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The efficacy of Vibrio proteolyticus No. 442 as a probiotic in preventing opportunistic infections caused by Vibrionaceae in tiger puffer, Takifugu rubripes, was evaluated. Two strains of Listonella anguillarum, IFO 13266T and Obama 5, were used as target bacteria. The antimicrobial activity of V. proteolyticus No. 442 peaks at approximately 25°C and is absent at temperatures at 15°C or above 35°C. This activity is also most pronounced at an oxygen concentration of 21% and is undetectable under anaerobic conditions. Using these characteristics, V. proteolyticus No. 442 and L. anguillarum IFO 13266 were co-cultured under aerobic and anaerobic conditions. The findings indicated that under aerobic conditions, L. anguillarum IFO 13266 was eradicated following a 48-hour incubation at 25°C, while under anaerobic conditions, both species maintained high bacterial densities. Consequently, the oral administration of the diets supplemented with V. proteolyticus No. 442 to juvenile tiger puffers for seven days resulted in a decrease in the density of Vibrionaceae (excluding V. proteolyticus) by two to three orders of magnitude in the gut, and by one to two orders of magnitude in the rearing water. Future research needs to investigate the long-term efficacy of V. proteolyticus as a probiotic, and its pathogenicity, fish growth, administration methods, and storage strategies. [ABSTRACT FROM AUTHOR]

The efficacy of DNA vaccine is associated closely with the expression of the antigen and the intensity of local immune responses. In our previous study, a recombinant DNA plasmid expressing the VAA protein (pVAA) of Listonella anguillarum has been proved to have a good protection against the infection of L. anguillarum. In the present study, the local immune responses eliciting by immunizing flounder with intramuscular (I.M.) injection of pVAA was investigated at the cellular and genetic level, the muscle at the injection site at 7th post vaccination day was sampled and analyzed by hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), flow cytometry (FCM), RNA sequencing (RNA-Seq)-based transcriptomics and RT-qPCR. Then variations on the specific antibodies in serum at 1st–6th post vaccination week and the relative percent survival rate (RPS) at following 14 days after challenge were measured. The H&E results showed that inflammatory cells and immune cells significantly increased at the injection site. The IHC using monoclonal antibody against T cell markers revealed that both CD4-1+ and CD4-2+ T lymphocytes were recruited to the injection site and FCM results showed that the proportion of CD4-1+ cells in pVAA immunized group was 28.6 %, in the control group was 8.7 %, and that of CD4-2+ cells in two groups was 21.2 % and 8.5 %, respectively. These results indicating that the proportion of CD4+ cells in the immune group was significantly increased compared with the control group. Moreover, there were 2551 genes differently expressed in pVAA immunized group, KEGG analysis showed the genes involved in the signal transduction and immune system, and surface markers for B-cells genes, T-cells and antigen presenting cells (APCs) genes were highly upregulated, suggesting the activation of the systemic immune responses. Antibody specific anti- L. anguillarum or anti-rVAA antibodies were significantly induced at 2nd post-immunization week, that reached a peak at 4–5th week. RPS in pVAA group was 53.85±3.64 %. In conclusion, pVAA induced effective local immune responses and then the systematic response. This probably is the main contribution of pVAA to effective protection against L. anguillarum. [ABSTRACT FROM AUTHOR]

Vibriosis caused by Listonella anguillarum was reported in several fish species from both fresh and saltwater conditions. This pathogen causes disease in rainbow trout, sea bass, and sea bream in Turkey, however, it has not been reported from meagre (Argyrosomus regius) before. Great loss of meagre was observed in the Recirculated Aquaculture System at the Faculty of Fisheries of Izmir Katip Celebi University, which had been transferred from a commercial hatchery for a nutrition experiment. Clinical signs of vibriosis were observed in infected fish, i.e. haemorrhage in the anal area and pectoral fins, mostly as tail ulcers. Petechial haemorrhages in the muscle, liver, peritoneal membranes and pyloric caeca were determined by necropsy. A Gramnegative, rod-shaped, motile bacterium was isolated, showing a positive reaction to oxidase, catalase and gelatin tests, and being sensitive to O/129. Biochemical identification tests and PCR amplifications identified the bacterium as Listonella anguillarum. In slide agglutination test with anti L. anguillarum O1 (ATCC43305) serum, all isolates were positive. The isolated bacteria was resistant to oxytetracycline, sensitive to enrofloxacin, flumequine, phosphomycin, furozulidone, kanamycin and oxolinic acid. In this study, the isolated bacteria from meagre were determined as Listonella anguillarum O1 with biochemical, moleculer identification and agglutination tests. [ABSTRACT FROM AUTHOR]

