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Opsonic effect of rainbow trout (Salmo gairdneri) antibody on phagocytosis of Yersinia ruckeri by trout leukocytes
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 7, pp. 253-259, 0145-305X/83/020253-07503.00/0 Printed in the USA.
1983.
OPSONIC EFFECT OF RAINBOW TROUT (SALMO GAIRDNERI) ANTIBODY ON PHAGOCYTOSIS OF YERSINIA RUCKERI BY TROUT LEUKOCYTES
B. R. Griffin 1 U.S. Fish and Wildlife Service National Fisheries Center - Leetown National Fish Health Research Laboratory Kearneysville, West Virginia 25430
ABSTRACT.
Partly purified peripheral blood leukocytes from normal rainbow trout (Salmo gairdneri) were used to study the influence of specific antibody on phagocytic uptake and intracellular killing of Yersinia ruckeri, a bacterial pathogen of trout. Specific antibody exerted a significant opsonic effect on the rate of phagocytic ingestion of the bacteria but did not affect the rate of intracellular killing. The results are discussed with reference to the current understanding of fish antibody function and phagocytosis by fish leukocytes.
INTRODUCTION Among protective effects ascribed to specific antibacterial antibody in mammals is its ability to opsonize homologous bacteria and thus enhance uptake by mononuclear and polymorphonuclear phagocytes (1-3). Phagocytosis of bacteria in fish has been reported (4-14), but studies of the effects of immunization on phagocytosis have produced differing results. Post (6) found an increased opsonic index in in vivo studies of rainbow trout (Saimo gairdneri) immunized with Aeromonas hydrophila. Avtalion and Shahrabani (13) reported that, although immunization enhanced intracellular killing of Staphylococcus aureus, it had no effect on the rate of ingestion of bacteria by cells of the whole blood of carp (Cyprinus carpio). Goncharov and Mikriakov (9) and Gonchorav (ii) found that phagocytosis of gram-negative bacteria in immunized carp proceeded at a rate twice that in nonimmunized controls. Tetz (i0) reported that phagocytosis of A. punctata (A. hydrophila) in carp blood was unaffected by immunization.
ipresent address: U.S. Fish and Wildlife Service, Fish Farming Experimental Station, P. O. Box 860, Stuttgart, Arkansas, 72160. 253
254
PHAGOCYTOSIS
BY TROUT LEUKOCYTES
After a review of the literature, it seems safe to are capable of engulfing bacteria, but that little that influence the process. The research presented an attempt to clarify the role of specific antibody bacteria by trout leukocytes.
Vol.
7, No. 2
say that fish phagocytes is known about factors here was undertaken as in phagocytosis of
METHODS Fish maintenance and treatment. Rainbow trout (Salmo gairdneri) from specific-pathogen-free stocks were used throughout these experiments as sources of immune serum, normal serum, and normal peripheral blood leukocytes. Adults averaging 15 to 25 cm in fork length, were maintained in fresh flowing spring water at 12°C. The fish were anesthetized for bleeding by adding tricaine methanesulfonate (Crescent Research Chemicals, Inc., Paradise Valley, Arizona*) to holding tank water. For production of specific antibody, trout were given a single l-mg in intraperitoneal injection of log-phase broth cultured, formalin killed Yersinia ruckeri cells that had been washed and resuspended in PBS. Immunized fish were bled 3 to 5 weeks later, and sera were collected and frozen at -80°C until use. All antisera had bacterial agglutination titers in excess of 1:64, whereas undiluted normal serum was negative. Peripheral blood leukocyte purification. Methods used for mammalian leukocyte separation employing Ficoll-Hypaque mixtures, based on the work of B~yum (15), were not usable with rainbow trout blood because separation of cells was poor and inconsistent. Caudal vein blood was drawn into an equal volume of 0.i M phosphate buffer, pH 7.2, containing i00 IU heparin/ml. The diluted blood was layered over 2.5-mi cushions of 21% Hypaque (Winthrop Laboratories, New York) in siliconized tubes (15 by 125 mm). The 21% Hypaque solution was prepared by diluting 50% stock with heparinized phosphate buffer. After centrifugation at 250 X g for 20 min at 8 to 10°C in a horizontal rotor centrifuge, the leukocyte band at the plasma: Hypaque interface was removed with a pasteur pipette and transferred to a fresh tube for washing, first with heparinized buffer, then with nonheparinized buffer. Cell densities were adjusted to between i and 3 X 107 cells/ml by suspension in RPMI-1640 (GIBCO, Grand Island, New York). Bacterial phagocytosis and intracellular killing. Procedures employed to assess in vitro phagocytosis and intracellular killing of bacteria by trout leukocytes were adapted from those described by van Furth and van Zwet (16). The bacterial culture was from a recent isolate of Yersinia ruckeri, the etiologic agent of enteric redmouth of salmonids. Log phase cells from culture in brain heart infusion (BHI) broth were washed in phosphate buffer and suspended in RPMI-1640 to a concentration giving an optical density of 0.4 at 525 nm. The working suspension prepared was a I:i0 dilution of this stock with RPMI-1640. The final suspension contained approximately 8 X 107 bacteria/ml, as determined by dilution plate counting. Before the bacteria and leukocytes were mixed, the bacteria were incubated 30 min in RPMI-1640 containing 10% complement-inactivated (42°C for 30 min) normal or immune trout serum. Equal 1-5 ml volumes of leukocytes and treated bacteria were then combined (bacteria:leukocyte ratio of 2 to 4) and gently shaken for 30 min at 20°C. The mixture was diluted with five volumes of cold RPMI-1640 to stop *Reference products.
to trade names does not imply government endorsement
of commercial
Vol.
7, No. 2
PHAGOCYTOSIS
BY TROUT LEUKOCYTES
255
phagocytosis and centrifuged at 400 X g for 4 min at 2°C; the leukocytes were then washed three times in cold RPMI-1640. The leukocytes were resuspended to the starting volume (3 ml) in 20°C RPMI-1640 containing 5% normal trout serum. Immediately (to estimate ingestion rate) and after 60 min at 20°C (to estimate intracellular killing rate), 0.5-ml samples were removed and diluted in 4.5 ml of ice-cold PBS containing 1% bovine serum albumin (PBSA). The leukocytes were disrupted by a 20-s exposure to ultrasound at 50 W with a probe type ultrasonifer (Sonifer Cell Disrupter, Model W1850, Ultrasonics, Inc., Plainview, New York). In control experiments this treatment caused complete disruption of leukocytes with no detectable effect on bacterial viability. Dilutions were made in PBSA and plate counts done on BHI agar plates. Identification of cell types. The cellular classification system described by Finn and Nielson (12) was used as a guide for identifying trout leukocytes. At the end of the phagocytosis experiments, a few slides were prepared from each test mixture. After slides--either simple smears or cytocentrifuge preparations--were dried, they were fixed by flooding with 3% buffered formalin for i0 s. Staining for peroxidase activity, as a more definitive means of identifying polymorphonuclear leukocytes (PMNs), was done as follows: aqueous 0.01 M KH2PO 4, pH 6.0, containing 0.008% 3,3'-dimethoxybenzidine (Eastman Chemical Co., Rochester, New York) and 0.003% H202 was applied as a primary stain; after 15 min the slides were rinsed with deionized water and stained with giemsa. Intracellular areas containing peroxidase were stained diffuse to granular brown, and nuclear and cytoplasmic areas gave typical Romanowsky staining reactions. Intracellular bacteria stained deep blue.
RESULTS The effect of specific antibody on the phagocytic uptake and subsequent intracellular killing of homologous bacteria was measured in individual tests with purified leukocytes from I0 unimmunized normal trout. Normal sera controls were run simultaneously. Ingestion of bacteria was greatly enhanced by specific antibody (Table i). The difference in P30 values obtained from immune versus normal serum was highly significant (P <.01). The apparent difference in the rate of intracellular killing (K60) was not significant. One experiment was done to compare effects of normal serum and no serum on phagocytosis. The results with normal serum were consistent with the other experiments: P30 = 0.6 and K60 = 115. The absence of serum in the test resulted in a marked reduction in uptake (P30 = 0.03) but there was little effect on killing (K60 = 102). When cells were counted on slide preparations from immune serum samples, approximately 27% of the leukocytes were phagocytic. Of these, 57% were mononuclear cells as seen in Figure i, and the remaining 43% were PMNs as seen in Figure 2. Differential staining showed that the phagocytic cells in Figure 1 were peroxidase negative with a faint-blue staining cytoplasm. Phagocytic cells in Figure 2 were peroxidase positive with an otherwise clear cytoplasm. Peroxidase negative mononuclear phagocytes engulfed greater numbers of bacteria (6.8/celi) than did peroxidase positive PMNs.
