Anguillarum Cells Research Articles

NKHs27, a sevenband grouper NK-Lysin peptide that possesses immunoregulatory and antimicrobial activity

As an effective and broad-spectrum antimicrobial peptide, NK-Lysin is attracted more and more attention at present. However, the functions and action mechanism of NK-Lysin peptides are still not comprehensive enough at present. In this study, a sevenband grouper (Hyporthodus septemfasciatus) NK-Lysin peptide, NKHs27, was identified and synthesized, and its biological functions were studied. The results indicated that NKHs27 shares 44.44%∼88.89% overall sequence identities with other teleost NK-Lysin peptides. The following antibacterial activity assay exhibited that NKHs27 was active against both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Listonella anguillarum, Vibrio parahaemolyticus and Vibrio vulnificus. Additionally, NKHs27 showed a synergistic effect when it was combined with rifampicin or erythromycin. In the process of interaction with the L. anguillarum cells, NKHs27 changed the cell membrane permeability and retained its morphological integrity, then penetrated into the cytoplasm to act on genomic DNA or total RNA. Then, in vitro studies showed that NKHs27 could enhance the respiratory burst ability of macrophages and upregulate immune-related genes expression in it. Moreover, NKHs27 incubation improved the proliferation of peripheral blood leukocytes significantly. Finally, in vivo studies showed that administration of NKHs27 prior to bacterial infection significantly reduced pathogen dissemination and replication in tissues. In summary, these results provide new insights into the function of NK-Lysin peptides in teleost and support that NKHs27, as a novel broad-spectrum antibacterial peptide, has potential applications in aquaculture against pathogenic infections.

Cinnamaldehyde exerts prophylactic and therapeutic effects against Vibrio anguillarum infection in Yesso scallop (Patinopecten yessoensis) by its direct antimicrobial activity and positive effect on the innate immunity

This study investigated the antimicrobial mechanism and immunoprotection of cinnamaldehyde (CNA) against Yesso scallop (Patinopecten yessoensis) pathogen Vibrio anguillarum (V. anguillarum). The results showed that V. anguillarum cells treated with CNA displayed an increased uptake of 1-N-phenylnaphthylamine and propidium iodide, as well as distinct morphological changes, which was indicative of its potent antibacterial activity against V. anguillarum via a membrane-active mechanism to kill the bacteria. Biosafety analysis showed that CNA exhibited no adverse effects on the hemocytes under the test concentrations. Most importantly, pre-treating, co-treating, and post-treating the scallop with CNA significantly increased the survival rate of the scallop after the injection of V. anguillarum. In vivo functional analysis further demonstrated that CNA could promote the clearance of bacteria in hemolymph. Further examination revealed that CNA had a significantly positive effect on the hemolymph immune parameters, contributing to the scallop's immune defense against pathogenic infections. In addition, CNA modulated the expressions of immune-associated genes like PGRP, Toll, NFκB, Defensin, Lysozyme, GPx, and Cu/Zn SOD as analyzed by a Real-Time PCR approach. Furthermore, it is interesting to note that pre-treatment with CNA mitigated a series of V. anguillarum-induced genes known to be responsible for antibacterial and anti-oxidant reactions. These results suggested that CNA exhibited potential for development as a new antibacterial strategy to combat scallop bacterial infections in aquaculture.

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.

Enniatin B metabolites in liver and tissues of gilthead sea bream (Sparus aurata)

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.

Comparison of 16SrDNA and toxR genes as targets for detection of Vibrio anguillarum in Dicentrarchus labrax kidney and liver

Vibrio anguillarum is a pathogen that causes high mortality in marine and freshwater fish. The aim of this study was to develop a real-time PCR assay for identification and quantification of V. anguillarum in fish tissue. The assay was carried out using two target genes, 1 6SrDNA and toxR, to evaluate the influence of differences in the operon copy number in quantitative assessment, both in pure cultures of V. anguillarum serovar O1 (strain 975/I), as a reference, and in the liver and kidney of a sea bass ( Dicentrarchus labrax) specimen. Real-time PCR analysis showed high specificity for both target genes, with a detection limit of approximately 1–10 bacterial cells per reaction in pure culture and 10/100 V. anguillarum cells per reaction in fish tissue, which corresponds to 2 × 10 2/2 × 10 3 cells g −1 fish tissue. Moreover, both genes showed high specificity but differing sensitivity due to the different operon copy number; as a result, it is possible to target the high copy number gene to improve sensitivity. Our results suggest that the protocol we tested can be used as a sensitive and specific molecular method for the detection of the fish pathogen V. anguillarum in fish tissue.

