Changes in serum concentration of a serum amyloid P-like pentraxin in Atlantic salmon, Salmo salar L., during infection and inflammation.

A serum amyloid P-like pentraxin has been isolated from Atlantic salmon. Salmo salar L., based on its calcium dependent binding to agarose. The subunits of approximately 37 kDa were all glycosylated and when covalently linked together formed a pentamer with disulphide bonds between all subunits. A specific rabbit antiserum raised against the pentameric form was used to follow changes in serum levels of the salmon pentraxin during infection with Aeromonas salar and inflammation induced by Escherichia coli LPS or killed A. salmonicida. The salmon pentraxin level in normal serum was in the range approximately 50-300 microg/ml. Unlike pentraxins from other species, the salmon pentraxin showed only moderate but significant increases or decreases in response to E. coli LPS or A. salmonicida infection, respectively. Although pentraxins and pentraxin-like proteins are evolutionary conserved, not all pentraxins are acute phase responders suggesting that their most ancestral function(s) are not related to acute phase induction.

Sialic acid in hemolymph and affinity purified lectins from two marine bivalves.

Sialic acids have been implicated in a variety of complex biological regulatory and signalling events and their functional importance is reflected by their presence in a wide variety of phyla. Potentially they may inhibit intermolecular and intercellular interactions. Lectins that exhibit specificity for sialic acid or sialoglycoconjugates are ubiquitous in the body fluids of invertebrates and this has supported the assumption that these lectins are involved in defense against microbes that express sialic acids on their surfaces. This biological function has also been inferred from the absence of sialic acids in lower invertebrates. However, most invertebrate lectins are heterogeneous and may also bind other ligands. The biological significance of the different carbohydrate specificities are not yet known. We have demonstrated the presence of sialic acids in hemolymph from two marine bivalves, the Pacific oyster Crassostrea gigas (approximately 15 micrograms ml-1) and the horse mussel Modiolus modiolus (48-100 micrograms ml-1) by several different assays. The sialic acid was mostly in free form. Affinity purified lectins from the horse mussel also contained bound sialic acids (2-5 mumol g-1). Oyster hemolymph stimulated the in vitro phagocytosis of bacteria by oyster hemocytes. The stimulation by hemolymph is facilitated by a dialyzable component, that apparently is active irrespective of the binding to sialic acid (BSM). Addition of sialic acid had no significant effect on the in vitro phagocytosis of bacteria by oyster hemocytes.

Atypical phosphorylcholine-reactive protein from Atlantic salmon, Salmo salar L.

A phosphorylcholine-reactive protein was isolated from serum of Atlantic salmon (Salmo salar L.) by affinity chromatography on a phosphorylcholine-conjugated Sepharose column followed by elution with phosphorylcholine. Based on the method used we describe the isolated protein as salmon phosphorylcholine-reactive protein (salmon PRP). Salmon PRP has calcium-independent binding to phosphorylcholine. The protein exists in a monomeric and dimeric form with molecular weight of approximately 80 and 160 kD, respectively. Separation of the protein preparation on SDS-PAGE under reducing conditions resulted in disappearance of the 80 and 160 kD bands and appearance of a major protein band of approximately 100 kD. The N-terminal amino acid sequences of the non-reduced 80 and 160 kD bands and the reduced 100 kD band were identical. Apart from the dimeric form, the molecular weight of salmon PRP and its appearance on SDS-PAGE is similar to human plasminogen. Comparison of the sequence in a protein database resulted in approximately 50% identity with human and bovine plasminogen. In addition, cross-reactivity between antibodies to human plasminogen and salmon PRP was demonstrated. Thus, salmon PRP appears to be different from other phosphorylcholine-reactive proteins which are mostly reported to be CRP-like proteins with calcium-dependent binding to phosphorylcholine, pentameric ring-structure and sequence homology between species. Whether salmon PRP is a new type of phosphorylcholine-binding protein with an unknown function or a plasminogen-like protein with binding specificity for phosphorylcholine calls for further investigation.

