Screening and characterisation of potentially pathogenic bacteria associated with Atlantic cod Gadus morhua larvae: bath challenge trials using a multidish system

In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10(6) and 10(4) CFU ml(-1). Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 106 CFU ml(-1), only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.     

Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae

Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 10(7) cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect.     

宁海地区香鱼弧菌病病原菌鉴定

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害,然而,病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌;采用回归感染试验确认病原菌,采用改进的寇氏法计算LD50;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌,它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明,ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明,细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高,为99.4%～99.5%,与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%;ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %,与其它弧菌科成员则低于75.6 %,系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Intraperitoneal vaccination of Atlantic cod, Gadus morhua with heat-killed Listonella anguillarum enhances serum antibacterial activity and expression of immune response genes

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 3 dpv, 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 7 dpv. 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 7 dpv, respectively. Significant upregulation in expression of pro-inflammatory cytokines, IL-1 beta and IL-8 was also observed in the vaccinated fish at 1 dpv. 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.     

Phage Therapy as an Approach to Prevent Vibrio anguillarum Infections in Fish Larvae Production

Fish larvae in aquaculture have high mortality rates due to pathogenic bacteria, especially the Vibrio species, and ineffective prophylactic strategies. Vaccination is not feasible in larvae and antibiotics have reduced efficacy against multidrug resistant bacteria. A novel approach to controlling Vibrio infections in aquaculture is needed. The potential of phage therapy to combat vibriosis in fish larvae production has not yet been examined. We describe the isolation and characterization of two bacteriophages capable of infecting pathogenic Vibrio and their application to prevent bacterial infection in fish larvae. Two groups of zebrafish larvae were infected with V. anguillarum (∼10⁶ CFU mL⁻¹) and one was later treated with a phage lysate (∼10⁸ PFU mL⁻¹). A third group was only added with phages. A fourth group received neither bacteria nor phages (fish control). Larvae mortality, after 72 h, in the infected and treated group was similar to normal levels and significantly lower than that of the infected but not treated group, indicating that phage treatment was effective. Thus, directly supplying phages to the culture water could be an effective and inexpensive approach toward reducing the negative impact of vibriosis in larviculture.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

蟹栖异阿脑虫寄生大菱鲆及其组织病理学研究

论文首次报道了蟹栖异阿脑虫(Mesanophrys carcini)在养殖大菱鲆体内的寄生情况以及所引起宿主的组织病理学变化。观察结果表明,感染的苗期大菱鲆的大部分器官组织包括皮肤、鳍、鳃、眼、脑、心脏、肝脏、肾脏、脾脏、胰腺和消化道等以及器官组织间隙中都发现了寄生的蟹栖异阿脑虫,属于全身性感染;而对于成鱼,蟹栖异阿脑虫主要寄生于皮肤、鳍和鳃等器官组织;严重感染者,蟹栖异阿脑虫可侵入其脑、肝脏、肾脏和心脏等器官组织,但数量不多。蟹栖异阿脑虫在大菱鲆体内的寄生能够引起病鱼各器官组织发生不同程度的组织病理学改变。该病在幼鱼和成鱼身上所表现出较一致的病理学特征为:鳃小叶呼吸上皮细胞增生;脑膜炎和脑组织液化性坏死;肾脏和脾脏造血组织坏死;肝脏中央静脉和窦状隙扩张;肌纤维发生凝固性坏死和眼的脉络膜充血、水肿;单核巨噬细胞浸润。研究认为,蟹栖异阿脑虫寄生引起的鳃损害使病鱼呼吸困难,窒息是病鱼发生死亡的主要原因,而虫体寄生导致的各器官组织变性,坏死,使得病鱼器官功能衰竭可加速鱼的死亡。另外,文中还探讨了蟹栖异阿脑虫入侵大菱鲆的可能途径,认为破损的皮肤和鳃是入侵的主要途径。研究结果告诉我们,在大菱鲆养殖过程中防止鱼体擦伤,保持池底清洁对于预防疾病的发生非常重要;对该病的治疗应采取外部杀虫和内部口服药物相结合的方式。     

Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua)

A growing concern for the high consumption of antibiotics in aquaculture has initiated a search for alternative methods of disease control. Improved resistance against infectious diseases can be achieved by the use of probiotics. Probiotics are live microorganisms supplemented in food or feed which give beneficial effects on the intestinal microbial balance. In the present study a dry feed containing lactic acid bacteria (Carnobacterium divergens) isolated from Atlantic cod (Gadus morhua)intestines was given to cod fry. After three weeks of feeding the fry was exposed to a virulent strain of Vibrio anguillarum. The death rate was recorded during further three weeks of feeding with lactic acid bacteria supplemented feed. A certain improvement of disease resistance was obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in surviving fish given feed supplemented with lactic acid bacteria. No obvious growth inhibition of V. anguillarum was observed in an in vitro mixed culture of this bacterium and the C. divergens isolated from cod intestines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe

16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence.     

Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe.

16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence.     

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.     

Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections

Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.     

The probiotic potential against vibriosis of the indigenous microflora of rainbow trout

The antibacterial properties of the indigenous microflora of rainbow trout ( Oncorhynchus mykiss Walbaum) and the potential use of inhibitory bacteria as fish probiotics were investigated. A total of 1018 bacteria and yeasts were isolated on tryptone soy agar (TSA) from skin, gills and intestine. Forty-five of these inhibited growth of the fish pathogenic bacterium Vibrio anguillarum in a well diffusion assay. The antagonism was most prominent among Pseudomonas spp., as 28 (66%) of the antagonistic bacteria belonged to this genus, despite constituting only 15% of the total tested flora. As pseudomonads are typically siderophore producers, chrome azurol S (CAS) agar was used as a semi-selective medium for isolation of antagonistic bacteria. On this medium, 75% of the iron-chelating strains were inhibitory to V. anguillarum . Eight strains out of a subset of 11 antagonists caused a 3–6 log unit reduction in the density of V. anguillarum [measured by polymerase chain reaction (PCR) detection in a most probable number (MPN) regimen] in a broth co-culture assay. Survival of rainbow trout infected with vibriosis was improved 13–43% by six out of nine antagonistic strains tested in vivo. All disease-protecting strains were pseudomonads, isolated from CAS plates, whereas two Carnobacterium spp. that were antagonistic in in vitro well diffusion assays did not alter the accumulated mortality of rainbow trout. The addition of live bacterial cultures to fish-rearing water may thus improve survival of the fish; however, in vitro antagonism could not completely predict an in vivo effect. Further studies on the underlying mechanism of activity are required to design appropriate selection criteria for fish probiotic bacteria.     

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.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species.

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Routes of entry of Piscirickettsia salmonis in rainbow trout Oncorhynchus mykiss.

Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has killed millions of farmed salmonids each year in southern Chile. The portal of entry for the pathogen was investigated by use of selected experimental infections in juvenile rainbow trout (12 g). The methods used were intraperitoneal injection, subcutaneous injection, patch contact on skin, patch contact on gills, intestinal intubation and gastric intubation. Cumulative mortalities at Day 33 post-inoculation were 98, 100, 52, 24, 24, and 2%, respectively. It was shown that intact skin and gills could be penetrated by P. salmonis. The high mortality obtained in subcutaneously injected fish indicated that skin injuries could facilitate the invasion of this pathogen. Results suggested that the main entry sites are through the skin and gills and that the oral route may not be the normal method by which P. salmonis initiates infection of salmonids.     

Diseases and Injuries Associated with Mortality of Hatchery Reared Baltic Cod (Gadus morhua L.) Larvae

A cod hatching plant was established in 1992 on the island of Bornholm in the Baltic Sea in order to elucidate the possibilites for restocking of cod fry in this brackishwater system. The disease prevalence in 3 batches of hatchery-reared yolksac larvae from the Baltic cod (Gadus morhua L.) was monitored during the posthatch period. High prevalences of bacteriosis/mycosis, lordosis/scoliosis, injuries and protozoan endoparasitism were recorded. Vibrio sp. and Vibrio anguillarum serovar 04, 06, 08 in addition to nontypable strains and saprolegniaceous fungi were isolated from the larvae. The dinoflagellate-like endoparasites were located in the yolksac of the cod larvae.     

Copper as an initiating factor of vibriosis (Vibrio anguillarum) in eel (Anguilla anguilla)

Eels ( Anguilla anguilla ) which were exposed to copper-contaminated fresh water (30–60 ρg Cu/I) died with signs of vibriosis ( Vibrio anguillarum). Eels kept in non-contaminated fresh water (<6ρgCu/I) remained healthy. V. anguillarum was shown to be present in the eels with symptoms of vibriosis. We suggest V. anguillarum is a common inhabitant of eels and copper can change a commensal association between fish and bacterium to one of pathogenicity.