Introduced European bison (Bison bonasus) in a confined forest district: A ten year parasitological survey.

In the year 2012 a total of seven individuals (one bull and six cows) of European bison (Bison bonasus) were introduced to a fenced 200 ha area in a forest district (Almindingen, Island Bornholm, Denmark) in the Baltic Sea. In 2017 the herd was supplemented by introduction of another bull. The animals all originated from a population in the Polish Bialowieza forest. Faecal samples were recovered with intervals and subjected to a copro-parasitological investigation (applying egg flotation, sedimentation and Baermann technique). In addition, parasites were sampled during necropsy of animals euthanized or found dead three years after introduction. The animals carried a range of parasite types including trematodes (liver fluke Fasciola and rumen fluke), cestodes (Moniezia), nematodes (Dictyocaulus viviparus, trichurids, and other gastrointestinal nematodes (GIN) including Trichostrongylus axei and Haemonchus contortus). Egg and larval morphology also suggested presence of GIN genera Nematodirus, Ostertagia and Cooperia. The trematodes did not establish a life cycle in the recipient area, as judged by examination of calves born in the new area, but the cestode and several nematode species occurred in these animals. Especially Dictyocaulus viviparus was successfully spread and occurred together with GIN at moderate to high infection intensities at most sampling points. The reproduction among bison in the herd was high during the first few years. The exact birth rate is unknown but up to 24 calves are believed to be born during the ten year study period. Mortality among adults and calves occurred and in early June 2022 the total number of live animals was 11 (seven males, three females, one calf). The possible impact of the parasite infections on the bison health and future wildlife infection status is discussed.

Avian schistosome species in Danish freshwater lakes: relation to biotic and abiotic factors.

Due to the increased prevalence of human infections with bird schistosome larvae (cercarial dermatitis) associated with bathing in Danish lakes, a nationwide survey of infected intermediate host snails was conducted in 2018-2020. Pulmonate snails (10,225 specimens) were collected from 39 freshwater lakes (in the four major geographic regions in Denmark) and subjected to shedding. Released schistosome cercariae were isolated and identified by polymerase chain reaction and sequencing whereby Trichobilharzia regenti, Trichobilharzia franki, Trichobilharzia szidati and Trichobilharzia anseri were recorded. Infections were primarily determined by biotic factors such as the presence of final host birds and intermediate host snails and water temperature was noted as an important abiotic parameter associated with the infection. No clear connection with other abiotic factors (conductivity, alkalinity, pH, nitrogen, phosphorous) was seen. The widespread occurrence of infected snails, when compared to previous investigations, suggests that climate changes at northern latitudes could be responsible for the increased risk of contracting cercarial dermatitis.

Nasal localization of a Pseudoterranova decipiens larva in a Danish patient with suspected allergic rhinitis.

Pseudoterranoviasis is a zoonotic disease caused by nematode larvae of species within the genus Pseudoterranova (seal worm, cod worm). Most infections are gastrointestinal, oesophageal or pharyngeal, but here we report a nasal infection. A 33-year-old patient suffering from rhinitis for 1.5 years recovered a worm larva from the nose. Diagnosis was performed by morphological and molecular characterization, showing the causative agent to be a third-stage larva of Pseudoterranova decipiens (sensu stricto). Various infection routes are discussed.

Quantitative trait loci (QTL) associated with resistance of rainbow trout Oncorhynchus mykiss against the parasitic ciliate Ichthyophthirius multifiliis.

