Veterinary Immunology andImmunopathology, 30 (1992) 419-429
419
Elsevier Science Publishers B.V., Amsterdam
Neutrophil, glass-adherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout Douglas P. Anderson a, Tadaaki M o r i t o m o b and Ricardo de G r o o t h c aU.S. Fish and Wildlife Service, National Fish Health ResearchLaboratory, Box 700, Kearneysville, WV25430, USA bNihon University, Laboratory offish Pathology, Kameino, Fufisawa, Kanagawa, Japan CDepartmentof Experimental Animal Morphology and Cell Biology, Agricultural University, Zodiac, P.O. Box 338, 6700 AH Wageningen, Netherlands (Accepted 16 January 1991 )
ABSTRACT Anderson, D.P., Moritomo, T. and de Grooth, R., 1992. Neutrophil, glass-adherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout. Vet. Immunol. Immunopathol., 30: 419-429. Neutrophil activity in rainbow trout (Oncorhynchus mykiss) is increased upon antigenic stimulation with the Yersinia ruckeri O-antigen bacterin. The characteristics of neutrophil attachment to glass and nitroblue tetrazolium (NBT) staining were used to determine the effectiveness of immunization programs with fingerling rainbow trout. Fish immunized by intraperitoneal injection with doses of 100, 10, or 1 #g of the bacterin showed the highest responses in that order in numbers of glass adherent, NBT-positive neutrophils. Studies on the kinetics of the occurrence of numbers of glass-adherent, NBT-positive staining cells from the fish injected with the l 0 #g dose showed the numbers of positive cells were largest on Day 2 after injection. The specific immune response was confirmed by demonstrating the presence of plaque-forming cells by the passive hemolytic plaque assay and the rise in humoral antibody titers by passive hemagglutination 12 days after injection. The effects of immunization in trout could be detected earlier by using the neutrophil glass adherence and NBT reduction assays than by using assays based on observations of the specific immune response. ABBREVIATIONS NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium" PBS, phosphate-buffered saline; PFC plaque-forming cells; SRBC, sheep red blood cells.
INTRODUCTION
Immunization o f fish against diseases is becoming a widely used technique in fish farms, hatcheries, and other intensive aquaculture facilities. The specific immune response to a bacterin such as Yersinia ruckeri can be detected as early as 7-10 days after immunization in fish held at 12 ° C by demonstrat-
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
420
D.P. ANDERSON ET AL.
ing plaque-forming cells (PFC). Low levels of humoral antibody also appear at this time and reach their highest titers about 28 days after immunization (Anderson et al., 1979). Sometimes fish are released into the environment a few days after immunization; consequently, methods are needed to detect the effectiveness of the immunogen as soon as possible. The specific immune response takes days to develop because it involves complex physiological pathways of selection and synthesis of tailored molecules such as antibody; however, the non-specific defense mechanisms are already in place and often need only to be activated or released. Thus, the non-specific defense mechanisms react faster after an immunization, injury or infection by microorganisms. Non-specific assays done with fish include demonstrating elevated activities of phagocytosis and chemotaxis of macrophages and monocytes (Weeks and Warinner, 1986); showing increases in certain blood proteins such as C-reactive protein and ceruloplasmin, which give indications of an inflammatory reaction (Fletcher and Baldo, 1974; Siwicki and Studnicka, 1986), or detecting the production of interferon (de Kinkelin and Dorson, 1982 ). Another important non-specific defense mechanism involves peripheral blood neutrophils. These cells become more