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.     

Association of a specific major histocompatibility complex class IIβ single nucleotide polymorphism with resistance to lactococcosis in rainbow trout,Oncorhynchus mykiss (Walbaum)

Major histocompatibility complex (MHC) loci encode glycoproteins that bind to foreign peptides and initiate immune responses through their interaction with T cells. MHC class II molecules are heterodimers consisting of α and β chains encoded by extremely variable genes; variation in exon 2 is responsible for the majority of observed polymorphisms, mostly concentrated in the codons specifying the peptide‐binding region. Lactococcus garvieae is the causative agent of lactococcosis, a warm‐water bacterial infection pathogenic for cultured freshwater and marine fish. It causes considerable economic losses, limiting the profitability and development of fish industries in general and the intensive production of rainbow trout, Oncorhynchus mykiss (Walbaum), in particular. The disease is currently controlled with vaccines and antibiotics; however, vaccines have short‐term efficacy, and increasing concerns regarding antibiotic residues have called for alternative strategies. To explore the involvement of the MHC class II β‐1 domain as a candidate gene for resistance to lactococcosis, we exposed 400 rainbow trout to naturally contaminated water. One single nucleotide polymorphism (SNP) and one haplotype were associated with resistance (P < 0.01). These results are promising for using MHC class IIβ as a molecular marker in breeding rainbow trout resistant to lactococcosis.     

Use of adjuvanted vaccines to lengthen the protection against lactococcosis in rainbow trout (Oncorhynchus mykiss)

In this work, we evaluated the effect of the inclusion of different adjuvants in the vaccine formulation against Lactococcus garvieae in comparison with an aqueous bacterin. The aqueous and non mineral oil adjuvanted vaccines (Montanide-ISA-763-A and Aquamun) yielded a good protection 4 weeks after the immunization (RPS of 82.6, 84.8 and 86.9, respectively). The protective rates obtained for the adjuvants Montanide-IMS-2212 (water based nanoparticles with immunostimulant) and Carbomer (high molecular weight polymer organic of polyacrylic acid) were 45.7% and 56.5%, respectively. We also evaluated the duration of protection confered by Aquamun adjuvanted-vaccine in comparison with the aqueous bacterin. Three months after vaccination, the RPS values obtained with the aqueous vaccine was 40% whereas with the Aquamun adjuvanted-vaccine the protection increased to values of 92%. In addition, a long lasting protection was observed with this adjuvanted vaccine with RPS values of 90% and 83.3% in the challenges performed at 6 and 8 months post-vaccination, respectively.     

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin‐killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin‐killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710‐1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60‐s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.     

Pre‐challenge and post‐challenge haemato‐immunological changes in Oreochromis niloticus (Linnaeus, 1758) fed argan oil against Lactococcus garvieae

The present study investigated the effects of argan oil, obtained from Argania spinosa, on pre‐ and post‐challenge immuno‐haematological and biochemical responses of Nile tilapia, Oreochromis niloticus. For this purpose, the fish were fed diets containing 0, 0.5%, 1% or 2% argan oil for 45 days. Following 45 days of feeding, fish were challenged with Lactococcus garvieae and mortality was recorded for 15 days. During the pre‐challenge period, significantly higher respiratory burst activity, total white blood cell (WBC), serum lysozyme activity and myeloperoxidase activity were determined in the argan oil‐fed groups. The serum glucose and cholesterol levels decreased whilst total protein and albumin did not change in the groups fed with argan oil‐supplemented diets. After challenge with Lactococcus garvieae, the percentage survival (%) was found to be the highest in the 1% and 2% argan oil‐supplemented feeding groups. Also, there was a significant increase in weight gain, specific growth rate and feed conversion ratio in those fish fed argan oil. The results of this study indicated that after the supplementation of fish diets with argan oil, especially at 1% and 2% concentrations, the immunological, haematological and biochemical values remained similar in both the pre‐ and post‐challenge periods and the immune response against L. garvieae in Nile tilapia was modulated.     

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.     

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.     

