Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout

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1 Fish & Shellfish Immunology 21 (26) 199e28 Respiratory burst activity of Atlantic cod (Gadus morhua L.) blood phagocytes differs markedly from that of rainbow trout Sami Nikoskelainen a, *, Ole Kjellsen b, Esa-Matti Lilius a, Merete Bjørgan Schrøder b a Department of Biochemistry and Food Chemistry, University of Turku, 214 Turku, Finland b Norwegian College of Fishery Science, University of Tromsø, 937 Tromsø, Norway Received 9 August 25; revised 4 October 25; accepted 14 November 25 Available online 1 January 26 Abstract In the present study we investigated the respiratory burst (RB) activity of Atlantic cod (Gadus morhua L.) blood phagocytes and we evaluated how the RB activity of cod blood cells differ from that of trout. The RB activities were measured directly from highly diluted whole blood as luminol-amplified chemiluminescence (CL) under various conditions. Studies regarding the blood dilutions for cod whole blood chemiluminescence measurements (WBCL) revealed that at a final blood dilution of 1.5 mlml ÿ1 or less the CL response was strictly proportional to the number of phagocytes. This range of blood dilution did not markedly differ from that of trout. However, the opsonisation capacity of cod plasma was markedly poorer. The RB activity of phagocytes was most active at 15 C when heterologous cod serum was used as a source of opsonin, whereas at final blood dilution of 8. mlml ÿ1 (when homologous cod plasma was at a higher concentration) the highest RB activity was observed at 1 C. Aeromonas salmonicida strain MT4 (As MT4) induced higher RB activity than the two known pathogens for cod, atypical A. salmonicida and Vibrio anguillarum. Cod blood phagocytes were more responsive to plastic surfaces and the adhesion response of phagocytes was partly inhibited but did not totally vanish even at a final gelatin concentration of.4%. Moreover, cod serum enhanced the adherence of phagocytes and cod blood phagocytes also showed slow spontaneous degranulation. Finally, within the tested anticoagulants (heparin, Na-citrate, EDTA) heparin treated blood phagocytes generated the highest RB activity. Ó 25 Elsevier Ltd. All rights reserved. Keywords: Atlantic cod; Gadus morhua L; Complement; Respiratory burst activity; Blood phagocytes; Luminol-amplified chemiluminescence; Aeromonas salmonicida; Vibrio anguillarum 1. Introduction Phagocytosis and complement comprise the innate defence against infectious diseases in fish and is a fundamental mechanism in teleosts. Phagocytosis is mediated by phagocytic cells such as neutrophils, monocytes and macrophages. Phagocytosing leukocytes of fish constitute the first line of defence against invading microbial pathogens [1], and for that reason RB activity studies of fish phagocytes has been widely used to assess the antimicrobial activity. * Corresponding author. Tel.: þ ; fax: þ address: sami.nikoskelainen@utu.fi (S. Nikoskelainen) /$ - see front matter Ó 25 Elsevier Ltd. All rights reserved. doi:1.116/j.fsi

2 2 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e28 During phagocytosis, phagocytes increase their consumption of molecular oxygen for respiratory burst (RB) activity. The antimicrobial defence mechanisms in mammalian phagocytic cells rely on their production of large quantities of reactive oxygen species (ROS), reactive nitrogen species (RNS) and antimicrobial enzymes such as cathepsin G and elastase [2e6]. Oxidant production begins with a cytoplasmic membrane-associated NADPH oxidase, where molecular oxygen undergoes a one-electron reduction to form superoxide anions (O 2 ÿ ). Superoxide anions can then form hydrogen peroxide (H 2 O 2 ) and several other ROS that serve as a substrate for the myeloperoxidase (MPO) reaction, in which a variety of highly toxic metabolites, including hypochlorous acid, are generated. Luminol-amplified chemiluminescence (CL) is widely used for the sensitive detection of reactive oxygen species (ROS) production in phagocytic cells. Recent studies have shown that the ingestion of bacteria can be measured directly from highly diluted blood of trout by the CL emission of blood phagocytes [7]. Additionally, the enhancing effect of specific antibodies, if present, on the ingestion of bacteria can also be determined by the CL measurements [8]. To the best of our knowledge the RB activity of cod has not been measured by the CL. The immune system of Atlantic cod (Gadus morhua L.) differs from other fish species because high levels of specific antibodies are not produced as a response to immunisation even if the cod attains a specific protection [9e11]. Basic studies of head kidney (HK) macrophages of cod have been carried out [12,13] showing that the cells were highly phagocytic and showed enhanced superoxide formation upon stimulation. The authors indicated that the cod macrophages respond to a lesser extent compared to macrophages from mammalian as well as other fish species. To get a better understanding of the immune mechanisms in cod it is necessary to establish methods for studying the functions of different cells and tissues involved in the defence reactions. Thus, the aim of this study was to establish a method for measuring the phagocytic activity of phagocytes from the whole blood of cod by luminol-amplified chemiluminescence (CL). 2. Materials and methods 2.1. Experimental fish Non-treated and healthy Atlantic cod (Gadus morhua L.) were obtained from Tromsø Research Station or caught from the sea. Wild cod were held in 2 l fibreglass tanks at temperatures between 8 and 11 C while the smaller farmed cod were held in 4 l fibreglass tanks at 1 C. All fish were fed with a commercial feed (Dana feed). The weight of the fish varied from 3 g to 3 kg Blood samples Blood samples were collected from the caudal vein of sacrificed or anaesthetised (in benzocaine, 4 mg l ÿ1 or metacaine, 1 mg l ÿ1 ) Atlantic cod. Blood was collected in EDTA tubes (BD vacutainer, Becton, Dickinson and Company, USA), and maintained at 4 C until use. For opsonisation of bacteria, blood samples were collected in serum tubes (BD vacutainer, Becton, Dickinson and Company, USA), and maintained at 4 C for at least 12 h until serum was separated and stored at ÿ2 C. When the effect of different anticoagulants on the RB activity was evaluated EDTA tubes, sodium heparin tubes or sodium citrate tubes (BD vacutainer, Becton, Dickinson and Company, USA) were used. The samples were assessed within 1 h after collection Bacteria and culture conditions Three different bacterial strains were used in this study. Aeromonas salmonicida strain MT4 (As MT4) is an avirulent A-layer negative strain [14] and was a gift from Dr Ian Bricknell (Marine Laboratory, Aberdeen, Scotland) whereas the A-layer positive atypical A. salmonicida 499 (aas 499) and the Vibrio anguillarum 4299 (serotype O2b) (Va) were provided by the Norwegian Veterinary Institute and are known to be virulent for Atlantic cod [15]. The bacteria stored at ÿ8 C were inoculated on to tryptic soy agar supplemented with 5% human blood and 1.5% NaCl (BA plates) and incubated for 3 days at 12 C(Va and aas 499) or at room temperature (RT) (As MT4). Further, Va was grown to mid-logarithmic phase in marine broth (MB-2216, Difco) at 12 C for 24 h, whereas the Aeromonas strains were grown to mid-logarithmic phase in Brain Heart Infusion broth (BHI, Difco) at 12 C for 48 h (aas 499) or overnight at 2 C(As MT4). Cells were harvested by centrifugation at 25 g and washed twice with Hank s balanced salt solution (HBSS), ph 7.4. After washing, bacteria were resuspended in the same