In May 2016, there was a disease outbreak among pufferfish (Takifugu rubripes) in a marine farm in Tianjin municipality, with the cumulative mortality rate reaching 45% within 7 days from the beginning of the outbreak. The main symptoms of the disease were abdominal and anal swelling of the diseased fish. Significant damage was also observed histopathologically in the intestine, liver, and kidney. The strain H008 was isolated from diseased kidneys, and the challenge test revealed that the same disease was diagnosed in pufferfish which suffered similarly high mortality rates. Using physiological biochemical tests combined with 16S rDNA sequence analysis, the strain was identified as Listonella anguillarum. Antimicrobial susceptibility tests showed that H008 was resistant to 10 of 15 antimicrobial agents tested. To the best of our knowledge, this is the first report of L. anguillarum causing disease in pufferfish. [ABSTRACT FROM AUTHOR]

Two fish pathogens, namely Listonella anguillarum (Vibrio anguillarum) and Vibrio vulnificus, which were beta-haemolytic, hemagglutinating with 1% D-mannose and hydroxamate siderophore producer were used in this study. The extracellular products (ECPs) of both species were of high enzymatic and haemolytic activities. SDS-PAGE analysis of ECPs of L. anguillarumand V. vulnificus revealed 4 band (20.7 to 47.5kDa) and 2 bands (30.1 to 46kDa) respectively. The cumulative mortalities in O. niloticus produced by crude ECPs of L. anguillarum (0.2ml/fish; 2.4mg protein /ml) and V. vulnificus (0.2ml/fish; 1.6mg protein /ml) were 90% and 80%. Heat treatment of ECPs of L. anguillarum and V. vulnificus at 56°C for 30 min reduced the mortalities to 30% and 20%, respectively. LD50 values in O. niloticus using crude and heat treated ECPs of L. anguillarum were 1.7 and 3.9 ^g protein/g fish; while those of V. vulnificus were 1.34 and 2.8 ^g protein /g fish, respectively. Injection of levamisole at a dose of 5mg/Kg fish, concurrently with ECPs of L. anguillarum and V. vulnificus resulted in subsidence of the mortality percentages from 90 to 40 and from 80 to 30, respectively. Ten laying hens were immunized with ECPs of L. anguillarum for preparation of specific egg immunoglobulins. The total protein content of IgY preparations collected from eggs of hens immunized with ECPs of L. anguillarum reached its peak 2 weeks post 3rd booster dose (3.820 ± 0.0700 g/dl). The molecular weight of separated protein bands of IgY preparations ranged from 183-191kDa. Fish injected (I/P) with specific anti ECPs IgY of L. anguillarum (4 mg/ fish) followed by I/P injection of ECPs of L. anguillarum (0.2 ml/fish; 2.4 mg protein /ml) showed a 10% mortality rate, while those challenged after receiving specific anti ECPs (100 mg/ kg B.W. orally) showed no mortalities.

Monocytes/macrophages (MO/MФ) play an important role in the response to infection in Plecoglossus altivelis (ayu). However, the role of transcription factors in the function of ayu MO/MФ is poorly understood. Here, we cloned the cDNA sequence of the Kruppel-like factor 2 (PaKLF2) gene from ayu. Phylogenetic analysis indicated that PaKLF2 was closest to that of Atlantic salmon ( Salmo salar ). Real time quantitative PCR (RT-qPCR) revealed that the PaKLF2 mRNA level was highest in the peripheral blood mononuclear cells among all tested tissues. The mRNA expression of PaKLF2 was upregulated in the head kidney, liver, spleen, and brain after Listonella anguillarum infection. Subsequently, PaKLF2 was expressed and purified to prepare anti-PaKLF2 antibodies. After L. anguillarum challenge, the PaKLF2 mRNA and protein levels were significantly upregulated in ayu MO/MФ. Moreover, PaKLF2 knockdown in MO/MФ resulted in the enhancement of cytokine production as well as phagocytotic and bactericidal capability. Therefore, PaKLF2 may modulate the immune response in ayu by suppressing the function of MO/MФ. [ABSTRACT FROM AUTHOR]