256
PHAGOCYTOSIS BY TROUT LEUKOCYTES
Vol.
7, No. 2
TABLE 1 Influence of Immune and Normal Serum on Phagocytosis and Intracellular Killing of Yersinia ruckeri by Rainbow Trout Leukocytes P30* Animal no.
Immune serum
K60 # Normal serum
Opsonic t index
Immune serum
Normal serum
1 2 3 4 5 6 7 8 9 i0
9.1 16.2 8.8 5.5 5.3 15.0 8.6 9.6 18.0 11.6
0.4 1.8 1.6 0.6 0.7 1.3 1.0 4.1 2.2 1.0
22.8 9.0 5.5 9.2 7.6 11.5 8.6 2.3 8.2 11.6
62 82 93 105 64 70 115 53 57 57
53 108 143 99 30 102 112 58 89 63
Mean
10.8
1.5
9.6
76
86
*P30 Phagocytic index = number of bacteria/leukocyte (X 10 -2 ) after 30 min contact. + Phagocytic index obtained with immune serum -Opsonic index = Phagocytic index obtained with normal serum. $K60 = Index of intracellular killing = percent of original viable cellassociated bacteria after 60 min incubation.
P
FIGURE i A peroxidase negative mononuclear phagocyte (arrow) with intracellular bacteria Adjacent to this cell are a lymphocyte and a peroxidase positive polymorphonuclear leukocyte. X i000.
Vol.
7, No. 2
PHAGOCYTOSIS BY TROUT LEUKOCYTES
257
FIGURE 2 A peroxidase positive polymorphonuclear leukocyte (arrow) with intracellular bacteria. The several adjacent cells are lymphocytes. X i000.
These observations were made on leukocyte slide preparations taken from samples containing bacteria in immune serum; phagocytes with engulfed bacteria were so rare in slide preparations from samples containing normal serum that estimates of the numbers and types of cells involved could not be obtained. DISCUSSION Bacterial phagocytosis by mammalian cells can be enhanced by specific antibody, by nonspecific normal-serum opsonins, by complement activation, and by the cellular condition of the immune animal through the action of its activated macrophages (1-3). I took several precautions to study, as nearly as possible, the specific effects of antibody on phagocytosis by fish cells. Leukocytes from unimmunized, seronegative, specific-pathogen-free animals, separated from erythrocytes and washed to remove the bulk of normal-serum associated opsonins, were used as a source of phagocytes. All sera were complement inactivated by heating at 42°C for 30 min, and normal serum controls were run in parallel with immune serum tests. The data from these experiments indicate that specific antibody exerts a significant opsonic effect on phagocytosis of homologous bacteria by normal trout phagocytes. In this regard, trout antibody appears to function in a manner similar to mammalian antibody. The results are not consistent with those of Avtalion and Shahrabani (13), who found that immunization did not influence the rate of uptake of S. a u r e u s in whole blood of carp. They suggested that the high titers of natural antibody (1:16-1:32) of normal carp used in the study may have been sufficient to permit maximum levels of phagocytosis. There may also have been species differences or temperature effects that are not yet understood.
258
PHAGOCYTOSIS BY TROUT LEUKOCYTES
Vol.
