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Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish
Veterinary immunology and immunopathology
Veterinary Immunology and Immunopathology
ELSEVIER
58 (1997) 181-190
Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish Craig A. Shoemaker AAgric~rlruml
Research Service,
a,b,Phillip H. Klesius a3*, John A. Plumb h
United States Department
h Department
of Fisheries
and Allied
Received
ofAgriculture.
P.O. Box 952, Auburn.
Laboratory,
Aquacultures,
13 August
Auburn
Fish Disease and Ptrrusitr
AL M330. Uniwrsitv.
1996: accepted 3 February
Rrsec~r~~ir
USA Auburn,
AL 36849. USA
1997
Abstract The role of peritoneal macrophages in immunity to enteric septicemia of catfish (ESC) after infection with live Edwardsiella ictuluri was investigated. Channel catfish macrophage-mediated bacteriocidal activity was dependent on the macrophage:bacteria ratio. Ratios of 1: 1 to 1: 12 exhibited significant differences (P I 0.05) in killing between macrophages from immune fish when compared to killing by macrophages from susceptible fish at 2.5 h. At 5 h, macrophages from immune fish were capable of effective killing (83.3%) at a I:24 effector:target ratio, whereas macrophages from susceptible fish killed significantly ( P I 0.05) less (56.9%). Macrophage bacteriocidal activity was significantly greater (P I 0.05) in macrophages from individual immune fish (93.4%) compared to macrophages from individual susceptible fish (85.4%). The kinetics of macrophage killing showed a linear increase in bacteriocidal activity from I to 3 h. Opsonization with immune serum enabled macrophages from immune fish to kill bacteria more effectively (93.8 vs. 75.9%) at 2.5 h. Opsonization of E. ictaluri with immune serum significantly suppressed the killing ability of macrophages from susceptible fish (46.2%) at 2.5 h. The results suggest that macrophages from fish immune to ESC had a greater capacity to kill E. icruluri than macrophages from susceptible fish especially when E. ictuluri were opsonized with anti-g. ictaluri antibody. 0 1997 Published by Elsevier Science B.V. Ke.vword.7:Macrophage; Bacteriocidal:
Edward~iella
Abbreviations: BHI, brain-heart infusion units; PBS. phosphate buffered saline
ictaluri:
Channel catfish: Immune: Susceptible
broth; ESC. enteric septicemia
of catfish: CFU. colony forming
* Corresponding author. 0165-2427/97/$17.00 PII
0 1997 Published
SOl65-2427(97)00026-3
by Elsevier Science B.V. All rights reserved.
182
C.A. Shoemaker et (11./ Veterinar?, Immunology and lmmunopatholog~
58 I19971 IHl-190
1. Introduction The role of macrophages in immunity to enteric septicemia of catfish (ESC), in channel catfish (Zctulurus punctatus), is unclear. Wise et al. ( 1993). Blazer (1991), Sheldon and Blazer (1991) and Blazer et al. (1989) examined phagocytosis and/or killing of Edwardsielfu icfaluri by macrophages from channel catfish fed experimental diets. These studies link dietary influences to E. ictuluri phagocytosis and killing by macrophages; however, they did not show the effects of other variables upon this ability of channel catfish macrophages. Other factors known to influence macrophage-mediated killing are disease susceptibility (immune or susceptible to ESC), time of incubation, and macrophage:bacteria ratios. These components are important in understanding immunity to ESC, a disease which results in $20-30 million losses annually by catfish farmers in the USA (Plumb and Vinitnantharat, 1993). The purpose of this study was to address the role of macrophages in immunity to ESC following infection with live E. ictuluri. We examined: (1) the kinetics of macrophage-mediated bacteriocidal activity in immune and susceptible fish, (2) the ratio of macrophage:bacteria, (3) the effects of opsonization with antibody positive serum on killing of E. ictuhtri. and (4) the effects of isolates of E. ictuluri on the bacteriocidal activity of macrophages from immune and susceptible fish.
2. Materials
and methods
2.1. Fish Channel catfish (17-25 cm total length) were held in 75 liter flow-through glass aquaria maintained at a temperature of 26 + 1°C. Two groups of catfish were utilized: 150 infected with live E. ictuluri AL-93-75 (immune) and 150 susceptible. Immune fish were survivors of immersion exposure and subsequent challenge (Table 1) with 2 X 10’ colony forming units (CFU) ml-’ E. ictuluri AL-93-75 at 28°C for 1 h (Klesius and
Table 1 Killing of E. ictaluri septicemia of catfish Ratio a
by macrophages
at 2.5 h and 5 h from channel catfish immune and susceptible
Mean percent killing h (SEM) ’ 5h
2.5 h
91.8 90.4 90.9 58.3 27.6
(0.09) (1.05) (1.10) (0.67) (6.90)
Immune
Susceptible
Immune 1:l 1:3 116 I:12 1:24
to enteric
a a a a ’
85.6 78.8 66.7 41.2 24.3
(I.1 1) (1.54) (0.90) (2.50) (3.90)
h b ’ b ,’
90.2 90.1 92.3 87.4 83.3
(0.39) (0.81) (0.82) (I .90) (3.60)
Susceptible a ,’ ” ,’ ”
85.6 89.8 88.8 70.4 56.9
(0.64) (0.87) (0.29) (3.50) (2.20)
b a h h h
*’The ratio is expressed as macrophage:bacterili.b Mean percent killing = [I -COD at time 2.5 or 5 h)/(OD at time O)]X 100.’ Means with different superscripts in the same row for each representative time are significantly different at P 5 0.05.
