Oral vaccination of Grass carp (Ctenopharyngodon idella) against Aeromonas veronii as a sustainable strategy for disease control and biodiversity conservation
DOI:
https://doi.org/10.5281/zenodo.18208272Keywords:
Grass carp, oral vaccine, Aeromonas veronii, fish oil adjuvant, immunityAbstract
Ctenopharyngodon idella (C. idella), a freshwater species of significant ecological and economic value, plays a vital role in aquaculture and aquatic ecosystem management. However, bacterial pathogens such as Aeromonas veronii (A. veronii) represent a major threat to fish survival, biodiversity, and sustainable aquaculture production. The current research assessed the protective potential of a monovalent oral vaccine formulated against A. veronii in grass carp (C. idella). Experimental feeds were formulated by two delivery methods: spraying and incorporation with either fish oil or mineral oil, included as adjuvants (10%). Healthy fingerlings (20 ± 5 g) were randomly assigned to five dietary groups: spray vaccine + mineral oil (SV-MO), incorporated vaccine + mineral oil (IV-MO), spray vaccine + fish oil (SV-FO), incorporated vaccine + fish oil (IV-FO), and unvaccinated control (C). The feeding trial lasted 60 days. After 56 days of dietary vaccination, vaccinated fish demonstrated markedly (p < 0.05) improved growth, antioxidant capacity, serum biochemical profiles (including total protein, albumin, and globulin) and immunological responses (lysozyme activity and antibody agglutination) relative to the control group. The dietary group IV-FO achieved the highest growth performance (12.7 ± 0.15a g) and exhibited superior immune responses relative to other treatments. Challenge with live A. veronii demonstrated that IV-FO provided the greatest protection (87%), with only 13% mortality and a relative percent survival (RPS) of 85%. Protection rates were comparatively lower in SV-FO (67%, RPS 62%), SV-MO (67%, RPS 62%), and IV-MO (54%, RPS 70%), while the control group displayed minimal protection (14%) with 86% mortality. The findings indicate that oral delivery of vaccines, particularly via incorporation with fish oil, provides an effective strategy to enhance fish resilience against bacterial infection. Fish oil acted as an immunomodulatory adjuvant that strengthened antibody production, balanced oxidative stress, and improved mucosal defense, whereas mineral oil mainly stabilized the antigen and prolonged its immune-stimulating effect. These differences explain the superior vaccine performance observed in the incorporated fish-oil formulation (IV-FO). By improving fish health and reducing disease-induced mortality, this approach supports sustainable aquaculture practices while contributing to biodiversity conservation in freshwater ecosystems.
References
Abu-Elala, N. M., Samir, A., Wasfy, M., & Elsayed, M. (2019). Efficacy of injectable and immersion polyvalent vaccine against streptococcal infections in broodstock and offspring of Nile tilapia (Oreochromis niloticus). Fish & Shellfish Immunology, 88, 293–300. * https://doi.10.1016/j.fsi.2019.02.042
Abu-Nor, N., Zamri Saad, M., Md Yasin, I. S., Salleh, A., Mustaffa Kamal, F., Matori, M. F., & Azmai, M. N. (2020). Efficacy of whole cell inactivated Vibrio harveyi vaccine against vibriosis in a marine red hybrid tilapia (Oreochromis niloticus × O. mossambicus) model. Vaccines, 8(4), 734. * https://doi.org/10.3390/vaccines8040734
Aebi, H. (1984). Catalase in vitro. In L. Packer (Ed.), Methods in enzymology (Vol. 105, pp. 121–126). Academic Press.
Amal, M. N., & Zamri Saad, M. (2011). Streptococcosis in tilapia (Oreochromis niloticus): A review. Pertanika Journal of Tropical Agricultural Science, 34, 195–206.
Amend, D. F. (1981). Potency testing of fish vaccines. Developments in Biological Standardization, 49, 447–454.
Anantasuk, N., Phurahong, T., Pumchan, A., Hirono, I., & Unajak, S. (2024). Molecular characterization and bivalent vaccine development of Aeromonas hydrophila and Aeromonas veronii in Nile tilapia (Oreochromis niloticus). Aquaculture, 590, 741042. * https://doi.org/10.1016/j.aquaculture.2024.741042Get rights and content
AOAC International. (2016). Official methods of analysis (20th ed., G. W. Latimer Jr., Ed.). AOAC International.
