doi:10.1111/jfd.12258
Journal of Fish Diseases 2015, 38, 107–112
Short communication The role of a specific antibody against Flavobacterium psychrophilum infection in ayu sweetfish, Plecoglossus altivelis altivelis (Temminck & Schlegel, 1846) G Kato1,2, K Suzuki3, T Sakai1, M Kawakami1, T Takano4, T Matsuyama4 and C Nakayasu1 1 2 3 4
Tamaki Laboratory, National Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan The Japan Society for the Promotion of Science, Tokyo, Japan Gunma Prefectural Fisheries Experimental Station, Gunma, Japan National Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan
Keywords: passive immunization, phagocytic rate, vaccine.
opsonization,
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD) and causes severe loss of aquaculture production of ayu sweetfish Plecoglossus altivelis altivelis (Temminck & Schlegel, 1846) in Japan (Wakabayashi, Toyama & Iida 1994). The characteristics of BCWD in ayu are different from those of BCWD in salmonid fishes. BCWD in ayu generally occurs when water temperature is 16 to 18 °C (Miwa & Nakayasu 2005), whereas that in salmonid fishes most frequently occurs in 4–10 °C (Shotts & Starliper 1999). Clinical signs of BCWD in ayu are anaemia and ulcerative lesions on the caudal peduncle (Iida & Mizokami 1996). However, erosion of fins and tail loss, which are typical clinical signs of BCWD in salmonid fishes (Nematollahi et al. 2003), are not observed in ayu. Ayu and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) that have recovered from BCWD are resistant to the reinfection (Kintsuji, Yamamoto & Ninomiya 2007; Lorenzen et al. 2010). High antibody titre is present in the serum of BCWD-recovered fish. LaFrentz et al. (2003) performed passive immunization of rainbow trout Correspondence C Nakayasu, Hiruta 224-1, Tamaki, Watarai, Mie 519-0423, Japan (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
107
using antiserum against F. psychrophilum and reported that a specific antibody has a critical protective role. In this study, passive immunization was performed to reveal the protective efficacy of a specific antibody against BCWD in ayu. The effects of opsonization by the antiserum on phagocytosis and of humoral bactericidal activity were also investigated. Flavobacterium psychrophilum strain GMA0330 (Arai et al. 2007) were cultured at 15 °C for 24 h in modified Cytophaga broth (Wakabayashi & Egusa 1974). Ayu (14.9 g average body weight) were injected (i.p.) with 2.2 9 105 colony-forming units (CFUs) F. psychrophilum. Fish were kept in 80-L tanks, and the water temperature was maintained at 15 °C. Serum was collected from the survivors at 3 weeks post-injection and was heat inactivated (45 °C for 30 min). Serum from PBS-injected fish was used as a negative control. Antibody titre (n = 5) was measured using a bacterial cell-binding ELISA with a mAb against ayu immunoglobulins (Kato et al. 2014). The antiserum was diluted to a concentration of 1:40. Flavobacterium psychrophilum (3.9 9 106 CFU) were suspended in the diluted serum and were incubated for 1 h at room temperature (RT). After washing with PBS, the mAb was added to the bacterial suspension and incubated for 1 h at
G Kato et al. The role of antibody in BCWD
Journal of Fish Diseases 2015, 38, 107–112
**
Absorbance (λ = 415 nm)
(a) 2.5
2
1.5
1
0.5
0
Cumulative survival (%)
(b)
Antiserum
Control serum
100 80 60 40 20 0 0
5
10
15
Days post-infection Antiserum
Control serum
PBS Figure 1 Antibody titre of the challenged fish and the control fish (a) and Kaplan–Meier survival curves for ayu passively immunized with antiserum, control serum or PBS (b). An asterisk indicates a statistically significant difference (P < 0.01).