Sealey, 19951. Susceptible fish were not exposed to E. ictaluri and were serologically (Klesius, 1993) and culturally negative for the pathogen. 2.2. Cell collection Macrophages from immune and susceptible fish were recruited to the peritoneal cavity by injecting the fish intraperitoneally with 0.25 ml of squalene (2,6,10,15.19.23hexamethyl-2.6.10,14,18,22-tetracosahexaenel 7- 10 days prior to harvest. Peritoneal macrophages were harvested using 20 ml cold sterile phosphate buffered saline (PBS) to wash cells from the cavity. Macrophage collections from groups (immune and susceptible) of six fish were harvested and pooled for use in triplicate assays. Macrophages were also collected from ten individual immune and susceptible fish to examine differences in bacteriocidal activity among individual fish. After cell collection, macrophage suspensions were centrifuged (400 X g) for 15 min, resuspended in complete channel catfish medium (Miller et al., 1994) and adjusted to 9 X lOh to I X 107. Approximately 0.9-0.95 X 10h macrophages were seeded per well in 96 well tissue culture plates and allowed to attach for 2 h. The experiment utilizing individual fish used 0.75-0.8 X IO” macrophages per well. Cells were washed following attachment with complete channel catfish medium and macrophage cultures were supplemented with 100 (*I of antibioticfree medium for the killing assay. 2.3. Bacterin and 0psoni:ation Ten isolates of E. ictaluri obtained from catfish with ESC were used. One isolate ( E. ictaluri AL-93-75) was used in the majority of the experiments. Eighteen hour bacterial cultures in brain-heart infusion (BHI) broth were adjusted to 3 X 10” CFU ml’, 20 ~1 of which (6 X IO6 CFU ml” > were added to obtain a 1:6 macrophage:bacteria ratio. Other ratios were adjusted accordingly. The opsonization experiment used 3 X IO* bacteria and serum containing E. ictaluri specific antibody as determined by FASTELISA (Klesius, 1993). A I:4 dilution of serum was made in PBS in which bacteria were incubated for 15 min before centrifugation and resuspension in BHI broth. 2.4. Killing assay The relationship between 3-(4,5-di-methylthiazoyl-2-yl) 2.5diphenyltetrazolium bromide (MTT) and CFU of E. ictaluri was established. For this bacteria 2.6 X 10” CFU to 6.3 X 10’ CFU were added per well of a microtiter plate. MTT (20 p.1) was added and the optical density was measured 5 min later on a Dynatech ELISA plate reader (Chantiliy, VA) at 620 nm. The bacteriocidal assay described by Peck (1985) and modified by Graham et al. (1988) was adapted. Edwardsiella ictaluri in BHI broth was added to cultured macrophages. Plates were then centrifuged at 150 X g for 5 min to bring bacteria into contact with macrophages. After centrifugation, the culture wells were aspirated and washed. Macrophages in the control (time 0) wells or test (time allowed for killing) wells were lysed with 50 pl of 0.2% Tween 20 to stop intracellular killing. The assays were conducted at 2.5 f 2°C. BHI broth was added for E. ictaluri
184
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et al. / Ve&rinaty
Immunologyand Immunoparhology58 (19971181-I 90
growth after macrophage lysis. Microtiter plates were shaken at 100 rpm on an orbital shaker and surviving bacteria were allowed to grow for 18 h. Reduction of MTT (20 p_l of a 10 mg ml-’ solution in distilled water; Graham et al., 1988) by E. ictaluri surviving within macrophages was used as a measure of intracellular killing. We also conducted an assay on use of antibiotic versus washing (Drevets et al., 1994) to eliminate extracellular bacteria. The assay was conducted as described above in triplicate with macrophages from susceptible fish and a 1:6 macrophage:bacteria ratio. A parallel assay was conducted as described above with the same macrophages and ratio; however, complete channel catfish medium (Miller et al., 1994) with gentamicin and penicillin/streptomycin was added after addition of bacteria. The antibiotic medium was allowed to remain in contact with macrophages for 5 min before washing three times with medium without antibiotics. The assays were then completed as described above. 2.5. Statistical
analysis
Data were analyzed by a one-way analysis of variance using Duncan’s multiple range test (SAS Institute Inc., 1985). Significant differences were determined at P I 0.05.