7, No. 2
Intracellular killing of bacteria by normal trout leukocytes was found to be a slow process and one that does not seem to be affected by opsonization with antibody. This observation is consistent with Finn and Nielson's (12) results with rainbow trout injected with S. aureus, in that intracellular destruction of bacteria was slow by mammalian standards. Avtalion and Shahrabani (13) found that blood cells from immunized carp were more efficient at intracellufar killing of S. aureus than were blood cells from nonimmunized carp. In their work however, Avtalion and Shahrabani (13) used whole blood and measured phagocytosis and intracellular killing of a nonpathogen of fish, S. aureus. The presence of complement in the whole blood may have enhanced the role of intracellular killing they observed. Fish leukocytes that have been reported to be phagocytic include lymphocytes (9,11,17,18), monocyte-macrophages (4,6,9,11,12,14,19,20), PMNs (6,7,11,12, 17), and eosinophils (5,7,17). Some of these reports are contradictory and there is confusion about the identity and function of fish leukocytes (21). Throughout the experiments reported here, the two cell types described were the only ones seen to be actively phagocytic. Identification of the blood monocyte (macrophage) as one of the principal phagocytes is consistent with other reports on fish phagocytes (4,8,12,14). A controversial point, however, is whether the PMN is a phagocytic cell (4,8,18,21). The most convincing work is that by Finn and Nielson (12), in which PMNs were active in phagocytosis of S. aureus. In spite of reports to the contrary, the peroxidase positive, phagocytic, leukocytes with multilobed nuclei observed in the present work, are considered to be PMNs. During these studies, however, a far greater number of cells with identical features showed no phagocytic activity. This lack of activity suggests that a certain stage of maturation is required before trout PMNs acquire phagocytic competence. REFERENCES
Macrophages and Immunity.
I.
NELSON, D. S. Inc., 1962.
2.
STOSSEL, T. P. 12, 83, 1975.
3.
CAMPBELL, P. A. Immunocompetent cells in resistance tions. Bacteriol. Rev. 40, 284, 1976.
4.
KATZ, M. Some interesting cells in the blood of a diseased silver salmon fingerling. Copeia 1950, 295.
5.
JAKOWSKA, S., and NIGRELLI, R. F. Localized responses in fish to experimental inflammation caused by pathogenic bacteria. Anat. Rec. 177, 526, 1953.
6.
POST, G.
Phagocytosis:
New York: John Wiley and Sons,
recognition and ingestion.
Semin. Hematol.
to bacterial
infec-
The immune response of rainbow trout to Aeromonas hydrophila.
Utah State Dep. Fish Game, Publ. 63-7, 1963. 7.
WATSON, L. J., SHECHMEISTER, I. L., and JACKSON, L. L. The haematology of goldfish, Carassius auratus. Cytologia 28, 118, 1963.
8.
KLONTZ, G. W., YASUTAKE, W. T., and ROSS, A. J. Bacterial diseases of the Salmonidae in the western United States: pathogenesis of furunculosis in rainbow trout. Am. J. Vet. Res. 27, 1455, 1966.
Vol. 7, No. 2
9.
PHAGOCYTOSIS BY TROUT LEUKOCYTES
259
GONCHAROV, G. D., and MIKRIAKOV. Study of the factors of the immunity of fish to a bacterial infection. (Etude des facteurs de l'immunite des poissons a une infection bacterienne.) Bull. Off. int. Epizoot. 69, 1373, 1968. (Translated from Russian.)
i0.
TETZ, V. I. Phagocytic reaction in carp. Izv. Gos. Nauchno-Issled. Inst. Ozern. Rechn. Rybn. Khoz. 69, 182, 1969.
ii.
GONCHAROV, G. D. Immunological reaction of carp kidney cells Report I. Phagocytosis of bacteria by carp kidney cells in vitro. (Immunologicheskaya reaktsiya kletok pochek karpa Soobschnenie I. Fagotsitoz bakterii kletkami pochek karpa in vitro.) Biol. Ynutr. Vod Inf. Byull. 8, 54, 1970. (Translated from Russian.)
12.
FINN, J. P., and NIELSON, N. O. The inflammatory response of rainbow trout. J. Fish Biol. 3, 463, 1971.
13.
AVTALION, R. R., and SHAHRABANI, R. Studies on phagocytosis in fish. In vitro uptake and killing of Staphylococcus aureus by peripheral leukocytes of carp (Cyprinus carpio). Immunology 29, 1181, 1975.
14.