injected (i.p.) with 50 lL pooled antiserum, pooled control serum or PBS. Twenty-four hours after passive immunization, fish were challenged with 1.3 9 105 CFU F. psychrophilum (i.p. injection). Fish were kept in 30-L tanks, and the water temperature was maintained at 18 °C. The numbers of dead fish were recorded for 15 days, and Kaplan–Meier survival curves were generated (Kaplan & Meier 1958). The log-rank test was used to compare the survival times for the passively immunized and the PBS-injected fish (Peto et al. 1977). The experiment was performed twice to confirm the reproducibility of the result, and the representative data are presented in this report. The pooled antiserum and the pooled control serum were each diluted in twofold dilutions (1:10–1:10240). Flavobacterium psychrophilum (3.9 9 106 CFU) were suspended in the diluted serum and then incubated for 1 h at 18 °C. The antibody titre of each dilution was measured using the bacterial cell-binding ELISA. Ayu immunoglobulins bound to the bacteria were detected using indirect immunofluorescence assay (IIFA; Kato et al. 2014). A one-way analysis of variance (ANOVA) was used to detect significant differences between treatments. Flavobacterium psychrophilum (3.9 9 106 CFU) were incubated with serially diluted antisera or with 1:10 diluted control serum as described above. Ayu (2.0 g average body weight, n = 30) were injected (i.p.) with 1.3 9 105 CFU of the serum-treated bacteria. The number of dead fish Table 1 Cumulative survival rate, calculated RPS value and P value by log-rank test in challenge test performed in this study Group
RT. A goat anti-mouse IgG antibody conjugated with HRP (Invitrogen) was added to the bacterial suspension and incubated for 30 min at RT. After washing, the bacterial cells were incubated with ABTS Peroxidase Substrate System (KPL) for 10 min at RT. Peroxidase Stop Solution (KPL) was added, and the absorbance (k = 415 nm) was measured. The mean value for five individuals was estimated, and statistically significant differences were determined using Student’s t-test. Equal volumes of the antiserum from the five fish were pooled and diluted to 1:4 with PBS. Ayu (2.0 g average body weight, n = 20) were Ó 2014 John Wiley & Sons Ltd
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Survival rate (survivors/total)
Passive immunization Antiserum 25.0% (15/20) Control serum 5.0% (19/20) PBS 5.0% (19/20) Challenge test with opsonized bacteria 1:10 60.0% (18/30) 1:40 37.7% (11/30) 1:160 40.0% (12/30) 1:640 23.3% (7/30) 1:2560 23.3% (7/30) 1:10240 16.7% (5/30) Control serum 20.0% (6/30) (1:10) NA, not applicable; NS, not significant. a Relative per cent survival (RPS) = {1 mortality (control)]} 9 100.
RPSa
Log-rank test
21.1 0 NA
P < 0.0001 NS NA
50.0 20.9 25.0 4.1 4.1 NA NA
P < 0.001 NS NS NS NS NS NA
[% mortality (treated)/%
G Kato et al. The role of antibody in BCWD
Journal of Fish Diseases 2015, 38, 107–112
Absorbance (λ = 415 nm)
(a) 2.5 2 1.5 1 0.5 0
Dilution Antiserum
(b)
Control serum
10
40
160
2560
10 240
Control
640
DAPI
FITC
Figure 2 Antibody titre of serially diluted antiserum. (a) Antibody titres for each dilution were measured using the bacterial cell-binding ELISA. (b) Ayu Igs binding to the surface of Flavobacterium psychrophilum was detected using immunofluorescence assay (IIFA). Arrowheads indicate agglutination and the Igs binding to the bacteria. Scale bar: 5 lm.
DAPI
FITC
was recorded for 20 days. Generation of the cumulative survival curve and the statistical analysis were performed as described above. Trunk kidney of uninfected ayu (4.3 g average body weight, n = 3) was teased and passed through a nylon mesh into RPMI-1640 (Life Technologies). Flavobacterium psychrophilum treated with the diluted antiserum were prepared as described above, added to 5.0 9 105 of the kidney cells and incubated at 18 °C for 30 min. The mixture was spread on a glass slide and stained with May–Grunwald–Giemsa stain. More than 1000 leukocytes (with or without phagocytized bacteria) were counted at random, and the phagocytic rate was Ó 2014 John Wiley & Sons Ltd
109
expressed as follows: phagocytic rate (%) = [number of leukocytes with phagocytized bacteria/number of observed leukocytes] 9 100. Serum was collected from uninfected fish (39.0 g average body weight, n = 3), as described above. Flavobacterium psychrophilum treated with diluted antiserum were prepared as described above and added to the uninfected fish serum without inactivation. The mixture was incubated at 18 °C for 6 h. The numbers of CFUs of the bacterial suspensions were counted by Miles– Misra method. Significant differences between treatments were assessed using a one-way ANOVA and Tukey’s post hoc test.
