Anguillarum Serotype Research Articles

Refining the Occurrence of Viral Encephalopathy and Retinopathy, Photobacteriosis, and Vibriosis in Connection with Seawater Physicochemical Parameters: A Five-Year Case Study

The marine environment and its physicochemical parameters play a pivotal role in the context of fish farming. Fundamental physicochemical factors of water, including temperature (T°C), salinity (S psu), dissolved oxygen (D.O. mg/l), and pH, exert a profound influence on the physiology of farmed aquatic animals and the composition of microbial communities. This study aimed to establish correlations between documented disease occurrences in sea bass intensive rearing and the prevailing physicochemical parameters of water. The objective was to analyse the significance of these parameters in disease outbreaks and develop a predictive model for the outbreak risk of key diseases. To this end, real-time physicochemical data were collected daily over five years (2011-2015) from two fish farms (A and B) located in the Gulf of Argostoli, island of Cephalonia, Western Greece. The focus was on incidences of Viral Encephalopathy and Retinopathy (VER), Photobacteriosis caused by Photobacterium damselae subsp. piscicida (PHDP) and Vibriosis caused by Vibrio anguillarum serotype O1 (VAO1). Statistically significant correlations were found between increasing water temperature and salinity and the occurrence of VER, PHDP, and VAO1 diseases. The reduction of dissolved oxygen, when combined with other parameters, synergistically triggered the manifestation of these three diseases. The study’s findings were discussed within the context of their potential to predict disease outbreaks based on fluctuations in the physicochemical parameters of water and anticipated climate change scenarios.

Evaluation of the protective efficiency of an autogenous Vibrio anguillarum vaccine in lumpfish (Cyclopterus lumpus) under controlled and field conditions in Atlantic Canada

Lumpfish (Cyclopterus lumpus) have become the predominant cleaner fish species used in North American salmon aquaculture. Vibrio anguillarum is a frequent pathogen of lumpfish in Atlantic Canada, and current vaccines against local isolates conferred low to moderate efficacy. This study evaluated the safety and efficiency of a V. anguillarum autogenous vaccine under controlled and field conditions. Two safety trials were conducted following the Canadian Food Inspection Agency (CFIA) regulations before field trial testing. The first safety trial was a common garden assay, and 250 PIT-tsgged lumpfish were used for five treatments per tank (PBS-negative control, Lab vaccine-positive control, autogenous intraperitoneal (IP) vaccine, autogenous dip vaccine, autogenous dip-IP boosted vaccine) in triplicates. Weight, gross pathology, and IgM titers were evaluated to determine the vaccine’s safety. After 10 weeks post-vaccination (wpv), lumpfish were bath-challenged with a lethal dose (1.24 x 106 CFU/mL) of V. anguillarum serotype O2. Survival rate, IgM titers, memory immune response, and cross-immune protection were evaluated. The second safety trial was conducted using a double vaccine dose in two groups, PBS and autogenous IP. The gross pathology score indicated a normal immune response without tissue damage, and no mortality nor fish health issues were observed in the immunized animals. The IP route of administration conferred the highest protection against the V. anguillarum challenge. The autogenous vaccine conferred long-term immunity but did not confer cross-protection against V. anguillarum serotype O1. To evaluate the efficiency of the autogenous vaccine under field conditions, 58,976 naïve lumpfish (8-10 g) were IP immunized with either the autogenous vaccine or the commercial Forte micro IV vaccine. After 7 wpv, the lumpfish were equally distributed into eight sea cages and co-cultivated with Atlantic salmon smolts (60,000 per cage). Lumpfish IgM titers, tissue bacterial loads, mortality, and sea lice count on salmon were monitored. The autogenous vaccine and Forte vaccine confer acceptable protection to the lumpfish in sea cages. No significant difference was observed between lumpfish vaccinated with autogenous and commercial vaccines under field conditions.

