Vibrio anguillarum is a species of Gram-negative bacteria with a curved-rod shape and one polar flagellum. Classified under three biotypes (A,B, and C), before scientists discovered that different strains of Vibrio anguillarum could be differentiated using serotypes. Vibrio anguillarum are halophiles that prefer warmer temperatures and neutral pH conditions. Vibrio anguillarum are able to compete for iron before the host can absorb it through iron acquisition mechanisms. It is an important pathogen of cultured salmonid fish, and causes the disease known as vibriosis or red pest of eels. This disease has the ability to impact brackish water, marine water, and freshwater species and may greatly impact cultured salmonid fish. Vibriosis has been observed in salmon, bream, eel, mullet, catfish, oysters, tilapia, and shrimp amongst others. The bacteria is most prevalent in late summer in salt or brackish water and gene transmission is mainly horizontal. Infection in humans is most commonly through the skin, but also through the mouth via contaminated food or water. It is widely distributed across the world. Vibrio anguillarum are damaging to the economy of aquaculture sector and fishing industries.
Vibrio anguillarum can be categorized into 23 different strains known as serotypes. O-serotype systems have been the most widely used method for dividing V. anguillarum into subgroups. Pedersen, Grisez et al. combined results from existing but differing serotype systems, to form the 23 O-serogroups that are now widely used.
These serotypes are characterized based on O antigen detection, using antisera, Western blots, and analysis on the varying lipopolysaccharide (LPS) profiles. Different serotypes exhibit different pathogenesis, and therefore have varying economic importance. Virulence factors vary between serotypes with serogroups O1 and O2 being the most common in diseased fish in aquaculture and in the wild, while serogroup O3 is the group often found in infected eels. The other serotypes are rarely found in diseased fish.
O1, O2 and O3 are generally considered the main and most significantly virulent serotypes of Vibrio anguillarum. However, there have been cases of O4 and O5 acting as the causative agent of vibriosis in aquatic organisms. An extreme case was seen in western Denmark where serotype O4 caused 100% loss of reared cod. O1 to O3 serotypes have two chromosomes which have been fully sequenced.
The genus Vibrio contains Vibrio anguillarum as well as other species that are important due to their ability to cause illness. Other pathogenic species of Vibrio include V. chloerae (which causes cholera), V. vulnificus (symptoms range from gastroenteritis, necrotizing wounds and septicemia) and V. Parahaemolyticus (primarily causes gastroenteritis).
Current research dictates that there are 23 different serotypes of Vibrio anguillarum. Of these serotypes, O1, O2, and O3 are known to play the most predominant role in generating vibriosis in infected species.
As early as 1718, Vibrio anguillarum was found on the coastline of continental Europe with mortality reports as early as 1893 referenced as the red disease in eels. In 1790, a red disease epidemic was described as approximately 40 tons of eels killed by an unknown pathogen (before the discovery of Vibrio anguillarum). In the late 1800s, large-scale fisheries suffered loss in fish yield from the mortality caused by the red outbreaks in Scandinavia. Before the early 1900s, many researchers mistook which bacterium was responsible for the red disease as other types of infection produced similar symptoms such as Aeromas, Atherynops, Fundulus, Gilichthys, Pseudomonas infection. It wasn't until the 8th edition of the Bergey's Manual of Determinative Bacteriology that the species Vibrio anguillarum was included. Before it was added to the manual, Vibrio anguillarum was identified as the same species as Beneckea anguillara, Vibrio piscism, and Pseudomonas ichthyodermis.
In the 1900s, Vibrio anguillarum was classified under three biotypes, A, B, and C. Type A are capable of producing acid without gaseous by-product from sucrose or mannitol and producing indole. Type B are incapable of reacting with sucrose or mannitol to produce gaseous by-product or indole. Type C are capable of producing acid from sucrose or mannitol but incapable of producing gaseous by-product or indole. It wasn't until the 1980s that Vibrio anguillarum became discriminated using serotypes. Serotypes were discovered when researchers found that Vibrio anguillarum isolates differed from one another by their cellular sugar compositions when screened using gas liquid chromatography.
Vibrio anguillarum were first noted in Canada on July 22, 1968 in Nanaimo, British Columbia. These V. anguillarum were initially observed in chum salmon and later observed in sockeye salmon. Despite less sockeye salmon dying as a result of V. anguillarum infection, nearly 50% of the chum salmon infected with V. anguillarum died within 96 hours of infection. Initial observations revealed that infected fish exhibited red markings located near their throat region as well as at the base of their paired fins.
Vibrio anguillarum have optimal growth temperatures between 30 °C and 34 °C. Growth rates are found to be increasing with temperature with a maximum growth temperature at 38.5 °C. They are Halophiles but growth is more dependent on temperature than salinity. Lethal temperatures and salinity are at values greater than 41 °C and 7 parts per hundred. The efficacy of Binary fission of Vibrio anguillarum cells are dependent on the pH levels of their surroundings. Binary fission is inhibited at pH greater than 9, disrupted at pH 6 or below, and most efficient at pH 7. They are more common in environments producing fertilized fish eggs with inhabitants such as larval fish or rotifers. An environment that contains divalent cations allow Vibrio anguillarum to thrive. V. anguillarum must adapt to its environment as changes in temperature and salinity as well as depleted nutrient source creates a stressful environment for the majority of its life cycle. Vibrio anguillarum must rely on sodium ions for survival in seawater during stressed starvation for long-term survival. When inside a host, Vibrio anguillarum can direct nutrients to itself through the use of a proton motive force. Environmental stress such as high temperature, osmotic stress, UV radiation, or oxidative stress may activate the rpoS gene which is responsible for genetic regulation of cellular response to environment stress.