McKINNEY, E. C., SMITH, S. B., HAINES, H. G., and SIGEL, M. M. Phagocytosis by fish cells. J. Reticuloendothel. Soc. 21, 89, 1977.
15.
BOYUM, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77, 1968.
16.
van FURTH, R., and van ZWET, T. L. In vitro determination of phagocytosis and intracellular killing by polymorphonuclear and mononuclear phagocytes. In: Handbook of Experimental Immunology, 2nd edition. D. M. Weir (Ed.) London: Blackwell Sci. Publ., 1973, p. 36.1.
17.
WEINREB, E. L., and WEINREB, S. A study of experimentally induced endocytosis in a teleost. I. Light microscopy of peripheral blood cell response. Zoologica (NY) 54, 25, 1969.
18.
KLONTZ, G. W. Haematological techniques and the immune response in rainbow trout. In: Diseases of Fish. L. E. Mawdesley-Thomas (Ed.) Symposia of the Zoological Society of London, London: Academic Press, 1972, p. 89-99.
19.
FERGUSON, H. W. Phagocytosis by the endocardial lining cells of the atrium of plaice (Pleuronecta platessa). J. Comp. Pathol. 85, 61, 1975.
20.
ELLIS, A. E., MUNRO, A. L. S., and ROBERTS, R. J. Defense mechanisms in fish. I. A study of the phagocytic system and the fate of intraperitoneally injected particulate material in the plaice (Pleuronectes platessa) J. Fish Biol. 8, 67, 1976.
21.
ELLIS, A. E. The leukocytes of fish: a review. 1977. Received : September, 1982 Accepted : November, 1982
J. Fish Biol. ii, 453,
I.
1983.
OPSONIC EFFECT OF RAINBOW TROUT (SALMO GAIRDNERI) ANTIBODY ON PHAGOCYTOSIS OF YERSINIA RUCKERI BY TROUT LEUKOCYTES
B. R. Griffin 1 U.S. Fish and Wildlife Service National Fisheries Center - Leetown National Fish Health Research Laboratory Kearneysville, West Virginia 25430
ABSTRACT.
Partly purified peripheral blood leukocytes from normal rainbow trout (Salmo gairdneri) were used to study the influence of specific antibody on phagocytic uptake and intracellular killing of Yersinia ruckeri, a bacterial pathogen of trout. Specific antibody exerted a significant opsonic effect on the rate of phagocytic ingestion of the bacteria but did not affect the rate of intracellular killing. The results are discussed with reference to the current understanding of fish antibody function and phagocytosis by fish leukocytes.
INTRODUCTION Among protective effects ascribed to specific antibacterial antibody in mammals is its ability to opsonize homologous bacteria and thus enhance uptake by mononuclear and polymorphonuclear phagocytes (1-3). Phagocytosis of bacteria in fish has been reported (4-14), but studies of the effects of immunization on phagocytosis have produced differing results. Post (6) found an increased opsonic index in in vivo studies of rainbow trout (Saimo gairdneri) immunized with Aeromonas hydrophila. Avtalion and Shahrabani (13) reported that, although immunization enhanced intracellular killing of Staphylococcus aureus, it had no effect on the rate of ingestion of bacteria by cells of the whole blood of carp (Cyprinus carpio). Goncharov and Mikriakov (9) and Gonchorav (ii) found that phagocytosis of gram-negative bacteria in immunized carp proceeded at a rate twice that in nonimmunized controls. Tetz (i0) reported that phagocytosis of A. punctata (A. hydrophila) in carp blood was unaffected by immunization.
ipresent address: U.S. Fish and Wildlife Service, Fish Farming Experimental Station, P. O. Box 860, Stuttgart, Arkansas, 72160. 253
254
PHAGOCYTOSIS
BY TROUT LEUKOCYTES
After a review of the literature, it seems safe to are capable of engulfing bacteria, but that little that influence the process. The research presented an attempt to clarify the role of specific antibody bacteria by trout leukocytes.
Vol.