Listonella anguillarum is a Gram-negative facultative anaerobic rod causing hemorrhagic septicemia in marine and rarely in freshwater fish. Succinate dehydrogenase (SDH) plays an important role in the tricarboxylic acid (TCA) cycle by oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. Recent studies indicate that central metabolic pathways, including the TCA cycle, contribute to bacterial virulence. However, the role of SDH in L. anguillarum virulence has not been studied. Here, we report in-frame deletion of the succinate dehydrogenase iron-sulfur protein (SDHB) and its role in L. anguillarum virulence in rainbow trout. To accomplish this goal, upstream and downstream regions of the L. anguillarum sdhB gene were amplified in-frame and cloned into a suicide plasmid. The chromosomal sdhB gene of L. anguillarum was deleted by homologous recombination. Virulence and immunogenicity of the L. anguillarum Δ sdhB mutant ( La Δ sdhB ) were determined in rainbow trout. Results show that La Δ sdhB was highly attenuated in rainbow trout, and fish immunized with La Δ sdhB displayed high relative survival rate after exposure to wild type L. anguillarum . These findings indicate SDH is important in L. anguillarum virulence in rainbow trout, and La Δ sdhB could be used as an immersion, oral, or injection vaccine to protect rainbow trout against vibriosis. [ABSTRACT FROM AUTHOR]

The research was aimed to assess the effect of dietary carvacrol (0.025% and 0.05%) on sea bass ( Dicentrarchus labrax) growth, immune response and resistance to Listonella anguillarum. Fish (69.2 ± 0.22 g) were fed the experimental diets for 9 weeks. Dietary carvacrol did not negatively affect fish survival, growth performance, feed intake and feed conversion ratio ( P > 0.05) nor carcass yield and viscerosomatic, hepatosomatic and mesenteric fat index ( P > 0.05). Serum and head kidney leucocytes were collected after 1, 4 and 8 weeks of feeding. Carvacrol significantly reduced serum proteins, immunoglobulins and lysozyme activity ( P < 0.01) and moderately increased phagocytosis and pinocytosis of head kidney macrophages. The release of reactive oxygen species by leucocytes was reduced in carvacrol-fed fish, even if significantly ( P < 0.05) only in those fed 0.05% carvacrol for 1 week. Dietary carvacrol did not significantly affect the aspecific immune response, although a potential antioxidant activity might be speculated. Moreover, feeding carvacrol provided an appreciable resistance to a challenge with L. anguillarum, when a bacterial dose lower than the Lethal Dose50 was used. Cumulative mortality in fish fed 0.025% carvacrol was significantly lower than that of untreated controls (75% Relative Per cent Survival). [ABSTRACT FROM AUTHOR]

Heavy metal pollution can affect the immune capability of organisms. We evaluated the effect of cadmium (Cd) on the defense responses of the Pacific oyster Crassostrea gigas to Listonella anguillarum challenge. The activities of several important defensive enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), acid phosphatase (ACP), Na, K -ATPase in gills and hepatopancreas, and phenoloxidase-like (POL) enzyme in hemolymph were assayed. In addition, the expression levels of several genes, including heat shock protein 90 (HSP90), metallothionein (MT), and bactericidal/permeability increasing (BPI) protein were quantified by fluorescent quantitative PCR. The enzyme activities of SOD, ACP, POL, and GPx in hepatopancreas, and the expression of HSP90 were down-regulated, whereas GPx activity in the gill, Na, K-ATPase activities in both tissues, and MT expression was increased in Cdexposed oysters post L. anguillarum challenge. However, BPI expression was not significantly altered by co-stress of L. anguillarum infection and cadmium exposure. Our results suggest that cadmium exposure alters the oysters' immune responses and energy metabolism following vibrio infection. [ABSTRACT FROM AUTHOR]

Heat shock proteins 70 kDa (HSP70) and apoptosis were investigated in thymus of sea bass juveniles ( Dicentrarchus labrax) subsequently to a vaccination against Listonella (syn. Vibrio) anguillarum. HSP70 expression was measured by immunohistochemistry and immunoenzymatic methods, resulting in increase in HSP70 after bath immunization and persistent in fish exposed to an intraperitoneal (i.p.) booster. The HSP70 increase in thymus was suggested as induction in lymphocytic cells, to be related to immune system stimulation after vaccination. However, a thymic recruitment of lymphocyte subpopulations, characterized by higher expression of HSP70, was also hypothesized after vaccination. No apparent relationships were found between HSP70 and apoptosis. In fact, the vaccination did not modulate the apoptosis response, as measured by TUNEL assay and by immunohistochemistry for active caspase-3 expression. The lack of apoptosis effects could be ascribed to the use of inactivated bacteria that appeared not able to interfere with programmed cell death mechanisms. This manuscript aims to contribute to the knowledge of some biochemical features underlying the immunization, with a particular emphasis on the modulation of HSP70. However, further parameters involved in innate/adaptative immunity and apoptosis pathways have to be taken into account to well establish the functional role of HSP70 in fish vaccination. [ABSTRACT FROM AUTHOR]

