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.     

Efficacy of in-feed probiotics against Aeromonas bestiarum and Ichthyophthirius multifiliis skin infections in rainbow trout (Oncorhynchus mykiss, Walbaum)

Aims:  The aim of this study was to assess the efficacy of in-feed probiotics as a preventive measure against skin infections caused by Aeromonas bestiarum and Ichthyophthirius multifiliis (Ich) in rainbow trout.
Methods and Results:  Fin rot was induced in fish by intradermal injection with 0·1 ml volumes containing 10⁵ cells per ml A. bestiarum at the base of the dorsal fin. Ich infections resulted from immersion in Ich-contaminated water. Each probiotic was administered orally [10⁸ cells per g feed for GC2 ( Aeromonas sobria ) and 10¹⁰ cells per g feed for BA211 ( Brochothrix thermosphacta )] for 14 days. Results showed that, after challenge with A. bestiarum , probiotics GC2 and BA211 led to 76% and 88% survival, respectively, in contrast to 22% survival for controls. Fish fed with probiotic GC2 had 100% survival after challenge with Ich compared with 2% for probiotic BA211 and 0% for controls. Analysis of innate immune responses revealed that probiotic GC2 promoted higher phagocytic activity, whereas probiotic BA211 led to enhanced respiratory burst activity.
Conclusion:  Of the two probiotics examined, GC2 was more effective in protecting against both fin rot and Ich. Each probiotic appeared to stimulate different pathways within the innate immune system.
Significance and Impact of the Study:  This is the first demonstration that probiotics can protect fish against surface infections. Furthermore, this is the first time a probiotic has been shown to protect against a eucaryotic pathogen, namely I. multifiliis .     

Environmental influences on cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two groups of wild (lakedwelling and anadromous), and a group of hatchery-reared Oncorhynchus mykiss (Walbaum) (rainbow trout) were sampled in order to measure cardiac morphometrics, haemoglobin concentration, and the DNA and protein concentration in cardiac muscle. A combination of these variables was used to distinguish wild fish from domestic ones.
The wild fish had significantly higher levels of haemoglobin (for male fish, 10.10 and 10.07 g 100 ml⁻¹ vs. 7.69 g d⁻¹) and larger relative ventricle mass (females, 0.091 and 0.099% ofbody mass vs. 0.073%; males, 0.108 and 0.134% vs. 0.102%; immatures, 0.086 and 0.094% vs. 0, 072%, respectively) than the domestic fish. The anadromous and domestic fish had significantly higher amounts of compact tissue when compared with lake fish (females, 43 and 47% of ventricle mass vs. 34%, respectively). Ventricle size distinguished wild fish from domestic fish, except male anadromous and male domestic fish which were distinguished only by haemoglobin and compact tissue values. Immature fishes from all groups had lower total protein levels in the ventricle, lower compact tissue levels, and less haemoglobin. Points regarding the potential environmental influences in determining these cardiovascular trends are discussed.     

Use of bacterial lipopolysaccharide (LPS) as an immunostimulant for the control of Aeromonas hydrophila infections in rainbow trout Oncorhynchus mykiss (Walbaum)

Aims: To determine the effect of lipopolysaccharide (LPS) for the prevention of infection by Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss Walbaum) fingerlings. Methods and Results: Rainbow trout fingerlings were fed with 0 mg (= controls), 1·875 mg, 3·75 mg, 7·5 mg and 15 mg of LPS per 100 g of commercial feed for 14 days before experimental challenge with A. hydrophila. The results revealed a reduction in mortalities to 5% in the two lowest doses and 15% in the group, which received 15 mg LPS per 100 g of feed, compared with 45% mortalities in the control. LPS exerted a powerful oxidative burst effect and was a potent mediator of phagocytic, lysozyme, bactericidal and antiprotease activities and total protein. However, whereas there were increases in specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) in LPS‐treated fish, the data were not significantly (P > 0·05) different. Conclusions: LPS was effective at preventing disease caused by A. hydrophila and in stimulating the innate immune response of rainbow trout. Significance and Impact of the Study: The results of this study highlight the role of LPS in fish disease control.     

Haemoglobin isomorph abundances in splenectomized rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum) were rendered acutely anaemic by immersion in phenylhydrazine HC1 at 5° C. Following splenectomy or sham operation erythropoiesis was induced by gradual warming. No consistent or significant differences in red cell numbers, haemoglobin, haematocrit, mean erythrocytic volume or haemoglobin or haemoglobin isomorph system organization were evident during or following recovery. It is unlikely, therefore, that reported acclimatory adjustments in haemoglobin isomorph abundances can be attributed to site-related differences in erythroid cell lineages.     

