Development of the Antibody Response to a Vaccine during Growth of Juvenile Japanese Amberjack

Abstract

We investigated the antibody dynamics at different growth stages of Japanese amberjack Seriola quinqueradiata to determine the timing of first IgM production. Vaccination trials of juveniles were then conducted using a formalin-inactivated Vibrio anguillarum serotype J-O-3 vaccine to characterize the changes in vaccine-specific IgM and efficacy at each growth stage. In whole-body analyses, although total IgM was slightly detected at 1 day post-hatching (dph), it was undetectable until 15 dph and then increased from 23 to 57 dph. Levels of IgM specific to V. anguillarum J-O-3 were significantly elevated in fish vaccinated at and after 60 dph. Relative percentage survival of vaccinated fish was 52.6% and 100% at 51 dph and ≥61 dph, respectively. These results suggest that the IgM response to the vaccine developed between 51 and 60 dph (body weight: 0.45 ± 0.04 to 1.15 ± 0.06 g; standard length: 2.68 ± 0.09 to 3.90 ± 0.08 cm), coinciding with the increase in vaccine efficacy. These data also suggest that development of the humoral immunity was delayed from the initiation of IgM production.

Immunoglobulins (Igs), also known as antibodies, are glycoproteins exclusively produced by B-lymphocytes (B cells) in response to foreign antigens, including pathogens that invade the body. Teleost fish produce multiple Ig-heavy chain isotypes including IgM that is the most abundant isotype in the serum of teleost fish (Salinas et al., 2011). Igs and the immune responses they induce affect the efficacy of vaccines in fish. Passive immunization tests conducted in previous studies demonstrated that fish serum raised against formalin-inactivated vaccines, which utilize formalin-killed cells (FKCs) of particular pathogens exerts a protective effect against pathogen infection in several fish species (LaFrentz et al., 2002; Matsuyama et al., 2018; Ooyama et al., 2002; Pasnik et al., 2006). Furthermore, a correlation between the titer of pathogen-specific IgM in serum and protective immunity against the pathogen was demonstrated using FKC-vaccinated fish (LaFrentz et al., 2002; Munang’andu and Evensen, 2019). Thus, IgM in the blood plays a prominent role in vaccine-induced immunity in fish.

Studies of the ontogeny of IgM in several fish species showed that IgM-bearing cells initially appear a few days to 2 months after hatching (Scapigliati et al., 1999). This IgM is then thought to be secreted by these cells and then transported to the blood, where it is involved in systemic responses (Piazzon et al., 2016). In our previous study, we demonstrated that juvenile Japanese amberjack, Seriola quinqueradiata, expressed lower levels of IgM in the serum than mature fish (Matsuura et al., 2024).

Seriola species, including Japanese amberjack, greater amberjack S. dumerili, and yellowtail amberjack S. aureovittata represent a major component of the Japanese aquaculture industry, accounting for ≥50% of total Japanese finfish aquaculture production (Matsuura et al., 2019). Collectively, these species have also been designated an important export product in Japan due to increased overseas demand. Demand for hatchery-reared juveniles of Seriola species relative to wild-caught juveniles has recently increased in Japan in response to efforts to reduce dependence on wild fish and promote sustainable production. For hatchery-reared juveniles, earlier vaccination is needed before fish are transferred from land-based farms to outdoor cages in which the risk of contact with pathogens is higher. However, the minimum age of Seriola species at which antibody production specific to the vaccine and vaccine efficacy would be expected has not been determined.

In the present study, we investigated the timing of antibody-related immunity development by analyzing the dynamics of IgM production in juvenile Japanese amberjack. We then performed vaccine trials in fish at different stages of growth using Vibrio anguillarum serotype J-O-3, a pathogenic bacteria to juvenile fish, in order to confirm the stage at which Japanese amberjack exhibit a vaccine-specific antibody response. Changes in the production of antibodies specific to antigens of V. anguillarum during fish growth were examined using fish vaccinated with FKCs of V. anguillarum J-O-3 at each growth stage. Vaccine efficacy was also measured by experimentally infecting fish with the bacteria and examining the relationship between antibody titer and vaccine efficacy.

