Secondary immune response of rainbow trout following repeated immersion vaccination

Teleosts are able to raise a protective immune response, comprising both innate and adaptive elements, against various pathogens. This is the basis for a widespread use of vaccines, administered as injection or immersion, in the aquaculture industry. It has been described that repeated injection vaccination of fish raises a secondary immune response, consisting of rapid, accelerated and increased antibody reaction. This study reports how rainbow trout responds to repeated immersion vaccination against yersiniosis (ERM) caused by the bacterial pathogen Yersinia ruckeri. It was found that rainbow trout does not raise a classical secondary response following repeated immersion vaccination. Serum antibody titres were merely slightly increased even after three immunizations, using 30‐s immersion into a bacterin consisting of formalin‐inactivated Y. ruckeri (serotype O1, biotypes 1 and 2), performed over a 3‐month period. The densities of IgM‐positive lymphocytes in spleen of fish immunized three times were increased compared to control fish, but no general trend for an increase with the number of immunizations was noted. The lack of a classical secondary response following repeated immersion vaccination may partly be explained by limited uptake of antigen by immersion compared to injection.     

Effect of quorum quenching bacteria on growth,virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout

Five N‐acyl homoserine lactone‐degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo‐C8‐HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo‐C8‐HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 10⁸ CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.     

Yersinia ruckeri—A threat not only to rainbow trout

Yersinia ruckeri (Y. ruckeri) can cause mortalities that are contributing to substantial economic losses in the rainbow trout (Oncorhynchus mykiss) aquaculture sector. Because of its most characteristic clinical signs, the disease in rainbow trout caused by this pathogen is called enteric redmouth disease. Although it is considered to affect mainly salmonids, there are reports in the available literature of isolating this bacterium from other fish species, both clinically healthy and diseased. The aim of this study was to analyse the available data concerning yersiniosis in non‐salmonid fish. The analysed data indicate that Y. ruckeri is a threat not only to rainbow trout. Some of the affected species have high commercial importance and mortalities may contribute to high economic losses. The disease symptoms may not be specific and can be different from those characteristic for enteric redmouth in trout, which may lead to misdiagnosis. Collected information indicates that infection with Y. ruckeri should be taken into account in the diagnostic procedures not only in salmonids.     

Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence-neutralizing activity

A lytic bacteriophage (φNC10) specific to serotype O1 Yersinia ruckeri has been identified and evaluated as a model to assess the potential use of bacteriophages and their products for disease control in aquaculture. Electron microscopy of purified φNC10 revealed a virion particle with a small (70 nm) polyhedral head and short tail. φNC10 infected only serotype O1 strains of Y. ruckeri and failed to bind a defined Y. ruckeri mutant strain lacking O1 lipopolysaccharides (O1-LPS), suggesting that φNC10 uses O1-LPS as its receptor. In addition, spontaneous φNC10-resistant mutants of Y. ruckeri exhibited defects in O1-LPS production and were sensitive to rainbow trout serum. Purified φNC10 displayed a polysaccharide depolymerase activity capable of degrading Y. ruckeri O1-LPS and thereby sensitizing Y. ruckeri to the bactericidal effects of rainbow trout serum. The φNC10-associated polysaccharide depolymerase activity also reduced the ability of Y. ruckeri cells to cause mortality following intraperitoneal injection into rainbow trout. These data demonstrate a potential utility of φNC10 and its associated polysaccharide depolymerase activity for Y. ruckeri disease prevention.     

Effect of combination of dietary Bacillus subtilis and trans‐cinnamic acid on innate immune responses and resistance of rainbow trout,Oncorhynchus mykiss to Yersinia ruckeri

The present study investigated the effects of combination of dietary Bacillus subtilis and trans‐cinnamic acid on serum biochemical parameters, innate immune responses and resistance of rainbow trout, Oncorhynchus mykiss to Yersinia ruckeri. Six experimental groups of fish with mean weights of 20.58 ± 0.35 g were used in the study. Five experimental groups of fish were fed diets containing Bacillus subtilis (10⁷ per gram) or a mix of the Bacillus subtilis (BS) and trans‐cinnamic acid (25 mg/kg‐25trcBS, 50 mg/kg‐50trcBS, 75 mg/kg‐75 trcBS, 150 mg/kg‐150 trcBS), whereas an additive‐free basal diet served as the control (Cont). In this study, an increase was observed in granulocyte percentage, respiratory burst activity, phagocytic activity, phagocytic index, myeloperoxidase activity and total antiprotease activity especially in fish fed with mix of the BS and trans‐cinnamic acid‐supplemented diets (p < .05). Moreover, at the end of the 20‐day challenge period the survival rates and antibody titre (p < .05), and relative per cent survival were higher in the BS group and all trcBS groups compared with control group. As a conclusion, the results in the present study show that feeding rainbow trout with diets containing a mix of B. subtilis and trans‐cinnamic acid over a 60‐day period might be sufficient for improving fish immune responses and disease resistance against Y. ruckeri.     