The parasitic ciliate Ichthyophthirius multifiliis has a low host specificity eliciting white spot disease (WSD) in a wide range of freshwater fishes worldwide. The parasite multiplies rapidly whereby the infection may reach problematic levels in a host population within a few days. The parasite targets both wild and cultured fish but the huge economic impact of the protozoan is associated with mortality, morbidity and treatment in aquacultural enterprises. We have investigated the potential for genetic selection of WSD-resistant strains of rainbow trout. Applying the DNA typing system Affymetrix® and characterizing the genome of the individual fish by use of 57,501 single nucleotide polymorphisms (SNP) and their location on the rainbow trout chromosomes, we have genetically characterized rainbow trout with different levels of natural resistance towards WSD. Quantitative trait loci (QTL) used for the selection of breeders with specific markers for resistance are reported. We found a significant association between resistance towards I. multifiliis infection and SNP markers located on the two specific rainbow trout chromosomes Omy 16 and Omy 17. Comparing the expression of immune-related genes in fish-with and without clinical signs-we recorded no significant difference. However, trout surviving the infection showed high expression levels of genes encoding IgT, T-cell receptor TCRß, C3, cathelicidins 1 and 2 and SAA, suggesting these genes to be associated with protection.

Primary immunization using low antigen dosages and immunological tolerance in rainbow trout.

Enteric redmouth disease (ERM), caused by the Gram negative enterobacterium Yersinia ruckeri, affects farming of salmonids, but vaccination against ERM confers a certain degree of protection dependent on the administration route. Recent studies on oral vaccination of rainbow trout suggest that immunological tolerance may be induced by primary immunization using a low antigen dosage. We have examined if low dosages of Y. ruckeri antigens, applied in feed or bath exposure over a prolonged period of time, leave rainbow trout more susceptible to infection. Groups of rainbow trout were immunized, either by immersion or feeding using different vaccine dosages, and subsequently challenged by live Y. ruckeri. Survival was recorded and immune reactions in surviving fish were evaluated (ELISA and qPCR). Trout, bath-vaccinated in a highly diluted vaccine or fed the same amount of bacterin in feed over 10 days, were not protected against Y. ruckeri challenge infection and in some cases these sub-optimally immunized fish experienced lower survival compared to non-primed controls. Genes encoding FoxP3 and immune-suppressive cytokines were down-regulated in fish vaccinated with a high antigen dosage when compared to groups exposed to low antigen dosages, suggesting a higher regulatory T cell activity in the latter fish groups. The study suggests that repeated exposure to low antigen concentrations induces some degree of immune tolerance in rainbow trout and we recommend application of high antigen dosages for primary immunization of trout.

Baltic cod endohelminths reflect recent ecological changes.

We suggest helminthological investigations of cod as a supplement to traditional biological and hydrographical methods for elucidation of ecological changes in the Baltic Sea. It is under discussion if oxygen deficit or seal abundance should explain the present critical situation of Baltic cod. A comparative investigation of endoparasitic helminths in Baltic cod (Gadus morhua), captured in the same marine habitat with an interval of 35 years (1983/2018) recorded 11 species of helminths comprising trematodes (Hemiurus luehei, Podocotyle atomon, Lepidapedon elongatum), nematodes (Contracaecum osculatum, Hysterothylacium aduncum, Capillaria gracilis, Cucullanus cirratus), cestodes (Bothriocephalus sp.) and acanthocephalans (Echinorhynchus gadi, Pomphorhynchus laevis, Corynosoma semerme). Significant prevalence and intensity increases were recorded for third-stage larvae of the nematode C. osculatum (liver location) and larvae of C. semerme (encapsulated in viscera). Both parasite species use grey seal as their final host, indicating the recent expansion of the Baltic seal population. A lower E. gadi intensity and an increased prevalence of L. elongatum of small cod (31-40 cm body length) suggest a lowered intake of amphipods (intermediate host) and elevated ingestion of polychaetes, respectively, but no significant changes were seen for other helminths.

Local immune depression in Baltic cod (Gadus morhua) liver infected with Contracaecum osculatum.