Antigenic variability of Vibrio anguillarum serotype O2a: A hurdle for vaccine efficacy against vibriosis in rainbow trout (Oncorhynchus mykiss)

Despite vaccination, outbreaks of vibriosis still occur in sea-reared rainbow trout in Denmark. Vibriosis outbreaks are caused mainly by V. anguillarum serotypes O1 and O2a, and bacterins of both serotypes are included in the commonly used vaccine against this disease in Danish aquaculture. However, while the strains belonging to serotype O1 are genetically similar, the strains belonging to serotype O2a are highly diverse. This work aimed first at examining how the antibody response and protection induced by bacterin-based vaccines were affected by the antigenic variability within V. anguillarum serotype O2a strains. Following vaccination of rainbow trout with either a commercial or an experimental vaccine, specific antibody reactivity in serum from vaccinated fish was examined by ELISA against 23 strains of V. anguillarum serotype O2a (VaO2a). The strains were divided into 4 distinct subgroups according to the observed detection pattern. Seven strains were strongly recognized only by sera from fish vaccinated with the experimental vaccine (EV-I antisera), while 13 other strains were primarily recognized by sera from fish vaccinated with the commercial vaccine (CV antisera). Two strains were recognized by both EV-I and CV antisera, but with intermediate reactivity, while one strain was not recognized at all. A partly similar recognition pattern was observed when purified lipopolysaccharide (LPS) was used as antigen in the examination of antibody reactivity in Western blotting. The level of protection was highly dependent on both the vaccine and the strain used for challenge and showed no consistent correlation with antibody reactivity. Secondly, we attempted to use a bacterin vaccine based on one of the V. anguillarum O2a strains intermediately recognized by both EV-I and CV antisera to investigate whether that could potentially provide protection across strain variability. The immunized fish did mount a cross-reactive antibody response, but protection still varied depending on the strain used for challenge.Interestingly, the grouping of strains according to antibody reactivity correlated not only with genotyping based on single nucleotides polymorphisms analysis (SNP) but also with variability in the accessory genome, indicating that presence or absence of protein antigens or proteins associated with the biosynthesis of antigenic epitopes may explain the observed distinct serological subgrouping within VaO2a strains by trout immune sera.In terms of vaccination against VaO2a, our results demonstrate that it is important to take (local) antigen variations into account when using bacterin-based vaccines but also that alternatives to traditional bacterin-based vaccines might be needed to induce protection against the highly virulent Vibrio anguillarum serotype O2a strains.

A Major QTL for Resistance to Vibrio anguillarum in Rainbow Trout.

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout (Oncorhynchus mykiss). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 107 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish – including survivors – and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.

Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

Vibriosis, a hemorrhagic septicemic disease caused by the bacterium Vibrio anguillarum, is an important bacterial infection in Danish sea-reared rainbow trout. Despite of vaccination, outbreaks still occur, likely because the vaccine is based on V. anguillarum strains from abroad/other hosts than rainbow trout. Information about the genetic diversity of V. anguillarum specifically in Danish rainbow trout, is required to investigate this claim. Consequently, the aim of the present investigation was to sequence and to characterize a collection of 44 V. anguillarum strains obtained primarily from vibriosis outbreaks in Danish rainbow trout. The strains were sequenced, de novo assembled, and the genomes examined for the presence of plasmids, virulence, and acquired antibiotic resistance genes. To investigate the phylogeny, single nucleotide polymorphisms were identified, and the pan-genome was calculated. All strains carried tet(34) encoding tetracycline resistance, and 36 strains also contained qnrVC6 for increased fluoroquinolone/quinolone resistance. But interestingly, all strains were phenotypic sensitive to both oxytetracycline and oxolinic acid. Almost all serotype O1 strains contained a pJM1-like plasmid and nine serotype O2A strains carried the plasmid p15. The distribution of virulence genes was rather similar across the strains, although evident variance among serotypes was observed. Most significant, almost all serotype O2 and O3 strains, as well as the serotype O1 strain without a pJM1-like plasmid, carried genes encoding piscibactin biosynthesis. Hence supporting the hypothesis, that piscibactin plays a crucial role in virulence for pathogenic strains lacking the anguibactin system. The phylogenetic analysis and pan-genome calculations revealed great diversity within V. anguillarum. Serotype O1 strains were in general very similar, whereas considerable variation was found among serotype O2A strains. The great diversity within the V. anguillarum serotype O2A genomes is most likely the reason why vaccines provide good protection from some strains, but not from others. Hopefully, the new genomic data and knowledge provided in this study might help develop an optimized vaccine against V. anguillarum in the future to reduce the use of antibiotics, minimize economic losses and improve the welfare of the fish.

