Effects of Antigens from Aeromonas-Salmonicida Ssp Achromogenes on Leukocytes from Primed and Unprimed Atlantic Salmon (Salmo-Salar L)

Fish & Shellfish Immunology (1995) 5, 493-504

Effects of a n t i g e n s f r o m Aeromonas salmonicida ssp. achromogenes on l e u k o c y t e s from primed and u n p r i m e d Atlantic s a l m o n (Salmo salar L.) SIGRIDURGUDMUNDSDOTTIR,BERGLJOTMAGNADOTTIRAND BJARNHEIDUR K. GUDMUNDSDOTTIR

Institute for Experimental Pathology, University of Iceland, Keldur, IS-112, Reykjav$k, Iceland (Received 8 March 1995, accepted in revised form 12 May 1995) The effects of extracellular antigens isolated from Aeromonas salmonicida ssp. achromogenes, were tested on leukocyte cultures from the head-kidney of Atlantic salmon (Salmo salar L.) fingerlings. Samples from vaccinated fingerlings were compared to samples from control fingerlings that were injected with incomplete Freunds adjuvant (IFA) or phosphate buffered saline (PBS). The antigen preparations used for the comparative studies were extracellular products (ECP) and a 20 kDa toxic metallo-caseinase (AsaP1). Lipopolysaccharide (LPS) isolated from the bacterium was tested on unvaccinated fingerlings only. All antigen preparations were mitogenic to cultures from unvaccinated fish. In cultures from vaccinated fish there were difficulties in discerning between mitogenic effects, adjuvant induced cell activation and possible specific responses to the antigens. ([} 1995 Academic Press Limited Key words:

Atlantic salmon, leukocyte proliferation assay, Aeromonas salmonicida ssp. achromogenes antigens.

I. I n t r o d u c t i o n C u r r e n t l y , the G r a m - n e g a t i v e b a c t e r i u m Aeromonas salmonicida is divided into four subspecies, i.e. ssp. salmonicida, achromogenes, masoucida a n d smithia (Holt et al., 1994). T h e s u b s p e c i e s salmonicida is r e f e r r e d to as the t y p i c a l b a c t e r i u m . T h e o t h e r three, as well as isolates of the b a c t e r i u m t h a t d e v i a t e f r o m d e s c r i p t i o n s of the four s u b s p e c i e s in a n u m b e r of b i o c h e m i c a l a n d p h y s i o l o g i c a l p r o p e r t i e s , a r e r e f e r r e d to as atypical. A t y p i c a l f u r u n c u l o s i s c a u s e d by A. salmonicida ssp. achromogenes is the m o s t h a r m f u l b a c t e r i a l disease in I c e l a n d i c a q u a c u l t u r e . O t h e r s u b s p e c i e s of A. salmonicida h a v e n o t b e e n i s o l a t e d in Iceland. K n o w l e d g e of the i m m u n e r e s p o n s e elicited in fish is of p r i m e i m p o r t a n c e in u n d e r s t a n d i n g disease as well as for the d e v e l o p m e n t of s u i t a b l e vaccines. In the m a j o r i t y of v a c c i n e trials in fish, efficacy h a s b e e n t e s t e d by c h a l l e n g i n g w i t h live b a c t e r i u m a n d / o r t h e h u m o r a l r e s p o n s e m o n i t o r e d by m e a s u r i n g specific a n t i b o d i e s . Studies on s a l m o n i d s , v a c c i n a t e d w i t h A. salmonicida ssp. salmonicida, h a v e s h o w n a p o o r c o r r e l a t i o n b e t w e e n a n t i b o d y r e s p o n s e a n d p r o t e c t i o n (Michel & F a i v r e , 1982; Ellis, 1988; M S y n e r et al., 1993; A a k r e et al., 493 1050 4648/95/070493+12 $12.00/0

~) 1995 Academic Press Limited

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S. GUDMUNDSDOTTIR ET AL.