adherent to tissue cell surfaces by the production of adhesion proteins that facilitate the migration from the capillaries to the injury sites (Kishimoto et al., 1989; Magnuson et al., 1989). In addition, neutrophils become more specialized in the production of oxygen radicals (O~-, O H - ) that are released in an oxidative burst to destroy invaders (Hassett and Cohen, 1989). The increased adherence characteristics of these cells can be analyzed by showing the degree of their stickiness and tendency to attach to glass surfaces. Nitroblue tetrazolium (NBT) can be used as a differential stain as the soluble dye changes from yellow to insoluble, dark blue granules of formazan in the cell cytoplasm in response to an increase in the levels of reactive oxygen species. Modifications of these assays with neutrophils have been used extensively in medical work to determine phagocytic dysfunction; for instance, chronic granulomatous disease in man results from a genetic deficiency in the cell membrane enzyme, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, so there is a decreased function in intracellular killing of microorganisms by oxygen radicals (Jabs et al., 1980; Caren and Larrick, 1989). In the following studies, we show that immunization by injection affects circulatory blood neutrophils by increasing glass adherent activities and the production of oxidative radicals. In the trout in this study, these changes are most apparent within 2 days after immunization, and can be detected by following the simple and rapid technique described here. Inasmuch as neutrophils have been described in many species of fish including rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri; Peters and Schwarzer, 1985 ), tilapia Oreochromis mossambicus (Doggett et al., 1987 ), Japanese eel Anguilla japonica (Moritomo et al,. 1988 ), and dogfish Squalus ancanthias
IMMUNIZATION EFFECTIVENESS IN RAINBOW TROUT
421
(Sherburne, 1974), these assays have application in determining levels of fish health. MATERIALS AND METHODS
Fish Rainbow trout were obtained from the White Sulphur Springs National Fish Hatchery, WV and transported to the National Fish Health Research Laboratory, Leetown, WV where they were held until they averaged 11.8 g (9.2 cm fork length ). Fish were held in fiberglass tanks holding 3401 of 11 + 1 °C spring water fed at 12 1 m i n - i. Control stock fish were held similarly. All trout were fed a commercial pelletized dry feed once a day.
Fig. 1. Three glass-adherent, NBl-posltlve blood neutrophils isolated from rainbow trout blood show the multi-lobed nuclei and dark blue granules of formazan after exposure to nitroblue tetrazolium dye. Scale bar represents 10/zm.
422
D.P. ANDERSON ET AL.
Antigen exposures
Groups of ten fish each were immunized with single intraperitoneal injections of 100, 10, or 1/tg of Yersinia ruckeri (Biotype II) O-antigen suspended in 0.1 ml phosphate-buffered saline (PBS) at pH 7.4. A parallel group of fish
-Q
!iii!ii~!!!ii!~!!~ ¸ i~ i~i!i!iii!iiiiiiii!ii!!!~i@ ¸ i~iI~~!i!~!!!!!!i!~ii!!i i ~!~~!~!~ii!~!~I!!!
Fig. 2. Glass-adherent NBT-positive neutrophils from rainbow trout. (a) Sample taken from a fish immunized by an injection 2 days earlier with the Y. ruckeri O-antigen bacterin. (b) Sample from a control fish injected with PBS. Scale bar represents 40 am.
IMMUNIZATION EFFECTIVENESS IN RAINBOW TROUT
42 3
was injected with 0.1 ml of PBS. The fish from each group were sampled for the presence of glass-adherent, NBT-positive cells 2 and 7 days after injection. A second experiment, designed to study the kinetics of the activation of neutrophils, was performed by injecting 70 fish with 10 gg of Y. ruckeri Oantigen. Ten fish each time were sampled on Days l, 2, 3, 7, 9, and 12 after injection. Groups of PBS and non-injected control fish were held similarly for comparisons.
