- Email: [email protected]
Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda
Accepted Manuscript Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda Hidehiro Kondo, Seangmin Chung, Eriko Hirosawa, Ikuo Hirono PII:
S1050-4648(18)30615-6
DOI:
10.1016/j.fsi.2018.09.071
Reference:
YFSIM 5592
To appear in:
Fish and Shellfish Immunology
Received Date: 30 May 2018 Revised Date:
19 September 2018
Accepted Date: 25 September 2018
Please cite this article as: Kondo H, Chung S, Hirosawa E, Hirono I, Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda, Fish and Shellfish Immunology (2018), doi: https://doi.org/10.1016/j.fsi.2018.09.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Adjuvant effects on protection and immune response of Japanese
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flounder immunized by the formalin-killed cells of Edwardsiella tarda
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Hidehiro Kondo*, Seangmin Chung*, Eriko Hirosawa, Ikuo Hirono
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*, equally contributed.
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Laboratory of Genome Science, Graduate School of Tokyo University of Marine
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Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan
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Corresponding author: Hidehiro Kondo
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TEL: +81-3-5463-0174. FAX: +81-3-5463-0174.
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E-mail: [email protected]
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Address: Laboratory of genome science, Tokyo University of Marine Science and
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Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan
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Abstract We evaluated the effects of Freund’s adjuvants (FCA/FIA) on protection and
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immune response of Japanese flounder Paralichthys olivaceus immunized by the
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formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA
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did not confer protection against the challenge, while they significantly induced higher
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antibody titers than that of FKC only. The suppression of FKC-dependent induction of
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interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression
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profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after
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FKC+FCA or +FIA. These results suggest that the mineral oil in Freund’s adjuvants
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might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.
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Introduction Edwardsiellosis causes economic losses in many cultured fish species in Japan
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including Japanese eel Aguilla japonica (Egusa et al. 1976), Japanese flounder
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Paralichthys olivaceus (Nakatsugawa et al. 1983) and Red sea bream Pagrus major
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(Yasunaga et al. 1982). The causative agent, Edwardsiella tarda, is an intracellular
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pathogen. The bacteria can survive in phagocytes and resist against bactericidal
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activities of the host defense system (Miyazaki and Kaige 1985, Iida and Wakabayashi
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1993, Srinivasa Rao et al. 2001).
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The formalin-killed cells (FKC) vaccine does not protect Japanese flounder against
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edwardsiellosis. Mekuchi et al. (1995) reported that the mortality of the fish immunized
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with the FKC, extracellular products or intracellular components of the bacteria was
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similar to the mortality of unimmunized fish. The relative percent survivals (RPSs) of
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Japanese flounder vaccinated with an attenuated strain or a natural avirulent isolate
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were significantly high (Cheng et al. 2010, Sun et al. 2010). On the other hand,
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vaccination with five avirulent strains provided only limited protection (Takano et al.
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2010). For example, the RPS obtained with one of these strains was only 45 %.
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Therefore, the effectiveness of a vaccine can strongly depend on the strain. Moreover,
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some subunit- or DNA vaccines against bacteria provided protection against bacterial
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challenge (Park et al. 2012).
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In aquaculture, vaccine adjuvants can modify the immune responses of fish. They
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may enhance antibody responses and provide a long-lasting protection against diseases.
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Freund's complete/incomplete adjuvants (FCA/FIA) have been extensively used in the
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immunological studies, in which they have been shown to stimulate both humoral and
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cell-mediated adaptive immune responses (Tafalla et al. 2013). FCA is a moisture of a
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mineral oil with heat-killed Mycobacteria cells, while FIA lacks the Mycobacteria cells.
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In E. tarda infection in Japanese flounder, a recombinant protein of major antigen Et49
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combined with FIA showed an RPS of 81 %, although the antigen by itself had an RPS
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of only 34 % (Jiao et al. 2010). In contrast, FCA/FIA had only limited effects in other
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fish species (Tafalla et al. 2013).
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In order to develop an effective vaccine by using adjuvants, it is necessary to
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understand how the adjuvants modulate fish immune responses. Here we elucidated the
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effects of the FCA/FIA on protection and immune response of Japanese flounder
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immunized by the E. tarda FKC. The adjuvants strongly enhanced antibody responses 2
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weeks post infection (w.p.i.). A microarray analysis revealed that FCA/FIA suppressed
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the upregulation of interferon-γ mRNA levels.