Third-stage larvae of the anisakid nematode Contracaecum osculatum infecting cod (Gadus morhua) liver elicit a host immune response involving both innate and adaptive factors, but the reactions differ between liver and spleen. Inflammatory reactions occur in both liver and spleen, but a series of immune effector genes are downregulated in liver infected with nematodes whereas these genes in spleen from the same fish are upregulated. A series of novel primer and probe sets targeting cod immune responses were developed and applied in a real-time quantitative polymerase chain reaction set-up to measure the expression of immune-relevant genes in liver and spleen of infected and uninfected cod. In infected liver, 12 of 23 genes were regulated. Genes encoding cytokines associated with inflammatory reactions (IL-1ß, IL-6, IL-8) were significantly upregulated, whereas genes encoding effector molecules, assisting the elimination of pathogens, C-reactive protein (CRP)-PII, hepcidin, lysozyme G1, lysozyme G2, C3 and IgDm, were significantly downregulated. The number of downregulated genes increased with the parasite burden. In spleen, 14 of 23 immune genes showed significant regulation and nine of these were upregulated, including genes encoding CRPI, CRPII, C3, hepcidin and transferrin. The general gene expression level was higher in spleen compared to liver, and although inflammation was induced in nematode-infected liver, the effector molecule genes were depressed, which suggests a worm-induced immune suppression locally in the liver.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis.

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 µg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 µg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 µg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.

Contracaecum osculatum and other anisakid nematodes in grey seals and cod in the Baltic Sea: molecular and ecological links.

Populations of grey seals (Halichoerus grypus), sprats (Sprattus sprattus) and cod (Gadus morhua) in the Baltic Sea are relatively stationary. The present work, applying classical and molecular helminthological techniques, documents that seals and cod also share a common parasite, the anisakid nematode Contracaecum osculatum, which uses seals as the final host and fish as transport hosts. Sequencing mitochondrial genes (COX1 and COX2) in adult worms from seals and third-stage larvae from livers of Baltic fish (sprats and cod), showed that all gene variants occur in both seals and fish. Other anisakid nematodes Pseudoterranova decipiens and Anisakis simplex are also found in both seals and cod in the Baltic Sea, but at much lower rates. The Baltic grey seal population was left at a critically low level (comprising a few hundred individuals) during the latter part of the 20th century, but since the year 2000 a marked increase in the population has been observed, reaching more than 40,000 individuals at present. Ecological consequences of the increased seal abundance may result from increased predation on fish stocks, but recent evidence also points to the influence of elevated parasitism on fish performance. Contracaecum osculatum larvae preferentially infect the liver of Baltic cod, considered a vital organ of the host. Whereas low prevalences and intensities in cod were reported during the 1980s and 1990s, the present study documents 100% prevalence and a mean intensity of above 80 worms per fish. Recent studies have also indicated the zoonotic potential of C. osculatum larvae in fish, following the consumption of raw or under-cooked fish. Therefore the present work discusses the impact of parasitism on the cod stock and the increasing risk for consumer health, and lists possible solutions for control.

Secondary immune response of rainbow trout following repeated immersion vaccination.

Teleosts are able to raise a protective immune response, comprising both innate and adaptive elements, against various pathogens. This is the basis for a widespread use of vaccines, administered as injection or immersion, in the aquaculture industry. It has been described that repeated injection vaccination of fish raises a secondary immune response, consisting of rapid, accelerated and increased antibody reaction. This study reports how rainbow trout responds to repeated immersion vaccination against yersiniosis (ERM) caused by the bacterial pathogen Yersinia ruckeri. It was found that rainbow trout does not raise a classical secondary response following repeated immersion vaccination. Serum antibody titres were merely slightly increased even after three immunizations, using 30-s immersion into a bacterin consisting of formalin-inactivated Y. ruckeri (serotype O1, biotypes 1 and 2), performed over a 3-month period. The densities of IgM-positive lymphocytes in spleen of fish immunized three times were increased compared to control fish, but no general trend for an increase with the number of immunizations was noted. The lack of a classical secondary response following repeated immersion vaccination may partly be explained by limited uptake of antigen by immersion compared to injection.

Effect of ES products from Anisakis (Nematoda: Anisakidae) on experimentally induced colitis in adult zebrafish.

