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Stability and Efficacy of Live-Attenuated Vibrio harveyi Vaccines Under Different Storage Conditions in Zebrafish (Danio rerio) Models
Corresponding Author(s) : Ina Salwany Md Yasin
Jurnal Ilmiah Perikanan dan Kelautan, Vol. 16 No. 2 (2024): JURNAL ILMIAH PERIKANAN DAN KELAUTAN
Abstract
Graphical Abstract
Highlight Research
- The LD50 (median lethal dose) of Vibrio parahaemolyticus and harveyi in zebrafish was determined to be 1 x 106 CFU/mL, while for V. alginolyticus it was found to be 1 x 105 CFU/mL.
- The LAVh vaccine demonstrated cross-protection against various pathogenic strains of Vibrio, leading to an average of 80% survival rate in vaccinated individuals.
- The analysis of the LAVh vaccine emphasized its versatility, as it can be quickly deployed and stored as a freeze-dried powder.
- The LAVh vaccine can be easily accessible and user-friendly in various aquaculture environments, such as offshore and remote farms.
Abstract
Vibriosis poses a significant threat to marine teleosts, causing substantial losses in the global aquaculture industry. Previous work in our lab led to the development of a live-attenuated V. harveyi vaccine (LAVh) candidate that targets the serine endoprotease gene with a three-point knockout and has shown promise in protecting against vibriosis. However, further investigation is necessary to evaluate the stability and efficacy of its various storage conditions for broader applications. This study aims to determine how well the three different LAVh vaccine storage (fresh, stale, and freeze-dried LAVh) worked against vibriosis. A total of 1000 adult zebrafish (Danio rerio) (mean weight: 0.20±0.5 g) were divided into four groups. Groups 1, 2, and 3 were intraperitoneally injected with different LAVh vaccine storage (fresh, stale, and freeze-dried, respectively), while Group 4 received 0.01 M phosphate-buffered saline (PBS) and served as the unvaccinated control. Fish were monitored for 21 days post-vaccination for safety, stability, efficacy, and antibody analysis. The results showed that a modest dosage of 1 x 104 CFU/mL of LAVh vaccine from all storage conditions provided 80% survival upon intraperitoneal challenge with pathogenic strains of pathogenic V. harveyi, V. alginolyticus, and V. parahaemolyticus. This dosage induced significant antibody production and conferred cross-protection against different Vibrio spp., indicating the LAVh vaccine’s potential for commercial application. The LAVh vaccine demonstrated high effectiveness and suitability for storage as a freeze-dried powder. This study might offer significant insights into practical strategies for reducing vibriosis, especially in aquaculture settings with limited infrastructure.
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- Bailone, R. L., Fukushima, H. C. S., Ventura Fernandes, B. H., De Aguiar, L. K., Corrêa, T., Janke, H., Grejo Setti, P., Roça, R. D. O., & Borra, R. C. (2020). Zebrafish as an alternative animal model in human and animal vaccination research. Laboratory Animal Research, 36(1):1-10.
- Ben Hamed, S., Tapia‐Paniagua, S. T., Moriñigo, M. Á., & Ranzani‐Paiva, M. J. T. (2021). Advances in vaccines developed for bacterial fish diseases, performance and limits. Aquaculture Research, 52(6):2377-2390.
- Chin, Y. K., Al-saari, N., Zulperi, Z., Mohd-Aris, A., Salleh, A., Silvaraj, S., Mohamad, A., Lee, J. Y., Zamri-Saad, M., & Ina-Salwany, M. Y. (2020). Efficacy of bath vaccination with a live attenuated Vibrio harveyi against vibriosis in Asian seabass fingerling, Lates calcarifer. Aquaculture Research, 51(6):389-399.
- Du, Y., Hu, X., Miao, L., & Chen, J. (2022). Current status and development prospects of aquatic vaccines. Frontiers in Immunology, 13(1):1-31.
- FAO. (2024). The State of World Fisheries and Aquaculture 2024. FAO.
- Huang, Z., Anokyewaa, M. A., Wang, J., Jian, J., & Lu, Y. (2022). Pathogenicity and antibiotic resistance analysis of Vibrio species found in coastal water at mainly beach of Shenzhen, China. Frontiers in Marine Science, 9(1):1-13.