7, No. 2
say that fish phagocytes is known about factors here was undertaken as in phagocytosis of
METHODS Fish maintenance and treatment. Rainbow trout (Salmo gairdneri) from specific-pathogen-free stocks were used throughout these experiments as sources of immune serum, normal serum, and normal peripheral blood leukocytes. Adults averaging 15 to 25 cm in fork length, were maintained in fresh flowing spring water at 12°C. The fish were anesthetized for bleeding by adding tricaine methanesulfonate (Crescent Research Chemicals, Inc., Paradise Valley, Arizona*) to holding tank water. For production of specific antibody, trout were given a single l-mg in intraperitoneal injection of log-phase broth cultured, formalin killed Yersinia ruckeri cells that had been washed and resuspended in PBS. Immunized fish were bled 3 to 5 weeks later, and sera were collected and frozen at -80°C until use. All antisera had bacterial agglutination titers in excess of 1:64, whereas undiluted normal serum was negative. Peripheral blood leukocyte purification. Methods used for mammalian leukocyte separation employing Ficoll-Hypaque mixtures, based on the work of B~yum (15), were not usable with rainbow trout blood because separation of cells was poor and inconsistent. Caudal vein blood was drawn into an equal volume of 0.i M phosphate buffer, pH 7.2, containing i00 IU heparin/ml. The diluted blood was layered over 2.5-mi cushions of 21% Hypaque (Winthrop Laboratories, New York) in siliconized tubes (15 by 125 mm). The 21% Hypaque solution was prepared by diluting 50% stock with heparinized phosphate buffer. After centrifugation at 250 X g for 20 min at 8 to 10°C in a horizontal rotor centrifuge, the leukocyte band at the plasma: Hypaque interface was removed with a pasteur pipette and transferred to a fresh tube for washing, first with heparinized buffer, then with nonheparinized buffer. Cell densities were adjusted to between i and 3 X 107 cells/ml by suspension in RPMI-1640 (GIBCO, Grand Island, New York). Bacterial phagocytosis and intracellular killing. Procedures employed to assess in vitro phagocytosis and intracellular killing of bacteria by trout leukocytes were adapted from those described by van Furth and van Zwet (16). The bacterial culture was from a recent isolate of Yersinia ruckeri, the etiologic agent of enteric redmouth of salmonids. Log phase cells from culture in brain heart infusion (BHI) broth were washed in phosphate buffer and suspended in RPMI-1640 to a concentration giving an optical density of 0.4 at 525 nm. The working suspension prepared was a I:i0 dilution of this stock with RPMI-1640. The final suspension contained approximately 8 X 107 bacteria/ml, as determined by dilution plate counting. Before the bacteria and leukocytes were mixed, the bacteria were incubated 30 min in RPMI-1640 containing 10% complement-inactivated (42°C for 30 min) normal or immune trout serum. Equal 1-5 ml volumes of leukocytes and treated bacteria were then combined (bacteria:leukocyte ratio of 2 to 4) and gently shaken for 30 min at 20°C. The mixture was diluted with five volumes of cold RPMI-1640 to stop *Reference products.
to trade names does not imply government endorsement
of commercial
Vol.
7, No. 2
PHAGOCYTOSIS
BY TROUT LEUKOCYTES
255
phagocytosis and centrifuged at 400 X g for 4 min at 2°C; the leukocytes were then washed three times in cold RPMI-1640. The leukocytes were resuspended to the starting volume (3 ml) in 20°C RPMI-1640 containing 5% normal trout serum. Immediately (to estimate ingestion rate) and after 60 min at 20°C (to estimate intracellular killing rate), 0.5-ml samples were removed and diluted in 4.5 ml of ice-cold PBS containing 1% bovine serum albumin (PBSA). The leukocytes were disrupted by a 20-s exposure to ultrasound at 50 W with a probe type ultrasonifer (Sonifer Cell Disrupter, Model W1850, Ultrasonics, Inc., Plainview, New York). In control experiments this treatment caused complete disruption of leukocytes with no detectable effect on bacterial viability. Dilutions were made in PBSA and plate counts done on BHI agar plates. Identification of cell types. The cellular classification system described by Finn and Nielson (12) was used as a guide for identifying trout leukocytes. At the end of the phagocytosis experiments, a few slides were prepared from each test mixture. After slides--either simple smears or cytocentrifuge preparations--were dried, they were fixed by flooding with 3% buffered formalin for i0 s. Staining for peroxidase activity, as a more definitive means of identifying polymorphonuclear leukocytes (PMNs), was done as follows: aqueous 0.01 M KH2PO 4, pH 6.0, containing 0.008% 3,3'-dimethoxybenzidine (Eastman Chemical Co., Rochester, New York) and 0.003% H202 was applied as a primary stain; after 15 min the slides were rinsed with deionized water and stained with giemsa. Intracellular areas containing peroxidase were stained diffuse to granular brown, and nuclear and cytoplasmic areas gave typical Romanowsky staining reactions. Intracellular bacteria stained deep blue.
