Aquaculture International

, Volume 27, Issue 1, pp 125–140 | Cite as

Vibrio harveyi protease deletion mutant as a live attenuated vaccine candidate against vibriosis and transcriptome profiling following vaccination for Epinephelus fuscoguttatus

  • Aslizah Mohd-Aris
  • Mohd-Zamri Saad
  • Hassan Mohd Daud
  • Mohd Termizi Yusof
  • Md Yasin Ina-SalwanyEmail author


Grouper aquaculture industries have a high risk of being inflicted by bacterial diseases such as vibriosis. Various types of vaccines for vibriosis have been studied throughout the years, yet the potential of live attenuated vaccines remains unsubstantial. Correspondingly, this study attempts to develop a Vibrio harveyi protease deletion mutant into a live attenuated vaccine candidate against vibriosis for Epinephelus fuscoguttatus. Site-directed mutagenesis (SDM) and allelic exchange replacement techniques are employed to synthesize genetically attenuated V. harveyi strain MVh-vhs. The evaluation on safety levels showed that MVh-vhs strain is safe when tested on E. fuscoguttatus. A 100% survival rate with no sign of vibriosis is indicated in fish challenged with the attenuated strain. In contrast, fish challenged with the parental strain showed obvious clinical signs of vibriosis. The median lethal dosage (LD50) of fish challenged with the parental strain is found at 106 CFU/fish. A single dose IP administration of the attenuated strain at 105 CFU/fish following a bacterial challenge at dose 108 CFU/fish is done 4 weeks post vaccination. The vaccinated fish show 52% relative percentage survival (RPS). The transcriptomic profiling following vaccination evoked the regulation of autophagosome pathway and the coagulation and complement cascade pathways as well as antigen processing and presentation pathways. As a conclusion, the V. harveyi attenuated strain MVh-vhs has significant potential to be applied as a live vaccine candidate against vibriosis for E. fuscoguttatus.


Epinephelus fuscoguttatus Fish immunity Live attenuated vaccine Vibriosis Vibrio harveyi 



The author would like to thank the Malaysian government (under SLAB-SLAI program) for supporting PhD study and Fisheries Society for awarding the AFS-Kanazawa Research Fellowship Grant to Aslizah Mohd Aris.

Funding information

This work was also supported by the grants provided by Ministry of Higher Institution via Higher Institution Centre of Excellence (HICoE) under vote no: 6369100 and Universiti Putra Malaysia Grant GP-IPB under vote number GP-IPB 9484102.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC), Universiti Putra Malaysia (UPM) under approval number UPM/IACUC/AUP/R059/2016.


