Publications by authors named "Per Walter Kania"

18 Publications

  • Page 1 of 1

Evidence of IgE-Mediated Cross-Reactions between and Proteins.

Pathogens 2021 Jul 28;10(8). Epub 2021 Jul 28.

Laboratory of Aquatic Pathobiology, Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark.

Fish consumers may develop allergic reactions following the ingestion of fish products containing nematode larvae within the genus . 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 -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 . 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 -sensitized patients when exposed to antigens. Sera from -sensitized patients confirmed the reaction with somatic antigens (SA) and excretory/secretory proteins (ES) from . Only anecdotal responses were obtained from other freshwater worm parasites. Consequently, it is suggested that -sensitized humans, especially patients with high levels of specific anti- antibodies, may react to proteins, possibly due to IgE-mediated cross-reactivity.
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http://dx.doi.org/10.3390/pathogens10080950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8399947PMC
July 2021

Morphological and molecular identification of Cryptocotyle lingua metacercariae isolated from Atlantic cod (Gadus morhua) from Danish seas and whiting (Merlangius merlangus) from the English Channel.

Parasitol Res 2021 Oct 27;120(10):3417-3427. Epub 2021 Aug 27.

Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Trematode larvae (metacercariae) causing black spot disease occur frequently in gills, fins, skin and the superficial muscle layers of marine fish. Species within the genus Cryptocotyle Lühe, 1899 are frequently associated with this disease. Descriptions of the metacercarial stage are relatively limited and none has hitherto been reported from fish from the English Channel. The present study reports the morphological and molecular identifications of encysted black spot-inducing parasites from whiting (Merlangius merlangus) and Atlantic cod (Gadus morhua) caught respectively from the north coast of France (English Channel) and from Danish sea waters. Metacercariae were characterised morphologically based on microscopic observations and molecularly using Sanger sequencing of fragments of the mitochondrial cox1 gene and rDNA ITS region. Morphological data were compared with available data in the literature. Phylogenetic trees including reference sequences were built to confirm morphological and molecular identifications. This survey constitutes the first description of C. lingua metacercariae in the English Channel ecosystems.
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http://dx.doi.org/10.1007/s00436-021-07278-6DOI Listing
October 2021

Inflammatory reactions in rainbow trout fins and gills exposed to biocides.

Dis Aquat Organ 2021 Aug 26;146:9-21. Epub 2021 Aug 26.

Laboratory of Aquatic Pathobiology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C., Denmark.

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-1β, 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.
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http://dx.doi.org/10.3354/dao03617DOI Listing
August 2021

Eye fluke effects on Danish freshwater fish: Field and experimental investigations.

J Fish Dis 2021 Jul 21. Epub 2021 Jul 21.

Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

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.
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http://dx.doi.org/10.1111/jfd.13496DOI Listing
July 2021

Trematode diversity reflecting the community structure of Danish freshwater systems: molecular clues.

Parasit Vectors 2021 Jan 12;14(1):43. Epub 2021 Jan 12.

Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Via Stigbøjlen 7, 1870, Frederiksberg C, Copenhagen, Denmark.

Background: Digenean trematodes are parasitic platyhelminths that use several hosts in their life cycles and are thereby embedded in various ecosystems affected by local environmental conditions. Their presence in a habitat will reflect the presence of different host species and, as such, they can serve as ecological indicators. Only limited information on the occurrence of trematodes and their link to other trophic levels in the Danish freshwater ecosystems is currently available.Therefore, the main aim of the present study was to increase our knowledge in this field.

Methods: Snails were sampled from 21 freshwater lakes in Denmark, following which shedding procedures were performed, cercariae were recoved and the released parasites were identified using molecular tools (PCR and sequencing).

Results: A total of 5657 snail hosts belonging to ten species were identified, revealing a highly diverse parasite fauna comprising 22 trematode species. The overall trematode prevalence was 12.6%, but large variations occurred between host species. The snail host Lymnaea stagnalis showed the highest prevalence and also exhibited the highest diversity, accounting for 47.6% of the species richness.

Conclusions: This survey contributes updated information on parasite-host relations and compatibility and may assist in describing the ecological structure of the investigated Danish freshwater ecosystems.
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http://dx.doi.org/10.1186/s13071-020-04536-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805065PMC
January 2021

A non-lethal method for detection of Bonamia ostreae in flat oyster (Ostrea edulis) using environmental DNA.

