The influence of concentration of inactivated Edwardsiella tarda bacterin and immersion time on antigen uptake and expression of immune-related genes in Japanese flounder (Paralichthys olivaceus)

Microbial Pathogenesis 103 (2017) 19e28

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The influence of concentration of inactivated Edwardsiella tarda bacterin and immersion time on antigen uptake and expression of immune-related genes in Japanese flounder (Paralichthys olivaceus) Yang Du a, Xiaoqian Tang a, Xiuzhen Sheng a, Jing Xing a, Wenbin Zhan a, b, * a

Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao 266003, China Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No.1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao 266071, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 October 2016 Received in revised form 11 November 2016 Accepted 6 December 2016 Available online 16 December 2016

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (109, 108, 107, 106 cfu ml
1) for 30, 60 and 90 min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6 h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6 h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 109-30, 109-60, 108-60, 108-90 and 108-90 groups than other groups (P < 0.05), especially the 108-60min group displayed the highest antigen uptaken. Meanwhile, the expression profiles of antigen recognization and presentation genes (MHCⅡa, TcRa, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1b, IL-6), heat shock protein 70 (HSP70) and c-type lysozyme were analyzed using real-time PCR. On the whole, the transcription levels of the eight genes exhibited to be higher in 107-90, 108 and 109 cfu ml
1 groups than other groups (P < 0.05), especially the 108-60 group displayed the highest up-regulation. These results demonstrated that immersion with formalininactivated E. tarda, especially under 108-60 min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Japanese flounder (Paralichthys olivaceus) Edwardsiella tarda Immersion vaccination Antigen uptake Gene expression

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.1. Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2. Preparation of inactivated E. tarda bacterin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.3. Immersion vaccination and sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4. Synthesis of cDNA and extraction of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.5. Detection of uptaken E. tarda in different tissues by qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.6. qPCR analysis of eight immune-related genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.7. Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

* Corresponding author. Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao 266003, China. E-mail address: [email protected] (W. Zhan). http://dx.doi.org/10.1016/j.micpath.2016.12.011 0882-4010/© 2016 Elsevier Ltd. All rights reserved.

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Y. Du et al. / Microbial Pathogenesis 103 (2017) 19e28

3.1. Quantification of E. tarda antigen uptake in different tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.2. The expression of MHCⅡa, TcRa and CD4-1 in different tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.3. The expression of IgM and IgT in different tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4. The expression of inflammatory cytokines IL-1b and IL-6 in different tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.5. The expression of heat shock protein (HSP70) and c-type lysozyme in different tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

1. Introduction Edwardsiella tarda is a common fish pathogen that causes hemorrhagic septicemia (also known as edwardsiellosis) in freshwater and marine fish worldwide, which also causes serious health problems in reptiles, shellfish, amphibians, birds, humans and mammals [1,2]. The disease resulted in extensive economic losses and became a concern to the aquaculture industry, especially in Japanese flounder (Paralichthys olivaceus) [3]. Although antibiotics were suggested to prevent the outbreak of E. tarda [4,5], the overuse of antibiotics can cause the emergence of antibiotic-resistant bacteria. Therefore, vaccines are considered as the most effective and secure strategy to control the infectious diseases in the fish industry [6]. Currently, intraperitoneal (i.p.) injection of oiladjuvanted vaccines, immersion immunization of inactivated whole bacterin vaccine and feeds-oral of recombinantly attenuated vaccine or DNA vaccine are the predominant methods of vaccination used to combat edwardsiellosis in Japanese flounder aquaculture [7e9]. Although injection vaccination could produce superior immune protection, it was not convenient for the operation, similarly, the most serious deficiency of the oral vaccine is that it is easily damaged. However, immersion vaccination not only evoke a mucosal immune response in fish, but confer adaptive immune responses [6,10,11]. It is note-worthy that the immersion immunization has outstanding advantages of minimal pain for fish, lower labor costs, less time necessary and operator safety, especially vaccinating large numbers of small size fish. However, immersion immunization does have drawbacks of the lower amount and unstable of antigen uptaken, thus in order to enhance the effect of immersion immunization, many factors including the level of inflicted stress, pH and salt concentration of the vaccine solution, the water temperature, the nature of antigen, the use of adjuvant, vaccine concentration and immersion time, which have been proved to influence immune efficacy [12e14], were needed to be considered and optimized. The amount of antigen uptaken will affect the immune efficacy, so which also could be an ideal indicator for evaluating the effect of immersion vaccination [15,16]. In recent years, the antigen uptaking post-immersion immune of fish has been paid great attention, and different type of antigens such as bovine serum albumin, fluorescent latex microspheres, fluorescein conjugated bacterin and inactivated virus were employed to investigate the amount and routes of antigen uptaken [17,18]. Studies have shown that the antigen uptaking mainly depended on the phagocytosis by mucosal tissues as gill, skin, lateral line and gastrointerstinal tract of teleost [19e21], and this process could be enhanced by the application of hyperosmotic immersion (HI), ultrasound mediated, multiple puncture instrument or combine adjuvant [22e25]. Till now, several methods were developed for detecting the antigen uptaken, such as plate culture, quantitative enzyme immunoassay, electron microscopy, a competitive ELISA or immunohistochemistry, in situ hybridization and 3D visualization method [26e30]. Among them,