The efficacy and side-effects of oil-based adjuvants emulsified Vibrio anguillarum bivalent inactivated vaccine in turbot (Scophthalmus maximus) under production mode

Turbot (Scophthalmus maximus) is an important commercial fish in China, and Vibrio anguillarum is a common pathogen of turbot. Oil emulsified vaccines have been widely used to protect fish against V. anguillarum with long-term immune responses in several fish species. Side-effects, such as adhesions and growth reduction caused by oil-based adjuvants, however, limit the application of oil emulsions in turbot. In this study, turbot V. anguillarum serotype O1 and O2 bivalent inactivated vaccines were emulsified as water-in-oil (W/O) emulsions with four kinds of oil-based adjuvants: Freund's incomplete adjuvant (FIA), MOBIL™ Marcol 52 (Marcol 52), SEPPIC Montanide™ ISA 763 A VG (ISA 763) and ISA 780 (ISA 780). Vaccine protection efficiency, fish adhesions, and growth were tested under production mode. The relative percent of survival (RPS) of W/O vaccines was all higher than that of the water-based (WB) vaccine at 7 days post vaccination. At 30 days post vaccination, the RPS of all vaccinated groups was over 90%, and most of the specific antibody titers of W/O vaccine-vaccinated fish were significantly higher than those of WB vaccines. Fish immunized with FIA, ISA 763, and ISA 780 adjuvants W/O vaccine showed serious tissue adhesions after vaccination, and their weight was significantly lower than control fish at 30 days post vaccination. Only slight or minor adhesions were observed in WB and Marcol 52 adjuvant vaccinated fish, which did not show growth reduction. These results indicate that Marcol 52 is a well-tolerated oil-based adjuvant for turbot, and could be used in vaccine development in further study.

Pharmacokinetics of chloramphenicol (CAP) in gilthead sea bream (Sparus aurata) and its in vitro activities against important bacterial fish pathogens

The pharmacokinetics of chloramphenicol (CAP) in gilthead sea bream (Sparus aurata), a warm water farmed fish species and its in vitro efficacy against important bacterial diseases of Mediterranean mariculture were investigated in this study. After an intravascular injection (10 mg/kg/fish), the distribution half-life (tl/2a) and the elimination half-life (tl/2ß) of CAP were calculated to be 1.6 and 69 h, respectively. Tissue penetration of CAP was found to be moderate since both the apparent volume of distribution of the drug at steady-state (V<i(ss)) and the apparent volume of the central compartment (Vc) were calculated to be 1.13 and 0.90 L/kg. The total clearance (CLp) of the drug was slow (0.022 L/kg/h). The minimum inhibitory concentration (MIC) values of CAP in distilled water supplemented with 2% NaCl against Vibrio anguillarum serotype lb, Photobacterium damsela subsp. piscicida, V alginolyticus and V ßuvialis were determined to be 4.78 pg/mL, while it was 0.60 pg/mL for V. damsela. The addition of 10 mM Ca2+ and 55 mM Mg2+ in the medium revealed MIC values of 19.13 pg/mL for V alginolyticus and V ßuvialis, whereas showed no effect for V anguillarum P. damsela subsp. Piscicida and V damsela. The results indicate that CAP displayed a satisfactory kinetic profile and it is eliminated fast from gilthead sea bream muscle; however, its high MIC values stress show a possible inefficacy against important bacterial pathogens of Mediterranean mariculture.