Argayosa, A. M., Santos, M. N., Argayosa, V. B., Pakingking, R. V., Jr., Buhian, W., Salvador, M. L., & Teh, R. E. (2024). Pathogenicity of Aeromonas veronii from Nile tilapia (Oreochromis niloticus) and efficacy of fish oral vaccine against motile aeromonad septicemia in tank trials. Aquaculture, 4(3), 163–179. * https://doi.org/10.3390/aquacj4030012
Asadi, M. S., Mirvaghefei, A. R., Nematollahi, M. A., Banaee, M., & Ahmadi, K. (2012). Effects of watercress (Nasturtium nasturtium) extract on selected immunological parameters of rainbow trout (Oncorhynchus mykiss). Open Veterinary Journal, 2, 32–39.
Assane, I. M., Gozi, K. S., Valladão, G. M., & Pilarski, F. (2019). Combination of antimicrobials to reduce their use in aquaculture: Thiamphenicol/florfenicol against Aeromonas hydrophila. Aquaculture, 507, 238–245.
* https://doi.org/10.1016/j.aquaculture.2019.04.021
Assefa, A., & Abunna, F. (2018). Maintenance of fish health in aquaculture: Review of epidemiological approaches for prevention and control. Veterinary Medicine International, 2018, 5432497. * https://doi.org/10.1155/2018/5432497
Aucouturier, J., Dupuis, L., & Ganne, V. (2001). Adjuvants designed for veterinary and human vaccines. Vaccine, 19(17), 2666–2672. * https://doi.org/10.1016/S0264-410X(00)00498-9
Bowman, J. P. (2005). Genus I. Aeromonas. In D. J. Brenner, N. R. Krieg, J. T. Staley, & G. M. Garrity (Eds.), Bergey’s manual of systematic bacteriology (2nd ed.). Springer.
Calder, P. C. (1998). Immunoregulatory and anti-inflammatory effects of n-3 polyunsaturated fatty acids. Brazilian Journal of Medical and Biological Research, 31, 467–490. * https://doi.org/10.1590/S0100-879X1998000400002
Chettri, J. K., Jaafar, R. M., Skov, J., Kania, P. W., Dalsgaard, I., & Buchmann, K. (2015). Booster immersion vaccination using diluted Yersinia ruckeri bacterin confers protection against ERM in rainbow trout. Aquaculture, 440, 1–5.
* https://doi.org/10.1016/j.aquaculture.2015.01.027
Citarasu, T., Sivaram, V., Immanuel, G., Rout, N., & Murugan, V. (2006). Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp Penaeus monodon with reference to haematological, biochemical and immunological changes. Fish & Shellfish Immunology, 21(4), 372–384. * https://doi.org/10.1016/j.fsi.2006.01.002
Cui, H., Hao, S., & Arous, E. (2007). A distinct cause of necrotizing fasciitis: Aeromonas veronii biovar sobria. Surgical Infections, 8(5), 523–528. * https://doi.org/10.1089/sur.2006.04
Dong, C., He, G., Mai, K., Zhou, H., & Xu, W. (2016). Palatability of water-soluble extracts and fishmeal replacement for juvenile turbot (Scophthalmus maximus). Journal of Ocean University of China, 15(3), 561–567. * https://doi.10.1007/s11802-016-2898-8
Doumas, B. T., Watson, W., & Biggs, H. G. (1971). Albumin standards and measurement of serum albumin with bromocresol green. Clinica Chimica Acta, 31(1), 87–96. * https://doi.10.1016/0009-8981(71)90365-2
Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11(1), 1–42. * https://doi.org/10.2307/3001478
Farias, T. H., Arijo, S., Medina, A., Pala, G., da Rosa Prado, E. J., Montassier, H. J., & de Andrade Belo, M. A. (2020). Immune responses induced by inactivated vaccine against Aeromonas hydrophila in pacu (Piaractus mesopotamicus). Fish & Shellfish Immunology, 101, 186–191. * https://doi.10.1016/j.fsi.2020.03.059
Firdaus Nawi, M., Yusoff, S. M., Yusof, H., Abdullah, S. Z., & Zamri Saad, M. (2014). Efficacy of feed-based adjuvant vaccine against Streptococcus agalactiae in Oreochromis spp. in Malaysia. Aquaculture Research, 45(1), 87–96. *https://doi.10.1111/j.1365-2109.2012.03207.x
Fraser, T. W., Hansen, T., Mayer, I., Skjæraasen, J. E., Glover, K. A., Sambraus, F., & Fjelldal, P. G. (2014). The effect of triploidy on vaccine side-effects in Atlantic salmon. Aquaculture, 433, 481–490. * https://doi.org/10.1016/j.aquaculture.2014.07.009
