Integrative analyses of probiotics, pathogenic infections and host immune response highlight the importance of gut microbiota in understanding disease recovery in rainbow trout (Oncorhynchus mykiss).

AIMS: Given the pivotal role played by the gut microbiota in regulating the host immune system, great interest has arisen in the possibility of controlling fish health by modulating the gut microbiota. Hence, there is a need to better understand of the host-microbiota interactions after disease responses to optimize the use of probiotics to strengthen disease resilience and recovery. METHODS AND RESULTS: We tested the effects of a probiotic feed additive in rainbow trout and challenged the fish with the causative agent for enteric red mouth disease, Yersinia ruckeri. We evaluated the survival, host immune gene expression and the gut microbiota composition. Results revealed that provision of probiotics and exposure to Y. ruckeri induced immune gene expression in the host, which were associated with changes in the gut microbiota. Subsequently, infection with Y. ruckeri had very little effect on microbiota composition when probiotics were applied, indicating that probiotics increased stabilisation of the microbiota. Our analysis revealed potential biomarkers for monitoring infection status and fish health. Finally, we used modelling approaches to decipher interactions between gut bacteria and the host immune gene responses, indicating removal of endogenous bacteria elicited by non-specific immune responses. CONCLUSIONS: We discuss the relevance of these results emphasizing the importance of host-microbiota interactions, including the protective potential of the gut microbiota in disease responses. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results highlight the functional consequences of probiotic-induced changes in the gut microbiota post infection and the resulting host immune response.

CK11, a Teleost Chemokine with a Potent Antimicrobial Activity.

CK11 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to both mammalian CCL27 and CCL28 chemokines, strongly transcribed in skin and gills in homeostasis, for which an immune role had not been reported to date. In the current study, we have demonstrated that CK11 is not chemotactic for unstimulated leukocyte populations from central immune organs or mucosal tissues but instead exerts a potent antimicrobial activity against a wide range of rainbow trout pathogens. Our results show that CK11 strongly inhibits the growth of different rainbow trout Gram-positive and Gram-negative bacteria, namely Lactococcus garvieae, Aeromonas salmonicida subsp. salmonicida, and Yersinia ruckeri and a parasitic ciliate Ichthyophthirius multifiliis Similarly to mammalian chemokines and antimicrobial peptides, CK11 exerted its antimicrobial activity, rapidly inducing membrane permeability in the target pathogens. Further transcriptional studies confirmed the regulation of CK11 transcription in response to exposure to some of these pathogens in specific conditions. Altogether, our studies related to phylogenetic relations, tissue distribution, and biological activity point to CK11 as a potential common ancestor of mammalian CCL27 and CCL28. To our knowledge, this study constitutes the first report of a fish chemokine with antimicrobial activity, thus establishing a novel role for teleost chemokines in antimicrobial immunity that supports an evolutionary relationship between chemokines and antimicrobial peptides.

Inflammatory reactions in rainbow trout fins and gills exposed to biocides.

Several biocides are widely used in rainbow trout aquaculture against various ectoparasites and ectobionts, but the inflammation induced in treated fish is less well described. Dose-response studies were conducted to elucidate the effects on rainbow trout (gills and fins) induced by a series of biocides including formalin, hydrogen peroxide (H2O2), peracetic acid (PAA) and the surfactant SPH6, which was isolated from the bacterium Pseudomonas H6. The compounds have documented antiparasitic effects, but the specific effects on fish needs further documentation. This study was performed over 24 h, and inflammatory reactions were evaluated in gills and fins. A dose-dependent effect was noted for expression of immune genes encoding for IL-1ß, TNFα, IFNγ, IL-10, IL-8, lysozyme, serum amyloid A (SAA), hepcidin, precerebellin and complement factor C3. PAA induced the strongest upregulation of cytokine and acute phase reactant genes followed by H2O2 and formalin. SPH6 showed a lower effect, and in several cases the compound induced downregulation of several genes. Gills showed a stronger response compared to fins. The mucous cell density in fins showed a range of changes which varied by compound. PAA, and to a lesser degree H2O2 and formalin, initially induced mucous cell hyperplasia, whereas SPH6 immediately decreased the number of cells containing mucus.

