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Initial Discovery and Background.
In 1936, two researchers named Frank Johnson and I.V. Shunk from the physiological laboratory at Princeton University and the Bacteriological Laboratory at the State College of the University of North Carolina described what was considered a new bioluminescent bacterial species. The morphology of the culture of Vibrios harveyi resembles that of many luminescent species including Achromobacter Fischeri. The new species was isolated from a dead amphipod belonging to Talorchestia. Although difficult to identify this bacteria, Johnson identified a unique characteristic–lack of or minimal growth and luminescence of V. harveyi on glycerol substrate with no acid production (2). Additionally, the original name for the species was proposed to be Achromobacter harveyi due to it's phenotypic similarity to other Achromobacters and paying homage to a great contributor to this project–E.N. Harvey (2).
V. harveyi belongs to the family Vibrionaceae of the class, Gammaproteobacteria and phylum, Proteobacteria (Zhang). The bacteria are marine fluorescent and gram-negative with closely related relatives in the Vibrios genus including V. campbellii, V. owensii, and V. rotiferanus; this core group of bacteria are all extremely phenotypically and genetically similar, making many tests and observations unable to discriminate between the species (4).
Attempts at Phenotypic and Genotypic Identification of V. harveyi.
A group of researchers performed tests to best identify V. harveyi phenotypically and genotypically. A summary of some phenotypic results can be found in Table 1. Evidently, there are no discriminatory results that aid in true identification of V. harveyi. The most prominent results are likely from Hydroxy L-prolin and N-Acetyl-D-galactosamine, both of which still have various expression in the strains (this is why the percentage is not 100%–not all of the strains exhibit it but compared to other species, V. harveyi has the highest results). Additionally, V. harveyi can produce ornithine decarboxylase, one of few phenotypic features that makes it distinguishable from V. campbelli. V. campbelli and V. harveyi have a DNA-DNA similarity of 69% and a 16S rRNA similarity better than 97% which is why both phenotypic and even some phenotypic tests make it difficult to identify V. harveyi (1). Figure 2 demonstrates the results from the most successful and discriminatory protocol for V. harveyi used in identification–forms of genetic fingerprinting.
Figure 3. Transmission electron micrograph of ML01 (V. harveyi) (4).
The bacterium appears to be a short rod with a single polar flagellum at a scale of one micrometer.
Figure 1. Images of V. harveyi.
Above in Figure 1 are images taken of strains of V. harveyi. (A) Growth of strain VIB 391 on marine agar 2216E. (B) Expressed luminescence of strain VIB 391. (C) Strain VIB 645 growth on TCBS agar. (D) VIB 645 cells from a marine broth culture under a scale of 1 micrometer (6).
Table 1. Results from Testing Characteristics of V. harveyi and related species.
The results of various different tests is listed in this table. Strains with variable results are in parentheses. (1)
Figure 2. A Dendrogram of Different Genetic Fingerprinting Methods. (1).
Above, three forms of genetic fingerprinting: FAFLP (fluorescent amplified fragment length polymorphism), GTG-PCR, and REP-PCR were attempted to better distinguish and correctly identify the species of Vibrios. An asterisk was used to identify noted hybridization in a studied strain and FAFLP was most effective in identifying the closely related species.
Figure 4. Pathogenic Mechanisms Employed by V. harveyi (6)
Pathogenic Mechanisms of V. harveyi
The mechanisms of pathogenesis for V. harveyi are hypothesized and gathered from many studies (primarily aquacultural) from around the world as summed up in Figure 4. Due to irregular and lack of truly discriminatory tests, some of the mechanisms as well as recorded symptoms and diseases brought on by V. harveyi should be taken slightly skeptically as there is still a level of uncertainty when defining V. harveyi across various studies and experiments. Nonetheless, it is still beneficial to note the mechanisms V. harveyi can take as an opportunistic pathogen as defined in Figure 4. Of those defined in the figure, luminescence and quorum sensing, formation of biofilms, extracellular products, and bacteriophage seem to be some of the most prominent and best understood.
Symptoms, Disease, and Immune Response to V. harveyi.