Gencer, Ö. (2025). Dietary innovation in blue crab aquaculture: Effects on performance and product quality. Aquaculture International, 33(3), 1–12. * https://doi. 10.1007/s10499-025-01899-9
Han, B., Xu, K., Liu, Z., Ge, W., Shao, S., Li, P., Yan, N., Li, X., & Zhang, Z. (2018). Oral yeast-based DNA vaccine confers effective protection from Aeromonas hydrophila infection on Carassius auratus. Fish & Shellfish Immunology, 84, 948–954. *https://doi.org/10.1016/j.fsi.2018.10.065
Ismail, M. S., Siti-Zahrah, A., Syafiq, M. R. M., Amal, M. N. A., & Firdaus-Nawi, M. (2016). Feed-based vaccination regime against streptococcosis in red tilapia (Oreochromis niloticus × O. mossambicus). BMC Veterinary Research, 12, 194. * https://doi.org/10.1186/s12917-016-0834-1
Jomova, K., Raptova, R., Alomar, S. Y., Alwasel, S. H., Nepovimova, E., Kuca, K., & Valko, M. (2023). Reactive oxygen species, toxicity, oxidative stress, and antioxidants: Chronic diseases and aging. Archives of Toxicology, 97(10), 2499–2574. * https:/doi.org/ 10.1007/s00204-023-03562-9
Kahiesh Esfandiari, M., Ina-Salwany, M., Aslah, M., Soltani, M., Sabili, A., Karimi, M., & Muthukrishnan, S. (2019). Growth performance, palatability and water stability of oral feed-based vaccines against Streptococcus agalactiae in red tilapia (Oreochromis sp.). Journal of Biochemical Technology, 10(2), 106–115.
Kalita, B., Pokhrel, H., Hussain, I. A., & Mohan, C. V. (2018). Kinetics of antibody titers and protection in Indian major carps to Aeromonas hydrophila antigen. International Journal of Current Microbiology and Applied Sciences, 7(9), 1461–1469. * https://doi.org/10.20546/ijcmas.2018.709.175
Kaur, A., Holeyappa, S. A., Bansal, N., Kaur, V. I., & Tyagi, A. (2020). Ameliorative effect of turmeric supplementation in feed of Labeo rohita challenged with pathogenic Aeromonas veronii. Aquaculture International, 28(3), 1169–1182. * https://doi.org/10.1007/s10499-020-00518-z
Kumar, V., Sahu, N. P., Pal, A. K., & Kumar, S. (2007). Immunomodulation of Labeo rohita juveniles due to dietary gelatinized and non-gelatinized starch. Fish & Shellfish Immunology, 23(2), 341–353. * https://doi.org/10.1016/j.fsi.2006.11.008
Legario, F. S., Choresca, C. H., Jr., Turnbull, J. F., & Crumlish, M. (2020). Isolation and molecular characterization of streptococcal species from farmed Nile tilapia in the Philippines. Journal of Fish Diseases, 43(12), 1431–1442. * https://doi.org/10.1111/jfd.13247
Magnadóttir, B. (2006). Innate immunity of fish: Overview. Fish & Shellfish Immunology, 20(2), 137–151. * https://doi.org/10.1016/j.fsi.2004.09.006
Mahmood, S., Rasool, F., Parveen, S., Ayub, A., Manzoor, K., Latif, M. D., Hussain, T., Younas, A., Rabbani, G., Hidait, N., & Anjum, K. M. (2024). Prevalence, antimicrobial profiling and molecular characterization of antimicrobial resistant genes of pathogenic bacteria detected in Channa marulius of the Indus riverine system in Pakistan. Pakistan Veterinary Journal, 44(4). . * https://doi.org/ 10.29261/pakvetj/2024.xxx
Mamun, M., Nasren, S., Abhiman, P., Rathore, S., Sowndarya, N., Ramesh, K., & Shankar, K. (2020). Effect of biofilm of Aeromonas hydrophila oral vaccine on growth and tissue histopathology in striped catfish. Indian Journal of Animal Research, 54(4), 563–569. * https://doi. 10.18805/ijar.B-3814
Manzoor, K., Rasool, F., Khan, N., Anjum, K. M., & Parveen, S. (2023). Prevalence and molecular detection of Edwardsiella tarda in cultured tilapia species of fish farms of Punjab in Pakistan and their postmortem examination. Pakistan Veterinary Journal, 43(2), 309–314. * https://doi.10.29261/pakvetj/2023.015
Maqsood, S., Samoon, M. H., & Singh, P. (2009). Immunomodulatory and growth promoting effect of dietary levamisole in Cyprinus carpio fingerlings against the challenge of Aeromonas hydrophila. Turkish Journal of Fisheries and Aquatic Sciences, 9(1), 111–120.
