Identification and characterization of lactic acid bacteria isolated from rainbow trout (Oncorhynchus mykiss,Walbaum 1792), with inhibitory activity against Vagococcus salmoninarum and Lactococcus garvieae

In this study, a total of 98 lactic acid bacteria isolated from rainbow trout intestines were screened for their probiotic properties. The isolates were tested for their ability to inhibit growth of Vagococcus salmoninarum and Lactococcus garvieae. Based on in vitro antagonism, 10 isolates were selected and evaluated pathogenicity in rainbow trout. Isolates were further investigated for hydrophobicity, bile salts and acid tolerance. These isolates were able to survive low pH and high bile concentrations and showed good adherence characteristics. Isolates were characterized phenotypically, and then, 16S rRNA gene sequence analysis was used for confirmation. Selected strains were administered orally at 10⁸ cfu/g feed, and fish were challenged with V. salmoninarum and L. garvieae. The fish fed with lactic acid bacteria supplemented diets did not improve protection against V. salmoninarum. However, administration of Lactococcus lactis subsp. lactis M17 2‐2 and Lactobacillus sakei 2‐3 resulted in a significant reduction in mortality due to L. garvieae when compared to the control fish. RPS values were calculated as 80 and 53% in fish fed with L. sakei 2‐3 and L. lactis subsp. lactis M17 2‐2, respectively. Our results suggest that these strains could provide an alternative for lactococcosis control in aquaculture.     

Experimental Lactococcus garvieae infection in rainbow trout,Oncorhynchus mykiss,Walbaum 1792: a comparative histopathological and immunohistochemical study

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young‐Adult IP groups and Young‐Adult IM groups, each contain 30 fish) and four control groups (Young‐Adult IP Control groups and Young‐Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976 . Fish in the IP groups were intraperitoneally administered an inoculate containing 10⁶ cfu mL^?1 bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10⁸ cfu mL^?1 bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin‐biotin‐peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.     

Effect of oregano (Origanum onites L.) essential oil on growth,lysozyme and antioxidant activity and resistance against Lactococcus garvieae in rainbow trout,Oncorhynchus mykiss (Walbaum)

The aim of this study was to investigate the effects of different levels of Origanum onites L. essential oil as feed additives on the growth performance, antioxidant activity and disease resistance of rainbow trout. Fish (26.05 ± 0.15 g) were fed the experimental diets supplemented with four different concentrations (0.125, 1.5, 2.5 and 3.0 mL kg^?1) of O. onites essential oil for 90 days. Fish fed diets containing essential oil of O. onites had significantly higher final weight than the control group. Feed conversion ratio in fish fed diets containing 1.5 and 3.0 mL kg^?1 essential oil of O. onites was improved than other treatments (P < 0.05). The lowest feed conversion efficiency ratio was recorded in the 0.125 mL kg^?1 group of O. onites. Antioxidant status of fish was assayed for levels of plasma superoxide dismutase (SOD) and plasma catalase (CAT) activity. Lysozyme activity in plasma was significantly higher in fish fed diet containing 3.0 mL kg^?1 essential oil of O. onites (P < 0.05). After 8 weeks of feeding, fish were challenged with Lactococcus garvieae and cumulative mortality was recorded over 15 days. Dietary administration of 0.125, 1.5 and 2.5 mL kg^?1 O. onites significantly reduced fish mortality (P < 0.05). The 3.0 mL kg^?1 diet showed no mortality after challenged with L. garvieae. These results suggested that the essential oil of O. onites could be applied as growth promoter and also improved disease resistance when added to rainbow trout feed.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum , A. hydrophila , A. hydrophila subsp. dhakensis , A. salmonicida subsp. salmonicida , A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10–25 bands, of which major bands were seen in the region of 32.5–47.5 and 62–83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83–173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.     

Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar , rainbow trout, Oncorhynchus mykiss , and turbot, Scophthalmus maximus , on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida . Four of these cultures, which were identified tentatively as A. hydrophila , Vibrio fluvialis , Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida . There was no indication of serum or mucus antibodies to A. salmonicida , but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the fish.     

Immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), with a low molecular mass fraction isolated from Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.     

Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout,Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F. psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28‐day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F. psychrophilum.     

Cellular components of probiotics control Yersinia ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.     

Covariation between diploid and triploid progenies from common breeders in rainbow trout, Oncorhynchus mykiss (Walbaum)

The covariation between diploid and triploid progenies from common breeders was investigated in various progeny-testing experiments where either dams or sires were sampled from rainbow trout, Oncorhynchus mykiss (Walbaum), stocks. Triploidiza tion was found to frequently reduce the performance in the traits studied: body length and weight, growth, coefficient of condition and pyloric caeca number. Triploidization also generated significant interactions with the parental breeding value. These interactions were caused in part by the familial variance not being the same in triploids as in diploids, but also by actual ranking differences between diploid and triploid familial performances. However, the effect of these interactions was minor as compared with the amount of variation common to both ploidy levels (genetic correlations averaged 0.7–0.9). Therefore, selection of diploid breeders appeared efficient enough for improving triploid progeny, unless family selection methods including triploid progeny testing were preferred for other reasons. Lastly, it was observed that variances from maternal origin tended to be larger in triploids, whereas variances from paternal origin tended to be smaller, as compared with diploids. This point was discussed referring to the genetic make-up of triploids and in the scope of dams and sires for selective breeding.     

Cellular responses of leucocytes isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Leucocytes were isolated from the gut of rainbow trout by collagcnase digestion. They were shown to be capable of producing a chemoattractant(s) for head kidney leucocytes and a macrophagc activating factor, following stimulation with calcium ionophore and T-cell mitogens, respectively. They also included low but significant numbers of phagocytes, as determined by phagocytosis of fluorescent latex microspheres and oxygen free radical production. However, migration of isolated gut cells in response to head kidney chemo-attractants was lacking. The significance of these results in relation to mucosal immunity is discussed.     

The effect of glycyrrhizin on rainbow trout, Oncorhynchus mykiss (Walbaum), leucocyte responses

Abstract. Treatment of rainbow trout macrophages with glycyrrhizin (GL), an aqueous extract of licorice, Glycyrrhiza glabra , enhanced their respiratory burst activity. Maximal effects were seen using concentrations of 10–100 μml⁻¹. GL also modulated trout lymphocytes, increasing proliferation responses to the mitogen phytohaemagglutinin some two-fold over a range of GL concentrations. In addition, GL elicited the release of a macrophage activating factor (MAF) from head kidney leucocytes, as assessed by the ability of generated supernatants to increase respiratory burst activity of target macrophages. MAF activity was most apparent using 100 μg ml⁻¹ GL to induce MAF release and a 48-h incubation period with the target macrophages. Finally, GL was shown to enhance the release of MAF in response to the mitogen concanavalin A. The possible use of GL as a stimulant of fish innate defences is discussed.     

Electrophoretic characterization of three hatchery-reared strains of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Significant differences were found in allelic frequency distribution among three strains of rainbow trout, Oncorhynchus mykiss (Walbaum). The isocitrate dehydrogenase ( IDH-3, 4 * 1.1.1.42) locus was fixed in two of the strains while the superoxide dismutase ( SOD-I * 1.15.1.1) locus was absent in one of the strains.
Similarity indices ranged from 0·973 to 0·988 on the scale of 1·000 among pairwise comparisons of the strains while Nei's genetic distance was 0·012 between Mount Lassen and Manx, 0·025 between Mount Lassen and Tagwerker and 0·027 between Manx and Tagwerker. Gene diversity was highest in the Mount Lassen strain, followed by Manx, with Tagwerker having the least. These differences in gene diversity were attributed to breeding and management practices used at the hatchery. The need for characterization of strains of rainbow trout, especially in crossbreeding programmes that aim to increase genetic variation, was emphasized by these results.