Inhibition of vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish

To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain Pseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect of P. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth of V. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not. P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1, 000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykiss Walbaum) to P. fluorescens AH2 at a density of 10(5) CFU/ml for 5 days before a challenge with V. anguillarum at 10(4) to 10(5) CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 10(7) CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12 degrees C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

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.     

Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.     

Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae

A study was conducted to determine the role of specific antibodies in immunity to Streptococcus agalactiae. Adult Nile tilapia (Oreochromis niloticus) were injected i.p. with tryptic soy broth as control or with S. agalactiae vaccine. Ninety days later, fish were challenged with 1.5x10(4)CFUS. agalactiae fish(-1). Blood was drawn from all fish 90d after vaccination and 25d after challenge, and the acquired serum was injected i.p. in fingerling Nile tilapia. These passively immunized fish were subsequently challenged 72h later with 1.5x10(4)CFUS. agalactiae fish(-1), and significantly less (P<0.0001) mortalities were noted among fish administered serum containing specific anti-S. agalactiae antibodies (0.0-10.0% mortalities) than in control groups (63.3-72.7% mortalities). Heat-inactivation of serum produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P<0.9455) or vaccinated-challenged fish (P<0.0781). Pre-challenge serum samples indicate that the passively immunized fish had significantly increased (P<0.0001) specific antibody levels over control fish. A highly significant (r(2)=0.5892; P<0.0001) correlation between increased pre-challenge specific serum antibody OD levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results of this study indicate that specific anti-S. agalactiae antibodies play a primary role in immunity to S. agalactiae in fish.     

Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and efficacy of protection induced by vaccination

Experimental bath challenge of juvenile and sub-adult Atlantic halibut with Vibrio anguillarum induced severe mortalities of 47 and 80%, respectively. However, animals vaccinated with a commercial V. anguillarum vaccine demonstrated excellent protection against the disease (100% RPS). This study also describes the gross pathology and histological changes associated with this infection. A loss of coordination, haemorrhage at the fin base and splenomegaly were frequent findings. Serum agglutinating activity demonstrated a rise following vaccination, the mean log2 titre rising from 3.8 to 8.4. This was associated with a significant rise in antibody-mediated complement killing ability of immune serum when compared to non-immune serum.     

Properties of Probiotics <Emphasis Type="Italic">Kocuria</Emphasis> SM1 and <Emphasis Type="Italic">Rhodococcus</Emphasis> SM2 Isolated from Fish Guts

This study characterized probiotics Kocuria SM1 and Rhodococcus SM2, which were recovered from the intestinal microbiota of rainbow trout (Oncorhynchus mykiss, Walbaum). The cultures were Gram-positive, non-motile, catalase-positive and oxidase-negative cocci or rods. Cell multiplication of SM1 and SM2 was observed at 4–37 °C (45 °C for SM1), in 0–20% (w/v) NaCl and at pH 2–11. The viability was not affected when exposed to pepsin at pH 2.0 and 3.0, and pancreatin at pH 8.0. Neither isolates were chrome azurol S-positive for siderophore production. Of the 19 common enzymes analysed using the API-ZYM system, only 8 were evident in the culture of SM1 compared to 11 enzymes for SM2. The secondary metabolites of both probiotics were inhibitory to Acinetobacter baumannii, Vibrio anguillarum and V. ordalii; SM2 inhibited Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. SM2 was resistant to penicillin and sulphatriad, out of six antimicrobial agents; SM1 was resistant to sulphatriad. These results suggest that Kocuria SM1 and Rhodococcus SM2 are able to grow over a wide range of temperature, salinity and pH, including in conditions that mimic the gastrointestinal environment of fish and produce extracellular enzymes that may have a role in the host digestive processes. Importantly, Rhodococcus SM2 displays a high degree of bacteriocinogenic potential against multi-drug-resistant human pathogens that have never been documented among the gut microbiota of fish.     

