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Moritella viscosais the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, butM. viscosahas not so far been isolated. In this study, turbot was shown to be sensitive toM. viscosainfection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containingM. viscosaas one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosaresponse, while elevated anti-M. viscosaantibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response againstAeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups. [ABSTRACT FROM AUTHOR]

Abstract Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine. [ABSTRACT FROM AUTHOR]

Abstract Cod, Gadus morhua L., of wild origin, were reared at different temperatures for 12 months. During this period, moribund and newly dead fish were examined and samples collected for bacteriology and histopathology. Atypical Aeromonas salmonicida was isolated from 10 individuals reared at or above 7 °C. The isolates were homogeneous with respect to biochemical and antibiogram characters and similar to the ssp. achromogenes National Collection of Industrial and Marine Bacteria, UK, type strain 1110 and reference strains that have been isolated from salmonids and haddock in Iceland. Histopathological analysis of the naturally infected cod showed typical ulceration associated with atypical A. salmonicida infection and also widespread granulomatous formations. One-year-old cod of farmed origin, kept at 9 °C, received intraperitoneal or intramuscular injection with different doses of atypical A. salmonicida , isolated from the above wild cod. Mortalities were monitored for 28 days and the LD50 calculated. The route of bacterial injection influenced the mortality rate and LD50 value and affected, to some extent, the pathological changes observed and humoral immune parameters. Pathological changes, including haemorrhage, early stages of granuloma formation and necrotic changes, were seen in several organs. Infection appeared to induce non-specific antibody activity against trinitrophenyl (TNP)-haptenated protein and may have activated the complement system. Specific antibody response against atypical A. salmonicida was not detected. [ABSTRACT FROM AUTHOR]

