Vibrio ordalii

For a more brief overview of V.ordalii and other fish diseases, check out these two websites:

Introduction

Vibrio ordalii is a bacterial fish pathogen that infects many salmonids in wild marine environments, specifically in Japan and in the Pacific Northwest of the United States. This organism is only one of a group of Vibrios (from Latin meaning, "that which vibrates or moves rapidly to and fro"), that cause fish disease. This pathogen, among other vibrios, is a serious concern to people because vibriosis is one of the most prevalent fish diseases caused by bacteria. Vibriosis is generally known as an acute bacterial septicemia and is responsible for massive fish die-offs every year. Mortalities in cultured fish alone may be higher than 50%, especially in young fish.

History

Historically, vibriosis is one of the oldest recognized infectious diseases of fish. Bonvari is our earliest record of having described "red pest" of eels, which we now know is caused by Vibrio. In 1893, a man by the name of Canestrini was the first person to isolate the organism. Bergman later denoted the organism V. anguillarum in 1909, anguillarum meaning "from eels".

Vibriosis was first reported in Japan in 1956 and in the PacificNorthwest of the United States in 1969.

Vibrio ordalii is a very young organism, taxonomically speaking.Until recently V.ordalii was thought of as another serotype of aclosely related organism, Vibrio anguillarum. Schiewe and Crosa published a paper in 1981 giving this organism it's own identity by naming it after a microbiologist interested in fish pathogens, E.J.Ordal. This identification was based on differences in histopathology, biochemical characteristics, and genetic composition.Because V.ordalii, as you will see, does not cause as much disease as it's sibling, V.anguillarum, it has not received as much attention in the way of research but we can always hope for the future. Thefollowing is a picture of V.anguillarum, which looks likeV.ordalii under the electron microscope.

Biochemical Characteristics

V.ordalii is:

Gram negative

Polarly flagellated

A curved rod

.5 X 1.5-2.5 micrometers long

Non-sporulating, nonencapsidated

Oxidase and Catalase positive

Aerobic or facultatively anaerobic metabolism

Carbohydrates fermented without the production of gas

G+C content of 43-44 moles%, but only has 58-69% homology with V. anguillarum

Contain protective LPS antigens that are heat stable

Even though V.ordalli and V.anguillarum are not largely genetically homologous, there is a need to distinguish between them when culturing them from fish. The following is a table that shows the phenotypic properties used to differentiate the two Vibrios.

Pathogenesis

Introduction

All marine fish are probably susceptible to at least one species of Vibrio. A major reason for the high prevalence of disease among salmonids is the amount of vibrios found in marine waters. The keyplayers in the vibrio classification of fish pathogens (or the ones that cause the most disease) are ubiquitous in the marine environment. Because V.ordalii has only recently been identified as a unique organism from V. anguillarum, scientists are only beginning to understand it's role in fish disease. Being able to distinguish between pathogens due to V.ordalii and V.anguillarum is important to scientists so the bacteria can be properly cultured, the fish can bet reated more accurately, and the proper prophylactic measures can be taken to prevent further infection.

Histopathology

Gross external and internal pathology is very close to that causedby V.anguillarum. The major difference with V.ordalii ishistopathological. While V.anguillarum is found mostly in the blood and blood producing organs, V.ordalii are less dispersed in the host and primarily infect cardiac and skeletal muscle, gill tissue, and the gastrointestinal tract, including the pyloric ceaca. The infection appears to begin in the descending intestines and rectum.This may be attributed to the low pH levels in the anteriorgastrointestinal tract when one considers that is has been determined that neither V.anguillarum nor V.ordalii can be cultivated on acidic media. Internal gross lesions produced by Vibriosis is similar tothat of other Gram negative bacteria. The liver and spleen become congested, the kidney swells, the intestines of the fish hemorrhage,and in advanced cases, the tissue of the internal organs may become necrotic. V.ordalii can also be distinguished by its tendency to form microcolonies within affected tissues and a slightly delayed septicemia.

None of the research literature has shown the effects of the LPS surface antigen. Bacterial "sloughing" of this antigen has been discovered, however. There are many examples within human systems where the pathogen causes disease by continually creating antigen,which creates a very large immune response (Staphylococcal aureus is one). This large immune response causes the pathology. Speculation may begin as to whether this is the mechanism by which the LPS antigen causes some pathology, but studies have not been done to deny or confirm this fact. We do know, however, the leukocytes in the blood and lymph decrease significantly with V.anguillarum and V.ordalii infection. Two main outer membrane proteins on the surface of V.ordalii have also been found to be antigenic. They are both approximately 40 kDa, heat labile, and may vary among the serogroups.They have been labeled as K and H antigens.