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Materials and methods
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Fish sampling and challenge test
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Fish, average total length of 12 cm, were intraperitoneally injected with phosphate
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buffered saline (PBS), 1 x 109 cells of FKC of E. tarda (strain NUS806), and 1 x 109
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cells of FKC mixed with FCA (FKC+FCA) or that with FIA (FKC+FIA). The injected
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fish were reared at 18 °C, and then spleen was sampled at 6 h and on 3 d after
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immunization. The blood was taken from the fish at 2 w.p.i. to be assessed by ELISA.
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Ten of the fish were intraperitoneally challenged with 1.5 x 105 cells of E. tarda at 4
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weeks after immunization and mortality was recorded.
72 Antibody titration using ELISA
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The antibody activities were measured by the method of Taechavasonyoo et al.
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(2013). Briefly, the ELISA plate (Thermo) was prepared by placing 109 cells of the FKC
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in 100 µl per well and incubated at 4 ºC overnight. The wells were washed three times
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with T-PBS (0.05% Tween 20 in PBS), covered with 100 µl of blocking solution (3%
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Skim milk) at room temperature for 2 h, washed three times with T-PBS, treated with
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100 µl of primary fish antisera diluted 20-times with PBS, incubated at room
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temperature for 1 h, washed three times with T-PBS, reacted with 1:3000 of
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anti-Japanese flounder IgM rabbit antiserum (Taechavasonyoo et al. 2013), washed
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three times with T-PBS, incubated with 1:1000 of anti-rabbit IgG conjugated with
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alkaline phosphatase (Sigma) to detect the rabbit IgG, washed five times, incubated
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with phosphatase substrate at room temperature for 30 min, and mixed with 50 µl of 3M
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NaOH to stop the reaction. The absorbance at 405 nm was measured with Multiskan FC
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Microplate Photometer (Thermo Scientific, Japan).
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Gene expression profiling by microarray
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Gene expression was profiled by the method of Kaneshige et al. (2016). Total RNA
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was extracted from spleen samples (n=3) using RNAiso Plus (Takara Bio, Japan) and
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purified with an RNeasy® Mini Kit (Qiagen, USA). RNA quality was evaluated using a
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2200 TapeStation (Agilent, USA). The microarray was analyzed using three samples in
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each group, and each sample was selected based on RNA quality. cDNA was
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synthesized from 200 ng of RNA and labeled with cyanine 3-CTP using a one-color
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microarray-based gene expression analysis following instructions of the manufacturer
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(Agilent, USA). The labeled cDNA was subsequently hybridized at 65 °C for 17 h.
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Microarrays were scanned with an Agilent G2565CA Microarray Scanner, and the
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images obtained from scanning were analyzed with Feature Extraction Software
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v9.5.3.1 (Agilent, USA). The data were normalized and analyzed using limma (Ritchie
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et al. 2015). The fold-change differences were calculated against the average of those
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before treatments. Genes with a fold change of at least 8.0 and p value less than 1e-5
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were considered to be differentially expressed.
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IFNγ and IL-12p35 genes expression profiling by qPCR
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The primers for the IFNγ, IL-12p35 and elongation factor 1 alpha (EF-1α) genes
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are shown in Supplemental Table. Total RNA was extracted from the spleen of each
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sample (n=5) using RNAiso (Takara, Japan) and 2 µg of total RNA was used for cDNA
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synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems,
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USA) according to the manufacturer's instructions. The cDNA samples were diluted
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5-times with distilled water. qRT-PCR was performed using 5 µl cDNA in a
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THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan) using an ABI7300 Real-time
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PCR system (Applied Biosystems, USA). The expression levels of the target genes were
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normalized to the expression level of an internal control gene (EF-1α) and the relative
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mRNA levels were calculated by the 2-∆∆Ct method (Livak and Schmittgen, 2001), and
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standardized to the average value of the PBS-injected group on the corresponding
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sampling points. The significance of differences between values on the standard and the
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others were evaluated by ANOVA followed by Dunnett’s post hoc test.
118 Results and Discussion
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Effects of Freund’s adjuvants on FKC vaccine
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Fish mortality was observed on day 4 after the challenge (Fig. 1A). All of the fish
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immunized with FKC+FCA died by 7 days after the challenge and all of the fish
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immunized with +FIA died by 6 days after the challenge. Unimmunized fish and fish
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immunized with FKC survived longer than those immunized with FKC+FCA and +FIA.
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Jiao et al. (2010) reported that the relative percent survival (RPS) of fish immunized
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with an antigen with FIA was 81 %, and that the RPS of fish immunized with only the
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antigen was 34 %. Tang et al (2010) also claimed that the antigens mixed with FIA
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showed higher RPS than the controls. In this study, we used FKC but not certain
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antigens, and thus the immune-response after the vaccination might be different from
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the response of those immunized with antigens mixed with FIA.
