AI-Driven Realtime Monitoring of Early Indicators for Ichthyophthirius multifiliis Infection of Rainbow Trout.

A novel video-based real-time system based on AI (artificial intelligence) was developed to detect clinical signs in fish exposed to pathogens. We selected a White Spot Disease model involving rainbow trout as the experimental animal and the parasitic ciliate Ichthyophthirius multifiliis as a pathogen. We compared two identical fish tank systems: one tank was infected by co-habitation, whereas the other tank was kept non-infected (sham infection). The two fish tanks were separately video monitored (full top and side view) during the course of infection, during which fish were removed whenever they developed clinical signs (direct visual inspection by the observer). Image analysis (object detection, classification and tracking) was used to track behavioural changes in fish (in every recorded video frame), focusing on movement patterns and spatial localisation. Initially, the two fish groups (infected and non-infected) exhibited similar behaviour and non-infected fish did not change behaviour during the 15 d observation period (from 5 d before infection until 10 dpi). At 4, 7, 8, 9 and 10 dpi some infected fish showed clinical signs (equilibrium disturbance, gasping and lethargy) and were removed from the experiment. Anorexia occurred from 5 dpi and a gradual progression of gasping behaviour was noted, whereas the frequency of fish flashing (rubbing/scratching against objects) was low. Equilibrium disturbances and the development of white spots in the skin appeared to be a much later (8-10 dpi at this temperature) indicator of infection. The video analysis showed a general distribution of non-infected fish in all parts of the fish tank during the entire experiment, whereas infected fish already at 4-5 dpi moved towards higher water currents in the top and bottom positions. This change of fish positioning within the tank appeared as a promising early indicator of infection. The study suggests that continuous monitoring of fish behaviour using AI can potentially optimise the timing of humane endpoints, indicate disease signs earlier and thereby improve animal welfare in both animal experimentation and in aquaculture settings.

Molecular and SEM studies on Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae).

Presenting new molecular and scanning electron microscope (SEM) features, this study gives additional data to the better knowledge of Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae), a parasite of the European catfish Silurus glanis Linnaeus, 1758 (Siluriformes, Siluridae) cultured in a commercial fish farm in Hungary. In addition, notes on the early development of sclerotized anchors are also provided. The main morphological difference of T. vistulensis compared to other congeneric species is associated with the male copulatory organ, which exhibits 5-7 loops in the middle of the penis length and a long open V-shaped sclerotized accessory piece, dividing terminally into two parts, securing the terminal part of the penis tube. The present study provides for the first time molecular characterization data based on the 2694 bp long nucleotide sequence of rDNA (ITS1, 5.8S, ITS2, and flanked with partial 18S and partial 28S) submitted in GenBank with the accession number OR916383. A phylogenetic tree based on ITS1 sequences supports a well-defined clade including T. vistulensis, forming a sister group with T. siluri, a species-specific monopisthocotylan parasite to S. glanis. The morphological characterization of T. vistulensis, especially for the male copulatory organ, together with the molecular data in the present study, extends knowledge about this monopisthocotylan species and provides new information for future phylogeny studies.

Transversotrema hafniensis n. sp. infection in Poecilia reticulata by cercariae released from Melanoides tuberculata in Denmark.

BACKGROUND: Exotic and ornamental fish are highly popular companion animals resulting in a significant transcontinental trade of fish, invertebrates and aquatic plants. A major issue is the diseases associated with these organisms, as they have a major impact on health of the fish in both public and private household aquaria. A secondary issue is the trade with these products, which potentially may expand the distribution area and spread a range of diseases to new habitats. RESULTS: We here describe how Poecilia reticulata (guppy), produced in a private household aquarium, were invaded by cercariae of an exotic trematode released by imported Melanoides tuberculata snails. The fish presented with severe clinical signs (tremor, flashing, scraping of body against objects). A standard parasitological examination and morphometric identification showed scale pocket infections with a digenean trematode species within the genus Transversotrema. Molecular identification by PCR, sequencing and phylogenetic analyses of a 2646 bp sequence encoding ribosomal RNA (partial 18 S, ITS1, 5.8 S, ITS2, partial 28 S) was performed. The 1107 bp sequence of mitochondrial DNA (cox1) showed that the parasite differed from previously described Transversotrema species in M. tuberculata. Morphometrics of adult and larval specimens of this isolate also differed from previously described freshwater species within the genus. The new species was described and is named after Copenhagen, for its geographic origin. CONCLUSIONS: The genus Transversotrema comprises a range of species, adapted to a microhabitat in scalepockets of teleosts. A combination of morphological and molecular characterization techniques has been shown to provide a good differentiation between species. The fish were not purchased from a pet shop but produced in the home aquarium. This indicated that an infection pressure existed in the aquarium, where the source of infection was found to be exotic intermediate host snails M. tuberculata, which originally were imported and purchased from a pet shop. The potential spread of fish diseases associated with trade of fish and snails to new geographic regions, where climate conditions are favourable, is discussed.