Eye fluke effects on Danish freshwater fish: Field and experimental investigations.

Eye flukes in fish are common in freshwater lakes. Fish become infected by the penetration of cercariae released from freshwater snails, and high infection pressures may be associated with mortalities in a Danish lake. Examination of two other freshwater lakes, combined with laboratory study, supported the notion. We investigated 77 freshwater fish from two lakes and the infection level suggested the occurrence of a high cercarial infection pressure in the Danish lakes. Dominant genera were Tylodelphys and Diplostomum covering a range of species identified by PCR and sequencing of the 18S (partial)-ITS1-5.8S-ITS2-28S (partial) of the rDNA. Cercariae of the prevalent species Diplostomum pseudospathaceum were used to infect zebrafish Danio rerio for the elucidation of short-term effects on the fish host. Zebrafish did not display abnormal behaviour when exposed to 200-400 cercariae, but a dosage of 600 and 1,000 cercariae/fish proved lethal. When fish were exposed to sublethal dosages, 19 out of 27 immune genes were significantly regulated and three genes encoding cytokine (IL 4/13B, IL-6 and IL-8) were upregulated at 3 hr post-infection (hpi), whereas others were downregulated especially at a later time point. We suggest that direct massive cercarial penetration of fish surfaces may be detrimental and may represent a threat to fish populations.

Morphological and molecular identification of Cryptocotyle lingua metacercariae isolated from Atlantic cod (Gadus morhua) from Danish seas and whiting (Merlangius merlangus) from the English Channel.

Trematode larvae (metacercariae) causing black spot disease occur frequently in gills, fins, skin and the superficial muscle layers of marine fish. Species within the genus Cryptocotyle Lühe, 1899 are frequently associated with this disease. Descriptions of the metacercarial stage are relatively limited and none has hitherto been reported from fish from the English Channel. The present study reports the morphological and molecular identifications of encysted black spot-inducing parasites from whiting (Merlangius merlangus) and Atlantic cod (Gadus morhua) caught respectively from the north coast of France (English Channel) and from Danish sea waters. Metacercariae were characterised morphologically based on microscopic observations and molecularly using Sanger sequencing of fragments of the mitochondrial cox1 gene and rDNA ITS region. Morphological data were compared with available data in the literature. Phylogenetic trees including reference sequences were built to confirm morphological and molecular identifications. This survey constitutes the first description of C. lingua metacercariae in the English Channel ecosystems.

Rainbow trout Oncorhynchus mykiss skin responses to salmon louse Lepeophtheirus salmonis: From copepodid to adult stage.

The marine crustacean Lepeophtheirus salmonis (salmon louse) is a common ectoparasite of wild and farmed salmonids. The parasite has a complex ontogeny comprising eight instars. The planktonic copepodid stage settles on host skin and pass through five instars to reach the adult stage. The present study comprises an experimental infestation of Oncorhynchus mykiss (rainbow trout) with salmon lice and describes histopathology and host immune responses in skin beneath the louse at multiple time points encompassing all louse developmental stages. Each fish was exposed to 80 infective copepodids, a mean no. of 32 parasites reached the preadult I stage whereas a mean no. of 11 parasites reached the adult stage. A progression in the severity of cutaneous lesions was observed, and levels of immune gene transcripts at the attachment site revealed a dynamic response, initially related to innate immunity. Later, immune cells accumulated in the dermis concomitant with a moderate decrease in levels of transcripts characteristic of both innate and adaptive immune responses. The present study also demonstrates that the cutaneous immune response was mainly induced at lice affected sites, while non-affected skin resembled the skin of untreated control. This indicates that the skin cannot be regarded as a uniform organ and requires careful sampling at all salmon louse stages.

Immune gene expression and genome-wide association analysis in rainbow trout with different resistance to Yersinia ruckeri infection.