1994). Reports on the antibody response in infections with other subspecies of A. salmonicida reveal a complex picture. Studies on Atlantic salmon, infected with A. salmonicida ssp. achromogenes, showed that repeated infections, in groups frequently treated with antibiotics, resulted in declining antibody titers as well as diminished specificity (Magnad6ttir & GuSmundsd6ttir, 1992; Magnad6ttir et al., 1995). Infection with an atypical A. salmonicida in carp (Cyprinus carpio) has been shown to suppress the humoral immune response (Evenberg et al., 1986; Pourreau et al., 1986) while experimental vaccination, with a preparation containing the same strain, demonstrated that immunity to ECP is important for protection (Evenberg et al., 1988). Evidently the pathogen-host interactions in A. salmonicida infections are complicated and there is a need for tests measuring immunological parameters other than antibodies. One such test evaluates cell proliferation in response to foreign antigen or the secondary response to a previously encountered antigen. Such proliferation is a feature of humoral as well as cell-mediated immunity. A few pathogens or pathogen derived antigens have been tested in proliferative assays in leukocyte cultures from fish. These include Vibrio anguillarum (Yui & Kattari, 1987; Thuvander, 1989), A. salmonicida ssp. salmonicida (Tatner, 1990; Reitan & Thuvander, 1991; Reitan & Thuvander, 1994; Marsden et al., 1994), atypical A. salmonicida isolated from carp (Evenberg et al., 1985; Pourreau et al., 1987), infectious salmon anaemia agent (Dannevig et al., 1993) and Ligula intestinalis (Taylor & Hoole, 1994). No reports of studies on the effects of A. salmonicida ssp. achromogenes or its antigens on leukocyte cultures exist in the literature. A. salmonicida ssp. salmonicida and ssp. achromogenes share certain cell associated antigens such as the A-layer protein and LPS (reviewed in Austin & Austin, 1993) but the exotoxins produced seem to be different (GuSmunsd6ttir et al., 1990; Ellis, 1991) which might be reflected in different immune responses. The aim of the present study was to investigate whether A. salmonicida ssp. achromogenes antigens could be used in a proliferation assay to evaluate specific immune responses in head-kidney leukocyte cultures from vaccinated fish. Cultures from unvaccinated fish were employed to test nonspecific effects of ECP, LPS and AsaP1 (GuSmundsd6ttir et al., 1990). The effects of ECP and AsaP1 on head-kidney leukocyte cultures from vaccinated and control injected Atlantic salmon were examined and the results compared to humoral responses and protection to challenge with live bacterium in the same groups.

II. M a t e r i a l s a n d M e t h o d s FISH

Atlantic salmon fingerlings with an average weight of 25 g were kept in tanks with continuously flowing well water at an ambient temperature of 10 + 3°C and fed commercial dry pellets with an automatic feeder. The fish came from The State Experimental Fish Farm (LaxeldisstS~in Kollafir~i) where atypical furunculosis has never been detected and were acclimatized for one week before vaccination.