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.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Identification of differentially expressed genes in parasitic phase Miamiensis avidus (Ciliophora: Scuticociliatia) using suppression subtractive hybridization

A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg microl(-1) for V anguillarum, 500 fg microl(-1) for P. salmonis, and 5 pg microl(-1) for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 x 10(5) CFU ml(-1) of V anguillarum, 1.26 x 10(4) CFU m(-1) of S. phocae, and 5.33 x 10(4) CFU ml(-1) of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 +/- 0.54 x 10(7) CFU g(-1) for V. anguillarum, 9.03 +/- 1.84 x 10(5) CFU g(-1) for S. phocae, 3.8 +/- 0.78 x 10(3) CFU mg(-1) for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of -1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simultaneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.     

Immune responses of zebrafish (Danio rerio) induced by bath-vaccination with a live attenuated Vibrio anguillarum vaccine candidate

A fish vaccine candidate, live attenuated Vibrio anguillarum, which can protect fish from vibriosis, was established in our laboratory. In this study, the protective immunological mechanism of live attenuated V. anguillarum was investigated in zebrafish as a model animal. After bath-vaccinated with the live attenuated strain, zebrafish were challenged with wild pathogenic strain to test the immunoprotection of the live attenuated strain. As the results, specific antibody response of fish against V. anguillarum was found to gradually increase during 28 days post-vaccination, and remarkable protection was showed with a high relative protection survival (RPS) of about 90%. Moreover, the vaccination changed the expressions of several immune-related genes in the spleens and livers of zebrafish. Among them, the expressions of pro-inflammatory factors such as IL-1 and IL-8 were tenderly up-regulated with about 3-4 fold in 1-7 days post-vaccination, while MHC II rose to a peak level of 4-fold in 7th day post-vaccination. These results gave some important messages about the mechanism of specific protection induced by live attenuated V. anguillarum and showed the availability of zebrafish model in the evaluation of the vaccine candidate.     

环境因子对鳗弧菌生物膜形成的影响

【背景】鳗弧菌是海产动物弧菌病的主要病原,在海水水域中广泛存在。鳗弧菌为了适应环境变化会生成生物膜,形成自我保护,对其防治是水产养殖行业的一大难题。【目的】探讨致病性鳗弧菌(Vibrio anguillarum)BYK0638生物膜的形成特性,为进一步研究鳗弧菌生物膜形成机制和致病机理提供参考。【方法】采用改良的微孔板法研究静置培养条件下鳗弧菌(V.anguillarum)BYK0638在96孔酶标板上的成膜情况,CCK-8法(Cell counting kit-8)定量检测生物膜中鳗弧菌的活力。【结果】鳗弧菌BYK0638能够在聚苯乙烯酶标板上形成稳定而明显的生物膜,其生物膜的OD450值在24 h达到峰值,60 h后趋于稳定;在107-108 CFU/m L范围内,鳗弧菌生物膜的OD450值显著高于其他试验组(P0.05);25°C时的生物膜OD450值显著高于其他温度生物膜的形成量;在p H 4.0-11.0范围内,当p H值为7.0时鳗弧菌形成的生物膜量最高,在p H值为3.0和12.0时鳗弧菌几乎不形成生物膜;在TSB培养基中加入0.03-2.00 mmol/L Ca Cl2,鳗弧菌生物膜形成量与未添加Ca Cl2对照组无显著性差异;在TSB培养基中加入0.03 mmol/L Mg Cl2,可促进鳗弧菌生物膜形成;Na Cl浓度为5%时,形成的生物膜OD450值最高;鳗弧菌在大黄鱼表皮黏液、肝脏、前肠、后肠组织提取液包被的96孔酶标板上形成的生物膜显著高于其他黏液和组织提取液包被组(P0.05)。【结论】致病性鳗弧菌BYK0638能形成稳定而明显的生物膜,其生物膜形成与外界环境因子变化有密切的关系,培养时间、初始菌浓度、温度、p H、Mg2+、盐度及不同组织和黏液等各种环境因子均能显著影响鳗弧菌生物膜的形成。     

Effect of relaxin on facilitation of parturition in dairy heifers

Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2 ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2 mg, 12 h apart; Group 2R, N = 7). Intervals (mean +/- s.e.) between the first injection of relaxin or PBS and calving were 64 +/- 17, 80 +/- 19 and 125 +/- 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P less than 0.01) and 1R (P less than 0.05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P less than 0.01). Body weights and ratios of males to females of the calves were similar (P greater than 0.05) between groups. Progesterone plasma concentrations decreased (P less than 0.01) earlier in Groups 1R and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2.7 +/- 1.1 ng/ml for Group 2R, 3.5 +/- 0.9 ng/ml for Group 1R, and 6.0 +/- 0.1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 +/- 2.2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0.3 +/- 0.01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.     