V. harveyi is linked to disease in a significant portion of warm-water fish and invertebrates including shrimp, hybrid groupers, bass, sharks, and countless others. In vertebrates, documented diseases caused by V. harveyi are as follows: eye disease, gastro-enteritis, necrotizing enteritis, nodules on operculum, scale drop and muscle necrosis, skin ulcers, tail rot disease, vasculitis. In invertebrates, diseases include: acute hepatopancreatic necrosis, bacterial white tail disease, black shell disease, Bolitas negricans, foot pustule disease, luminous virbriosis, skin ulcerfication, white patch disease, white spot on the foot (6). Several mechanisms have been identified as effective in increasing the chance of survival of a marine animal post-infection including: utilization of immune-related genes like heat shock protein 70 gene and a class 1a gene of major histocompatibility complex (MHC) from spleen and kidney of Scophthalmus maximus. In another study apoptosis was induced in cultured fish cells (6).
Virulence Factors and Management of Disease.
Virulence factors such as lipopolysaccharides (LPS), proteases, hemolysins, capacity to bind iron, bacteriophage interaction, formation of biofilms, and quorum sensing are recognized factors in the pathogenicity of V. harveyi. (6). Due to the multiple pathogenic pathways and mortality rate from infection, both natural and aquacultural populations can be gravely affected, emphasizing the need for management and control of infection. Unfortunately, like some pathogenic studies, usage of antibiotics has lead to the development and spread of antibiotic resistance; consequently, future management should rely more on preventative measures rather than measures that cure disease. Of these measures, bacteriophage therapy–creating mixtures of phages resulting in bacterial inhibition and the effects of disease–biological control, dietary supplements (slightly altering and boosting the immune system to better fight disease or bacterial invasion), inhibition of quorum sensing, and various kinds of vaccines (6) have all been used in studies and experiments to provide aquacultural practice with some hope of preventative measures, avoiding mass mortality of their populations.
Some Pathogenic Cases of V. harveyi
V. harveyi and Monocultured Sea Horses.
V. harveyi can wreak havoc on marine animals and is generally pathogenic in behavior. One study focused on a particular case of the near 50% decline in populations near waters closest to Asian countries in which sea horse populations were in popular demand, yet becoming scarce. The Aquaculture Department of the Southeast Asian Fisheries Center in the Philippines decided to interfere and create a breeding program of the sea horse Hippocampus kuda for fear of extinction. Under 1% mortality was observed per day and of those that died, they shared symptoms of loss of color or discoloration in the upper body, slow and weak movement, with some experiencing depressed abdomens and pale kidneys upon dissection (5). Bacterial samples were taken from some of the deceased H. kuda with samples purified and isolated. Twelve colonies were chosen for study with eleven of the twelve selected being V. harveyi. The pathway of colonies from the kidney–those being V. harveyi--demonstrated a septic course of infection and further supports symptoms such as discoloration and abdominal depression were due to V. harveyi. (5), which acted as a primary pathogen in mortalities of monocultured H. kuda. This study was eye opening to other aquacultural species, being attributed to infection in Panaeous monodon (tiger shrimp), Lates calcarifer (sea bass), as well as specific mass mortality in a few species of sea horse (5). Overall, mass mortality and rapid infection caused by V. harveyi is taxing and detrimental to aquaculture as well as our oceans if infection is at a high rate and widespread.
V. harveyi affects Aquaculture again - Skin Ulcer Disease Caused by ML01.
A recently bred cross of Epinephelus fuscoguttatus (female) and Epinephelus lanceolatus (male) has produced a hybrid grouper prized for its high economic value, delicious meat, rapid growth, and resilience to many diseases. The successful cross and mariculture cultivation of this fish occurred in 2008 in China and has been marketed high since (4). Due to its relatively new existence, little is known about bacteria or viruses that the grouper is susceptible to. Researchers subjected healthy juveniles to bacterial infection by immerson, immersion after dermal abrasion (seen in Figure 5 b), and intraperitoneal injection. 2 weeks after infection, an isolated strain of bacteria, ML01 caused mass mortality in these juveniles either by injection or immersion after dermal abrasion. Morphology, 16S rDNA sequencing, multilocus sequence analysis, and profile index identification identified this strain as V. harveyi (4). The results suggest the causation of skin ulcer disease in these juveniles was primarily by V. harveyi. Additionally the study tested susceptibility to antibiotics, finding this isolate–ML01, V. harveyi--was susceptible to ceftriaxone, minocycline, and doxycycline and ulcers were examineed and deemed not to be caused by parasites or fungi (4). This information may be useful in management and prevention of mass mortality in aquaculture by V. harveyi in a variety of species.