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495

BACTERIAANDBACTERIALANTIGENS Three A. salmonicida ssp. achromogenes isolates (265-87, M108-91 and $24-92) originating from diseased Atlantic salmon on three Icelandic fish farms, were used in the study. The isolates were homogeneous as regards the following characteristics: Gram-negative short rods, non-motile, facultative anaerobic; produced indole and acid from sucrose; failed to produce gas from glucose and to degrade aesculin; were resistant to the antibiotics ampicillin and cefaloridin; were autoaggregating; showed delayed production of brown pigment and excreted a 20 kDa toxic metallo-caseinase, AsaP1. The vaccines used were an autogenous bacterin, Iceland Biojec. OO (IBOO), produced by BioMed Inc., Seattle, USA, from the Icelandic isolate M108-91, containing mineral oil adjuvant, and an experimental vaccine (K-15) prepared at our laboratory by fermentation of isolate 265-87. K-15 contained formalinkilled bacterial cells and ECP with chemically inhibited enzyme activity, emulsified in an equal volume of incomplete Freund's adjuvant (IFA). Controls received PBS or PBS emulsified in an equal volume of IFA. Extracellular products (ECP) were prepared from isolate 265-87, by the cellophane overlay method as described by Gubmundsdbttir et al. (1990). Protein concentration was determined by the Bradford protein assay (Bradford, 1976) and the preparation aliquoted and stored at - 20 ° C. AsaP1 was isolated from an ECP preparation of isolate 265-87 as previously described by Gubmundsdbttir et al. (1990). Briefly, anion-exchange chromatography was followed by gel-filtration using Mono Q HR 5]5 and Superose 12 HR 10130 columns respectively and the fast protein liquid chromatography (FPLC) system from Pharmacia. Protein concentrations of fractions collected were determined by the Bradford protein assay, the purified enzyme aliquoted and stored at - 2 0 ° C. The fraction used for all leukocyte proliferation assays contained 150 ~g m l - 1 protein and 0-18 eu m l - 1 lipopolysaccharide (LPS) as determined in the L i m u l u s amoebocyte lysate test (E-Toxate, Sigma). LPS was isolated from a formalinized whole cell preparation of isolate 265-87, by the butanol-water extraction procedure followed by trypsin digestion (Koga et al., 1985). The preparation contained 4800 eu ml-1 (Limulus amoebocyte lysate test, E-Toxate, Sigma). SDS-PAGE ANALYSIS

SDS-PAGE separation of ECP, LPS and AsaP1 was carried out using the MiniProtean II system from Bio-Rad according to the manufacturer's instructions. Ten gl samples were applied to the gel, containing 0.1-10-0~g protein reduced with 5% mercaptoethanol in 125 mM Tris buffer (pH 6.8) containing 2% SDS. The stacking gel was 4-5% and the separation gel 14% acrylamide (Sigma, A7168). The gel was stained using BioRad silver staining kit for proteins. IMMUNIZATION AND CHALLENGE TESTS

Fifty fingerlings were included in each of four test-groups. Prior to injection, the fish were anaesthetized in benzocaine at a concentration of 40 mg 1-1 . The vaccines, as well as the control injections, were administered

S. GUDMUNDSD6TTIRE T AL.

496

T a b l e 1. Experimental design No.

Total number

Injected with

Dose (ml)

PBS IFA K-15 IBOO

0.1 0.1 0.1 0.2

50 50 50 50

No. sampled

challenged

6 weeks post-vacc,

12 weeks post-vacc,

12 weeks post-vacc.

10 10 10 10

10 10 10 10

30 30 30 30

intra-peritoneally, 0.1 ml for all groups except IBOO, where 0"2ml was injected according to the producers instructions. The experimental design is shown in Table 1. Test-groups were maintained in two tanks, one for groups maintained for 6 weeks and the ot her for groups maintained for 12 weeks. Challenge was by i nt r a m us c ul a r injection of 5000 cfu (colony forming units) of isolate $24-92 per fish. Deaths were recorded for three weeks post challenge. SAMPLING

Prior to sampling, the fish were anaesthetized as above and blood samples for antibody analysis were t a ke n from the caudal aorta and allowed to clot at 15 ° C for 4 h and then overnight at 4 ° C, centrifuged at 2000 g for 10 min and serum collected. Head-kidney samples were aseptically removed and transferred to test tubes containing 5 ml of Hanks' balanced salt solution (HBSS) with antibiotics and kept on ice. LEUKOCYTE ISOLATION

Head-kidney samples were t hr us t t h r o u g h a steel mesh to produce single cell suspensions. The suspensions were transferred to conical test tubes and kept on ice for 5 min, allowing the tissue remains to form a pellet. The s u p er n atan ts were transferred to similar tubes and spun at 300 g for 10 rain at 4 ° C after which s uper na t a nt s were discarded and the cells gently suspended in 0"75% ammonium chloride solution in 1.7 mM Tris buffer (pH 7-2, 20 ° C). The tubes were kept at room t e m p e r a t u r e for 5 min until red blood cells had lysed and then spun at 300 g for 10 min at 4 ° C, The cells were washed twice with HBSS containing 100 iu penicillin m l - 1 and 50 iu streptomycin m l -1. After the second wash, the cells were counted and resuspended in Leibowitz-15 medium (Gibco) with 5% foetal calf serum (FCS, Myoclone, Gibco), 2 mM glutamine and penicillin and streptomycin as described above. The cell suspensions (1"5 x 105 cells in 200~1 of medium per well) were seeded in flat-bottom tissue-culture plates (Nunc) for the proliferation assay. LEUKOCYTE PROLIFERATION ASSAY