the quantitative PCR (qPCR) has enabled us to determine the amount of uptaken antigen in a more convenient and sensitive way [15,16,31]. Our previous work has demonstrated that the relative percent survival (RPS), sIgþ cell proliferation and specific antibodies level of Japanese flounder (Paralichthys olivaceus) were easily affected by the concentration of inactivated E. Tarda bacterin and immersion time [13]. However, little information relating to the influence of vaccine concentration and immersion time on antigen uptake and expression of immune-related genes has been reported. Therefore, in the present work, a quantitative PCR (qPCR) was developed to investigate the amounts of antigen uptake in various tissues of Japanese flounder immersed in a formalin-inactivated E. tarda vaccine, and the expression profiles of eight immunerelated immune genes antigen recognization and presentation genes (MHCⅡa, TcRa, CD4-1), immunoglobulin (IgM, IgT), inflammatory cytokines (IL-1b, IL-6), heat shock protein 70 (HSP70) and ctype lysozyme were also examined using RT-qPCR. 2. Materials and methods 2.1. Fish Healthy Japanese flounders (average length was 15 ± 2 cm) were obtained from a fish farm in Shandong province, China. The fish were quarantined in a 500 L tank supplied with UV and bio-filters treated recirculating water at optical laboratory conditions (dissolved oxygen 6.0 ± 0.5 mg L
1; temperature 20 ±1  C). Fish were fed twice a day with commercial dry food pellets at 3% body weight. The fish were pathogen-free and had no history of untoward mortalities or abnormalities, which was confirmed by histopathology and molecular analysis for bacterial, parasitic and viral pathogens. After acclimation to laboratory conditions for two weeks, fish were randomly divided into 13 groups for the immersion vaccination experiments. 2.2. Preparation of inactivated E. tarda bacterin The strain of E. tarda HC01090721 stored in saline with 15% glycerol at
80  C, which was isolated from the ascites of Japanese flounder and proved to be a pathogenic strain by infection experiment previously by our laboratory [32]. The E. tarda bacterin was prepared according to the procedures described previously [13]. Briefly, frozen stocks were directly inoculated onto brain heart infusion (BHI) agar plate and cultured at 37  C for 48 h, and then selected single clones and transferred into BHI broth for shaking incubation at 37  C until the optical density (OD) at 600 nm reached 1.0. Then, the bacteria were harvested and washed with 0.01 M phosphate buffered saline (PBS, pH 7.2) by centrifuging at 8000g for 10 min at 4  C. The bacterial cell numbers was calculated directly using an Accuri C6 flow cytometer (BD). The bacterial suspension with the concentration of 2.5  109 cfu ml
1 was treated with 0.5% formalin (V/V) for 72 h at 4  C, and inactivation of