3. Results 3. I. Infection The mortality of channel catfish after initial exposure to E. ictaluri AL-93-75 were 34.0, 40.0 and 40.0% in the three replicates (57 of 150 fish died). Fish that survived initial infection were challenged 51 days after initial infection. No deaths due to ESC resulted after second challenge (mortality rate, 0.0%). One fish died after second challenge but E. ictaluri was not isolated upon culture of the kidney. 3.2. MTT reduction and antibiotics A linear relationship between reduction of MTT and CFU of E. ictaluri was found. In the experiments to determine if antibiotics were more effective at eliminating extracellular E. ictaluri than washing, bacteria were eliminated in control wells (i.e. no time allowed for bacteriocidal activity by macrophages) in which antibiotic was added. Bacteria were also eliminated in test wells in which 2.5 h was allowed for bacteriocidal activity to occur. Edwardsiella ictaluri grew in the control wells when washing was used to eliminate extracellular bacteria and bacteriocidal activity of the triplicate tests at 2.5 h was 68% (range 62.0-75.1%). 3.3. Effector:target
ratio
Bacteriocidal activity of macrophages from fish immune and susceptible septicemia of catfish was demonstrated at 2.5 and 5 h (Table 1). Bacteriocidal
to enteric activity of
C.A. Shoemaker et al. / Veterinary Immunology and Immunopathology 58 CIY97) IKI- /YO Table 2 Killing of E. icraluri Cl:9 macrophage:bacteria susceptible to enteric septicemia of catfish
ratio) by macrophages
from ten individual
IX.5
fish immune and
Mean percent killing a at 2.5 h
Group
Immune fish Susceptible fish
Mean CSEM) b
Range
93.4 (0.95) il 85.4 (2.54) h
89.5-96.0 73.0-94.0
’ Mean percent killing = [I -COD at time 2.5 g)/(OD at time 0 h)] x 100. b Means with different superscripts are significantly different at P 5 0.05.
macrophages was dependent on the macrophage:bacteria ratio. Ratios of I :l to I: 12 exhibited significant differences in killing ability between macrophages from immune and susceptible fish at 2.5 h (Table 1). Macrophages from immune and susceptible fish were not effective at killing E. ict&.b at high ratios (1:24): 27.6% and 24.3%. respectively (Table 1). At 5 h, macrophages from both groups of fish were able to kill E. ictuluri at higher effector:target ratios; however, macrophages from immune fish killed significantly more bacteria (83.3%) than macrophages from susceptible fish (56.9%) at the 1:24 ratio (Table 1). 3.4. hzdir!iduals Because previously conducted challenge studies indicate individuals may vary in their resistance to E. ictaluri challenge, we investigated whether macrophages from individual catfish may vary in their bacteriocidal activity. Macrophages were collected from ten individual fish from each group (immune and susceptible) to assess the ability of macrophages to kill E. ictuluri. Due to a decrease in macrophage numbers obtained from individual fish, the effector:target ratio was 1:9 rather than 1:6. Time of killing was held constant at 2.5 h. A significant difference was found between the killing ability of
Table 3 Killing of E. ictaluri by macrophages (I:6 phagocyte:bacteria ratio) from channel catfish immune susceptible to enteric septicemia of catfish at various times of incubation before macrophape lysis Time th)
0.5
1.o I.5 2.5 3.0 5.0 7.0
Mean percent killing a (SEMI b Immune fish
Susceptible
34.3 30.2 66.5 75.9 95.2 91.9 94.6
7.0 (3.60) h 24.8 (8.20) il 40.6 (3.20) h 55.9 (2.40) h 85.6 (1.60) h 86.6 (2.50) ’ 92.7 ( 1.20) a
(0.15) (3.30) (6.20) (6.30) (0.05) (0.09) (1.90)
a a a * a a a
” Mean percent killing = [I -COD time of test)/(OD time O)]X 100. ” Means with different superscripts in the same row are significantly different
fish
at P < 0.05
and
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C.A. Shoemaker et al. / Veterinary Immunology and Immunopathology
Table 4 Effect of opsonization on killing of E. icfnfuri (I:6 macrophage:bacteria catfish immune and susceptible to enteric septicemia of catfish Time (h)
ratio) by macrophages
from channel
Mean percent killing ’ (SEM) ’ Immune fish
1.5 2.5 3.0 5.0
58 (19971 181~ 190
Susceptible
Non-opsonized
Opsoniaed
66.4 75.9 95.2 91.9
23.5 93.8 95.1 95.4
(6.15) (6.30) (0.05) (0.95)
a h d ‘I
(1.05) (1.20) (2.05) (0.05)
h ’ a a
fish
Non-opsonized
Opsonized
40.6 55.9 85.5 86.6
17.1 46.2 64.2 78.8
(3.20) (1.36) (1.55) (2.54)
h ’ ’ b
a Mean percent killing = [ 1 - (OD time of test)/(OD time O)] X 100. ’ Means with different superscripts in the same row are significantly different
(7.45) (3.35) (2.05) (2.95)
c ’ ’ ’
at P I 0.05
macrophages collected from immune fish (93.4%) and susceptible fish (85.4%) (Table 2). In addition, a broader range of killing ability (73-94%) was observed between susceptible individuals compared to immune individuals. 3.5. Kinetics Macrophages from immune fish showed significantly greater levels of bacterial killing from 1.5 to 3 h when compared to the macrophages from susceptible fish (Table 3). The killing ability of both sets of macrophages were similar at 5 and 7 h. This similarity may be explained by macrophage activation due to elicitation of cells into the peritoneal cavity. However, macrophages from three groups of susceptible fish (six each) were harvested without elicitation (i.e. peritoneal cavity washed without squalene injection) and the mean percent killing at 2.5 h (target:effector ratio, 1:6) was 83% (range 76-89%).