For antigen stimulation, 20 ~1 of ECP, AsaP1 or LPS diluted in Leibowitz-15 medium were added to triplicate wells. The ECP concentrations tested were

EFFECTS OF ANTIGENS ON SALMON LEUKOCYTES

497

0.01, 0-1, 0"5, and 2.5]1g protein m1-1 and 0"001, 0"01, 0"1 and 0"5~g protein ml - 1 for AsaP1. LPS was used at 12-0 eu ml - 1 (endotoxin units). Control wells received 20111 of media. If isolated leukocytes were too few for all tests planned, the highest and the lowest concentrations of ECP were excluded. The culture plates were wrapped in polythene bags and incubated at 15 ° C. After four days, 3H-thymidine (1 ~Ci per well) was added, the plates incubated at room tem pe r at ur e for 16 h and t hen frozen at - 20 ° C. After thawing, the cells were harvested on glass fibre filters (Skatron) and dried at 37 ° C. Radioactivity was measured in a liquid scintillation counter (Packard TRICARB 1600 CA) using ULTIMA GOLD scintillation fluid (Packard). Results were expressed as Acpm, i.e. the value obtained when means of counts per minute (cpm) of triplicate control wells are subtracted from mean cpm of test wells. DOUBLESANDWICHANTI-ECPELISA The assay has previously been described in detail (Magnad6ttir & Gubmundsdbttir, 1992). Briefly, ELISA trays (Nunc) were coated overnight with 100/A per well of ECP (10~g protein m l - 1 ) washed, blocked with BSA and overlaid with 100gl of test sera diluted 1:100 in fresh PBS. After an overnight incubation at 4 ° C, bound antibody was detected in three steps: (a) polyclonal mouse antibody to salmon IgM incubated for 1 h at 37° C; (b) peroxidase labelled rabbit antibody to mouse immunoglobulins (Dako) incubated for 1 h at 37 ° C; and (c) colour development with OPD substrate (Sigma) incubated for 20 min at 37 ° C. Between steps, wells were washed three times with PBS containing 0'05% Tween-20. STATISTICAL ANALYSIS

Groups were compared with One Fa ct or Anova or a Chi-square test and paired samples by Kendall's r a n k coefficient. Arithmetic means and standard deviations were calculated according to standard procedures. III. R e s u l t s MITOGENICPROPERTIESOF AEROMONAS SALMONICIDA SSP.ACHROMOGENES ANTIGENS Fig. 1. shows a silver stained SDS-PAGE gel of ECP, LPS and AsaP1, purified from A. s a l m o n i c i d a ssp. achromogenes. The ECP separated into a multitude of components whereas the LPS preparat i on included two bands, 12 and 45 kDa. The AsaP1 pr e pa r at i on showed a strong 20 kDa band and a weak one at approximately 32 kDa. All preparations were mitogenic to head-kidney leukocyte cultures from u n t r e a t e d salmon fingerlings. Results from cultures of leukocytes from five u n t r e a t e d fish, showing the highest means obtained for each preparation, are shown in Table 2. Mitogenic effects of ECP and AsaP1 on head-kidney leukocyte cultures from fish injected i.p. with PBS (unprimed controls) 6 weeks post injection are shown in Fig. 2. ECP stimulation values peaked at 0"5/~g protein m l - 1 and AsaP1 values for the two highest concentrations tested, 0.1 and 0"5 pg protein

498

S. GUDMUNDSDOTTIRE T AL.

1

2

3

4

Fig. 1. Silver stained SDS-PAGE gel of A. salmonicida ssp. achromogenes antigens. Lane 1: molecular weight standards (kDa); Lane 2: ECP, Lane 3: LPS, Lane 4: AsaP1. Table 2. Effects of antigen preparations from A. salmonicida ssp. achromogenes on head-kidney leukocyte cultures from untreated salmon fingerlings. ECP: 0-5 ttg protein ml-1; AsaPl: 0-5/lg protein m l - 1; LPS: 12 eu ml - 1. The Acpm values shown are mean values for 5 fish Antigen

Acpm

± 1 S.D.