Atlantic salmon were vaccinated against Aeromonas salmonicida ssp. achromogenes (Asa) by injection with three vaccines developed in our laboratory and an autogeno us bacterin (IcelandBiojec. OO, lBOO) produced by a commercial vaccine producer. The humoral antibody responses to bacterial antigens were monitored by ELISA and Western blotting. The fish were challenged by infection with Ma 6 and 12 weeks post-vaccination. Protection was induced in all groups of vaccinated fish. The protection achieved was time-dependent. The autogenous bacterin, IBOO, induced a protective immune response later than our experimental vaccines. All the vaccines tested induced specific antibody response that increased between 6 and 12 weeks after vaccination. The antibody response was mainly directed against the A- layer protein. but antibodies to other bacterial components were also detected. Significant correlation was obtained between the antibody titre to extracellular Ma antigens, induced by the different vaccine preparations, and survival of vaccinated fish challenged by a virulent Ma strain. Furthermore, the detection of antibodies directed against an extracellular toxic metallo-caseinase, AsaP1, in fish sera correlated with protection. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are lipid bilayer vesicles which are released from cells and play multifaceted roles in cellular communication in health and disease. EVs can be isolated from various body fluids, including serum and plasma, and are usable biomarkers as they can inform health status. Studies on EVs are an emerging research field in teleost fish, with accumulating evidence for important functions in immunity and homeostasis, but remain to be characterised in most fish species, including halibut. Protein deimination is a post-translational modification caused by a conserved family of enzymes, named peptidylarginine deiminases (PADs), and results in changes in protein folding and function via conversion of arginine to citrulline in target proteins. Protein deimination has been recently described in halibut ontogeny and halibut serum. Neither EV profiles, nor total protein or deiminated protein EV cargos have yet been assessed in halibut and are reported in the current study. Halibut serum EVs showed a poly-dispersed population in the size range of 50-600 nm, with modal size of EVs falling at 138 nm, and morphology was further confirmed by transmission electron microscopy. The assessment of EV total protein cargo revealed 124 protein hits and 37 deiminated protein hits, whereof 15 hits were particularly identified in deiminated form only. Protein interaction network analysis showed that deimination hits are involved in a range of gene regulatory, immune, metabolic and developmental processes. The same was found for total EV protein cargo, although a far wider range of pathways was found than for deimination hits only. The expression of complement component C3 and C4, as well as pentraxin-like protein, which were identified by proteomic analysis, was further verified in EVs by western blotting. This showed that C3 is exported in EVs at higher levels than C4 and deiminated C3 was furthermore confirmed to be at high levels in the deimination-enriched EV fractions, while, in comparison, C4 showed very low detection in deimination-enriched EV fractions. Pentraxin was exported in EVs, but not detected in the deimination-enriched fractions. Our findings provide novel insights into EV-mediated communication in halibut serum, via transport of protein cargo, including post-translationally deiminated proteins., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Peptidylarginine deiminases (PADs) are a family of phylogenetically conserved calcium-dependent enzymes which cause post-translational protein deimination. This can result in neoepitope generation, affect gene regulation and allow for protein moonlighting via functional and structural changes in target proteins. Extracellular vesicles (EVs) carry cargo proteins and genetic material and are released from cells as part of cellular communication. EVs are found in most body fluids where they can be useful biomarkers for assessment of health status. Here, serum-derived EVs were profiled, and post-translationally deiminated proteins and EV-related microRNAs are described in 5 ceataceans: minke whale, fin whale, humpback whale, Cuvier's beaked whale and orca. EV-serum profiles were assessed by transmission electron microscopy and nanoparticle tracking analysis. EV profiles varied between the 5 species and were identified to contain deiminated proteins and selected key inflammatory and metabolic microRNAs. A range of proteins, critical for immune responses and metabolism were identified to be deiminated in cetacean sera, with some shared KEGG pathways of deiminated proteins relating to immunity and physiology, while some KEGG pathways were species-specific. This is the first study to characterise and profile EVs and to report deiminated proteins and putative effects of protein-protein interaction networks via such post-translationald deimination in cetaceans, revealing key immune and metabolic factors to undergo this post-translational modification. Deiminated proteins and EVs profiles may possibly be developed as new biomarkers for assessing health status of sea mammals., Competing Interests: Declaration of competing interest The authors declare no conflicting interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, leading to functional and structural changes in target proteins. Protein deimination can cause the generation of neo-epitopes, affect gene regulation and also allow for protein moonlighting and therefore facilitate multifaceted functions of the same protein. PADs are furthermore a key regulator of cellular release of extracellular vesicle (EVs), which are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and EVs were assessed in sera of two seal species, grey seal and harbour seal. We report a poly-dispersed population of serum-EVs, which were positive for phylogenetically conserved EV-specific markers and characterised by transmission electron microscopy. A number of deiminated proteins critical for immune and metabolic functions were identified in the seal sera and varied somewhat between the two species under study, while some targets were in common. EV profiles of the seal sera further revealed that key microRNAs for inflammation, immunity and hypoxia also vary between the two species. Protein deimination and EVs profiles may be useful biomarkers for assessing health status of sea mammals, which