Y. Du et al. / Microbial Pathogenesis 103 (2017) 19e28

the bacteria was confirmed by incubating the solution on BHI agar at 37  C. The inactivated cells were harvested and washed three times with sterilized PBS by centrifuging at 8000  g for 10 min at 4  C. After last wash, adjusted the bacterin suspension concentration to 1.0  109 cfu ml
1 and -20  C for later use. The safety of the bacterin was checked by culturing the cells on BHI agar at 37  C for 72 h and no bacterin grow on the agar plate. Meanwhile, intraperitoneally injecting 0.5 ml of the collected bacterin into each healthy Japanese flounder at a density of 1.0  108 cfu ml
1, and no any adverse reactions have been found until the 20th day. 2.3. Immersion vaccination and sampling The vaccine solution was prepared by diluting the stored inactivated E. tarda to a concentration of 106, 107, 108, 109 cfu ml
1 respectively with normal seawater. Then, 30 fish in each group were immersed in the 50 L tanks under the four bacterin solutions for 30, 60 and 90 min respectively, and then washed in the clear seawater and immediately transferred back to recirculating aquaria system. The fish without immersion vaccination were set as control. During the immersion vaccination, aeration was given all the time. Some of the Japanese flounders exhibited a little stress and active treated in the 109 cfu ml
1 solution, whereas no obvious stress response was observed in the other groups. Following, 3 fish were randomly anesthetized with MS-222 (Sigma, USA) and sampled at 3, 6, 12, 18, 24, 48, 72 and 120 h from vaccinated and control groups post immersion. Before sampling, each fish was washed three times with phosphate-buffered saline (PBS) to remove mucus. Then using sterile scalpel to take the gills, skin, spleen and kidney separately, and then place into tubes containing RNA/DNAlater and stored at
80  C for subsequent RNA and DNA extraction. The samples from the three fish were mixed together using as one replicate, and each group had three replicates. 2.4. Synthesis of cDNA and extraction of DNA Equal portions of four tissues were taken and total RNA was isolated using TRizol (Sigma, USA). 1000 ml of Trizol was added to every sample and disrupted the tissues by sonication on ice. The quantity and concentration of the RNA were detected by NanoDrop ND-8000 spectrophotomete, and the integrity of RNA was evaluated on a 1.5% agarose gel. To ensure complete removal of genomic DNA, 1 mg of total RNA was incubated with 1 unit of DNase I for 15 min at room temperature. Complementary DNA (cDNA) was synthesized with Reverse Transcriptase M-MLV kit (TaKaRa, China) according to manufacturer's instructions with Oligo (dT18) primers and the synthesized cDNA was stored at
80  C. For isolation of DNA, all the sampled tissues were washed three times in the PBS to prevent the bacterin from adhering to the surfaces. Total DNA of each tissue, weighing 30 mg, was extracted using a Tiangen DNA tissue Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol, and then dissolved in ultrapure water. The DNA concentration was measured and adjusted to 50 ng/ml using NanoDrop ND-1000 (NanoDrop Technologies, USA). 2.5. Detection of uptaken E. tarda in different tissues by qPCR The establishment of quantitative real time PCR (qPCR) standard curve for quantifying the uptake of E. tarda in the four tissues of Japanese flounder was performed as described in Gao et al. [33]. 2 mg bacterial DNA was extracted from 3.3  108 cfu ml
1 of E. tarda using DNeasy Blood & Tissue Kit (Qiagen, Germany), and diluted in 10-fold serial as templates to generate standard curve using the primers: Eta2-351 and Edwsp-780r [34], equivalent to the bacteria

