Bergljót Magnadóttir

Immunological Control of Fish Diseases

All metazoans possess innate immune defence system whereas parameters of the adaptive immune system make their first appearance in the gnathostomata, the jawed vertebrates. Fish are therefore the first animal phyla to possess both an innate and adaptive immune system making them very interesting as regards developmental studies of the immune system. The massive increase in aquaculture in recent decades has also put greater emphasis on studies of the fish immune system and defence against diseases commonly associated with intensive fish rearing. Some of the main components of the innate and adaptive immune system of fish are described. The innate parameters are at the forefront of immune defence in fish and are a crucial factor in disease resistance. The adaptive response of fish is commonly delayed but is essential for lasting immunity and a key factor in successful vaccination. Some of the inherent and external factors that can manipulate the immune system of fish are discussed, the main fish diseases are listed and the pathogenicity and host defence discussed. The main prophylactic measures are covered, including vaccination, probiotics and immunostimulation. A key element in the immunological control of fish diseases is the great variation in disease susceptibility and immune defence of different fish species, a reflection of the extended time the present day teleosts have been separated in evolution. Future research will probably make use of molecular and proteomic tools both to study important elements in immune defence and prophylactic measures and to assist with breeding programmes for disease resistance.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) I. The effects of environmental temperature

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.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) II. The effects of size and gender under different environmental conditions

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.

A comparison of total and specific immunoglobulin levels in healthy Atlantic salmon (Salmo salar L.) and in salmon naturally infected with Aeromonas salmonicida subsp. achromogenes

Healthy Atlantic salmon and salmon with a history of chronic natural Aeromonas salmonicida subsp. achromogenes infection were compared with respect to total serum protein and the concentration and specificity of serum immunoglobulin. The immunoglobulin level was measured using competitive ELISA and the specific antibody activity against Aeromonas salmonicida subsp. achromogenes was measured using double sandwich ELISA. Significant elevation of serum protein and immunoglobulin concentration was observed in the infected salmon compared with the healthy fish. This was accompanied by weak anti-A. salmonicida activity in the infected fish which seemed to contribute to the raised immunoglobulin level to only a limited degree.

Study of the humoral response of Atlantic salmon (Salmo salar L.), naturally infected with Aeromonas salmonicida ssp. achromogenes

The humoral antibody response of healthy Atlantic salmon and of two groups of salmon, naturally infected with Aeromonas salmonicida ssp. achromogenes, was examined in some detail. One diseased group was chronically infected and the other recently infected. It was found that the humoral response of these two infected groups was quite different. The chronically infected fish showed poor specific response to the causative agent whereas the recently infected salmon produced strong specific antibody response. The chronically infected fish showed evidence of increased unspecific response including an elevated level of natural antibodies. The specific humoral response of the recently infected fish was primarily directed against two cell-associated antigens of the A. salmonicida ssp. achromogenes bacterium, the A-layer protein and the o-polysaccharide component of LPS. In the chronically infected fish the humoral response was primarily directed against the A-layer protein.

Natural antibodies of cod (Gadus morhua L.): specificity, activity and affinity.

Natural antibodies are present in the serum of vertebrates regardless of antigenic stimulation. Characteristic activity is commonly detected against haptenated proteins, single stranded DNA and thyroglobulin. Natural antibodies are believed to provide an instant protection against pathogens of a broad specificity and to participate in homeostasis. Cod is a poor antibody responder but shows a relatively high level of natural antibodies against haptenated proteins. In this project the specificity, activity and affinity of natural antibodies was studied in different groups of cod and the effects of age/size, environmental temperature, immunisation and infection examined. Antigen driven selection of natural antibodies was also studied in one group of cod. The results demonstrated a broad and yet characteristic specificity, primarily directed against haptenated proteins and possible food antigens. The antibody activity increased with increasing age and at higher temperature whereas immunostimulation by immunisation or infection resulted in variable response. The affinity index of natural antibodies of cod generally did not correlate with changes in the antibody activity but it was in the same range as the affinity index of acquired cod antibodies and that of some mammalian monoclonal acquired antibodies. Analysis of antigen driven antibody selection showed that the natural antibody repertoire of individual cod was heterogeneous with respect to its affinity for haptenated protein.

Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

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.

Is Apolipoprotein A-I a regulating protein for the complement system of cod (Gadus morhua L.)?

Apolipoproteins are a heterogenic class of lipid-associated proteins found in the plasma and other body fluids of vertebrates. Apolipoprotein is a constituent of the hydrophilic coat that surrounds the lipids and different types are characteristic for different lipoprotein densities. Apolipoprotein A-I (ApoLP A-I) is the major protein component of high density lipoproteins (HDL) [1–3]. ApoLP A-I is an activator of the lecithin-cholesterol-acyl-transferase (LCAT), a plasma enzyme involved in cholesterol metabolism [1]. Several other functions have been attributed to ApoLP A-I. It is, for example, involved in the binding of LPS (lipopolysaccharide) [4], antiviral activity has been described [5,6] and ApoLP A-I isolated from carp (Cyprinus carpio) has been shown to have heparin binding activity implicated in nerve regeneration processes [7]. A regulatory role in the complement system has also been discovered, ApoLP A-I primarily acting as an inhibitor of the membrane attack complex, C5b-9 [8–11]. The complement system of cod (Gadus morhua L.) has been studied at our institute in recent years [12,13]. The initial emphasis was on isolating and characterizing the complement component C3, the central component of the three complement pathways, and on the production of a specific anti-C3 antibody [13]. This was tackled in three ways: (1) Purification of C3 was attempted, based on the well documented ability of C3 to bind to zymosan, an insoluble preparation of yeast (Saccharomyces cerevisiae) [14] and to MacroGard (MG), an insoluble yeast cell wall, beta-1,3-and beta-1,6-linked glucan. For the preparation of zymosan-absorbed serum proteins, zymosan prepared at our laboratory was used, washed in complement fixation test buffer (prepared from tablets, Oxoid, UK) containing 0.1% gelatine (CFT-G). This was then mixed with cod serum and the suspension agitated overnight at 4 (C. After centrifugation, the supernatant was discarded, the zymosan was washed with CFT-G buffer and bound proteins eluted with distilled water. The eluted proteins were collected after centrifugation and concentrated by filter centrifugation. The protein yield was about 50–100 μg ml 1 serum.

Light chain variable region diversity in Atlantic cod (Gadus morhua L.).

This study was undertaken to determine if a lack of V(L) domain variability could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod V(L) regions was studied in 33 cDNA and two genomic clones. The variability of the CDRs was estimated by the Shannon entropy method and compared with that in other species. It was found to be lowest in the little skate (Raja erinacea), higher in cod, and highest in Xenopus and mouse. While the variability of the CDRs is slightly lower in cod than in Xenopus and mouse, it is spread over broader areas of the amino acid sequence. The length of CDR1 and CDR3 in cod is equal to or exceeds that found in skate, Xenopus, chicken and mammals. Isoelectric points and hydrophobicity vary substantially among the studied Ig light chain domains. Thus, neither the length, nor the variability, nor the physicochemical properties (pI and hydrophobicity) of the L chain CDRs can explain the absence of antibody response to immunization in cod.

Isolation of two C-reactive protein homologues from cod (Gadus morhua L.) serum.

Pentraxins are important molecules in innate defence and play a role in the acute phase response of both mammals and fish. Isolation of cod pentraxins by affinity chromatography using phosphorylcholine agarose revealed two pentraxin-like proteins, referred to as PI and PII proteins. These varied in their overall charge, pentameric and subunit molecular size, glycosylation and N-terminal amino acid sequences. The PI protein was homologous with the CRP-like pentraxin previously described in cod whereas the PII protein was a new CRP homologue, which was characterized by substantial individual heterogeneity with regard to subunit size and relative density. The results indicate considerable genetic variations in the cod pentraxins.