Selective breeding programmes involving marker assisted selection of innately pathogen resistant strains of rainbow trout rely on reliable controlled infection studies, extensive DNA typing of individual fish and recording of expression of relevant genes. We exposed juvenile rainbow trout (6 h bath to 2.6 × 105 CFU mL-1) to the fish pathogen Yersinia ruckeri serotype O1, biotype 2, eliciting Enteric Red Mouth Disease ERM, and followed the disease progression over 21 days. Cumulative mortality reached 42% at 12 days post challenge (dpc) after which no disease signs were recorded. All fish were sampled for DNA-typing (50 k SNP chip, Affymetrix®) throughout the course of infection when they showed clinical signs of disease (susceptible fish) or at day 21 when fish showed no clinical signs of disease (survivors - resistant fish). Genome-wide association analyses of 1027 trout applying single nucleotide polymorphisms (SNPs) as markers revealed an association between traits (susceptible/resistant) and certain regions of the trout genome. It was indicated that multiple genes are involved in rainbow trout resistance towards ERM whereby it is considered a polygenic trait. A corresponding trout group was kept as non-exposed controls and a comparative expression analysis of central innate and adaptive immune genes in gills, spleen and liver was performed for three fish groups: 1) moribund trout exhibiting clinical signs 7 dpc (CS), 2) exposed fish without clinical signs at the same sampling point (NCS) and 3) surviving fish at 21 dpc (survivors). Immune genes encoding inflammatory cytokines (IL-1ß, IL-2A, IL-6A, IL-8, IL-10A, IL-12, IL-17A/F2A, IL-17C1, IL-17C2, IL-22, IFNγ, TNFα), acute phase reactants (SAA, C3, cathelicidins, lysozyme) were expressed differently in CS and NCS fish. Correlation (negative or positive) between expression of genes and bacterial load suggested involvement of immune genes in protection. Down-regulation of adaptive immune genes including IgDm, IgDs, IgT and TCR-ß was seen primarily in CS and NCS fish whereas survivors showed up-regulation of effector molecule genes such as cathelicidins, complement and lysozyme suggesting their role in clearing the infection. In conclusion, SNP analyses indicated that ERM resistance in rainbow trout is a multi-locus trait. The gene expression in surviving fish suggested that several immune genes are associated with the trait conferring resistance.

Gill amoebae from freshwater rainbow trout (Oncorhynchus mykiss): In vitro evaluation of antiparasitic compounds against Vannella sp.

Gill parasitic infections challenge farming of rainbow trout (Oncorhynchus mykiss, Walbaum) in freshwater facilities. Apart from flagellates (Ichthyobodo, (Pinto) and ciliates (Ichthyophthirius (Fouquet), Ambiphrya (Raabe), Apiosoma (Blanchard), Trichodinella (Sramek-Husek) and Trichodina (Ehrenberg)), we have shown that amoebae are prevalent in Danish trout farms. Gills were isolated from farmed rainbow trout in six fish farms (conventional and organic earth pond and recirculated systems) and placed on non-nutrient agar (NNA) moistened with modified Neff's amoeba saline (AS) (15°C). Gill amoebae from all examined fish colonized the agar and were identified based on morphological criteria showing species within the genera Trinema (Dujardin) (family Trinematidae), Vannella (Bovee) (family Vannellidae). In addition, hartmannellid amoebae were recorded. We established a monoculture of Vannella sp., confirmed the genus identity by PCR and sequencing and performed an in vitro determination of antiparasitic effects (dose-response studies) of various compounds including sodium chloride (NaCl), hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant. All amoebae were killed in concentrations of 16.90 mg/ml (garlic), 17.90 mg/ml (oregano), NaCl (7.5 mg/ml), hydrogen peroxide (100 µg/ml), peracetic acid (0.03 µg/ml), formaldehyde (25 µg/ml) and the Pseudomonas H6 surfactant (250 µg/ml).