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.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

The 'jellied' or 'mushy' condition of fish in the North Atlantic and North Pacific fisheries: Characteristics, causes and consequences.

The North Atlantic and North Pacific commercial fisheries report problematic occurrences of 'jellied' or 'mushy' fish. These fish exhibit an abnormally soft and jelly-like musculature that attains a mushy consistency when cooked. The condition affects several economically important species, and is commonly termed 'jellied condition' or, specifically for halibut, 'mushy halibut syndrome'. The inferior quality of 'jellied' or 'mushy' products reduces the market value considerably, leading to wastage and losses in the fisheries. The syndrome is associated with an abnormally high moisture content and lowered protein of the white skeletal musculature, particularly the fillets. Alterations in lipid content varies depending on species investigated. In some fish species, myxozoan infections can induce similar fillet alterations, but studies on 'jellied' or 'mushy' meat indicate a non-infectious myopathy. Several hypotheses have been launched to explain the jelly-like syndrome, including dietary deficiencies, spawning exhaustion, environmental circumstances, genetics and adaptive physiology. This review provides a comprehensive overview of the characteristics of the 'jellied' or 'mushy' syndrome including a discussion of plausible etiologies and applicable mitigation strategies. The main conclusion is that the syndrome may involve two different etiologies dependent on species and location, but new studies are needed to verify past findings and rule out alternative explanations. A growing human population and an increasing demand for food requires efficient utilization of all harvested fish products. Expanded and updated knowledge is vital to reduce food waste and losses related to 'jellied' or 'mushy' fish in catches, and promote sustainable quota usage. We advocate for further research into the syndrome, including prevalence in commercial fish stocks and correlational studies with focus on association with biological parameters, chemical composition, infectious agents, geographic distribution and seasonal variation.

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.

Reproductive strategies of the parasitic flatworm Thaparocleidus vistulensis (Siwak, 1932) (platyhelminthes, monogenea) infecting the European catfish Silurus glanis Linnaeus, 1758.

The life cycle of Thaparocleidus vistulensis (Siwak, 1932), a host-specific monogenean parasite of European catfish (Silurus glanis Linnaeus, 1758), was investigated by detailed observation of infection dynamics, egg development, hatching rate and in vitro survival rates of the parasite at different life stages at 23 °C. A total of 30 naive fingerlings were infected in three exposure trials by co-habitation with donor fish carrying adult parasites. Two fish were dissected every two days during the 10-day experimental period to explore the development of larvae and juvenile parasites on the host gills. Freshly laid eggs by adult monogeneans were collected and observed daily under a light microscope until hatching. A total of 445 eggs were collected and distributed into wells of 96-well microtiter plates containing filtered fish tank water to determine their hatching rates. A similar method was used to investigate the survival rates of isolated parasites at different developmental stages (larvae, juveniles, and adults). T. vistulensis populations on the European catfish in fish tanks increased markedly within ten days, dependent on the severity of the initial infection levels of the donor fish. The first eggs hatched three to four days after oviposition, and the hatching rate peaked on the fifth day (89.7%). The survival rate for freely swimming oncomiracidia without host was 7.4% after five days, whereas isolated juvenile and adult parasites showed a higher dependence of host contact (survival rates three days post-isolation of 0.9% and 1.6%, respectively). The data allows prediction of parasite-host dynamics and may improve control of gill-disease in cultured European catfish stocks in fish farms.