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Effects of Freund’s adjuvants on the antibody titer
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Antibody levels in fish immunized with FKC, FKC+FCA or FKC+FIA were
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significantly higher than those in the PBS group (Fig. 1B). Moreover, the FKC+FCA
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and FKC+FIA groups showed higher antibody levels than the FKC group. Although the
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antibody levels in the fish immunized with FKC+FCA and +FIA were higher than those
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in the other groups, these fish were susceptible to the bacterial challenge (see Fig. 1A).
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The specific antibody, which opsonize the bacteria, enhance adhesion of E. tarda to
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phagocytes, where the bacteria is able to survive and replicate (Srinivasa Rao et al.
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2001). Therefore, the specific antibodies elicited by the FKC with the adjuvants did not
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confer to the protection against the bacteria.
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Gene expression profiles after the vaccination
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The gene expression profiles in the spleen were compared between fish immunized
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with FKC+FCA and fish immunized with only FKC. The microarray data were
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deposited into the gene expression omnibus database under accession number
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GSE111115. In the samples at 6 h after FKC immunization, the intensities of 222 spots
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were at least 8- fold higher (p < 105) than they were before immunization, while in the
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samples at 6 h after FKC+FCA immunization, the intensities of 213 spots were at least
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8- fold higher. Similarly, the intensities of 214 spots were higher at 3 d after
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immunization. The mRNA levels of some cytokines (CC chemokine, interleukin (IL)-1β
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and IL-6) increased at 6 h in the FKC immunized group (Table 1). Almost all of the
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spots showed similar patterns between the FKC and FKC+FCA immunization (data not
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shown). In Japanese flounder, the genes induced by the injection of certain bacterial
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FKC were not significantly different, while the magnitude of up- or down regulation
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was distinct between the bacterial species (Kondo et al. 2014). Although mycobacteria
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cell components in FCA have been thought to influence gene expression in mammals
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(Behr et al. 2015, Ishikawa et al. 2017), they may have limited effects in fish.
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Only the intensities of the spots corresponding to interferon γ (IFNγ) were
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significantly higher in the FKC-immunized fish, but they were not significantly
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different in the FKC+FCA group (Table 1). It should be noted that only the IFNγ gene
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was differently regulated between FKC and FKC+FCA groups. The IFNγ mRNA level
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at 6 h after FKC immunization was significantly higher than it was in the other groups,
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while the levels at on 3d were not significantly changed (Fig. 1C). The IFNγ mRNA
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levels at 6 h also did not increase in FKC+FIA group. Therefore, regulation of the IFNγ
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gene expression might be caused by mineral oil forming an emulsion but not by the
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cellular components of mycobacteria.
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In mammals, interleukin (IL)-12 upregulates IFNγ transcription. IL-12 is an
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inflammatory cytokine, composed of p35 and p40 subunits (Watford et al. 2003,
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Zundler and Neurath 2015). Because the microarray did not have spots for the IL-12
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subunits, we further investigated changes IL-12p35 subunit mRNA levels by qPCR. As
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in the case of IFNγ, the IL-12p35 mRNA level at 6 h after FKC immunization was
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significantly higher than the other groups (Fig. 1D). Interestingly, at 3 d, the IL-12p35
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mRNA level was significantly lower in the FKC+FCA-immunized fish than in the
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control. In the amberjack, FKC vaccine with recombinant IL-12 showed effective
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against the intracellular bacterium Nocardia seriolae (Matsumoto et al. 2017). IL-12
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and IFNγ regulate the balance between the Th1 and Th2 responses, which act against
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intracellular bacteria and regulate antibody production, respectively (Watford et al. 2003,
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Zundler and Neurath, 2015), and induce cell mediated immunity (Matsumoto et al.
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2017). Therefore, the fish immunized with FKC+FCA/FIA, which showing lower IL-12
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and IFNγ mRNA levels might show lower cell mediated immune responce. In conclusion, adding Freund's adjuvants to the vaccines resulted in higher antibody
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titers and, and suppress the upregulation of IL-12 and IFNγ mRNA levels. Our data
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raise the possibility that the signaling pathways involved in IL-12 and IFNγ induction
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might be distinct from the other inflammatory pathways. Because IL-12 and IFNγ are
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important in the Th1 response, further studies are needed to characterize the signaling
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pathways regulating IL-12 and IFNγ expression to develop effective vaccines against
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intracellular bacteria.
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This research was supported in part by grants from JSPS KAKENHI Grant Number
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Acknowledgements
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Legends for figures
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Figure 1 (A) Cumulative survival of vaccinated fish after the challenge with E. tarda.
