Development of Vibrio harveyi protease deletion mutant as live attenuated vaccine against vibriosis in Epinephelus fuscoguttatus (Forsskal, 1775)
Groupers aquaculture industries have faced a high risk of bacterial diseases such as Vibriosis. Various types of vaccine have been studied including attenuated vaccine. However, the potential of live attenuated vaccine is still understudied. This study was conducted to develop a live attenuate...
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| Format: | Thesis |
| Language: | English |
| Published: |
2018
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/69203/1/IB%202018%2010%20IR.pdf |
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| Summary: | Groupers aquaculture industries have faced a high risk of bacterial diseases such as
Vibriosis. Various types of vaccine have been studied including attenuated vaccine.
However, the potential of live attenuated vaccine is still understudied. This study was
conducted to develop a live attenuated protease derivative from pathogenic Vibrio
harveyi strain Vh1 as a live vaccine candidate using site-directed mutagenesis (SDM) and
allelic exchange replacement techniques. In SDM, an overlapping PCR was performed to
generate a deletion in a specific amino acid residue that represents a catalytic site of the
protease gene. Seven mutant variants consisted of single deletion catalytic sites (DelD,
DelH, and DelS), double deletion catalytic sites (DelDDelH, DelDDelS and DelHDelS)
and triple mutation catalytic sites (DelDDelHDelS) were successfully developed.
Evaluation of protease activity showed DelDDelHDelS recombinant recorded highest
relative protease activity. A mutagenesis plasmid was further constructed using pRE112
as suicide plasmid, prior to the transformation into E. coli SM10pir. The resulting
mutagenesis plasmid (pRE112-DelDDelHDelS) was integrated into the chromosome of
V. harveyi strain Vh1 by employing a combined mixed broth-membrane filter technique
for conjugation and the allelic replacement technique. Subsequently, the attenuated strain
was designated as V. harveyi attenuated strain MVh-vhs and was further used as live
vaccine candidate throughout this study. In vivo study was performed in Epinephelus
fuscoguttatus to evaluate the safety and pathogenicity level of the attenuated strain. Fish
challenged with the parental strain showed obvious clinical signs of Vibriosis such as
hemorrhages on the ventral abdominal pelvic area, external lesion and white
depigmentation in skin. The median lethal dosage (LD50) in fish challenged with the
parental strain was found at 106 CFU/fish. Histopathological analysis showed the
presence of immunological response activity in both fish treatment. In contrast, 100%
survival with no indication of vibriosis was detected in fish challenged with the
attenuated strain. Vaccination with the attenuated strain in juvenile E. fuscoguttatus was
performed using a single dose IP administration at 105 CFU/fish. The bacterial challenge
was done after four weeks post vaccination with the pathogenic V. harveyi strain Vh1 at
dosage concentration of 108 CFU/fish following IP administration. Fish vaccinated with the attenuated strain showed 52% relative percentage survival (RPS). De novo
transcriptomic analysis revealed that the vaccinated E. fuscoguttatus with the attenuated
strain conferred both innate and adaptive immunity. The innate adaptive involved in
regulation of the autophagosome pathway and coagulation and complement cascade
pathway. Adaptive immunity relies on the regulation of antigen processing and
presentation pathway. In addition, V. harveyi attenuated strain MVh-vhs possessed an
unmarked gene deletion and the attenuation properties were found stable after 15 in vitro
passages. As a conclusion, the V. harveyi attenuated strain MVh-vhs has significant
potential to be applied as a live vaccine candidate against vibriosis in E. fuscogutattus. |
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