Validation of a QTL associated with resistance to Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Vibriosis is a bacterial disease in fish caused by the Gram negative bacterium Vibrio anguillarum with severe impact on rainbow trout (Oncorhynchus mykiss) farming. Sustainable control methods should be developed and we here show that marker assisted selective breeding of fish naturally resistant to the disease is feasible. We have validated the use of a single nucleotide polymorphism (SNP) marker SNP AX-89,945,921 (QTL on chromosome 21). The QTL was previously found associated with resistance to vibriosis and described following a genome wide association analysis (GWAS) of trout exposed to the bacterium. For this validation spawners were genotyped by use of the 57 K Axiom®Trout Microarray (Affymetrix) and homozygous male fish carrying the allele with the SNP AX-89,945,921 were then selected and used to fertilize eggs from outbred female trout resulting in fish all carrying the SNP (QTL-fish). Control fish (non-QTL fish) were produced by fertilizing the same batch of eggs by use of male parents negative for the SNP. The fish were exposed in freshwater to V. anguillarum (water bath infection) at 19 C°. A total of 900 fish were challenged in a common garden set-up in triplicate. A bacterial solution of V. anguillarum (serotype O1) was added to each of three freshwater fish tanks, each with 150 QTL and 150 non-QTL fish. Fish were tagged by tail fin cut (upper/lower) to discern the two groups, whereafter fish were monitored around the clock to detect disease signs and remove moribund fish. Clinical vibriosis developed within two days in non-QTL-fish (overall morbidity of 70%). QTL fish developed clinical signs later and the morbidity was significantly lower and did not reach 50%. Rainbow trout farming may benefit from using the QTL associated with higher resistance towards vibriosis. The effect may be optimized in the future by use of both male and female parents homozygous for the marker allele.

Seals, fish, humans and parasites in the Baltic: ecology, evolution and history.

Evolutionary and ecological processes affecting the interactions between hosts and parasites in the aquatic environment are at display in the Baltic Sea, a young and ecologically unstable marine ecosystem, where fluctuating abiotic and biotic factors affect the parasitofauna in fish. The dynamic infections of Baltic cod, a subpopulation of the Atlantic cod (Gadus morhua Linnaeus), with third stage anisakid nematode larvae of Pseudoterranova decipiens (Krabbe, 1878) and Contracaecum osculatum (Rudolphi, 1802) have increased following a significant increase of the Baltic grey seal Halichoerus grypus (Fabricius) population in the region. Cod serves as a paratenic host and marine mammals, pinnipeds, are definitive hosts releasing parasite eggs, with faeces, to the marine environment, where embryonation and hatching of the third stage larva take place. The parasite has no obligate intermediate hosts, but various invertebrates, smaller fish and cod act as paratenic hosts transmitting the infection to the seal. Contracaecum osculatum has an impact on the physiological performance of the cod, which optimises transmission of the larva from fish to seal. Thus, a muscle mass decrease of nearly 50% may result from heavy C. osculatum infections, probably amplified by a restricted food availability. The muscle atrophy is likely to reduce the escape reactions of the fish when meeting a foraging seal. In certain regions, where fish and seals are restricted in their migration patterns, such as the semi-enclosed Baltic Sea, the predation may contribute to a severe cod stock depletion. The parasites are zoonotic and represent a human health risk, when consumers ingest insufficiently heat- or freeze-treated infected products. Marked infections of the cod were previously reported during periods with elevated seal populations (late 19th and middle 20th century) and various scenarios for management of risk factors are evaluated in an evolutionary context.

Flavobacterium psychrophilum: Response of Vaccinated Large Rainbow Trout to Different Strains.

BACKGROUND: Although Flavobacterium psychrophilum is recognized as the causative pathogen of rainbow trout fry syndrome (RTFS), often resulting in high fry mortality, it is also responsible for bacterial cold water disease (BCWD) in large and older rainbow trout (Oncorhynchus mykiss). These older fish do not experience high mortality, but sustain, through the shedding of bacteria, a constant infection pressure at farm level, which exposes fry to an unnecessary infection risk. We have produced and assessed the immunogenicity of an experimental injection BCWD vaccine, which may be used to decrease the shedding of bacteria from older fish. METHODS: A total of 800 fish were i.p.-injected: 200 fish received the bacterin with adjuvant, 200 fish received the bacterin alone, 200 fish received adjuvant alone and 200 fish were injected with physiological saline. Blood samples were taken at day 0 and at three different time points (4, 8 and 14 weeks) post-vaccination. Plasma antibody levels were measured by ELISA for reactivity against both the homologous F. psychrophilum vaccine strain (serotype Fd) and heterologous strains (serotype Th). RESULTS: Significantly elevated antibody titers were found against all serotypes in vaccinated fish. Welfare parameters associated with the vaccination process were evaluated by analyzing trout plasma samples for six different biochemical parameters, but no adverse effects associated with injection were indicated. CONCLUSIONS: The study suggests that an injection vaccine containing formalin-inactivated whole cells of F. psychrophilum (serotype Fd), adjuvanted with FIA, may also induce protection against heterologous strains. We advocate for, as the next step, the performance of field trials evaluating if the vaccination of older rainbow trout will (1) reduce the infection pressure in farms, (2) elevate the general health level in all groups and (3) minimize F. psychrophilum infection in fry at farm level. This may reduce the need for the administration of antibiotics in all age classes.