Table 5 Killing of different E. ictahtri isolates at 2.5 h by macrophages immune and susceptible to enteric septicemia of catfish Isolate
S94-0872 s94-0732 S94-07 I6 S94-0694 SY4-0629 s94-1051 AL-93-75 S94-0873 s94- 1034 SY4-1017
(I :6 macrophage:bacteria
Mean percent killing ’ (SEMI h Immune fish
Susceptible
fish
95.5 95.3 97.5 96.0 96.0 89.3 83.1 95.2 80.7 87.3
96.6 (0.88) 95.2(1.4X)” 94.5 (0.47) 91.4C5.17)” 83.0 (2.35) 66.3 (0.05) 54.4 (6.10) 55.5 (9.45) 52.8 (5.36) 37.5 (4.59)
a
(0.17) (1.02) (0.22) (0.15)’ (0.40) (3.29) (1.92)’ (0.21) (I .90) (6.00)
il d 9 li a a ” a
a Mean percent killing = [l -(OD time 2.5 h)/(OD time O)] X 100. h Means with different superscripts in the same row are significantly
,I h ’ * h h h
different at P I 0.05.
ratio) from fish
3.6. 0psoni;ution Opsonization of E. ictuluri with immune serum was carried out to determine its effect on macrophage-mediated bacteriocidal activity (Table 4). Macrophages from immune fish incubated with opsonized bacteria were able to kill more effectively (93.8%) at 2.5 h than non-opsonized bacteria incubated with the same macrophages (75.9%). Similar levels of killing were obtained at 3 and 5 h with macrophages from Opsonization of E. ictaluri with serum immune fish regardless of opsonization. significantly suppressed the killing ability of macrophages from susceptible fish at 1.5. 3 and 5 h (Table 4). 3.7. Effect
of E. ictaluri isolate
Macrophage-mediated bacteriocidal activity of susceptible fish was effected by E. ictaluri isolate phagocytized. Four of the ten different E. ictuluri isolates were readily killed by macrophages from both susceptible and immune fish (Table 5). In contrast for the remaining six isolates examined, the killing capacity by macrophages from immune fish was enhanced significantly over the killing capacity of macrophages from susceptible fish. Macrophages from susceptible fish showed a greater difference in their killing capacity (38-97%) for different isolates of E. ictaluri than killing capacity (81-98%) by macrophages from immune fish.
4. Discussion Antonio and Hedrick (1994) and Klesius and Sealey (1995) in independent studies indicate that antibody alone does not protect against ESC. Channel catfish that survive ESC are immune to challenge (Antonio and Hedrick, 1994; Klesius and Sealey. 1995). Our challenge data support that finding. The present study demonstrated that live exposure enhanced the bacteriocidal activity of macrophages. especially for opsonized E. ictaluri. The use of antibiotics in assays to measure bacteriocidal activity has been questioned (Drevets et al., 1994). They suggest gentamicin. a membrane-impermeant antibiotic. can enter macrophages when phagocytosis occurs. They demonstrated that gentamicin influenced macrophage bacteriocidal activity against Listeria monocytogenes. Our results demonstrate that gentamicin can contribute to bacteriocidal activity of channel catfish macrophages and that washing should be used to remove extracellular bacteria rather than antibiotics. The number of bacteria to macrophage is important in relation to macrophage bacteriocidal activity. Our data indicate that macrophages from fish surviving exposure to live E. ictaIuri are capable of killing large numbers of bacteria per macrophage at 2.5 h. Neither macrophages from immunized or susceptible fish were able to kill high levels of bacteria (1:24 ratio) in vitro. Macrophages from immune fish were able to kill a larger number of bacteria (1:24 ratio) at 5 h. Graham et al. (1988) working with rainbow trout Oncorhvnchus mvkiss and the bacteria Aeromonas salmonicida, demonstrated the
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C.A. Shoemaker et al. / Veterinary Immunology and Immunopathology 58 (I 9971 181- I90
importance of the effector:target ratio. Their work indicated macrophage-mediated bacterial killing occurs at ratios of 15 and above using the same assay system with head kidney macrophages. Oliver et al. (1986) found similar ratios when examining A. salmonicida killing by elicited peritoneal macrophages from brook trout Salvelinus fontinalis. These results indicate there is a level of bacteria which can overwhelm macrophage bacteriocidal activity. Sheldon and Blazer (199 1) used a 1: 10 effector:target ratio in conducting their assays and showed differences in macrophage function in relation to diet. They attempted to show that immunization with formalin-killed E. ictaluri stimulated macrophage bacteriocidal activity. However, killed vaccines do not always protect channel catfish against ESC, presumably because killed vaccines do not elicit an effective cell mediated response. Sheldon and Blazer (1991) did not present data demonstrating that their fish immunized with killed E. ictuluri were immune to ESC by a challenge experiment. Macrophage-mediated bacteriocidal activity was greater in immune fish than in susceptible fish (using macrophages pooled from six fish of each group). We wanted to determine if bacteriocidal activity by macrophages was different between individuals. Levels of macrophage killing between individual immune and susceptible fish differed at a 1:9 effector:target ratio. Killing levels in the immune individuals were 89% or greater, indicating that channel catfish that were infected with E. ictaluri have enhanced macrophage bacteriocidal activity. Variation in bacteriocidal activity was greater in susceptible individuals. The reason for this finding is unknown. However, in Brucella abortus infections, a genetic factor has been implicated in attachment and killing of bacteria by macrophages from cattle (Campbell et al., 1994). Fish to fish variation may account for the wide range of macrophage-mediated bacteriocidal activity. Differences in bacteriocidal activity may explain why some susceptible fish can eliminate E. ictuluri and others can not. The bacterial killing capacity of macrophages from susceptible fish was effected by the isolate of E. ictaluri phagocytized. Sixty percent of the isolates tested were significantly more resistant to bacterial killing capacity of macrophages from susceptible fish. Significantly enhanced bacterial killing of these isolates was demonstrated by macrophages from immune fish. If the isolate was readily killed by macrophages of susceptible fish then no enhanced bacterial killing capacity by macrophages from immune fish was demonstrated. Again, the results showed that bacterial killing capacity by macrophages from susceptible fish was more variable than killing capacity of macrophages from immune fish. Oliver et al. (1986) used glycogen to elicit brook trout peritoneal macrophages. These macrophages were capable of killing avirulent strains of A. salmonicida but not virulent strains. We suspect that the ten isolates of E. ictuluri used in our study differ in either their surface composition or their resistance to antimicrobial compounds of catfish macrophages. We did not attempt to determine these differences only that differences existed in E. ictuluri isolates previously believed to be homogeneous (Bertolini et al., 1990). Graham and Fletcher (1992) suggest that killing ability of fish macrophages occurs between 1 and 2 h of contact with bacteria, with little or no increase in killing beyond this point. Bacteriocidal activity of macrophages from immune and susceptible channel catfish increased in a linear fashion from I to 3 h, with macrophages from immune fish
C.A. Shoemaker
et al. / Veterinary Immunology and Immunopnthologv
58 C1997) 1816I90
IX9
having a higher killing ability. At longer incubation time, the mean percentage killing between the groups was similar. An explanation for the similarity may be activation of macrophages due to elicitation of cells into the peritoneal cavity by squalene. However. susceptible fish macrophages harvested without elicitation by stimulant showed bacteriotidal activity similar to macrophages of the susceptible group injected with squalene. This similarity suggests macrophages present in the peritoneal cavity of naive fish are capable of intracellular killing of E. icfuluri. The number of peritoneal macrophages in non-elicited fish is much lower than in elicited fish (data not shown). Squalene elicits macrophages to the peritoneal cavity and this accumulation may activate some cells. However, squalene should do so in a non-specific manner. In mice. thioglycolate elicited peritoneal macrophages were shown to be as active as resident peritoneal macrophages based on intracellular killing ability of Sruphylococcus epiclermidis (Leijh et al., 1984). Studies on salmonids suggest that only modified adjuvants containing formalin killed bacteria can be used to elicit and activate peritoneal macrophages (Oliver et al., 1986; Graham et al.. 1988). Opsonization of bacteria with antibody has been shown to increase levels of macrophage killing in other vertebrates. However, this is apparently dependent on the pathogen. In rainbow trout, opsonization with anti-A. salmonicidu (Michel et al.. 199 I) and anti-Y. ruckeri antibodies (Griffin, 1983) has been demonstrated. Opsonization increased bacteriocidal activity of macrophages in channel catfish which had been infected with E. ictaluri. Opsonization of E. ictuluri limited killing by macrophages from naive fish. This suggests that E. ictaluri specific antibody, which is present on opsonized bacterial surfaces, may hinder engulfment of E. ictaluri by macrophages from susceptible fish. Apparently, activated macrophages may be more capable of binding and killing opsonized bacteria. Macrophages from E. ictuluri exposed fish were clearly shown to kill bacteria when the bacteria was either opsonized or non-opsonized. The response was greater in macrophages from immune fish incubated with opsonized bacteria. Klesius ( 1992) stated that immunity to ESC is dependent on an effective immune response because catfish that recover from infection are immune to the disease. The mortality data presented earlier demonstrates this relationship. Not only do findings of this experiment demonstrate the importance of the macrophage in immunity, but also, indicate the importance of the relationship between the humoral and cellular immune response in channel catfish to ESC. This study shows a significant role for macrophages can be observed in acquired cellular resistance against ESC. The kinetics of macrophage killing from naive fish suggest macrophages are important in resistance against primary infection. Live exposure to E. ictuluri results in an increase in macrophage-mediated bacteriocidal activity which may be important in acquired immunity to ESC.
Acknowledgements The authors wish to thank Dr. David Wise (Mississippi State Agricultural Experiment Station, Stoneville, MS) for providing some bacterial cultures used in the experiment.