G Kato et al. The role of antibody in BCWD
Journal of Fish Diseases 2015, 38, 107–112
(a) 100
Cumulative survival (%)
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60
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2560 10 240 Control
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2.0E+05
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The mean antibody titre for the survivors in the challenged group was significantly higher than that for the control group (P < 0.01, Fig. 1a). In Ó 2014 John Wiley & Sons Ltd
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Figure 3 Effects of opsonization of the bacteria on the host immune responses. Kaplan–Meier survival curves for ayu injected with opsonized bacteria (a). Phagocytic rate of kidney leukocytes against opsonized bacteria (b). CFUs of the opsonized bacteria after treatment with normal serum (c). Different letters represent significant differences between treatments (P < 0.05). The error bars represent the standard deviation of the mean.
passive immunization study, cumulative per cent survival for the antiserum group was 25%, whereas those for the control groups were 5%
Journal of Fish Diseases 2015, 38, 107–112
(Fig. 1b). Relative per cent survival (RPS) is shown in Table 1. The differences in survival times for the antiserum group and the two control groups were significantly different (P < 0.0001). The antiserum titre value was approximately equal to the control titre value at the 1:10240 (Fig. 2a). A microscopic examination revealed that agglutination occurred when the bacteria were treated with antiserum diluted to 1:10–1:160 (Fig. 2b, upper panels). The IIFA revealed that ayu immunoglobulins bound to bacterial surfaces at the same antiserum dilutions (Fig. 2b, lower panels). The values for cumulative per cent survival were higher in the 1:10, 1:40 and 1:160 antiserum groups compared with the control group (Fig. 3a). RPS values are shown in Table 1. The survival time of the 1:10 antiserum group was significantly longer, compared with the survival time of the control group (P < 0.001). Approximately 44% of the leukocytes phagocytized opsonized bacteria in the 1:10 antiserumtreated group, but the phagocytic rate was only 3% in the control group (Fig. 3b). No significant increase in phagocytic rate occurred in the 1:160to 1:10240-treated groups. There were 1.1 9 105 CFU viable F. psychrophilum in the control serum group, compared with 1.4 9 104 CFU viable bacteria in the 1:10 antiserum-treated group (Fig. 3c). Compared with the control, the bactericidal activity of the 1:10240 antiserum-treated group was not significantly different. The phagocytic rate increased after opsonization. Flavobacterium psychrophilum can be degraded in the phagosome after phagocytosis (Wiklund & Dalsgaard 2003). In mammals, pathogenic bacteria opsonized with IgG are trapped by the Fc receptor, which is located on the phagocytic cells. Fc receptors and corresponding signal pathways are also present in teleost fish, including ayu (Lu et al. 2013). Immunoglobulins also mediate the recognition between phagocytes and antigens (Secombes & Fletcher 1992). Neutrophils are abundant (60–80% of total cell count) in ayu kidney leukocytes (Moritomo et al. 2003) and display unusually high respiratory burst activities (Serada et al. 2005). These results suggest that opsonization with antibody and Fc receptordependent phagocytosis by neutrophils may be critical for the elimination of bacteria during BCWD in ayu. The complement system present in fish serum is essential for humoral defence against bacterial Ó 2014 John Wiley & Sons Ltd
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G Kato et al. The role of antibody in BCWD
pathogens. In this study, opsonization stimulated the bactericidal activity of the humoral component. This result suggests that the classical complement pathway is also associated with the elimination of F. psychrophilum by the ayu immune system. In contrast, F. psychrophilum isolated from rainbow trout are resistant to the complement activity present in rainbow trout serum (Wiklund & Dalsgaard 2002). The resistance of pathogenic bacteria to serum components is mediated by the presence of LPS and a polysaccharide capsule (Boesen et al. 1999). A difference in F. psychrophilum serotypes has been reported (Wakabayashi et al. 1994; Izumi & Wakabayashi 1999). This difference may be important for the differences in resistance of each isolate against host serum components. In conclusion, specific antibody production may be important for immunity against F. psychrophilum in ayu, by accelerating the phagocytic and bactericidal activity of serum. Acknowledgement This work was supported in part by a Grant-inAid (Number 12J04812) for Fellows of the Japan Society for the Promotion of Science. Publication History Received: 21 February 2014 Revision received: 14 April 2014 Accepted: 16 April 2014
This paper was edited and accepted under the Editorship of Professor Ron Roberts. References Arai H., Morita Y., Izumi S., Katagiri T. & Kimura H. (2007) Molecular typing by pulsed-field gel electrophoresis of Flavobacterium psychrophilum isolates derived from Japanese fish. Journal of Fish Diseases 30, 345–355. Boesen H.T., Pedersen K., Larsen J.L., Koch C. & Ellis A.E. (1999) Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide. Infection and Immunity 67, 294–301. Iida Y. & Mizokami A. (1996) Outbreaks coldwater disease in wild ayu and pale chub. Fish pathology 31, 157–164. Izumi S. & Wakabayashi H. (1999) Further study on serotyping of Flavobacterium psychrophilum. Fish Pathology 34, 89–90. Kaplan E.L. & Meier P. (1958) Nonparametric estimation from incomplete observations. Journal of the American Statistical Association 53, 457–481.