Response to low osmotic stress in a fish pathogen, Vibrio anguillarum

Low osmotic stress responses of the fish pathogen, Vibrio anguillarum, were investigated and characterized with regard to changes in cell density and cation type in natural lake water (aged lake water, ALW) and MilliQ water (MQW). This pathogen showed higher survival (% CFU) in ALW than in MQW at all the examined cell densities (106–109 CFU ml−1). Higher resistance in ALW was partially due to divalent cations such as Ca2+ and Mg2+ present in ALW. It was also observed that addition of the supernatant, which was obtained from the pre-suspension of V. anguillarum cells in ALW, significantly increased the survival of the pathogen in ALW. This indicated that the extracellular substance(s) released from the pathogen also played an effective role(s) for survival in ALW. Thus, divalent cations and cell density assist the pathogen, V. anguillarum, to overcome the low osmotic stress in natural freshwater environments.

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate 55Fe3+ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of 55Fe3+ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.

Kinetics of adhesion of Listonella anguillarum to the mucus of gilt-head seabream, and the implication of surface components

The kinetics of adhesion of Listonella anguillarum cells to skin, intestinal and gill mucus suspensions of gilt-head seabream was studied. A Langmuir adsorption isotherm was calculated, and the results obtained indicated that this microorganism showed a saturation kinetics. The pretreatment of the mucus suspensions with a surface extract obtained from L. anguillarum cells promoted a very important inhibition of the number of adhered bacteria to the different mucus suspensions. The potential role of surface structures on the adhesion of L. anguillarum to mucus of gilt-head seabream is discussed.

Development of a PCR-based method for the detection of Listonella anguillarum in fish tissues and blood samples.

A PCR assay for detection and identification of the fish pathogen Listonella anguillarum was developed. Primers amplifying a 519 bp internal fragment of the L. anguillarum rpoN gene, which codes for the factor sigma54, were utilized. The detection limit of the PCR using L. anguillarum pure cultures was approximately 1 to 10 bacterial cells per reaction. For tissue or blood samples of infected turbot Scophthalmus maximus, the detection limit was 10 to 100 L. anguillarum cells per reaction, which corresponds to 2 x 10(3) to 2 x 10(4) cells g(-1) fish tissue. Our results suggest that this PCR protocol is a sensitive and specific molecular method for the detection of the fish pathogen L. anguillarum.

Physiological State of Vibrio anguillarum, a Fish Pathogen, under Starved and Low-Osmotic Environments.

Vibriosis caused by Vibrio anguillarum serotype J-O-1 seriously affects the freshwater fish ayu (Plecoglossus altivelis) in Lake Biwa, Japan. Survival patterns of V. anguillarum were investigated in aged lake water (ALW) supplemented with or without 0.75% NaCl. It was found that 0.1-1.0% of V. anguillarum cells maintained the ability to form colonies even after 6 weeks in 0.75% NaCl-ALW. Under the same starved conditions, MPN counts with liquid medium were 100 times higher than CFU counts. When exposed to sterilized aged lake water without NaCl, V. anguillarum entered a non-culturable state within half a day. As the mineralization activity of non-culturable cells was still 0.1-5.4% of that in the culturable phase, this physiological state can be described as viable but non-culturable. However, all attempts to return to a culturable state including re-infection were un-successful. The non-culturable cells in ALW lost all pathogenicity in fish. Since a longer exposure to ALW resulted in less mineralization activity, the non-culturable state of V. anguillarum cells in freshwater seems to reflect a phase of decay leading to cell death. However, certain environmental factors such as coldness and microaerobiosis seem to help the pathogen to survive longer in freshwater without a loss of culturability.