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(B) Antibody titers at 2 weeks after vaccination with FKC, FKC+FCA or FKC+FIA.
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Serum samples were collected from 5 fish/group, diluted 1:20, and then analyzed by
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ELISA. Each point represents an individual fish and black bar indicate the average of
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each group. Different letters represent significant differences as determined by Tukey's
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HSD (P < 0.05). (C, D) Expressions of IFNγ (C) and IL-12p35 (D) genes in the spleen
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from 3 fish/group at 6 h and on 3 d after PBS, FKC, FKC+FCA and FKC+FIA injection.
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Expression is expressed as Log2 values, relative to the average of the PBS group. Each
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point represents an individual fish and black bar indicate the average of each group.
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Asterisk indicates a significance difference by one-way ANOVA (P < 0.05).
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FKC 6h Product name
CUST_12226_PI426483990
AB427185.1
CUST_12932_PI426483990
a
CUST_351_PI426483993 CUST_48_PI426483993
b
CUST_49_PI426483993
b
a
Log2FC
P_value
CC chemokine-like
4.8
6.1E-06
AB427185.1
CC chemokine-like
6.4
3.3E-06
AB427185.1
CC chemokine-like
6.0
BAG50577.1
Interferon gamma
FKC 3d
FKC+FCA 3d
Log2FC
P value
Log2FC
P value
Log2FC
P value
4.7
7.6E-06
1.4
9.4E-02
1.6
9.9E-03
5.9
6.8E-06
2.4
1.8E-02
1.6
9.9E-03
6.2E-05
5.5
1.5E-04
1.8
1.8E-01
2.1
9.2E-03
6.4
3.9E-07
1.9
1.6E-02
2.1
8.8E-03
2.6
4.1E-02
5.9
3.1E-06
1.3
1.7E-01
1.3
1.9E-01
4.2
1.3E-03
5.6
4.3E-06
1.2
2.0E-01
1.2
1.9E-01
3.2
7.2E-03
5.8
3.7E-05
5.6
5.5E-05
2.7
2.3E-02
2.6
4.1E-02
7.4
5.9E-06
7.3
6.8E-06
3.9
2.2E-03
0.9
1.8E-01
4.1
4.9E-06
2.6
4.5E-04
0.6
3.3E-01
3.1
3.3E-04
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Acc. No. a
FKC+FCA 6h
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Table List of cytokines up-regulated after the treatments
Interferon gamma
CUST_9251_PI426483990
BAG50577.1
Interferon gamma
CUST_7075_PI426483990
c
AB720983.1
Interleukin 1 beta
CUST_9569_PI426483990
c
AB720983.1
Interleukin 1 beta
DQ267937.1
Interleukin 6
BAF91496.1
M17 homologue
4.4
1.7E-06
5.3
2.7E-07
0.5
3.5E-01
2.2
1.6E-02
BAF91496.1
M17 homologue
4.6
7.4E-07
5.6
1.2E-07
0.9
1.8E-01
2.5
5.2E-03
CUST_290_PI426483993 CUST_10972_PI426483990 CUST_310_PI426483993
d
d
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BAG50577.1 b
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Probes indicated by the same alphabet encode the same gene products. The values were compared with those before the treatments, and the Log2
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transformed fold-change values (Log2FC) and P value were represented. both Log2FC > 3.0 and P value < 1.0E-05 were indicated by gray.
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(A)
(B)
100
FKC+FIA *
60 50 30 10 0 1
2
3
4
5
6
7
8
Days post challenge
(C)
9
10
a
FKC +FCA
FKC +FIA
b
1.0
c
c
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a
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Absorbance (405 nm)
FKC+FCA *
0.5
0.0
Before PBS injection
FKC
(D)
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*
* p < 0.05
8 6
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4 2
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0
PBS FKC
FKC FKC +FCA +FIA
6h
PBS FKC FKC FKC +FCA +FIA
3d
Relative IL-12p35 mRNA levels (Log 2)
Survival rate (%)
FKC
80
20
Relative IFNγ mRNA levels (Log 2)
2.0
PBS
90
-2
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Fig. 1
5
* p < 0.05 4
*
3 2 1 0
*
-1 -2 PBS FKC
FKC FKC +FCA +FIA
6h
PBS FKC FKC FKC +FCA +FIA
3d
ACCEPTED MANUSCRIPT Highlights •
Effects of adjuvants (FCA, FIA) on protection and immune response of Japanese flounder were evaluated. Freund’s adjuvants enhanced antibody responses but did not confer protection.