Sci Rep 2020 09 30;10(1):16143. Epub 2020 Sep 30.

Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Science, University of Copenhagen, Stigbøjlen 7, 1870, Frederiksberg C, Denmark.

Surveillance and diagnosis of parasitic Bonamia ostreae infections in flat oysters (Ostrea edulis) are prerequisites for protection and management of wild populations. In addition, reliable and non-lethal detection methods are required for selection of healthy brood oysters in aquaculture productions. Here we present a non-lethal diagnostic technique based on environmental DNA (eDNA) from water samples and demonstrate applications in laboratory trials. Forty oysters originating from Limfjorden, Denmark were kept in 30 ppt sea water in individual tanks. Water was sampled 6 days later, after which all oysters were euthanized and examined for infection, applying PCR. Four oysters (10%) were found to be infected with B. ostreae in gill and mantle tissue. eDNA purified from the water surrounding these oysters contained parasite DNA. A subsequent sampling from the field encompassed 20 oysters and 15 water samples from 5 different locations. Only one oyster turned out positive and all water samples proved negative for B. ostreae eDNA. With this new method B. ostreae may be detected by only sampling water from the environment of isolated oysters or isolated oyster populations. This non-lethal diagnostic eDNA method could have potential for future surveys and oyster breeding programs aiming at producing disease-free oysters.
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http://dx.doi.org/10.1038/s41598-020-72715-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527985PMC
September 2020

Gill amoebae from freshwater rainbow trout (Oncorhynchus mykiss): In vitro evaluation of antiparasitic compounds against Vannella sp.

J Fish Dis 2020 Jun 15;43(6):665-672. Epub 2020 Apr 15.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Gill parasitic infections challenge farming of rainbow trout (Oncorhynchus mykiss, Walbaum) in freshwater facilities. Apart from flagellates (Ichthyobodo, (Pinto) and ciliates (Ichthyophthirius (Fouquet), Ambiphrya (Raabe), Apiosoma (Blanchard), Trichodinella (Sramek-Husek) and Trichodina (Ehrenberg)), we have shown that amoebae are prevalent in Danish trout farms. Gills were isolated from farmed rainbow trout in six fish farms (conventional and organic earth pond and recirculated systems) and placed on non-nutrient agar (NNA) moistened with modified Neff's amoeba saline (AS) (15°C). Gill amoebae from all examined fish colonized the agar and were identified based on morphological criteria showing species within the genera Trinema (Dujardin) (family Trinematidae), Vannella (Bovee) (family Vannellidae). In addition, hartmannellid amoebae were recorded. We established a monoculture of Vannella sp., confirmed the genus identity by PCR and sequencing and performed an in vitro determination of antiparasitic effects (dose-response studies) of various compounds including sodium chloride (NaCl), hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant. All amoebae were killed in concentrations of 16.90 mg/ml (garlic), 17.90 mg/ml (oregano), NaCl (7.5 mg/ml), hydrogen peroxide (100 µg/ml), peracetic acid (0.03 µg/ml), formaldehyde (25 µg/ml) and the Pseudomonas H6 surfactant (250 µg/ml).
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http://dx.doi.org/10.1111/jfd.13162DOI Listing
June 2020

Injection Vaccines Formulated with Nucleotide, Liposomal or Mineral Oil Adjuvants Induce Distinct Differences in Immunogenicity in Rainbow Trout.

Vaccines (Basel) 2020 Feb 25;8(1). Epub 2020 Feb 25.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1870 Frederiksberg, Denmark.

Protection facilitated by the widespread use of mineral oil adjuvanted injection vaccines in salmonid fish comes with adverse effects of varying severity. In this study, we characterized the immunological profiles of two alternative vaccine formulations, both with proven efficacy and an improved safety profile in rainbow trout. Experimental injection vaccines were prepared on an identical whole-cell bacterin platform and were formulated with CpG oligodeoxynucleotides, a liposomal (CAF01) or a benchmark mineral oil adjuvant, respectively. A naïve group, as well as bacterin and saline-injected groups were also included. Following administration, antigen-specific serum antibody titers, the tissue distribution of immune cell markers, and the expression of immune-relevant genes following the in vitro antigenic restimulation of anterior kidney leukocytes was investigated. Immunohistochemical staining suggested prolonged antigen presentation for the particulate formulations and increased mucosal presence of antigen-presenting cells in all immunized fish. Unlike the other immunized groups, the CAF01 group only displayed a transient elevation in specific antibody titers and immunohistochemical observations, and the transcription data suggest an increased role of cell-mediated immunity for this group. Finally, the transcription profile of the CpG formulation approached that of a T profile. When compared to the benchmark formulation, CAF01 and CpG adjuvants induce slight, but distinct differences in the resulting protective immune responses. This is important, as it allows a broader immunological approach for the future development of safer vaccines.
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http://dx.doi.org/10.3390/vaccines8010103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157222PMC
February 2020