Vibrio anguillarum outbreaks have been demonstrated to correlate with temperature increases and warm weather systems. As global temperatures continue to increase as a result of climate change, research examining how this will impact Vibrio anguillarum abundances are essential.
Vibrio anguillarm can remain virulent in freshwater conditions but their motility is hindered since it enhanced with increasing salinity. Vibrio anguillarum are not dependent on sodium or potassium ions for nicotinamide adenine dinucleotide hydride (NADH) oxidase functionality which allows it to remain virulent in freshwater conditions.
Vibrio anguillarum strains can produce one of two iron-scavenging siderophores, anguibactin and vanchrobactin under iron-limiting conditions. Anguibactin is only synthesized in some serotype O1 strains that have a virulence plasmid encoding the genes needed for siderophore biosynthesis and the transport proteins, while vanchrobactin is encoded in the chromosome of all serotype O2 strains and certain serotype O1 strains that do not have the virulence plasmid. These siderophores are used to gather or sequester iron from the environment and are subsequently taken up by the cell and released into its cytoplasm or modified by V. anguillarum. This allows V. anguillarum and other fish pathogens to bypass the defense mechanisms of fish taking up iron before pathogens do, giving them a chance to compete for iron in the environment.
Like other gram-negative bacteria, siderophore production is negatively regulated by the ferric uptake regulator protein (Fur), which represses downstream iron acquisition genes in the presence of sufficiently high intracellular iron concentration. Another avenue of negative repression would be through the antisense RNA, RNAα that bind to the mRNA of outer membrane and periplasmic iron transporter genes, fatA and fatB to repress their translation, whereby its production is mediated by Fur.
Downstream genes for iron acquisition for anguibactin includes the outer membrane transporter genes FatA, the periplasmic transporter lipoprotein FatB, the ATP-binding cassette (ABC) transporter, formed by a complex consisting of FatC and FatD, as well as the ATPase FatE. Anguibactin first enters through the outer membrane via FatA, then is transported through the periplasm by FatB, where it then binds and enters through the ABC transporter complex of FatC and FatD and then through FatE, where it finally enters the cytoplasm. Vanchrobactin uses a similar set of genes as anguibactin, but they are named Fvt A, B, C, D, and E. However, FvtE can be distinguished from FatE, where FvtE can be used to take in both anguibactin and vanchrobactin.
A third serotype, O3 has demonstrated to have two chromosomally-encoded, iron-regulated outer membrane heme receptor proteins that increase the affinity for iron under iron-limiting conditions, one of which is called HuvS. This protein is a substitute for the heme receptor protein HuvA on serotypes O1 and O2, and has an identifical function, but the HuvS protein of serotype O3 does not appear to have a competitive advantage.
Pathogen and virulence
Vibrio anguillarum can either attach to the host surface cells by absorbing their mucus or by penetrating through the epithelial and vascular tissue. The adhesiveness of V. anguillarum is dependent on the functionality of their exopolysaccharide which is encoded by the hfq gene. Exopolysaccharide transport system utilizes the fish's (host) natural mucus shedding mechanism by its integument system to remain attached to the host. The adhesive ability is not dependent on the flagella but in order for V.anguillarum, the flagella's function must be maintained. The presence of a flagellum enables species as the Vibrio anguillarum to infect other organisms with greater efficiency. Research has found that flagellum may assist with adhesion and/or mobility which are important in virulence as they may enable infective species to dominate. Within the sheath of a V. anguillarum's flagellum, antigens, such as lipopolysaccharides (LPS), may be stored. LPS, in turn, may assist V. anguillarum with infection following entry into the target species. After invasion, V. anguillarum will adhere and colonize in the host's gut where bacteria has the highest replication yield.
When Vibrio anguillarum are under constant state of stressed survival and conditional variations in environment or seasonal weather, they will rarely infects organisms. Infection by V. anguillarum will rapidly progress and cause symptoms as fast as 2 days after initial exposure. It does so by using a series of genetic virulence factors to aggressively penetrate a host which could be lethal 5 days after infection or 2 days in certain larvae cultures. Susceptibility of host organism to be cooperatively infected by other pathogens increases when infected by V. anguillarum.
The host's mouth, gills, anus, nasal cavity, epidermis, and eyes all have been regions of external vibriosis symptoms which causes the initial site of entry of Vibrio anguillarum to still be up for debate. During the earliest stages of infection, gills contain the most abundance of Vibrio anguillarum but only being exposed to the gills is not enough to cause a systemic infection. V. anguillarum have been shown to survive the acidic environments in fish's stomach for a couple of hours but oral consumption rarely causes infection. It is generally understood for the pathogen to enter the host through the mucosal surfaces such as surface skin and fins and penetrating through the epithelial or vascular tissue.