3 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e28 21 buffer. To study the effect of gelatin on the RB activity, the bacteria were finally resuspended in HBSS containing.1e.4% gelatin (g-hbss). The optical density of bacterial suspensions was adjusted to.5.1 at 6 nm, which is equivalent to ÿ5 1 8 CFU ml ÿ1. Opsonised bacteria (OB) were prepared of strain As MT4. Following growth, cells were washed and prepared in calcium and magnesium free HBSS (CMF-HBSS). Then, the suspension of bacteria was divided into two portions and fresh pooled (n ¼ 3) normal cod serum was added to one tube (OB, final serum concentration of 1%) and an equal volume of CMF-HBSS to the other one (non-opsonised bacteria, NOB). Both suspensions were incubated at 1 C with slow agitation for 1 h. The cells were washed twice with g-hbss (.1%) and the number of bacteria adjusted to OD 6 ¼.5.5 with g-hbss which is equivalent to CFU ml ÿ Zymosan Six hundred milligrams of zymosan (Sigma, St. Louis, USA) was mixed with 3 ml of HBSS and boiled in a water bath for 2 min. After cooling, the zymosan was washed twice with HBSS and finally resuspended in 3 ml of HBSS to a final concentration of 2 mg ml ÿ1. Boiled non-opsonised zymosan (NOZ) was stored at ÿ7 C in aliquots of 5 ml until use. For opsonisation, NOZ was mixed with 1% of pooled normal cod sera diluted in CMF-HBSS and incubated at 1 C for 1 h. After incubation, the opsonised zymosan (OZ) was washed twice with CMF-HBSS buffer and finally resuspended in 5 ml CMF-HBSS to obtain the final concentration of 2 mg ml ÿ Respiratory burst assays In order to determine the effect of blood dilution on the RB activity, the RB of blood phagocytes was induced with opsonised As MT4 (OB) and non-opsonised As MT4 (NOB) in g-hbss, ph 7.4. Blood diluted in g-hbss, 1 ml of 1 mm luminol in.2 M borate buffer, ph 9., and 5 ml of bacterial suspension were combined in microtitre plate wells (White Cliniplate, Thermo Electron, Finland) and the final volume was adjusted with g-hbss buffer to obtain final blood dilutions ranging from 1.5 ml ml ÿ1 to 13.5 ml ml ÿ1 in a final volume of 3 ml. The CL emission of the blood phagocytes was measured immediately with a luminometer (Plate Chameleon, Hidex, Finland) for at least 3 h at 9 C to obtain a kinetic curve for each sample. To investigate the effect of gelatin and serum concentrations as well as the effect of bacterial strains on the RB activity, As MT4, aas 499 or Va (w ÿ per well) were prepared either in.1e.4% g-hbss or HBSS and were added to the microtitre plate wells (White Cliniplate, Thermo Electron, Finland). Before the blood samples, diluted either in HBSS or g-hbss, were added to microplate wells, 1 ml of 1 mm luminol and different concentrations of pooled normal cod sera (NS), or heat inactivated (at 56 C) pooled sera (HI) diluted in either HBSS or g-hbss, were added to the wells to obtain a final volume of 3 ml and a final blood dilution of 1:65 (1.5 mlml ÿ1 ). The CL emission of blood phagocytes was measured with a luminometer (Plate Chameleon, Hidex, Finland) for at least 3 h at temperatures of 4 C, 1 C and 15 C to obtain a kinetic curve. 3. Results 3.1. The effect of blood dilution We have previously investigated the effect of blood dilution on the respiratory burst (RB) activity of rainbow trout. The RB activity of trout phagocytes appeared to be most active at their maintaining temperature and for that reason trout measurements were conducted at 18 C [16]. For cod RB assays, a temperature of 1 C, the maintaining temperature, was chosen. As with the earlier RB assays of trout the RB activity of cod blood phagocytes was induced in parallel with opsonised (OB) and non-opsonised (NOB) As MT4 cells. Additionally,.1% of gelatin was included in all measurements to decrease the first peak of expected biphasic response (adhesion and ingestion responses) [7].At a final blood concentration of 2. mlml ÿ1 or less, practically no RB activity was detected when cod phagocytes were stimulated with NOB. However, at the same blood dilutions phagocytes generated RB activity when cells were stimulated with OB (Fig. 1A). To determine a blood dilution where the plasma proteins and the number of blood cells do not interfere with the CL response, the peak values of RB activities of cod were plotted as a function of blood