Skin immune response of rainbow trout (Oncorhynchus mykiss) experimentally exposed to the disease Red Mark Syndrome.

Vet Immunol Immunopathol 2019 May 23;211:25-34. Epub 2019 Mar 23.

Unit for Fish and Shellfish Diseases, National Institute of Aquatic Resources, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.

Red Mark Syndrome (RMS) is a skin disease reported from farmed rainbow trout. Since the turn of the millennium it has been spreading through Europe. RMS is probably a bacterial disease caused by a Midichloria-like organism (MLO). It is non-lethal and causes little obvious changes in appetite or behavior but results in red hyperaemic skin lesions, which may lead to economic losses due to downgrading. Here we transfer RMS to naïve specific pathogen free (SPF) fish by cohabitation with RMS-affected seeder fish. During disease development we characterize local cellular immune responses and regulations of immunologically relevant genes in skin of the cohabitants by immunohistochemistry and qPCR. Skin samples from SPF controls and cohabitants (areas with and without lesions) were taken at 18, 61, 82 and 97 days post-cohabitation. Gene expression results showed that lesions had a Th1-type profile, but with concurrent high expression levels of all three classes of immunoglobulins (IgD, IgM and IgT). The marked local infiltration of IgD + cells in the skin lesions as well as a highly up-regulated expression of the genes encoding sIgD and mIgD indicate that this immunoglobulin class plays an important role in skin immunity in general and in RMS pathology in particular. The co-occurrence of an apparent B cell dominated immune reaction with a Th1-type profile suggests that the local production of antibodies is independent of the classical Th2 pathway.
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http://dx.doi.org/10.1016/j.vetimm.2019.03.008DOI Listing
May 2019

Association between adaptive immunity and neutrophil dynamics in zebrafish (Danio rerio) infected by a parasitic ciliate.

PLoS One 2018 11;13(9):e0203297. Epub 2018 Sep 11.

Section of Parasitology and Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

The protective immune response in zebrafish (Danio rerio) against the parasitic ciliate Ichthyophthirius multifiliis, targeting host skin, fins and gills, comprises an accelerated and manifold elevated immunoglobulin gene expression as well as a significantly elevated number of neutrophils at infected sites. Experimental fish were subjected to a primary I. multifiliis infection followed by a series of secondary exposures before they were challenged by a high dosage of infective theronts. Immunized fish responded immediately with a protective response suggesting existence of immunological memory whereas fish exposed to the parasite for the first time obtained a marked infection. The primary response to infection was dominated by expression of genes encoding acute phase reactants and inflammatory cytokines as well as recruitment of neutrophils at infected locations. Immunized fish showed a significantly upregulated immunoglobulin gene expression following challenge, which indicates existence of a secondary response effected by antibodies. Both responses induced a significantly elevated expression of the Th2 signature cytokine Il13. The increased presence of neutrophils in immunized fish suggests that innate cell mediated immunity supplements or influence the protective response against the parasite.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203297PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133357PMC
February 2019

Comparative susceptibilities and immune reactions of wild and cultured populations of Caspian trout Salmo trutta caspius to VHSV.

Dis Aquat Organ 2018 06;128(3):187-201

Department of Biology, Faculty of Science, University of Guilan, Rasht 4199613776, Iran.