Possible effect of lesser galangal (Alpinia officinarum) extracts encapsulated into mesoporous silica nanoparticles on the immune status of rainbow trout (Oncorhynchus n)

Using plant extracts as immunostimulants in aquaculture has prove effectiv in resisting infectious disease, in addition to being safe and inexpensive. The present study is concerned about the prospective mode of action of using lesser galangal extract (Alpinia officinarum) encapsulated into amine surface functionalized mesoporous silica nanoparticles (MSN) in order to elevate the immune status of rainbow trout (Oncorhynchus mykiss). Fish were randomly divided into 6 groups and fed for 4 weeks with commercial diets supplemented with 0% (control), 0.5% and 1.5% of lesser galangal extract, and with previous concentrations encapsulated with MSN (0.5% + MSN, 1.5% + MSN) and MSN (without extract). The effects of the feeding trial on the innate humoural immune parameters (total protein, myeloperoxidase content, lysozyme antiproteases and bactericidal activities) in rainbow trout were examined. Also, the influence of dietary supplement on some immune‐related genes in rainbow trout head kidney (TNF, IL‐8, IL‐1b, LYZ2 and CD4) after challenge with Yersinia ruckeri was examined. The results revealed enhancement in immune parameters in all treatment groups compared to the control, especially in the fish group fed with 1.5% + MSN which showed the highest significant difference (p < .05) in total protein, lysozyme and antiproteases activities. Also, feeding lesser galangal extract encapsulated into MSN led to an increase in myeloperoxidase content and bactericidal activity. An improvement in the expression of immune‐related genes has been recorded in fish groups fed doses of lesser galangal extract and lesser galangal extract encapsulated into MSN compared to the control or to the group fed with MSN only. Particularly, the group fed with 0.5% + MSN showed a significant up‐regulation in most of the immune‐related genes. The current investigation supports using lesser galangal extract encapsulated into MSN in fish diets as a supplement to enhance the immune response of rainbow trout and elevate its resistance to infectious diseases.     

Effects of dietary humic acid on growth performance,haemato‐immunological and physiological responses and resistance of Rainbow trout,Oncorhynchus mykiss to Yersinia ruckeri

This study investigated the effects of dietary humic acid sodium salt on growth performance, haemato‐immunological and physiological responses, and resistance of rainbow trout, Oncorhynchus mykiss to Yersinia ruckeri. The experimental fish were divided into four groups; three of them were fed with humic acid incorporated diets (0.3% H3, 0.6% H6, 1.2% H12) and an additive free basal diet served as the control. Growth performance and haematological parameters of rainbow trout were not affected by humic acid supplemented diets (p > 0.05). However, dietary humic acid especially with 0.6% incorporation significantly increased stomach pepsin, intestinal trypsin and lipase activities p < 0.05. Following 60 days of feeding trial, fish were challenged with Yersinia ruckeri for 20 days. At the end of the challenge period, significantly higher (p < 0.05) survival rates were found in the 6% humic acid group compared to all other experimental treatment. Thus humic acid might replace antibiotics in diets for rainbow trout to control yersiniosis.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.     