The presence of copper can be an initiating factor in the infection of eels and aquaculture are 50% more susceptible to infection when stressed by copper.
The Centers for Disease Control and Prevention recently reported the first known V. anguillarum-induced human fatality in history. The patient —a female senior-citizen— was exposed to the pathogen in the United States along the coast of Maine during the summer season. She was taking immunosuppressant drugs for an unrelated medical condition which likely aided in V. anguillarum's survival within the host patient. The pathogen was believed to have entered the patient through either: a) the epidermis of her leg which resulted in visual necrosis, b) through seafood that was consumed at a local restaurant, or c) via the aid of a horsefly vector by which she was bitten. This tragic story should be evaluated seriously in parallel to climate change and modern medical decisions.
Clinical signs and diagnosis
Multiple haemorrhages in the body and skin changes signifying systemic involvement occur. Splenomegaly (enlargement of spleen) may be evident in young fish. Diagnosis relies on culture of V. anguillarum and the use of monoclonal antibodies. V. anguillarum causes haemorrhagic septicaemia in a broad range of fish, which can presents clinically as expohthalmia (bulging eyes), whitening of the corneas, softening of the spleen, and large hemorrhages on the liver and the base of the fins. As stated by Hickey et al., symptoms may include: flesh rot, internal and external ulceration, lethargy, appetite loss, necrosis, erythema, boil formation upon muscle tissue, visual lesions, abdominal distension, petechia, sheathing of arteries and circulatory haemorrhage, and eventually death.
Treatment and control
Treatment measures such as the addition of soluble mixture halquinol have been shown to inhibit growth of Vibrio anguillarum in the presence of fish. Another treatment includes increasing the temperature beyond its tolerance range to 44 °C for 3 minutes or 47.5 °C for 2 minutes. The addition of 1 mg/L ozone has been shown to completely destroy the presence of Vibrio anguillarum. Mortality from Vibrio anguillarum infection can be reduced with the continual addition of anti-microbial peptides (cecropin-bee melittin hybrid peptide, CEME and pleurocidin amide). Mortality can also be reduced with changes to aquaculture feed such as adding selenium, antibodies from hen egg yolks, or oregano-derived carvacrol as feed additives. The addition of antagonistic bacteria can be used to prevent the attachment of Vibrio anguillarum enterocytes. The use of probiotics such as V. alginolyticus, Vagococcus fluvialis, Pseudomonas fluorescens AH2, and extracellular proteins from Kocuria spp., and Rhodococcus spp. has shown to reduce mortality by Vibrio anguillarum infection.
Vaccinations have also shown to work by administering Vibrio anguillarum killed by heat or formaldehyde into fish cultures. Other ways to administer dead Vibrio anguillarum is by adding it into feed as wet-packed whole cell or lyophilized whole-cell bacterin. Another way is to place fish cultures into muscle and vaccine containing immersion solution before sending off to aquaculture pools. Monoclonal anti-idiotype antibodies as surrogate antigen can be used instead of administering dead Vibrio anguillarum. Another type of vaccine is through the use of adhesion proteins from the outer-membrane of Aeromonas hydrophila. Other vaccine types include the addition of live-attenuated Vibrio anguillarum that are mutated or plasmid free Vibrio anguillarum.
Oral vaccines have been proven to be ineffective due to the fact that the gastric fluid in the upper intestine of fish will inactivate components in the vaccine. However, previous research examining resistance found that when Atlantic cod fry were administered Carnobacterium divergens the fry became resistant to infection by V. anguillarum.
Other treatment options include antibiotics such as oxytetracycline, erythromycin, chloramphenicol, triomethoprim, and carbencillin. Antibiotic resistant strains of Vibrio anguillarum are already present due to the high amounts of antibiotic required for treatment. The use of probiotics bacteria such as Roseobacter, Pseudomonas fluorescens, and Kocuria are able to prevent vibriosis in aquaculture.
The economic wellbeing of larviculture and aquacultural markets worldwide can be impacted by Vibrio anguillarum as this species may drastically increase mortality rates in freshwater and marine water organisms such as salmon. Fisheries and Oceans Canada reported that Canada exported an estimated $6.6 billion worth of seafood and fish products in 2016 alone. Meanwhile, more than $1 billion in GDP was generated by the Canadian aquaculture market in 2015 and employed approximately 72,000 workers.
The expansion of aquatic farming has led to increases in the number of fish being harvested for public consumption. As a result, V. anguillarum abundances have increased as their host populations have also increased. The increased prevalence has induced viral mortality in aquaculture which may cause up to 100% loss in aquatic organism farming. These losses have costed Japan as high as $18–30 million annually.
Internationally, Vibrio anguillarum have been reported to pose major concerns to the European aqua cultural market as well as the current aqua culture markets in Asian countries including China, Japan, and Taiwan. China, in particular, it is estimated to be responsible for supplying the world market with more than two thirds of all of its aquacultural products.
- Vibrio fischerii
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- Vibrio vulnificus
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