- Jørgensen, L. von G. (2020). Zebrafish as a model for fish diseases in aquaculture. Pathogens, 9(8):1-20.
- Laith, A. A., Abdullah, M. A., Nurhafizah, W. W. I., Hussein, H. A., Aya, J., Effendy, A. W. M., & Najiah, M. (2019). Efficacy of live attenuated vaccine derived from the Streptococcus agalactiae on the immune responses of Oreochromis niloticus. Fish and Shellfish Immunology, 90(7):235-243.
- Lan, N. G. T., Dong, H. T., Vinh, N. T., Senapin, S., Shinn, A. P., Salin, K. R., & 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, 1441(1):1-11.
- Liu, X., Jiao, C., Ma, Y., Wang, Q., & Zhang, Y. (2018). A live attenuated Vibrio anguillarum vaccine induces efficient immunoprotection in Tiger puffer (Takifugu rubripes). Vaccine, 36(11):1460-1466.
- Mohamad, A., Mursidi, F.-A., Zamri-Saad, M., Amal, M. N. A., Annas, S., Monir, M. S., Loqman, M., Hairudin, F., Al-saari, N., & Ina-Salwany, M. Y. (2022). Laboratory and Field Assessments of Oral Vibrio Vaccine Indicate the Potential for Protection against Vibriosis in Cultured Marine Fishes. Animals, 12(2):1-15.
- 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 Newly Developed Feed-Based Whole-Cell Polyvalent Vaccine against Vibriosis, Streptococcosis and Motile Aeromonad Septicemia in Asian Seabass, Lates calcarifer. Vaccines, 9(4):1-22.
- Mohd-Aris, A., Saad, M. Z., Daud, H. M., Yusof, M. T., & Ina-Salwany, M. Y. (2019). Vibrio harveyi protease deletion mutant as a live attenuated vaccine candidate against vibriosis and transcriptome profiling following vaccination for Epinephelus fuscoguttatus. Aquaculture International, 27(3-4):125-140.
- Mondal, H., & Thomas, J. (2022). A review on the recent advances and application of vaccines against fish pathogens in aquaculture. Aquaculture International, 30(4):1971-2000.
- Monir, M. S., Yusoff, S. M., Mohamad, A., & Ina-Salwany, M. Y. (2020). Vaccination of Tilapia against Motile Aeromonas Septicemia: A Review. Journal of Aquatic Animal Health, 32(2):65-76.
- Nazarudin, M.F., Shamsudin, M.N., & Yaakob, H.A.M. (2013). Scanning and Transmission Electron Microscopy Evaluation of The Effects and Efficiency of Formulated L. Lactis Cell Suspended in Skim Milk in The Presence of Starch and Gellan Gum as Excipients. Australian Journal of Basic and Applied Sciences, 7(2):343–349.
- Nehlah, R., Firdaus-Nawi, M., Nik-Haiha, N.Y., Karim, M., Zamri-Saad, M., & Ina-Salwany, M.Y. (2017). Recombinant vaccine protects juvenile hybrid grouper, Epinephelus fuscoguttatus × Epinephelus lanceolatus, against infection by Vibrio alginolyticus. Aquaculture International, 25(6):2047–2059.
- Pang, H., Chang, Y., Zheng, H., Tan, H., Zhou, S., Zeng, F., Hoare, R., Monaghan, S. J., Wang, N., & Ding, Y. (2022). A live attenuated strain of HY9901ΔvscB provides protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus). Aquaculture, 546(1):1-12.
- Pillai, S., Kobayashi, K., Michael, M., Mathai, T., Sivakumar, B., & Sadasivan, P. (2021). John William Trevan’s concept of Median Lethal Dose (LD50/LC50) – more misused than used. Journal of Pre-Clinical and Clinical Research, 15(3):137-141.
- Reed, L. J., & Muench, H. (1938). A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology, 27(3):493-497.
- Smyrli, M., Anka, I. Z., Koutsoni, O., Dotsika, E., Kyriazis, I. D., Pavlidis, M., & Katharios, P. (2022). Development of autogenous vaccines for farmed European seabass against Aeromonas veronii using zebrafish as a model for efficacy assessment. Fish & Shellfish Immunology, 123(4):381-387.