Journal of Fish Diseases 2015, 38, 107–112
Kato G., Sakai T., Suzuki K., Yamaguchi K., Takano T., Matsuyama T. & Nakayasu C. (2014) Antigenic proteins of Flavobacterium psychrophilum recognized by ayu Plecoglossus altivelis antisera. Diseases of Aquatic Organisms 108, 103–112. Kintsuji H., Yamamoto M. & Ninomiya K. (2007) The resistance of ayu Plecoglossus altivelis recovered from bacterial coldwater disease against reinfection with Flavobacterium psychrophilum. Fish Pathology 42, 159–161. LaFrentz B.R., LaPatra S.E., Jones G.R. & Cain K.D. (2003) Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease and rainbow trout fry syndrome. Journal of Fish Diseases 26, 377–384. Lorenzen E., Brudeseth B.E., Wiklund T. & Lorenzen N. (2010) Immersion exposure of rainbow trout (Onchorhynchus mykiss) fry to wild type Flavobacterium psychrophilum induces no mortality, but protects against later intraperitoneal challenge. Fish and Shellfish Immunology 28, 440–444. Lu J.X., Hang X.Y., Yin L., He Y.Q., Chen J., Shi Y.H. & Li C.H. (2013) Sequencing of the first ayu (Plecoglossus altivelis) macrophage transcriptome and microarray development for investigation of the effect LECT2 on macrophages. Fish and Shellfish Immunology 34, 497–504. Miwa S. & Nakayasu C. (2005) Pathogenesis of experimentally induced bacterial cold water disease in ayu Plecoglossus altivelis. Diseases of Aquatic Organisms 67, 93–104. Moritomo T., Serata K., Teshirogi K., Aikawa H., Inoue Y., Itou T. & Nakanishi T. (2003) Flow cytometric analysis of the neutrophil respiratory burst of ayu, Plecoglossus altivelis: comparison with other fresh water fish. Fish and Shellfish Immunology 15, 29–38. Nematollahi A., Decostere A., Pasmans F. & Haesebrouck F. (2003) Flavobacterium psychrophilum infections in salmonid fish. Journal of Fish Disease 26, 563–574.
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Peto R., Pike M.C., Armitage P., Breslow N.E., Cox D.R., Howard S.V., Mantel N., McPherson K., Peto J. & Smith P.G. (1977) Design and analysis of randomized clinical trials requiring prolonged observation of each patient: II. Analysis and examples. British Journal of Cancer 35, 1–39. Secombes C.J. & Fletcher T.C. (1992) The role of phagocytes in the protective mechanisms of fish. Annual Review of Fish Diseases 2, 53–71. Serada K., Moritomo T., Teshirogi K., Itou T., Shibashi T., Inoue Y. & Nakanishi T. (2005) Comparison of respiratory burst activity of inflammatory neutrophils in ayu (Plecoglossus altivelis) and carp (Cyprinus carpio). Fish and Shellfish Immunology 19, 363–373. Shotts E.B. & Starliper C.E. (1999) Flavobacterium diseases: columnaris disease, cold-water disease and bacterial gill disease. In: Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections (ed. by P.T.K. Woo & D.W. Bruno), pp. 559–576. CABI Publishing, New York. Wakabayashi H. & Egusa S. (1974) Characteristics of myxobacteria associated with some freshwater fish diseases in Japan. Bulletin of the Japanese Society of Scientific Fisheries 40, 751–757. Wakabayashi H., Toyama T. & Iida T. (1994) A study on serotyping of Cytophaga psychrophila isolated from fishes in Japan. Fish Pathology 29, 101–104. Wiklund T. & Dalsgaard I. (2002) Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro. Fish and Shellfish Immunology 12, 141–153. Wiklund T. & Dalsgaard I. (2003) Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro. Fish and Shellfish Immunology 15, 387–395.