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References Antonio, D.B. and Hedrick. R.P., 1994. Effects of the corticosteroid kenalog on carrier state of juvenile channel catfish exposed to Edwardsieflu icraluri. J. Aquat. Anim. Health., 6: 44-52. Bertolini. J.M.. Cipriano, R.C., Pyle. SW. and McLaughlin, J.J.A., 1990. Serological investigation of the fish pathogen Edwardsielfo ictafuri. cituse of enteric septicemia of catfish. J. Wildlife Dis.. 26: 246-352. Blazer, VS., Ankley. G.T. and Finco-Kent. D., 1989. Dietary influences on disease resistance factors in channel catfish. Dev. Comp. Immunol.. 13: 43-48. Blazer. VS.. 1991. Piscine macrophage function and nutritional influences: a review. J. Aquat. Anim. Health.. 3: 87-93. Campbell. G.A.. Adams, L.G. and Sowa. B.A.. 1994. Mechanisms of binding of Brucellu abortus to mononuclear phagocytes from cows naturally resistant or susceptible to brucellosis. Vet. Imm. Immunopathol., 41: 295-306. Drevets, D.A.. Canono, B.P.. Leenen, P.J.M. and Campbell. P.A.. 1994. Gentamicin kills intracellular Listeria monocytogenes. Infect. Imm.. 62: 2222-2228. Graham. S. and Fletcher, T.C., 1992. The role of phagocytes in the protective mechanisms of fish. Ann. Rev. Fish Dis.. 2: 53-71. Graham, S., Jefferies. A.H. and Secombes. C.J., 1988. A novel assay to detect macrophage bactericidal activity in fish: factors influencing the killing of Aeromonas salmonicidu. J. Fish Dis.. I I: 389-396. Griffin, B.R., 1983. Opsonic effect of rainbow trout (Salmu gairdnrri) antibody on phagocytosis of Yersinia nrckeri by trout leukocytes. Dev. Comp. Immunol., 7: 253-259. KIesius, P., 1992. immune system of channel catfish: an overture on immunity to Edwodsiefftr ictafuri. Ann. Rev. Fish Dis., 2: 325-338. Klesius. P., 1993. Rapid enzyme-linked immunosorbant tests for detecting antibodies to Edword.rie//a ictahrri in channel catfish Zctahruspunctatususing exoantigen. Vet. Imm. Immunopathol., 36: 359-368. Klesius, P.H. and Sealey. W.M., 1995. Characteristics of serum antibody in enteric septicemia of catfish. J. Aquat. Anim. Health., 7: 205-210. Leijh. P.C.J., vanZwet, T.L., terKuile, M.N. and vanFurth. R.. 1984. Effect of thioglycolate on phagocytic and microbial activities of peritoneal macrophages. Infect. Imm.. 46: 448-452. Michel. C., Dorson. M. and Faivre, B., 1991. Opsonizing activity of anti-Aemmorrrr.v solmonicida antibodies after inactivation of complement in rainbow trout. Ann. Rech. Vet. 22: 51-58. Miller, N.W., Chinchar. V.G. and Clem, L.W.. 1994. Development of leukocyte cell lines from channel catfish (Ictafurus puncratusl. J. Tis. Cult. Meth., 16: 117-123. Oliver, G., Eaton, C.A. and Campbell. N.. 1986. Interaction between Aeromonas safmonicida and peritoneal macrophages of brook trout (Sul~elinusfontina~is). Vet. Imm. Immunopathol.. 12: 223-234. Peck, R., 1985. A one plate assay for macrophage bactericidal activity. J. Imm. Meth. 82: 131-140. Plumb. J.A. and Vinitnantharat, S.. 1993. Vaccination of channel catfish. lctofunrs punctatus (Rafinesque)~ by immersion and oral booster against Edwnrdsielfu ictafuri. J. Fish Dis.. 16: 65-71. SAS Institute Inc., 1985. SAS user’s guide: statistics, version 5 edn. SAS Institute. Cary. North Carolina. Sheldon. W.M. and Blazer, VS., 1991. Influence of dietary lipid and temperature on bactericidal activity ot channel catfish to Edwarrfsiefio ictufuri. J. Aquat. Anim. Health.. 3: 87-93. Wise, D.J., Tomasso. J.R., Schwedler, T-E., Blazer, V.S. and Gatlin III. D.M.. 1993. Effect of vitamin E on the immune response of channel catfish to Edwardsiello ictafrrri. J. Aquat. Anim. Health.. 5: 183-188.
Veterinary Immunology and Immunopathology
ELSEVIER
58 (1997) 181-190
Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish Craig A. Shoemaker AAgric~rlruml
Research Service,
a,b,Phillip H. Klesius a3*, John A. Plumb h
United States Department
h Department
of Fisheries
and Allied
Received
ofAgriculture.
P.O. Box 952, Auburn.
Laboratory,
Aquacultures,
13 August
Auburn
Fish Disease and Ptrrusitr
AL M330. Uniwrsitv.
1996: accepted 3 February
Rrsec~r~~ir
USA Auburn,
AL 36849. USA
1997
Abstract The role of peritoneal macrophages in immunity to enteric septicemia of catfish (ESC) after infection with live Edwardsiella ictuluri was investigated. Channel catfish macrophage-mediated bacteriocidal activity was dependent on the macrophage:bacteria ratio. Ratios of 1: 1 to 1: 12 exhibited significant differences (P I 0.05) in killing between macrophages from immune fish when compared to killing by macrophages from susceptible fish at 2.5 h. At 5 h, macrophages from immune fish were capable of effective killing (83.3%) at a I:24 effector:target ratio, whereas macrophages from susceptible fish killed significantly ( P I 0.05) less (56.9%). Macrophage bacteriocidal activity was significantly greater (P I 0.05) in macrophages from individual immune fish (93.4%) compared to macrophages from individual susceptible fish (85.4%). The kinetics of macrophage killing showed a linear increase in bacteriocidal activity from I to 3 h. Opsonization with immune serum enabled macrophages from immune fish to kill bacteria more effectively (93.8 vs. 75.9%) at 2.5 h. Opsonization of E. ictaluri with immune serum significantly suppressed the killing ability of macrophages from susceptible fish (46.2%) at 2.5 h. The results suggest that macrophages from fish immune to ESC had a greater capacity to kill E. icruluri than macrophages from susceptible fish especially when E. ictuluri were opsonized with anti-g. ictaluri antibody. 0 1997 Published by Elsevier Science B.V. Ke.vword.7:Macrophage; Bacteriocidal:
Edward~iella
Abbreviations: BHI, brain-heart infusion units; PBS. phosphate buffered saline
ictaluri:
Channel catfish: Immune: Susceptible
broth; ESC. enteric septicemia
of catfish: CFU. colony forming
* Corresponding author. 0165-2427/97/$17.00 PII
0 1997 Published
SOl65-2427(97)00026-3
by Elsevier Science B.V. All rights reserved.
182
C.A. Shoemaker et (11./ Veterinar?, Immunology and lmmunopatholog~
58 I19971 IHl-190
1. Introduction The role of macrophages in immunity to enteric septicemia of catfish (ESC), in channel catfish (Zctulurus punctatus), is unclear. Wise et al. ( 1993). Blazer (1991), Sheldon and Blazer (1991) and Blazer et al. (1989) examined phagocytosis and/or killing of Edwardsielfu icfaluri by macrophages from channel catfish fed experimental diets. These studies link dietary influences to E. ictuluri phagocytosis and killing by macrophages; however, they did not show the effects of other variables upon this ability of channel catfish macrophages. Other factors known to influence macrophage-mediated killing are disease susceptibility (immune or susceptible to ESC), time of incubation, and macrophage:bacteria ratios. These components are important in understanding immunity to ESC, a disease which results in $20-30 million losses annually by catfish farmers in the USA (Plumb and Vinitnantharat, 1993). The purpose of this study was to address the role of macrophages in immunity to ESC following infection with live E. ictuluri. We examined: (1) the kinetics of macrophage-mediated bacteriocidal activity in immune and susceptible fish, (2) the ratio of macrophage:bacteria, (3) the effects of opsonization with antibody positive serum on killing of E. ictuhtri. and (4) the effects of isolates of E. ictuluri on the bacteriocidal activity of macrophages from immune and susceptible fish.
