Vibrio anguillarum

Vibrio anguillarum is a species of prokaryote that belongs to the family Vibrionaceae, genus Vibrio. V. anguillarum is typically 0.5 - 1 μm in diameter and 1 - 3 μm in length. It is a gram-negative, comma-shaped rod bacterium that is commonly found in seawater and brackish waters. It is polarly flagellated, non-spore-forming, halophilic, and facultatively anaerobic. V. anguillarum has the ability to form biofilms. V. anguillarum is pathogenic to various fish species, crustaceans, and mollusks.

Vibrio anguillarum can grow at temperatures as low as 5 °C but peaks at 37 °C, and favors saline and slightly basic water for growth. V. anguillarum was shown to be penicillin-resistant when tested with Rosco Neo-sensitabs System against antibiotics novobiocin and penicillin. In lab cultures, colonies get up to 1mm after 24 hours of incubation and 4-5mm after a week of incubation. Young colonies appear yellow and turn brown as they get older. When grown in broth, growth starts in the upper part of the test tube and reaches the bottom over two days. Cultures start as lightly turbid but develop into films and deposits in later stages.

Discovery
The discovery and understanding of Vibrio anguillarum has evolved over time through the contributions of various researchers.

Canestrini's observations (1893)
In 1893, Canestrini made pioneering observations on epizootics among migrating eels (Anguilla vulgaris), noting their association with a bacterium he termed Bacillus anguillarum. Canestrini meticulously documented the clinical signs exhibited by infected eels, laying the groundwork for further investigations into the pathogenic nature of this bacterium.

Bergman's description (1909)
Expanding upon Canestrini's work, Bergman's description in 1909 provided a comprehensive account of Vibrio anguillarum as the etiological agent responsible for the 'Red Pest of eels' in the Baltic Sea. Bergman's observations detailed the clinical manifestations of the disease in infected eels, explaining the pathological changes associated with V. anguillarum infection. His work not only confirmed the pathogenicity of this bacterium but also underscored its significance as a major threat to aquatic organisms in marine environments.

Disease description
Research by Gunnar Holt provided crucial insights into the emergence of Vibrio anguillarum as a pathogen in Norwegian coastal waters. Until 1964, V. anguillarum had not been associated with fish disease in Norway. However, Holt documented epizootic outbreaks of vibriosis in rainbow trout reared in seawater, causing substantial mortality in affected populations. Holt's investigations revealed a range of disease manifestations associated with vibriosis, including sudden mortality and varied pathological findings upon necropsy. These findings highlighted the severity and diversity of symptoms observed in affected fish populations, emphasizing the need for further research into disease prevention and control strategies.

Biochemistry
In addition to basic, saline water, Vibrio anguillarum can grow on MacConkey agar and TCBS agar. Larsen (1983) tested the hemolysis of V. anguillarum by measuring growth in an agar base with 5% citrated calf blood; hemolysis was observed just beneath the colonies and in a semitransparent zone surrounding the colonies.

In general, different Vibrio anguillarum strains respond similarly to various biochemical tests. Larsen (1983) tested V. anguillarum fermentation of various carbohydrates and glycosides. Most V. anguillarum strains were found to be able to ferment glucose, fructose, galactose, mannitol, mannose, maltose, sucrose, trehalose, dextrin, glycogen, chitin and ONPG. No fermentation reactions were found in xylose, adonitol, dulcitol, rhamnose, inositol, melezitose, raffinose, and inulin. Only a few V. anguillarum strains were found to ferment lactose, melibiose, aesculin, and salicin.

In tests with amino acids, proteins, lipids, and other compounds, most or all V. anguillarum strains showed positive activity with arginine dihydrolase, indole (tryptophan deaminase), catalase, oxidase, nitrate, and hemolysin, lipase and various proteins. Fish pathogen strains of V. anguillarum showed positive reactions in VP, 2,3-butanediol, citrate, NH4/glucose medium, and gluconate but not environmental strains.

Iron uptake systems
Vibrio anguillarum has multiple iron uptake systems, including TonB-dependent transporters and outer membrane receptors. V. anguillarum also has an iron sequestering system that allows it to sequester iron from haem and haem-containing proteins.

Vibrio anguillarum produces siderophores anguibactin and vanchrobactin, which are small molecules used to scavenge and transport iron. Siderophores are important virulence factors for V. anguillarum because they enable the bacteria to obtain iron from the host and evade the host’s immune system, essentially allowing the bacteria to compete with the host for iron and establish an infection. The genes involved in the biosynthesis and uptake of these siderophores are located on the virulence plasmid of V. anguillarum.

After the secreted siderophore binds to iron, the chelated iron complex is transported to the cytosol. The complex then binds to FatA receptors on the outer membrane and is transported into the cell. FatB/FatC/FatD receptors are also involved in iron transport between the periplasm and cytosol. The iron uptake system is negatively controlled by the Fur protein, which is chromosomally encoded and represses transcription by binding to and bending the DNA. The iron uptake system is further controlled by plasmid-encoded regulators: AngR and TAFr.

Genome
Vibrio anguillarum has two circular chromosomes, and many strains have a virulence plasmid. The number of protein-coding genes can vary by strain, but on average chromosome one has 1891 genes and chromosome two has 479 genes. A study on Vibrio anguillarum NB10Sm, a pathogenic serotype O1 strain, found 329 essential genes, 95 domain-essential genes, and 25 essential genes not found in other Vibrio species.