•
Freund’s adjuvants suppressed the induction of IFNγ and IL12p35 mRNA expression
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after the FKC vaccination.
S1050-4648(18)30615-6
DOI:
10.1016/j.fsi.2018.09.071
Reference:
YFSIM 5592
To appear in:
Fish and Shellfish Immunology
Received Date: 30 May 2018 Revised Date:
19 September 2018
Accepted Date: 25 September 2018
Please cite this article as: Kondo H, Chung S, Hirosawa E, Hirono I, Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda, Fish and Shellfish Immunology (2018), doi: https://doi.org/10.1016/j.fsi.2018.09.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Adjuvant effects on protection and immune response of Japanese
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flounder immunized by the formalin-killed cells of Edwardsiella tarda
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Hidehiro Kondo*, Seangmin Chung*, Eriko Hirosawa, Ikuo Hirono
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*, equally contributed.
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Laboratory of Genome Science, Graduate School of Tokyo University of Marine
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Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan
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Corresponding author: Hidehiro Kondo
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TEL: +81-3-5463-0174. FAX: +81-3-5463-0174.
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E-mail: [email protected]
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Address: Laboratory of genome science, Tokyo University of Marine Science and
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Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan
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Abstract We evaluated the effects of Freund’s adjuvants (FCA/FIA) on protection and
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immune response of Japanese flounder Paralichthys olivaceus immunized by the
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formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA
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did not confer protection against the challenge, while they significantly induced higher
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antibody titers than that of FKC only. The suppression of FKC-dependent induction of
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interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression
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profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after
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FKC+FCA or +FIA. These results suggest that the mineral oil in Freund’s adjuvants
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might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.
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Introduction Edwardsiellosis causes economic losses in many cultured fish species in Japan
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including Japanese eel Aguilla japonica (Egusa et al. 1976), Japanese flounder
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Paralichthys olivaceus (Nakatsugawa et al. 1983) and Red sea bream Pagrus major
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(Yasunaga et al. 1982). The causative agent, Edwardsiella tarda, is an intracellular
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pathogen. The bacteria can survive in phagocytes and resist against bactericidal
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activities of the host defense system (Miyazaki and Kaige 1985, Iida and Wakabayashi
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1993, Srinivasa Rao et al. 2001).
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The formalin-killed cells (FKC) vaccine does not protect Japanese flounder against
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edwardsiellosis. Mekuchi et al. (1995) reported that the mortality of the fish immunized
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with the FKC, extracellular products or intracellular components of the bacteria was
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similar to the mortality of unimmunized fish. The relative percent survivals (RPSs) of
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Japanese flounder vaccinated with an attenuated strain or a natural avirulent isolate
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were significantly high (Cheng et al. 2010, Sun et al. 2010). On the other hand,
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vaccination with five avirulent strains provided only limited protection (Takano et al.
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2010). For example, the RPS obtained with one of these strains was only 45 %.
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Therefore, the effectiveness of a vaccine can strongly depend on the strain. Moreover,
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some subunit- or DNA vaccines against bacteria provided protection against bacterial
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challenge (Park et al. 2012).
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In aquaculture, vaccine adjuvants can modify the immune responses of fish. They
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may enhance antibody responses and provide a long-lasting protection against diseases.
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Freund's complete/incomplete adjuvants (FCA/FIA) have been extensively used in the
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immunological studies, in which they have been shown to stimulate both humoral and
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cell-mediated adaptive immune responses (Tafalla et al. 2013). FCA is a moisture of a
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mineral oil with heat-killed Mycobacteria cells, while FIA lacks the Mycobacteria cells.
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In E. tarda infection in Japanese flounder, a recombinant protein of major antigen Et49
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combined with FIA showed an RPS of 81 %, although the antigen by itself had an RPS
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of only 34 % (Jiao et al. 2010). In contrast, FCA/FIA had only limited effects in other
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fish species (Tafalla et al. 2013).
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In order to develop an effective vaccine by using adjuvants, it is necessary to
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understand how the adjuvants modulate fish immune responses. Here we elucidated the
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effects of the FCA/FIA on protection and immune response of Japanese flounder
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immunized by the E. tarda FKC. The adjuvants strongly enhanced antibody responses 2
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weeks post infection (w.p.i.). A microarray analysis revealed that FCA/FIA suppressed
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the upregulation of interferon-γ mRNA levels.
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Materials and methods
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Fish sampling and challenge test
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Fish, average total length of 12 cm, were intraperitoneally injected with phosphate
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buffered saline (PBS), 1 x 109 cells of FKC of E. tarda (strain NUS806), and 1 x 109
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cells of FKC mixed with FCA (FKC+FCA) or that with FIA (FKC+FIA). The injected
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fish were reared at 18 °C, and then spleen was sampled at 6 h and on 3 d after
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immunization. The blood was taken from the fish at 2 w.p.i. to be assessed by ELISA.