Effect of catecholamine stress hormones (dopamine and norepinephrine) on growth,swimming motility,biofilm formation and virulence factors of Yersinia ruckeri in vitro and an in vivo evaluation in rainbow trout

In this study, we evaluated the impact of the catecholamines on growth, swimming motility, biofilm formation and some virulence factors activities of pathogenic Yersinia ruckeri. Norepinephrine and dopamine (at 100 µM) significantly increased the growth of Y. ruckeri in culture media containing serum. An increase in swimming motility of the pathogen was found following the exposure to the hormones; however, no effect was seen on caseinase, phospholipase and haemolysin productions. Further, antagonists for the catecholamine receptors were observed to block some of the influences of the catecholamines. Indeed, the effects of catecholamines were inhibited by chlorpromazine (the dopaminergic receptor antagonist) for dopamine, labetalol (α‐and β‐adrenergic receptor antagonist) and phenoxybenzamine (the α‐adrenergic receptor antagonist) for norepinephrine, but propranolol (the β‐adrenergic receptor antagonist) showed no effect. Pretreatment of Y. ruckeri with the catecholamines resulted in a significant enhancement of its virulence towards rainbow trout and the antagonists could neutralize the effect of the stress hormones in vivo. In summary, our results show that the catecholamines increase the virulence of Y. ruckeri which is pathogenic to trout through increasing the motility, biofilm formation and growth.     

Experimental infection by Yersinia ruckeri O1 biotype 2 induces brain lesions and neurological signs in rainbow trout (Oncorhynchus mykiss)

Pathological manifestations in rainbow trout (Oncorhynchus mykiss) following experimental waterborne infection with Yersinia ruckeri serotype O1 biotype 2 (strain 07111224) were investigated. Rainbow trout were exposed to 8 × 10⁷ CFU/ml of Y. ruckeri by bath for 6 hr, and mortality was then monitored for 22 days post‐infection (dpi). Organs were sampled at 3 dpi and also from moribund fish showing signs of severe systemic infection such as bleeding, exophthalmia or erratic swimming behaviour. Y. ruckeri was observed in the meninges and diencephalon of the brain, and lamina propria of olfactory organ at 3 dpi. At 12 dpi, Y. ruckeri had spread throughout the brain including cranial connective tissues and ventricles and the infection was associated with haemorrhages and an infiltration with leucocytes. Y. ruckeri infection and associated with leucocyte infiltration were observed at 13 dpi. In conclusion, Y. ruckeri strain 07111224 causes encephalitis in the acute phase of infection, which could explain why Y. ruckeri‐affected fish show exophthalmia and erratic swimming known as signs of ERM.     

Effects of Panax ginseng extract in practical diets for rainbow trout (Oncorhynchus mykiss) on growth performance,immune response and resistance to Yersinia ruckeri

In a preliminary in vitro study, a Panax ginseng extract exhibited an evident antibacterial activity against Yersinia ruckeri and Lactococcus garvieae and affected the respiratory burst and proliferation of rainbow trout Oncorhynchus mykiss leukocytes. Subsequently, the effects of a dietary ginseng extract supplementation on growth, blood biochemical profile, innate immune response and resistance against Y. ruckeri infection were investigated in vivo in rainbow trout juveniles. Four experimental diets were obtained by adding 0.0%, 0.01%, 0.02%, 0.03% of ginseng ethanolic extract to a commercial feed. Each diet was fed to triplicate groups of fish (mean body weight 30.5 ± 0.15 g) at 1% of body weight day^?1 for 10 weeks. The dietary supplementation with ginseng extract did not affect growth performance, feed utilization, biometric traits and fish whole body composition (P > 0.05). No major changes due to graded levels of ginseng extract in the diet were observed in blood biochemical parameters except for increasing plasma triglycerides and non‐esterified fatty acids in fish fed diets including 0.01% and 0.02% of extract (P < 0.05). The innate immune response was barely modulated by the dietary addition of ginseng extract. Serum lysozyme and leukocytes respiratory burst activities were just slightly increased in fish fed all the ginseng extract‐supplemented diets compared with controls, whereas serum antiproteases and leukocyte MPO were not affected (P > 0.05). The dietary administration of ginseng extract induced a reduction in mortality of rainbow trout infected with Y. ruckeri, although no significant differences between treated and control groups were observed (P > 0.05).     

Microencapsulation of a putative probiotic Enterobacter species,C6‐6, to protect rainbow trout,Oncorhynchus mykiss (Walbaum), against bacterial coldwater disease

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD), which has a major impact on salmonid aquaculture globally. An Enterobacter species, C6‐6, isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum), has been identified as a potential probiotic species providing protection against BCWD. This study examined the effects of alginate microencapsulation on the protective efficacy of C6‐6 against BCWD in vivo when administered to rainbow trout fry orally or by intraperitoneal (IP) injection. Viable C6‐6 bacteria were microencapsulated successfully, and this process (microencapsulation) did not significantly deteriorate its protective properties as compared to the administration of non‐microencapsulated C6‐6 bacteria. Both oral and IP delivery of C6‐6 achieved significantly better protection than control treatments that did not contain C6‐6 bacteria. The highest relative percent survival (RPS) resulted from IP delivery (71.4%) and was significantly greater than the highest oral RPS (38.6%). Successful intestinal colonization was not critical to protective effects of C6‐6. The study showed that C6‐6 administration, with or without encapsulation, was a viable choice for protecting fry from BCWD especially when administered intraperitoneally.     