- Soto, E., Brown, N., Gardenfors, Z. O., Yount, S., Revan, F., Francis, S., Kearney, M. T., & Camus, A. (2014). Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia. Fish and Shellfish Immunology, 41(2):593-599.
- Sudheesh, P. S., & Cain, K. D. (2016). Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion vaccine. Fish and Shellfish Immunology, 56(9):169-180.
- Tan, H., Da, F., Lin, G., Wan, X., Cai, S., Cai, J., & Qin, Q. (2022). Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus). Fish and Shellfish Immunology, 124(5):543–551.
- Triga, A., Atinuke, Z., Smyrli, M., Fenske, L., & Katharios, P. (2024). Virulence and pangenome analysis of Vibrio harveyi strains from Greek and Red Sea marine aquaculture. Aquaculture, 587(11):1-11.
- Vinoj, G., Jayakumar, R., Chen, J. C., Withyachumnarnkul, B., Shanthi, S., & Vaseeharan, B. (2015). N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection. Fish and Shellfish Immunology, 42(1):204-212.
- Wan, Q., Zhai, S., Chen, M., Xu, M., & Guo, S. (2024). Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection. Microbial Pathogenesis, 189(4):1-11.
- Wang, J., Wang, S., Zhang, J., Zhu, C., Chen, S., & Zhou, Q. (2024). Integrated transcriptomic and metabolomic analysis provides insights into the responses to Vibrio infection in Plectropomus leopardus. Aquaculture, 587(11):1-10.
- Yang, B., Zhi, C., Li, P., Xu, C., Li, Q., & Liu, S. (2024). Genomic selection accelerates genetic improvement of resistance to Vibriosis in the Pacific oyster, Crassostrea gigas. Aquaculture, 584(8):1-9.
- Ye, N., Wu, H., & Zhang, Y. (2016). Maternal transfer and protection role in zebrafish (Danio rerio) offspring following vaccination of the brood stock with a live attenuated Vibrio anguillarum vaccine. Aquaculture Research, 47(11):3667-3678.
- Zhang, W., Chen, L., Feng, H., Wang, J., Zeng, F., Xiao, X., Jian, J., Wang, N., & Pang, H. (2022). Functional characterization of Vibrio alginolyticus T3SS regulator ExsA and evaluation of its mutant as a live attenuated vaccine candidate in zebrafish (Danio rerio) model. Frontiers in Veterinary Science, 9(1):1-18.
- Zhang, Z. H., Wu, H. Z., Xiao, J. F., Wang, Q. Y., Liu, Q., & Zhang, Y. X. (2014). Booster vaccination with live attenuated Vibrio anguillarum elicits strong protection despite weak specific antibody response in zebrafish. Journal of Applied Ichthyology, 30(1):117-120.
- Zhou, J., Yu, J., & Chu, Q. (2024). Comparative transcriptome analysis reveals potential regulatory mechanisms of genes and immune pathways following Vibrio harveyi infection in red drum (Sciaenops ocellatus). Fish & Shellfish Immunology, 146(3):1-11.
- Zupičić, I. G., Oraić, D., Križanović, K., & Zrnčić, S. (2024). Whole genome sequencing of Vibrio harveyi from different sites in the Mediterranean Sea providing data on virulence and antimicrobial resistance genes. Aquaculture, 581(5):1-11.
References
Bailone, R. L., Fukushima, H. C. S., Ventura Fernandes, B. H., De Aguiar, L. K., Corrêa, T., Janke, H., Grejo Setti, P., Roça, R. D. O., & Borra, R. C. (2020). Zebrafish as an alternative animal model in human and animal vaccination research. Laboratory Animal Research, 36(1):1-10.
Ben Hamed, S., Tapia‐Paniagua, S. T., Moriñigo, M. Á., & Ranzani‐Paiva, M. J. T. (2021). Advances in vaccines developed for bacterial fish diseases, performance and limits. Aquaculture Research, 52(6):2377-2390.