Studies on the antibody response and side effects after intramuscular and intraperitoneal injection of Atlantic lumpfish (Cyclopterus lumpus L.) with different oil‐based vaccines

Atlantic lumpfish (Cyclopterus lumpus L.) is used as a biological delousing agent for sea lice (Lepeophtheirus salmonis K.) infestations in Norwegian aquaculture. Here, we present a study on the antibody response and vaccine side effects after intramuscular and intraperitoneal injection of lumpfish with two vaccines. Both vaccines contained bacterial antigens from atypical Aeromonas salmonicida A-layer types V and VI, Vibrio anguillarum serotype O1 and Moritella viscosa sp., but one vaccine contained a vegetable oil-based adjuvant, while the other contained a mineral oil-based adjuvant. Intramuscular injection of the mineral oil-based vaccine caused a high acute mortality of fish within 48hr after immunization. Intraperitoneal injection of the mineral oil-based vaccine resulted in a lower severity of intra-abdominal side effects than the vegetable oil-based vaccine. Intramuscular injection of the mineral oil-based vaccine resulted in a significantly higher antibody response against A.salmonicida when compared to controls and the vegetable oil-based vaccine group. The antibody response was poor against V.anguillarum and M.viscosa for all groups. Our results indicate that intramuscular injection of oil-based vaccines might be feasible for providing immunological protection for Atlantic lumpfish against bacterial diseases, especially atypical A.salmonicida, but more work is required to identity optimal adjuvants.

Prevention of sea bass (Dicentrarchus labrax, L. 1758) photobacteriosis and vibriosis. Long term efficacy study of intraperitoneally administered bivalent commercial vaccines

The long term efficacy of different bivalent commercial vaccine formulations against Vibriosis and Photobacteriosis in sea bass (Dicentrarchus labrax, L. 1758) was investigated. Duplicate whole cages in the field were intraperitoneally vaccinated with either an adjuvanted (AJ) or an aqueous (AV) commercial vaccine or with sterile 2% NaCl. All groups of fish were monitored over a period of 12months in respect to natural disease outbreaks, performance and the condition of the abdominal cavity. At certain time-points post-vaccination, fish collected from the field were experimentally infected with either Vibrio (Listonella) anguillarum serotype O1 or Photobacterium damselae subsp. piscicida. In addition, blood samplings were performed for the isolation of sera in order to analyze the quantitative characteristics of the humoral immune response of vaccinated fish.No disease outbreak in the field was noted during the course of the study, for both pathogens under investigation. In the laboratory-based efficacy tests, both commercial products conferred significant protection in comparison to the controls. In 5 consecutive experimental infections with V. anguillarum O1 the RPSs calculated for each commercial vaccine were as follows: day 54 post-vaccination (dpv), 38.0 & 23.0; 96dpv, 90.2 & 81.8; 163dpv, 30.0 & 10.0; 230dpv, 67.6 & 48.1; 306dpv, 13.0 & 47.0, for the AJ and AV formulations, respectively. In 4 consecutive experimental infections with Ph. damselae subsp. piscicida the RPSs calculated were as follows: 82dpv, 0.0 & 0.0; 142dpv, 62.5 & 37.5; 209dpv, 35.0 & 26.0; 287dpv -21.0 & 31.0, for the AJ and AV formulations, respectively. The statistical evaluation of all the efficacy data collected over a 12-month period showed that the adjuvanted vaccine offered better protection against vibriosis. In contrast, there was no statistical difference between the commercial products in the protection against photobacteriosis. The vaccines did not affected negatively the survival of vaccinated fish (i.e. cumulative mortalities 10months post-vaccination in the field were as follows: AJ: 2.41%, AV: 2.93%, Control: 1.78%). Fish vaccinated with the adjuvanted vaccine developed an aseptic granulomatous peritonitis and mild adhesions in the abdominal cavity in contrast to the aqueous vaccine where a mild attraction of leukocytes was the only observation. Vaccinated fish groups reached higher weights in comparison to the controls, while FCR and SGR between groups for the whole period were not statistically significant. The adjuvanted commercial vaccine provoked the production of significantly higher specific antibody titers against each of the pathogens in comparison to the other experimental groups. These higher antibody titers corresponded well to the overall performance of the adjuvanted vaccine. Results are discussed in view to the performance of these products and parameters that may influence it.

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model.

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.

Expression, secretion and bactericidal activity of type VI secretion system in Vibrio anguillarum.