  1. Amend DF (1981) Potency testing of fish vaccines. In: Anderson DP, Hennessen H (eds) Fish biologies: serodiagnostics and vaccines development in biological standardization. Karger, Basel, pp 447–454Google Scholar
  2. Austin B, Austin DA (2007) Bacterial fish pathogens: diseases of farmed and wild fish, 4th edn. Praxis Publishing Ltd, United KingdomGoogle Scholar
  3. Bondad-Reantaso MG, Subasinghe RP, Richard Arthur J, Ogawa K, Chinabut S, Adlard R, Tan Z, Shariff M (2005) Disease and health management in Asian aquaculture. Vet Parasitol 132(3–4):249–272CrossRefGoogle Scholar
  4. Cai SH, Lu YS, Wu ZH, Jian JC (2013) Cloning, expression of Vibrio alginolyticus outer membrane protein-OmpU gene and its potential application as vaccine in crimson snapper, Lutjanus erythropterus Bloch. J Fish Dis 36(8):695–702CrossRefGoogle Scholar
  5. Crotzer VL, Blum JS (2010) Autophagy and adaptive immunity. Immunology 131:9–17CrossRefGoogle Scholar
  6. Delgado CL, Wada N, Rosegrant MW, Meijer S, Ahmed M (2003) The future of fish: issues and trends to 2020. Washington DC, USA: International Food Policy Research Institute and Worldfish Center, pp 1–5Google Scholar
  7. FAO (2014) The state of world fisheries and aquaculture 2014: opportunities and challenge. Food and Agriculture Organization of the United Nations, RomeGoogle Scholar
  8. Fathin-Amirah M, Ina-Salwany MY, Zamri-Saad M, Amal MNA, Siti Zahrah A (2015) Antigenicity analysis of outer membrane proteins (OMPs) of Vibrio sp isolated from diseased tiger grouper for vaccine development. In: Proceedings of the TIIKM’s 2nd International Conference on Fisheries and Aquaculture. Colombo, Sri LankaGoogle Scholar
  9. Finney DJ (1952) Probit analysis. Cambridge University Press, CambridgeGoogle Scholar
  10. Gauger E, Smolowitz R, Uhlinger K, Casey J, Gómez-Chiarri M (2006) Vibrio harveyi and other bacterial pathogens in cultured summer flounder, Paralichthys dentatus. Aquaculture 260:10–20CrossRefGoogle Scholar
  11. Håstein T, Gudding R, Evensen Ø (2005) Bacterial vaccines for fish—an update of the current situation worldwide. Dev Biologicals 121. pp 55–74Google Scholar
  12. Hu Y-H, Deng T, Sun B-G, Sun L (2012) Development and efficacy of an attenuated Vibrio harveyi vaccine candidate with cross protectivity against Vibrio alginolyticus. Fish Shellfish Immunol 32:1151–1161Google Scholar
  13. Ingmer H, Brøndsted L (2009) Proteases in bacterial pathogenesis. Res Microbiol 160:704–710CrossRefGoogle Scholar
  14. Lan MZ, Peng X, Xiang MY, Xia ZY, Bo W, Jie L, Li XY, Jun ZP (2007) Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.). Fish Shellfish Immunol 23:521–530CrossRefGoogle Scholar
  15. Ma Y, Zhang Y, Zhao D (2008) Polyvalent attenuated live vaccine for preventing and curing vibriosis of cultivated fish. US Patent 0274136 A1Google Scholar
  16. Male D (2014) Immunology: an illustrated outline. In: Garland science, 1st edn. Taylor and Francis Group, USGoogle Scholar
  17. Mohd-Aris A, Ina-Salwany MY, Zamri-Saad M, Daud HM, Alipiah NM (2016) Molecular characterization of Vibrio harveyi virulence-associated serine protease and outer membrane protein genes for vaccine development. Int J Biosci 8(3):10–28CrossRefGoogle Scholar
  18. Patel DH, Wi SG, Bae HJ (2009) Modification of overlap extension PCR: a mutagenic approach. Indian J Biotechnol 8:183–186Google Scholar
  19. Sung YY, MacRae TH, Sorgeloos P, Bossier P (2011) Stress response for disease control in aquaculture. Rev Aquac 3:120–137CrossRefGoogle Scholar
  20. Toranzo AE, Santos Y, Barja JL (1996) Immunization with bacterial antigens: Vibrio infections. Dev Biol Stand 90:93–105Google Scholar
  21. Wang Y-D, Huang S-J, Chou H-N, Liao W-L, Gong H-Y, Chen J-Y (2014) Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity related complement pathway in Epinephelus coioides. BMC Genomics 15:1102CrossRefGoogle Scholar
  22. Yang D, Liu Q, Yang M, Wu H, Wang Q, Xiao J, Zhang Y (2012) RNA-seq liver transcriptome analysis reveals an activated MHC-I pathway and an inhibited MHC-II pathway at early stage of vaccine immunization in zebrafish. BMC Genomics 13:319CrossRefGoogle Scholar
  23. Zhang W-W, Sun K, Cheng S, Sun L (2008) Characterization of DegQVh, a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain. Appl Environ Microbiol 74(20):6254–6262CrossRefGoogle Scholar
  24. Zorrilla I, Chabrillon M, Arijo S, Diaz-Rosales P, Martinez-Manzanares E, Balebona MC, Morinigo MA (2003) Bacteria recovered from diseased cultured gilthead sea bream (Sparus aurata L.) in South Western Spain. Aquaculture 218:11–20CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Aslizah Mohd-Aris
    • 1
    • 2
  • Mohd-Zamri Saad
    • 1
    • 3
  • Hassan Mohd Daud
    • 1
    • 4
  • Mohd Termizi Yusof
    • 5
  • Md Yasin Ina-Salwany
    • 1
    • 6
    Email author
  1. 1.Laboratory of Marine Biotechnology (MARSLAB), Institute of BioscienceUniversiti Putra MalaysiaSerdangMalaysia
  2. 2.School of Biology, Faculty of Applied SciencesUniversiti Teknologi MARAKuala PilahMalaysia
  3. 3.Department of Veterinary Pathology and Microbiology, Faculty of Veterinary MedicineUniversiti Putra MalaysiaSerdangMalaysia
  4. 4.Department of Veterinary Clinical Studies, Faculty of Veterinary MedicineUniversiti Putra MalaysiaSerdangMalaysia
  5. 5.Department of Microbiology, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSerdangMalaysia
  6. 6.Department of Aquaculture, Faculty of AgricultureUniversiti Putra MalaysiaSerdangMalaysia

Personalised recommendations