Passive immunisation of fish against vibriosis, comparison of intraperitoneal, oral and immersion routes

Passive immunisation of fish was conducted to determine whether anti- Vibrio anguillarum whole sera (AVA) and affinity-purified AVA raised in sheep, rabbits and rainbow trout ( Oncorhynchus mykiss) were persistent when injected and orally administered into rainbow trout. These responses were compared with active immunisation by immersion in, and intraperitoneal (i.p.) injection with, formalin-killed V. anguillarum cells. Sheep and rabbit AVA were detected in rainbow trout sera for up to 70 days (half-life ∼21 days) after i.p. injection as determined by an enzyme-linked immunosorbent assay (ELISA). The relative percentage survival (RPS) of passively immunised rainbow trout challenged with virulent V. anguillarum after an injection was comparable to that of active immunisation by immersion after 1 month post-immunisation (p.i). Affinity-purified sheep and rabbit AVA exhibited the same protective potential as whole serum in rainbow trout. Rabbit and sheep immune sera diluted 1:8 and 1:50, respectively, provided equivalent protection as undiluted fish immune serum. An active immune response against passively acquired heterologous immunoglobulins was demonstrated by ELISA, with responses against sheep AVA being less than those against rabbit AVA. Rainbow trout given purified sheep AVA conjugated to LTB (the GM-1-binding subunit of Escherichia coli heat-labile toxin) and administered orally had an RPS of 37.5% at 15 days and 27% at 1 month p.i. In contrast, fish given sheep AVA conjugated to TraT (an internal membrane of E. coli) or in micellar form with Quil-A had RPSs of only 18.7 and 6.2%, respectively, after 15 days, and 13.3 and 0% after 1 month, respectively. The protection conferred by immune sera was shown to be due to the immunoglobulin component alone. Heat inactivation of the complement in sera had no effect on the potency of immune sera.

Survival, persistence and proliferation of Vibrio anguillarum in juvenile turbot, Scophthalmus maximus (L.), intestine and faeces.

The aim of this study was to determine whether orally administered V. anguillarum cells could survive passage through the intestinal tract of feeding turbot, and thereafter, proliferate in the released faeces. In addition, the growth of the pathogen in turbot faeces in the presence of a Carnobacterium sp. with inhibitory effects against a number of bacterial fish pathogens was studied. It was found that V. anguillarum cells survived the acidic environment of the stomach for several hours, persisted in the intestine and readily proliferated in the released faeces. The antagonistic Carnobacterium sp. inhibited the growth of V. anguillarum during co-culture in turbot faecal extracts. From the results, it was concluded that the turbot intestinal tract and faeces can serve as an enrichment site for V. anguillarum, and the use of intestinal bacteria with antagonistic activity against vibrios may be used to reduce the load of fish pathogenic vibrios in turbot hatcheries.

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate 55Fe 3+ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of 55Fe 3+ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.

Immunostimulating effect of growth hormone: in-vivo administration of growth hormone in rainbow trout enhances resistance to Vibrio anguillarum infection.

In-vivo administration of purified (nGH) and recombinant (rGH) chum salmon, Oncorhynchus keta, growth hormone enhanced the survival of rainbow trout, O. mykiss, against virulent Vibrio anguillarum. Macrophages obtained from rainbow trout that were treated in vivo with either GH preparation showed enhanced stimulation of the chemiluminescent responses induced by opsonized V. anguillarum cells and increased phagocytic activities. However, the serum bactericidal activity against V. anguillarum was not enhanced in fish injected with nGH or rGH. These findings show that in-vivo administration of either GH preparation can effectively prime macrophages and increase the resistance to V. anguillarum in rainbow trout.

Response to low osmotic stress in a fish pathogen, Vibrio anguillarum

Low osmotic stress responses of the fish pathogen, Vibrio anguillarum, were investigated and characterized with regard to changes in cell density and cation type in natural lake water (aged lake water, ALW) and MilliQ water (MQW). This pathogen showed higher survival (% CFU) in ALW than in MQW at all the examined cell densities (10 6–10 9 CFU ml −1). Higher resistance in ALW was partially due to divalent cations such as Ca 2+ and Mg 2+ present in ALW. It was also observed that addition of the supernatant, which was obtained from the pre-suspension of V. anguillarum cells in ALW, significantly increased the survival of the pathogen in ALW. This indicated that the extracellular substance(s) released from the pathogen also played an effective role(s) for survival in ALW. Thus, divalent cations and cell density assist the pathogen, V. anguillarum, to overcome the low osmotic stress in natural freshwater environments.