The type VI secretion system (T6SS) was recently shown to modulate quorum sensing and the stress response in Vibrio anguillarum serotype O1 strain NB10. It is not known whether there is a functionally active T6SS in other serotypes of V. anguillarum. Here, homologues to T6SS cluster VtsEFGH and hemolysin-coregulated protein (Hcp)-encoding genes were found to be prevalent and conserved in clinical isolates of V. anguillarum from fish, including four O1 and five non-O1 serotype strains. Unexpectedly, only the non-O1 serotype strains expressed VtsEFGH and Hcp under laboratory and marine-like conditions, in contrast to the serotype O1 strains. This suggested that the V. anguillarum non-O1 serotype strains tested have constitutive expression of T6SS. Examination of a representative non-O1 strain, MHK3, showed that Hcp production was growth phase dependent and that maximum Hcp production was observed in the exponential growth phase. Moreover, Hcp production by MHK3 was most active under warm marine-like conditions. Further examination revealed a correlation of the constitutive expression of T6SS with bactericidal activity against Escherichia coli and Edwardsiella tarda. The work presented here suggests that the constitutive expression of T6SS provides V. anguillarum with advantage in microbial competition in marine environments.

Induction of mild enterocolitis in zebrafish Danio rerio via ingestion of Vibrio anguillarum serovar O1.

Vibrio anguillarum is the etiological agent of a fatal hemorrhagic disease known as vibriosis that affects a wide range of fish species, causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of our study was to evaluate the most effective way to induce mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using 2 methods of administration of V. anguillarum serotype O1 to adult zebrafish Danio rerio: via intraperitoneal injection and via ingestion of infected Artemia nauplii. The results showed that the intraperitoneal administration often caused massive fish death due to severe systemic involvement. In our experiments, the effect of intraperitoneal infection was evident 48 h post infection, with cumulative mortality within 7 d post infection with severe histopathological changes in kidney hematopoietic tissue and in the intestine. In contrast, oral infection via Artemia did not show systemic involvement and only a moderate degree of inflammatory influx of the mucosa, a partial recovery at 12 d post infection, and no mortality. For these reasons, oral infection with live food appears to be the most effective method to induce mild enteritis with a local inflammatory response.

Prevention of fish photobacteriosis. Comparison of the efficacy of intraperitoneally administered commercial and experimental vaccines

Two commercial multivalent vaccines against vibriosis, caused by Vibrio anguillarum serotype(s) and photobacteriosis, caused by Photobacterium damsela subsp. piscicida, one with oil adjuvant (AJ) and the other,being an aqueous solution (AV), and an experimental monovalent (Ph. damselae subsp. piscicida) vaccine inactivated with formalin or heat, namely EVF and EVH, were tested in laboratory trials on sea bass (Dicentrarchus labrax) in respect to their efficacy against experimentally induced photobacteriosis. The first trial aiming at high bacterial pressure was carried out 34 days post-vaccination and resulted in 90% mortalities in the control. The relative per cent survival (RPS) of vaccinated fish was calculated at 24, 3.7, 0 and 0 for the AJ, AV, EVF and EVH formulations, respectively. The second trial aiming at medium bacterial pressure was carried out 49 days post-vaccination and resulted in 45% mortalities in the control. The relative per cent survival (RPS) of vaccinated fish was calculated at 100, 92.7, 77.8 and 66.7 for the AJ, EVF, EVH and AV, formulations, respectively. Apparently, under both these high and medium bacterial pressure conditions, the commercial vaccine AJ performed better than the commercial vaccine AV, while under medium pressure there was no statistical difference between the performance of EVF and AJ. The measurement of specific antibody titers in sera collected from all fish groups 49 days post-vaccination, showed high levels in the fish vaccinated with the AJ vaccine, almost three times lower levels for the AV and EVF vaccines and even lower levels for the EVH vaccine. Results are discussed in respect to the choices mariculture companies have in selecting a commercial vaccine against photobacteriosis and possible alternatives, which, if commercially developed, may reduce vaccine cost.

The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid–encoded system of Vibrio anguillarum?