Inflammatory bowel disease (IBD) in developed countries is linked with elevated hygienic standards. One of the several factors involved in this question may be reduced exposure to the immunomodulatory effects of parasitic helminths. Several investigations on treatment of mice and humans with helminth-derived substances have supported this notion, but underlying mechanisms remain unclear. This study therefore dissects to what extent a series of immune-related genes are modulated in zebrafish with experimentally induced colitis following exposure to excretory-secretory (ES) products isolated from larval Anisakis, a widely distributed fish nematode. Adult zebrafish intrarectally exposed to the colitis-inducing agent TNBS developed severe colitis leading to 80% severe morbidity, but if co-injected (ip) with Anisakis ES products, the morbidity rate was 50% at the end of the experiment (48 hours post-exposure). Gene expression studies of TNBS-treated zebrafish showed clear upregulation of a range of genes encoding inflammatory cytokines and effector molecules and some induction of genes related to the adaptive response. A distinct innate-driven immune response was seen in both TNBS and TNBS + ES groups, but expression values were significantly depressed for several important pro-inflammatory genes in the TNBS + ES group, indicating protective mechanisms of Anisakis ES compounds on intestinal immunopathology in zebrafish.

Extrusion of Contracaecum osculatum nematode larvae from the liver of cod (Gadus morhua).

Baltic cod livers have during recent years been found increasingly and heavily infected with third-stage larvae of Contracaecum osculatum. The infections are associated with an increasing population of grey seals which are final hosts for the parasite. Heavy worm burdens challenge utilization and safety of the fish liver products, and technological solutions for removal of worms are highly needed. We investigated the attachment of the worm larvae in liver tissue by use of histochemical techniques and found that the cod host encapsulates the worm larvae in layers of host cells (macrophages, fibroblasts) supported by enclosures of collagen and calcium. A series of incubation techniques, applying compounds targeting molecules in the capsule, were then tested for their effect to induce worm escape/release reactions. Full digestion solutions comprising pepsin, NaCl, HCl and water induced a fast escape of more than 60% of the worm larvae within 20 min and gave full release within 65 min but the liver tissue became highly dispersed. HCl alone, in concentrations of 48 and 72 mM, triggered a corresponding release of worm larvae with minor effect on liver integrity. A lower HCl concentration of 24 mM resulted in 80% release within 35 min. Water and physiological saline had no effect on worm release, and 1% pepsin in water elicited merely a weak escape reaction. In addition to the direct effect of acid on worm behaviour it is hypothesised that the acid effect on calcium carbonate in the encapsulation, with subsequent release of reaction products, may contribute to activation of C. osculatum larvae and induce escape reactions. Short-term pretreatment of infected cod liver and possibly other infected fish products, using low acid concentrations is suggested as part of a technological solution for worm clearance as low acid concentrations had limited macroscopic effect on liver integrity within 35 min.

Rainbow trout (Oncorhynchus mykiss) immune response towards a recombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliis.

The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n-terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I-antigen in future vaccine trials.

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model.

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.

Effect of hydrogen peroxide and/or Flavobacterium psychrophilum on the gills of rainbow trout, Oncorhynchus mykiss (Walbaum).

The immune response and morphological changes in the gills of rainbow trout fry after immersion in hydrogen peroxide (H2O2), Flavobacterium psychrophilum or combined exposure were examined. The gills were sampled 4, 48, 125 and 192 h after exposure, and the regulation of expression of the following genes was investigated using qPCR: IgT, IgM, CD8, CD4, MHC I, MHC II, IL-4/13A, TcR-ß, IL-10, IL-1ß, IL-17, SAA and FoxP3. Bacteria were not observed in haematoxylin-and-eosin-stained gill tissue, but the presence of F. psychrophilum 16S rRNA was detected using qPCR. The 16S rRNA levels were correlated with gene expression. Although pretreatment with H2O2 before immersion in F. psychrophilum did not significantly alter the amount of bacteria found in the gill, the immune response was influenced: exposure to F. psychrophilum resulted in a negative correlation with expression of IL-17c1, MHC I and MHC II, while pretreatment with H2O2 resulted in a positive correlation with IL-4/13A and IgM. Exposure to either H2O2 or F. psychrophilum influenced the regulation of gene expression and damaged tissue. Exposure to both combined altered the immune response to infection and postponed healing of gill tissue.