Materials and Methods

Ethics statement

All fish used for husbandry and sampling in the study were handled in accordance with the policy designated by the Institutional Animal Care and Use Committee of the Fisheries Technology Institute. All animal experiments were approved by the same committee (no. 22003).

Fish

Hatchery-reared juvenile Japanese amberjack were produced at Kamiura Field Station, Fisheries Technology Institute. Stock juveniles were reared in 60-kL tanks with running water at 20–23°C and aeration until used in experiments. Feeding began on 3 dph. Stock fish at 3–16 dph, 17–22 dph, 23–26 dph, 27–34 dph, and 35 dph or later were fed rotifer Brachionus plicatilis sp. complex, rotifer with brine shrimp Artemia sp., brine shrimp, brine shrimp with dry pellets, or dry pellets twice each day, respectively. Some stock fish were used for IgM quantification at 1, 8, 15, 23, 30, 37, 43, 51, 57, 60, 72, 81, and 92 dph. The fish were not fed on the day crude extracts of whole tissue or serum were prepared. The size of each fish, including body weight and standard length (SL), was recorded before preparation of samples (Table 1). Fish were used for vaccine trials at 51, 60, 72, 81, and 92 dph. Groups of vaccinated fish were maintained in different 30-L tanks in running water at 25°C with aeration and fed dry pellets twice each day, except on the day of vaccination or other experiments, including blood collection. The size of fish used in the trials was recorded before vaccination (Table 1). A subgroup of fish in each vaccination group was maintained in different 30-L tanks in running water at 25°C with aeration for further experimental infection tests. The fish were not fed on the day of experimental infection or the day after.

Table 1. Size of juvenile fish for samplings

Purpose: Preparation of crude extract derived from whole fish

dph	Body weight (mg [*] or g)	SL (mm)	Number of fish or pools of fish analyzed by IgM ELISA
1	ND	ND	6 (pool samples, 30 fish/pool)
8	ND	ND	4 (pool samples, 50 fish/pool)
15	3.95 ± 0.09*	5.57 ± 0.07	10
23	0.02 ± 0.01	7.51 ± 0.28	10
30	0.04 ± 0.00	14.35 ± 0.53	10
37	0.10 ± 0.01	21.23 ± 0.64	10
43	0.26 ± 0.02	26.70 ± 0.73	10
51	0.39 ± 0.05	32.00 ± 1.23	10
57	0.95 ± 0.08	38.90 ± 1.29	10

ND = not determined

 

Purpose: Serum preparation and vaccine trials

dph	Body weight (g)	SL (mm)	Number of fish examined in following experiments
			ELISA of total IgM before vaccination	ELISA of V. anguillurum –specific IgM	Experimental infection after vaccination
51	0.45 ± 0.04	26.83 ± 0.94	7	4 (initial) or 10 (Others)	20
60	1.15 ± 0.06	39.00 ± 0.80	10	10	20
72	3.97 ± 0.35	60.80 ± 2.12	10	10	20
81	7.26 ± 0.44	72.61 ± 1.07	10	10	20
92	13.82 ± 1.23	89.64 ± 1.84	10	10	20

Bacteria

Vibrio anguillarum V03-17 (serotype J-O-3) isolated from the kidney of diseased Japanese amberjack farmed in Ehime Prefecture, western Japan, was used in the study. The isolate was cultured in tryptic soy broth (BD) with 1.5% NaCl as described in the section below.