Anisakid nematode larvae in the liver of Atlantic cod Gadus morhua L. from West Greenland.

Anisakid nematode larvae occur frequently in the liver of Atlantic cod, but merely few infection data from cod in waters around Greenland exist. The present study reports the occurrence of third-stage anisakid larvae in the livers of 200 Atlantic cod caught on fishing grounds along the West coast of Greenland (fjord systems of Maniitsoq) in May, June, August and September 2017. Classical and molecular helminthological techniques were used to identify the nematodes. A total of 200 cod livers were examined, and 194 were infected with third-stage nematode larvae (overall prevalence of infection 97%) with a mean intensity of 10.3 (range between 1 and 44 parasites per fish). Prevalences recorded were 96% for Anisakis simplex (s.l.), 55% for Pseudoterranova decipiens (s.l.) and 8% for Contracaecum osculatum (s.l.). Sequencing the mtDNA cox2 from 8 out of 23 these latter larvae conferred these to C. osculatum sp. B. A clear seasonal variation was observed, with a rise in A. simplex (s.l.) and P. decipiens (s.l.) occurrence in June and August and a decline in September. The study may serve as a baseline for future investigations using the three anisakids as biological indicators in Greenland waters.

Effect of oral booster vaccination of rainbow trout against Yersinia ruckeri depends on type of primary immunization.

Vaccination of rainbow trout against Enteric Redmouth Disease (ERM) caused by Yersinia ruckeri can be successfully performed by administering vaccine (a bacterin consisting of formalin killed bacteria) by immersion, bath or injection. Booster immunization is known to increase the protection of fish already primed by one of these vaccination methods. Oral vaccination of trout (administering vaccine in feed) is an even more convenient way of presenting antigen to the fish but the effect of an oral booster has not previously been described in detail. The present work describes to what extent protection may be enhanced by oral boostering following priming with different administration methods. The study confirms that vaccination by 30 s dip into a bacterin (diluted 1:10) may confer a significant protection compared to non-vaccinated fish. The immunity may be optimized by booster immunization either provided as dip (most effective), bath (less effective) or orally (least effective). Oral immunization may be used as booster after dip but applied as a single oral application it induced merely a slight and statistically non-significant response. It is noteworthy that primary oral immunization followed by an oral booster vaccination showed a trend for an even weaker response. It should be investigated if continued exposure to a low antigen concentration - as performed by two oral immunizations - may induce tolerance to the pathogen and thereby leave the fish more vulnerable.

Transcriptomic analysis of Baltic cod (Gadus morhua) liver infected with Contracaecum osculatum third stage larvae indicates parasitic effects on growth and immune response.

High infection levels due to third-stage larvae of the anisakid nematode Contracaecum osculatum have been documented in cod from the eastern part of the Baltic sea during the latest decades. The nematode larvae mainly infect the liver of Baltic cod and prevalence of infection has reached 100% with a mean intensity up to 80 parasites per host in certain areas and size classes. Low condition factors of the cod have been observed concomitant with the rise in parasite abundance suggesting a parasitic effect on growth parameters. To investigate any association between parasite infection and physiological status of the host we performed a comparative transcriptomic analysis of liver obtained from C. osculatum infected and non-infected cod. A total of 47,025 predicted gene models showed expression in cod liver and sequences corresponding to 2084 (4.43%) unigenes were differentially expressed in infected liver when compared to non-infected liver. Of the differentially expressed unigenes (DEGs) 1240 unigenes were up-regulated while 844 unigenes were down-regulated. The Gene Ontology (GO) enrichment analysis showed that 1304 DEGs were represented in cellular process and single-organism process, cell and cell part, binding and catalytic activity. As determined by the Kyoto Encyclopedia of Gene and Genomes (KEGG) Pathways analysis, 454 DEGs were involved in 138 pathways. Ninety-seven genes were related to metabolic pathways including carbohydrate, lipid, and amino acid metabolism. Thirteen regulated genes were playing a role in immune response such as Toll-like receptor signaling, NOD-like receptor signaling, RIG-I-like receptor signalling and thirty-six genes were associated with growth processes. This indicates that the nematode infection in Baltic cod may affect on molecular mechanisms involving metabolism, immune function and growth.

