Dendritic cells (DCs) are leukocytes specialized in antigen presentation. As competent stimulators of naive T lymphocytes, they link the innate and adaptive immune responses of vertebrates. The RAG-mediated adaptive immune system appeared approximately 500 million years ago in jawed fish and a number of studies suggest that DCs exist in bony and cartilaginous fish. However, the exact role of DCs in the fish immune system is not determined and questions remain as to whether a cell type truly homologous to DCs in homeotherms does exist. My project aimed to identify potential DCs surface markers (CD209A and LAMP3) in rainbow trout (Oncorhynchus mykiss) leukocytes for evaluation of the expression patterns by qRT-PCR under different conditions and stimuli, in vitro and in vivo. Another goal was to validate and evaluate the specificity of a produced anti-trout CD209A polyclonal antibody to further characterise antigen presenting cells (APCs) in fish. The methodology was to look for up-regulation of the predicted markers together with other markers known to be expressed by DCs in mammals and to evaluate at the mRNA and protein expression level after in vitro stimulation of trout primary leukocytes with trout rIL-4/13. Trout CD209A and LAMP3 mRNA was expressed in the main lymphoid organs of fish and could be modulated with microbial mimics. Upon in vitro stimulation of trout primary leukocytes with trout rIL-4/13, trout CD209A mRNA expression was up-regulated together with both CD83 and the MHC class II chain known to be expressed by mammalian DCs. In addition, CD209A protein expression was highly induced by trout rIL-4/13. Taken together, these results suggests that the characterisation of DCs in trout with tools such as transcript evaluation of surface markers and the anti-trout CD209A antibody, could help to more precisely define these leukocyte subsets. These findings could have further impact on fish vaccine improvements and be of importance for the aquaculture industry, by optimising stimulation of adaptive immunity.

Saprolegnia is a fresh water fish parasite responsible for significant economic losses in the aquaculture industry worldwide. The disease caused by this organism is termed saprolegniosis, being characterized by a mycosis-like infection of gills and fish skin. Without sustainable treatments available for controlling or preventing saprolegniosis, it has become a significant problem especially for salmon farming, frequently affecting the fish after vaccination. Little is known regarding how the fish immune system responds to infection and such information is vital for developing future treatments and preventive measures to saprolegniosis. To study the immune response of Atlantic salmon to Saprolegnia, a detailed immune profile of experimentally infected presmolts was performed by analyzing the expression levels of several immunity-related genes. Infected fish exhibit a very strong inflammatory response while the majority of genes associate with the adaptive immunity were found to be down regulated. The mechanisms behind this response were then investigated: It was discovered that the cell wall of Saprolegnia can be recognized by fish immune cells, triggering an inflammatory response. A protease secreted by the parasite that has the ability to degrade fish antibodies was identified and, for the first time in an oomycete, the production of prostaglandin E2 was characterized, a molecule that was later shown to potentiate inflammatory responses while suppressing host adaptive immunity genes. Lastly two metabolic pathways of the oomycete were explored as novel targets for control, prostaglandin and sterol metabolism by the use of cyclooxygenase and CYP51 inhibitors, respectively.

No description available.

With the culture of marine fish species increasing, interest in ways to combat infection in these species has grown. One such avenue is the search for novel antimicrobials. Antimicrobial peptides (AMPs) are host derived molecules with a significant role in innate immune responses and have been studied extensively in mammals. Well known AMP families include β-defensins, cathelicidins, liver expressed antimicrobial peptides and, specific to fish, the piscidins. While these molecules have been found in some fish species mining of expressed sequence tag (EST) databases was carried out to identify them in other fishes. In this study we have found new members of the β-defensin, cathelicidin and piscidin families of AMPs, while also identifying new hepcidins and LEAP-2 molecules in a diverse range of fish species. The rest of this thesis focused on one major AMP family, the cathelicidins, which in fish have been mainly identified in salmonids. They are stored in secondary granules of neutrophils as inactive pro-peptides and when infection occurs, a cleavage event releases the active mature form that effects microbicidal activity. Characterization of the cathelicidin genes present in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) was undertaken. A number of ESTs with significant homology to cathelicidins were found in Atlantic cod. Primers were designed to amplify these sequences by PCR in both gadoid species, and the gene products were cloned and sequenced. One and two cathelicidin genes were identified in cod (gmCath1) and haddock (maCath1; maCath2), respectively, which differ in the length of their active mature peptides. Gadoid cathelicidins were constitutively expressed in many tissues and were upregulated by a range of immunostimulants. Analysis of the gmCath1 promoter revealed numerous putative transcription factor binding sites, some of which were used in an expression vector construct to show upregulation of gene expression when stimulated with the viral RNA mimic Poly I:C. Synthetic and recombinant gadoid cathelicidins were produced but did not appear to display antimicrobial activity, so this requires further investigation. The discovery of new members of the major AMP families in such a diverse range of fish species bodes well for future research into the use of these antimicrobials as potential disease control molecules in the aquaculture industry. Particularly for Atlantic cod this is crucial as the major cod farms in Norway are experiencing difficulties with bacterial infections for which there is no vaccine at present.

No description available.

No description available.

Graduation date: 1997

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 4 uppsatser.

The epidemiology of Aeromonas salmonicida subsp. salmonicida in the marine environment was investigated. Nutrient resuscitation and infectivity studies did not support a previous claim of dormancy in A. salmonicida and validated the use of colony-forming units (cfu) in survival studies. Survival of A. salmonicida in seawater was assessed and found to be of short duration «10 days). Survival of the bacterium in non-sterile sediment, obtained from beneath a salmon cage, appeared to be limited. The minimum infective dose of A. salmonicida to Atlantic salmon in short duration (1-3 days) bath exposure in sea water was 10' cfu ml-I. Prolonged exposure for three weeks resulted in infection with 102 cfu ml- I. Intragastric intubation of the bacterium established infection with doses >105 cfu. Shedding of A. salmonicida from infected salmon was 105-108 cfu/fish/hr. Survival and shedding results were combined in a computer model. A. salmonicida was predicted to travel >6 km suspended within the water column of a sea loch. Covert infection in freshwater farmed salmon was assessed by ELISA and the standard stress test. Results indicated that ELISA may be useful as a routine monitor of furunculosis infection. The efficacy of dot-blot immunoassay was found to be 108 cfu A. salmonicida in fish kidney tissue. Rainbow trout (Oncorhynchus mykiss) and salmon mucus were not found to inhibit the growth of A. salmonicida supporting recent evidence that fish skin is a site of carriage. In vitro studies suggested that trout serum proteins do not confer protection from fish antibody on A. salmonicida in covert infections. Preliminary work was undertaken to develop a specific DNA probe for A. salmonicida which will allow its detection in environmental samples and carrier fish. A gene library of A. salmonicida was constructed in lambda gtll and screened for "A"-protein with antibodies.

The present study is mainly a descriptive account of the food and feeding of the two Scottish powan populations from Loch Lomond and Loch Eck. Quantitative and qualitative comparisons of powan diet are made. Seasonal variations of diet are determined for the two populations and some aspects of the planktivorous feeding of adult pawan in Loch Lomond are examined. The partitioning of the ingested energy into somatic and non-somatic growth is considered. Factors affecting the feeding of the two populations are discussed.