4 22 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e A B Concentration of blood (µl/ml) Normal serum opsonized bacteria (N=8) Non opsonised bacteria (N=8) Normal serum opsonized bacteria (N=8) Non opsonized bacteria (N=8) C D 1 RLU int 1, Blood concentration (µl/ml) Fig. 1. The effect of blood dilution on the respiratory burst (RB) activity of cod blood phagocytes. The RB activity of cod blood phagocytes was induced in parallel with opsonised (A) and non-opsonised (B) Aeromonas salmonicida MT4 cells (w bacteria per well) at blood dilutions ranging from 1.5 to 13.5 ml ml ÿ1. The RB activity of blood phagocytes was measured for 3 h at 1 C. Following kinetic measurements the peak values of RB activities were plotted as a function of blood dilution (C). All results represent the mean value of eight cod. (D) A plot of blood dilution and RB activity of rainbow trout measured under similar conditions at 18 C. concentration (Fig. 1C) similarly as the peak values of RB activities of trout (Fig. 1D) [16]. At a blood concentration of 6. mlml ÿ1 or higher, the NOB also induced CL response (Fig. 1C). Because the CL response of phagocytes induced with OB showed a linear correlation with the concentration of blood at a final blood dilution of 2. mlml ÿ1 or less, it was concluded that whole blood chemiluminescence (WBCL) measurements of cod should be conducted at these dilutions or less The effects of serum and gelatin concentration on the respiratory burst activity The opsonisation efficiency of plasma of cod appeared to be low at 1 C since the increased volume of blood in the reaction did not markedly increase the magnitude of RB activity induced by NOB. Therefore, we next examined the effect of serum on the RB activity measurements at 15 C. Surprisingly, a RB response was observed in the presence of cod serum even in the absence of target bacteria (Fig. 2A). Cod serum enhanced the adhesion response dosedependently and gelatin did not totally abolish the adhesion response (Fig. 2C). Addition of target bacteria abolished this adhesion response (Fig. 2B) and caused a second peak which appeared with a peak time of 2e3 min which we expected to be the response to ingestion. To evaluate the starting point of ingestion, duplicate samples were combined with cytochalasin D (final concentration of 3 mm), a known agent to inhibit cell movements and phagocytosis. From these measurements it was estimated that, similarly as in [7], the CL response generated before 15 min was a response to adhesion (data not shown). Gelatin markedly decreased the magnitude of the second peak of ingestion (Fig. 2D). When we plotted the CL responses as a function of serum concentration,.3% of cod serum induced the highest ingestion response (Fig. 2E).

5 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e A no bacteria no gelatin B 1.7 x 1 7 bacteria/well no gelatin Concentration of serum 8 %.3% 6 1.% 1.7% C no bacteria.4% of gelatin 1.7 x 1 7 bacteria/well.4% of gelatin D E 12.4% gelatin Absent gelatin ,,3,6,9 1,2 1,5 1,8 Serum concentration (%) Fig. 2. The effect of serum concentration on the ingestion and adhesion responses of cod blood phagocytes at 15 C. The RB activity of cod blood phagocytes was measured at a final blood dilution of 1.5 mlml ÿ1 and the phagocytes were activated without (A and C) or with (B and D) Aeromonas salmonicida MT4 cells (w bacteria per well). Additionally, the measurements were performed without (A and B) or with (C and D).4% of gelatin and in the presence of various concentrations of cod serum (shown in the figure). All curves represent the mean values of six cod. Cod blood phagocytes appeared to be more adhesive or responsive to plastic surfaces than those of trout phagocytes and we next sought to determine the concentration of gelatin needed to abolish the adhesion response. Following kinetic WBCL measurements, the peak CL responses of adhesion and ingestion processes of individual measurements were plotted as a function of gelatin concentration. It was observed that gelatin did not totally inhibit the adhesion response at any tested concentrations. Routinely,.4% gelatin was included in the assays.