Vaccine trials

FKCs of V. anguillarum V03-17 isolate were used as the formalin-inactivated vaccine in trials in this study. Bacteria were cultured in tryptic soy broth with 1.5% NaCl overnight at an agitation rate of 130 rotations per minute at 25°C, followed by inactivation by incubation in 0.3% formalin overnight. The FKCs were washed 3 times with phosphate-buffered saline (PBS) by centrifugation at 2,000 ×g for 10 min at 4°C to remove formalin. Fifty microliters of the preparation containing 5.0 × 10⁷ colony forming units (CFU) of bacteria was injected intraperitoneally into 40 juvenile fish at 51, 60, 72, 81, and 92 dph, and the vaccinated fish were maintained for 3 weeks before further experiments, including serum preparation and experimental infection. The same volume of PBS instead of FKCs was injected into fish used as a control vaccination group. All fish were anesthetized using 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation) before vaccination.

Preparation of crude extract derived from whole fish

Crude extracts of whole fish at 1, 8, 15, 23, 30, 37, 43, 51, and 57 dph were prepared according to the method described by Castillo et al. (1993). Naïve juvenile fish were euthanized using 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation) and then homogenized using a Potter-Elehjem tissue grinder on ice in PBS supplemented with protease inhibitor cocktail (cOmplete, Sigma-Aldrich Japan). The homogenates were centrifuged at 8,400 ×g for 5 min at 4°C, and the supernatant was harvested. Extracts of fish at 1 and 8 dph were prepared from pools of 30 and 50 fish, respectively, because the body weight of the individual fish was unmeasurable.

Serum preparation

Serum samples were prepared from naïve fish at 51, 60, 72, 81, and 92 dph, in addition to vaccinated fish. Samples could not be prepared for fish younger than 51 dph due to the difficulty of blood collection. For vaccinated fish, serum preparation was carried out 3 weeks after vaccination. These fish were anesthetized using 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation), and blood was collected using capillary tubes from the caudal peduncle, which was severed using a scalpel (FEATHER Safety Razor). The collected blood was allowed to stand for 1 h at room temperature to promote coagulation and then centrifuged at 1,000 ×g for 5 min at 4°C. The serum was harvested from the resulting supernatant and used for further analyses.

ELISA for IgM quantification

IgM in whole tissue of fish or serum was quantified using a sandwich ELISA (sensitivity: <0.12 ng/mL (Matsuura et al., 2024)). Briefly, appropriately diluted crude extracts of tissue or serum were loaded into wells of an ELISA plate (Nunc MaxiSorp plate, ThermoFisher Scientific Japan) coated with 1 μg/mL of polyclonal antibody against IgM. A peroxidase-conjugated monoclonal antibody (mAb) against IgM was then applied. The amount of IgM contained in the samples was determined based on a calibration curve prepared using a 2-fold serial dilution of purified IgM.

ELISA for measuring vaccine-specific IgM titer

Levels of vaccine-specific IgM were measured using a direct ELISA. To prepare the immobilization antigen for the ELISA, V. anguillarum V03-17 cells grown on tryptic soy agar plates were sonicated to extract bacterial antigens, which were suspended in PBS at 10 mg/mL. The sonicated bacteria were centrifuged at 20,000 ×g for 5 min, and the resulting supernatant was diluted 100-fold in PBS and applied to the wells of a Nunc MaxiSorp plate (ThermoFisher Scientific Japan) and incubated at 4°C overnight. The antigen solution was then removed, and the wells were blocked with 5% skim milk (FUJIFILM Wako Pure Chemical Corporation) in Tris-buffered saline with 0.05% Tween20 (TBS-T) for 1 h at room temperature. After removal of milk from the wells, serum samples diluted 20-fold in 1% skim milk were added to the wells, and the plate was incubated for 1 h at room temperature. The plate was then washed five times with TBS-T, and peroxidase-conjugated mAb against Japanese amberjack IgM (Matsuura et al., 2024) pre-diluted to 0.25 μg/mL in 1% skim milk was loaded onto the plate as the detection antibody. The plate was incubated for 1 h at room temperature and then washed five times with the same buffer. Substrate solution including 3,3′,5,5′-tetramethylbenzidine (Seracare Life Sciences) was finally loaded onto the plate and incubated for 10 min at room temperature for color development. The colorimetric signals were measured at 450 nm using a microplate reader (PerkinElmer) after stopping the enzyme reaction by the addition of 1 M phosphoric acid.