face environmental challenges, including opportunistic infection, pollution and shifting habitat due to global warming., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The complement system is a critical part of teleost immune defences, with complement component C4 forming part of the classical and lectin pathways. Cod C4-like protein was isolated from plasma, specific antibodies generated and C4-like protein was assessed in cod sera, mucus and in extracellular vesicles (EVs) from serum and mucus. Higher levels of C4-like protein were detected in serum- than mucus-derived EVs. Post-translational deimination, caused by conversion of arginine into citrulline, can affect protein structure and function. Here we detected deiminated forms of C4-like protein in cod serum and at lower levels in mucus. C4-like protein was also found in deiminated form at low levels in EVs from both serum and mucus. C4-like protein was assessed by immunohistochemistry in cod larvae and detected in a range of organs including in liver, kidney, gut, muscle, skin and mucus, as well as in neuronal tissues of the brain, spinal cord and eye. This abundance of C4-like protein during early development may indicate roles in tissue remodelling, in addition to immune defences. The presence of deiminated C4-like protein in serum and mucosa, as well as in EVs, may suggest C4 protein moonlighting via post-translational deimination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Extracellular vesicles are released from cells and participate in cell communication via transfer of protein and genetic cargo derived from the parent cells. EVs play roles in normal physiology and immunity and are also linked to various pathological processes. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes with physiological and pathophysiological roles. PADs cause post-translational protein deimination, resulting in structural and, in some cases, functional changes in target proteins and are also linked to EV biogenesis. This study describes for the first time EVs isolated from cod mucosa. Mucosal EVs were characterised by electron microscopy, nanoparticle tracking analysis and EV-specific surface markers. Cod mucosal EVs were found to carry PAD, complement component C3 and C-reactive proteins. C3 was found to be deiminated in both whole mucus and mucosal EVs, with some differences, and further 6 deiminated immune and cytoskeletal proteins were identified in EVs by LC-MS/MS analysis. As mucosal surfaces of teleost fish reflect human mucosal surfaces, these findings may provide useful insights into roles of EVs in mucosal immunity throughout phylogeny., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Post-translational protein deimination is mediated by peptidylarginine deiminases (PADs), which are calcium dependent enzymes conserved throughout phylogeny with physiological and pathophysiological roles. Protein deimination occurs via the conversion of protein arginine into citrulline, leading to structural and functional changes in target proteins. In a continuous series of early halibut development from 37 to 1050° d, PAD, total deiminated proteins and deiminated histone H3 showed variation in temporal and spatial detection in various organs including yolksac, muscle, skin, liver, brain, eye, spinal cord, chondrocytes, heart, intestines, kidney and pancreas throughout early ontogeny. For the first time in any species, deimination of complement components C3 and C4 is shown in halibut serum, indicating a novel mechanism of complement regulation in immune responses and homeostasis. Proteomic analysis of deiminated target proteins in halibut serum further identified complement components C5, C7, C8 C9 and C1 inhibitor, as well as various other immunogenic, metabolic, cytoskeletal and nuclear proteins. Post-translational deimination may facilitate protein moonlighting, an evolutionary conserved phenomenon, allowing one polypeptide chain to carry out various functions to meet functional requirements for diverse roles in immune defences and tissue remodelling., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Peptidylarginine deiminases (PADs) are calcium dependent enzymes with physiological and pathophysiological roles conserved throughout phylogeny. PADs promote post-translational deimination of protein arginine to citrulline, altering the structure and function of target proteins. Deiminated proteins were detected in the early developmental stages of cod from 11 days post fertilisation to 70 days post hatching. Deiminated proteins were present in mucosal surfaces and in liver, pancreas, spleen, gut, muscle, brain and eye during early cod larval development. Deiminated protein targets identified in skin mucosa included nuclear histones; cytoskeletal proteins such as tubulin and beta-actin; metabolic and immune related proteins such as galectin, mannan-binding lectin, toll-like receptor, kininogen, Beta2-microglobulin, aldehyde dehydrogenase, bloodthirsty and preproapolipoprotein A-I. Deiminated histone H3, a marker for anti-pathogenic neutrophil extracellular traps, was particularly elevated in mucosal tissues in immunostimulated cod larvae. PAD-mediated protein deimination may facilitate protein moonlighting, allowing the same protein to exhibit a range of biological functions, in tissue remodelling and mucosal immune defences in teleost ontogeny., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pentraxins are fluid phase pattern recognition molecules that form an important part of the innate immune defence and are conserved between fish and human. In Atlantic cod (Gadus morhua L.), two pentraxin-like proteins have been described, CRP-I and CRP-II. Here we show for the first time that these two CRP forms are post-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect to tissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed in liver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II is strongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types in immune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. The presence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possibly facilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions and thus meeting different functional requirements in different tissues. The presented findings may further current understanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as in various human pathologies, including autoimmune diseases, amyloidosis and cancer., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4beta chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.