Caspian trout Salmo trutta caspius is an endangered subspecies of brown trout Salmo trutta which is native to the Caspian Sea. Restocking programmes have been established, but recent introduction of the rhabdovirus viral haemorrhagic septicaemia virus (VHSV) into Iranian rainbow trout farms connected to waterbodies supporting wild Caspian trout may represent an additional threat to the declining stock. The susceptibility of wild and cultured populations of this endemic subspecies was demonstrated by performing controlled VHSV infection experiments (both by bath and injection challenges). Subsequently, VHSV infection in exposed fish was confirmed (CPE and quantitative PCR), virus levels were measured, and regulation of immune genes in exposed fish was investigated with a focus on the genes encoding IL-8, IFNγ, TGFβ, TNFα, SAA, C3-4, CD8α, IgM, MHC I, MHC II, iNOS and IGF-1. The presence of IgM-, CD8α- and MHC II-positive cells in host organs was visualized by immunohistochemistry. Both wild and cultured trout strains proved to be VHSV-susceptible following experimental challenge, but the mortality curves and associated regulation of immune-related genes differed between the 2 trout types. Implications of the results for future management of Caspian trout populations are discussed.
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http://dx.doi.org/10.3354/dao03231DOI Listing
June 2018

Immunomodulatory effects of excretory/secretory compounds from Contracaecum osculatum larvae in a zebrafish inflammation model.

PLoS One 2017 24;12(7):e0181277. Epub 2017 Jul 24.

Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of C. osculatum ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181277PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524353PMC
September 2017

Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss.

PLoS One 2017 9;12(2):e0171944. Epub 2017 Feb 9.

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17-30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171944PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300222PMC
August 2017

Antigen Uptake during Different Life Stages of Zebrafish (Danio rerio) Using a GFP-Tagged Yersinia ruckeri.

PLoS One 2016 12;11(7):e0158968. Epub 2016 Jul 12.

Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark.

Immersion-vaccines (bacterins) are routinely used for aquacultured rainbow trout to protect against Yersinia ruckeri (Yr). During immersion vaccination, rainbow trout take up and process the antigens, which induce protection. The zebrafish was used as a model organism to study uptake mechanisms and subsequent antigen transport in fish. A genetically modified Yr was developed to constitutively express green fluorescent protein (GFP) and was used for bacterin production. Larval, juvenile and adult transparent zebrafish (tra:nac mutant) received a bath in the bacterin for up to 30 minutes. Samples were taken after 1 min, 15 min, 30 min, 2 h, 12 h and 24 h. At each sampling point fish were used for live imaging of the uptake using a fluorescence stereomicroscope and for immunohistochemistry (IHC). In adult fish, the bacterin could be traced within 30 min in scale pockets, skin, oesophagus, intestine and fins. Within two hours post bath (pb) Yr-antigens were visible in the spleen and at 24 h in liver and kidney. Bacteria were associated with the gills, but uptake at this location was limited. Antigens were rarely detected in the blood and never in the nares. In juvenile fish uptake of the bacterin was seen in the intestine 30 min pb and in the nares 2 hpb but never in scale pockets. Antigens were detected in the spleen 12 hpb. Zebrafish larvae exhibited major Yr uptake only in the mid-intestine enterocytes 24 hpb. The different life stages of zebrafish varied with regard to uptake locations, however the gut was consistently a major uptake site. Zebrafish and rainbow trout tend to have similar uptake mechanisms following immersion or bath vaccination, which points towards zebrafish as a suitable model organism for this aquacultured species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158968PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942034PMC
July 2017

Occurrence of Diplostomum pseudospathaceum Niewiadomska, 1984 and D. mergi Dubois, 1932 (Digenea: Diplostomidae) in Danish freshwater snails: ecological and molecular data.

Folia Parasitol (Praha) 2013 May;60(2):177-80

Laboratory of Aquatic Pathobiology, Section of Biomedicine, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Freshwater pulmonate snails from three locations in Lake Furesø north of Copenhagen were screened for infection with furcocercariae (by shedding in the laboratory) and recovered parasite larvae were diagnosed by molecular methods (by performing PCR of rDNA and sequencing the internal transcribed spacer [ITS] region). Overall prevalence of infection in snails was 2%. Recovered cercariae from Lymnaea stagnalis (Linnaeus) were diagnosed as Diplostomum pseudospathaceum Niewiadomska, 1984 (prevalence 4%) and cercariae from Radix balthica (Linnaeus) as D. mergi (Dubois, 1932) (prevalence 2%). Pathogen-free rainbow trout were then exposed to isolated cercariae and infection success and site location of metacercariae in these fish were determined. Infection experiments confirmed that both species could infect rainbow trout with the eye lens as infection site for the metacercarial stage although infection success differed. Combination of molecular and biological assays may contribute to improvement of our knowledge on diagnosis, distribution and biology of diplostomids in fish.
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http://dx.doi.org/10.14411/fp.2013.020DOI Listing
May 2013

Tissue specific uptake of inactivated and live Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss): visualization by immunohistochemistry and in situ hybridization.