Chin, Y. K., Al-saari, N., Zulperi, Z., Mohd-Aris, A., Salleh, A., Silvaraj, S., Mohamad, A., Lee, J. Y., Zamri-Saad, M., & Ina-Salwany, M. Y. (2020). Efficacy of bath vaccination with a live attenuated Vibrio harveyi against vibriosis in Asian seabass fingerling, Lates calcarifer. Aquaculture Research, 51(6):389-399.
Du, Y., Hu, X., Miao, L., & Chen, J. (2022). Current status and development prospects of aquatic vaccines. Frontiers in Immunology, 13(1):1-31.
FAO. (2024). The State of World Fisheries and Aquaculture 2024. FAO.
Huang, Z., Anokyewaa, M. A., Wang, J., Jian, J., & Lu, Y. (2022). Pathogenicity and antibiotic resistance analysis of Vibrio species found in coastal water at mainly beach of Shenzhen, China. Frontiers in Marine Science, 9(1):1-13.
Jørgensen, L. von G. (2020). Zebrafish as a model for fish diseases in aquaculture. Pathogens, 9(8):1-20.
Laith, A. A., Abdullah, M. A., Nurhafizah, W. W. I., Hussein, H. A., Aya, J., Effendy, A. W. M., & Najiah, M. (2019). Efficacy of live attenuated vaccine derived from the Streptococcus agalactiae on the immune responses of Oreochromis niloticus. Fish and Shellfish Immunology, 90(7):235-243.
Lan, N. G. T., Dong, H. T., Vinh, N. T., Senapin, S., Shinn, A. P., Salin, K. R., & 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, 1441(1):1-11.
Liu, X., Jiao, C., Ma, Y., Wang, Q., & Zhang, Y. (2018). A live attenuated Vibrio anguillarum vaccine induces efficient immunoprotection in Tiger puffer (Takifugu rubripes). Vaccine, 36(11):1460-1466.
Mohamad, A., Mursidi, F.-A., Zamri-Saad, M., Amal, M. N. A., Annas, S., Monir, M. S., Loqman, M., Hairudin, F., Al-saari, N., & Ina-Salwany, M. Y. (2022). Laboratory and Field Assessments of Oral Vibrio Vaccine Indicate the Potential for Protection against Vibriosis in Cultured Marine Fishes. Animals, 12(2):1-15.
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 Newly Developed Feed-Based Whole-Cell Polyvalent Vaccine against Vibriosis, Streptococcosis and Motile Aeromonad Septicemia in Asian Seabass, Lates calcarifer. Vaccines, 9(4):1-22.
Mohd-Aris, A., Saad, M. Z., Daud, H. M., Yusof, M. T., & Ina-Salwany, M. Y. (2019). Vibrio harveyi protease deletion mutant as a live attenuated vaccine candidate against vibriosis and transcriptome profiling following vaccination for Epinephelus fuscoguttatus. Aquaculture International, 27(3-4):125-140.
Mondal, H., & Thomas, J. (2022). A review on the recent advances and application of vaccines against fish pathogens in aquaculture. Aquaculture International, 30(4):1971-2000.
Monir, M. S., Yusoff, S. M., Mohamad, A., & Ina-Salwany, M. Y. (2020). Vaccination of Tilapia against Motile Aeromonas Septicemia: A Review. Journal of Aquatic Animal Health, 32(2):65-76.
Nazarudin, M.F., Shamsudin, M.N., & Yaakob, H.A.M. (2013). Scanning and Transmission Electron Microscopy Evaluation of The Effects and Efficiency of Formulated L. Lactis Cell Suspended in Skim Milk in The Presence of Starch and Gellan Gum as Excipients. Australian Journal of Basic and Applied Sciences, 7(2):343–349.
Nehlah, R., Firdaus-Nawi, M., Nik-Haiha, N.Y., Karim, M., Zamri-Saad, M., & Ina-Salwany, M.Y. (2017). Recombinant vaccine protects juvenile hybrid grouper, Epinephelus fuscoguttatus × Epinephelus lanceolatus, against infection by Vibrio alginolyticus. Aquaculture International, 25(6):2047–2059.