21

number ranging from 3.3  108 to 3.3  10
1 cfu ml
1. RNase free water was used as negative control, and the PCR assay was performed in triplicate for each dilution using Roche480 real-time PCR system (LightCycler®480, USA). The up-taking of E. tarda bacterin in four tissues was performed by qPCR in total reaction volumes of 20 ml containing 2 ml of the tissues DNA extraction, 10 ml of SYBR Green (Roche, Sweden), 1 ml of primers (10 mM), and 7 ml of RNasefree water. The cycling protocol consisted of an initial PCR predenaturation step at 94  C for 5 min, followed by 45 cycles of denaturation at 94  C for 30 s, annealing at 60  C for 30 s and extension at 72  C for 10 s. DNA melting-curve analysis ensured that the desired amplicon was specifically detected. Finally, the linear relationship between the logarithm of inactivated E. tarda cells number (cfu ml
1) and Ct value was generated, the linear equation was y ¼
3.515x þ 43.78 (R2 ¼ 0.994). When Ct values (y) were obtained, the x values (log10Number of E. tarda) could be calculated according to the equation. The detection sensitivity of the qPCR was determined based on the highest dilution that resulted in a detectable amplification signal. 2.6. qPCR analysis of eight immune-related genes The expression profiles of eight immune-related genes including (MHC II a, TcRa, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1b and IL-6), heat shock protein (HSP70) and c-type lysozyme were investigated by using a Roche480 real-time PCR system (LightCycler 480, USA). The specific primers are shown in Table 1, which were identified by sequencing of all the products. The cDNA concentrations of different tissues were adjusted to 100 ng ml
1. Triplicate reactions were performed for every sample, and each reaction well contained 10 ml of SYBR Green I Master, 0.4 ml each of forward and reverse primers (10 mM), 2 ml of diluted cDNA and 7.2 ml of RNase-free water to a volume of 20 ml. The thermal cycling profile consisted of an initial denaturation at 95  C for 30 s, followed by 45 cycles of denaturation at 95  C for 5 s and extension at 60  C for 30 s. An additional temperatureramping step was utilized to produce melting curves of the reaction from 65  C to 95  C. The expression level of eight genes in blank control individuals was defined as 1.18S primers were used as endogenous control to correlate for potentially different loading amounts of RNA added to the RT-PCR reaction and for variation in cDNA synthesis efficiencies. Table 1 Primers used for amplification of specific gene products. Primers name

Primer sequences (50 e30 )

Accession

18sRNA-F 18sRNA-R Eta2-351 Edwsp-780r MHCⅡa-F MHCⅡa TcRa-F TcRa-R CD4-1-F CD4-1 IgM-F IgM-R IgT-F IgT-R IL-1b-F IL-1b-R IL-6-F IL-6-R HSP70-F HSP70-R C-type lysozyme-F C-type lysozyme-R

50 -GGTCTGTGATGCCCTTAGATGTC-30 50 -AGTGGGGTTCAGCGGGTTAC-30 50 -TAGGGAGGAAGGTGTGAA-30 50 - CTCTAGCTTGCCAGTCTT-30 50 -ACAGGGACGGAACTTATCAACG-30 50 -TCATCGGACTGGAGGGAGG-30 50 -GGTCTGATGCTTCACAGTGTGAG-30 50 -ACCGCCGGATCTTTCTTCA-30 50 -CCAGTGGTCCCCACCTAAAA-30 50 -CACTTCTGGGACGGTGAGATG-30 50 -ACAAAAGCCATTGTGAGATCCA-30 50 -TTGACCAGGTTGGTTGTTTCAG-30 50 -TAATTGTTCAGTAACTCATGCCG-30 50 -GATTGAAGTGTTCCTATGCGTCT-30 50 -CAGCACATCAGAGCAAGACAACA-30 50 -TGGTAGCACCGGGCATTCT-30 50 -CAGCTGCTGCAAGACATGGA-30 50 -GATGTTGTGCGCCGTCATC-30 50 -TCCTCATGGGTGACACTTCG-30 50 -TTGTCCTTGGTCATGGCTCT-30 50 -TGTCATTGTGGCGATCAAATG-30 50 -GCTCCGATCCCGTTTGG-30

EF126037

AY997530 AB053406 AB643634 AF226284 KX174301 AB720983 DQ267937 AF053059 AB050469

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2.7. Data analysis RT-PCR data were analyzed using MX Pro-Mx3000P Multi-plex Quantitative PCR system Software and the relative expression ratio (R) of mRNA was calculated according to the formula 2
△△Ct ¼ 2
(△Ct(test)
△Ct(18S)) [35]. The data were normalized for each gene against those obtained for 18S rRNA [36]. The results are presented as means with standard deviations of fold increase/ decrease from three fish at each time point. The statistical software orange 8.0 was used for creating graphs.