Transcriptomic analysis of immunity in rainbow trout (Oncorhynchus mykiss) gills infected by Ichthyophthirius multifiliis.

The parasite Ichthyophthirius multifiliis infecting skin, fins and gills of a wide range of freshwater fish species, including rainbow trout, is known to induce a protective immune response in the host. Although a number of studies have reported activation of several immune genes in infected fish host, the immune response picture is still considered incomplete. In order to address this issue, a comparative transcriptomic analysis was performed on infected versus uninfected rainbow trout gills and it showed that a total of 3352 (7.2%) out of 46,585 identified gene sequences were significantly regulated after parasite infection. Of differentially expressed gene sequences, 1796 genes were up-regulated and 1556 genes were down-regulated. These were classified into 61 Gene Ontology (GO) terms and mapped to 282 reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Infection of I. multifiliis induced a clear differential expression of immune genes, related to both innate and adaptive immunity. A total of 268 (6.86%) regulated gene sequences were known to take part in 16 immune-related pathways. These involved pathways related to the innate immunity such as the Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration. Elevated transcription of genes encoding the TLR 8 gene and chemokines (CCL4, CCL19, CCL28, CXCL8, CXCL11, CXCL13, CXCL14) was recorded indicating their roles in recognition of I. multifiliis and subsequent induction of the inflammatory response, respectively. A number of upregulated genes in infected gills were associated with antigen processing/presentation and T and B cell receptor signaling (including B cell marker CD22 involved in B cell development). Overall the analysis supports the notion that I. multifiliis induces a massive and varied innate response upon which a range of adaptive immune responses are established which may contribute to the long lasting protection of immunized rainbow trout.

A pentavalent vaccine for rainbow trout in Danish aquaculture.

Mariculture in Denmark is based on production of rainbow trout grown two years in fresh water followed by one growth season in sea cages. Although the majority of rainbow trout are vaccinated against the most serious bacterial pathogens - Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Yersinia ruckeri, by the use of commercially available vaccines, disease outbreaks requiring treatment with antibiotics still occur. The present study tested the potential of a new experimental multicomponent vaccine that is based on local bacterial strains, isolated from rainbow trout in Danish waters, and thus custom-designed for Danish rainbow trout mariculture. The vaccination with the multicomponent vaccine resulted in protection against three relevant bacterial diseases (yersiniosis, furunculosis, vibriosis) under experimental conditions. We showed that i.p. injection of the vaccine induced specific antibody responses in trout against the different bacterial antigens and regulated expression of genes encoding SAA, C3, IL-1ß, IL-6, IL-8, IgD and MHCII.

Endoparasitic helminths in Baltic salmon Salmo salar: ecological implications.

Parasites in fish are ecological indicators, as they reflect the host's migration routes, feeding behavior and immune status. We performed a parasitological investigation of sea-running Baltic salmon to study the use of parasites as indicators for this fish stock. The host-a strain of Atlantic salmon Salmo salar-has been isolated for several millennia in the semi-enclosed brackish Baltic Sea, with limited migration to and from the North Sea. Twenty-four salmon (total body weight: 4.2-14.2 kg; total body length: 80-105 cm) were caught by spoon bait in the southern Baltic Sea during feeding migrations, necropsied shortly afterwards and internal organs subjected to parasitological investigation focusing on endoparasitic helminths. The pyloric region was heavily parasitized by the cestode Eubothrium crassum (100% prevalence; intensity: 97-273 parasites per infected fish), reflecting a diet of smaller pelagic fishes. The stomach contained the hemiurid digeneans Brachyphallus crenatus (95.8% prevalence; intensity: 8-151) and Hemiurus luehei (58.3% prevalence; intensity: 2-13), indicating a diet of clupeids. Schistocephalus solidus (25% prevalence; intensity: 1-2), liberated from ingested sticklebacks, the acanthocephalan Echinorhynchus truttae (54% prevalence; intensity: 1-13) and the adult nematode Hysterothylacium aduncum (29% prevalence; intensity: 1-13) were found in the intestine. The liver was parasitized by third-stage nematode larvae of Contracaecum osculatum (45.8% prevalence; intensity: 1-4), but these were growth-stunted and encapsulated. The parasite fauna differs markedly from salmon in North Atlantic waters, and the lack of purely marine parasite species indicates that the Baltic salmon has remained in the Baltic Sea during its life history.