Characterization of Weissella ceti infections in Brazilian rainbow trout,Oncorhynchus mykiss (Walbaum), farms and development of an oil-adjuvanted vaccine

Weissella ceti is an emerging bacterial pathogen that affects rainbow trout, Oncorhynchus mykiss (Walbaum), farms. The aims of this study were to genotype W. ceti strains isolated from distinct geographical origins and to determine the efficacy of an oil-adjuvanted vaccine against the disease. Between 2010 and 2012, outbreaks were recorded in five Brazilian farms, and 34 W. ceti isolates were genetically characterized by repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequences PCR and pulsed-field gel electrophoresis. Two different W. ceti vaccines were tested: an aqueous-based whole-cell inactivated vaccine (bacterin) and oil-adjuvanted vaccine. Their efficacy was evaluated in rainbow trout at 30 and 60 days post-vaccination (d.p.v.). W. ceti was found to be a highly homogeneous population in Brazil, with clonally related genotypes. Oil-adjuvanted vaccine exhibited the best (P < 0.05) protection against disease, reaching relative percentage survival (RPS)values of 92% at 30 and 60 d.p.v. Bacterin resulted in RPS values of 67% and 58% at day 30 and 60, respectively. The oil-adjuvanted vaccine provided effective protection against W. ceti infection in rainbow trout.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

Comparison of pulsed‐field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae

Biochemical test, pulsed‐field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC‐PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC‐PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216‐6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC‐PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC‐PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods.     

Effects of alternative dietary oils on immune response,expression of immune‐related genes and disease resistance in juvenile Nile tilapia,Oreochromis niloticus

The expression of immune‐related genes and immune responses to Aeromonas hydrophila were investigated on Oreochromis niloticus (6.07 ± 0.07 g), by feeding them six different diets for 8 weeks to apparent satiation. Diets contained fish oil (60g/kg FO), virgin coconut oil (60g/kg VCO) and corn oil (60g/kg CO) as sole lipids or blends of 30g/kg FO + 30g/kg VCO (3FVCO), 30g/kg FO + 30g/kg CO (3FCO) and 30g/kg VCO + 30g/kg CO (VO). Fish fed 3FCO recorded higher final weight, percentage weight gain (%WG) and specific growth rate (%SGR) but not significantly higher than all other groups. Triglyceride was higher in fish fed 3FCO than 3FVCO and CO (p ? 0.05), whereas total immunoglobulin (TIg) was not significant (p ? 0.05) between groups. Lysozyme activity was significantly higher in fish fed diet CO while groups FO, 3FCO and VCO recorded the least activities (p ? 0.05). Although alternative complement activity (ACH₅₀), complement proteins (C3 and C4), was not influenced, antibody titre production was significantly higher in fish fed diet 3FCO and lower in group CO. mRNA expression of IL‐1β was significantly upregulated in fish fed VO while the expression of C‐type lysozyme and TGF‐β was not significantly influenced across treatments, although group fed FO recorded higher expression levels, respectively. Lower mortalities of fish were recorded in groups fed 3FCO and VO after 14 days postchallenge with A. hydrophila disease indicating the enhancing effects of vegetable oils to boost immune response and resistance to disease. The study concludes that alternative lipid sources with high polyunsaturated fatty acids (PUFAs‐ALA and LA) (CO) and blend of saturated fatty acids (SFA)(VCO) can partially and or exclusively replace FO with an improved effect on tilapia and resistance to A. hydrophila in tilapia.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Studies on the antibody response and side effects after intramuscular and intraperitoneal injection of Atlantic lumpfish (Cyclopterus lumpus L.) with different oil‐based vaccines

Atlantic lumpfish (Cyclopterus lumpus L.) is used as a biological delousing agent for sea lice (Lepeophtheirus salmonis K.) infestations in Norwegian aquaculture. Here, we present a study on the antibody response and vaccine side effects after intramuscular and intraperitoneal injection of lumpfish with two vaccines. Both vaccines contained bacterial antigens from atypical Aeromonas salmonicida A‐layer types V and VI, Vibrio anguillarum serotype O1 and Moritella viscosa sp., but one vaccine contained a vegetable oil‐based adjuvant, while the other contained a mineral oil‐based adjuvant. Intramuscular injection of the mineral oil‐based vaccine caused a high acute mortality of fish within 48 hr after immunization. Intraperitoneal injection of the mineral oil‐based vaccine resulted in a lower severity of intra‐abdominal side effects than the vegetable oil‐based vaccine. Intramuscular injection of the mineral oil‐based vaccine resulted in a significantly higher antibody response against A. salmonicida when compared to controls and the vegetable oil‐based vaccine group. The antibody response was poor against V. anguillarum and M. viscosa for all groups. Our results indicate that intramuscular injection of oil‐based vaccines might be feasible for providing immunological protection for Atlantic lumpfish against bacterial diseases, especially atypical A. salmonicida, but more work is required to identity optimal adjuvants.     