Protective effect of herbal and probiotics enriched diet on haematological and immunity status of Oplegnathus fasciatus (Temminck & Schlegel) against Edwardsiella tarda

This study determines the effect of diet enriched with the herb Baical skullcap Scutellaria baicalensis, and/or probiotics Lactobacillus sakei BK19 in rock bream, Oplegnathus fasciatus (32 ± 3 g) against Edwardsiella tarda. The changes in haematological parameters, innate immune response, and disease resistance were investigated after 1, 3, and 6 weeks. The white blood cell count (WBC: 10(4) mm(-3)), red blood cell count (RBC: 10(6) mm(-3)), and haemoglobin (Hb: g dl(-1)) levels significant increased (P < 0.05) with mixed diet on 3rd and 6th week and probiotics enriched diet on 6th week. The haematocrit (Ht: %) level significantly increased (P < 0.05) when fed with mixed diet on weeks 1-6. Interestingly, in mixed diet group the lymphocytes (LYM), monocytes (MON), and neutrophils (NEU) significantly increased from week 1-6. The eosinophils (EOS) significantly increased in all the treated groups. In the probiotics or mixed diet groups the total protein (TP: g dl(-1)) increased significantly on weeks 3 and 6. The serum lysozyme activity significantly was enhanced in all the treated groups indicating an increase in the innate immunity level. Serum complement, antiprotease activities, reactive oxygen species (ROS) and reactive nitrogen species (RNS) production significantly increased from week 1-6 with mixed diet. The maximum protection against E. tarda was recorded in mixed diet group with a minimum cumulative mortality of 20% and a high relative percent survival (RPS) of 72.84. In the probiotics and herbal diet groups the cumulative mortality was 25% and 35% and RPS was 68.63 and 59.42, respectively. This study indicates that administration of probiotics or mixed diets can effectively minimize the mortality and restore the altered hematological parameters and enhancing the innate immunity in O. fasciatus against E. tarda.     

Detection of infection and susceptibility of different Pacific salmon stocks (Oncorhynchus spp.) to the haemoflagellate Cryptobia salmositica

The haematocrit centrifugation technique, modified by keeping the haematocrit tubes cold (between 1 and 10 C), was sensitive for detecting light infections of Cryptobia salmositica (as few as 75 flagellates per ml of blood). In wet mount preparations, infections lighter than 7.5 X 10(3) flagellates per ml of blood could not be detected consistently. Different Pacific salmon stocks from British Columbia demonstrated differences in susceptibility to C. salmositica in experimental studies using laboratory reared juvenile fish. Oncorhynchus keta and Oncorhynchus tshawytscha from the Big Qualicum River stocks (Vancouver Island), and Oncorhynchus nerka from the Fulton River stock (Skeena River system), were all equally susceptible and suffered high mortalities at low exposures (100 flagellates in 0.1 ml physiological saline inoculated intraperitoneally per fish). Oncorhynchus nerka from the Weaver Creek stock (Fraser River system) was the most resistant with no mortalities even at exposures of 10(6) flagellates (in 0.1 ml physiological saline) per fish. Oncorhynchus kisutch seemed to be slightly less resistant than the Weaver Creek O. nerka, but fewer than 16% of the inoculated fish died. Oncorhynchus kisutch from the Big Qualicum River seemed to be slightly more resistant than O. kisutch from the Capilano River stock (a coastal river near Vancouver), with fewer mortalities and lighter infections when the experiments were terminated. Differences in susceptibility are believed to be associated with innate, genetically transmitted resistance.     

