H. L. (2012). Prevalence of antibiotic resistance genes in the environments of five South Asian countries. Environmental Science and Technology, 46: 11067-11074.

Soveral, L. F., Corrêa, C. R. and Furuya, W. M. (2022). Immune gene expression as a biomarker of vaccine efficacy in aquaculture fish. Aquaculture, 557: 738308.

Sun, H. X., Xie, Y. and Ye, Y. P. (2009). Advances in saponin-based adjuvants. Vaccine, 27: 1787-1796.

Swain, P., Nayak, S. K., Nanda, P. K. and Dash, S. (2007). Biological effects of bacterial lipopolysaccharide (endotoxin) in fish: a review. Fish and Shellfish Immunology, 22: 89-105.

Tammas, I., Bitchava, K. and Gelasakis, A. I. (2024). Advances in vaccine adjuvants for teleost fish: implications for aquatic welfare and the potential of nanoparticle-based formulations. Vaccines, 12(12): 1347.

Tancredo, K. R., Hashimoto, D. T., da Cruz, C., Matushima, E. R. and Pádua, S. B. (2015). Blood collection techniques for immunological studies in teleost fish. Veterinary Research Communications, 39: 181-186.

Thakkar, S. G. and Cui, Z. R. (2017). Stimulating mucosal and systemic immune responses against an inactivated vaccine. Pharmaceutical Research, 34: 1491-1505.

Tort, L., Balasch, J. and Mackenzie, S. (2003). Fish immune system. A crossroads between innate and adaptive responses. Inmunología, 22(3): 277-286.

Uchuwittayakul, A., Thompson, K. D., Thangsunan, P., Phaksopa, J., Buncharoen, W., Saenphet, K., Kumwan, B., Meachasompop, P., Saenphet, S., Wiratama, N., Mahatnirunkul, T., Kantha, P., Suree, N. and Srisapoome, P. (2025). Evaluation of a hydrogel platform for encapsulated multivalent Vibrio antigen delivery to enhance immune responses and disease protection against vibriosis in Asian seabass (Lates calcarifer). Fish and Shellfish Immunology, 160: 110230.

Vazzana, M., Vizzini, A., Salerno, G., Di Bella, M. L., Celi, M., & Parrinello, N. (2008). Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax. Tissue & Cell, 40(2), 89–94. https://doi.org/10.1016/j.tice.2007.09.008.

Vasudeva Rao, Y., Das, B. K., Jyotyrmayee, P. and Chakrabarti, R. (2006). Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish and Shellfish Immunology, 20: 263-273.

Vinay, T. N., Kim, Y. J., Jung, M. H., Kim, W. S., Kim, D. H. and Jung, S. J. (2014). Inactivated vaccine against viral hemorrhagic septicemia (VHS) emulsified with Montanide ISA 763 A VG adjuvant provides long-term protection in olive flounder. Vaccine, 32: 4762-4769.

Wang, W. and Singh, M. (2011). Selection of adjuvants for enhanced vaccine potency. World Journal of Vaccines, 1: 33-78.

Wang, Y., Chen, X., Liu, W. and Zhang, S. (2024). Tissue-specific HSP gene expression in teleost fish under biotic stress: transcriptomic insights and immune implications. Aquaculture Research, 55: 4112-4125.

Wang, Z., Chou, Y. C., Hsu, J. L. and Chen, S. C. (2023). Current status and developments in fish vaccination for aquaculture. Reviews in Aquaculture, 15: 1070-1091.

Xiao, T., Zhou, L., Gui, L. and Li, M. (2010). Cystatin B in turbot enhances macrophage-mediated bacterial killing. Fish and Shellfish Immunology, 29: 764-770.

Yang, H., Song, L., Qu, Y., Jiang, M. and Li, Y. (2016). Turbidimetric method for lysozyme activity in fish with modification for microplate reader. Aquaculture, 462: 54-58.

Yang, Y. Y., Jiang, T. F., Liu, W. B. and Li, X. F. (2020). Tissue-specific effects of Glycyrrhiza uralensis on heat shock protein expression in Asian seabass. Aquaculture Research, 51: 4527-4535.

Yasuike, M., Kondo, H., Hirono, I. and Aoki, T. (2007). Difference in gene expression profile of Japanese flounder, Paralichthys olivaceus by stimulation with lipopolysaccharide (LPS)-induced expression of immune-related genes. Fish and Shellfish Immunology, 23: 1339-1351.

Zavasnik-Bergant, T. (2008). Cystatin protease inhibitors and immune functions. Frontiers in Bioscience-Landmark, 13(12): 4625-4637.

Zhu, Y., Lin, Q., Chen, J., Wu, H. and Zhang, X. (2024). Genome-wide identification, molecular characterisation, and expression analysis of the HSP70 and HSP90 gene families in Thamnaconus septentrionalis. International Journal of Molecular Sciences, 25(11): 5706.