Abstract: The effect of vaccination on immune parameters of European sea bass, Dicentrarchus labrax, is not fully established, as well as surveyed throughout rearing till the commercial size. Furthermore, available information on the possible role of booster treatments is scarce. Sea bass juveniles were vaccinated against Listonella anguillarum using a commercial bivalent formulation administered by immersion (priming: 95dph; booster: 165dph) or by immersion (priming: 95dph; booster: 165dph) and subsequent i.p. injection (booster: 233dph). Serum specific IgM and numbers of IgM+ cells in head kidney and spleen evidenced B-cell responses mainly after the immersion booster, accompanied by increased TcR-β transcripts and leucocyte respiratory burst. Immune enhancement was confirmed by the protection towards i.p. challenges with a virulent strain. RPS accounted for >70% in fish immersion-boosted and near 100% in fish further boosted i.p. Differently from usual farm practices, this innovative vaccination protocol proved to be highly effective. Booster treatments are therefore strongly recommended. [Copyright &y& Elsevier]

Abstract: The effect of Coriolus versicolor extract supplemented diets on innate immune response and disease resistance in kelp grouper, Epinephelus bruneus against Listonella anguillarum, is reported. Kelp grouper were divided into four groups of 25 each and fed with C. versicolor enriched diets at 0% (control), 0.01%, 0.1%, and 1.0% level. After 30 days of feeding, all fish were injected interaperitoneally (i.p.) with 50 μl of L. anguillarum (4.7 × 107 CFU) to investigate the immune parameters at weeks 1, 2, and 4. The reactive oxygen species and reactive nitrogen species production were significantly enhanced in fish fed with 0.1% and 1.0% supplementation diets from weeks 1–4 when compared to the non enriched diet fed and infected control. The phagocytic activity significantly increased with 0.1% and 1.0% diets on weeks 2 and 4. The leucocyte myeloperoxidase content, lysozyme activity, and total protein level significantly increased when fed with 0.1% and 1.0% supplementation diets from weeks 1–4. The cumulative mortality was 35% and 45% in 1.0% and 0.1% enriched diet fed groups whereas it was 55% and 80% in 0.01% and 0% groups respectively. The present results suggest that diets enriched with C. versicolor at 0.1% or 1.0% level positively enhance the innate immune system and affords protection from L. anguillarum. [Copyright &y& Elsevier]

Abstract: Suppressive subtractive hybridization (SSH) was employed to identify differentially expressed genes in ayu (Plecoglossus altivelis) associated with Listonella anguillarum infection. 800 random clones were selected from forward and reverse subtractive libraries and 787 were successfully sequenced. After assembling, 105 contigs and 414 singletons were finally obtained, some of which were immune-related genes. A real-time quantitative PCR (RT-qPCR) analysis of the expression patterns of 28 transcripts showed that the false-positive rate was approximately 7.1%. Furthermore, Wap65-2 was overexpressed in Escherichia coli, purified and used for antiserum preparation. Western blot analysis revealed that serum Wap65-2 of ayu significantly increased after bacterial infection, suggesting that it was a positive acute-phase protein (APP). [Copyright &y& Elsevier]

Abstract: The anti-lipopolysaccharide factor (ALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724bp, consisting of an open reading frame (ORF) of 363bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53–65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional α-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonella anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris, indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture. [Copyright &y& Elsevier]

Abstract: The health effects of the flame retardant polybrominated diphenyl ethers (PBDEs) in fish are not well understood. To determine the potential effects of this ubiquitous contaminant class on fish health, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha) were fed a diet that reflected the PBDE congeners found in the stomach contents of subyearling Chinook salmon collected from the highly urbanized and industrialized lower Willamette River in the Columbia River Basin of North America. The diet, consisting of five PBDE congeners (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154), was fed to the salmon at 2% of their body weight in food per day for 40 days. Two concentrations of the diet (1× and 10× PBDE) were fed to the salmon. The 1× PBDE diet reflected the concentration of PBDEs (190ng PBDEs/g food) found in the stomach contents of juvenile subyearling Chinook salmon; the 10× diet was prepared at 10 times that concentration. The fish were then exposed to the marine bacterial pathogen Listonella anguillarum to assess susceptibility to infectious disease. Juvenile Chinook salmon fed the 1× PBDE diet were more susceptible to L. anguillarum than salmon fed the control diet. This suggests that juvenile salmonids in the lower Willamette River exposed to PBDEs may be at greater risk for disease than nonexposed juvenile salmonids. In contrast, salmon that consumed the 10× PBDE diet were not more susceptible to the pathogen than salmon fed the control diet. The mechanisms for the dichotomous results observed in disease susceptibility between salmon fed the 1× and 10× PBDE diets are currently not known but have also been observed in other species exposed to PBDEs with respect to immune function. [Copyright &y& Elsevier]

