Anguillarum Cells Research Articles

A Comparison of Pathological Changes Caused by Vibrio anguillarum and Its Extracellular Products in Rainbow Trout(Oncorhynchus mykiss).

Some hematological parameters and histopathological changes in the liver, spleen, kidney, gills, gut, skin, muscle and heart of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with virulent Vibrio anguillarum or with V. anguillarum extracellular products (ECPs) were compared.The number of circulating red blood cells, the hematocrit and hemoglobin values decreased in the fish injected with either bacteria or ECPs compared with controls. The erythrocyte sedimentation rate increased in both groups of injected fish; however, the serum protein levels were similar to those observed in control fish.Light and electron microscopic observations of samples obtained from fish injected with V. anguillarum cells or its ECPs revealed similar histopathological changes in most tissues. Both groups of fish showed degeneration and necrosis of hepatocytes, vasodilation of the glomerular capillaries, cloudy swelling and hyaline droplet degeneration of the epithelial tubular cells, edematous gills, desquamation of the intestinal mucosa and necrosis of the muscle fibers at the site of injection. However, the bacterium multiplied extensively in the kidney and spleen where it caused more acute alterations than ECPs.Based on these results, it was concluded that the extracellular products play an important role in the virulence mechanisms of V. anguillarum.

Isolation and partial characterization of turbot ( Scophthalmus maximus) immunoglobulins

1. 1. An IgM-like immunoglobulin was isolated from the serum of turbot ( Scophthaimus maximus) by both fast protein liquid chromatography (FPLC) on Superose® 6HR columns and affinity chromatography on Mensep® protein A cartridges. 2. 2. Molecular weight of the native Ig was estimated to be 835 kD by SDS-PAGE and 820 kD on the basis of gel filtration. 3. 3. Purity was verified by SDS-PAGE (under non-reducing and reducing conditions) and by immunoelectrophoresis and immunoblotting of antisera raised against the isolated Ig fraction in mice: only minor protein contaminants were observed. 4. 4. Both SDS-PAGE under reducing conditions and immunoblotting also indicated that the heavy and light chains of the isolated turbot Ig are of about 78 and 27 kD, respectively. 5. 5. Total circulating Ig level was estimated by single radial immunodiffusion using a polyclonal anti-turbot Ig serum to be about 4.8 mg/ml. 6. 6. The same antiserum was used to verify that the purified proteins were Igs, in an ELISA test using serum from Vibrio mguillarum -immunized turbot as primary antibody and immobilized V. anguillarum cells as antigen. 7. 7. Finally, our data indicate that about 25% of turbot Igs bind to Mem-Sep® protein A.

Agglutinin activity in pacific oyster ( Crassostrea gigas) hemolymph following in vivo Vibrio anguillarum challenge

Hemolymph from the Pacific oyster ( Crassostrea gigas) contains lectins that agglutinate horse (Gigalin E) and human (Gigalin H) erythrocytes. The gigalins also agglutinate bacteria, including Vibrio anguillarum, and were adsorbed from oyster hemolymph at different temperatures by living, heat-killed, and freeze-dried V. anguillarum cells. Baseline activities of the two gigalins were established by measuring their activities in oyster hemolymph over a period of 4 years. A normal distribution of Gigalin H activity (mean titer 139) was found, whereas the distribution of Gigalin E activity in the same samples was skew (mean titer 512). No covariance was observed between the two agglutinin activities. Increased lectin activity above this baseline was found in oysters exposed for varying time intervals to V. anguillarum at different seasons and temperatures over a period of 2 years. Such exposure resulted in an increase in activity (titer) of four- to nine-fold for Gigalin E and three- to seven-fold for Gigalin H when compared with controls, and in augmentation in the hemolymph of a protein with the same electrophoretic mobility as affinity-purified oyster lectins (gigalins). Challenge with either living or heat-killed bacteria resulted in a significant increase of Gigalin E activity, whereas results for Gigalin H were variable. Oysters challenged with bacteria were observed to filter normally with open shells during the experiments. Also, no increase was found in hemolymph calcium that could indicate anoxia following bacterial challenge (0.49 ± 0.004 mg mL -1) compared to unexposed oysters (0.50 ± 0.001 mg mL -1). Increase in the concentration of free amino acids in oyster hemolymph was observed following exposure to bacteria (15.05 mM) and anaerobiosis (13.51 mM) compared to controls (9.06 mM), and changes (in mol %) of individual amino acids differed considerably between hemolymph from animals challenged with bacteria and animals kept anaerobic. The augmented lectin activity in oyster hemolymph, following in vivo exposure to increased bacteria in the seawater, suggests their involvement in enhancing bacterial clearance and defense in the oyster.