Experimental infection tests

Twenty fish vaccinated at each growth stage were experimentally infected with V. anguillarum V03-17 to evaluate vaccine efficacy. Colonies of bacteria grown on a tryptic soy agar plate (BD) for 24 h were suspended in PBS at 10 mg/mL and then diluted 100-fold in the same buffer; the diluted bacterial suspension was then injected intraperitoneally into fish of each group. Cumulative mortality among the infected fish was recorded daily for 1 week. The number of CFU of bacteria used for each test was determined before the injection. Vaccine efficacy was estimated based on the relative percentage survival (RPS) calculated according to the method of Amend (1981), and differences in survival time were analyzed using the log-rank test (Bland and Altman, 2004).

Statistical analyses

Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc.). Data for body weight, SL, and amount of IgM are shown as mean ± standard error of the mean (SEM).

Results

Dynamics of IgM production in different growth stages in the whole body of naïve juvenile Japanese amberjack

No IgM was detected in pooled samples of fish at 1 and 8 dph, except for one sample at 1 dph, which showed 0.12 μg IgM/g fish. No IgM was detected in any fish at 15 dph, but some fish at 23 and 30 dph exhibited trace amounts of IgM, at 0.01 ± 0.01 and 0.03 ± 0.01 μg IgM/g fish, respectively. The IgM level rapidly increased as the fish grew, with values of 0.27 ± 0.03, 0.94 ± 0.21, 2.14 ± 0.20, and 3.18 ± 0.56 μg IgM/g fish at 37, 43, 51, and 57 dph, respectively (Fig. 1A). Moderate positive correlations between IgM level and both body weight and SL were observed in fish from 1 to 57 dph (Fig. 1B and 1C, p < 0.0001, Pearson correlation coefficient).

Fig. 1. Dynamics of IgM production in whole fish during growth of Japanese amberjack.

(A) Change in total IgM level in crude extract of whole naïve fish. IgM levels were measured using individual fish at 15, 23, 30, 37, 43, 51, and 57 dph. For fish at 1 and 8 dph, the levels were measured from pools of 30 and 50 individual fish, respectively, because the body weight of the individual fish was unmeasurable. IgM levels were normalized to the body weight of the individual or total weight of the pooled sample. Statistical significance was determined using one-way ANOVA, followed by Dunnett’s multiple comparisons test (****p < 0.0001); n = 6 (1 dph), 4 (8 dph), 10 (15, 23, 30, 37, 43, 51, 57 dph). Mean ± SEM. (B, C) Correlation between IgM level and body weight (B) or SL (C) of the fish. Data were evaluated using Pearson correlation coefficient and linear regression analyses.

Dynamics of serum IgM level in naïve juvenile Japanese amberjack at different growth stages

Serum IgM levels were measured in naïve juvenile fish to investigate the development of IgM-related systemic immunity at different growth stages. The level remained low until 72 dph and then dramatically increased from 81 dph, with values of 169.8 ± 24.2 and 245.2 ± 31.2 μg/mL at 81 and 92 dph, respectively (Fig. 2A). Moderate positive correlations between IgM level and both body weight and SL were observed in fish from 51 to 92 dph (Fig. 2B and 2C, p < 0.0001, Pearson correlation coefficient).

Fig. 2. Dynamics of IgM level in the serum during growth of Japanese amberjack.

(A) Serum IgM level in naïve fish at 51, 60, 72, 81, and 92 dph. Statistical significance was determined using one-way ANOVA, followed by Dunnett’s multiple comparisons test (***p < 0.001; ****p < 0.0001); n = 7 (51 dph), 10 (others). Mean ± SEM. (B, C) Correlation between the IgM level and body weight (B) or SL (C) of the fish. Data were evaluated using Pearson correlation coefficient and linear regression analyses.

