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 .     

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.     

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.     

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.     

Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases.

Clinical and macroscopic findings (anorexia, lethargy, loss of orientation and exophthalmia) indicate that Streptococcus iniae and Lactococcus garvieae infections of trout share some common features, but histopathology reveals notable differences between the 2 diseases. Meningitis and panophthalmitis are the main lesions among S. iniae infected trout, whereas L. garvieae infection results in a hyperacute systemic disease. Differences in the LD50s of the 2 pathogens and the sudden onset of signs and death correlate with the histopathological findings, indicating the severity of L. garvieae infection of trout.     

Resistance to beta-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss).

Bacterial infections caused by members of the genus Aeromonas, with a relatively high antibiotic resistance, are among the most common and troublesome diseases of fish raised in ponds with recirculation systems. In this study, carried out at an experimental aquaculture station in northern Portugal, 51 strains identified as belonging to the genus Aeromonas were isolated from 20 rainbow trout (Oncorhynchus mykiss) skin and kidney samples, as well as from raceway water samples. Macro- and microscopic examination of the fish tissues revealed lesions or cellular alterations in skin and kidney that seemed to correlate with the presence of those isolates. The sensitivity of all isolated strains to different groups of beta-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) was evaluated using the disc diffusion method. The highest rates of resistance were to amoxicillin, carbenicillin and ticarcillin. Unexpected resistance to imipenem, an antibiotic of clinical usage, was also detected, which suggests that resistance may have been transferred to the Aeromonas population from the environment.     

The auxotrophic aroA mutant of Aeromonas hydrophila as a live attenuated vaccine against A. salmonicida infections in rainbow trout (Oncorhynchus mykiss)

An auxotrophic aroA mutant of the Aeromonas hydrophila AG2 strain is a live attenuated vaccine against A. hydrophila infection in rainbow trout (Oncorhynchus mykiss). The protection conferred by the live attenuated vaccine against A. salmonicida strains is reported here, and several parameters of the specific and non-specific immune response in vaccinated trout were characterised. Vaccination with a dose of 10(7)cells/fish of the aroA mutant elicited significant protection against the Hooke and DK30 strains of A. salmonicida (relative percent survival RPS >60%). This cross-protection correlated moderately with the activation of the humoral and cellular specific immune responses, which show cross-reactivity against antigens shared by the two bacterial species, and a moderate increase in the lysozyme and antiprotease activities in the serum of vaccinated trout.     

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10? CFU g?1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.     

Antibody increases phagocytosis and killing of Lactococcus garvieae by rainbow trout (Oncorhynchus mykiss, L.) macrophages

The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.     

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.     

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.     

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.     

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.     

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.     

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.     

A blue variant in the rainbow trout, Oncorhynchus mykiss Walbaum

A blue variant of the rainbow trout, which appeared in a French fish farm, displayed an iridescent body color that was cobalt blue on the back, lighter on the undersides, and silvery on the belly and which held up to adult stage. This color was supposed to result from a Tyndall effect involving a structural arrangement of melanin pigments because it disappeared when it was associated with a depigmenting gene. This blue variant appeared to be governed by an autosomal recessive gene. Blue fry survival and body weight were about 25% less than those of wild-type sibs, but no major problem was observed in further breeding performances, including reproduction. These features do not correspond with those of the blue variants previously described in the rainbow trout.     

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.     

Immunization of rainbow trout Oncorhynchus mykiss against Discocotyle sagiffata (Monogenea)

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with 2 different Discocotyle sagittata extracts dissolved in PBS and subsequently exposed to controlled infection. Immunization resulted in significantly reduced (p < 0.0001) worm intensities in > 50% of vaccinated fish (response arbitrarily defined as parasite burdens < mean control intensity - 1 SD), irrespective of the immunization regime (different parasite extracts, dosing and application schedules) employed. The protective effect of worm extract applied in Freund's complete adjuvant (FCA) did not differ significantly from extract given in PBS. Vaccination with embryonated parasite eggs extract and with FCA alone did not result in partial immunity, suggesting the observed protective effect is specific. Immunized fish had significantly higher specific antibody titres at the time of dissection (as determined by ELISA) than both naive and control fish. Overall, a significant negative correlation was found between antibody titres and worm burdens, suggesting immunoglobulins are implicated in mediating partial immunity. Western blot tests indicated the 2 different worm extracts used to immunize fish share antigens, but each one primarily induced recognition of a distinct band (30 and 38 kDa). Immunization seems to promote a shift between 2 equilibria, rather than progressively increasing protection. This would explain why boosting did not increase immunity, and why 2 different extracts primarily inducing recognition of 2 distinct antigens provide similar degrees of protection. Although several other non-specific and cellular factors are likely to be involved in controlling parasite numbers, it cannot be excluded that antibodies could be involved in mediating the observed partial immunity.