Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging the genus Vibrio affecting many marine and fresh water fishes. The disease characterized by septicemia, dermal ulceration, ascitis and haematopiotic necrosis.

1. Vibriosis in Marine Fish
D.D.T.T. Darshana Senarathna
Aquaculture Health Management
AARM/FAB, School of Environment, Resources and Development
Asian Institute of Technology
A B C
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2. Outline
Introduction
Causative agent
Host factors
Disease pattern (Epidemiology)
Pathogenesis
Diagnosis
Control Measures
D 2

3. Introduction
• Recognized as early as 1718
• Migrating eels Anguilla vulgaris in 1817
• A systemic bacterial infection
• Both wild and farmed marine fishes
• Economic losses in aquaculture industry
• North America, Europe and Asia (Japan 1963)
(Colwell and Grimes, 1984)
(Mancuso et al., 2015)
(Sindermann, 1984).
RED
DISEASE
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4. Causative agent
• Multilocus sequence analysis (MLSA)
ftsZ, gapA, gyrB, mreB, pyrH, recA and
TopA gene sequences from 96 taxa
• Vibrio harveyi, V. vulnifcus, V.
parahaemolyticus, V. alginolyticus,
V. anguillarum, most common marine
pathogenic species
Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Vibrionales
Family: Vibrionaceae
Genus: Vibrio
(Sawabe et al., 2007).
(Higuera et al., 2013).
Vibrio anguillarum
Infect more than 50 species of fish in temperate region
Model microorganism to study the pathogenesis of vibriosis
4

5. Characteristics
• Gram-negative
• Facultative anaerobic
pathogens
• Opportunistic organisms
• Oxidase positive, reduce nitrate
to nitrite, ferment D-fructose,
maltose and glycerol.
• Thermos-dependent bacteria
• Many species are halophiles
straight or
comma-shaped
rod bacteria
Polar flagella enclosed
within a sheath
(Raszl et al., 2016)
(Vezzulli et al., 2015)
sediment, water column
and various aquatic plants.
non-spore-
forming
Electron micrograph
of V. anguillarum
E
5

6. • 1. Capsular polysaccharides
• 2. Adhesive factors
• 3. Cytotoxins
• 4. Chemotaxis and motility
• 5. An iron uptake system
• Extracellular products with
proteolytic or haemolytic
activity.
Virulent Factors,
Macrophage
Outer
membrane
Inner
membrane
Pilli
Flagella
motor
Flagella
Heme
Heme
receptor
Iron
1
2
3
4
5
(Deng et al., 2020)
(N. Mohamad, et al. 2019)
6

7. Host factors
• Susceptible hosts; salmon, turbot, sea bass, sea bream,
cod, eel, ayu, and groupers
• Susceptible stages; All age classes, high mortality among
the young fry
• Carriers; wild aquatic birds and both cultured and wild
marine fish
• Contaminated inputs and water from upstream
farms, Infected eggs, juveniles & broodstock
introduce Vibrio spp.
(Fernández–Delgado et al., 2016)
(Tendencia and Lavilla-Pitogo, 2004)
F
Sea bass fry dead with vibriosis
7