RESULTS The effect of specific antibody on the phagocytic uptake and subsequent intracellular killing of homologous bacteria was measured in individual tests with purified leukocytes from I0 unimmunized normal trout. Normal sera controls were run simultaneously. Ingestion of bacteria was greatly enhanced by specific antibody (Table i). The difference in P30 values obtained from immune versus normal serum was highly significant (P <.01). The apparent difference in the rate of intracellular killing (K60) was not significant. One experiment was done to compare effects of normal serum and no serum on phagocytosis. The results with normal serum were consistent with the other experiments: P30 = 0.6 and K60 = 115. The absence of serum in the test resulted in a marked reduction in uptake (P30 = 0.03) but there was little effect on killing (K60 = 102). When cells were counted on slide preparations from immune serum samples, approximately 27% of the leukocytes were phagocytic. Of these, 57% were mononuclear cells as seen in Figure i, and the remaining 43% were PMNs as seen in Figure 2. Differential staining showed that the phagocytic cells in Figure 1 were peroxidase negative with a faint-blue staining cytoplasm. Phagocytic cells in Figure 2 were peroxidase positive with an otherwise clear cytoplasm. Peroxidase negative mononuclear phagocytes engulfed greater numbers of bacteria (6.8/celi) than did peroxidase positive PMNs.
256
PHAGOCYTOSIS BY TROUT LEUKOCYTES
Vol.
7, No. 2
TABLE 1 Influence of Immune and Normal Serum on Phagocytosis and Intracellular Killing of Yersinia ruckeri by Rainbow Trout Leukocytes P30* Animal no.
Immune serum
K60 # Normal serum
Opsonic t index
Immune serum
Normal serum
1 2 3 4 5 6 7 8 9 i0
9.1 16.2 8.8 5.5 5.3 15.0 8.6 9.6 18.0 11.6
0.4 1.8 1.6 0.6 0.7 1.3 1.0 4.1 2.2 1.0
22.8 9.0 5.5 9.2 7.6 11.5 8.6 2.3 8.2 11.6
62 82 93 105 64 70 115 53 57 57
53 108 143 99 30 102 112 58 89 63
Mean
10.8
1.5
9.6
76
86
*P30 Phagocytic index = number of bacteria/leukocyte (X 10 -2 ) after 30 min contact. + Phagocytic index obtained with immune serum -Opsonic index = Phagocytic index obtained with normal serum. $K60 = Index of intracellular killing = percent of original viable cellassociated bacteria after 60 min incubation.
P
FIGURE i A peroxidase negative mononuclear phagocyte (arrow) with intracellular bacteria Adjacent to this cell are a lymphocyte and a peroxidase positive polymorphonuclear leukocyte. X i000.
Vol.
7, No. 2
PHAGOCYTOSIS BY TROUT LEUKOCYTES
257
FIGURE 2 A peroxidase positive polymorphonuclear leukocyte (arrow) with intracellular bacteria. The several adjacent cells are lymphocytes. X i000.
