Title:
Evaluation of immune response to OTC in red seabreams
Members’ names:
Su-Min Baka, Fumiya Takahashib, Suzuki Satorub, Eun-Young Kimc,d, Hisato Iwatab∗
Affiliations:aDepartment of Advanced Toxicology Research, Korea Institute of Toxicology, 141 Gajeong-ro,Yuseong-gu, Daejeon, 34114, Republic of Koreab

Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama, 790-8577, Japanc

Department of Life and Nanopharmaceutical Science, Kyung Hee University,26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of KoreadDepartment of Biology, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul,02447, Republic of Korea
Aim:
Increasing seafood consumption results in intensive aquacultural system, which has beenaccompanied by an increased prevalence of the fish disease. The antibiotic oxytetracycline (OTC)
is commonly used to prevent and treat bacterial diseases in fish. However, overuse of OTC has
led to side effects, such as immune alteration. In view of this, the aim of present study is to reveal
to immune effects by OTC administration in aquacultured red seabream (Pagrus major).
Procedure
Our previous study done the in vivo OTC administration to the red seabream juveniles. Threegroups were established followed OTC administration concentration and sampling of immune
organ spleen and liver were collected followed by OTC feeding-withdrawal days cycle.
Three experimental groups were established: control group (fed corn oil-supplemented food) andexperimental groups (40 and 178 mg OTC per body kg feed, respectively). Each oral
administration (total twice administration each for seven days), we collected spleen, and liverfrom each group of red seabream juveniles at the three-sampling time (at day 8, 43, and 70; after
the first fed period, the first and second fed period, and after two-times fed and withdrawal period,respectively) (Figure 1). Using the liver and spleen from the OTC administrated red seabreamjuveniles, measured the expression level of immune-related genes by quantitative real-time PCR(qRT-PCR) and compare with the alteration of immune cells distributions by immunophenotyping.
① In vivo OTC administration to the red seabream juveniles was performed. Three groups wereestablished followed OTC administration concentration (0, 40 and 178 mg∙kg-1 diet), andthree sampling times. Liver and spleen were collected followed by OTC feeding-withdrawal
day cycle (Figure 1).

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Figure. 1 Experimental groups and three-sampling schedules.
② Primer design (Table 1):◼

Immunoglobulin M (IgM; membrane-bound IgM; mIgM and secreted IgM; sIgM)◼

One reference gene: Elongation factor 1α (Ef-1α)

Table 1. Primers used for Real time qPCR
Gene nameGeneGeneabbreviation

BankPrimer sequences (5’→3’)(F) CTGTCAAGGAAATCCGTCGTElongation factor 1α Ef-1α

AF184170

Immunoglobulin M

mIgM

KX599199

Interleukin-1βsIgM

JQ811851(R) TGACCTGAGCGTTGAAGTTG(F) GCTATGGAGGCGGAGGAAGATAACA(R) GCAGAGTGATGAGGAAGAGAAGGATGAA(F) ACCTCAGCGTCCTTCAGTGTTTATGATGCC(R) CAGCGTCGTCGTCAACAAGCCAAGC③ Real-time qPCR: to evaluate the differences of immune related gene’s expression level,measured the expression level of immune-related genes by qRT-PCR in liver and spleen.
Normalized using Ef-1α gene expression level.
④ The immunophenotyping: Leukocytes were successfully isolated from blood and spleen fromeach red seabream samples. The used fish antibodies successfully detected the anti-CD4,anti-CD8a and anti-IgM expressed immune cells in red seabream. Spleen and blood
lymphocytes were gated on FS & SS dot plot and lymphocytes were analyzed with antibody.
The CD4-positive helper T cells, CD8α-positive cytotoxic T cells and IgM-positive B cells
STAT3-positive T helper17 (Th17) cells and FOXP3-positive T regulatory (Treg) cells weredetected and compare each group.
Result:
The immunophenotyping results shown that there are no significant differences between OTC
treated and control groups due to the high individual differences in blood leukocytes andsplenocytes (Figure 2 and 3). Overall, OTC treatment might have no effects on the immune cell
populations after first OTC administered red seabream juveniles.

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Figure 2. The immunophenotyping results in blood leukocytes. Summary graphs (n = 6 pergroup) of B and T cell frequencies (mean ± SD) are shown. The average percentages (%) of CD8αpositive T cells (A), IgM-positive B cells (B), CD4-positive T cells, STAT3-positive Th17 cells
and FOXP3-positive Treg cells were represented with SD.

Figure 3. The immunophenotyping results in splenocytes. Summary graphs (n = 6 per group)of B and T cell frequencies (mean ± SD) are shown. The average percentages (%) of CD8αpositive T cells (A), IgM-positive B cells (B), CD4-positive T cells, STAT3-positive T cells andFOXP3-positive T cells were represented with SD.
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In order to examine the effect of oxytetracycline on Immunoglobulin M genes, RT-qPCR wasperformed (Figure 4). The RT-qPCR results shown that there are no significant differences
between OTC treated and control groups due to the high individual differences. Overall, OTC
treatment might have no effects on the immune cell populations after first OTC administered red
seabream juveniles.

Figure 4. Relative expression levels of immunoglobulin M genes from OTC administrated
red seabream. Relative expression levels of mIgM (A) and sIgM (B) from liver and mIgM (C)and sIgM (D) from spleen were represented with SD (n = 3 per group). Asterisk indicatedsignificantly difference compare to the control group at same sampling point using one-way
analysis of variance followed by Dunnett’s multiple comparison test (p < 0.05). ND represented
that are not detectable due to the under the detection limit.
Publication/conference presentation:
In progress:◼

With improved data, this study would be presented with other researchers in theconference and published as a paper.

4 / 5Perspectives in future:
This study could contribute to◼

The understanding of the immunomodulatory effects in OTC administrated fish◼

Provides basic immunological knowledge on fish disease prevention for thedevelopment of immunoactive agents◼

The establishment of appropriate regulations and management of OTC usage inaquaculture.

◼

Solving the problem of drug-resistant bacteria occurrence caused by misuse and abuseof antibiotics

Also, by reducing the use of antibiotics, environmental and ecological side effects such as residual
effects could be alleviated.

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