An established hybridoma clone producing a monoclonal antibody against Vibrio anguillarum.

Vibrio anguillarum is a pathogenic microorganism of vibriosis, an infectious disease found in various fish species. A mouse hybridoma clone, named C5, that produced a monoclonal antibody to V. anguillarum was established. The specific reaction of C5 antibody with V. anguillarum was confirmed by the pre-adsorption effect of the V. anguillarum cells in ELISA and a cell immunoprecipitation experiment. Western blotting analysis indicated that the C5 antibody recognized a high molecular weight substance extracted from cells with detergents.

Is the turbot, Scophthalmus maximus (L.), intestine a portal of entry for the fish pathogen Vibrio anguillarum?

AbstractThe role of the intestinal tract in Vibrio anguillarum infection of turbot, Scophthalmus maximus (L.), fingerlings was investigated in two in vivo models and the possible mechanisms involved were studied in vitro. Viable V. anguillarum cells were detected in spleens from more than 50% of the fish administered the pathogen orally or rectally, suggesting that the intestinal tract is a portal of entry for V anguillarum. In transmission electron micrographs, V. anguillarum‐ like cells were seen close to the rectal epithelium, suggesting penetration of the mucus layer, but no epithelial cell penetration or endocytosis was evident. Attachment to intact turbot intestines was investigated, and 80% or more of the bacterial cells still remained attached after serial washings. A significantly higher number of cells attached to rectal segments than to the other intestinal segments. In vitro, V. anguillarum cells did not adhere specifically to intestinal mucus, but rather accumulated close to intestinal mucus interfaces and subsequently penetrated them. It is proposed that the intestinal ttact of tutbot is a portal of entry for V. anguillarum and that the cells penetrate the intestinal mucus overlaying the epithelial cells.

Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid.

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa proFatB precursor protein.

Kinetics of antibody production against Vibrio anguillarum antigens in turbot

We evaluated the kinetics of antibody production by turbot ( Scophthalmus maximus L.) in response to intraperitoneal (i.p.) inoculation with Vibrio anguillarum (ATCC strain 19264) cells in the presence or absence of Freund's complete adjuvant. Antibody production was monitored by enzyme-linked immunosorbent assay (ELISA), using and anti-turbot Ig monoclonal antibody produced in our laboratory and three bacterial antigen preparations (whole cells, a whole-cell extract and O-antigen). Our results demonstrate: (a) that maximal antibody response can be achieved with a single inoculation in the presence of adjuvant, and is reached within 45 days; (b) that in the absence of adjuvant, an i.p. booster is required to achieve a maximal response; and (c) that the whole-cell extract (a cytosolic fraction obtained by sonication and subsequent ultracentrifugation at 100 000× g for 30 min) is the best antigen for determination by ELISA of anti- V. anguillarum antibodies in turbot.

The detection of fish pathogenVibrio anguillarum in water and fish using a species-specific DNA probe combined with membrane filtration

The marine bacteriumVibrio anguillarum causes disease in fish worldwide and is particularly devastating in aquaculture. Little is known about the ecology ofV. anguillarum in the environment and how this may relate to the pathogenicity of this organism. Combining membrane filtration and a species-specific DNA probe, culturableV. anguillarum cells were detected in water from three habitats and in chinook salmon (Onchorynchus tshawytscha) tissue samples. Results show that different marine habitats have a marked effect on cell numbers and that water temperature may play a role in the culturability and distribution ofV. anguillarum. Vibrio anguillarum was detected from the gills of salmon within 24 h of transfer of fingerlings from freshwater to seawater, with cell numbers reaching a concentration of 1.9 × 10(2) cells g(-1) tissue 28 days post transfer.Vibrio anguillarum cell numbers were low in the colon throughout the study, andV. anguillarum was not detected in healthy kidney samples. The methodology reported in this paper allows the accurate quantification of culturableV. anguillarum cells and has allowed a preliminary study of the ecology of this species.