6 24 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e The effect of assay temperature and bacterial number on the respiratory burst activity Despite the fact that cod blood phagocytes showed RB activity at 1 and 15 C it was unclear whether these tested assay temperatures were optimal for cod blood phagocytes. Therefore, we assessed the RB activity of cod blood phagocytes at 4 C and additionally, we tested whether the number of target bacteria had any effect on the RB activity (Fig. 3). The RB activity was induced with As MT4 cells together with.3% of heterologous normal cod sera and.4% of gelatin at a final blood concentration of 1.5 ml ml ÿ1 (Fig. 3AeC) and without.3% of normal cod sera at a final blood concentration of 8. ml ml ÿ1 (Fig. 3DeF). The peak times of the CL responses were dependent on assay temperature and the shortest peak time was observed at 15 C. Perhaps surprisingly, cod blood phagocytes showed a RB response at all tested assay temperatures even without target bacteria (Fig. 3). Addition of bacteria per well, however, at least doubled the magnitude of RB compared to samples without bacteria. The luminol amplified CL has been shown to be nearly completely dependent on MPO [17e21] and, moreover, on the release of MPO from the azurophil granules [22e24]. Luminol-amplified CL leads to emission of light both intra-and extracellularly. The results therefore indicated a spontaneous slow degranulation of cod phagocytes that was probably a consequence of adherence of cells to the microplate and this differed kinetically from that of previously seen adhesion responses of trout and cod (see Fig. 2) and was markedly decreased at lower assay temperatures. In order to investigate the effect of assay temperature and homologous plasma on the spontaneous slow degranulation of phagocytes, WBCL measurements were also conducted at a final blood concentration of 8. mlml ÿ1 (1:125) at 4, 1 and 15 C(Fig. 3DeF). This blood dilution was chosen because at a blood concentration of 8. mlml ÿ1 the CL A B C Number of bacteria (x1 6 bacteria/well) D E F Number of bacteria (x1 6 bacteria/well) Fig. 3. The effect of assay temperature and number of target bacteria on the whole blood chemiluminescence response (WBCL) of cod blood phagocytes. The respiratory burst (RB) activities were measured at final blood dilutions of 1.5 ml ml ÿ1 (AeC) and 8. ml ml ÿ1 (DeF). (A) and (D) represent measurements performed at 4 C, (B) and (E) represent measurements at 1 C and (C) and (F) represent measurements at 15 C. The RB activity of phagocytes was induced with varying numbers of Aeromonas salmonicida MT4 cells shown in the figure in the presence of.3% of normal cod serum (AeC) with a final concentration of.4% of gelatin and in the absence of cod serum (DeF). All curves represent the mean values of six cod.

7 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e28 25 response induced with NOB showed marked in situ opsonisation. In contrast to measurements performed with 1.5 ml ml ÿ1 (1:65) of blood, homologous plasma (8. mlml ÿ1 of blood) had no opsonising effect at 4 C and therefore, no differences were detected between spontaneous slow degranulation (without bacteria) compared to samples containing bacteria. At 1 C, the velocity and magnitude of RB activity were markedly accelerated and increased compared to spontaneous slow degranulation indicating opsonisation of target bacteria by homologous plasma factors The effect of anticoagulants and bacterial strain on RB activity In order to evaluate the effect of various anticoagulants on the RB activity of phagocytes, blood samples from the same cod were collected into EDTA, heparin or citrate tubes. The RB activities of blood phagocytes were stimulated with various bacterial strains (see Section 2) at a final blood dilution of 1:65 in the presence of.4% of gelatin and.3% of cod serum at 15 C. Heparin always gave at least 25% higher CL response compared to EDTA or citrate indicating that cod blood phagocytes were activated in the heparin (Fig. 4A), as also observed by us with human phagocytes [25]. In addition, the concentration of EDTA in tubes is w6 mm when the tube is filled with blood. However, we have diluted blood samples at least 1-fold, making the final concentration of EDTA less than 6 mm in our samples and such a low concentration of EDTA should not have any contribution in our RB assays. No marked differences were seen between EDTA (Fig. 4B) or citrate treated blood (Fig. 4C). The non-pathogenic strain of A. salmonicida (MT4) induced higher RB response compared to the known pathogenic strain of A. salmonicida (aas 499) for cod. The smallest RB response was seen when phagocytes were activated with V. anguillarum A B C MT4 aas Va Fig. 4. The effect of anticoagulant and bacterial strain on the respiratory burst activity of cod blood phagocytes. The RB activity of cod blood phagocytes was measured at a final blood dilution of 1.5 ml ml ÿ1. The RB activity of phagocytes was induced with different bacterial strains shown in the figure (21 7 bacteria well ÿ1 ) in the presence of.3% of normal cod serum with a final concentration of.4% of gelatin. Panel A; heparin treated blood, Panel B; EDTA treated blood and Panel C; citrate treated blood. All curves represent mean values of four cod.