The ontogeny of drinking in the rainbow trout, Oncorhynchus mykiss (Walbaum)

Drinking was measured by the rate of uptake of tritiated dextran in fresh water by the alevins and fry of rainbow trout at various stages of development during a 40-day post-hatch period. Drinking increased almost S-fold during the initial 16 day yolk-sac stage. Drinking rate increased most between 16 and 23 days, the transitional period between yolk-sac absorption and first feeding. The maximum weight-specific drinkingrateof 3.24μlg⁻¹ h⁻¹ recorded for 40-day-old fry was higher than previously recorded for adults. Abrupt transfer from an adaptation temperature of 10 to 19° C increased drinking significantly (Q₁₀= 1.2), but sudden transfer to 1 1‰ salinity sea water caused a substantial fall in drinking rate in 23-day fry. A 24-h period of adaptation to 1 1‰ and 15‰ salinity restored drinking to a rate similar to that in fresh water. The water drunk by 31-day fry fed to satiation was initially higher than by unfed fry, but drinking rates subsequently fell below the control level. The results are discussed in terms of osmoregulation and the uptake of dissolved or suspended substances by the intestinal tract for the purpose of immunization.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Enhanced Aeromonas salmonicida bacterin uptake and side effects caused by low frequency sonophoresis in rainbow trout (Oncorhynchus mykiss)

Low frequency sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances, due to the modification of the stratum corneum lipid bilayer, in focal skin applications in mammals. Based on these findings, LFS has been suggested as a potential technology to be used for enhancement in immersion fish vaccination. In contrast to mammals where LFS is applied to discrete regions of the skin, in fish the whole individual needs to be exposed for practical purposes. The current study evaluated the impact of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative real time PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by behavioural examination, histology and blood serum analysis. The sonication intensity of 171 mW/cm² was enough for increasing skin permeability, but caused heavy erratic swimming and gill haemorrhages. Sonication intensities as low as 105 mW/cm² did not modify skin permeability and enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. Following sonication, the gill permeability for the bacterin decreased after 20 min and 120 min by factor 3 and 2, respectively. However, during sonication, erratic swimming of the fish raised some concerns. Further reduction of the sonication intensity to 57 mW/cm² did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by factor 3. In addition, a decreasing albumin–globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity without significant side effects.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

Effectiveness of bivalent vaccines against Aeromonas hydrophila and Lactococcus garvieae infections in rainbow trout Oncorhynchus mykiss (Walbaum)

Lactococcus garvieae and Aeromonas hydrophila are bacterial pathogens affecting salmonids and other fish species and cause of heavy losses in aquaculture. Diseases caused by these bacteria can be controlled satisfactory by immunization using monovalent vaccines. In this study, the protective efficacy of two bivalent vaccines against L. garvieae and A. hydrophila was evaluated in rainbow trout (Oncorhynchus mykiss). Bivalent formulations, containing formalin-inactivated bacteria, were prepared as an aqueous bacterin and as an adjuvanted vaccine using montanide ISA-763. Protection against L. garvieae and A. hydrophila was tested at day 30 and 90 post-vaccination. High levels of protection were achieved for the aqueous and adjuvanted bivalent vaccines against L. garvieae (RPS of 100% and 95.3%) and A. hydrophila (RPS of 100% and 95.3%) at day 30 post-vaccination. Significant differences (p < 0.05) were found between the RPS at days 30 and 90 post-immunization with a decrease in the protection levels for the aqueous bivalent vaccine against L. garvieae (RPS 76.2%) and A. hydrophila (RPS 85%), but not for the adjuvanted vaccine (RPS of 90% against L. garvieae and 95% against A. hydrophila). In addition, high antibody levels were observed in the vaccinated fish at day 15 post-immunization using both vaccines. Our results demonstrate that these bivalent vaccines can effectively protect rainbow trout against L. garvieae and A. hydrophila and could offer an appropriate strategy to prevent these infections in rainbow trout farms.     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Development of immunity in rainbow trout (Oncorhynchus mykiss, Walbaum) to Aeromonas hydrophila after the dietary application of garlic

The development and duration of immune protection against Aeromonas hydrophila infections with garlic as an immunostimulant in rainbow trout Oncorhynchus mykiss was studied. Rainbow trout fingerlings of 14 g average weight were fed with 0 g (=Control), 0.5 g and 1.0 g of garlic 100 g(-1) of feed for 14 days. Physiological factors, biochemical, immunological, haematological parameters and electrolyte indices were evaluated after a further 14, 21 and 28 days before challenge with Aeromonas hydrophila. Fourteen days after the cessation of feeding with garlic, mortality rates of 12% (relative percent survival [RPS] = 86%) and 16% (RPS = 80%) were recorded in groups which received 0.5 g and 1.0 g of garlic 100 g(-1) of feed, respectively, compared to 84% mortalities in the controls. The corresponding RPS 21 days after ending the feeding regime was 75% and 68, respectively. One week later, the RPS had dropped to 55% and 46% in the groups fed with 0.5 g and 1.0 g garlic 100 g(-1) of feed, respectively.     

Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii

The taxonomy of marine vibrios has changed rapidly over the last two decades, and a wealth of new species have been identified. Many Vibrio species are pathogenic to fish and crustaceans; however, little is known about the virulence of many of the novel species. The purpose of this study was to determine the ability of various recent isolates of vibrios to cause disease in rainbow trout (Oncorhynchus mykiss, Walbaum) and crustacea, i.e. Artemia nauplii. Of 56 isolates, representing 26 species of Enterovibrio, Photobacterium and Vibrio, obtained from a diversity of healthy and diseased aquatic animal hosts and water samples from many geographical locations, Vibrio brasiliensis, V. coralliilyticus, V. ezurae, V. fortis, V. kanaloaei, V. neptunius, V. rotiferianus and V. tubiashii were pathogenic to rainbow trout and Artemia nauplii with mortalities of up to 100%. The extracellular products of these pathogenic isolates were harmful to the animal models. In contrast, cultures of Enterovibrio norvegicus, E. coralii, Photobacterium rosenbergii, Vibrio campbellii, V. chagasii, V. cyclitrophicus, V. gallicus, V. gigasii, V. hepatarius, V. hispanicus, V. lentus, V. nereis, V. pacini, V. pomeroyi, V. shilonii, V. superstes, V. tasmaniensis and V. xuii demonstrated either non- or low virulence in the animal models.     

Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida

Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1beta, IL-8, TNF-alpha and IFN-gamma was increased in the proximal intestine. TGF-beta was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1beta, IL-8, TNF-alpha and TGF-beta. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.     

Inflammatory response of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) larvae against Ichthyophthirius multifiliis

At hatching, the immune system of the rainbow trout larva is not fully developed. The larva emerges from the egg and is exposed to the aquatic freshwater environment containing pathogenic organisms. At this early stage, protection from disease causing organisms is thought to depend on innate immune mechanisms. Here, we studied the ability of young post-hatch rainbow trout larvae to respond immunologically to an infection with Ichthyophthirius multifiliis and also report on the localization of 5 different immune relevant molecules in the tissue of infected and uninfected larvae. Quantitative RT-PCR (qPCR) was used to analyze the genetic regulation of IL-1β, IL-8, IL-6, TNF-α, iNOS, SAA, cathelicidin-2, hepcidin, IL-10, IL-22, IgM and IgT. Also, a panel of 5 monoclonal antibodies was used to investigate the presence and localization of the proteins CD8, SAA, MHCII, IgM and IgT. At 10 days (84 degree days) post-hatching, larvae were infected with I. multifiliis and sampled for qPCR at 3, 6, 12, 24, 48 and 72 h post-infection (p.i.). At 72 h p.i. samples were taken for antibody staining. The first of the examined genes to be up-regulated was IL-1β. Subsequently, IL-8 and cathelicidin-2 were up-regulated and later TNF-α, hepcidin, IL-6, iNOS and SAA. Immunohistochemical staining showed presence of CD8 and MHCII in the thymus of both infected and non-infected larvae. Staining of MHCII and SAA was seen at sites of parasite localization and weak staining of SAA was seen in the liver of infected larvae. Staining of IgT was seen at site of infection in the gills which may be one of the earliest adaptive factors seen. No positive staining was seen for IgM. The study illustrates that rainbow trout larvae as young as 10 days (84 degree days) post-hatch are able to regulate important immune relevant cytokines, chemokines and acute phase proteins in response to infection with a skin parasitizing protozoan parasite.     

Behavioural fever boosts the inflammatory response in rainbow trout Oncorhynchus mykiss

Behavioural fever, manifested as an increased preferred temperature, was shown in rainbow trout Oncorhynchus mykiss following an injection of bacterial lipopolysaccharide. Simulated behavioural fever, through a 2·5° C water temperature rise following bacterial lipopolysaccharide injection, enhanced the expression of the cytokine interleukin-1β, in comparison with an untreated group held at the initial temperature. The present findings show that an important mediator in the immune response can be boosted through behavioural fever in fishes.     

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.