Pang, H., Chang, Y., Zheng, H., Tan, H., Zhou, S., Zeng, F., Hoare, R., Monaghan, S. J., Wang, N., & Ding, Y. (2022). A live attenuated strain of HY9901ΔvscB provides protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus). Aquaculture, 546(1):1-12.
Pillai, S., Kobayashi, K., Michael, M., Mathai, T., Sivakumar, B., & Sadasivan, P. (2021). John William Trevan’s concept of Median Lethal Dose (LD50/LC50) – more misused than used. Journal of Pre-Clinical and Clinical Research, 15(3):137-141.
Reed, L. J., & Muench, H. (1938). A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology, 27(3):493-497.
Smyrli, M., Anka, I. Z., Koutsoni, O., Dotsika, E., Kyriazis, I. D., Pavlidis, M., & Katharios, P. (2022). Development of autogenous vaccines for farmed European seabass against Aeromonas veronii using zebrafish as a model for efficacy assessment. Fish & Shellfish Immunology, 123(4):381-387.
Soto, E., Brown, N., Gardenfors, Z. O., Yount, S., Revan, F., Francis, S., Kearney, M. T., & Camus, A. (2014). Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia. Fish and Shellfish Immunology, 41(2):593-599.
Sudheesh, P. S., & Cain, K. D. (2016). Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion vaccine. Fish and Shellfish Immunology, 56(9):169-180.
Tan, H., Da, F., Lin, G., Wan, X., Cai, S., Cai, J., & Qin, Q. (2022). Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus). Fish and Shellfish Immunology, 124(5):543–551.
Triga, A., Atinuke, Z., Smyrli, M., Fenske, L., & Katharios, P. (2024). Virulence and pangenome analysis of Vibrio harveyi strains from Greek and Red Sea marine aquaculture. Aquaculture, 587(11):1-11.
Vinoj, G., Jayakumar, R., Chen, J. C., Withyachumnarnkul, B., Shanthi, S., & Vaseeharan, B. (2015). N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection. Fish and Shellfish Immunology, 42(1):204-212.
Wan, Q., Zhai, S., Chen, M., Xu, M., & Guo, S. (2024). Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection. Microbial Pathogenesis, 189(4):1-11.
Wang, J., Wang, S., Zhang, J., Zhu, C., Chen, S., & Zhou, Q. (2024). Integrated transcriptomic and metabolomic analysis provides insights into the responses to Vibrio infection in Plectropomus leopardus. Aquaculture, 587(11):1-10.
Yang, B., Zhi, C., Li, P., Xu, C., Li, Q., & Liu, S. (2024). Genomic selection accelerates genetic improvement of resistance to Vibriosis in the Pacific oyster, Crassostrea gigas. Aquaculture, 584(8):1-9.
Ye, N., Wu, H., & Zhang, Y. (2016). Maternal transfer and protection role in zebrafish (Danio rerio) offspring following vaccination of the brood stock with a live attenuated Vibrio anguillarum vaccine. Aquaculture Research, 47(11):3667-3678.
Zhang, W., Chen, L., Feng, H., Wang, J., Zeng, F., Xiao, X., Jian, J., Wang, N., & Pang, H. (2022). Functional characterization of Vibrio alginolyticus T3SS regulator ExsA and evaluation of its mutant as a live attenuated vaccine candidate in zebrafish (Danio rerio) model. Frontiers in Veterinary Science, 9(1):1-18.
Zhang, Z. H., Wu, H. Z., Xiao, J. F., Wang, Q. Y., Liu, Q., & Zhang, Y. X. (2014). Booster vaccination with live attenuated Vibrio anguillarum elicits strong protection despite weak specific antibody response in zebrafish. Journal of Applied Ichthyology, 30(1):117-120.
Zhou, J., Yu, J., & Chu, Q. (2024). Comparative transcriptome analysis reveals potential regulatory mechanisms of genes and immune pathways following Vibrio harveyi infection in red drum (Sciaenops ocellatus). Fish & Shellfish Immunology, 146(3):1-11.
Zupičić, I. G., Oraić, D., Križanović, K., & Zrnčić, S. (2024). Whole genome sequencing of Vibrio harveyi from different sites in the Mediterranean Sea providing data on virulence and antimicrobial resistance genes. Aquaculture, 581(5):1-11.