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.

Control of parasitic diseases in aquaculture.

Finfish aquaculture in freshwater and marine environments is continuously expanding globally, and the potential for a substantial further increase is well documented. The industry is supplying fish products for human consumption to the same extent as capture fisheries, and new fish species for domestication are still being selected by the industry. The challenge faced by all aquacultured species, classical and novel, is the range of pathogens associated with each new fish type. A fish host in its natural environment carries a series of more or less specific parasites (specialists and generalists). Some of these show a marked ability to propagate in aquaculture settings. They may then elicit disease when infection intensities in the confined aquaculture environment reach high levels. In addition, the risk of transmission of parasites from aquaculture enterprises to wild fish stocks adds to the parasitic challenge. Control programmes of various kinds are needed and these may include chemotherapeutants and medicines as the farmer's first and convenient choice, but mechanical, biological, immunological and genetic control methods are available solutions. New methods are still to be developed by scrutinizing the life cycle of each particular parasite species and pin-pointing the vulnerable stage to be targeted. As parasites exhibit a huge potential for adaptation to environmental changes, one must realize that only one approach rarely is sufficient. The present work therefore elaborates on and advocates for implementation of integrated control strategies for diseases caused by protozoan and metazoan parasites.

Small, charged proteins in salmon louse (Lepeophtheirus salmonis) secretions modulate Atlantic salmon (Salmo salar) immune responses and coagulation.

Little is known about glandular proteins secreted from the skin- and blood-feeding ectoparasite salmon louse (Lepeophtheirus salmonis). The labial gland has ducts extending into the oral cavity of the lice, and the present study aimed to identify novel genes expressed by this gland type and to investigate their role in modulation of host parameters at the lice feeding site. Five genes associated with labial gland function were identified and named Lepeophteirus salmonis labial gland protein (LsLGP) 1-4 and 1 like (LsLGP1L). All LsLGPs were predicted to be small charged secreted proteins not encoding any known protein domains. Functional studies revealed that LsLGP1 and/or LsLGP1L regulated the expression of other labial gland genes. Immune dampening functions were indicated for LsLGP2 and 3. Whereas LsLGP2 was expressed throughout the parasitic life cycle and found to dampen inflammatory cytokines, LsLGP3 displayed an increased expression in mobile stages and appeared to dampen adaptive immune responses. Expression of LsLGP4 coincided with moulting to the mobile pre-adult I stage where hematophagous feeding is initiated, and synthetic LsLGP4 decreased the clotting time of Atlantic salmon plasma. Results from the present study confirm that the salmon louse secretes immune modulating and anti-coagulative proteins with a potential application in new immune based anti-salmon louse treatments.

Neutrophils and aquatic pathogens.

INTRODUCTION: Neutrophilic granulocytes are short-lived cells continuously circulating in the vascular system of vertebrates. They play a basic and decisive role in the innate immune defence of the hosts against all types of pathogenic microorganisms. METHODS: Based on a literature study, the functions of neutrophils and cells with similar functions are described. The study places special emphasis on organisms in the aquatic environment and the pathogens occurring in that particular environment. RESULTS: The evolutionary origin of this specific cell type is not clear, but its most basic traits (recognition of foreign elements, extracellular trap release, phagocytosis and elimination of ingested material) are found in phagocytes in members of evolutionary ancient invertebrate groups spanning from amoebae, sponges, sea-anemones, mollusks (snails and mussels), arthropods (crustaceans and insects) to echinoderms (sea stars and sea urchins). Their functions as innate immune sentinels and effector cells in these groups are well described. Neutrophilic granulocytes with elongated and lobed nuclei (possibly allowing cell movements through narrow extracellular spaces and leaving space for phagosomes) occur in vertebrates including fish, amphibians, reptiles, birds and mammals although the morphology of the nucleus, stainability of cytoplasmic granula, and the antimicrobial armament vary among groups. Following the pathogen invasion of a fish host, the neutrophils migrates from the vascular system into the infection focus. They apply their PRRs (including TLRs) to recognize the invader as non-self, produce netosis by casting extracellular chromatin containing traps in the microenvironment. These nets assist the immobilization of invading microbes and prevents their further spread. The cells attach to and engulf the microbes by phagocytosis, whereafter they eliminate the pathogen in phagolysosomes equipped with a range of killing mechanisms and attract, by release of chemokines, additional immune cells (monocytes, macrophages and lymphocytes) to the site of invasion. Their role in innate immunity of fish hosts towards aquatic pathogens has been elucidated by in vivo and in vitro studies. Neutrophils interact with virus (e.g. IPNV and VHSV), bacteria (e.g. Aeromonas, Vibrio, Edwardsiella, Mycobacterium and Renibacterium) and parasites, including monogeneans (Gyrodactylus), cestodes (Diphyllobothrium), trematodes (Diplostomum) and ciliates (Ichthyophthirius and Philasterides). Despite the decisive function of neutrophils in innate immunity and early protection, the excessive production of ROS, RNS and NETs may lead to pathological disturbances in the host, which are exacerbated if the pathogens evolve immune evasion mechanisms. CONCLUSION: Neutrophils in aquatic organisms play a central role in innate immunity but may serve as a toll and a support in acquired protection. The strong impact of the cellular reactions not only on pathogen but also on host tissues emphasizes that an optimal immune reaction is balanced, involves targeted and specific effector mechanisms, which leaves a minimum of collateral damage in host organs.