Effect of growth on the production of vaccine-specific IgM in juvenile Japanese amberjack

Levels of serum IgM specific to V. anguillarum (J-O-3) in fish vaccinated at 60 dph or later increased significantly after vaccination, whereas specific IgM levels increased only slightly in fish vaccinated at 51 dph (p = 0.3562 vs PBS control). The increase in IgM level was most remarkable in fish vaccinated at 81 or 92 dph. The levels in all naïve fish before vaccination (initial) and fish in the PBS control group were low, clearly indicating that the increase was the result of vaccination (Fig. 3).

Fig. 3. Induction of V. anguillurum–specific IgM in serum of Japanese amberjack juveniles vaccinated at different growth stages.

Serum was collected from fish vaccinated with V. anguillurum FKCs or PBS control fish for 3 weeks and analyzed. Serum collected from fish before vaccination was also analyzed as a control (initial). Statistical significance was determined using two-way ANOVA, followed by Tukey’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001); n = 4 (initial samples prepared from fish at 51 dph) or 10 (other samples except for the initial samples at 51 dph). Mean ± SEM.

Effect of fish growth on vaccine protection a gainst vibriosis

All fish vaccinated with FKCs at 60, 72, 81, and 92 dph survived experimental infection, whereas fish vaccinated at 51 dph were only partially protected. The survival time of fish vaccinated at 51, 60, 72, 81, and 92 dph was significantly higher than that of control fish (p < 0.0001, Fig. 4A-E). The RPS of fish vaccinated on or after 60 dph was 100%, whereas fish vaccinated at 51 dph exhibited an RPS rate of 52.6%.

Fig. 4. Change in the protective effect of the vaccine against V. anguillurum in Japanese amberjack juveniles at different growth stages.

Vaccinated fish (V. anguillurum FKC [●]; PBS control [○]) at 51 (A), 60 (B), 72 (C), 81 (D), or 92 (E) dph were experimentally infected with 1.8 × 10⁶, 1.5 × 10⁵, 2.4 × 10⁶, 1.5 × 10⁶, or 1.5 × 10⁶ CFU of live V. anguillarum, respectively, and the cumulative mortality of the infected fish was recorded each day for a week (n = 20).

No abnormal behaviors or deaths caused by the vaccine were observed in any of the vaccination groups during the observation period, indicating the vaccine did not cause any adverse reactions that could affect the experimental infection tests.

Discussion

In the present study, we elucidated the timing of the vaccine-related immune response mediated by IgM in juvenile Japanese amberjack. The timing of the response was delayed a few weeks from the time IgM production was first confirmed. This is the first study to demonstrate the exact stage of fish growth during which the immune response to a vaccine develops.

We demonstrated that the maturity of the fish affects the efficacy of the vaccine relative to the titer of the V. anguillurum–specific antibody. The effects of vaccination in terms of both the induction of specific IgM and development of full protection were observed in fish vaccinated on or after 60 dph. The IgM response to the vaccine that conferred protection appeared to develop between 51 and 60 dph (body weight: 0.45 ± 0.04 to 1.15 ± 0.06 g, SL: 2.68 ± 0.09 to 3.90 ± 0.08 cm) and became stronger as the age of fish at the time of vaccination increased. Vaccine trials in small juvenile fish have been reported in previous studies, although the involvement of systemic antibody reactions in conferring protection has not been demonstrated. Gudmundsdottir et al. (2009) demonstrated that an immersion vaccine for V. anguillarum did not enhance IgM specific to V. anguillarum antigens in cod weighing 1.2 ± 0.4 g (40–50 dph, as estimated based on the results of another study (Rosenlund and Halldórsson, 2007)). Another group (Angelidis et al., 2006) reported that an immersion vaccine only weakly enhanced the titer of agglutinating antibodies against the target bacteria, even in more-developed juvenile sea bass, Dicentrarchus labrax, weighing 3.3 ± 0.2 g (~117 dph, as estimated based on the results of another study (dos Santos et al., 2000)). Except for our present study, there are no reports in the literature of studies examining antibody responses to vaccines administered via the intraperitoneal route in juvenile fish. Further studies in other fish species are thus needed to confirm the timing of the development of vaccine-related immune responses in teleosts.