Matsuura, Y., Terashima, S., Takano, T., & Matsuyama, T. (2019). Current status of fish vaccines in Japan. Fish & Shellfish Immunology, 95, 236–247. * https://doi.org/10.1016/j.fsi.2019.09.031
Mohamad, A., Zamri-Saad, M., Amal, M. N. A., Al-Saari, N., Monir, M. S., Chin, Y. K., & Md Yasin, I. S. (2021). Vaccine efficacy of a feed-based polyvalent vaccine against vibriosis, streptococcosis and MAS in Asian seabass (Lates calcarifer). Vaccines, 9(4), 368. * https://doi.10.3390/vaccines9040368
Mohd Ali, N. S., Ngalimat, M. S., Zamri-Saad, M., Azmai, M. N. A., Salleh, A., Zulperi, Z., & Md Yasin, I. S. (2024). Stability characterizations of feed-based bivalent vaccine containing inactivated Streptococcus agalactiae and Aeromonas hydrophila against streptococcosis and Aeromonas infections in red hybrid tilapia (Oreochromis sp.). Archives of Microbiology, 206(11):444. *https:// https://doi.10.1007/s00203-024-04166-2
Monir, M. S., Yusoff, S. B., Zulperi, Z. B., Hassim, H. B., Mohamad, A., Ngoo, M. S., & Ina-Salwany, M. Y. (2020). Haemato-immunological responses and feed-based bivalent vaccine effectiveness against Streptococcus iniae and Aeromonas hydrophila in hybrid red tilapia. BMC Veterinary Research, 16, 1–4. * https://doi.10.1186/s12917-020-02443-y
Mubeen, N., Abbas, F., Hafeez-ur-Rehman, M., Amin, I., Jalil, S., & Can, E. (2025a). Immunopotential induced in culturable carps fed on edible bivalent vaccine against Aeromonas veronii and Aeromonas hydrophila. Aquaculture Research, 2025(1), 3462669. * https://doi.org/10.1155/are/3462669
Mubeen, N., Abbas, F., Hafeez-ur-Rehman, M., Crumlish, M., Mahboob, H., Akmal, M., Sadiqa, A., Alam, T.M. & Jalil, S. (2025b). Effectiveness of feed-based monovalent Aeromonas vaccine in farmed carp. Microorganisms, 13(8), 1903. * https://doi.org/10.3390/microorganisms13081903
Nasr-Eldahan, S., Attia Shreadah, M., Maher, A. M., El-Sayed Ali, T., & Nabil-Adam, A. (2024). New vaccination approach using formalin-killed Streptococcus pyogenes vaccine on the liver of Oreochromis niloticus fingerlings. Scientific Reports, 14(1), 18341. * https://doi.org/10.1038/s41598-024-67198-0
Nayak, D. K., Mishra, B. K., & Mishra, K. P. (2004). Effects of some pesticides on carbohydrate metabolism of the air-breathing fish Clarias batrachus. Journal of Applied Sciences and Environmental Management, 8(1), 59–62.