The complement systems of fish are well developed and play an important role in the innate immune response. Complement C3 is the central protein of all three activation pathways and is the major opsonin of the complement system and essential for the generation of the membrane attack complex. A 1548 bp part of complement component C3 was isolated from a halibut liver cDNA library by immunoscreening. The deduced amino acid sequence showed that this part of halibut C3 contained key amino acids for factor H, I and properdin binding as well as two N-glycosylation sites. Digoxigenine labelled mRNA probes were synthesised and the transcription of C3 was monitored in three larval stages at 206, 430 and 1000 degrees d (30, 50 and 99 days post hatching), by in situ hybridisation. C3 mRNA was detected in muscle, liver, brain, chondrocytes, spinal cord, eye, intestines, oesophagus and kidney. These findings are in accordance with a former immunohistochemical study on halibut C3 protein ontogeny, indicating that C3 is indeed locally expressed in many organs from the youngest stages on. Complement may thus be linked to the formation and generation of different organs during development and play an important role in the early immune response of halibut larvae.

The innate immune system is the only defence weapon of invertebrates and a fundamental defence mechanism of fish. The innate system also plays an instructive role in the acquired immune response and homeostasis and is therefore equally important in higher vertebrates. The innate system's recognition of non-self and danger signals is served by a limited number of germ-line encoded pattern recognition receptors/proteins, which recognise pathogen associated molecular patterns like bacterial and fungal glycoproteins and lipopolysaccharides and intracellular components released through injury or infection. The innate immune system is divided into physical barriers, cellular and humoral components. Humoral parameters include growth inhibitors, various lytic enzymes and components of the complement pathways, agglutinins and precipitins (opsonins, primarily lectins), natural antibodies, cytokines, chemokines and antibacterial peptides. Several external and internal factors can influence the activity of innate immune parameters. Temperature changes, handling and crowding stress can have suppressive effects on innate parameters, whereas several food additives and immunostimulants can enhance different innate factors. There is limited data available about the ontogenic development of the innate immunological system in fish. Active phagocytes, complement components and enzyme activity, like lysozyme and cathepsins, are present early in the development, before or soon after hatching.

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.

The complement system is important both in the innate and adaptive immune response, with C3 as the central protein of all three activation pathways. Apolipoprotein A-I (ApoLP A-I), a high-density lipoprotein (HDL), has been shown to have a regulatory role in the complement system by inhibiting the formation of the membrane attack complex (MAC). Complement has been associated with apoptotic functions, which are important in the immune response and are involved in organ formation and homeostasis. mRNA probes for cod C3 and ApoLP A-I were synthesized and in situ hybridisation used to monitor the ontogenic development of cod from fertilised eggs until 57 days after hatching. Both C3 and ApoLP A-I transcription was detected in the central nervous system (CNS), eye, kidney, liver, muscle, intestines, skin and chondrocytes at different stages of development. Using TUNEL staining, apoptotic cells were identified within the same areas from 4 to 57 days posthatching. The present findings may suggest a role for C3 and ApoLP A-I during larval development and a possible role in the homeostasis of various organs in cod.

The aim of this study was to monitor the ontogenic development of innate immune parameters of cod (Gadus morhua L.) and to determine the presence of maternal IgM. The general protein composition and enzyme activity was also studied. At intervals, samples were collected of fertilized cod eggs and larvae from 3 days after fertilization until 57 days after hatching. Cell lysates were prepared and analysed by Western blotting using antibodies prepared against cod IgM, the complement component C3 and C-reactive protein (CRP) as well as against cod serum proteins and haemoglobin. Antibodies against salmon cathepsins and against several mammalian proteins of immunological significance were also used. Maternal IgM was not detected but C3 and the closely associated apolipoprotein A-I were present from the time of embryo organogenesis. C-reactive protein was not detected and none of the antibodies against mammalian immune parameters cross-reacted with the cod material. Protein and proteomic analysis showed that the major proteins of the egg samples were vitellogenin derived maternal proteins. Other non-vitellogenin maternal proteins, not yet identified, were also detected in the fertilized eggs. Cathepsin was present in all samples, but other enzyme activity was restricted to larval samples from 4 days after hatching when feeding had commenced. Haemoglobin was not detected until 10 days after hatching.

The complement systems of fish are well developed and play an important role in the immune response. Complement C3 is the central protein of all three activation pathways, being the major opsonin of the complement system and essential for the generation of the membrane attack complex. Very little is known about the development of the complement system in fish. In this study, we detect the presence of C3 in halibut larvae from the age of 37 degrees d post hatching until 1050 degrees d (i.e. 5-99 d post hatching) using immunohistochemistry with specific antibodies produced against the beta-chain of halibut C3. At the different stages of larval maturation, C3 was detectable in yolksac, muscle, liver, brain, chondrocytes, spinal chord, eye, heart, stomach, intestines, oesophagus, pharynx and kidney. Our findings suggest a role of complement in the formation and generation of different organs, besides its important functions in the immune defence against invading pathogens., (Copyright 2004 Elsevier Ltd.)

The complement system in fish is well developed and plays an important role in the immune response. Very little is known about the ontogeny of C3 in fish and no study has previously been done on the development of C3 in teleosts. In this study we have detected the presence of C3 in cod larvae from the age of 1 day post hatching (p.h.) till 57 days p.h., using immunohistochemistry. The specific primary antibodies used, were produced against the beta-chain of cod C3. Immunostaining on cod larvae sections revealed that C3 is detectable in the yolksac membrane from day 1 p.h., and in liver, brain, kidney and muscle from day 2 p.h. C3 was also detected in other organs such as eye, notochord, stomach, intestines, pancreas, heart and gills at different stages of cod larval development. These findings suggest that complement is not only important in immune defence against invading pathogens but may also play a role in the formation and generation of different organs.