ECP AsaP1 LPS Control

20 750 12 608 20 468 844

± 3658 ± 4045 + 4998 ± 440

ml - 1, w e r e identical. W i t h b o t h a n t i g e n p r e p a r a t i o n s , t h e r e w e r e c o n s i d e r a b l e i n d i v i d u a l v a r i a t i o n s as s h o w n by t h e s t a n d a r d d e v i a t i o n values. CELLULAR RESPONSES 6

AND 12 WEEKSPOST-VACCINATION

Six w e e k s p o s t - v a c c i n a t i o n (results n o t shown), t h e E C P a n d A s a P 1 stimul a t i o n v a l u e s for c u l t u r e s f r o m v a c c i n a t e d fish a n d fish r e c e i v i n g I F A did n o t differ s i g n i f i c a n t l y f r o m t h e v a l u e s for t h e P B S i n j e c t e d c o n t r o l g r o u p s h o w n in Fig. 2. Twelve weeks post-vaccination the highest stimulation value obtained with E C P w a s for t h e I B O O v a c c i n a t i o n g r o u p a t a p r o t e i n c o n c e n t r a t i o n of 0-5/~g m l - 1 a n d for t h e K-15 v a c c i n a t e d g r o u p w i t h 0.1/tg m l - 1 (Fig. 3). Sufficient n u m b e r s of l e u k o c y t e s c o u l d n o t be i s o l a t e d f r o m all t h e fish a n d t h e r e f o r e t h e h i g h e s t a n d l o w e s t E C P c o n c e n t r a t i o n s w e r e t e s t e d on c u l t u r e s f r o m five i n d i v i d u a l s i n s t e a d of ten. Differences b e t w e e n g r o u p s w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t for a n y c o n c e n t r a t i o n tested.

EFFECTS OF ANTIGENS ON SALMON LEUKOCYTES

499

16 000 14 000 12 000 10 000 8000 6000 4000 2000 0

T 0.001

0.01 0.1 0.5 ECP/AsaP1, pg m1-1

2.5

Fig. 2. Responses to ECP [] and AsaP1 [] in head-kidney leukocyte cultures from Atlantic salmon fingerlings, injected with PBS 6 weeks earlier. Results are expressed as the arithmetic mean of Acpm 4- 1 S.D. for 10 fish. 16 000 14 000 12 000 10 000 8000 6000

t~

4000 2000 0

0.01

0.1 0.5 ECP pg m1-1

2.5

Fig. 3. ECP-induced head-kidney leukocyte stimulation in cultures from controlinjected (PBS [3, IFA [3) and vaccinated (K-15 @, IBOO II) fingerlings 12 weeks post vaccination. Results are expressed as the arithmetic mean of Acpm 4- 1 S.D. for 10 fish in each group except for the highest and lowest concentrations where 5 fish were included. For AsaP1 stimulated cultures, twelve weeks post-vaccination, there was a s i g n i f i c a n t difference in s t i m u l a t i o n v a l u e s b e t w e e n t h e I B O O v a c c i n a t e d g r o u p a n d t h e PBS c o n t r o l g r o u p for all c o n c e n t r a t i o n s t e s t e d e x c e p t the l o w e s t (Fig. 4). T h e K-15 v a c c i n a t e d g r o u p differed significantly f r o m t h e PBS g r o u p o n l y a t a AsaP1 c o n c e n t r a t i o n of 0 " l # g m l - 1 . C o m p a r i s o n of s t i m u l a t i o n v a l u e s o b t a i n e d for c u l t u r e s f r o m the v a c c i n a t e d g r o u p s a n d the I F A i n j e c t e d c o n t r o l g r o u p did n o t r e v e a l s i g n i f i c a n t differences for a n y c o n c e n t r a t i o n tested.