Comparative susceptibilities and immune reactions of wild and cultured populations of Caspian trout Salmo trutta caspius to VHSV.

Caspian trout Salmo trutta caspius is an endangered subspecies of brown trout Salmo trutta which is native to the Caspian Sea. Restocking programmes have been established, but recent introduction of the rhabdovirus viral haemorrhagic septicaemia virus (VHSV) into Iranian rainbow trout farms connected to waterbodies supporting wild Caspian trout may represent an additional threat to the declining stock. The susceptibility of wild and cultured populations of this endemic subspecies was demonstrated by performing controlled VHSV infection experiments (both by bath and injection challenges). Subsequently, VHSV infection in exposed fish was confirmed (CPE and quantitative PCR), virus levels were measured, and regulation of immune genes in exposed fish was investigated with a focus on the genes encoding IL-8, IFNγ, TGFß, TNFα, SAA, C3-4, CD8α, IgM, MHC I, MHC II, iNOS and IGF-1. The presence of IgM-, CD8α- and MHC II-positive cells in host organs was visualized by immunohistochemistry. Both wild and cultured trout strains proved to be VHSV-susceptible following experimental challenge, but the mortality curves and associated regulation of immune-related genes differed between the 2 trout types. Implications of the results for future management of Caspian trout populations are discussed.

Host size-dependent anisakid infection in Baltic cod Gadus morhua associated with differential food preferences.

A significant increase in the infection level of Baltic cod Gadus morhua with the anisakid nematode larvae Contracaecum osculatum and Pseudoterranova decipiens has been recorded during recent years due to the expanding local population of grey seals Halichoerus grypus, which act as final hosts for these parasites. Here, we report from an investigation of 368 cod (total length [TL] 6-49 cm; caught in ICES Subdivision 25) that the infection level of juvenile cod (TL 6-30 cm) with larvae of C. osculatum and P. decipiens is absent or very low, whereas it increases drastically in larger cod (TL 31-48 cm). A third nematode Hysterothylacium aduncum was rarely found. The study indicates that the prey animals for large cod act as transport hosts for the parasite larvae. Analyses of stomach contents of cod caught in the same area (2007-2014) showed that small benthic organisms (including polychaetes Harmothoë sarsi) are preferred food items by small cod, the isopod Saduria entomon is taken by all size classes, and sprat Sprattus sprattus are common prey items for cod larger than 30 cm. Parasitological investigations (microscopic and molecular analyses) of H. sarsi (100 specimens) and S. entomon (40 specimens) did not reveal infection in these invertebrates, but 11.6% of sprat (265 specimens examined) was shown to be infected with 1-8 C. osculatum third stage larvae per fish. Analyses of sprat stomach contents confirmed that copepods and cladocerans are the main food items of sprat. These observations suggest that the C. osculatum life cycle in the Baltic Sea includes grey seals as final hosts, sprat as the first transport host and cod as second transport host. It may be speculated that sprat obtain infection by feeding on copepods and/or cladocerans, which could serve as the first intermediate hosts. One cannot exclude the possibility that the size-dependent C. osculatum infection of cod may contribute (indirectly or directly) to the differential mortality of larger cod (>38 cm) compared to smaller cod (<30 cm) recently recorded in the Baltic cod population.

Molecular diversity of avian schistosomes in Danish freshwater snails.