Journal of Fish Diseases 2015, 38, 107–112
Short communication The role of a specific antibody against Flavobacterium psychrophilum infection in ayu sweetfish, Plecoglossus altivelis altivelis (Temminck & Schlegel, 1846) G Kato1,2, K Suzuki3, T Sakai1, M Kawakami1, T Takano4, T Matsuyama4 and C Nakayasu1 1 2 3 4
Tamaki Laboratory, National Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan The Japan Society for the Promotion of Science, Tokyo, Japan Gunma Prefectural Fisheries Experimental Station, Gunma, Japan National Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan
Keywords: passive immunization, phagocytic rate, vaccine.
opsonization,
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD) and causes severe loss of aquaculture production of ayu sweetfish Plecoglossus altivelis altivelis (Temminck & Schlegel, 1846) in Japan (Wakabayashi, Toyama & Iida 1994). The characteristics of BCWD in ayu are different from those of BCWD in salmonid fishes. BCWD in ayu generally occurs when water temperature is 16 to 18 °C (Miwa & Nakayasu 2005), whereas that in salmonid fishes most frequently occurs in 4–10 °C (Shotts & Starliper 1999). Clinical signs of BCWD in ayu are anaemia and ulcerative lesions on the caudal peduncle (Iida & Mizokami 1996). However, erosion of fins and tail loss, which are typical clinical signs of BCWD in salmonid fishes (Nematollahi et al. 2003), are not observed in ayu. Ayu and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) that have recovered from BCWD are resistant to the reinfection (Kintsuji, Yamamoto & Ninomiya 2007; Lorenzen et al. 2010). High antibody titre is present in the serum of BCWD-recovered fish. LaFrentz et al. (2003) performed passive immunization of rainbow trout Correspondence C Nakayasu, Hiruta 224-1, Tamaki, Watarai, Mie 519-0423, Japan (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
107
using antiserum against F. psychrophilum and reported that a specific antibody has a critical protective role. In this study, passive immunization was performed to reveal the protective efficacy of a specific antibody against BCWD in ayu. The effects of opsonization by the antiserum on phagocytosis and of humoral bactericidal activity were also investigated. Flavobacterium psychrophilum strain GMA0330 (Arai et al. 2007) were cultured at 15 °C for 24 h in modified Cytophaga broth (Wakabayashi & Egusa 1974). Ayu (14.9 g average body weight) were injected (i.p.) with 2.2 9 105 colony-forming units (CFUs) F. psychrophilum. Fish were kept in 80-L tanks, and the water temperature was maintained at 15 °C. Serum was collected from the survivors at 3 weeks post-injection and was heat inactivated (45 °C for 30 min). Serum from PBS-injected fish was used as a negative control. Antibody titre (n = 5) was measured using a bacterial cell-binding ELISA with a mAb against ayu immunoglobulins (Kato et al. 2014). The antiserum was diluted to a concentration of 1:40. Flavobacterium psychrophilum (3.9 9 106 CFU) were suspended in the diluted serum and were incubated for 1 h at room temperature (RT). After washing with PBS, the mAb was added to the bacterial suspension and incubated for 1 h at
G Kato et al. The role of antibody in BCWD
Journal of Fish Diseases 2015, 38, 107–112
**
Absorbance (λ = 415 nm)
(a) 2.5
2
1.5
1
0.5
0
Cumulative survival (%)
(b)
Antiserum
Control serum
100 80 60 40 20 0 0
5
10
15
Days post-infection Antiserum
Control serum
PBS Figure 1 Antibody titre of the challenged fish and the control fish (a) and Kaplan–Meier survival curves for ayu passively immunized with antiserum, control serum or PBS (b). An asterisk indicates a statistically significant difference (P < 0.01).
