The early ontogenetic development of immune cells and organs in teleosts.

Fully developed teleosts possess a highly developed immune system comprising both innate and adaptive elements, but when hatching from the egg, the yolksac larva is still at an ontogenetically incomplete stage with regard to physiological, including immunological, functions. The immune system in these young fish stages is far less developed when compared to the youngs appearing from reptile and avian eggs and from most mammals at parturition. In those vertebrate groups the early ontogenetic development of the fetus is highly protected. The lack of a fully developed immune system in yolksac larvae of fish is critical, because this stage encounters a potentially hostile and infectious aquatic environment. The strong selective pressure on the immune system of the yolksac larva and the youngest fry stages explains the existence of a multi-facetted innate system, which is protecting the young fish stages against viral, bacterial and parasitic infections. The sequential development of immune cells and organs depends on host species and its environmental setting. However, a strong armament comprising innate cells (neutrophilic granulocytes, macrophages) and molecules (receptors, lectins, complement, AMPs and constitutively expressed immunoglobulins) protect the earliest stages. The adaptive immune elements, including T-cells and B-cells, occur gradually in headkidney, spleen, thymus, tonsils, bursa equivalent (if present) and mucosa associated lymphoid cells. A functional protective response following immunization occur later.

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.

Whole-genome association study searching for QTL for Aeromonas salmonicida resistance in rainbow trout.

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, has extensive negative effects on wild and farmed salmonids worldwide. Vaccination induces some protection under certain conditions but disease outbreaks occur even in vaccinated fish. Therefore, alternative disease control approaches are required to ensure the sustainable expansion of rainbow trout aquaculture. Selective breeding can be applied to enhance host resistance to pathogens. The present work used genome-wide association study (GWAS) to identify quantitative trait loci (QTL) associated with A. salmonicida resistance in rainbow trout. A total 798 rainbow trout exposed to A. salmonicida by bath challenge revealed 614 susceptible and 138 resistant fish. Genotyping was conducted using the 57 K single nucleotide polymorphism (SNP) array and the GWAS was performed for survival and time to death phenotypes. We identified a QTL on chromosome 16 and located positional candidate genes in the proximity of the most significant SNPs. In addition, samples from exposed fish were examined for expression of 24 immune-relevant genes indicating a systematic immune response to the infection. The present work demonstrated that resistance to A. salmonicida is moderately heritable with oligogenic architecture. These result will be useful for the future breeding programs for improving the natural resistance of rainbow trout against furunculosis.

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.

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.

Contracaecum osculatum (sensu lato) infection of Gadus morhua in the Baltic Sea: inter- and intraspecific interactions.

The subpopulation of Atlantic cod, Gadus morhua, in the eastern part of the Baltic Sea has experienced a significant increase in infections with anisakid nematode larvae of the species Contracaecum osculatum sensu lato (s.l.) since the year 2000. The life cycle of the parasite includes seals and especially the grey seal, Halichoerus grypus, as final hosts, carrying the adult nematodes in the stomach, crustaceans (copepods, amphipods) as first intermediate hosts and various fish species (clupeids, sandeel) including cod as second intermediate/paratenic hosts. Cod with a body length below 28 cm are generally non-infected but experience increasing infection levels when they switch to a piscine diet (infected intermediate/paratenic hosts). We present an overall frequency distribution analysis of worms in 166 cod (body length 30-49 cm) collected in the spawning area over the last 5 years. It shows a fit to the negative binomial distribution, a prevalence of infection of 89.8%, a mean intensity of 29.3 parasites per fish (range 1-377) and a variance/mean ratio of 59.2 (≫1), indicating overdispersion. We present measurements of the adult Contracaecum osculatum (s.l.) specimens in the seal stomach and show that the parasites reach a maximum length of 6.6 cm (females) and 5.8 cm (males). L3s in sprat have a total length from 1to 11 mm whereas the larvae in cod liver are 3-27 mm. A decreasing mean worm length associated with high worm densities in cod (number of nematodes per liver) was recorded. Possible explanations might include timing of feeding on infected intermediate/paratenic hosts, intraspecific competition (crowding) between larvae in cod and host responses (indicated by a significant antibody production in cod against C. osculatum (s.l.) antigens). A significant negative correlation between infection intensity and muscle mass of cod was found, suggesting parasite-induced down-regulation of growth factors in cod.

A Major QTL for Resistance to Vibrio anguillarum in Rainbow Trout.

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout (Oncorhynchus mykiss). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 107 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish - including survivors - and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.