IgM production began in juvenile Japanese amberjack after 23–30 dph (body weight: 3.95 ± 0.09 to 21.14 ± 9.73 mg, SL: 0.56 ± 0.01 to 0.75 ± 0.03 cm). A significant increase in the titer of V. anguillurum–specific IgM in the serum and full protection due to vaccination were confirmed in fish vaccinated on or after 60 dph, as mentioned above. These discrepancies in timing suggested that development of the immune response mediated by circulating IgM was delayed a few weeks from the time at which IgM production first began.

We also demonstrated that both IgM production and the titer of circulating IgM in the blood increased as the fish grew. The results of our analysis of IgM production dynamics agree with those of a previous study in sea bass, which showed that whole-body total IgM begins to increase with fish growth at 35 dph (Breuil et al., 1997). Other studies examining the ontogeny of humoral immunity in various fish species have focused on the timing of initial IgM production. Magnadóttir et al. (2005) reported that IgM production is generally initiated earlier in freshwater fish than saltwater fish. IgM production begins in rainbow trout, Oncorhynchus mykiss and channel catfish, Ictalurus punctatus at 1 week after hatching (Castillo et al., 1993; Petrie-Hanson and Ainsworth, 2001). In saltwater fish, IgM production is delayed several weeks (2–3 weeks in sea bass (Breuil et al., 1997) and 8–9 weeks in Atlantic cod, Gadus morhua (Schrøder et al., 1998)). These reports agree with our study in Japanese amberjack (23 dph). In contrast to the initiation of IgM production, the IgM level in serum began to increase much later (81 dph), as observed in our previous study (Matsuura et al., 2024). Aside from our study, no other studies have reported the dynamics of serum IgM associated with growth in juvenile fish; thus, further research in other species is required to confirm the timing of systemic humoral immune response development in teleosts.

A trace amount of IgM was detected in one pooled sample of juveniles at 1 dph, which was thought to be derived from maternal IgM transferred to the offspring. Transfer of the maternal IgM has been reported in both freshwater and saltwater fish species (Mozambique tilapia, Oreochromis mossambicus (Takemura, 1993); rainbow trout (Castillo et al., 1993), red seabream, Pagrus major (Kanlis et al., 1995); and sea bass (Breuil et al., 1997)). Maternal IgM is thought to disappear as the yolk absorption process is completed (Swain and Nayak, 2009); thus, further analyses using samples at earlier stages of development, including fertilized eggs, are required.

Although antibodies play a crucial role in systemic immune responses to eliminate pathogens, few studies have investigated the dynamics of antibody responses in juvenile fish. This is the first report describing the development of the IgM response to a vaccine in juvenile fish. Vaccines for Japanese amberjack in Japan are currently only certified for fish that weigh ≥10 g due to a lack of information regarding development of the immune response and the efficacy of vaccines in juvenile fish (Matsuura et al., 2019). Our present study provides critical information that will help elucidate the appropriate timing for effective vaccination and makes an important contribution to disease control in juvenile fish.

Acknowledgements

We thank all staff members of the Marine Fisheries Research and Development Center for providing Japanese amberjack juveniles. We also thank Dr. Tomokazu Itano, Ehime Prefectural Fish Disease Control Center, Ehime, Japan, for providing V. anguillarum V03-17. This study was supported by the Fisheries Technology Institute, Japan Fisheries Research and Education Agency, and funded by the Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry, and Fisheries of Japan.

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