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Ten of the fish were intraperitoneally challenged with 1.5 x 105 cells of E. tarda at 4
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weeks after immunization and mortality was recorded.
72 Antibody titration using ELISA
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The antibody activities were measured by the method of Taechavasonyoo et al.
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(2013). Briefly, the ELISA plate (Thermo) was prepared by placing 109 cells of the FKC
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in 100 µl per well and incubated at 4 ºC overnight. The wells were washed three times
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with T-PBS (0.05% Tween 20 in PBS), covered with 100 µl of blocking solution (3%
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Skim milk) at room temperature for 2 h, washed three times with T-PBS, treated with
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100 µl of primary fish antisera diluted 20-times with PBS, incubated at room
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temperature for 1 h, washed three times with T-PBS, reacted with 1:3000 of
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anti-Japanese flounder IgM rabbit antiserum (Taechavasonyoo et al. 2013), washed
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three times with T-PBS, incubated with 1:1000 of anti-rabbit IgG conjugated with
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alkaline phosphatase (Sigma) to detect the rabbit IgG, washed five times, incubated
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with phosphatase substrate at room temperature for 30 min, and mixed with 50 µl of 3M
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NaOH to stop the reaction. The absorbance at 405 nm was measured with Multiskan FC
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Microplate Photometer (Thermo Scientific, Japan).
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Gene expression profiling by microarray
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Gene expression was profiled by the method of Kaneshige et al. (2016). Total RNA
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was extracted from spleen samples (n=3) using RNAiso Plus (Takara Bio, Japan) and
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purified with an RNeasy® Mini Kit (Qiagen, USA). RNA quality was evaluated using a
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2200 TapeStation (Agilent, USA). The microarray was analyzed using three samples in
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each group, and each sample was selected based on RNA quality. cDNA was
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synthesized from 200 ng of RNA and labeled with cyanine 3-CTP using a one-color
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microarray-based gene expression analysis following instructions of the manufacturer
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(Agilent, USA). The labeled cDNA was subsequently hybridized at 65 °C for 17 h.
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Microarrays were scanned with an Agilent G2565CA Microarray Scanner, and the
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images obtained from scanning were analyzed with Feature Extraction Software
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v9.5.3.1 (Agilent, USA). The data were normalized and analyzed using limma (Ritchie
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et al. 2015). The fold-change differences were calculated against the average of those
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before treatments. Genes with a fold change of at least 8.0 and p value less than 1e-5
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were considered to be differentially expressed.
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IFNγ and IL-12p35 genes expression profiling by qPCR
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The primers for the IFNγ, IL-12p35 and elongation factor 1 alpha (EF-1α) genes
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are shown in Supplemental Table. Total RNA was extracted from the spleen of each
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sample (n=5) using RNAiso (Takara, Japan) and 2 µg of total RNA was used for cDNA
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synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems,
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USA) according to the manufacturer's instructions. The cDNA samples were diluted
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5-times with distilled water. qRT-PCR was performed using 5 µl cDNA in a
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THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan) using an ABI7300 Real-time
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PCR system (Applied Biosystems, USA). The expression levels of the target genes were
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normalized to the expression level of an internal control gene (EF-1α) and the relative
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mRNA levels were calculated by the 2-∆∆Ct method (Livak and Schmittgen, 2001), and
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standardized to the average value of the PBS-injected group on the corresponding
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sampling points. The significance of differences between values on the standard and the
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others were evaluated by ANOVA followed by Dunnett’s post hoc test.
118 Results and Discussion
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Effects of Freund’s adjuvants on FKC vaccine
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Fish mortality was observed on day 4 after the challenge (Fig. 1A). All of the fish
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immunized with FKC+FCA died by 7 days after the challenge and all of the fish
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immunized with +FIA died by 6 days after the challenge. Unimmunized fish and fish
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immunized with FKC survived longer than those immunized with FKC+FCA and +FIA.
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Jiao et al. (2010) reported that the relative percent survival (RPS) of fish immunized
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with an antigen with FIA was 81 %, and that the RPS of fish immunized with only the
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antigen was 34 %. Tang et al (2010) also claimed that the antigens mixed with FIA
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showed higher RPS than the controls. In this study, we used FKC but not certain
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antigens, and thus the immune-response after the vaccination might be different from
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the response of those immunized with antigens mixed with FIA.