Abstract: Vaccines are commonly used in salmonid aquaculture as a method of disease prevention. Although there is a substantial amount of published research regarding the immunological and physiological effects following the injection of different polyvalent vaccines and DNA vaccines, there are no published reports examining the physiological and immunological effects of concurrent vaccine injection, which is the situation encountered in aquaculture. Using key immunological parameters such as lysozyme activity and specific antibody titres we examined the short-term activation of the immune response of cultured Atlantic salmon (Salmo salar L.) following concurrent injection with a traditional, polyvalent, oil-adjuvanted vaccine (AV) and an IHNV-specific DNA vaccine (DV). Our results indicate that different aspects of the innate and adaptive immune responses are influenced in either a positive or negative manner. While concurrent vaccine injection elicited an increase in lysozyme activity, changes in antibody titre (Ab) were antigen specific. The production of anti-Aeromonas salmonicida Abs was significantly greater in the combined vaccine group at 296 degree days post-vaccine injection (dd pvi), while the production of anti-Listonella anguillarum Abs was significantly greater at 106 dd pvi in the combined vaccine group. Of even greater interest was the apparent delay in production of IHNV-specific neutralizing antibodies (NAb) when the DV was injected concurrently with the polyvalent AV. The results indicated that concurrent injection of a polyvalent oil-AV and a DV can be beneficial to the production of antibodies; however, the specific anti-viral response may be delayed. [Copyright &y& Elsevier]

Abstract: Listonella anguillarum 90-11-287 and Vibrio splendidus DMC-1 were incorporated in the rotifer Brachionus plicatilis, which was subsequently maintained under larval rearing conditions to determine the residence time of both pathogens in rotifers. Real-time PCR was applied to specifically detect and quantify both pathogens. L. anguillarum colonized rotifers more efficiently than V. splendidus and both pathogenic strains were released from rotifers to seawater, after infected rotifers were transferred to rearing tanks. V. splendidus grew and became predominant in the seawater of tanks. Both pathogens remained in rotifer or seawater enough time to infect fish larvae, but their different behaviour could determine different infection patterns, preferentially by ingestion of prey or by active intake or contact with surrounding seawater. The effect of L. anguillarum and V. splendidus on the bacterial community associated with rotifers and seawater of rearing tanks was analysed by DGGE of PCR-amplified 16S rDNA fragments. The bacterial community of rotifers did not present a marked species dominance. The incorporation of L. anguillarum or V. splendidus did not reduce bacterial diversity and shifts could be explained by bacterial exchange between rotifers and seawater. Main bacterial groups were identified by sequencing the DNA extracted from Marine Agar (MA) bacterial isolates and DGGE excised bands. Only 2 DGGE bands corresponded to bacteria isolated from MA plates, suggesting that bacterial groups present in rotifers may not be easily cultivable. The bacterial community of rotifers was composed by Gram negative bacteria belonging to α-Proteobacteria (Ruegeria spp), γ-Proteobacteria (Alteromonas alvinellae, Marinobacter sp, an Oceanospirillaceae bacterium and Pseudoalteromonas sp), Cytophaga–Flexibacter–Bacteroides group (Polaribacter dokdonensis, Roseivirga spongicola and Tenacibaculum soleae) and a Gram positive bacterium (Microbacterium sp). [Copyright &y& Elsevier]

Abstract: The suppressors of cytokine signaling 2 (SOCS2) has been identified as negative feedback inhibitors for various cytokines signaling via the JAK/STAT pathway. In the present studies, the cDNA of Eriocheir sinensis SOCS2 (designated as EsSOCS2) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsSOCS2 was of 2535bp, consisting of an open reading frame (a ORF) of 1071bp encoding a polypeptide of 357 amino acids. The deduced amino acid sequence of EsSOCS2 shared 55–62% similarity with other SOCS2 family members. There were three typical conserved SOCS family domains in EsSOCS2, including an N-terminal ESS formed from a single amphipathic helix, a central SH2 domain with a classic phosphotyrosine (pY) site and a C-terminal SOCS box. The sequence and structural similarity of EsSOCS2 with SOCS2 proteins from other organisms indicated that EsSOCS2 should be a new member of the SOCS2 family. Phylogenetic analysis revealed that EsSOCS2 was clustered with SOCS2 from the other invertebrates, and fell into the group of type II SOCS subfamily as a sister branch to CIS and SOCS2 from vertebrate, suggesting the great divergence of SOCS2 of vertebrate from invertebrate and complex evolution of SOCS2 family members. The mRNA transcript of EsSOCS2 could be detected by semi-quantitative RT-PCR in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad. The mRNA expression of EsSOCS2 in haemocytes was up-regulated to 3.5-fold at 8h after Listonella anguillarum challenge, 3-fold and 3.5-fold at 4 and 6h, respectively, after Micrococcus luteus challenge. These results collectively suggested that EsSOCS2 could be induced by bacteria challenge, and it was involved in the immune defense responses in E. sinensis. [Copyright &y& Elsevier]

Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of <1.0×105 colony forming units (CFU) mL−1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay (IFA) was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature. [ABSTRACT FROM AUTHOR]

The present investigation determined the antibacterial activities in the sera of both cultured and wild Atlantic cod Gadus morhua. Serum samples from both groups of fish were quantified for total protein, and their effects against fish pathogens Listonella anguillarum, Aeromonas salmonicida and Yersinia ruckeri were determined using co-incubation assay. Total serum protein concentrations were not significantly different between farmed and wild Atlantic cod. Sera of cultured Atlantic cod significantly decreased growth of L. anguillarum by at least two-log10 reductions in bacterial count, while those of the wild Atlantic cod were able to inhibit the growth of all three fish bacterial pathogens. The present study showed that sera from wild fish possess broader antibacterial activities than cultured Atlantic cod and that these could provide an explanation for the differences regarding their immunity to bacterial infections as well as their health status. [ABSTRACT FROM AUTHOR]

Abstract: Serum-mediated reduction in bacterial count and expression of a number of immune response genes in the blood of Atlantic cod, Gadus morhua were investigated following intraperitoneal vaccination with heat-killed Listonella (Vibrio) anguillarum. Blood was collected from the caudal vein of both vaccinated and non-vaccinated (PBS-injected) fish at 0, 1, 3, 7 and 10 days post-vaccination (dpv). Serum protein concentration and antibacterial activity of the serum samples were determined. Whole blood was used for semi-quantitative RT-PCR of immune-related genes. Total serum protein was not significantly different between the vaccinated and non-vaccinated groups. Sera from the vaccinated fish significantly reduced L. anguillarum count on 3dpv, with reductions of at least 2 log colony forming units per ml (CFU/ml) relative to the non-vaccinated fish. Expression of antibacterial genes, bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin was significantly upregulated in the vaccinated fish, with maximum expression within 7dpv. Cytotoxic-related and cell-mediated immunity genes such as, apolipoprotein A-I and the non-specific cytotoxic cell receptor protein (NCCRP-1) had maximum expression at 3 and 7dpv, respectively. Significant upregulation in expression of pro-inflammatory cytokines, IL-1β and IL-8 was also observed in the vaccinated fish at 1dpv. The upregulation of immune response genes following vaccination provides valuable information in the understanding of immune mechanisms against vibriosis in Atlantic cod particularly on the acute phase response during bacterial infection. [Copyright &y& Elsevier]

Abstract: Microbially-derived diseases cause economic losses in aquaculture on global scale. Since many marine invertebrates and plants have evolved potent defence mechanisms against microbial epibionts, such as bacteria, viruses and fungi, there is increasing interest in these organisms as a source for natural antifouling and antibacterial substances. In this context, marine macroalgae are considered as a promising source for new antimicrobial agents. In this study, we investigated antibacterial effects of hexane and methanol extracts of the macroalgae Mastocarpus stellatus, Laminaria digitata and Ceramium rubrum on 12 marine and 7 prominent fish pathogenic bacteria at ecologically realistic concentrations of macroalgal secondary metabolites. Antibacterial activity was determined with a highly sensitive growth inhibition assay that records the fluorescence intensity of stained bacterial DNA. The assay allowed the detection of bacterial growth inhibition at and below the tissue level concentration of algal metabolites. Tests were carried out with algae collected in November 2006 and in March 2007 to investigate seasonal variation of antibacterial activities. Methanol extracts of C. rubrum at 10 mg dry weight per ml and hexane extracts of L. digitata at 31 mg dry weight per ml evoked strong antibacterial activities and inhibited almost all tested bacteria. There was no clear distinction between antibacterial effects of algae collected in November 2006 and March 2007. The growth inhibition of bacteria by algal extracts was frequently higher than 100%. This result indicated that bacteria must have been lysed during the exposure to algal extract components. Therefore, the extracts presumably evoked not only a bacteriostatic but also bacteriolytic mode of action. The assays revealed different susceptibilities of the bacterial phyla under investigation to algal extracts. Gram-positive marine Bacillaceae were generally more susceptible than Gram-negative marine Vibrionaceae. Listonella anguillarum, Pseudomonas anguilliseptica and Aeromonas salmonicida were the most susceptible fish pathogenic bacteria. [Copyright &y& Elsevier]