Many Vibrio anguillarum serotype O1 strains carry 65-kb pJM1-type plasmids harboring genes involved in siderophore anguibactin biosynthesis and transport. The anguibactin system is an essential factor for V. anguillarum to survive under iron-limiting conditions, and as a consequence, it is a very important virulence factor of this bacterium. Our comparative analysis of genomic data identified a cluster harboring homologs of anguibactin biosynthesis and transport genes in the chromosome of Vibrio harveyi. We have purified the putative anguibactin siderophore and demonstrated that it is indeed anguibactin by mass spectrometry and specific bioassays. Furthermore, we characterized two genes, angR and fatA, in this chromosome cluster that, respectively, participate in anguibactin biosynthesis and transport as determined by mutagenesis analysis. Furthermore, we found that the V. harveyi FatA protein is located in the outer membrane fractions as previously demonstrated in V. anguillarum. Based on our data, we propose that the anguibactin biosynthesis and transport cluster in the V. anguillarum pJM1 plasmid have likely evolved from the chromosome cluster of V. harveyi or vice versa.

Synthesis and antibacterial activity of conjugates between norfloxacin and analogues of the siderophore vanchrobactin

From synthetic functionalized analogues of vanchrobactin, a siderophore produced by the fish pathogenic bacteria Vibrio anguillarum serotype O2, several vanchrobactin analogues–norfloxacin conjugates were obtained and their antimicrobial activities against the wild-type and mutant strains of Vibrio anguillarum serotype O2 have been determined.

Comparative Proteomics of two Vibrio anguillarum Serotype O1 Strains with Different Virulence Phenotypes

Vibrio anguillarum is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal hemorrhagic septicemia in marine fish due to the action of virulence-associated proteins. In our study, V. anguillarum MN and 3010 were identified as serotype O1 by AFLP analysis, and the virulence of V. anguillarum MN was shown 50-fold higher than that of the strain 3101 by LD(50) tests with Japanese flounder Paralichthys olivaceus. Nine spots were noted as differentially expressed proteins by comparing the cellular and extracellular protein profiles of V. anguillarum MN and 3101. Mass spectrometry results showed OmpU and PrtV were highly expressed in the virulent strain MN but lowly expressed in the less virulent strain 3101. Expression level confirmed by semiquantitative RT-PCR showed that ompU and prtV were indeed highly expressed in the virulent strain MN. Together with similar amino acid sequences of both OmpU and PrtV in V. anguillarum MN and 3101, our study indicated that the expression level of OmpU and PrtV may be associated with the virulence of V. anguillarum.

Carnobacterium maltaromaticum vs. Vibrio (Listonella) anguillarum in the midgut of Atlantic cod (Gadus morhua L.): an ex vivo study

The present investigation addresses whether the midgut (MG) of Atlantic cod (Gadus morhua L.) is an infection route for Vibrio (Listonella) anguillarum serotype 02 β and if Carnobacterium maltaromaticum, a probiotic bacterium, can out-compete the pathogen and modulate the autochthonous MG microbiota. This was investigated by using an ex vivo method the intestinal sac, utilized previously in studies on Atlantic cod and Atlantic salmon (Salmo salar L.). Exposure of the MG to V. (L.) anguillarum did not reveal any cell damage indicating that the MG does not appear to be an infection route for V. (L.) anguillarum in healthy Atlantic cod. This finding together with previous observations on Atlantic cod and Atlantic salmon indicate that intestine as an infection route might vary between these two species. When the MG was exposed to C. maltaromaticum, no cell damage or cellular disruptions were observed. As budding from the apices of microvilli was observed in all treatments exposed to bacteria, we suggest that budding might be involved in the primary barrier against bacterial infection. However, to clarify this hypothesis, further studies are needed. Exposure of the MG to the probiotics and pathogenic bacteria indicated that C. maltaromaticum, to some extent, is able to out-compete V. (L.) anguillarum but the topic merits further investigation. Analysis of the MG microbiota after sterile saline solution and bacterial exposure indicates that bacteria related to Staphylococcus sciuri belong to the autochthonous gut microbiota in Atlantic cod.