Microb Pathog 2013 Jun-Jul;59-60:33-41. Epub 2013 Apr 10.

Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.

Understanding of uptake and invasion routes of Yersinia ruckeri, causing Enteric Red Mouth Disease (ERM) in rainbow trout (Oncorhynchus mykiss), is essential for improved understanding of the pathogenicity and immune response mechanisms associated this disease. The present work shed light on areas of invasion in rainbow trout by the use of immunohistochemistry and in situ hybridization techniques. Fish were exposed to live or formalin inactivated bacteria and samples were subsequently taken for histology from various outer and inner surfaces. We applied a specific monoclonal antibody and specific oligonucleotide probes binding to Y. ruckeri (serotype O1, biotype 2) in tissue sections and were able to demonstrate a tissue specific uptake of this bacterium (both formalin inactivated and live form). Uptake and subsequent translocation dynamics at various surfaces demonstrated different site specific propensities between the formalin inactivated and live bacterial organisms. Lateral lines, dorsal fin, epidermis and gastro-intestinal tract mucosal tissue were the primary areas where bacterial uptake was demonstrated readily after exposure. The fate of internalized bacterial organisms within the host suggested that central immune organs are involved in the final antigen processing.
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http://dx.doi.org/10.1016/j.micpath.2013.03.001DOI Listing
November 2013

Approaches towards DNA vaccination against a skin ciliate parasite in fish.

PLoS One 2012 7;7(11):e48129. Epub 2012 Nov 7.

Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark.

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR β, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048129PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492342PMC
April 2013

Immunomodulatory effects of dietary β-1,3-glucan from Euglena gracilis in rainbow trout (Oncorhynchus mykiss) immersion vaccinated against Yersinia ruckeri.

Fish Shellfish Immunol 2012 Jul 21;33(1):111-20. Epub 2012 Apr 21.

Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Potential immunostimulatory effects of orally administered β-glucan were investigated in combination with immersion vaccination against enteric redmouth disease caused by Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). A linear, unbranched and pure (purity ≥98%) β-1,3-glucan (syn. paramylon) from the alga Euglena gracilis was applied at an inclusion level of 1% β-glucan in feed administered at a rate of 1% biomass day(-1) for 84 consecutive days. Fish were vaccinated after two weeks of experimental feeding and bath challenged with live Y. ruckeri six weeks post-vaccination. Blood and head kidney were sampled at day 0, 13 (1 day pre-vaccination), 15, 55, 59 (day 3 post-challenge (p.c.)), 70 and 84. Vaccination induced significantly increased survival p.c., whereas the β-glucan had no effect on survival in either unvaccinated or vaccinated fish. Expression in head kidney of genes related to the acute phase response, i.e. interleukin-1β (IL-1β), serum amyloid A (SAA), precerebellin, and hepcidin, was significantly different in vaccinated fish receiving β-glucan compared to vaccinated controls at day 3 p.c., while no effect of β-glucan was observed among unvaccinated fish. Significant interaction between β-glucan and vaccination was found for the regulation of IL-1β, tumour necrosis factor-α, interferon-γ, SAA, precerebellin and hepcidin p.c. For SAA, the significant effect of β-glucan in vaccinated fish persisted at day 14 p.c. and 28 p.c. The difference in gene expression among vaccinated fish was mainly observed as down-regulations in vaccinated, β-glucan fed fish compared to up-regulations or no regulation in vaccinated controls. Slightly increased levels of plasma lysozyme activity were found in fish (both unvaccinated and vaccinated) receiving β-glucan at day 3 p.c. compared to control fed groups. This was associated with a faster clearance of Y. ruckeri in unvaccinated fish receiving β-glucan. In contrast to the trend towards a beneficial effect of β-glucan on plasma lysozyme activity, a trend towards suppression of plasma antibodies was seen in both unvaccinated and vaccinated fish receiving β-glucan. However, the effects of β-glucan were not reflected in the survival curves, and the differences seen in plasma lysozyme activity and antibody levels may have counteracted and set off each other as well as counteracted any potential effect represented by the differences in gene expression found.
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http://dx.doi.org/10.1016/j.fsi.2012.04.009DOI Listing
July 2012
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