Abstract: The goal of fish vaccination today is to protect fish against multiple bacterial fish pathogens simultaneously using polyvalent vaccines. However, many immunological processes such as antigenic cross-reaction, antigenic competition, affinity maturation and antigen-induced suppression may affect the specificity, avidity and level of antibodies. Consequently, the biological function of antibodies may be markedly different from that predicted by conventional serologic tests. Here, we investigated the effects of vaccination and composition of vaccine on the plasma antibody levels, biological function of antibodies in opsonophagocytosis as well as the effects of vaccination on the blood leucocyte counts. Rainbow trout were vaccinated with saline or with two different polyvalent, mineral oil-adjuvanted vaccines. Vaccine 1 contained Aeromonas salmonicida, Listonella anguillarum and both Th and Fd serotypes of Flavobacterium psychrophilum antigens and vaccine 2 contained A. salmonicida, L. anguillarum and only Fd serotype of Fl. psychrophilum. The antibody-mediated opsonophagocytosis was determined as the respiratory burst (RB) activity of blood monocytes and granulocytes against the tested bacterial antigens. Three weeks after vaccination both vaccine groups and the control group showed increased RB activity against all bacterial strains. However, the increase in RB activities was non-specific and originated from the increased number of circulating granulocytes and monocytes. On the other hand, at 6 weeks post-vaccination both specific antibodies and antibody-dependent opsonophagocytosis appeared in both vaccine groups. However, the composition of the vaccine had a marked effect on the magnitude of specific responses. The Fd+Th vaccine enhanced the target specific opsonophagocytosis, to a lesser extent than the Fd vaccine. Both polyvalent vaccines appeared to mainly affect the numbers of circulating monocytes and our results suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis. Our results also suggest that the presented opsonophagocytic assay is an advantageous method to predict vaccine efficiency and that the number, and properties, of bacterial antigens in polyvalent vaccines should be carefully selected in order to avoid inhibitory effects of antigens on the specific response of fish. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to investigate the distribution of Listonella anguillarum in a Japanese flounder (Paralichthys olivaceus) hatchery. A total of 2704 isolates were obtained from the developing fish, live diets and artificial feeds of Japanese flounder and their rearing water, 439 of which were identified as L. anguillarum by the combining incubation on thiosulfate-citrate-bile salt-sucrose (TCBS) agar at 35 °C overnight with polymerase chain reaction (PCR) detection for the VAH1 hemolysin gene. L. anguillarum was detected in all seven rotifer samples, with densities of 2.5×103 to 4.6×106 colony forming units (CFU) g−1. Both the analyzed samples of Nannochloropsis oculata contained this bacterium at densities of 1.6×104 to 1.4×105 CFU g−1. L. anguillarum was detected in only one of four samples of Artemia nauplii with a density of 4.8×105 CFU g−1 (35%) and it was not detected in the two analyzed artificial feed samples. L. anguillarum was detected in 11 of 18 specimens of larval and juvenile Japanese flounder at densities of 5.0×101 to 7.4×105 CFU g−1, while it was not detected in the two analyzed egg specimens of Japanese flounder. These results indicate that L. anguillarum associated with the developing Japanese flounder is likely derived from rearing water and live diets such as rotifers. Further, it is strongly suggested that L. anguillarum is a transient bacterium of the intestinal microflora for the Japanese flounder but is a permanently indigenous one for the Japanese flounder hatcheries. [Copyright &y& Elsevier]

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum, an important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic effect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates significantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized 108 CFU g−1 of this isolate for 15 days. An other group of similar characteristics received the non-supplemented commercial diet. After the challenge, the mortality of the fish receiving the diet supplemented with the potential probiotic was significantly lower than that observed in the groups of fish receiving the non-supplemented commercial diet. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the distribution of Listonella anguillarum in the rearing water, fish and diets (rotifers) of Japanese flounder ( Paralichthys olivaceus). A total of 793 isolates were obtained from the seed production environment of Japanese flounder and 175 out of them were identified as L. anguillarum by biochemical characterization, polymerase chain reaction (PCR) detection for VAH1 haemolysin gene and phylogenetic analysis of 16S ribosomal deoxyribonucleic acids (rDNA) sequences. These results strongly suggested that L. anguillarum is rapidly and accurately identified by the combination of incubation on thiosulphate–citrate–bile salt–sucrose agar at 35°C overnight and PCR detection for the VAH1 haemolysin gene. All flounder specimens and all rotifer samples harboured L. anguillarum at high densities of 6.9× 103–6.3× 105 colony forming units (CFU) g−1 and 1.5× 104–2.3× 106 CFU g−1, respectively, while as low as 5.0× 100–2.0× 101 CFU mL−1 of L. aguillarum were detected in only two of 11 seawater samples, even though no vibriosis occurred in larval and juvenile flounder of tanks. This fact strongly suggests that L. anguillarum is an inhabitant in the seed production environments of Japanese flounder. [ABSTRACT FROM AUTHOR]