injected (i.p.) with 50 lL pooled antiserum, pooled control serum or PBS. Twenty-four hours after passive immunization, fish were challenged with 1.3 9 105 CFU F. psychrophilum (i.p. injection). Fish were kept in 30-L tanks, and the water temperature was maintained at 18 °C. The numbers of dead fish were recorded for 15 days, and Kaplan–Meier survival curves were generated (Kaplan & Meier 1958). The log-rank test was used to compare the survival times for the passively immunized and the PBS-injected fish (Peto et al. 1977). The experiment was performed twice to confirm the reproducibility of the result, and the representative data are presented in this report. The pooled antiserum and the pooled control serum were each diluted in twofold dilutions (1:10–1:10240). Flavobacterium psychrophilum (3.9 9 106 CFU) were suspended in the diluted serum and then incubated for 1 h at 18 °C. The antibody titre of each dilution was measured using the bacterial cell-binding ELISA. Ayu immunoglobulins bound to the bacteria were detected using indirect immunofluorescence assay (IIFA; Kato et al. 2014). A one-way analysis of variance (ANOVA) was used to detect significant differences between treatments. Flavobacterium psychrophilum (3.9 9 106 CFU) were incubated with serially diluted antisera or with 1:10 diluted control serum as described above. Ayu (2.0 g average body weight, n = 30) were injected (i.p.) with 1.3 9 105 CFU of the serum-treated bacteria. The number of dead fish Table 1 Cumulative survival rate, calculated RPS value and P value by log-rank test in challenge test performed in this study Group
RT. A goat anti-mouse IgG antibody conjugated with HRP (Invitrogen) was added to the bacterial suspension and incubated for 30 min at RT. After washing, the bacterial cells were incubated with ABTS Peroxidase Substrate System (KPL) for 10 min at RT. Peroxidase Stop Solution (KPL) was added, and the absorbance (k = 415 nm) was measured. The mean value for five individuals was estimated, and statistically significant differences were determined using Student’s t-test. Equal volumes of the antiserum from the five fish were pooled and diluted to 1:4 with PBS. Ayu (2.0 g average body weight, n = 20) were Ó 2014 John Wiley & Sons Ltd
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Survival rate (survivors/total)
Passive immunization Antiserum 25.0% (15/20) Control serum 5.0% (19/20) PBS 5.0% (19/20) Challenge test with opsonized bacteria 1:10 60.0% (18/30) 1:40 37.7% (11/30) 1:160 40.0% (12/30) 1:640 23.3% (7/30) 1:2560 23.3% (7/30) 1:10240 16.7% (5/30) Control serum 20.0% (6/30) (1:10) NA, not applicable; NS, not significant. a Relative per cent survival (RPS) = {1 mortality (control)]} 9 100.
RPSa
Log-rank test
21.1 0 NA
P < 0.0001 NS NA
50.0 20.9 25.0 4.1 4.1 NA NA
P < 0.001 NS NS NS NS NS NA
[% mortality (treated)/%
G Kato et al. The role of antibody in BCWD
Journal of Fish Diseases 2015, 38, 107–112
Absorbance (λ = 415 nm)
(a) 2.5 2 1.5 1 0.5 0
Dilution Antiserum
(b)
Control serum
10
40
160
2560
10 240
Control
640
DAPI
FITC
Figure 2 Antibody titre of serially diluted antiserum. (a) Antibody titres for each dilution were measured using the bacterial cell-binding ELISA. (b) Ayu Igs binding to the surface of Flavobacterium psychrophilum was detected using immunofluorescence assay (IIFA). Arrowheads indicate agglutination and the Igs binding to the bacteria. Scale bar: 5 lm.
DAPI
FITC
was recorded for 20 days. Generation of the cumulative survival curve and the statistical analysis were performed as described above. Trunk kidney of uninfected ayu (4.3 g average body weight, n = 3) was teased and passed through a nylon mesh into RPMI-1640 (Life Technologies). Flavobacterium psychrophilum treated with the diluted antiserum were prepared as described above, added to 5.0 9 105 of the kidney cells and incubated at 18 °C for 30 min. The mixture was spread on a glass slide and stained with May–Grunwald–Giemsa stain. More than 1000 leukocytes (with or without phagocytized bacteria) were counted at random, and the phagocytic rate was Ó 2014 John Wiley & Sons Ltd
109
expressed as follows: phagocytic rate (%) = [number of leukocytes with phagocytized bacteria/number of observed leukocytes] 9 100. Serum was collected from uninfected fish (39.0 g average body weight, n = 3), as described above. Flavobacterium psychrophilum treated with diluted antiserum were prepared as described above and added to the uninfected fish serum without inactivation. The mixture was incubated at 18 °C for 6 h. The numbers of CFUs of the bacterial suspensions were counted by Miles– Misra method. Significant differences between treatments were assessed using a one-way ANOVA and Tukey’s post hoc test.