8 26 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e A OZ NOZ blood B NOZ zymosan treated plasma+noz zymosan treated+hi plasma+noz HI plasma+noz blood blood+plasma C plasma+bacteria HI plasma+bacteria zymosan treated plasma+bacteria zymosan treated+hi plasma+bacteria Blood blood+plasma Fig. 5. Zymosan induced respiratory burst (RB) of cod blood phagocytes and the effect of zymosan treatment and heating of serum on the opsonising effect of serum. (A) The RB activity of cod blood phagocytes was measured at a final blood dilution of 1.5 ml ml ÿ1 and the RB activity was induced with serum opsonised (OZ) and non-opsonised (NOZ) zymosan. (B) and (C) show the boosting effects of.3% of heat inactivated plasma (HI) and.3% of plasma treated with zymosan on the RB activity of phagocytes induced by NOZ (B) and by Aeromonas salmonicida MT4 cells (C). All curves represent mean value of four cod The effect of zymosan and heat inactivation of plasma on the RB activity Zymosan was an efficient cell activator and induced approximately a 1-fold higher CL response than bacteria (Fig. 5A and C). Opsonisation of zymosan with serum induced a higher CL response and increased the velocity of the RB compared to non-opsonised zymosan. We also evaluated how the zymosan treatment of cod plasma affects the opsonisation properties of plasma. Plasma samples of cod were incubated with zymosan for 6 min at 1 C. Thereafter the zymosan was removed by centrifugation and the RB activity of blood phagocytes was stimulated with NOZ or bacteria together with zymosan treated plasma (final concentration of.6% of plasma). Moreover, half of the zymosan and normal plasma samples were heated in a water bath at 56 C for 3 min to destroy the complement activity before use as a source of opsonins. Heat inactivation or zymosan treatment of plasma had no major effect on the RB activity when the blood phagocytes were activated with NOZ (Fig. 5B). However, when similarly treated plasma samples were used to boost MT4 induced RB activity, zymosan treatment had only a minor effect on the RB activity whereas heat inactivation significantly reduced the RB activity of phagocytes (Fig. 5C). 4. Discussion The aim of this study was to investigate the respiratory burst (RB) activity of cod blood phagocytes and evaluate how factors such as assay temperature, volume of opsonins, anticoagulants, effector target (E:T) ratio and properties of target bacteria affect the RB activity of cod blood phagocytes. Whole blood chemiluminescence (WBCL) measurements revealed that the final blood concentration of cod must be less than 2. mlml ÿ1 in order to avoid the opsonising effect of homologous plasma proteins, and light absorbing effects of blood cells. This observation is in congruence with our recent observation with rainbow trout [16]. Accordingly, the CL response is strictly proportional to the number of phagocytes at blood concentrations less than 2. ml ml ÿ1 also in cod.

