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.

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.

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.

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.

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.

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.

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.

Location and elimination of Anisakis simplex third stage larvae in Atlantic herring Clupea harengus L.

We here describe the location of anisakid third stage larvae in Atlantic herring Clupea harengus L. caught in the North Sea in August 2023. We further demonstrate how industrial processing (mechanical gutting, removal of entrails, head, tail, hypaxial anterior musculature and vertebral column) reduces the overall infection and worm load in the musculature. The isolated anisakid larvae were identified as Anisakis simplex sensu stricto by a combination of morphometrics and molecular methods (PCR of rDNA and mtDNA, sequencing, BLAST analysis). As a baseline we examined a total of 75 specimens of freshly caught and ungutted herring and showed a positive correlation between host size (fish length and weight) and infection level. The overall prevalence of infection was 84 %, the mean intensity 11.3 (range 1-38 parasites per fish) and the abundance 9.52. The main part of the overall worm population was associated with stomach and pyloric caeca in the body cavity (77 %) and only 5 % was found in the musculature. Larvae occurred in the hypaxial part of the musculature (21), the epaxial part (7 worms) and the caudal part (5 worms). The prevalence of muscle infection was 28 % and the mean intensity 1.6 (range 1-5) parasites per fish and abundance 0.44 parasites per fish. In order to assess the effect of industrial processing on worm occurrence in the fish we examined a total of 67 specimens of herring, from exactly the same batch, but following processing. This included removal of organs in the body cavity, cutting the lower part of the hypaxial segment but leaving the right and left musculature connected by dorsal connective tissue. Five out of these fish carried one larva (prevalence 7.5%, mean intensity 1, abundance 0.07 larvae per fish), and these worms were located in the ventral part of the anterior musculature (2), in the central part of the anterior musculature (2) and one larva in the central part of the caudal musculature. The industrial processing reduced the overall occurrence (abundance) of worms in the fish from 9.52 to 0.07 (136 times reduction) and the occurrence in the musculature from 0.44 to 0.07 (6.28 times reduction). The overall prevalence was reduced from 84 % to 7.5 % (11.2 times reduction). Muscle infection prevalence fell from 28 % to 7.5 % (3.7 times reduction). We then followed another batch of herring following a marinating process (11% NaCl for 24 h and subsequent incubation in acetic acid and vinegar) by artificially digesting the flaps during week 1-8. Although a total of 31 larvae were recovered from 144 fish examined no live nematode larvae were isolated. The importance of fish handling, processing and marination for consumer safety is discussed.

Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss).

Excretory/secretory (ES) products are molecules produced by parasitic nematodes, including larval Anisakis simplex, a parasite occurring in numerous marine fish hosts. The effects of these substances on host physiology have not been fully described. The present work elucidates the influence of ES substances on the fish immune system by measuring immune gene expression in spleen and liver of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with ES products isolated from A. simplex third stage larvae. The overall gene expression profile of exposed fish showed a generalized down-regulation of the immune genes tested, suggesting a role of ES proteins in immunomodulation. We also tested the enzymatic activity of the ES proteins and found that lipase, esterase/lipase, valine and cysteine arylamidases, naphthol-AS-BI-phosphohydrolase and α-galactosidase activities were present in the ES solution. This type of hydrolytic enzyme activity may play a role in nematode penetration of host tissue. In addition, based on the notion that A. simplex ES products may have an immune-depressive effect (by minimizing immune gene expression) it could also be suggested that worm enzymes directly target host immune molecules which would add to a decreased host immune response and increased worm survival.

Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae.