G Kato et al. The role of antibody in BCWD
Journal of Fish Diseases 2015, 38, 107–112
(a) 100
Cumulative survival (%)
80
60
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40
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2560 10 240 Control
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a
a
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The mean antibody titre for the survivors in the challenged group was significantly higher than that for the control group (P < 0.01, Fig. 1a). In Ó 2014 John Wiley & Sons Ltd
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Figure 3 Effects of opsonization of the bacteria on the host immune responses. Kaplan–Meier survival curves for ayu injected with opsonized bacteria (a). Phagocytic rate of kidney leukocytes against opsonized bacteria (b). CFUs of the opsonized bacteria after treatment with normal serum (c). Different letters represent significant differences between treatments (P < 0.05). The error bars represent the standard deviation of the mean.
passive immunization study, cumulative per cent survival for the antiserum group was 25%, whereas those for the control groups were 5%
Journal of Fish Diseases 2015, 38, 107–112
(Fig. 1b). Relative per cent survival (RPS) is shown in Table 1. The differences in survival times for the antiserum group and the two control groups were significantly different (P < 0.0001). The antiserum titre value was approximately equal to the control titre value at the 1:10240 (Fig. 2a). A microscopic examination revealed that agglutination occurred when the bacteria were treated with antiserum diluted to 1:10–1:160 (Fig. 2b, upper panels). The IIFA revealed that ayu immunoglobulins bound to bacterial surfaces at the same antiserum dilutions (Fig. 2b, lower panels). The values for cumulative per cent survival were higher in the 1:10, 1:40 and 1:160 antiserum groups compared with the control group (Fig. 3a). RPS values are shown in Table 1. The survival time of the 1:10 antiserum group was significantly longer, compared with the survival time of the control group (P < 0.001). Approximately 44% of the leukocytes phagocytized opsonized bacteria in the 1:10 antiserumtreated group, but the phagocytic rate was only 3% in the control group (Fig. 3b). No significant increase in phagocytic rate occurred in the 1:160to 1:10240-treated groups. There were 1.1 9 105 CFU viable F. psychrophilum in the control serum group, compared with 1.4 9 104 CFU viable bacteria in the 1:10 antiserum-treated group (Fig. 3c). Compared with the control, the bactericidal activity of the 1:10240 antiserum-treated group was not significantly different. The phagocytic rate increased after opsonization. Flavobacterium psychrophilum can be degraded in the phagosome after phagocytosis (Wiklund & Dalsgaard 2003). In mammals, pathogenic bacteria opsonized with IgG are trapped by the Fc receptor, which is located on the phagocytic cells. Fc receptors and corresponding signal pathways are also present in teleost fish, including ayu (Lu et al. 2013). Immunoglobulins also mediate the recognition between phagocytes and antigens (Secombes & Fletcher 1992). Neutrophils are abundant (60–80% of total cell count) in ayu kidney leukocytes (Moritomo et al. 2003) and display unusually high respiratory burst activities (Serada et al. 2005). These results suggest that opsonization with antibody and Fc receptordependent phagocytosis by neutrophils may be critical for the elimination of bacteria during BCWD in ayu. The complement system present in fish serum is essential for humoral defence against bacterial Ó 2014 John Wiley & Sons Ltd
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pathogens. In this study, opsonization stimulated the bactericidal activity of the humoral component. This result suggests that the classical complement pathway is also associated with the elimination of F. psychrophilum by the ayu immune system. In contrast, F. psychrophilum isolated from rainbow trout are resistant to the complement activity present in rainbow trout serum (Wiklund & Dalsgaard 2002). The resistance of pathogenic bacteria to serum components is mediated by the presence of LPS and a polysaccharide capsule (Boesen et al. 1999). A difference in F. psychrophilum serotypes has been reported (Wakabayashi et al. 1994; Izumi & Wakabayashi 1999). This difference may be important for the differences in resistance of each isolate against host serum components. In conclusion, specific antibody production may be important for immunity against F. psychrophilum in ayu, by accelerating the phagocytic and bactericidal activity of serum. Acknowledgement This work was supported in part by a Grant-inAid (Number 12J04812) for Fellows of the Japan Society for the Promotion of Science. Publication History Received: 21 February 2014 Revision received: 14 April 2014 Accepted: 16 April 2014
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