Third-stage nematode larvae of Contracaecum osculatum from Baltic cod (Gadus morhua) elicit eosinophilic granulomatous reactions when penetrating the stomach mucosa of pigs.

Third-stage larvae of the anisakid nematode Contracaecum osculatum were recovered from livers of Atlantic cod (Gadus morhua) caught in the Baltic Sea (June 2014) and used for experimental infection of two pigs (one male and one female). Each pig received 215 larvae by oral infection (feeding with minced cod liver containing live nematode larvae). Pigs were euthanized after 5 days, necropsied, and subjected to parasitological investigation. A total of 12 larvae were found penetrating the mucosa of the ventricle (7 in the female pig and 5 in the male pig) eliciting a granulomatous reaction at the penetration site. Four non-attached larvae were found in the female pig stomach and one in the male pig. Petechial bleeding was observed at several locations in the ventricular mucosa where larvae were located. Histological examination of the stomach mucosa revealed a massive cellular infiltration (giant cells, lymphocytes, macrophages, granulocytes, and fibroblast like cells) around the penetrating larva. Mononuclear and polymorphonuclear cells containing eosinophilic granulae were particularly prominent in the granulomas. Reactions correspond to reactions in pigs following experimental infection with the human pathogenic anisakid larvae Anisakis sp. and Pseudoterranova sp. which suggests that C. osculatum might have a zoonotic potential as well.

Evaluation of the immune response in rainbow trout fry, Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide.

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion-based challenge with or without prior exposure to hydrogen peroxide (H2O2). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post-infection. Separately, both H2O2 and F. psychrophilum influenced gene expression, and pre-treatment with H2O2 influenced the response to infection with F. psychrophilum. Pre-treatment with H2O2 also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H2O2-treated fish in the early phase of infection was indicated, but H2O2 exposure did not affect antibody levels 50 days post-infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre-treated with H2O2 relative to the F. psychrophilum group. The results show that a single pre-treatment with H2O2 impairs the response against F. psychrophilum and may intensify infection.

Epidermal response of rainbow trout to Ichthyobodo necator: immunohistochemical and gene expression studies indicate a Th1-/Th2-like switch.

Infections with the parasitic flagellate Ichthyobodo necator (Henneguy, 1883) cause severe skin and gill disease in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) juveniles. The epidermal disturbances including hyperplasia and mucous cell exhaustion caused by parasitization are known, but no details on specific cellular and humoral reactions have been presented. By applying gene expression methods and immunohistochemical techniques, further details of immune processes in the affected skin can be presented. A population of I. necator was established in the laboratory and used to induce an experimental infection of juvenile rainbow trout. The course of infection was followed by sampling for parasite enumeration, immunohistochemistry (IHC) and quantitative PCR (qPCR) on days 0, 5, 9 and 14 post-infection. IHC showed a significant increase in the occurrence of IgM-positive cells in the skin of the infected fish, whereas IgT-positive cells were eliminated and the number of CD8-positive cells declined. qPCR studies supported the IHC findings showing a significant increase in IgM and a decrease in the CD8 gene expression. In addition, genes encoding innate immune genes such as lysozyme, SAA and cathelicidin 2 were up-regulated. Expression of cytokines (IL-1ß, IL-4/13A, IL-6, IL-8, IL-10), the cell marker CD4 and the transcription factor GATA3 showed a significant increase after infection. Cytokine profiling including up-regulation of IL-4/13A and IL-10 genes and transcription factor GATA3 connected to the proliferation of IgM producing lymphocytes suggests a partial shift towards a Th2 response associated with the I. necator infection.