2. Materials
and methods
2.1. Fish Channel catfish (17-25 cm total length) were held in 75 liter flow-through glass aquaria maintained at a temperature of 26 + 1°C. Two groups of catfish were utilized: 150 infected with live E. ictuluri AL-93-75 (immune) and 150 susceptible. Immune fish were survivors of immersion exposure and subsequent challenge (Table 1) with 2 X 10’ colony forming units (CFU) ml-’ E. ictuluri AL-93-75 at 28°C for 1 h (Klesius and
Table 1 Killing of E. ictaluri septicemia of catfish Ratio a
by macrophages
at 2.5 h and 5 h from channel catfish immune and susceptible
Mean percent killing h (SEM) ’ 5h
2.5 h
91.8 90.4 90.9 58.3 27.6
(0.09) (1.05) (1.10) (0.67) (6.90)
Immune
Susceptible
Immune 1:l 1:3 116 I:12 1:24
to enteric
a a a a ’
85.6 78.8 66.7 41.2 24.3
(I.1 1) (1.54) (0.90) (2.50) (3.90)
h b ’ b ,’
90.2 90.1 92.3 87.4 83.3
(0.39) (0.81) (0.82) (I .90) (3.60)
Susceptible a ,’ ” ,’ ”
85.6 89.8 88.8 70.4 56.9
(0.64) (0.87) (0.29) (3.50) (2.20)
b a h h h
*’The ratio is expressed as macrophage:bacterili.b Mean percent killing = [I -COD at time 2.5 or 5 h)/(OD at time O)]X 100.’ Means with different superscripts in the same row for each representative time are significantly different at P 5 0.05.
Sealey, 19951. Susceptible fish were not exposed to E. ictaluri and were serologically (Klesius, 1993) and culturally negative for the pathogen. 2.2. Cell collection Macrophages from immune and susceptible fish were recruited to the peritoneal cavity by injecting the fish intraperitoneally with 0.25 ml of squalene (2,6,10,15.19.23hexamethyl-2.6.10,14,18,22-tetracosahexaenel 7- 10 days prior to harvest. Peritoneal macrophages were harvested using 20 ml cold sterile phosphate buffered saline (PBS) to wash cells from the cavity. Macrophage collections from groups (immune and susceptible) of six fish were harvested and pooled for use in triplicate assays. Macrophages were also collected from ten individual immune and susceptible fish to examine differences in bacteriocidal activity among individual fish. After cell collection, macrophage suspensions were centrifuged (400 X g) for 15 min, resuspended in complete channel catfish medium (Miller et al., 1994) and adjusted to 9 X lOh to I X 107. Approximately 0.9-0.95 X 10h macrophages were seeded per well in 96 well tissue culture plates and allowed to attach for 2 h. The experiment utilizing individual fish used 0.75-0.8 X IO” macrophages per well. Cells were washed following attachment with complete channel catfish medium and macrophage cultures were supplemented with 100 (*I of antibioticfree medium for the killing assay. 2.3. Bacterin and 0psoni:ation Ten isolates of E. ictaluri obtained from catfish with ESC were used. One isolate ( E. ictaluri AL-93-75) was used in the majority of the experiments. Eighteen hour bacterial cultures in brain-heart infusion (BHI) broth were adjusted to 3 X 10” CFU ml’, 20 ~1 of which (6 X IO6 CFU ml” > were added to obtain a 1:6 macrophage:bacteria ratio. Other ratios were adjusted accordingly. The opsonization experiment used 3 X IO* bacteria and serum containing E. ictaluri specific antibody as determined by FASTELISA (Klesius, 1993). A I:4 dilution of serum was made in PBS in which bacteria were incubated for 15 min before centrifugation and resuspension in BHI broth. 2.4. Killing assay The relationship between 3-(4,5-di-methylthiazoyl-2-yl) 2.5diphenyltetrazolium bromide (MTT) and CFU of E. ictaluri was established. For this bacteria 2.6 X 10” CFU to 6.3 X 10’ CFU were added per well of a microtiter plate. MTT (20 p.1) was added and the optical density was measured 5 min later on a Dynatech ELISA plate reader (Chantiliy, VA) at 620 nm. The bacteriocidal assay described by Peck (1985) and modified by Graham et al. (1988) was adapted. Edwardsiella ictaluri in BHI broth was added to cultured macrophages. Plates were then centrifuged at 150 X g for 5 min to bring bacteria into contact with macrophages. After centrifugation, the culture wells were aspirated and washed. Macrophages in the control (time 0) wells or test (time allowed for killing) wells were lysed with 50 pl of 0.2% Tween 20 to stop intracellular killing. The assays were conducted at 2.5 f 2°C. BHI broth was added for E. ictaluri
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growth after macrophage lysis. Microtiter plates were shaken at 100 rpm on an orbital shaker and surviving bacteria were allowed to grow for 18 h. Reduction of MTT (20 p_l of a 10 mg ml-’ solution in distilled water; Graham et al., 1988) by E. ictaluri surviving within macrophages was used as a measure of intracellular killing. We also conducted an assay on use of antibiotic versus washing (Drevets et al., 1994) to eliminate extracellular bacteria. The assay was conducted as described above in triplicate with macrophages from susceptible fish and a 1:6 macrophage:bacteria ratio. A parallel assay was conducted as described above with the same macrophages and ratio; however, complete channel catfish medium (Miller et al., 1994) with gentamicin and penicillin/streptomycin was added after addition of bacteria. The antibiotic medium was allowed to remain in contact with macrophages for 5 min before washing three times with medium without antibiotics. The assays were then completed as described above. 2.5. Statistical
analysis
Data were analyzed by a one-way analysis of variance using Duncan’s multiple range test (SAS Institute Inc., 1985). Significant differences were determined at P I 0.05.
3. Results 3. I. Infection The mortality of channel catfish after initial exposure to E. ictaluri AL-93-75 were 34.0, 40.0 and 40.0% in the three replicates (57 of 150 fish died). Fish that survived initial infection were challenged 51 days after initial infection. No deaths due to ESC resulted after second challenge (mortality rate, 0.0%). One fish died after second challenge but E. ictaluri was not isolated upon culture of the kidney. 3.2. MTT reduction and antibiotics A linear relationship between reduction of MTT and CFU of E. ictaluri was found. In the experiments to determine if antibiotics were more effective at eliminating extracellular E. ictaluri than washing, bacteria were eliminated in control wells (i.e. no time allowed for bacteriocidal activity by macrophages) in which antibiotic was added. Bacteria were also eliminated in test wells in which 2.5 h was allowed for bacteriocidal activity to occur. Edwardsiella ictaluri grew in the control wells when washing was used to eliminate extracellular bacteria and bacteriocidal activity of the triplicate tests at 2.5 h was 68% (range 62.0-75.1%). 3.3. Effector:target
ratio
Bacteriocidal activity of macrophages from fish immune and susceptible septicemia of catfish was demonstrated at 2.5 and 5 h (Table 1). Bacteriocidal
to enteric activity of
C.A. Shoemaker et al. / Veterinary Immunology and Immunopathology 58 CIY97) IKI- /YO Table 2 Killing of E. icraluri Cl:9 macrophage:bacteria susceptible to enteric septicemia of catfish
ratio) by macrophages
from ten individual
IX.5
fish immune and
Mean percent killing a at 2.5 h
Group
Immune fish Susceptible fish
Mean CSEM) b
Range
93.4 (0.95) il 85.4 (2.54) h
89.5-96.0 73.0-94.0
’ Mean percent killing = [I -COD at time 2.5 g)/(OD at time 0 h)] x 100. b Means with different superscripts are significantly different at P 5 0.05.