Complement component C3 was isolated from the plasma of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.). Fast protein liquid chromatography (FPLC) techniques, involving ion exchange and gel filtration columns, were used. The purified proteins were analysed by SDS-PAGE which showed a two-chain structure, alpha- and beta-chains, as seen in higher vertebrates. Both proteins had intra-chain thioesters located within their alpha-chains and N-terminal amino acid sequencing confirmed their identity with reference to known C3 amino acid sequences from other species. Specific antibodies were prepared against cod and halibut C3 and tested in Western blotting on sera and purified C3. The proteolytic fragmentation of C3 was tested with trypsin, pepsin, papain and the extracellular product (ECP) from the bacterium Aeromonas salmonicida ssp. achromogenes (Asa). Both trypsin and papain were successful in cleaving C3 whereas pepsin and ECP had no effect. Carbohydrate moieties were detected in the alpha- and beta-chains of cod and halibut C3 and N-linked oligosaccharides were removed from the C3 with PNGase treatment, revealing a difference in C3 glycosylation between the two species.

The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass. The optimum assay temperature was determined for each species and different red blood cell donors were tested. The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared. Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 degrees C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 degrees C. The haemolytic activity of halibut sera was inactivated at 45 degrees C while sea bass sera were inactivated at 56 degrees C. The haemolytic activity of halibut sera was significantly reduced during short-term storage at -80 degrees C, whereas the sea bass sera maintained fairly good activity after 1-year storage at -80 degrees C. EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species. Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect. Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.

The carbohydrate moiety of cod serum IgM was analysed using oligosaccharide sequencing techniques. The carbohydrate moiety constituted about 10% of the molecular weight of cod IgM, was associated with the constant region of the heavy chains (Fc), and was composed of N-linked complex type oligosaccharides. Considerable heterogeneity was observed. Sixteen different glycan structures were identified, over 60% were sialylated and 40% contained core fucose. The carbohydrate moiety of cod IgM was shown to provide protection against protease digestion, and partial deglycosylation abolished the antigen binding property of natural cod anti-TNP-BSA antibody.

The immune response of cod (Gadus morhua L.) is unusual in that specific antibody response is limited or absent. In the present study cod was immunised with haptenated and non-haptenated protein antigen at two different temperatures and the antibody response monitored over a period of 18 months. Other humoral parameters of immunological importance were also analysed, namely total immunoglobulin concentration, anti-protease and spontaneous haemolytic activity. No specific antibody response was detected but increased activity of non-specific anti-TNP antibodies was observed 10-12 weeks after immunisation, irrespective of the antigen used. This antibody activity was attributed to the adjuvant used (FCA) and did not cross react with other antigens tested. Other parameters were probably not influenced by the immunisation but seasonal fluctuations were indicated. The immunoglobulin level appeared to peak in August-September and the anti-protease activity and the haemolytic activity in October-January.

The effects of size and gender on several humoral immune parameters in cod were examined under different environmental conditions. Serum samples were collected from wild cod of different sizes. Two samplings were undertaken: In the spring in relatively cold waters off the north west coast of Iceland and in the fall in relatively warm waters off the west coast of Iceland. Most of the parameters increased with increasing cod size, except the haemolytic activity which decreased. Higher serum protein levels were seen in cod sampled in the fall than in the spring. In cod sampled in the spring there was an apparent difference between specimens < 75 cm in length and the larger specimens with respect to haemolytic activity and iron concentration. None of the parameters were influenced by the gender of the cod.

The effects of environmental temperature on certain humoral immune parameters in Atlantic cod (Gadus morhua L.) were studied. Serum samples were collected from captive cod, of wild origin, kept at different temperatures for 12 months. It was found that immunoglobulin and natural antibody levels increased with increasing temperature whereas the total serum protein concentration, anti-protease activity, iron concentration, unsaturated and total iron binding capacity decreased with increasing temperature. Haemolytic activity and percentage iron saturation also tended to decrease with increasing temperature although this was not statistically significant.