Distribution and prevalence of T. bryosalmonae in Austria: A first survey of trout from rivers with a shrinking population

The first evidence of proliferative kidney disease (PKD) in an Austrian river (the River Kamp) was documented in 2016, and no information on the PKD infection status of trout in other rivers was available. Since then, brown trout (Salmo trutta fario) and rainbow trout (Oncorhynchus mykiss) have been collected from rivers in Upper and Lower Austria for different diagnostic purposes. In this study, we summarize the recent findings of a first survey concerning the distribution of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), from these samples. Between September 2015 and October 2017, a total of 280 brown trout and 39 rainbow trout were collected from 21 rivers in the provinces of Upper and Lower Austria. T. bryosalmonae was detected by PCR of kidney tissue in 17 of 21 sampled rivers and in 138 of 280 brown trout as well as in 11 of 39 rainbow trout. Pathological signs of PKD (e.g., hypertrophy of the kidney) were observed in 33 analysed brown trout and six rainbow trout samples. No correlations between fish infected by T. bryosalmonae and the parameters size and age class, condition factor, geological origin of the streams and distribution within the river course were found, while positively tested fish are significantly increased at sampling sites exceeding water temperatures of 15°C for median periods of 115 days. The prevalence within the affected streams or stream sections is highly variable, and in single rivers, infection rates of up to 90% are confirmed.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

Effects of oral passive immunization against somatostatin‐14 on growth performance,body composition and IgY delivery in rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio)

Egg‐yolk antibodies (IgY) against somatostatin‐14 (anti‐SST‐14) were evaluated as orally administered, growth promotants in gastric rainbow trout (Oncorhynchus mykiss) and agastric common carp (Cyprinus carpio) in an 8‐week feeding trial. Feeding groups were compared with fish which did not receive anti‐SST‐14 IgY. Growth responses and IgY plasma contents of the blood were assessed. In contrast to rainbow trout, oral anti‐SST‐14 addition to carp significantly improved protein efficiency ratio (PER, 1.02 ± 0.04) and protein productive value (PPV, 26.7 ± 0.91) after 56 days (P < 0.05) compared to the negative control (PER = 0.91 ± 0.02; PPV = 22.9 ± 0.66). IgY was undetectable in plasma of rainbow trout after oral administration, indicating that gastric degradation of the fed IgY makes this application route for growth promotion in gastric trout challenging.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Epizootics in farm-raised channel catfish, Ictalurus punctatus (Rafinesque), caused by the enteric redmouth bacterium Yersinia ruckeri

In the winters of 1995 and 1996, unusual disease outbreaks occurred on two separate channel catfish farms in Arkansas, USA. Affected fish exhibited extraordinary haemorrhaged rings around the eyes and raised haemorrhaged areas overlying the frontal foramens. Other signs included abnormal swimming, lethargy, loss of equilibrium, and exophthalmia. Bacterial isolates from the moribund fish were identified as Yersinia ruckeri by biochemical tests, no lysis by the Hafnia-specific bacteriophage 1672, and Y. ruckeri-specific growth patterns on Shotts-Waltman media. Fingerling catfish injected intraperitoneally with the bacterial isolate at 7.8 × 10⁶ bacteria fish^?1 developed lesions characteristic of the epizootics at 13, 18, and 22 °C and a biochemically identical isolate was recovered. Fingerling rainbow trout injected with the channel catfish isolate at 1 × 10⁵ bacteria fish^?1 and held at 20 °C developed signs typical of enteric redmouth by 4 days post-inoculation and were moribund by 5 days post-inoculation. Some differences of clinical signs occurred between experimentally infected rainbow trout and channel catfish. Clinical and biochemical similarities between infections of Y. ruckeri and many warmwater pathogens in affected catfish may lead to incorrect diagnosis of ERM infections.