500

S. GUDMUNDSDOTTIR ET AL. 16 000 14 000 12 000 10 000 8000 6000 4000 2000 0

0.001

0.01 0.1 AsaP1 pg m1-1

0.5

Fig. 4. AsaP1 induced head-kidney l e u k o c y t e s t i m u l a t i o n in c u l t u r e s from controlinjected (PBS [3, I F A O) and v a c c i n a t e d (K-15 D, IBOO I ) fingerlings 12 weeks post vaccination. Results are expressed as the a r i t h m e t i c m e a n of Acpm ± 1 S.D. for 10 fish in each group. 2.0

2.0

L (a)

_ (b)

1.8 1.8 I 1.6

1.6

1.4

1.4

1.2

1.2

~ 1.o

1.0

L

0

0.8

0.8

0.6

0.6

0.4

0.4 0.2

0

PBS IFA K-15 IBOO 6 weeks post-vaccination

0

PBS IFA K-15 IBOO 12 weeks post-vaccination

Test-group Fig. 5. H u m o r a l a n t i b o d i e s to ECP m e a s u r e d by E L I S A in serum samples (diluted 1:100) from control injected (PBS, IFA) and v a c c i n a t e d (K-15, IBOO) fingerlings 6(a) and 12(b) weeks post vaccination. Results are expressed as the a r i t h m e t i c m e a n of OD492 values ± 1 S.D. for 10 fish in each group. ANTI-ECP ANTIBODIES

The vaccinated groups showed a good antibody response 6 weeks postvaccination and the response continued to increase for the remaining 6 weeks of the experiment (Fig. 5). Anti-ECP ELISA values and Acpm values for ECP or AsaP1 stimulated cultures were compared for individual fish in all groups. A significant

EFFECTS OF ANTIGENS ON SALMON LEUKOCYTES

501

association was not found in any group, neither 6 nor 12 weeks postvaccination, using Kendall's rank coefficient. CHALLENGE

Survival in the challenge test, 12 weeks post vaccination, was 0% in the PBS group, 4% in the IFA group, 48% in the IBOO vaccinated group and 60% in the K-15 vaccinated group. The vaccinated groups differed significantly from the control groups, but not from each other, in a chi-square test.

IV. D i s c u s s i o n The effects of antigen preparations from A. salmonicida ssp. achromogenes on leukocyte cultures from Atlantic salmon have not been reported previously. In the present study, proliferative responses to an ECP preparation were observed in all groups tested. In cultures of leukocytes from unprimed fish, the response was clearly mitogenic. This effect was due to LPS and AsaP1, both of which were highly mitogenic and present in ECP, although mitogenic effects of unidentified factors cannot be excluded. The depression of cpm values seen with the highest ECP concentration tested could either be due to a true inhibitory or toxic effect or indicate that the peak of proliferation was reached earlier in these cultures. Future work should include kinetic studies of ECP at different concentrations, especially in view of the work of Pourreau et al. (1987), who demonstrated that ECP from an atypical A. salmonicida strain isolated from carp was able to inhibit PHA stimulated blastogenesis in carp head-kidney leukocytes. AsaP1, the toxic metallo-caseinase of A. salmonicida ssp. achromogenes (GuSmundsdSttir et al., 1990) emerged in the present study as a powerful mitogen of Atlantic salmon leukocytes. These results cannot be attributed to LPS impurities as the highest AsaP1 concentration tested for proliferative properties (0"5~g m l - 1) contained as little as 0-0006 eu m l - 1 LPS. Inhibitory effects were not seen in leukocyte cultures for the AsaP1 concentrations tested. Further studies need to be done on AsaP1 to define the mechanism of this activity. AsaP1 may be a mitogen that acts directly on a leukocyte subpopulation or it could exert its effects through accessory cells either by stimulating production of cytokines or in a superantigenic fashion. Mitogenic effects of ECP from other subspecies of A. salmonicida have been described previously (Pourreau et al. 1987; Tatner, 1990; Reitan & Thuvander, 1991; Reitan & Thuvander, 1994; Marsden et al., 1994) but mitogenic properties of a toxic protease from a bacterial fish pathogen have not been reported. Several microbial toxins are known to be powerful tools in activation as well as counteraction of host-defence mechanisms (KSnig et al., 1991). A proteolytic enzyme from a Gram-negative bacterium, Pseudomonas aeruginosa, exhibited mitogenic as well as inhibitory effects upon clones of human and murine T-lymphocytes and appeared to be the principal immunogen as well (Parmely et al., 1984). Some of the most potent mitogens known are extracellular toxins of the Gram-positive bacteria Staphylococci and Streptococci (Alouf et al.,