Obaldo, L. G., Divakaran, S., & Tacon, A. G. (2002). Method for determining physical stability of shrimp feeds in water. Aquaculture Research, 33(5), 369–377. * https://doi.org/10.1046/j.1365-2109.2002.00681.x
Paul, P., Adikesavalu, H., Banerjee, S., & Abraham, T. J. (2015). Antibiotic resistant motile aeromonads induced septicemia in Philippine catfish Clarias batrachus (Linnaeus, 1758). Croatian Journal of Fisheries, 73(4), 170–175. * https://doi.org/10.14798/73.4.844
Peepim, T., Dong, H., Senapin, S., Khunrae, P., & Rattanarojpong, T. (2016). Epr3: Conserved immunogenic protein among Aeromonas spp. inducing antibody response in Nile tilapia. Aquaculture, 464, 399–409. * https://doi.org/10.1016/j.aquaculture.2016.07.022
Poline, M. E., Babila Jasmine, J., Annalakshmi, T., Xavier Innocent, B., & Krishnamoorthy, S. (2023). Efficacy of Aeromonas hydrophila vaccine on biochemical parameters of freshwater fish Channa striatus. International Journal of Zoological Investigations, 9(2), 810–820. * https://doi.10.33745/ijzi.2023.v09i02.091
Pridgeon, J. W., Klesius, P. H., & Yildirim-Aksoy, M. (2013). Development of live attenuated bacterial vaccines via resistance selection. Vaccine, 31(18), 2222–2230. * https://doi.org/10.1016/j.vaccine.2013.03.004
Queipo-Ortuño, M. I., De Dios Colmenero, J., Macias, M., Bravo, M. J., & Morata, P. (2008). Boiling method for bacterial DNA template preparation and PCR inhibition by IgG. Clinical and Vaccine Immunology, 15(2), 293–296. * https://doi.org/10.1128/CVI.00270-07
Rahman, M. M., Somsiri, T., Tajima, K., & Ezura, Y. (2004). Distribution of Aeromonas spp. in aquatic animals in Southeast Asia. Pakistan Journal of Biological Sciences, 7(2), 258–268. * http://doi.org/10.3923/pjbs.2004.258.268
Rasmussen-Ivey, C. R., Hossain, M. J., Odom, S. E., Terhune, J. S., Hemstreet, W. G., Shoemaker, C. A., Zhang, D., Xu, D. H., Griffin, M. J., Liu, Y. J., Figueras, M. J., Santos, S. R., Newton, J. C., & Liles, M. R. (2016). Classification of a hypervirulent Aeromonas hydrophila pathotype responsible for epidemic outbreaks in warm-water fishes. Frontiers in Microbiology, 7, 1615. *https:// doi: 10.3389/fmicb.2016.01615
Reinhold, J. G. (1953). Manual for determination of serum total protein, albumin and globulin fractions by Biuret method. In M. Reiner (Ed.), Standard Methods of Clinical Chemistry (Vol. 1, pp. 88–89). Academic Press.
Reyes, M., Ramírez, C., Ñancucheo, I., Villegas, R., Schaffeld, G., Kriman, L., Gonzalez, J., & Oyarzun, P. (2017). Oral DNA vaccination against IPNV using in-feed delivery provides protection in Atlantic salmon. Vaccine, 35(4), 626–632. * https://doi.org/10.1016/j.vaccine.2016.12.013
Rohani, M. F., Islam, S. M., Hossain, M. K., Ferdous, Z., Siddik, M. A., Nuruzzaman, M., Padeniya, U., Brown, C., & Shahjahan, M. (2022). Probiotics, prebiotics and synbiotics improve aquafeed functionality and fish performance. Fish & Shellfish Immunology, 120, 569–589. * https://doi.org/10.1016/j.fsi.2021.12.037
Ruckenstein, E., & Zeng, X. (1997). Macroporous chitin affinity membranes for lysozyme separation. Biotechnology and Bioengineering, 56(6), 610–617. * https://doi.org/10.1002/(SICI)1097-0290(19971220)56:6<610::AID-BIT3>3.0.CO;2-Q
Salas-Leiton, E., Anguis, V., Martín-Antonio, B., Crespo, D., Planas, J. V., Infante, C., Cañavate, J. P., & Manchado, M. (2010). Effects of stocking density and feeding on growth and immunity gene expression in Senegalese sole. Fish & Shellfish Immunology, 28(2), 296–302. * https://doi.org/10.1016/j.fsi.2009.11.006
Singh, V., Chaudhary, D. K., & Mani, I. (2012). Molecular characterization and modeling of 16S rRNA secondary structure from Aeromonas veronii. International Journal of Applied Biology and Pharmaceutical Technology, 3(1), 253–260.