macrophages was dependent on the macrophage:bacteria ratio. Ratios of I :l to I: 12 exhibited significant differences in killing ability between macrophages from immune and susceptible fish at 2.5 h (Table 1). Macrophages from immune and susceptible fish were not effective at killing E. ict&.b at high ratios (1:24): 27.6% and 24.3%. respectively (Table 1). At 5 h, macrophages from both groups of fish were able to kill E. ictuluri at higher effector:target ratios; however, macrophages from immune fish killed significantly more bacteria (83.3%) than macrophages from susceptible fish (56.9%) at the 1:24 ratio (Table 1). 3.4. hzdir!iduals Because previously conducted challenge studies indicate individuals may vary in their resistance to E. ictaluri challenge, we investigated whether macrophages from individual catfish may vary in their bacteriocidal activity. Macrophages were collected from ten individual fish from each group (immune and susceptible) to assess the ability of macrophages to kill E. ictuluri. Due to a decrease in macrophage numbers obtained from individual fish, the effector:target ratio was 1:9 rather than 1:6. Time of killing was held constant at 2.5 h. A significant difference was found between the killing ability of
Table 3 Killing of E. ictaluri by macrophages (I:6 phagocyte:bacteria ratio) from channel catfish immune susceptible to enteric septicemia of catfish at various times of incubation before macrophape lysis Time th)
0.5
1.o I.5 2.5 3.0 5.0 7.0
Mean percent killing a (SEMI b Immune fish
Susceptible
34.3 30.2 66.5 75.9 95.2 91.9 94.6
7.0 (3.60) h 24.8 (8.20) il 40.6 (3.20) h 55.9 (2.40) h 85.6 (1.60) h 86.6 (2.50) ’ 92.7 ( 1.20) a
(0.15) (3.30) (6.20) (6.30) (0.05) (0.09) (1.90)
a a a * a a a
” Mean percent killing = [I -COD time of test)/(OD time O)]X 100. ” Means with different superscripts in the same row are significantly different
fish
at P < 0.05
and
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Table 4 Effect of opsonization on killing of E. icfnfuri (I:6 macrophage:bacteria catfish immune and susceptible to enteric septicemia of catfish Time (h)
ratio) by macrophages
from channel
Mean percent killing ’ (SEM) ’ Immune fish
1.5 2.5 3.0 5.0
58 (19971 181~ 190
Susceptible
Non-opsonized
Opsoniaed
66.4 75.9 95.2 91.9
23.5 93.8 95.1 95.4
(6.15) (6.30) (0.05) (0.95)
a h d ‘I
(1.05) (1.20) (2.05) (0.05)
h ’ a a
fish
Non-opsonized
Opsonized
40.6 55.9 85.5 86.6
17.1 46.2 64.2 78.8
(3.20) (1.36) (1.55) (2.54)
h ’ ’ b
a Mean percent killing = [ 1 - (OD time of test)/(OD time O)] X 100. ’ Means with different superscripts in the same row are significantly different
(7.45) (3.35) (2.05) (2.95)
c ’ ’ ’
at P I 0.05
macrophages collected from immune fish (93.4%) and susceptible fish (85.4%) (Table 2). In addition, a broader range of killing ability (73-94%) was observed between susceptible individuals compared to immune individuals. 3.5. Kinetics Macrophages from immune fish showed significantly greater levels of bacterial killing from 1.5 to 3 h when compared to the macrophages from susceptible fish (Table 3). The killing ability of both sets of macrophages were similar at 5 and 7 h. This similarity may be explained by macrophage activation due to elicitation of cells into the peritoneal cavity. However, macrophages from three groups of susceptible fish (six each) were harvested without elicitation (i.e. peritoneal cavity washed without squalene injection) and the mean percent killing at 2.5 h (target:effector ratio, 1:6) was 83% (range 76-89%).
Table 5 Killing of different E. ictahtri isolates at 2.5 h by macrophages immune and susceptible to enteric septicemia of catfish Isolate
S94-0872 s94-0732 S94-07 I6 S94-0694 SY4-0629 s94-1051 AL-93-75 S94-0873 s94- 1034 SY4-1017
(I :6 macrophage:bacteria
Mean percent killing ’ (SEMI h Immune fish
Susceptible
fish
95.5 95.3 97.5 96.0 96.0 89.3 83.1 95.2 80.7 87.3
96.6 (0.88) 95.2(1.4X)” 94.5 (0.47) 91.4C5.17)” 83.0 (2.35) 66.3 (0.05) 54.4 (6.10) 55.5 (9.45) 52.8 (5.36) 37.5 (4.59)
a
(0.17) (1.02) (0.22) (0.15)’ (0.40) (3.29) (1.92)’ (0.21) (I .90) (6.00)
il d 9 li a a ” a
a Mean percent killing = [l -(OD time 2.5 h)/(OD time O)] X 100. h Means with different superscripts in the same row are significantly
,I h ’ * h h h
different at P I 0.05.
ratio) from fish
3.6. 0psoni;ution Opsonization of E. ictuluri with immune serum was carried out to determine its effect on macrophage-mediated bacteriocidal activity (Table 4). Macrophages from immune fish incubated with opsonized bacteria were able to kill more effectively (93.8%) at 2.5 h than non-opsonized bacteria incubated with the same macrophages (75.9%). Similar levels of killing were obtained at 3 and 5 h with macrophages from Opsonization of E. ictaluri with serum immune fish regardless of opsonization. significantly suppressed the killing ability of macrophages from susceptible fish at 1.5. 3 and 5 h (Table 4). 3.7. Effect
of E. ictaluri isolate
Macrophage-mediated bacteriocidal activity of susceptible fish was effected by E. ictaluri isolate phagocytized. Four of the ten different E. ictuluri isolates were readily killed by macrophages from both susceptible and immune fish (Table 5). In contrast for the remaining six isolates examined, the killing capacity by macrophages from immune fish was enhanced significantly over the killing capacity of macrophages from susceptible fish. Macrophages from susceptible fish showed a greater difference in their killing capacity (38-97%) for different isolates of E. ictaluri than killing capacity (81-98%) by macrophages from immune fish.