502

S. GUt)MUNDSDOTTIR ET AL.

1991). These proteins stimulate T cells through accessory cells and this interaction has been shown to be superantigenic in nature. The vaccines, tested in the present study, induced specific antibodies that increased from 6 to 12 weeks following vaccination. Comparison of paired samples, i.e. humoral responses and proliferation of leukocytes in the presence of ECP or AsaP1, did not reveal a significant correlation between these parameters in any group. This indicates that in addition to antibody producing cells other cell populations were proliferating and that low responses in one test cannot be interpreted as a sign of low immunocompetence for that individual. In cell proliferation studies on leukocyte cultures from the vaccinated groups, there were difficulties in discerning between mitogenic effects, adjuvant induced cell activation and possible specific effects of the antigens. Six weeks after vaccination, differences in cell stimulation tests between vaccinated groups and controls were insignificant. Twelve weeks after vaccination, cultured cells from the IBOO vaccinated groups stimulated with AsaP1 responded significantly better than cultures from the corresponding PBS control group. Possible adjuvant effects could not be evaluated accurately in this group since the adjuvant used in t h a t vaccine was not available. As it is an oil based adjuvant, its effects can be expected to be similar to the effects observed for IFA where high stimulation values made it impossible to discern specific from unspecific effects in the K-15 group. This stimulatory effect of IFA was not reflected in the challenge test but these results have to be interpreted with caution since 100% mortality in the PBS group shows a high infection load and a lower dose might have given different results. In a recent study, heat-inactivated ECP from A. salmonicida ssp. salmonicida was tested in cultures of head-kidney leukocyte from vaccinated rainbow trout (Marsden et al., 1994). In comparison to the present study, especially noteworthy differences were the rapid decline of ECP stimulation in the vaccinated group and the absence of stimulation by ECP in the IFA control group. Whether this is because of differences in the preparation and/or the nature of ECPs from the two subspecies, or because different species and size of fish reared at different temperatures were used, cannot be determined here. Further studies on the effects of IFA on salmonid leukocytes reared under different conditions could be of interest. In conclusion, ECP showed mitogenic effects on unprimed leucocytes in vitro, and LPS and the toxic metallo-caseinase AsaP1 were also mitogenic to unprimed cells. Specific antigen stimulation in leukocyte cultures from the vaccinated groups was possibly masked by the mitogenic effects of ECP and AsaP1 as well as by adjuvant induced cell activation. Neither preparation was therefore suitable for tests of specific cellular proliferation in response to A. salmonicida ssp. achromogenes in head-kidney leukocyte cultures 6 or 12 weeks following vaccinations. Studies on interactions of the antigens with defined populations of cells are needed to get a clearer picture of the pathogen-host relationship. The authors wish to thank GuSmundur P~tursson, SigurbjSrg I~orsteinsdSttir, GuSmundur JShann Arason and ValgerSur Stein~SrsdSttir for their help, and The

EFFECTS OF ANTIGENS ON SALMON LEUKOCYTES

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Icelandic Council of Science, The Icelandic Agriculture Production Board and The National Research Council for financial support.

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