Basic immune response mechanisms in vertebrates against helminths are still poorly understood. Fish-nematode models may prove valuable for elucidation of this question. In this study we orally challenged rainbow trout (Oncorhynchus mykiss) with larvae of Anisakis simplex (Nematoda: Anisakidae) and subsequently investigated the expression of 18 immune relevant genes in spleen and liver 1, 4 and 8days post infection (d.p.i.). Gene expression data were analysed with regard to the infection status of the challenged rainbow trout at the time of necropsy; "worms rejected" (÷worms), "worms present" (+worms) and a combined group consisting of samples pooled from both previous groups (÷/+worms). No significant regulation of cytokine genes was recorded but fish which had rejected worms up-regulated the CD4 gene (6.1-fold change, 8d.p.i.) in liver. The gene encoding CD8 was significantly down-regulated 24h post challenge in livers in fish still carrying worms (2.7-fold change) but not in the worm-free group. The immunoglobulin gene IgM was significantly down-regulated (2.9-fold change, 8d.p.i.) in liver samples from the +worms group. Complement factor C3 and precerebellin genes were significantly up-regulated twofold in liver samples from infected fish 4d.p.i. Significant up-regulation of the acute-phase protein SAA was observed in all three groups and in both tissues. To our knowledge, this is the first study to describe the expression of immune genes in a fish host challenged with live nematode larvae.

Comparative infectivity of three larval nematode species in three different salmonids.

Host specificity of parasites may depend both on ecological and physiological factors. Basic descriptions of the susceptibility/resistance of fish to specific nematodes are needed in order to reveal mechanisms in the host-parasite relation. Rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and Atlantic salmon (Salmo salar) were orally infected with larval stages of three different anisakid nematodes-Hysterothylacium aduncum, Contracaecum osculatum, and Anisakis simplex-and parasite survival and location was subsequently recorded for up to 14 days post infection (dpi). H. aduncum was most prevalent and numerous in brown trout 2 dpi, but a large proportion of the worms were recovered dead. No tissue penetration was observed. Rainbow trout exhibited the highest susceptibility to C. osculatum larvae at 2, 7, and 14 dpi. Mean intensities and mean abundances were lower in brown trout and salmon at all time points. The pyloric cecum was penetrated in rainbow trout on two occasions. A. simplex larvae established more successfully in salmon compared to rainbow trout; brown trout exhibited the highest natural resistance. Mean intensity and mean abundance was highest in rainbow trout at 2 and 7 dpi, but not after 14 days. A range of tissues, including muscle and liver, were found penetrated by larvae, but the pyloric ceca were the preferred microhabitat for Anisakis in both rainbow trout and salmon. It can be concluded that closely related salmonids differ in susceptibility towards different anisakid larvae and these parasites select different microhabitats in the hosts. The physiological basis for this specificity is discussed.

Myxobolus groenlandicus n. sp. (Myxozoa) distorting skeletal structures and musculature of Greenland halibut Reinhardtius hippoglossoides (Teleostei: Pleuronectidae).

A specimen of Greenland halibut Reinhardtius hippoglossoides (Walbaum, 1792) caught on the west coast of Greenland (Qasigiannguit) was found to possess serious pathological changes in the body musculature. A series of cartilaginous cylindrical structures organized symmetrically at the position of the proximal pterygiophores had changed the musculature and produced irreversible distortions (cavities and holes) in the fillet of the processed fish, leaving it with no value for the industry. Histopathological investigation showed that these structures consisted of hypertrophic cartilage containing numerous myxospore-producing plasmodia. Morphometric and molecular analyses of the parasites showed that both spore morphology and rDNA sequences complied with characteristics of the genus Myxobolus, but no full affiliation with a known species could be found. The parasite is a previously undescribed species, and the name Myxobolus groenlandicus n. sp. is assigned to this new myxobolid.

Responses towards eyefluke (Diplostomum pseudospathaceum) in different genetic lineages of rainbow trout.

Marker-assisted selective breeding of fish with higher levels of resistance towards specific pathogens may improve fish health, but the impact of host genotype on susceptibility to multiple pathogen infections is still poorly investigated. This study examined the resistance in rainbow trout Oncorhynchus mykiss towards infection with the eye fluke Diplostomum pseudospathaceum. We used genetically selected rainbow trout, carrying SNPs associated with resistance towards the parasitic ciliate Ichthyophthirius multifiliis, and exposed the fish to eye fluke cercariae. We showed that fish partly resistant to I. multifiliis were more susceptible to eye fluke invasion. The expression of immune relevant genes (encoding innate and adaptive factors) was also affected as these genotypes responded less strongly to a secondary fluke infection. The complexity of genome architecture in disease resistance towards multiple pathogens is discussed.

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies.

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.