Sampling fish and NBT assays Fish were sampled by severing the caudal peduncle of each fish and withdrawing clean blood into two heparinized capillary hematocrit tubes. The tubes were held at room temperature while other fish were sampled. Within 15 min after sampling, one drop of blood from each capillary tube was placed onto a 22-mm square coverslip. The coverslips were placed individually in humid chambers (60 m m petri dishes with a wet paper towel) and incubated for 30 min at room temperature (22°C) to allow the neutrophils to stick to the glass. After incubation, the coverslips were gently washed with PBS (pH 7.4 ) and the cells were transferred upside down to a microscope slide containing a 50/~1 drop of 0.2% filtered NBT solution (Sigma Chemicals, St. Louis,
140120-
~ +
100
-
80-
~eO-
40n~
20o
2 DAYS AFTER
7 INJECTION
Fig. 3. Numbers of glass-adherent, NBT-positive blood neutrophils from rainbow trout injected with doses of 100 (t-'l), lO (~t), or l #g ( m ) of Y. ruckeri O-antigen. (11) shows the results from the negative control sample. Error bars represent SE (n = 10). *denotes significant differences between groups of fish injected with the bacterin compared to controls injected with PBS
(P
419
Elsevier Science Publishers B.V., Amsterdam
Neutrophil, glass-adherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout Douglas P. Anderson a, Tadaaki M o r i t o m o b and Ricardo de G r o o t h c aU.S. Fish and Wildlife Service, National Fish Health ResearchLaboratory, Box 700, Kearneysville, WV25430, USA bNihon University, Laboratory offish Pathology, Kameino, Fufisawa, Kanagawa, Japan CDepartmentof Experimental Animal Morphology and Cell Biology, Agricultural University, Zodiac, P.O. Box 338, 6700 AH Wageningen, Netherlands (Accepted 16 January 1991 )
ABSTRACT Anderson, D.P., Moritomo, T. and de Grooth, R., 1992. Neutrophil, glass-adherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout. Vet. Immunol. Immunopathol., 30: 419-429. Neutrophil activity in rainbow trout (Oncorhynchus mykiss) is increased upon antigenic stimulation with the Yersinia ruckeri O-antigen bacterin. The characteristics of neutrophil attachment to glass and nitroblue tetrazolium (NBT) staining were used to determine the effectiveness of immunization programs with fingerling rainbow trout. Fish immunized by intraperitoneal injection with doses of 100, 10, or 1 #g of the bacterin showed the highest responses in that order in numbers of glass adherent, NBT-positive neutrophils. Studies on the kinetics of the occurrence of numbers of glass-adherent, NBT-positive staining cells from the fish injected with the l 0 #g dose showed the numbers of positive cells were largest on Day 2 after injection. The specific immune response was confirmed by demonstrating the presence of plaque-forming cells by the passive hemolytic plaque assay and the rise in humoral antibody titers by passive hemagglutination 12 days after injection. The effects of immunization in trout could be detected earlier by using the neutrophil glass adherence and NBT reduction assays than by using assays based on observations of the specific immune response. ABBREVIATIONS NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium" PBS, phosphate-buffered saline; PFC plaque-forming cells; SRBC, sheep red blood cells.
INTRODUCTION
Immunization o f fish against diseases is becoming a widely used technique in fish farms, hatcheries, and other intensive aquaculture facilities. The specific immune response to a bacterin such as Yersinia ruckeri can be detected as early as 7-10 days after immunization in fish held at 12 ° C by demonstrat-
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00
420
D.P. ANDERSON ET AL.
ing plaque-forming cells (PFC). Low levels of humoral antibody also appear at this time and reach their highest titers about 28 days after immunization (Anderson et al., 1979). Sometimes fish are released into the environment a few days after immunization; consequently, methods are needed to detect the effectiveness of the immunogen as soon as possible. The specific immune response takes days to develop because it involves complex physiological pathways of selection and synthesis of tailored molecules such as antibody; however, the non-specific defense mechanisms are already in place and often need only to be activated or released. Thus, the non-specific defense mechanisms react faster after an immunization, injury or infection by microorganisms. Non-specific assays done with fish include demonstrating elevated activities of phagocytosis and chemotaxis of macrophages and monocytes (Weeks and Warinner, 1986); showing increases in certain blood proteins such as C-reactive protein and ceruloplasmin, which give indications of an inflammatory reaction (Fletcher and Baldo, 1974; Siwicki and Studnicka, 1986), or detecting the production of interferon (de Kinkelin and Dorson, 1982 ). Another important non-specific defense mechanism involves peripheral blood neutrophils. These cells become more adherent to tissue cell