Simplified processing method of banana (Musa acuminata) peels possess the improvement in immunological responses of Macrobrachium rosenbergii

Musa acuminate peel extract as an immunostimulant, administrated in the diets of Macrobrachium rosenbergii, and an assessment of a simplified process to develop low‐cost feed additives are conducted in this study. The products obtained serially, during the processes of hot‐water banana peel extraction, were administrated in the diets of M. rosenbergii, including dried banana peel powder (DBP) at 8 g/kg, hot‐water‐treated banana peel (HBP) at 8 g/kg and hot‐water extract of banana peel (BPE) at 2 g/kg during the 32 days of the feeding trial. Total haemocyte count, different haemocyte count, respiratory bursts; and activity of superoxide dismutase, glutathione peroxidase, phenoloxidase and transglutaminase, as well as an accelerated haemolymph clotting time, significantly increased in M. rosenbergii fed with diets containing HBP and BPE, 32 days post‐feeding. Furthermore, the phagocytic activity and clearance efficiency of prawns against Lactococcus garvieae infection increased significantly. The respective relative survival percentages of prawns fed with HBP, and BPE containing diets after 32 days of feeding trial were 29.2% and 41.7% against L. garvieae infection for 144 hours, and 50%, and 50% against hypothermal stress for 96 hours of exposure. We may therefore conclude that HBP, obtained from a simplified procedure, without centrifugation and lyophilization, may strategically promote tolerance to hypothermal stress, and enhance immunity and resistance to L. garvieae infection.     

Development of a selective and differential medium for capsulated Lactococcus garvieae

A selective and differential medium termed ‘LG agar’ was developed for the isolation and presumptive identification of Lactococcus garvieae that results in black colonies with red halos. In this study, all 14 strains of L. garvieae and only 9 of the 148 strains representing 38 other species were able to grow on the LG agar. The nine viable strains on LG agar plates (including Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Vibrio fluvialis, Vibrio furnissii, Vibrio mimicus and Vibrio salmonicida) were further differentiated from L. garvieae by various colours or colony features. Colonies isolated from the mixing culture and the infected giant sea perch using LG agar plates were all positively identified as L. garvieae by conventional tests and 16S rDNA sequencing. Furthermore, LG agar discriminated capsulated strains of L. garvieae, which were believed to be correlated with pathogens of fish and shellfish, from non‐capsulated ones by colony appearances. The specificity and differentiating ability of LG agar suggest that this medium displays considerable potential for primary isolation and presumptive identification of L. garvieae from pathological and environmental samples.     

Immunization of rainbow trout Oncorhynchus mykiss (Walbaum) with a crude lipopolysaccharide extract from Flavobacterium psychrophilum

Control methods for Flavobacterium psychrophilum are limited and oftentimes ineffective; hence, research efforts have focused on vaccine development. This study tested the hypothesis that a crude lipopolysaccharide (LPS) extract from F. psychrophilum will elicit a protective immune response in rainbow trout Oncorhynchus mykiss (Walbaum) against F. psychrophilum challenge. Rainbow trout (mean weight, 3 g) were immunized intraperitoneally with the following treatment and control preparations: 10 μg of crude LPS with or without Freund's complete adjuvant (FCA), 25 μg of crude LPS with or without FCA and saline with or without FCA. Immunization of fish with 10 or 25 μg of crude LPS/FCA resulted in significant antibody responses against F. psychrophilum using ELISA with a whole‐cell lysate as the coating antigen, but only minimal levels of protection were conferred following F. psychrophilum challenge at 14 weeks post immunization. Western blot analyses demonstrated that fish exhibited antibodies specific for low‐molecular mass proteins present in the crude LPS extract, but did not exhibit antibodies specific for F. psychrophilum LPS. The results indicate that higher immunization doses and/or the use of an alternative extraction method that yields larger LPS molecules (23–70 kDa) may be necessary to elicit specific antibody responses against F. psychrophilum LPS.     

Rainbow trout (Oncorhynchus mykiss) immune response towards a recombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliis

The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n‐terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I‐antigen in future vaccine trials.