Avian schistosomes are widespread parasites of snails and waterfowl and may cause cercarial dermatitis (swimmer's itch) in humans, a disease that is frequently reported in European countries. These parasites are known to occur in Denmark, but here, we applied a new approach using molecular tools to identify the parasites at species level. In order to do that, 499 pulmonate freshwater snails (Radix sp., Lymnaea stagnalis, Stagnicola sp. and Planorbarius corneus) were sampled from 12 lakes, ponds, and marshes in the greater Copenhagen area. Avian schistosome cercariae were identified by microscopy and subjected to molecular investigation by sequencing and phylogenetic analysis of the 5.8S and ITS2 ribosomal DNA for species identification. Additionally, snail hosts belonging to the genus Radix were identified by sequencing and phylogenetic analysis of partial ITS2 ribosomal DNA. Three out of 499 snails shed different species of Trichobilharzia cercariae: Trichobilharzia szidati was isolated from L. stagnalis, Trichobilharzia franki from Radix auricularia and Trichobilharzia regenti from Radix peregra. In the light of the public health risk represented by bird schistosomes, these findings are of concern and, particularly, the presence of the potentially neuro-pathogenic species, T. regenti, in Danish freshwaters calls for attention.

Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout.

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.

Effects of adjuvant Montanide™ ISA 763 A VG in rainbow trout injection vaccinated against Yersinia ruckeri.

Enteric redmouth disease (ERM) caused by the fish pathogen Yersinia ruckeri is a major threat to freshwater production of rainbow trout (Oncorhynchus mykiss) throughout all life stages. Injection vaccination of rainbow trout against Y. ruckeri infection has been shown to confer better protection compared to the traditionally applied immersion vaccination. It may be hypothesized, based on experience from other vaccines, that adjuvants may increase the protective level of ERM injection vaccines even more. Controlled comparative vaccination studies have been performed to investigate effects of the oil adjuvant Montanide™ ISA 763 A VG (Seppic) when added to an experimental Y. ruckeri bacterin (containing both biotype 1 and 2 of serotype O1). A total of 1000 fish with mean weight 19 g was divided into five different groups (in duplicated tanks 2 × 100 fish per group) 1) non-vaccinated control fish (NonVac), 2) fish injected with a commercial vaccine (AquaVac(®) Relera™) (ComVac), 3) fish injected with an experimental vaccine (ExpVac), 4) fish injected with an experimental vaccine + adjuvant (ExpVacAdj) and 5) fish injected with adjuvant alone (Adj). Injection of the experimental vaccine (both adjuvanted and non-adjuvanted) induced a significantly higher antibody (IgM) level, increased occurrence of IgM(+) cells in spleen tissue and significant up-regulation of several immune genes. Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvant as the challenge produced 100% mortality in the NonVac group, 60% mortality in both of ComVac and Adj groups and only 13 and 2.5% mortalities in the ExpVac and the ExpVacAdj groups, respectively.

Characterization of serum amyloid A (SAA) in rainbow trout using a new monoclonal antibody.

Serum amyloid A (SAA) is an integral part of the innate immune response in mammals and considered to be important during the acute phase response. The present study was undertaken to elucidate the role of SAA protein in the innate immune response of rainbow trout. A monoclonal antibody raised against a recombinant peptide of rainbow trout SAA was characterized using Western blot, dot blot, ELISA and immunohistochemistry. SAA association with high density lipoprotein (HDL) complicated band identification in Western blot, but delipidization of the SAA-HDL isolate highly increased the quality of reaction in the western blot. Rainbow trout fry (87 days post hatch) infected with Yersinia ruckeri showed a significant up-regulation of the SAA gene at 72 h post infection with an increase until 96 h post infection. Non-significant up-regulations were seen at earlier time points i.e. 4 and 24 h. The expression pattern of SAA significantly correlated to the immunohistochemical analysis of the infected fry. A weak staining was seen in liver tissue at 4 h post infection which increased in intensity during the course of infection i.e. 24, 72 and 96 h post infection.