Experiments have shown that pathogenesis is very similar in experimentally infected fish and infected fish found in the wild. The pathology is also very similar across almost all species of Salmonids. A picture is shown below of an acute necrotizing myositis in Atlantic salmon with vibriosis. The arrow shows a large number of bacteria that may be seen surrounding one fiber.

Septicemia

Both V.anguillarum and V.ordalii cause septicemia (of course, this is the definition of a fish Vibriosis infection), but infection due to V.anguillarum tends to cause a very acute septicemia. V.ordalii does not present as serious of a systemic infection. In fact, bacterial plate counts taken by Ransom, et al, showed the following results:

Host species	V.anguillarum	V.ordalii
Coho	2.1 X 10^8 viable cells/mL	2.94 X 10^4 viablecells/mL
Chinook	2.4 X 10^8 viable cells/mL	1.5 X 10^6 viable cells/mL

This data clearly shows that the titer of V.ordalii is much lower in the blood of an infected fish than that of V. anguillarum.

Leukopenia

An interesting pathological characteristic that distinguishesV.ordalii is its' ability to cause serious leukopenia in fish. According to an experiment performed by Ransom, et al, decreases in white blood cell counts (80-95% decrease in circulating leucocytes) correlated with the presence of bacteria in the blood of diseased fish. Chinook salmon with more 1 X 10^6 cells of V.ordalii /mL ofblood had leucocyte counts that ranged from 1.6 X 10^3 to 7.2 X 10^3cells/mL, while fish with fewer bacteria in the blood had leucocytecounts between 1.1 X 10^4 and 6.2 X 10^4 cells/mL. Leukocyte counts from fish that were not infected showed a range from 3.4 X 10^4 to 7X 10^4 cells/mL.

Leucocyte decreases is obviously going to hinder the fish's ability to fight off infection, especially when considering the immediate innate response upon infection is to phagocytose the fishvia macrophages and neutrophils. Therefore this disease is a realdanger to the fish because when the pathogen bypasses the immunesystem, the fish has signed an innate immunity death certificate, soto speak. Secondary infection will now become a real threat if the fish doesn't die from Vibriosis.

What about plasmid pMJ101?

The plasmid pJM1 has shown to be an extremely important virulence plasmid in V. anguillarum. Because V.ordalii and V.anguillarum were thought to be the same species, pJM1 was sought out in V.ordalli, but not found. Instead, an entirely different plasmid, pMJ101, was found that bore almost no homology to pMJ101. The molecular basis of the plasmid was characterized by Bidinost, et al, and a map of theplasmid is shown below.

The function of the plasmid is not well known. Bidinost, et al,was able to show that the plasmid is approximately 30 Kb, which is fairly large. The plasmid is also present in the cell in high copies,and requires DnaA transcription.

Epizootology

Host Range

As was mentioned previously, almost all marine fish are probablysusceptible to at least one species of Vibrios. Interestingly,V.ordalii and V.anguillarum have been shown to be a part of the normal alimentary microflora of healthy fish, both wild and cultivated. Pathogenesis, which will be discussed below is largely a function of environmental stress.

Water-borne infection is the primary means of transmission between fish. Vibrios are shed from the vent and open lesions into the water.The location of Vibrio infection from the water to the fish isusually through the integument with the gills probably being a very common entry site. V.ordalii is a little different than V.anguillarumin that the pathogen can only be isolated from infected or moribund fish. Also, V.ordalii, along with V.salmonicida tends to infect only salmonids.

Geographic Range

V.ordalii is almost exclusively found in marine waters of the Pacific Northwest of the United States and Japan. Outbreaks have also been recorded in fresh-water species, usually associated with the feeding of marine fish offal.

The Role of Environment in Infection

Vibriosis is a classical example of a stress-borne disease. Losses caused by vibriosis are higly dependent on the severity of the environmental stress that predisposed the outbreak, varying from acute to chronic. Salmonids tend to come down with Vibriosis when they have been moved from fresh water to seawater.

In the wild marine environment, Vibriosis normally occurs in fish in shallow waters in later summer, when temperatures are high. In farmed fishes the disease can occur at almost any time of the year, although it is again most frequent in the summer months. This occurs because warmer temperatures allows gaseous oxygen to come out of the water. Less dissolved oxygen in the water will then be available tot he fish. In the summer months, the oxygen level may become low enough to stress the fish, predisposing it to Vibriosis infection.

As was mentioned previously, some Vibrios may be a part of the healthy, normal flora of the fish. Crowding, organic pollution, and other stressors can precipitate outbreaks. Strains may also vary in virulence, and some strains can cause disease without any predisposing factors. Exposure to certain concentrations of metals also increases susceptibility to the disease.