uptaken at 3 h p.i. were significantly higher than that at 6 h p.i. among different immersion groups. In contrast, the amounts of uptaken cells were increased from 3 to 6 h p.i. in spleen and kidney. In gills, 108-60 group resulted in the highest amount of uptaken cells at 3 h p.i., followed by the 109-30, 109-60, 109-90 and 108-90 groups. In skin, the highest amount of uptaken cells was detected in 109-60 group at 3 h p.i., followed by the 108-60, 109-30 and 109-90 groups. In spleen and kidney, the highest amounts of uptaken cells were detected in the groups of 108-60, 109-30 and 106-90, followed by 108-90 and 109-90 groups.

3. Result 3.2. The expression of MHCⅡa, TcRa and CD4-1 in different tissues 3.1. Quantification of E. tarda antigen uptake in different tissues The uptaken amounts of inactivated E. tarda cells in gills, skin, spleen and kidney were determined by quantitative PCR in 12 experimental groups at 3 and 6 h post immersion (p.i). As shown in Fig. 1, the amounts of E. tarda cells uptaken by gill and skin were much higher than that in spleen and kidney within 6 h post immersion, and the antigen amount uptaken by gill was even almost 10 times higher than that by the skin. With the extension of immersion time, the amounts of E. tarda cells uptaken by the four detected tissues in 106 and107 cfu ml
1 groups were increased, however, the uptaken amounts were decreased in 108 and109 cfu ml
1 groups. In gill and skin, the amounts of cells

After immersion vaccination, the expression levels of MHCⅡa, TcRa and CD4-1 genes were significantly up-regulated in the four tissues examined, although they were much higher expressed in the gill and skin than in spleen and kidney shown in Fig. 2. A quicker response of the three genes was also found in gill and skin against immersion vaccination than that in spleen and kidney, the peak expression levels of the three genes mainly occurred at 24 h p.i. in the gill and skin, but at 48 h p.i. in the spleen and kidney. Among different immersion groups, the highest expression levels of MHCⅡa, TcRa and CD4-1 in gill and skin were observed in 108-60 group, followed by the 109-30, 109-60, 109-90 and 108-90 groups, which were significantly higher than those of the other groups.

Fig. 1. The cell numbers of inactivated E. tarda uptaken by gill (A), skin (B), spleen (C) and kidney (D) of Japanese flounder at 3 and 6 h post immersion. Values were means ± SE, and different letters denoted significant difference among different immersion groups at the same sampling time (P < 0.05).

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Fig. 2. Expressions profiles of MHCⅡa, TcRa and CD4-1 genes in the gill, skin, spleen and kidney of Japanese flounder post immersion vaccination with inactivated E. tarda. The data are presented as the means ± SEM of three fish, the length of the color bars indicate the extent of upregulated expressions of the genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.3. The expression of IgM and IgT in different tissues The expression profile of IgM and IgT genes were significantly upregulated in the four detected tissues of Japanese flounder post vaccination as shown in Fig. 3. On the whole, the IgM expressed much higher in spleen and kidney than that in gill and skin, on the contrary, the IgT genes were expressed much higher in gill and skin than that in spleen and kidney. The expression peak time of IgM appeared at 24e48 h p.i. in the gill and skin, but in spleen and kidney occurred at 72e120 h p.i. Whereas, a quicker response of IgT gene was found in gill and skin against immersion vaccination than that in spleen and kidney, the peak expression levels mainly occurred at 24 h p.i. in the gill and skin, but at 120 h p.i. in the spleen and kidney. Among different immersion groups, the highest expression levels of IgM in spleen and kidney were observed in 108-60 group, followed by the 109-30, 109-60, 109-90 and 108-90 groups, which were significantly higher than that of the other groups. While, the highest expression levels of IgT in gill were observed in 108-60 group.