Sotomayor-Gerding, D., Troncoso, J. M., Díaz-Riquelme, K., Torres-Obreque, K. M., Cumilaf, J., Yañez, A. J., & Rubilar, M. (2022). Microencapsulation of Piscirickettsia salmonis antigens for fish oral immunization: optimization and stability studies. Polymers, 14(23), 5115. * https://doi.org/10.3390/polym14235115
Sughra, F., Hafeez-ur-Rehman, M., Abbas, F., Altaf, I., Aslam, S., Ali, A., Khalid, M., Mustafa, G., & Azam, S. M. (2021). Evaluation of oil-based inactivated Aeromonas hydrophila vaccine in Indian major carps: Immune response, challenge, growth and histopathology. Aquaculture Reports, 21, 100885. * https://doi.org/10.1016/j.aqrep.2021.100885
Sukenda, Carman, O., Rahman, R., Hidayatullah, D., & Yumaidawati, N. (2017). Vaccination of Nile tilapia broodstock with whole cell vaccine and fry resistance to Aeromonas hydrophila. Indonesian Journal of Aquaculture, 16(2), 268–276. *https://doi.org/10.19027/jai.16.2.268-276
Sun, Y. I., Oberley, L. W., & Li, Y. (1988). A simple method for clinical assay of superoxide dismutase. Clinical Chemistry, 34(3), 497–500. *https://doi.org/10.1093/clinchem/34.3.497
Swain, P., Dash, S., Sahoo, P. K., Routray, P., Sahoo, S. K., Gupta, S. D., Meher, P. K., & Sarangi, N. (2007). Non-specific immune parameters of brood Labeo rohita and seasonal variation. Fish & Shellfish Immunology, 22(1–2), 38–43. * https://doi.org/10.1016/j.fsi.2006.03.010
Thirumalaikumar, E., Lelin, C., Sathishkumar, R., Vimal, S., Anand, S. B., Babu, M. M., & Citarasu, T. (2021). Oral delivery of pVAX-OMP and pVAX-hly DNA vaccine using Cs-TPP nanoparticles in Labeo rohita against Aeromonas hydrophila. Fish & Shellfish Immunology, 115, 189–197. * https://doi.org/10.1016/j.fsi.2021.06.004
Tkachenko, H., Kurhaluk, N., Grudniewska, J., & Andriichuk, A. (2014). Tissue-specific responses of oxidative stress biomarkers and antioxidant defenses in rainbow trout Oncorhynchus mykiss during a vaccination against furunculosis. Fish Physiology and Biochemistry, 40(4), 1289–1300. * https://doi. 10.1007/s10695-014-9924-9
Ullah, M., Rasool, F., Khan, N., Ali, S., & Sheikh, A. A. (2023). Antibiotic resistance and its gene profile in Escherichia coli isolated from diseased farm-raised carps in Punjab, Pakistan. Pakistan Veterinary Journal, 43(3), 470–476. * https://doi.10.29261/pakvetj/2023.041
Wang, Q. C., Ji, W., & Xu, Z. (2020). Current use and development of fish vaccines in China. Fish & Shellfish Immunology, 96, 223–234. * https://doi.org/10.1016/j.fsi.2019.12.010
Watts, J. E., Schreier, H. J., Lanska, L., & Hale, M. S. (2017). Rising antimicrobial resistance in aquaculture: Sources, sinks and solutions. Marine Drugs, 15(6), 158. * https://doi.org/10.3390/md15060158
Wendel, A. (1981). Glutathione peroxidase. In Methods in Enzymology (Vol. 77, pp. 325–333). Academic Press.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Journal of Wildlife and Biodiversity
This work is licensed under a Creative Commons Attribution 4.0 International License.
JWB applies the 