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.

Toxicity of the antiparasitic lipopeptide biosurfactant SPH6 to green algae, cyanobacteria, crustaceans and zebrafish.

A lipopeptide with biosurfactant properties produced by the bacterium Pseudomonas H6 (SPH6) has antiparasitic effects and may serve as an alternative to chemotherapeutants against aquatic pathogens in aquaculture. We have elucidated its ecotoxicological potential by short-term standardized tests, including a growth rate inhibition test with algae (Raphidocelis subcapitata), a lethality test on the cyanobacteria Phormidium autumnale, a lethality test using crustaceans (Daphnia magna), a fish embryo acute toxicity test and a fish acute toxicity test using zebrafish (Danio rerio). The decrease of the biosurfactant concentration in zebrafish test water during 24 h was measured. The toxicity for crustaceans was highest (LC50 = 20 mg/L), followed by the test with the zebrafish embryo (LC50 = 27 mg/L). The juvenile zebrafish fish (complete mortality occurred between 40 and 80 mg/L), the cyanobacteria (LC50 = 80 mg/L) and the green algae (EC50 = 170 mg/L) showed higher tolerance. The determination of SPH6 concentrations in fish tank (up to 50% elimination over 24 h) suggested that the compound may become adsorbed to tank walls, absorbed by fish or degraded. Further studies should determine its impact under different environmental settings (e.g. temperature) relevant for different branches of the aquaculture sector.

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.

Cercarial Dermatitis at Public Bathing Sites (Region Zealand, Denmark): A Case Series and Literature Review.

During recent years, we have observed an increasing occurrence of cercarial dermatitis in Denmark. We here describe 5 new cases from 2019 to 2020 associated with bathing in lakes Esrum sø, Furesø, and Ringen with emphasis on clinical symptoms and their relation to previous exposure to bird schistosome cercariae. In 2020, 2 patients from Furesø suffered from different severity of clinical symptoms after morning bathing in the same lake. We suggest that the differential symptoms may be explained by primary versus secondary exposure to the immunogenic pathogen.

Evidence of IgE-Mediated Cross-Reactions between Anisakis simplex and Contracaecum osculatum Proteins.

Fish consumers may develop allergic reactions following the ingestion of fish products containing nematode larvae within the genus Anisakis. Sensitized patients may cross-react with proteins from insects, mites and mollusks, leading to allergic reactions even in the absence of the offending food. Potential cross-reactivity in Anisakis-allergic patients with larval proteins from other zoonotic parasites present in freshwater and sea fish should be investigated due to an increasing occurrence in certain fish stocks, particularly Contracaecum osculatum. In this work, we evaluated IgE-cross reactions by in vivo (skin prick tests with parasites extracts) and in vitro methods (IgE-ELISA and IgE-immunoblot). In vivo skin prick tests (SPT) proved the reactivity of Anisakis-sensitized patients when exposed to C. osculatum antigens. Sera from Anisakis-sensitized patients confirmed the reaction with somatic antigens (SA) and excretory/secretory proteins (ES) from C. osculatum. Only anecdotal responses were obtained from other freshwater worm parasites. Consequently, it is suggested that Anisakis-sensitized humans, especially patients with high levels of specific anti-Anisakis antibodies, may react to C. osculatum proteins, possibly due to IgE-mediated cross-reactivity.