4. Discussion Antonio and Hedrick (1994) and Klesius and Sealey (1995) in independent studies indicate that antibody alone does not protect against ESC. Channel catfish that survive ESC are immune to challenge (Antonio and Hedrick, 1994; Klesius and Sealey. 1995). Our challenge data support that finding. The present study demonstrated that live exposure enhanced the bacteriocidal activity of macrophages. especially for opsonized E. ictaluri. The use of antibiotics in assays to measure bacteriocidal activity has been questioned (Drevets et al., 1994). They suggest gentamicin. a membrane-impermeant antibiotic. can enter macrophages when phagocytosis occurs. They demonstrated that gentamicin influenced macrophage bacteriocidal activity against Listeria monocytogenes. Our results demonstrate that gentamicin can contribute to bacteriocidal activity of channel catfish macrophages and that washing should be used to remove extracellular bacteria rather than antibiotics. The number of bacteria to macrophage is important in relation to macrophage bacteriocidal activity. Our data indicate that macrophages from fish surviving exposure to live E. ictaIuri are capable of killing large numbers of bacteria per macrophage at 2.5 h. Neither macrophages from immunized or susceptible fish were able to kill high levels of bacteria (1:24 ratio) in vitro. Macrophages from immune fish were able to kill a larger number of bacteria (1:24 ratio) at 5 h. Graham et al. (1988) working with rainbow trout Oncorhvnchus mvkiss and the bacteria Aeromonas salmonicida, demonstrated the
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importance of the effector:target ratio. Their work indicated macrophage-mediated bacterial killing occurs at ratios of 15 and above using the same assay system with head kidney macrophages. Oliver et al. (1986) found similar ratios when examining A. salmonicida killing by elicited peritoneal macrophages from brook trout Salvelinus fontinalis. These results indicate there is a level of bacteria which can overwhelm macrophage bacteriocidal activity. Sheldon and Blazer (199 1) used a 1: 10 effector:target ratio in conducting their assays and showed differences in macrophage function in relation to diet. They attempted to show that immunization with formalin-killed E. ictaluri stimulated macrophage bacteriocidal activity. However, killed vaccines do not always protect channel catfish against ESC, presumably because killed vaccines do not elicit an effective cell mediated response. Sheldon and Blazer (1991) did not present data demonstrating that their fish immunized with killed E. ictuluri were immune to ESC by a challenge experiment. Macrophage-mediated bacteriocidal activity was greater in immune fish than in susceptible fish (using macrophages pooled from six fish of each group). We wanted to determine if bacteriocidal activity by macrophages was different between individuals. Levels of macrophage killing between individual immune and susceptible fish differed at a 1:9 effector:target ratio. Killing levels in the immune individuals were 89% or greater, indicating that channel catfish that were infected with E. ictaluri have enhanced macrophage bacteriocidal activity. Variation in bacteriocidal activity was greater in susceptible individuals. The reason for this finding is unknown. However, in Brucella abortus infections, a genetic factor has been implicated in attachment and killing of bacteria by macrophages from cattle (Campbell et al., 1994). Fish to fish variation may account for the wide range of macrophage-mediated bacteriocidal activity. Differences in bacteriocidal activity may explain why some susceptible fish can eliminate E. ictuluri and others can not. The bacterial killing capacity of macrophages from susceptible fish was effected by the isolate of E. ictaluri phagocytized. Sixty percent of the isolates tested were significantly more resistant to bacterial killing capacity of macrophages from susceptible fish. Significantly enhanced bacterial killing of these isolates was demonstrated by macrophages from immune fish. If the isolate was readily killed by macrophages of susceptible fish then no enhanced bacterial killing capacity by macrophages from immune fish was demonstrated. Again, the results showed that bacterial killing capacity by macrophages from susceptible fish was more variable than killing capacity of macrophages from immune fish. Oliver et al. (1986) used glycogen to elicit brook trout peritoneal macrophages. These macrophages were capable of killing avirulent strains of A. salmonicida but not virulent strains. We suspect that the ten isolates of E. ictuluri used in our study differ in either their surface composition or their resistance to antimicrobial compounds of catfish macrophages. We did not attempt to determine these differences only that differences existed in E. ictuluri isolates previously believed to be homogeneous (Bertolini et al., 1990). Graham and Fletcher (1992) suggest that killing ability of fish macrophages occurs between 1 and 2 h of contact with bacteria, with little or no increase in killing beyond this point. Bacteriocidal activity of macrophages from immune and susceptible channel catfish increased in a linear fashion from I to 3 h, with macrophages from immune fish
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IX9
having a higher killing ability. At longer incubation time, the mean percentage killing between the groups was similar. An explanation for the similarity may be activation of macrophages due to elicitation of cells into the peritoneal cavity by squalene. However. susceptible fish macrophages harvested without elicitation by stimulant showed bacteriotidal activity similar to macrophages of the susceptible group injected with squalene. This similarity suggests macrophages present in the peritoneal cavity of naive fish are capable of intracellular killing of E. icfuluri. The number of peritoneal macrophages in non-elicited fish is much lower than in elicited fish (data not shown). Squalene elicits macrophages to the peritoneal cavity and this accumulation may activate some cells. However, squalene should do so in a non-specific manner. In mice. thioglycolate elicited peritoneal macrophages were shown to be as active as resident peritoneal macrophages based on intracellular killing ability of Sruphylococcus epiclermidis (Leijh et al., 1984). Studies on salmonids suggest that only modified adjuvants containing formalin killed bacteria can be used to elicit and activate peritoneal macrophages (Oliver et al., 1986; Graham et al.. 1988). Opsonization of bacteria with antibody has been shown to increase levels of macrophage killing in other vertebrates. However, this is apparently dependent on the pathogen. In rainbow trout, opsonization with anti-A. salmonicidu (Michel et al.. 199 I) and anti-Y. ruckeri antibodies (Griffin, 1983) has been demonstrated. Opsonization increased bacteriocidal activity of macrophages in channel catfish which had been infected with E. ictaluri. Opsonization of E. ictuluri limited killing by macrophages from naive fish. This suggests that E. ictaluri specific antibody, which is present on opsonized bacterial surfaces, may hinder engulfment of E. ictaluri by macrophages from susceptible fish. Apparently, activated macrophages may be more capable of binding and killing opsonized bacteria. Macrophages from E. ictuluri exposed fish were clearly shown to kill bacteria when the bacteria was either opsonized or non-opsonized. The response was greater in macrophages from immune fish incubated with opsonized bacteria. Klesius ( 1992) stated that immunity to ESC is dependent on an effective immune response because catfish that recover from infection are immune to the disease. The mortality data presented earlier demonstrates this relationship. Not only do findings of this experiment demonstrate the importance of the macrophage in immunity, but also, indicate the importance of the relationship between the humoral and cellular immune response in channel catfish to ESC. This study shows a significant role for macrophages can be observed in acquired cellular resistance against ESC. The kinetics of macrophage killing from naive fish suggest macrophages are important in resistance against primary infection. Live exposure to E. ictuluri results in an increase in macrophage-mediated bacteriocidal activity which may be important in acquired immunity to ESC.
Acknowledgements The authors wish to thank Dr. David Wise (Mississippi State Agricultural Experiment Station, Stoneville, MS) for providing some bacterial cultures used in the experiment.
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