9 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e28 27 At a final blood concentration of w3 ml ml ÿ1 or higher, the WBCL response induced by non-opsonised bacteria (NOB) started to increase. This is due to the increased amount of opsonising plasma factors in the reaction which opsonise target bacteria in situ [16]. Interestingly, NOB induced WBCL response never reached the same magnitude of WBCL induced by OB as observed with trout (Fig. 1D) or human phagocytes [25]. Under similar measurement conditions with trout blood phagocytes, the CL response induced with NOB reaches the magnitude of OB at a final blood dilution of 6. mlml ÿ1 showing that at this blood concentration the amount of plasma factors (w.3% of plasma concentration) is sufficient to fully opsonise NOB in situ. The result obtained with cod phagocytes indicates that the opsonisation capacity of cod plasma is poorer compared to rainbow trout. Unlike the blood and head kidney phagocytes of rainbow trout [7], blood phagocytes of cod generated RB activity even in the absence of bacteria. This observation suggests that the blood phagocytes of cod are more adhesive on the plastic surfaces than trout phagocytes and that the cod phagocytes are directly activated by the wall of the microplates. Moreover, cod serum seemed to enhance the spontaneous slow degranulation of phagocytes as can be seen in Fig. 2. Contrary to trout blood phagocytes, bacteria induced a relatively good CL response even without any opsonins. This observation indicates that factors other than the classical opsonin receptors (Ig, complement) play major roles in the recognition and phagocytic processes in cod. The non-specific CL responses were not always recorded and seemed to be dependent on individual cod. Whether this response is regulated by the stress level of cod or whether this is dependent on the expression of receptors of phagocytes remains to be investigated. Another interesting finding compared to trout was that, unlike trout, the heat inactivated serum sometimes gave an extremely high and rapid CL response even in the absence of target bacteria (data not shown). We assume that this finding is related to lipid contents or generation of anaphylactic compounds during the heat inactivation of the sera. Oxidation of polyunsaturated fatty acids generates peroxides that are known to induce RB activity of phagocytes [26]. Serum samples that had been stored at ÿ2 C for several months induced similar extraordinary CL responses. These findings indicate that cod WBCL assays should be performed with fairly fresh serum or plasma to avoid non-specific activation of phagocytes. It is widely agreed that the generation of specific antibodies in cod following vaccination is different compared to other fish species as only limited antibody titres are raised in cod sera after vaccination [9e11]. Because no purified cod antibodies against the tested bacterial strains are available we were not able to investigate the effect of specific antibodies on the RB activity. The Aeromonas salmonicida strain MT4 is an avirulent mutant lacking the A-layer which is a vital virulence factor [14]. This bacterium induced higher RB activity than Vibrio anguillarum and an atypical A. salmonicida, both known pathogens for cod. This might be because the virulent strains may be ingested to a lesser extent by cod phagocytes. The magnitude of the RB activity of cod blood is at least double compared to trout when the cells are activated with OZ or NOZ under similar measurement conditions, and the velocity of the RB is faster. This suggests that the RB capacity of cod phagocytes is significantly higher compared to rainbow trout cells or, the number of blood phagocytes is greater in cod blood, which we indeed do not know, yet. Trout are tetraploid and have larger blood cells than many other species. Therefore, there may be more phagocytes per unit volume of blood in cod. This observation, together with the fact that the phagocytes of cod are more rapidly activated than the cells of trout and the observation that infected cod develop major granulomas in various tissues [27], emphasize the importance of cod phagocytes against infectious diseases. We additionally believe that the extracellular degranulation might play some role in the bactericidal activity of cod phagocytes. Acknowledgements This study was supported by Norsk-finsk kulturfond Hanasaari. We thank K. Niittymäki and J. Haaslahti (Hidex OY, Finland) for providing material support for this study. References [1] Secombes CJ. The nonspecific immune system: cellular defence. In: Iwama G, Nakanishi T, editors. The fish immune system. San Diego, CA: Academic Press; p. 63e13.