These observations were made on leukocyte slide preparations taken from samples containing bacteria in immune serum; phagocytes with engulfed bacteria were so rare in slide preparations from samples containing normal serum that estimates of the numbers and types of cells involved could not be obtained. DISCUSSION Bacterial phagocytosis by mammalian cells can be enhanced by specific antibody, by nonspecific normal-serum opsonins, by complement activation, and by the cellular condition of the immune animal through the action of its activated macrophages (1-3). I took several precautions to study, as nearly as possible, the specific effects of antibody on phagocytosis by fish cells. Leukocytes from unimmunized, seronegative, specific-pathogen-free animals, separated from erythrocytes and washed to remove the bulk of normal-serum associated opsonins, were used as a source of phagocytes. All sera were complement inactivated by heating at 42°C for 30 min, and normal serum controls were run in parallel with immune serum tests. The data from these experiments indicate that specific antibody exerts a significant opsonic effect on phagocytosis of homologous bacteria by normal trout phagocytes. In this regard, trout antibody appears to function in a manner similar to mammalian antibody. The results are not consistent with those of Avtalion and Shahrabani (13), who found that immunization did not influence the rate of uptake of S. a u r e u s in whole blood of carp. They suggested that the high titers of natural antibody (1:16-1:32) of normal carp used in the study may have been sufficient to permit maximum levels of phagocytosis. There may also have been species differences or temperature effects that are not yet understood.
258
PHAGOCYTOSIS BY TROUT LEUKOCYTES
Vol.
7, No. 2
Intracellular killing of bacteria by normal trout leukocytes was found to be a slow process and one that does not seem to be affected by opsonization with antibody. This observation is consistent with Finn and Nielson's (12) results with rainbow trout injected with S. aureus, in that intracellular destruction of bacteria was slow by mammalian standards. Avtalion and Shahrabani (13) found that blood cells from immunized carp were more efficient at intracellufar killing of S. aureus than were blood cells from nonimmunized carp. In their work however, Avtalion and Shahrabani (13) used whole blood and measured phagocytosis and intracellular killing of a nonpathogen of fish, S. aureus. The presence of complement in the whole blood may have enhanced the role of intracellular killing they observed. Fish leukocytes that have been reported to be phagocytic include lymphocytes (9,11,17,18), monocyte-macrophages (4,6,9,11,12,14,19,20), PMNs (6,7,11,12, 17), and eosinophils (5,7,17). Some of these reports are contradictory and there is confusion about the identity and function of fish leukocytes (21). Throughout the experiments reported here, the two cell types described were the only ones seen to be actively phagocytic. Identification of the blood monocyte (macrophage) as one of the principal phagocytes is consistent with other reports on fish phagocytes (4,8,12,14). A controversial point, however, is whether the PMN is a phagocytic cell (4,8,18,21). The most convincing work is that by Finn and Nielson (12), in which PMNs were active in phagocytosis of S. aureus. In spite of reports to the contrary, the peroxidase positive, phagocytic, leukocytes with multilobed nuclei observed in the present work, are considered to be PMNs. During these studies, however, a far greater number of cells with identical features showed no phagocytic activity. This lack of activity suggests that a certain stage of maturation is required before trout PMNs acquire phagocytic competence. REFERENCES
Macrophages and Immunity.
I.
NELSON, D. S. Inc., 1962.
2.
STOSSEL, T. P. 12, 83, 1975.
3.
CAMPBELL, P. A. Immunocompetent cells in resistance tions. Bacteriol. Rev. 40, 284, 1976.
4.
KATZ, M. Some interesting cells in the blood of a diseased silver salmon fingerling. Copeia 1950, 295.
5.
JAKOWSKA, S., and NIGRELLI, R. F. Localized responses in fish to experimental inflammation caused by pathogenic bacteria. Anat. Rec. 177, 526, 1953.
6.
POST, G.
Phagocytosis:
New York: John Wiley and Sons,
recognition and ingestion.
Semin. Hematol.
to bacterial
infec-
The immune response of rainbow trout to Aeromonas hydrophila.
Utah State Dep. Fish Game, Publ. 63-7, 1963. 7.
WATSON, L. J., SHECHMEISTER, I. L., and JACKSON, L. L. The haematology of goldfish, Carassius auratus. Cytologia 28, 118, 1963.
8.
KLONTZ, G. W., YASUTAKE, W. T., and ROSS, A. J. Bacterial diseases of the Salmonidae in the western United States: pathogenesis of furunculosis in rainbow trout. Am. J. Vet. Res. 27, 1455, 1966.
Vol. 7, No. 2
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