Occurrence of Listonella anguillarum-like bacteria was monitored in grow out ponds of the black tiger shrimp Penaeus monodon in 1996 and 1997. L. anguillarum-like bacteria were determined in source seawater and sediment, pond water and sediment, and in the shrimp tissue. L. anguillarum-like bacteria density was lower in source seawater than in shrimp culture pond water. Water, sediment, and shrimp tissue in the probiotic-treated ponds contained lower L. anguillarum-like bacteria densities than those in the untreated (control) ponds. Forty different isolates of L. anguillarum-like bacteria were identified, and their antibiotic sensitivity, pathogenicity, and plasmid profiles were determined. Pathogenicity experiments revealed that LD50 values of 9×105 and 9×104 colony forming units (CFU) ml−1 of L. anguillarum-like bacteria produced mortality of intramuscular injected P. monodon and Penaeus indicus, respectively. Antibiotic resistance studies showed that 40 isolates of L. anguillarum-like bacteria were 100% resistant to Ampicillin, 80% to Chlortetracycline, and sensitive to Erythromycin (60%) and Gentamycin (70%). Plasmid profile analysis revealed that among the 40 isolates of L. anguillarum-like bacteria, only seven harboured plasmids ranging from 1.8 to 18.5 kb in size; the remaining 33 were devoid of plasmids. [Copyright &y& Elsevier]

Mx genes are inducible by Type I interferons and are involved in antiviral defences. A commercially available vibrio bacterin, intended for immersion vaccination, was shown to be a potent inducer of Mx gene expression in Atlantic salmon parr following intraperitoneal injection. The response was dose and temperature dependent. At 10 °C and 10 times concentration the bacterin induced Mx response kinetics similar to that induced by poly I:C.At 10 °C, enhanced Mx responses were detected from days 1 to 9 with both 1 times (1×) and 10 times (10×) concentrated bacterin, with a tendency for a higher response to the concentrated bacterin on days 1 and 3. Basal levels of Mx mRNA were detected on day 12 after injection to both concentrations. The response induced by poly I:C was higher on day 1 and it was still present at day 12, with basal levels being reached on day 18. At 6 °C, there was a more definitive dose effect of the vibrio bacterin and the Mx response was delayed in comparison to that at 10 °C. Increased Mx expression did not appear until day 6 and with the 1× dose it had disappeared by day 9. However, the 10× dose continued to induce Mx at day 12, disappearing by day 18.The Mx response to the purified Listonella anguillarum lipopolysaccharide (LPS) and DNA in fish held at 10 °C showed some differences in the rate of onset. The response to DNA was faster, beginning on day 1 compared with day 3 for the LPS. The response to DNA peaked on day 3 while for LPS the peak was on day 9. However, the response to both components had disappeared by day 12. The response kinetics to the L. anguillarum DNA was essentially similar to the 10× dose of the vibrio bacterin and to poly I:C at 10 °C. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: A more rapid and powerful response against repeated exposure of same pathogen in vertebrates is usually considered as the reflection of immunological memory, but it is not well understood in invertebrates. In the present study, the temporal expression profiles of Chlamys farreri peptidoglycan recognition protein-S1 (CfPGRP-S1) gene after two challenges of Listonella anguillarum were examined to evaluate priming response in scallops. The up-regulation of CfPGRP-S1 mRNA occurred 3h earlier, and the expression level was significant higher (P <0.05), after the second challenge than that after the first challenge. The preliminary results provided new insights into invertebrate immunological memory, and they also would be helpful to develop strategies for disease control. [Copyright &y& Elsevier]

The article focuses on the study conducted for the Listonella anguillarum, a Vibrionaceae bacterium and is responsible for causing vibriosis in numerous marine and freshwater fish in Japan. Accordingly, the study shows that these bacteria normal attack and inhabits during the seed production of the marine species. It notes that the member of the Vibrionaceae are normally seen in the intestinal tracts of marine fish thus it presumes that Listonella anguillarum is also widely distributed in the intestinal tract of wild marine species. Moreover, the details of the said study are also presented, along with the data gathered during the investigation.