10 28 S. Nikoskelainen et al. / Fish & Shellfish Immunology 21 (26) 199e28 [2] Hyslop PA, Hinshaw DB, Scraufstatter IU, Cochrane CG, Kunz S, Vosbeck K. Hydrogen peroxide as a potent bacteriostatic antibiotic: implications for host defense. Free Radic Biol Med 1995;19(1):31e7. [3] Campbell EJ, Silverman EK, Campbell MA. Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity. J Immunol 1989;143(9):2961e8. [4] Belaaouaj A. Neutrophil elastase-mediated killing of bacteria: lessons from targeted mutagenesis. Microbes Infect 22;4(12):1259e64. [5] Ellis AE. Immunity to bacteria in fish. Fish Shellfish Immunol 1999;9(4):291e38. [6] Neumann NF, Stafford JL, Barreda D, Ainsworth AJ, Belosevic M. Antimicrobial mechanisms of fish phagocytes and their role in host defense. Dev Comp Immunol 21;25(8e9):87e25. [7] Nikoskelainen S, Verho S, Airas K, Lilius E-M. Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish. Dev Comp Immunol 25;29(6):525e37. [8] Verho S, Järvinen S, Nikoskelainen S, Lilius E-M. Biological effect of vaccination can be assessed directly from diluted whole blood of rainbow trout using homologous blood phagocytes as immunosensors. Fish Shellfish Immunol 25;19(2):175e83. [9] Espelid S, Røedseth OM, Jøergensen T. Vaccination experiments and studies of the humoral immune responses in cod, Gadus morhua L., to four strains of monoclonal-defined Vibrio anguillarum. J Fish Dis 1991;14(2):185e97. [1] Pilströem L, Petersson A. Isolation and partial characterization of immunoglobulin from cod (Gadus morhua L.). Dev Comp Immunol 1991;15(3):143e52. [11] Schrøder MB, Espelid S, Jøergensen T. Two serotypes of Vibrio salmonicida isolated from diseased cod (Gadus morhua L.); virulence, immunological studies and vaccination experiments. Fish Shellfish Immunol 1992;2(3):211e21. [12] Sørensen KK, Sveinbjørnsson B, Dalmo RA, Smedsrød B, Bertheussen K. Isolation, cultivation and characterization of head kidney macrophages from Atlantic cod, Gadus morhua L. J Fish Dis 1997;2:93e17. [13] Steiro K, Johansen A, Gildberg A, Bøgwald J. Optimising of culture conditions and stimulation of head kidney macrophages from Atlantic cod, Gadus morhua L. J Fish Dis 1998;21(5):335e44. [14] Lamas J, Ellis AE. Atlantic salmon (Salmo salar) neutrophil responses to Aeromonas salmonicida. Fish Shellfish Immunol 1994;4(3): 21e19. [15] Mikkelsen H, Schrøder MB, Lund V. Vibriosis and atypical furunculosis vaccines; efficacy, specificity and side effects in Atlantic cod, Gadus morhua L. Aquaculture 24;242(1e4):81e91. [16] Nikoskelainen S, Bylund G, Lilius E-M. Effect of environmental temperature on rainbow trout (Oncorhynchus mykiss) immunity. Dev Comp Immunol 24;28(6):581e92. [17] Cohen MS, Shirley PS, DeChatelet LR. Further evaluation of luminol-enhanced luminescence in the diagnosis of disorders of leukocyte oxidative metabolism: role of myeloperoxidase. Clin Chem 1983;29(3):513e5. [18] Aniansson H, Stendahl O, Dahlgren C. Comparison between luminol- and lucigenindependent chemiluminescence of polymorphonuclear leukocytes. Acta Pathol Microbiol Immunol Scand 1984;92(6):357e61. [19] Dahlgren C, Stendahl O. Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1983;39(2):736e41. [2] Briheim G, Stendahl O, Dahlgren C. Intra- and extracellular events in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1984;45(1):1e5. [21] Gerber CE, Kuci S, Zipfel M, Niethammer D, Bruchelt G. Phagocytic activity and oxidative burst of granulocytes in persons with myeloperoxidase deficiency. Eur J Clin Chem Clin Biochem 1996;34(11):91e8. [22] Stendahl O, Andersson T, Dahlgren C, Magnusson KE. Defective chemiluminescence response in differentiated HL6 cells due to impaired degranulation. Biochim Biophys Acta 1986;881(3):43e6. [23] Edwards SW, Hart CA, Davies JM, Pattison J, Hughes V, Sills JA. Impaired neutrophil killing in a patient with defective degranulation of myeloperoxidase. J Clin Lab Immunol 1988;25(4):21e6. [24] Dahlgren C, Follin P, Johansson A, Lock R, Orselius K. Localization of the luminol-dependent chemiluminescence reaction in human granulocytes. J Biolumin Chemilumin 1989;4(1):263e6. [25] Lilius E-M, Nuutila J. Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasma. Chemiluminescence 25;2, (in press). [26] Cheung K, Archibald AC, Robinson MF. The origin of chemiluminescence produced by neutrophils stimulated by opsonized zymosan. J Immunol 1983;13(5):2324e9. [27] Magnadottir B, Bambir SH, Gudmundsdottir BK, Pilström L, Helgason S. Atypical Aeromonas salmonicida infection in naturally and experimentally infected cod, Gadus morhua L. J Fish Dis 22;25(1):583e97.