Immune response of zebrafish (Danio rerio) against a newly isolated bacterial pathogen Aeromonas hydrophila

A strain of Aeromonas hydrophila associated with unusual mortalities in zebrafish (Danio rerio) culture facilities was isolated, identified and characterized. In challenge experiments, adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection with viable bacteria and its extracellular products (ECPs) reaching very high mortalities in a few hours. The infection, by the viable bacteria or the ECPs, caused cell death in kidney, due to the cytotoxic and haemolytic activities of the bacterial ECPs. Moreover, the infection affected the release of oxygen (ROS) and nitrogen (NO) reactive free radicals. To determine if this A. hydrophila infection induces an inflammatory response, mRNA expression levels of tumour necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and inducible nitric oxide (iNOS) were assessed by real time PCR. The expression levels of TNFalpha, IL-1beta and IFNgamma were upregulated in the kidneys of infected zebrafish with viable bacteria, heat-killed bacteria and ECPs. Expression levels of iNOS were upregulated by ECPs. Mortality rate (LD(50)) and histopathology were also determined.     

Specific and natural antibody response of cod juveniles vaccinated against Vibrio anguillarum

The purpose of the present study was to study specific and natural antibody levels in individual cod juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and the performance of different groups to bath challenge by the bacterium tested. Antibody responses to V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant antibody response. Both groups showed increased levels of natural antibodies while levels were low in other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while fish that received a second vaccination showed the best protection. It was not ascertained whether there is a long term difference between the effects of immersion versus i.p. injection as a booster method. Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest mortalities show that neither could have been used to predict protection given by the vaccines tested.     

Efficacy of orally administered flumequine in the treatment of vibriosis caused by Listonella anguillarum in atlantic cod Gadus morhua

The efficacy of orally administered flumequine in the treatment of experimentally induced vibriosis in Atlantic cod Gadus morhua was investigated. Cod (mean +/- SD, 120 +/- 30 g) were randomly distributed to twelve tanks and bath challenged for 1 h with Listonella anguillarum serotype O2alpha, strain HI-610, using a dose of 9.2 x 10(6) CFU ml(-1). At 3 d post-challenge, medication was introduced in 10 of the groups at doses of 2.5, 5, 10, 15 and 25 mg flumequine kg(-1) body weight d(-1) in duplicate. The medication was administered on Days 1, 2, 4, 6, 8, and 10 after the initiation of treatment. In challenged unmedicated fish, mortality started on Day 4 post-challenge, reaching a final cumulative mortality of 82% at Day 18. In the medicated groups, mortality started on Days 3 to 5 post-challenge, reaching final cumulative mortalities of 42, 49, 37, 37 and 23% respectively for the fish treated with 2.5, 5, 10, 15 and 25 mg flumequine kg(-1) body weight d(-1). Survival of medicated fish in all groups was significantly greater than in challenged unmedicated fish (p < 0.001). Twenty-four h following the final medication, HPLC analysis found a linear relationship between doses and mean concentrations of the drug in plasma, muscle and liver.     

Infectivity of a Scottish isolate of Piscirickettsia salmonis for Atlantic salmon Salmo salar and immune response of salmon to this agent

A Scottish isolate of Piscirickettsia salmonis (SCO-95A), previously shown by intraperitoneal injection to have a lethal dose (LD50) of < 2 x 10(3) infectious rickettsial units, was tested for virulence by bath challenge, surface application to the skin, or dorsal median sinus injection. Atlantic salmon Salmo salar post-smolts were used in all experiments, and exposure to 1 x 10(5) tissue culture infective doses (TCID) of P. salmonis ml(-1) for 1 h in a bath challenge resulted in only 1 mortality, 18 d later, in 10 exposed fish. Application of 2.5 x 10(6) TCID of P. salmonis SCO-95A to paper discs on the skin failed to induce any mortalities within 42 d. Intraperitoneally, fish were administered vaccines containing 10(9) heat-inactivated (100 degrees C, 30 min) or 10(9) formalin-inactivated P. salmonis SCO-95A in adjuvant, with a control group receiving phosphate-buffered saline (PBS) in adjuvant. After an induction period of over 6 mo fish were challenged by injection of P. salmonis into the dorsal median sinus. Mortalities in the control group reached 81.8% and the heat-inactivated and formalin-inactivated vaccines gave significant protection from P. salmonis, with relative percentage survivals of 70.7 and 49.6%, respectively. The nature of the protective antigen is unknown, but could be lipopolysaccharide or a heat-stable outer membrane protein. Fish that survived a dorsal median sinus challenge of P. salmonis or were cohabitants showed a strong immune response to P. salmonis.