The effect of triploidy on phagocytosis, leucocyte migration, antibody and complement levels of ayu, Plecoglossus altivelis

In order to clarify the effects of triploidy, phagocytosis, agglutinating antibody titres, complement activity of the serum, chemotaxis and phagocytosis of kidney leucocytes were determined in triploid and diploid, immunised and nonimmunised ayu. Formalin-killed Vibrio anguillarum cells were used as the immunogen. Leucocytes from control triploid ayu showed slightly higher phagocytosis than the diploid ones, but after immunisation no significant differences were detected in agglutinating antibody titres, complement activity and phagocytic activity between triploid and diploid ayu.

Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.

Fluctuation in the number of bacterial cells in organs of vaccinated fish after artificial challenge.

The distribution of Vibrio anguillarum cells in fish challenged by live organisms was surveyed in the blood, spleen, kidney, liver, bile, and intestinal tracts, in order to explain the mechanism of the protection in fish vaccinated by immersion or injection method, and non-vaccinated fish. In the case of intraperitoneal injection with 105 cells, the organism appeared in each organ of vaccinated fish and non-vaccinated fish by 30min after challenge. The number of organisms in each organ of vaccinated fish fluctuated 10h after challenge; However, the organisms were completely eliminated 72h after challenge. On the other hand, the number of organisms in each organ of non-vaccinated fish increased with time, and all fish died by 72h after challenge. In the vaccinated fish exposed to 107 cells/ml, the challenge organism was completely eliminated from the blood within 4h, in each organ except the intestinal tracts within 48h, and in the intestinal tracts within 72h after challenge. Whereas, challenge organisms increased in the blood and each organ of non-vawinated Ssh in course of time, reaching from 105 to 107 cells/g or ml 72h after challenge.

Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1

Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.

Modification of the hyperosmotic infiltration method of vaccination against vibriosis in cultured ayu, Plecoglossus altivelis Temminck and Schlegel

Abstract. A vaccine solution of a formalin‐killed culture of Vibrio anguillarum cells was observed to be toxic to young ayu when administered by the hyperosmotic infiltration method. The toxin was present in the culture broth. After the toxin was removed from the broth by centrifugation, the fish were dipped in 5.32% NaCl solution for 2 min and then in a solution containing precipitated cells for 3 min. The immunized fish were protected against vibriosis when challenged one month after immersion. The bacterin was administered to ayu by a further two methods, both using lyophilized whole cells of formalin‐killed V. anguillarum. In one method, the fish were placed in a 5.32% NaCl solution for 2 min and then in a solution containing lyophilized cells at 2 g/l of well water for 3 min (two‐step immersion). In the other method, the fish were placed in a 5.32% NaCl solution containing lyophilized cells also at 2 g/l for 3 min (one‐step immersion). A high level of protection against artificial challenge was achieved with either method. No agglutinating antibodies to V. anguillarum were detected in either the serum or mucus of fish dipped in a vaccine solution, a supernatant, or a precipitated solution, one month after immersion. On the other hand, serum titres were detected in fish vaccinated by injection, although no titres were detected in mucus. LD50 values are presented for the virulence of the V. anguillarum strain. Compared to the original strain, virulence increased after the third passage in ayu, but decreased after the thirteenth passage in medium.

Immersion vaccination and water-borne challenge of ayu (Plecoglossus altivelis) against vibriosis.