3.4. The expression of inflammatory cytokines IL-1b and IL-6 in different tissues IL-1b and IL-6 were expressed increase in the four tissues after immersion vaccination, and the mean transcript levels was higher in gill as shown in Fig. 4. There appeared two expressed peak of IL1b in gills, spleen and kidney at 3e6 h p.i. and 24e48 h p.i, and 10930, 109-60, 109-90 and 108-90 groups own much higher transcript level than other groups, and 108-60 displayed the highest upregulated in gill, spleen and kidney, but a quicker response was observed in 109-30, 109-60, 109-90 groups. Moreover, the results of the IL-6 expression shown two peaks only in skin at 3e6 h p.i. and 24e48 h p.i. The transcript level also increased with the immersion time extent in 107 and 106, in contrast, decline with the immersion time prolong in 109 and 108, and the highest expression was in 10860 in the four tissues. The peak time appeared earlier in 109-30, 10960, 109-90 groups than other groups in gill and kidney.

Fig. 3. Expressions profiles of IgM and IgT genes in the gill, skin, spleen and kidney of Japanese flounder post immersion vaccination with inactivated E. tarda. The data are presented as the means ± SEM of three fish, the length of the color bars indicate the extent of upregulated expressions of the genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Expressions profiles of IL-1b and IL-6 genes in the gill, skin, spleen and kidney of Japanese flounder post immersion vaccination with inactivated E. tarda. The data are presented as the means ± SEM of three fish, the length of the color bars indicate the extent of upregulated expressions of the genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. The expression of heat shock protein (HSP70) and c-type lysozyme in different tissues The expressions of the HSP70 and c-type lysozyme genes in four tissues all peaked at 18e48 h p.i, and then gradually decreased (Fig. 5). The mean expression levels of the HSP70 gene in the high concentration groups (109 cfu ml
1) were much higher than other group, with the highest levels of expression observed in the 109-90 group in gill. While, the significant up-regulated of C-loysome mRNA in the four tissues, and the peak time were at 3e12 h p.i in gills, skin and kidney, which was earlier than spleen of 24e48 h. The highest transcript level was found in 108-60 and 109-30 group in kidney, followed by the 109-60, 109-90 and 108-90 groups, which were significantly higher than that of the other groups, whereas there was no significant difference among 109-60, 109-90 and 10890 (P > 0.05). 4. Discussion Immersion vaccination for fish was usually performed in a

concentrated solution for a short period of time, which could also be conducted in a dilute suspension for a prolonged immersion time [12,37,38]. The present work showed that the Japanese flounder immersed for a short time in a high bacterin solution uptook more antigen than that immersed in low bacterin solutions for a long time, and long immersion time did cause a significantly higher of uptaken antigen amount in Japanese flounder immersed in 106 and 107 cfu ml
1 vaccine solution than that of short immersion time, but the antigen uptaking would exhibit a decline with a further increased immersion time in the high bacterin solutions (108 and 109 cfu ml
1). These results indicated that a proper combination of vaccine concentration and immersion time would best activate the phagocytosis of mucosal epithelia, which might has an adjuvant activity of facilitating antigen entry and induced a stronger immune response, but 109-90 immersion treatment might beyond the tolerance of Japanese flounder and resulted in a loss of capacity to respond to vaccination. Therefore, it is of great importance to determine the optimal combination of vaccine concentration and immersion time, our previous work showed that 108-60 immersion treatment could evoke strongest immune response of

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Fig. 5. Expressions profiles of heat shock protein (HSP70) and c-type lysozyme genes in the gill, skin, spleen and kidney of Japanese flounder post immersion vaccination with inactivated E. tarda. The data are presented as the means ± SEM of three fish, the length of the color bars indicate the extent of upregulated expressions of the genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Japanese flounder and produced a highest relative protective survival (RPS) [13], which also induced the highest amount of antigen uptake in the present study. All these results demonstrated that vaccine concentration and immersion time would significantly influence the amount of antigen uptake, which might have a close relationship with the effects of immersion vaccination. It was reported that foreign antigens are trapped by the secondary circulatory systems of gills, skin and lateral line and gastrointerstinal tract in immersion vaccination, and then spread further to the pillar capillaries [30]. Our quantitative analyses showed that the antigen amount uptaken in the mucosalassociated tissues was significantly higher than spleen and kidney after immersion vaccination, and the amount uptook in gill was even much higher than that in skin, which suggested that gill is the main route for the antigen entry, and this view was supported by several previous studies [27,39,40]. However, several studies showed that the skin was the main route for antigen entry [41,12]. We speculated that these contradictory findings might be associated with the different species of fish, types of antigen and methods