Figure 5. Skin Ulcer Disease in Hybrid Grouper (4).
(a) Juvenile that naturally suffered from skin ulcers. (b) Juvenile infected with strain ML01 through immersion infection followed by dermal abrasion (Shen 2017).
Future Environmental-Friendly Approach to the Prevention of V. harveyi in Populations–Bacteriophages.
V. harveyi thrive at hatch sites of brine shrimp, becoming threats to any organism feeding at those sites, including the shrimp. In years prior to this experiment, chemotherapy and disinfecting were key to reducing mortality due to V. harveyi and other related species; yet these treatments negatively impact the shrimp and the surrounding environment. In this study, target bacterial species were Vibrio paraphaemolyticus and Vibrio harveyi which phage therapy was aimed to treat in brine shrimp with particular focus on survival and hatching. Bacteriophages were chosen from a collection called the CICIMAR phage collection, two phages, F8 and F12 of Leviviridae and Podoviridae families were chosen due to their lytic nature towards V. harveyi (3). Three phages for V. parahaemolyticus was chosen for the same properties as phages chosen for V. harveyi. Brine shrimp at cyst incubation and larval development were exposed to challenge tests to the bacterium and then washed and sterilized prior to treatment with bacteriophage or cocktail. After sixteen hours of being infected, the brine shrimp were observed microscopically to determine the degree of hatching (3). As seen in Figure 6, for both hatching and survival rate, the bacteriophage cocktails targeted at V. harveyi appear to be extremely effective. In graph B, hatching reaches 100% prior to 24 hours after infection and the bacteriophage cocktail appears to be effective as nearly 90-95% of the shrimp treated survive. This study offers promising results with little costs as it preserves the quality of the shrimp, does less harm, if any, to the environment, and is able to increase both survival and hatching rate which would likely mitigate mass mortality. Future experiments should be run on other invertebrates and vertebrates to determine if these bacteriophages are broadly effective in mitigating mass mortality and widespread disease in aquaculture populations as well as if the bacteriophages cause harm to other types of organisms.
Figure 6. Hatching and Survival of Brine Shrimp Post-Infection and Treated with Bacteriophages (3).
To the left is the graph of hatching percentage over time with shrimp exposed to V. harveyi at (B). To the right are histograms indicating the survival rate of the shrimp challenged by V. harveyi (B).
Cited References.
(1) Gomez-Gil, B., Soto-Rodriguez, S., Garcia-Gasca, A., Roque, A., Vazquez-Juarez, R., Thompson, F. L., & Swings, J. (2004). Molecular identification of vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiology (Society for General Microbiology), 150(6), 1769-1777. https://doi.org/10.1099/mic.0.26797-0
(2) Johnson, F. H., & Shunk, I. V. (1936). An interesting new species of luminous bacteria. Journal of Bacteriology, 31(6), 585-593.
(3) Quiroz-Guzmán, E., Peña-Rodriguez, A., Vázquez-Juárez, R., Barajas-Sandoval, D. R., Balcázar, J. L., & Martínez-Díaz, S. F. (2018). Bacteriophage cocktails as an environmentally-friendly approach to prevent vibrio parahaemolyticus and vibrio harveyi infections in brine shrimp (artemia franciscana) production. Aquaculture, 492, 273-279. https://doi.org/10.1016/j.aquaculture.2018.04.025
(4) Shen, G. M., Shi, C. Y., Fan, C., Jia, D., Wang, S. Q., Xie, G. S., Li, G. Y., Mo, Z. L., & Huang, J. (2017). Isolation, identification and pathogenicity of vibrio harveyi, the causal agent of skin ulcer disease in juvenile hybrid groupers epinephelus fuscoguttatus × Epinephelus lanceolatus. Journal of Fish Diseases, 40(10), 1351-1362. https://doi.org/10.1111/jfd.12609
(5) Tendencia, E. A. (2004). The first report of vibrio harveyi infection in the sea horse hippocampus kuda bleekers 1852 in the philippines. Aquaculture Research, 35(13), 1292-1294. https://doi.org/10.1111/j.1365-2109.2004.01109.x
(6) Zhang, X.-H., He, X., & Austin, B. (2020). Vibrio harveyi: A serious pathogen of fish and invertebrates in mariculture. Marine Life Science & Technology, 2(3), 231–245. https://doi.org/10.1007/s42995-020-00037-z