Sialic acid-binding lectin with antibacterial activity from the horse mussel: further characterization and immunolocalization.

A heterogeneous sialic acid-binding lectin with affinity for bacterial LPS was isolated and partially characterized from hemolymph of the horse mussel Modiolus modiolus.(1) Using two-dimensional electrophoresis with immobilized pH gradients, the lectin revealed three subunits with different molecular weight and isoelectric points (pI); Mr14 (pI approximately 5.1 and approximately 5.5), 17.5 (pI approximately 5.5) and 20 (pI approximately 4.9) kDa. The affinity purified lectin existed in its native state as aggregates, and by stepwise centrifugation it could be fractionated into molecular entities with distinct specificities towards human and/or horse erythrocytes (modiolin H and/or E activity, respectively). While the medium size entities (range < or = 30 and < 100 kDa) exhibited only modiolin E activity and the lowest size entities (range < or = 5 and < 10 kDa) demonstrated only modiolin H activity, the largest aggregates (> or = 100 kDa-)expressed both activities. Antibacterial activity of the lectin has been observed against various marine bacteria, whereas the whole hemolymph was less effective. The lectin exhibited strong antibacterial effect against all tested strains of Vibrio anguillarum, Vibrio salmonicida, Vibrio viscosus, Vibrio wodanis, and Vibrio ordalii, slight effect on Aeromonas salmonicida salmonicida and Shewanella putrefaciens, and no inhibitory effect with Alteromonas sp. Hemolymph of the horse mussel demonstrated no antibacterial effect against A. salmonicida salmonicida, Alteromonas sp., Sh. putrefaciens and some strains of V. anguillarum, but slight effects against some strains of V. anguillarum and both strains of V. ordalii, and more predominantly against V. wodanis, V. salmonicida and V. viscosus. These results indicate that the lectin plays a role in elimination of bacteria. Circulating hemocytes were demonstrated to be the source of the lectins since granules of the hemocytes were immunoreactive to anti-hemolymph lectin antibody and protein A/gold labelling.

A comparative study of pentraxin-like proteins in different fish species.

Pentraxins are a family of pentameric serum proteins that have been conserved in evolution and share sequence homology, similar subunit assembly and the capacity for calcium-dependent ligand binding. The classical pentraxins are human C-reactive protein (CRP) and serum amyloid P component (SAP). The sequence homology and gene organization indicate that they arose from a gene duplication of an ancestral pentraxin gene. They are usually isolated based on their affinity for phosphorylcholine and agarose, respectively. We have used this method for isolation of pentraxin-like proteins from normal serum of Atlantic salmon (Salmo salar), common wolffish (Anarhichas lupus), cod (Gadus morhua) and halibut (Hippoglossus hippoglossus). Although pentraxin structures have not been verified, the isolated proteins all appear to be pentraxin-like based on their binding specificity, molecular weight of subunits, cross-reactivity with antibodies to human pentraxins and N-terminal amino acid sequences. However, with the described method only one pentraxin-like protein was detected in each of the fish species.

A heterogeneous sialic acid-binding lectin with affinity for bacterial LPS from horse mussel (Modiolus modiolus) hemolymph.

A sialic acid-binding lectin that agglutinates a variety of erythrocytes and bacteria and react with sialoconjugates and purified lipopolysaccharides from marine vibrios has been affinity purified from hemolymph of the horse mussel Modiolus modiolus using Bovine submaxillary mucin conjugated to CNBr-activated Sepharose 4B. The lectin demonstrated heterogeneous activity, and at least two main entities were partially characterized, and are referred to as modiolin H and modiolin E activities for the agglutination of human and horse (equine) erythrocytes, respectively. Only modiolin E activity required calcium ions for hemagglutination. The M. modiolus lectin was mainly specific for NeuAc, although the lectin demonstrated a broader range of specificity, similarly to the Limulus polyphemus lectin. The purified lectin was a glycoprotein, and in the native state existed as aggregates with M(r) in the range of 100-1,300 kDa as observed by gradient-gel electrophoresis and gel filtration on Biogel and Superose. SDS-PAGE under reducing conditions revealed three subunits of M(r) 14, 17.5 and 20 kDa. Various marine bacteria adsorbed the hemagglutinating activities of the M. modiolus lectin. Purified LPS preparations from various pathogenic marine vibrios were also effective inhibitors, in particular for modiolin E activity. These results indicate that the lectin play a role in recognition of bacteria.