8. Disease pattern (Epidemiology)
Transmission mechanism
• water column - natural transmission
medium
• Horizontally - open lesions or as
secretion in the faeces of infected fish/
carriers.
Morbidity and Mortality – 30% - 100%
Geographical distributions – worldwide
(North America, Europe and Asia)
Mechanical factors
• Skin damage
• High stocking density
• Rough handling
• Transferring newly
caught wild fish into
enclosed systems
Risk
Factors
Environmental factors
• Warmer months
• Poor water quality
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9. Pathogenesis
• routes of entry - Ingestion and invasion
via gill or epidermis cut and Gastro-
intestinal tract
• Virulence factors and Extracellular
products
Pathogenic Vibrio
in the marine
environment
(Ceccarelli and Colwell, 2014)
(Xu et al., 2017).
 Highly plastic genomes
 Horizontal virulent gene transfer
 Plasmids and insertion sequence
common region (ISCR); mobile
genetic elements
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10. External
Weight loss, red spots, swollen, rotting of fins, dark skin lesions
Internal
Organs appear enlarged, the lesion associated with gastroenteritis, ascites
2. Clinical aspects
(Deng et al., 2020)
(N. Mohamad, et al. 2019)
Diagnosis (Level 1)
1. Abnormal behaviors
Loss of appetite, abnormal swimming behavior
with head floating near the surface of water.
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11. Level 1:Gross signs
Ulceration
Exophthalmia
Scale drops
Necrosis
Inflammation
Haemorrhagic of
liver and kidney
Pale liver
A
A
B
A
C
A
B
B
A
C
Hybrid grouper
B Asian Sea bass
C Red snapper
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12. Histopathology
• Hepatic lesions; congestion,
haemorrhage, swollen hepatocytes
with vacuolation and perivascular
hepatocyets
• Kidneys; hyaline degeneration and
necrosis of the convoluted tubules,
necrosis of the glomeruli,
haemorrhage, congestion and
presence of inflammatory cells
I II
III IV
VI
V
I. myocardial haemorrhage, II. hyperplasia (hp) of the
secondary lamellae leading to their fusion in the gill, III.
congestion in the Kidney, IV. haemorrhage in the kidney
parenchyma, V. MMCs (mmc) in the kidney, VI. necrotic
changes in kidney tissue
(I Frans et al. 2011)
Diagnosis (Level 2)
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13. Level 2:
Hybrid grouper Epinephelus lanceolatus × E. fuscoguttatus infected with Vibrio spp.
A) Severe to moderate congestion (c), generalize micro focal necrosis (n), generalize vacuolation of hepatocytes (v) in the liver
B) Mild tubular necrosis (n) and haemorrhage (h) in the kidney
C) Severe congestion (c) in the brain (H&E, 100×)
(N. Mohamad, et al. 2019)
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14. Diagnosis (Level 3)
Improved plate count on mTCBS. (a) Colonies on thiosulfate‐citrate‐bile
salts‐sucrose agar (TCBS) (standard medium). (b) Colonies onto mTCBS
(modified medium) (Tagliavia et al.,2019)
TCBS culture + PCR amplification and sequencing
Ribotyping - rRNA-based
phylogenetic analyses
RAPD - Random
Amplification of
Polymorphic DNA
ERIC-PCR - Enterobacterial
Repetitive Intergenic
Consensus (ERIC) PCR
(N. Mohamad, et al., 2019)
16S rRNA
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15. • A fluorescently labelled monoclonal antibody/ DAPI (4¢,6-diamidino-2-
phenylondole) double staining technique was developed to detect V.
anguillarum
• Latex agglutination-based assay (BIONOR Mono-Va-kit)
• Recently, an ELISA-based (Bionor AQUARAPIDVa test) and a magnetic
particle enzyme immunoassay (Bionor AQUAEIA-Va test)
• Loop-mediated isothermal amplification method (LAMP)
(Notomi et al. 2000)
(Gonzalez et al. 2004).
(Miyamoto & Eguchi 1997) 15

16. Control Measures
Prevention
Biosecurity/husbandry practices
• High quality fingerlings
• Appropriate clean-up
• Disinfection and dry-out procedures
• Regular changing of cage culture net
• Water quality management
• Optimum stocking density
• Appropriate chemical utilization methods
• Dietary supplementation of vitamins and
immunostimulants
(N. Mohamad, et al. 2019) 16

17. Control Measures
Treatments
Therapeutic treatment
• Antibiotics; Oxytetracycline, tetracycline,
quinolones, nitrofurans, potentiated
sulfonamides, trimethoprim, sarafloxacin,
flumequine and oxolinic acid
Prophylactic treatments
• Inactivated vaccines and DNA vaccines
AlphaJect 2000™, Aqua-Vac™Vibrio-Pasteurella
and Aquavac Vibrio Oral®, Vibrio vaccine, ALPHA
MARINETM
Fish vaccination
(N. Mohamad, et al. 2019) 17