for quantifying antigen uptake. Moreover, the amount of antigen was declined from 3 h p.i. to 6 h p.i. in gill and skin, but increased in spleen and kidney, which indicated the antigen was firstly uptaken by the mucosal-associated tissues and then transported to the spleen and kidney after immersion vaccination. Interestingly, the highest antigen amount was detected at 3 h p.i. in gill under 108-60 immersion treatment, which also induced the quickest increasing of antigen amount in spleen and kidney, this result suggested that the antigen transportation would be influenced by the immersion vaccine concentration and immersion time. In addition, the amount of uptaken antigen in gill was increased with the extension of immersion time in 109 cfu ml
1 vaccine solution, but declined in skin, spleen and kidney, we speculate that immunization with a long time immersion in high concentration vaccine might result in anoxia and stress to fish or the immune organs maybe have effluxmediated resistance mechanism to antigen, which further to hindered the antigen entre into blood circulation system. MHC II, CD4-1 and TcRa are important antigen presentation and recognition molecules involved in immune signal transduction

Y. Du et al. / Microbial Pathogenesis 103 (2017) 19e28

after pathogen invasion. These three genes showed a significant upregulation in the mucosal tissues and reached their peak levels in a short time after immersion, and the 108-60 group exhibited significantly higher gene expressions than that of the other groups. In contrast, expression levels of the three immune genes was increased slowly in the spleen and kidney after immersion, which were in accordance with the trend of antigen uptaking in the spleen and kidney. Our results demonstrated that the amount of antigen uptaken influenced the expression of the three antigen presentation related genes. The traditional view of antigen presentation is that MHCⅡis responsible for presenting the treated exogenous antigen to CD4þ T cells and TcRa participate in helping lymphocyte T recognize the complex of antigen recognition peptide-MHCII to activate and regulate humoral and cellular immune response [42,43]. This result may be explained by the fact that presentation of antigens is governed by endocytosis mechanisms, which determined the intracellular routing of the endocytosed antigens. Immunoglobulins are the major part of the adaptive immune system of jawed vertebrates [44], and it has been postulated that IgM plays a key role in system response and IgT represents a single autonomous immune compartment in the mucosal immunity of fish [45]. The present work showed that IgM were up-regulated much higher in spleen and kidney than that in gill and skin after vaccination, but reached the peak time much earlier in gill and skin than that in spleen and kidney. In accordance, our previous work showed that the specific antibodies in mucus were increased rapidly but lower level than that in serum, which indicates that IgM was mainly involved in system immune response and the mucosal tissues were the main first organs to capture antigens and produce specific antibodies post immersion vaccination. Research demonstrated that, apart from being mainly involved in mucosal immunity, IgT is also involve in systemic immunity [45,46]. This work observed that IgT expressed significantly higher in gill and skin than that in spleen and kidney, which indicates immersion vaccination was mainly induce the mucosal immune response. Moreover, under 108-60, the expression levels of the two genes, the sIgþ response and the specific antibodies in serum and mucus were much higher than other groups, which suggested that an optimal immersion would facilitate antigen uptaking and evoking mucosal and systematic immune response in Japanese flounder. Induction of inflammation is a key factor for evoking an adaptive immune response in mucosal tissues, where foreign substances such as dietary proteins and pathogens are abundant [47,48]. IL-1b could induce IL-6 production and show a coordination role to promote the immune response. In the present study, it was observed that the inflammatory cytokine genes (IL-1b and IL-6) were up-regulated in the four tissues, and expressed significantly higher in gills than other three tissues post immersion. These results support the finding that the gill is much more important for antigen uptaking after immersion vaccination. However, a quicker response of the two genes was observed under the highconcentration conditions, and the transcription levels were increased with the extension of immersion time. It was reported that IL-1b/IL-6 play an important role in starting the inflammation, leukocyte phagocytosis activity and in respiratory burst after stimulation by lipopolysaccharide and gram-negative bacteria [49e51]. Thus, the results might indicate fish suffered stress in high-concentration with a long time immersion, which further could induce a series of physiological reaction such as respiratory burst, inflammation and inflammation-related genes expression. Heat shock proteins (HSP) are intracellular molecules that are sensitive to various stimulation-stress, which could be immunoregulatory agents participate in immune defense during the pathogen infection and mediate a range of essential housekeeping and cytoprotective functions [52e54]. Results showed that the

