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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.

Single nucleotide polymorphisms (SNPs) on rainbow trout chromosomes Omy16 and Omy17 are associated with a lower parasitic load and delayed-time-to-death following exposure to the parasitic ciliate Ichthyophthirius multifiliis causing white spot disease (WSD). We have evaluated the application of two quantitative trait loci (QTL) represented by two of these SNPs for practical breeding purposes. Homozygous males served as parent fish securing offspring with at least one allele (heterozygous and homozygous fish) associated with higher resistance (QTL fish). We measured the infection levels and time to morbidity/mortality in QTL fish and in non-QTL fish (male parent fish negative for the SNPs) following exposure to infective theronts. We conducted hexaplicate challenge trials (common garden experimental set-up in each tank) and recorded the development of trophonts (white spots) in the fish epidermis and associated morbidity in all six fish tanks and in both fish groups. QTL fish showed a significantly lower infection and delayed development of WSD morbidity. Analyses of SNP locations on the trout chromosomes Omy 16 and Omy 17 may in the future indicate genes associated with higher natural protection. Evidence points at immune factors, physiological functions, mucus production and regulatory elements (lncRNA and pseudogenes).

A parasitological investigation of Danish farmed freshwater (180 fish) and marine rainbow trout (170 fish), Oncorhynchus mykiss, was performed with an emphasis on the occurrence of zoonotic parasites. Reliability of the parasitological methods applied was assessed by examining fish from a stock of wild Atlantic cod Gadus morhua caught in the Sound (Øresund). While wild fish carried a number of zoonotic parasites, we found that farmed fish, both in the fresh and marine environment, were free of these parasites. Non-zoonotic trout parasites found were eye-flukes Diplostomum paracaudum, D. pseudospathaceum and Eubothrium crassum in maricultured rainbow trout. The acanthocephalan Echinorhynchus truttae and Diplostomum sp. were found in freshwater trout. In contrast, wild cod were infected with five different nematode species (Anisakis simplex, Contracaecum osculatum, Pseudoterranova decipiens, Hysterothylacium aduncum, Cucullanus cirratus), three trematode species (Lepidapedon elongatum, Cryptocotyle lingua, Hemiurus communis), one copepod species (Lernaeocera branchialis) and one myxozoan (Myxobolus aeglefini). The risk factors associated with aquaculture production in an infectious aquatic environment are analyzed and reasons for absence of zoonotic parasites in aquacultured trout are pinpointed. Based on the analysis recommendations for management practices securing parasite freedom in aquacultured fish are given.

Four specimens of mallard (Anas platyrhynchos) shot by local hunters (December 2020 to January 2021 along the eastern coastline of the island of Bornholm in the Baltic Sea) were diagnosed with a heavy load of sarcocysts in the musculature. Morphometric and molecular diagnosis based on rDNA (18 S, ITS1, 28 S) of parasites recovered from two of the birds revealed the causative pathogen to be Sarcocystis rileyi. We further present novel sequences for the entire 5.8 S and ITS2 for this species. Elongate cysts (mean length 5.25 (SD 0.6) mm, width 1.37 (SD 0.2) mm) were recorded in all parts of the striated skeletal musculature of the birds. The main part (72%) of the 2585 cysts in one female mallard was located in the outer superficial pectoral musculature, with 11% in the inner pectoral musculature. Minor but significant parts were found in the dorsal, ventral abdominal, neck and head, legs, hand and arm (wing) musculature. No cysts were found in the smooth musculature. Each cyst contained a median of 3.2 mio bradyzoites indicating that more than 8 billion bradyzoites are available for infection of one or more predators/scavengers ingesting the bird. Bradyzoites (median length 13.5 μm (range 12.1–14.5) and median width 2.66 μm (range 2.1–3.3)) were highly resistant to proteinase treatment, which secures the passage through the stomach of the predator to its intestine where wall penetration takes place. One of the birds was ringed (tagged) in Sweden Island Øland in the Baltic Sea two years before being shot. This is documenting immigration of mallards from northern locations. The parasite species was originally described in North America in 1893 and was commonly reported in this region during the 20th century but not in Europe. Recent cases from Norway, Finland, Lithuania, Poland, UK and Hungary suggest that the species may be spreading geographically. Experienced duck hunters with a 40 years record of hunting on the island reported that this type of infection unprece

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-1β, 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 immu

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.

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.

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.

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.

The name of Contracaecum osculatum and how the parasite was described.

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 (>

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 (>