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Effects of Freund’s adjuvants on the antibody titer
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Antibody levels in fish immunized with FKC, FKC+FCA or FKC+FIA were
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significantly higher than those in the PBS group (Fig. 1B). Moreover, the FKC+FCA
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and FKC+FIA groups showed higher antibody levels than the FKC group. Although the
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antibody levels in the fish immunized with FKC+FCA and +FIA were higher than those
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in the other groups, these fish were susceptible to the bacterial challenge (see Fig. 1A).
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The specific antibody, which opsonize the bacteria, enhance adhesion of E. tarda to
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phagocytes, where the bacteria is able to survive and replicate (Srinivasa Rao et al.
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2001). Therefore, the specific antibodies elicited by the FKC with the adjuvants did not
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confer to the protection against the bacteria.
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Gene expression profiles after the vaccination
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The gene expression profiles in the spleen were compared between fish immunized
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with FKC+FCA and fish immunized with only FKC. The microarray data were
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deposited into the gene expression omnibus database under accession number
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GSE111115. In the samples at 6 h after FKC immunization, the intensities of 222 spots
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were at least 8- fold higher (p < 105) than they were before immunization, while in the
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samples at 6 h after FKC+FCA immunization, the intensities of 213 spots were at least
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8- fold higher. Similarly, the intensities of 214 spots were higher at 3 d after
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immunization. The mRNA levels of some cytokines (CC chemokine, interleukin (IL)-1β
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and IL-6) increased at 6 h in the FKC immunized group (Table 1). Almost all of the
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spots showed similar patterns between the FKC and FKC+FCA immunization (data not
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shown). In Japanese flounder, the genes induced by the injection of certain bacterial
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FKC were not significantly different, while the magnitude of up- or down regulation
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was distinct between the bacterial species (Kondo et al. 2014). Although mycobacteria
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cell components in FCA have been thought to influence gene expression in mammals
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(Behr et al. 2015, Ishikawa et al. 2017), they may have limited effects in fish.
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Only the intensities of the spots corresponding to interferon γ (IFNγ) were
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significantly higher in the FKC-immunized fish, but they were not significantly
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different in the FKC+FCA group (Table 1). It should be noted that only the IFNγ gene
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was differently regulated between FKC and FKC+FCA groups. The IFNγ mRNA level
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at 6 h after FKC immunization was significantly higher than it was in the other groups,
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while the levels at on 3d were not significantly changed (Fig. 1C). The IFNγ mRNA
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levels at 6 h also did not increase in FKC+FIA group. Therefore, regulation of the IFNγ
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gene expression might be caused by mineral oil forming an emulsion but not by the
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cellular components of mycobacteria.
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In mammals, interleukin (IL)-12 upregulates IFNγ transcription. IL-12 is an
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inflammatory cytokine, composed of p35 and p40 subunits (Watford et al. 2003,
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Zundler and Neurath 2015). Because the microarray did not have spots for the IL-12
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subunits, we further investigated changes IL-12p35 subunit mRNA levels by qPCR. As
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in the case of IFNγ, the IL-12p35 mRNA level at 6 h after FKC immunization was
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significantly higher than the other groups (Fig. 1D). Interestingly, at 3 d, the IL-12p35
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mRNA level was significantly lower in the FKC+FCA-immunized fish than in the
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control. In the amberjack, FKC vaccine with recombinant IL-12 showed effective
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against the intracellular bacterium Nocardia seriolae (Matsumoto et al. 2017). IL-12
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and IFNγ regulate the balance between the Th1 and Th2 responses, which act against
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intracellular bacteria and regulate antibody production, respectively (Watford et al. 2003,
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Zundler and Neurath, 2015), and induce cell mediated immunity (Matsumoto et al.
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2017). Therefore, the fish immunized with FKC+FCA/FIA, which showing lower IL-12
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and IFNγ mRNA levels might show lower cell mediated immune responce. In conclusion, adding Freund's adjuvants to the vaccines resulted in higher antibody
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titers and, and suppress the upregulation of IL-12 and IFNγ mRNA levels. Our data
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raise the possibility that the signaling pathways involved in IL-12 and IFNγ induction
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might be distinct from the other inflammatory pathways. Because IL-12 and IFNγ are
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important in the Th1 response, further studies are needed to characterize the signaling
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pathways regulating IL-12 and IFNγ expression to develop effective vaccines against
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intracellular bacteria.
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This research was supported in part by grants from JSPS KAKENHI Grant Number
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Acknowledgements
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Legends for figures
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Figure 1 (A) Cumulative survival of vaccinated fish after the challenge with E. tarda.