Serotypes
Strains are categorized into O serotype, since O-antigens were found to be the most specific surface antigens. There are 23 known serotypes of Vibrio anguillarum, O1 through O23, but only serotypes O1, O2, and O3 are known to be pathogenic.

pJM1
The pJM1 virulence plasmid and pJM1-like plasmids allow strains of Vibrio anguillarum that carry it to survive in environments with low levels of bioavailable iron, like inside of a fish, by releasing iron from molecules that sequester it such as transferrin and lactoferrin. The pJM1 plasmid has approximately 65 Kbp and a G+C content of 42.6%. pMJ1 plasmids from different host species and geographical regions generally have low amounts of variation. One study found almost all serotype O2 and O3 strains, as well as the serotype O1 strain without a pJM1-like plasmid, carried genes encoding the biosynthesis of the siderophore piscibactin.

Pathogenicity
Vibrio anguillarum can infect many species of fresh water and marine fish, as well as bivalves, and crustaceans. In fish, V. anguillarum infection can cause hemorrhagic septicemia called vibriosis. V. anguillarum is more virulent at cooler temperatures, potentially influenced by the fact that piscibactin production is favored at lower temperatures. Chemotactic mobility via flagella is necessary for the virulence of V. anguillarum in water. The discovery of a metalloprotease with mucinase activity, and a severe reduction in virulence in its absence, suggest its use in penetrating the host fish’s protective mucus layer. V. anguillarum also possesses genes for several hemolysins, which are thought to be the main contributor to hemorrhaging in fish with vibriosis.

Vibrio anguillarum is capable of colonizing and growing in the gastrointestinal tract of fish, utilizing intestinal mucus as a nutrient. Clinical signs of vibriosis include skin ulcers, hemorrhages, sepsis, and systemic infections. Vibriosis outbreaks are a significant concern in global aquaculture due to their impact on fish health and the development of antibiotic resistance, which can lead to significant economic losses in aquaculture. Control measures for V. anguillarum in aquaculture include hygiene practices, vaccination, and the use of antibiotics in some cases. Inactivated whole-cell vaccines are available, but there is a need for more effective and safer subunit vaccines.

Vibrio anguillarum is known to produce an extracellular protease called empA metalloprotease which plays a role in its pathogenesis. This protease enzyme is encoded in the empA gene in V. anguillarum. This gene is induced when cells are at high density and incubated in gastrointestinal mucus, and expressed during the stationary phase when V. anguillarum cells are incubated. EmpA expression is regulated by multiple factors, including cell density, gastrointestinal mucus, quorum sensing (QS) signals such as quorum-sensing molecules, and the alternative sigma factor RpoS. EmpA metalloprotease is a main factor involved in tissue damage and destruction during infection in salmonids, similar to other proteases produced by pathogenic bacteria. Conditioned cells from an empA mutant strain were found to induce protease activity which suggests the presence of an unidentified autoinducer.

Although typically not associated with disease in humans, in 2017 an immunocompromised woman died in hospital from sepsis and multiorgan failure and laboratory tests confirmed the presence of Vibrio anguillarum in her blood.

Ecology
Vibrio anguillarum is a ubiquitous marine bacterium found in various aquatic environments worldwide, particularly in marine coastal ecosystems. Its ecology is closely linked to its ability to infect and colonize a range of aquatic organisms, including fish, shellfish, and crustaceans.

Impact on aquaculture
The presence of Vibrio anguillarum poses a significant threat to aquaculture operations, particularly those focused on fish farming. Vibriosis outbreaks can result in substantial economic losses due to mortality and decreased productivity. The economic burden of preventing and treating vibriosis can be considerable, as it often involves the use of antibiotics, vaccines, and other management strategies. Additionally, the loss of valuable fish stocks can have long-term implications for the sustainability of aquaculture businesses.

Environmental factors
The behavior of Vibrio anguillarum is intricately linked to environmental factors, including temperature, iron availability, and water conditions, which play pivotal roles in its pathogenicity and disease management.

Temperature
Temperature is a critical environmental factor influencing the virulence and expression of virulence factors in Vibrio anguillarum. Despite its optimal growth temperature of around 25–34 °C, Vibrio anguillarum exhibits temperature-dependent variations in virulence. This temperature-dependent expression of virulence factors underscores the significance of understanding how environmental cues shape the pathogenicity of Vibrio anguillarum, particularly in the context of aquaculture practices conducted in varied temperature regimes.

Iron availability
The presence of iron, a vital nutrient crucial for both bacterial growth and virulence, plays a significant role in regulating the expression of virulence factors in Vibrio anguillarum. When iron levels are low, Vibrio anguillarum undergoes significant metabolic adjustments, leading to an increase in the expression of genes associated with virulence. Notably, genes linked to siderophore systems like vanchrobactin and piscibactin are particularly active under conditions of iron scarcity, with piscibactin showing heightened transcription at lower temperatures. This heightened activity of siderophore systems contributes to the increased virulence of Vibrio anguillarum in colder environments, illustrating the intricate relationship between iron availability, temperature, and the expression of virulence factors in determining the severity of the disease.

Water conditions
The aquatic environment significantly influences Vibrio anguillarum ecology and the control of vibriosis outbreaks in aquaculture. Factors like salinity, nutrient availability, water flow, oxygen levels, and biofilm presence affect Vibrio anguillarum 's survival, growth, and virulence, impacting disease spread among aquatic organisms. Effective management of water quality parameters, including salinity levels and nutrient levels, is crucial for regulating Vibrio anguillarum populations and mitigating vibriosis risks in aquaculture settings. Diligent monitoring and maintenance of optimal water conditions are vital aspects of disease control strategies, fostering the well-being and productivity of aquaculture operations while reducing the impact of bacterial pathogens.