surfaces by the production of adhesion proteins that facilitate the migration from the capillaries to the injury sites (Kishimoto et al., 1989; Magnuson et al., 1989). In addition, neutrophils become more specialized in the production of oxygen radicals (O~-, O H - ) that are released in an oxidative burst to destroy invaders (Hassett and Cohen, 1989). The increased adherence characteristics of these cells can be analyzed by showing the degree of their stickiness and tendency to attach to glass surfaces. Nitroblue tetrazolium (NBT) can be used as a differential stain as the soluble dye changes from yellow to insoluble, dark blue granules of formazan in the cell cytoplasm in response to an increase in the levels of reactive oxygen species. Modifications of these assays with neutrophils have been used extensively in medical work to determine phagocytic dysfunction; for instance, chronic granulomatous disease in man results from a genetic deficiency in the cell membrane enzyme, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, so there is a decreased function in intracellular killing of microorganisms by oxygen radicals (Jabs et al., 1980; Caren and Larrick, 1989). In the following studies, we show that immunization by injection affects circulatory blood neutrophils by increasing glass adherent activities and the production of oxidative radicals. In the trout in this study, these changes are most apparent within 2 days after immunization, and can be detected by following the simple and rapid technique described here. Inasmuch as neutrophils have been described in many species of fish including rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri; Peters and Schwarzer, 1985 ), tilapia Oreochromis mossambicus (Doggett et al., 1987 ), Japanese eel Anguilla japonica (Moritomo et al,. 1988 ), and dogfish Squalus ancanthias
IMMUNIZATION EFFECTIVENESS IN RAINBOW TROUT
421
(Sherburne, 1974), these assays have application in determining levels of fish health. MATERIALS AND METHODS
Fish Rainbow trout were obtained from the White Sulphur Springs National Fish Hatchery, WV and transported to the National Fish Health Research Laboratory, Leetown, WV where they were held until they averaged 11.8 g (9.2 cm fork length ). Fish were held in fiberglass tanks holding 3401 of 11 + 1 °C spring water fed at 12 1 m i n - i. Control stock fish were held similarly. All trout were fed a commercial pelletized dry feed once a day.
Fig. 1. Three glass-adherent, NBl-posltlve blood neutrophils isolated from rainbow trout blood show the multi-lobed nuclei and dark blue granules of formazan after exposure to nitroblue tetrazolium dye. Scale bar represents 10/zm.
422
D.P. ANDERSON ET AL.
Antigen exposures
Groups of ten fish each were immunized with single intraperitoneal injections of 100, 10, or 1/tg of Yersinia ruckeri (Biotype II) O-antigen suspended in 0.1 ml phosphate-buffered saline (PBS) at pH 7.4. A parallel group of fish
-Q
!iii!ii~!!!ii!~!!~ ¸ i~ i~i!i!iii!iiiiiiii!ii!!!~i@ ¸ i~iI~~!i!~!!!!!!i!~ii!!i i ~!~~!~!~ii!~!~I!!!
Fig. 2. Glass-adherent NBT-positive neutrophils from rainbow trout. (a) Sample taken from a fish immunized by an injection 2 days earlier with the Y. ruckeri O-antigen bacterin. (b) Sample from a control fish injected with PBS. Scale bar represents 40 am.
IMMUNIZATION EFFECTIVENESS IN RAINBOW TROUT
42 3
was injected with 0.1 ml of PBS. The fish from each group were sampled for the presence of glass-adherent, NBT-positive cells 2 and 7 days after injection. A second experiment, designed to study the kinetics of the activation of neutrophils, was performed by injecting 70 fish with 10 gg of Y. ruckeri Oantigen. Ten fish each time were sampled on Days l, 2, 3, 7, 9, and 12 after injection. Groups of PBS and non-injected control fish were held similarly for comparisons.
Sampling fish and NBT assays Fish were sampled by severing the caudal peduncle of each fish and withdrawing clean blood into two heparinized capillary hematocrit tubes. The tubes were held at room temperature while other fish were sampled. Within 15 min after sampling, one drop of blood from each capillary tube was placed onto a 22-mm square coverslip. The coverslips were placed individually in humid chambers (60 m m petri dishes with a wet paper towel) and incubated for 30 min at room temperature (22°C) to allow the neutrophils to stick to the glass. After incubation, the coverslips were gently washed with PBS (pH 7.4 ) and the cells were transferred upside down to a microscope slide containing a 50/~1 drop of 0.2% filtered NBT solution (Sigma Chemicals, St. Louis,
140120-
~ +
100
-
80-
~eO-
40n~
20o
2 DAYS AFTER
7 INJECTION
Fig. 3. Numbers of glass-adherent, NBT-positive blood neutrophils from rainbow trout injected with doses of 100 (t-'l), lO (~t), or l #g ( m ) of Y. ruckeri O-antigen. (11) shows the results from the negative control sample. Error bars represent SE (n = 10). *denotes significant differences between groups of fish injected with the bacterin compared to controls injected with PBS
(P