Iron(iii) complexation by Vanchrobactin, a siderophore of the bacterial fish pathogen Vibrio anguillarum

The bacterial fish pathogen Vibrio anguillarum serotype O2 strain RV22 produces the mono catecholate siderophore Vanchrobactin (Vb) under conditions of iron deficiency. Vb contains two potential bidentate coordination sites: catecholate and salicylate groups. The iron(III) coordination properties of Vb is investigated in aqueous solutions using spectrophotometric and potentiometric methods. The stepwise equilibrium constants (log K) for successive addition of Vb dianion to a ferric ion are 19.9; 13.3, and 9.5, respectively, for an overall association constant of 42.7. Based on the previous results, we estimated the equilibrium concentration of free iron(III) under physiological conditions for pH 7.4 solution containing 10(-6) M total iron and 10(-5) M total Vb as pFe = 20 (=-log[Fe(3+)]). The Vb model compounds catechol (Cat) and 2,4-dihydroxy-N-(2-hydroxyethyl)benzamide (Dhb) have also been examined, and the obtained results show that the interaction of the whole system of Vb that contains the ferric-chelating groups of both Dhb and Cat, is synergically greater than the separate parts; i.e. Vb is the best chelating agent either in acid or basic media. In summary, bacteria employing Vb-mediated iron transport thus are able to compete effectively for iron with other microorganisms within which they live.

Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod ( Gadus morhua L.) juveniles

The purpose of this study was to investigate the efficacy of three monovalent and a trivalent vibriosis dip vaccines in juvenile Atlantic cod ( Gadus morhua L.), examine whether the responses were specific and study the expression of selected immune genes after dip vaccination. In addition, the study addressed whether the deviating isolates of Vibrio anguillarum serotype O2 belongs to another sero-subgroup than the previously established sero-subgroups O2a, O2b and O2c. Rabbit V. anguillarum serotype O2 antiserum adsorbed with V. anguillarum O2a O-antigen was shown, by both ELISA and immunoblotting, to still contain serotype O2 specific antibodies. Cod V. anguillarum serotype O2 antiserum reacted only with isolate of homologous serotype and not with heterologous sero-subgroups. This indicates that the deviating V. anguillarum O2 isolates represent a new sero-subgroup differing from sero-subgroup O2a. The monovalent vaccines included formalin inactivated cultures of V. anguillarum sero-subgroup O2a, O2b or serotype O2, while the trivalent vaccine contained all three sero-subgroups. Cod mounted high protection 7 weeks post dip vaccination with monovalent vaccines when challenged with homologous isolates and significantly lower when challenged with heterologous isolates, regardless of sero-subgroups. The trivalent vaccine resulted in efficient protection against all sero-subgroups tested. Dip vaccination of cod juveniles did not result in detectable antibody production or alteration in gene expression of the heavy chain of IgM and IgD. In the trivalent vaccine group expression of IFNγ and IL-12p40 were significantly up-regulated 3 days post vaccination. However, in groups vaccinated against V. anguillarum sero-subgroups O2b or O2, IL-12p40 and IFNγ gene expression were slightly increased 3 and 55 days post vaccination, respectively.

Role of the pJM1 plasmid‐encoded transport proteins FatB, C and D in ferric anguibactin uptake in the fish pathogen Vibrio anguillarum

Vibrio anguillarum serotype O1 is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal haemorrhagic septicemia in marine and fresh water fish due to the action of the anguibactin iron uptake system encoded by the virulence plasmid pJM1. This plasmid harbours the genes for the biosynthesis of the siderophore anguibactin and the ferric anguibactin transport proteins FatD, C, B and A encoded in the iron transport operon. The FatA protein is the outer membrane receptor for the ferric siderophore complex and the FatB lipoprotein provides the periplasmic domain for its internalization, whereas the FatC and D proteins are located in the cytoplasmic membrane and might play a role as part of the ABC transporter for internalization of the ferric siderophore. In this work we demonstrate the essential role of these two inner membrane proteins in ferric anguibactin transport and that the lipo-protein nature of FatB is not necessary for ferric anguibactin transport.