Indigenous bacteria in hemolymph and tissues of marine bivalves at low temperatures.

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8 degrees C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 x 10 to 5.6 x 10 bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6 degrees C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 x 10), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 x 10 bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4 degrees C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10 bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 x 10 was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8 degrees C. Increased bacterial numbers in seawater at 4 degrees C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.

Agglutinin activity in Pacific oyster (Crassostrea gigas) hemolymph following in vivo Vibrio anguillarum challenge.

Hemolymph from the Pacific oyster (Crassostrea gigas) contains lectins that agglutinate horse (Gigalin E) and human (Gigalin H) erythrocytes. The gigalins also agglutinate bacteria, including Vibrio anguillarum, and were adsorbed from oyster hemolymph at different temperatures by living, heat-killed, and freeze-dried V. anguillarum cells. Baseline activities of the two gigalins were established by measuring their activities in oyster hemolymph over a period of 4 years. A normal distribution of Gigalin H activity (mean titer 139) was found, whereas the distribution of Gigalin E activity in the same samples was skew (mean titer 512). No covariance was observed between the two agglutinin activities. Increased lectin activity above this baseline was found in oysters exposed for varying time intervals to V. anguillarum at different seasons and temperatures over a period of 2 years. Such exposure resulted in an increase in activity (titer) of four- to nine-fold for Gigalin E and three- to seven-fold for Gigalin H when compared with controls, and in augmentation in the hemolymph of a protein with the same electrophoretic mobility as affinity-purified oyster lectins (gigalins). Challenge with either living or heat-killed bacteria resulted in a significant increase of Gigalin E activity, whereas results for Gigalin H were variable. Oysters challenged with bacteria were observed to filter normally with open shells during the experiments. Also, no increase was found in hemolymph calcium that could indicate anoxia following bacterial challenge (0.49 +/- 0.004 mg mL-1) compared to unexposed oysters (0.50 +/- 0.001 mg mL-1). Increase in the concentration of free amino acids in oyster hemolymph was observed following exposure to bacteria (15.05 mM) and anaerobiosis (13.51 mM) compared to controls (9.06 mM), and changes (in mol %) of individual amino acids differed considerably between hemolymph from animals challenged with bacteria and animals kept anaerobic. The augmented lectin activity in oyster hemolymph, following in vivo exposure to increased bacteria in the seawater, suggests their involvement in enhancing bacterial clearance and defense in the oyster.

Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae.

The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy. Larvae were hatched and kept in filtered seawater or in seawater with penicillin and streptomycin. The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments. Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus. The posterior region of the intestinal lumen appeared completely occluded with bacteria. The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp. dominated in larvae exposed to antibiotics. The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics. Thus, a dramatic change in the microflora resulted from incubation with antibiotics. Nonpigmented yeasts were detected in both larval groups. Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated. These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae. The possible significance for larval health and nutrition is discussed.

Bacterial Colonization of Cod (Gadus morhua L.) and Halibut (Hippoglossus hippoglossus) Eggs in Marine Aquaculture.

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibrio fischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.

Trimethylamine oxide: a terminal electron acceptor in anaerobic respiration of bacteria.

Trimethylamine oxide (TMAO) stimulated both the anaerobic growth rate and the growth yield of Proteus NTHC 153. The molar growth yield from glucose and pyruvate in tryptone/yeast extract medium doubled in the presence of TMAO, and the organism grew anaerobically on the non-fermentable substrates L-lactate and formate when TMAO was added to the medium. We conclude that TMAO stimulated growth by serving as a terminal electron acceptor in an oxidative phosphorylation process.