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(B) Antibody titers at 2 weeks after vaccination with FKC, FKC+FCA or FKC+FIA.
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Serum samples were collected from 5 fish/group, diluted 1:20, and then analyzed by
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ELISA. Each point represents an individual fish and black bar indicate the average of
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each group. Different letters represent significant differences as determined by Tukey's
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HSD (P < 0.05). (C, D) Expressions of IFNγ (C) and IL-12p35 (D) genes in the spleen
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from 3 fish/group at 6 h and on 3 d after PBS, FKC, FKC+FCA and FKC+FIA injection.
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Expression is expressed as Log2 values, relative to the average of the PBS group. Each
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point represents an individual fish and black bar indicate the average of each group.
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Asterisk indicates a significance difference by one-way ANOVA (P < 0.05).
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FKC 6h Product name
CUST_12226_PI426483990
AB427185.1
CUST_12932_PI426483990
a
CUST_351_PI426483993 CUST_48_PI426483993
b
CUST_49_PI426483993
b
a
Log2FC
P_value
CC chemokine-like
4.8
6.1E-06
AB427185.1
CC chemokine-like
6.4
3.3E-06
AB427185.1
CC chemokine-like
6.0
BAG50577.1
Interferon gamma
FKC 3d
FKC+FCA 3d
Log2FC
P value
Log2FC
P value
Log2FC
P value
4.7
7.6E-06
1.4
9.4E-02
1.6
9.9E-03
5.9
6.8E-06
2.4
1.8E-02
1.6
9.9E-03
6.2E-05
5.5
1.5E-04
1.8
1.8E-01
2.1
9.2E-03
6.4
3.9E-07
1.9
1.6E-02
2.1
8.8E-03
2.6
4.1E-02
5.9
3.1E-06
1.3
1.7E-01
1.3
1.9E-01
4.2
1.3E-03
5.6
4.3E-06
1.2
2.0E-01
1.2
1.9E-01
3.2
7.2E-03
5.8
3.7E-05
5.6
5.5E-05
2.7
2.3E-02
2.6
4.1E-02
7.4
5.9E-06
7.3
6.8E-06
3.9
2.2E-03
0.9
1.8E-01
4.1
4.9E-06
2.6
4.5E-04
0.6
3.3E-01
3.1
3.3E-04
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Acc. No. a
FKC+FCA 6h
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Table List of cytokines up-regulated after the treatments
Interferon gamma
CUST_9251_PI426483990
BAG50577.1
Interferon gamma
CUST_7075_PI426483990
c
AB720983.1
Interleukin 1 beta
CUST_9569_PI426483990
c
AB720983.1
Interleukin 1 beta
DQ267937.1
Interleukin 6
BAF91496.1
M17 homologue
4.4
1.7E-06
5.3
2.7E-07
0.5
3.5E-01
2.2
1.6E-02
BAF91496.1
M17 homologue
4.6
7.4E-07
5.6
1.2E-07
0.9
1.8E-01
2.5
5.2E-03
CUST_290_PI426483993 CUST_10972_PI426483990 CUST_310_PI426483993
d
d
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BAG50577.1 b
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Probes indicated by the same alphabet encode the same gene products. The values were compared with those before the treatments, and the Log2
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transformed fold-change values (Log2FC) and P value were represented. both Log2FC > 3.0 and P value < 1.0E-05 were indicated by gray.
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(A)
(B)
100
FKC+FIA *
60 50 30 10 0 1
2
3
4
5
6
7
8
Days post challenge
(C)
9
10
a
FKC +FCA
FKC +FIA
b
1.0
c
c
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a
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Absorbance (405 nm)
FKC+FCA *
0.5
0.0
Before PBS injection
FKC
(D)
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*
* p < 0.05
8 6
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0
PBS FKC
FKC FKC +FCA +FIA
6h
PBS FKC FKC FKC +FCA +FIA
3d
Relative IL-12p35 mRNA levels (Log 2)
Survival rate (%)
FKC
80
20
Relative IFNγ mRNA levels (Log 2)
2.0
PBS
90
-2
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Fig. 1
5
* p < 0.05 4
*
3 2 1 0
*
-1 -2 PBS FKC
FKC FKC +FCA +FIA
6h
PBS FKC FKC FKC +FCA +FIA
3d
ACCEPTED MANUSCRIPT Highlights •
Effects of adjuvants (FCA, FIA) on protection and immune response of Japanese flounder were evaluated. Freund’s adjuvants enhanced antibody responses but did not confer protection.
•
Freund’s adjuvants suppressed the induction of IFNγ and IL12p35 mRNA expression
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after the FKC vaccination.