Abstract
We engineered probiotic bacteria, Lactobacillus plantarum, to produce specific double-stranded (ds)RNA for viral inhibition in penaeid shrimp. When supplemented in feed, the developed strain should offer specific viral disease resistance and still remain other beneficial effects on shrimp health against other bacterial pathogens. The pWH1520 expression vector was constructed to encode hairpin RNA targeting shrimp yellow head virus (YHV) in L. plantarum. The quantity of YHV-specific dsRNA (dsYHV) was approximately 1.66 ± 0.25 ng from 7 × 1011 CFU of L. plantarum. Feeding shrimp with the probiotic expressing dsYHV (at 1011 CFU g−1 feed) for 5 days prior to YHV challenge provided shrimp partial protection against YHV. In vitro agar well diffusion analysis revealed that both wild-type and transformed L. plantarum inhibited growth of Vibrio parahaemolyticus, the shrimp pathogen responsible for acute hepatopancreatic necrosis disease (AHPND) and early mortality syndrome (EMS). When tested in shrimp by immersion method, both L. plantarum strains (at 107 CFUs ml−1 seawater) also provided shrimp protection against V. parahaemolyticus. In conclusion, this study suggested the potential of the dsRNA-expressed L. plantarum for promoting shrimp health against the pathogenic virus. In addition, engineering L. plantarum for dsRNA production did not alter the probiotic’s intrinsic antibacterial property, and therefore, the developed strain can be used to protect shrimp against the important bacterial pathogen, V. parahaemolyticus causing AHPND/EMS.
Abbreviations
- bp:
-
base pair
- CFU:
-
Colony Forming Unit
- °C:
-
Degree centigrade
- ml:
-
Milliliter
- μg:
-
Microgram
- ng:
-
Nanogram
- OD:
-
Optical density
- PCR:
-
Polymerase chain reaction
- RT-PCR:
-
Reverse transcriptase-polymerase chain reaction
- qRT-PCR:
-
Quantitative-reverse transcriptase-polymerase chain reaction
- rpm:
-
Revolution per minute
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Acknowledgements
This project is supported by the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative to V.S., and Young Scientist and Technologist Program (YSTP) to S.S. We gratefully acknowledge Dr. Kallaya Sritunyalucksana-Dangtip, Shrimp-virus interaction laboratory, BIOTEC, NSTDA and Ms. Duangnate Isarangkul Na Ayudhaya (Department of Microbiology, Faculty of Science, Mahidol University) for providing us the AHPND/EMS-causing V. parahaemolyticus strain and the pWH1520 vector, respectively.
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Since the Ethical Principles and Guidelines for the Use of Animals of the National Research Council of Thailand (1999) apply to vertebrates only and there is no official standard for invertebrates, we followed the guidelines of the Australian, New South Wales state government for the humane harvesting of fish and crustaceans (http://www.dpi.nsw.gov.au/agriculture/livestock/animal-welfare/general/fish/shellfish) with respect to details regarding the transport of the shrimp from the breeding centers to the laboratory and for their laboratory maintenance. With respect to processing the shrimp, the salt water/ice slurry method was used as recommended in the Australian guidelines.
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Thammasorn, T., Jitrakorn, S., Charoonnart, P. et al. Probiotic bacteria (Lactobacillus plantarum) expressing specific double-stranded RNA and its potential for controlling shrimp viral and bacterial diseases. Aquacult Int 25, 1679–1692 (2017). https://doi.org/10.1007/s10499-017-0144-z
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DOI: https://doi.org/10.1007/s10499-017-0144-z