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transcript level of HSP70 was increased with increasing the concentration of vaccine and with prolonging the immersion time in the four tissues, but more expressed acute in 109 cfu ml
1 than other concentrations, and the highest expression level was found in 109-90 group in the four tissues, which indicates 109 cfu ml
1 was not the appropriate concentration to vaccination for this concentration might exceed the phagocytic capacity of mucosal tissues or caused stress to fish. Lysozyme widely exist in fish mucus, blood serum, phagocytic cells and mononuclear cells, and plays an important role in antibacterial infection [55,56]. In the present work, a faster expression of c-type lysozyme was seen in gill, skin and kidney than that in spleen, and the highest expression level was detected in kidney, this may be related to the characteristic of the organization differences in expression. While, the expression level of c-type lysozyme was increased with increasing the vaccine concentration and immersion time and quickly reached the peak time in gill, and the highest expression level was found in 109-90 group, but the highest transcript level was observed in 108-60 group in other tissues, which might indicate the high concentration-stress also could induce the expression of c-type lysozyme in gill and 108-60 was the optimal moderate condition for Japanese flounder vaccination. Formalin-inactivated E. tarda was proved to effectively induce higher cellular and humoral responses and relative protection survival (RPS) in Japanese flounder from our previous work, with the strongest immune responses in the 108-60 min group [13]. The present study showed that proper concentration and immersion time treatment (e.g. 108-60 min) could enhance antigen uptake and expression levels of immune-related genes in Japanese flounder, which suggested that the antigen uptake has a close relationship with the efficacy of immersion vaccination. Acknowledgement This study was supported by the National Natural Science Foundation of China (31672685; 31302216; 31672684; 31472295), National Basic Research Program of China (2012CB114406), Taishan Scholar Program of Shandong Province, Science and Technology Development Project of Shandong Province (2014GNC111015) and The Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02). References [1] T. Matsuyama, T. Kamaishi, N. Ooseko, K. Kurohara, T. Iida, Pathogenicity of motile and non-motile Edwardsiella tarda to some marine fish, Fish. Pathol. 40 (2005) 133e136. [2] B.R. Mohanty, P.K. Sahoo, Edwardsiellosis in fish: a brief review, J. Biosci. 32 (2007) 1331e1344. [3] T.T. Xu, X.H. Zhang, Edwardsiella tarda: an intriguing problem in aquaculture, Aquaculture 431 (2014) 129e135. [4] W.D. Waltman, E.B. Shotts, Antimicrobial susceptibility of Edwardsiella tarda from the United States and Taiwan, Vet. Microbiol. 12 (1986) 277e282. [5] R.B. Clark, P.D. Lister, J.M. Janda, In vitro susceptibilities of Edwardsiella tarda to 22 antibiotics and antibiotic-beta-lactamase-inhibitor agents, Diagn. Micro Infect. Dis. 14 (1991) 173e175. [6] K.P. Plant, S.E. Lapatra, Advances in fish vaccine delivery, Dev. Comp. Immunol. 35 (2011) 1256e1262. [7] S.R. Kwon, E.H. Lee, Y.K. Nam, S.K. Kim, K.H. Kim, Efficacy of oral immunization with Edwardsiella tarda ghosts against edwardsiellosis in olive flounder (Paralichthys olivaceus), Aquaculture 269 (2007) 84e88. [8] Z.L. Mo, P. Xiao, Y.X. Mao, Y.X. Zou, B. Wang, J. Li, Y.L. Xu, P.J. Zhang, Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.), Fish. Shellfish Immunol. 23 (2007) 521e530. [9] Y. Sun, C.S. Liu, L. Sun, Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain, Vaccine 28 (2010) 6603e6608.

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