In a Vibrio anguillarum strain passaged through ayu (Plecoglossus altivelis), the LD50 value against ayu by the water-borne challenge method did not change from that of the original strain freshly isolated from diseased fish. The ayu were reproducibly infected with the passaged strain by the water-borne method in which the fish were first exposed to 5.32% NaCl solution and then to a suspension of V. anguillarum cells. The symptoms in ayu infected by this challenge method were observed to be very similar to the symptoms of a natural vibriosis infection. The LD50 value against fish exposed first to NaCl solution and then to the cell suspension was lower than that of fish exposed to only the cell suspension. This challenge method, incorporating initial exposure to a 5.32% NaCl solution followed by exposure to a Vibrio cell suspension, provided a clear difference in mortality between vaccinated and non-vaccinated fish.High level protection against vibriosis was observed in fish vaccinated by immersion in V. anguillarum cells which had been formalinized, washed, and pelleted or which had been formalinized, washed, and lyophilized.

魚病菌Ｖｉｂｒｉｏ ａｎｇｕｉｌｌａｒｕｍに対するＰｓｅｕｄｏｍｏｎａｓ ｓｐ．の溶菌および致死作用

A Pseudomonas sp. (L 1-7) capable of lysing Vibrio anguillarum, important pathogenic organism of cultured fish, was isolated from pond water for fish culture and tank water for plankton culture. The high yield of lytic factor in the supernatant of strain L 1 culture was obtained by shaking culture at 25°C for 24 hours in the medium containing 1% meat extract, 0.5% polypepton, 0.4% yeast extract and 0.3% glucose at pH 7.0. The activity of the lytic factor was highest at around 45°C and a little lower at 30°C; it was inactivated by heating at 50°C for 4 hours or at 60°C for 15 minutes. Lytic factor produced by strain L 1-7 was found to show varying degree of activity against various gram negative organisms other than V. anguillarum, depending upon the difference in strains. Gram positive organisms such as Bacillus subtilis and the other Bacillus organisms were lysed more or less, whereas Streptococcus faecalis and S. faecium strains were completely insensitive to the lytic factor. It is then considered that no correlation exists between the lytic action of the organisms and gram reaction of the organisms examined. The culture supernatant of strain L 1 was not only able to lyse the V. anguillarum cells, but also found to show remarkable lethal action.

Outer membrane proteins induced under conditions of iron limitation in the marine fish pathogen Vibrio anguillarum 775.

Cells of the marine fish pathogen Vibrio anguillarum 775 harboring a plasmid associated with virulence can grow unaffected in the presence of iron-binding compounds such as transferrin. In contrast, the growth of isogenic plasmidless derivatives is inhibited by the presence of iron chelators. Radioactive from (55Fe3+) uptake experiments indicate that this plasmid-linked ability of V. anguillarum cells to grow under conditions of iron limitation is indeed due to a more rapid and efficient iron uptake mediated by the virulence plasmid. In addition, V. anguillarum cells growing under iron limitation show at least two novel outer membrane proteins. One of them, a 86,000-dalton protein we called OM2, is inducible only in those cells in which the virulence plasmid is present.

Bath‐immunization—a practical and non‐stressing method of vaccinating sea farmed rainbow trout Salmo gairdneri Richardson against vibriosis

Abstract. A non‐stressing easily applicable vaccination method against vibriosis in rainbow trout is described. The vaccine consisted of trypsin digested Vibrio anguillarum cells and was administered through a 2 h bath treatment, giving adequate protection in challenge experiments with the same V. anguillarum strain.

Vibrio anguillarum as a cause of disease in winter flounder (Pseudopleuronectes americanus).

Vibrio anguillarum was repeatedly isolated from skin and muscle lesions of the winter flounder, Pseudopleuronectes americanus. The dermal lesions, usually accompanied by fin necrosis, included petechiae and ecchymoses in their acute phase and frank ulceration in their more chronic manifestations. Anemia, as evidenced by lowered hematocrit values and increased renal hematopoiesis, was present. The disease was reproduced experimentally by the intradermal injection of as few as 640 V. anguillarum cells. The differential identification of the V. anguillarum isolates from aeromonads, plesiomonads, and other marine vibrios was described and discussed.