18. Treatments (Cont.)
Biocontrol agents
• application of bacteria and phage (virus) -
Vagococcus fluvialis and Bacillus subtilis
• Bacteriophages belong to Siphoviridae family
• disrupting the quorum sensing – Macro algae,
micro algae, aquatic sponges
Plants products
• plant extracts as immunostimulants
• Essential oil (EO), acetone and butanolic
(Sorroza et al., 2012)
(Touraki et al., 2012)
SEM image of Bacillus subtilis
G
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19. References
Sources of diagrams
A. V. Anguillarum in seabass, exophthalmos lesions (picture by M. Isern), workshop,
vibriosis in aquaculture, 16th EAFP Conference, Finland, 2013
B. V. vulnificus, Science photo library
C. Colony of V. anguillarum PT-24 cultured on nutrient agar
D. Vibrio parahaemolyticus, HD image
E. Electron micrograph of V. anguillarum775 showing single polar flagellum. Shadowed
preparation x 10,000. (Micrograph by Dr J.H. Crosa.)
F. Dead Seabass fries by V. anguillarum, Photographs by Dr. Panos Varvarigos
G. Bacillus subtilis, Science photo library
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20. Bibliography
• Colwell, R.R., Grimes, D.J., 1984. Vibrio diseases of marine fish populations. Helgoländer Meeresun. 37, 265–
287.
• Mancuso, M., Genovese, M., Guerrera, M.C., Casella, G., Genovese, L., Piccolo, G., Maricchiolo, G., 2015. First
episode of vibriosis in wild specimens of Pagellus bogaraveo (Brünnich, 1768) in the Mediterranean Sea. Cah.
Biol. Mar. 56, 355–361.
• Sindermann, C.J., 1984. Disease in marine aquaculture. Helgoländer Meeresun. 37 (1),505.
• Sawabe, T., KitaTsukamoto, K., Thompson, F.L., 2007. Inferring the evolutionary history of Vibrios by means of
multilocus sequence analysis. J. Bacteriol. 189 (21), 7932–7936
• Higuera, G., Bastías, R., Tsertsvadze, G., Romero, J., Espejo, R.T., 2013. Recently discovered Vibrio anguillarum
phages can protect against experimentally induced vibriosis in Atlantic salmon, Salmo salar. Aquaculture 392,
128–133
• Raszl, S.M., Froelich, B.A., Vieira, C.R., Blackwood, D.A., Noble, R.T., 2016. Vibrio parahaemolyticus and Vibrio
vulnificus in South America: water, seafood, and human infections. J. Appl. Microbiol. 121 (5), 1201–1222.
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21. • Vezzulli, L., Pezzati, E., Stauder, M., Stagnaro, L., Venier, P., Pruzzo, C., 2015. Aquatic ecology of the
oyster pathogens Vibrio splendidus and Vibrio aestuarianus. Environ. Microbiol. 17, 1065–1080
• Deng et al., 2020, Prevalence, virulence genes, and antimicrobial resistance of Vibrio species
isolated from diseased marine fish in South China, https://doi.org/10.1038/s41598-020-71288-0
• N. Mohamad, et al., 2019, Vibriosis in cultured marine fishes: a review,
https://doi.org/10.1016/j.aquaculture.2019.734289
• Fernández–Delgado, M., Sanz, V., Giner, S., Suárez, P., Contreras, M., Michelangeli, F., García–
Amado, 2016. Prevalence and distribution of Vibrio spp. in wild aquatic birds of the Southern
Caribbean Sea, Venezuela, 2011-12. J. Wildl. Dis. 52 (3), 621–626.
• Tendencia, E.A., Lavilla-Pitogo, C.R., 2004. Chapter 2. Bacterial diseases. In: Nagasawa, K., Cruz-
Lacierda, E.R. (Eds.), Diseases Of Cultured Groupers. Aquaculture Department, Southeast Asian
Fisheries Development Center, Tigbauan, Iloilo, Philippines.
• Ceccarelli, D., Colwell, R.R., 2014. Vibrio ecology, pathogenesis, and evolution. Front. Microbiol. 5,
256.
• Xu, Y., Wang, C., Zhang, G., Tian, J., Liu, Y., Shen, X., Feng, J., 2017. ISCR2 is associated with the
dissemination of multiple resistance genes among Vibrio spp. and Pseudoalteromonas spp.
isolated from farmed fish. Arch. Microbiol. 199 (6), 891–896.
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22. • Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N. & Hase T. (2000)
Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28, e63i–e63vii.
• Miyamoto N. & Eguchi M. (1997) Direct detection of a fish pathogen, Vibrio anguillarum serotype
J-O-1, in freshwater by fluorescent antibody technique. Fisheries Science 63, 253–257.
• Gonzalez S.F., Osorio C.R. & Santos Y. (2004) Evaluation of the AQUARAPID-Va, AQUAEIA-Va and
dot-blot assays for the detection of Vibrio anguillarum in fish tissues. Journal of Fish Diseases 27,
617–621.
• Sorroza, L., Padilla, D., Acosta, F., Román, L., Grasso, V., Vega, J., Real, F., 2012. Characterization of
the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus
labrax) against vibriosis by Vibrio anguillarum. Vet. Microbiol. 155 (2), 369–373
• Touraki, M., Karamanlidou, G., Karavida, P., Chrysi, K., 2012. Evaluation of the probiotics Bacillus
subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in
European sea bass larvae (Dicentrarchus labrax, L.). World J. Microbiol. Biotechnol. 28 (6), 2425–
2433.
• I France et al., 2011, Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and
prevention, doi:10.1111/j.1365-2761.2011.01279.x
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23. THANK YOU!