Reservoirs of Infection, the Good and theBad

The bad news is that the infected fish themselves are carriers of this pathogen, of course the higher the population of fish in a designated area, the higher the titer of the pathogen in the water.When fish are then subjected to infection by the pathogen, the number of organisms in the environment will go up exponentially with each afflicted fish.

The good news is that the pathogen can't be transmitted vertically, or a female may not pass the bacteria to her eggs.

Detection, Laboratory Diagnosis, and Prevention

Detection and Diagnosis

External signs of a V.ordalii infection include anorexia,darkening, and gross lesions. Internal signs should be commensurate with the pathologies described above (localized skeletal and Gill lesions/necrosis). The kidney and lesions should then be sampled forthe pathogen because V.ordalii may be a part of healthy skin orgastrointestinal microflora, whose presence wouldn't necessarily indicate that this pathogen was the causative agent of infection. An image depicting this procedure is shown below.

This figure shows, however not very well, an anterior kidneybiopsy technique. A needle is inserted through the medial membraneof the gill chamber and into the kidney. Confirmation that kidneymaterial has been obtained is indicated by the presence of melanocytes.

A presumptive diagnosis can then be made after culturing the organism on 1% NaCl media at 30 degress C. (High salt tolerance is characteristic of Vibrios. However, very high salt contents inhibit the growth of V.ordalii. Therfore TCBS, thiosulphate citrate bile salt sucrose is a commonly used selective media for Vibrios, but won't allow the V.ordalii growth.) Presumptive diagnosis includes apositive reaction in a motility tube, a positive cytochrome oxidasereaction, and fermentation with no gas production in a glucose O/F media.

A confirmatory diagnosis would then be needed to show that this agent, is definitely a Vibrio, and to distinguish what strain this bacteria is. This diagnosis is based on a sensitivity to a vibriostatic agent 0/129 (2,4-diamino 6,7-diisopropyl pteridine phosphate) and novobiocin, in reference to genus. Identification of strain is determined serologically via a couple of different methods. Some of those methods include immunobead assays, ELISA, a slide agglutination test, or a foreign antibody test. Chen, et.al, was responsible for developing an antibody that recognizes most species of infectious Vibrios. An antibody was also developed that recognizes V.ordalii and V.anguillarum 02. This antibody has been used extensively to study the attachment of Vibrios to fish cells.

Prevention

Two of the most common methods used to control V.ordalii outbreaks have been antibiotics and immunization.

Antibiotics are generally used when outbreaks of Vibriosis are occuring. The problem, however, is that the most effective method of administering antibiotics is orally. Fish suffering from Vibriosis generally tend to become anorexic and probably will not eat food containing the antibiotic. Some of the antibiotics available to control this pathogen are oxytetracycline, sulfonamides, and oxolinicacid. There is increasing resistance to these drugs, usually mediated by plasmids so new antibiotics are always being sought out.

Killed whole cell vaccines have been developed that have been very successfully in the prevention of V.ordalii infection. Only one dose of the vaccine is required for proper immunization. The vaccine may be given orally, by injection, or more commonly through immersion and spray techniques where the fish is immersed or sprayed with a solution that contains the antibiotic.

Bibliography

Actis , L.A., Tolmasky, M.E., Crosa, J.H. Fish Diseases and Disorders, Volume 3: Viral, Bacterial, and Fungal Infections (eds P.T.K. Woo and D.W. Bruno) 1999 pgs 523-558

Bidinost, Carla, Crosa, Jorge H., Actis, Luis A. 1994. Localization of the Replication Region of the pMJ101 Plasmid fromVibrio ordalii. Plasmid 31:242-250

Bruno, David W., Hasting, Trevor S., Ellis, Anthony E. 1986. Histopathology, bacteriology, and experimental transmission ofcold-water vibriosis in Atlantic salmon Salmo salar. Diseasesof Aquatic Organisms 1:163-168

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Chen, D., Hanna, P.J., Altmann, K., Smith, A., Moon, P.,Hammond, L.S. 1992. Development of Monoclonal Antibodies ThatIdentify Vibrio Species Commonly Isolated from Infections ofHumans, Fish, and Shellfish. Applied and Environmental Microbiology58:3694-3700

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Ransom, D.P., C.N. Lannan, J.S. Rohevec, and J.L. Fryer. 1984. Comparison of histopathology caused by Vibrio anguillarum andVibrio ordalii in three species of Pacific Salmon. Journal ofFish Diseases. 7:107-116.

Reno, P.W., Fryer, J.L., Bartholomew, J.L. MB492 Diseases ofFish Lecture Outline; Spring Term 2000

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Schiewe, M. H., J.H. Crosa et E.J. Ordal. 1977.Deoxyribonucleic acid relationships among marine Vibrios pathogenicto fish. Can. J. Microbiology. 23:954-958