Int. J. Aquat. Biol. (2019) 7(4): 211-217 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2019 Iranian Society of Ichthyology Original Article Effects of florfenicol on skin mucus immune parameters and immune related genes expression in zebrafish (Danio rerio) Narmin Ezatrahimi1, Siyavash Soltanian*1, Mostafa Akhlaghi1, Seyed Hossein Hoseinifar2 1Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran 2Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. s Article history: Received 3 June 2019 Accepted 24 August 2019 Available online 2 5 August 2019 Keywords: Florfenicol Immune Mucus Gene expression Zebrafish Abstract: Florfenicol (FF) is a common, inexpensive antibiotic with relatively low toxicity. The present study investigates the possible effects of Florfenicol on cutaneous mucus immune parameters and immune related genes expression in Danio rerio. After two weeks adaptation, fish were stocked in experimental units at density of 20 fish per aquaria and fed with experimental diets containing 0, 10, 30 and 50 mg/kg FF per for 11 days. Evaluation of immune parameters at the end of trial showed that the control group had a significantly lower level of lysozyme activity when compared with those treated with antibiotic. However, the skin mucus total immunoglobulins level of fish fed diets containing antibiotics did not show any significant difference compared to the control group. The highest expression level of TNF-α, IL1β and lysozyme was observed in fish treated with 10 mg/kg FF while the lowest expression was noticed in those fish treated with 30 and 50 mg/kg FF. The present results indicate the relatively positive effects of this antibiotic on the immune system of zebrafish, and it seems that the appropriate dosage of the drug can serve as an immunostimulant for zebrafish. Introduction Fishes are vulnerable to a vast variety of pathogenic and non-pathogenic micro-organisms. As aquaculture is developing and new methods of culture are developed, there is an increase in incidence rates of bacterial, viral, parasitic diseases as well as environmental problems. To deal with stressful conditions, fish develop adaptation mechanism. In fact, through development of such mechanism, natural or homeostatic status of body can be maintained (Makvandi et al., 2011). A complex set of specific and non-specific immune parameters concerning stress response in fish have been identified (Ellis, 2001). Primary response to pathogens is formed by the primary defense system which include the skin mucus, skin, gills, gastrointestinal tract as well as blood components, including natural killer cells and phagocytes (Anbarasu and Chandran, 2001). The mucus layer consists of glycoprotein, proteoglycan and proteins that, besides providing physical *Correspondence: Siyavash Soltanian DOI: https://doi.org/10.22034/ijab.v7i4.631 E-mail: siyavashsoltanian@yahoo.com protection, contain secretory compounds such as agglutinins, lysines, lysozymes, non-specific precipitators, acute phase protein. Also, the presence of natural anti-bodies provide defensive-chemical role against environmental micro-organisms (Soltani, 2008). Fish are commonly exposed to chemicals released into aquatic system which per se can disrupt innate and adaptive immune system (Yousefi and Hoseinifar, 2018). Antibiotics have significant roles in controlling diseases. Besides, in aquaculture they have formerly been used as growth stimulators, antibacterial agents, and for a variety of other purposes (Masahiro et al., 1999; Li et al., 2012). However, due to their residual effects on the fish and shrimp tissues and consumers' lack of interest, application of antibiotics is not recommended in aquaculture (Khajeali, 2012). The interaction between medications and lymphoid tissues may affect the performance and balance of immune system causing unintentional negative effects such as 212 Ezatrahimi et al./ Florfenicol effect on zebrafish immunity immunosuppression, uncontrolled cell proliferation, changes in defensive mechanisms of host against pathogens, and even neoplasia (Ren et al., 2014). Several immunomodulatory effects of anti-bacterial and other medications have been reported (Lunden et al., 1999). In recent years, several reports on antibiotic resistance in pathogens raised a concern about fisheries and public health, hence, emphasizing identification and application of new antibacterial compounds to deal with such bacterial diseases (Park et al., 2008). Florfenicol (FF) is a broad-spectrum antibiotic, uses for treatment of various infections, which has been confirmed in food-producing animals due to low price and relatively low toxicity (Carty et al., 2008). In addition to merits of chloramphenicol such as wide spectrum effects and high tissue infiltration, FF has a longer elimination half-life. On the other hand, due to substitution of fluorine with a hydroxyl group in its chemical structure, florfenicol is more resistant to enzymatic inactivation, putting effects on a wider spectrum of chloramphenicol-resistant bacterial strains (Anadon et al., 2008). Despite an increase in use of FF, some studies document its toxicity (Hu et al., 2014). Therefore, it is essential to determine its effects on mucosal immune level of fishes as their primary defense mechanism against pathogens. Zebrafish (Danio rerio) has recently been introduced as a model for rapid analysis of gene performance (Zon et al., 2005). It is widely used in different laboratories for genetic research (Haffter et al., 1996; Driever et al., 1996). Hence, the present study aims to investigate the effects of florfenicol on the fish mucosal immunity as the primary defensive mechanism against pathogens. Materials and Methods Fish and experimental design: A total of 240 zebrafish were purchased from a private farm. Each experimental unit (84 L aquarium) contained 20 fish. Prior to the study, a 2-week adaptation period with laboratory conditions was considered. They were fed a commercial food Biomar (BioMar SAS, Nersac, France). Throughout the study, water temperature, pH, and dissolved oxygen were monitored daily and maintained at optimum rates. FF was purchased from the Rooyan Company and added to the basal diet. The experiment was designed as completely randomized with four treatments (three experimental and one control) repeated in triplicates. The feeding trial was lasted for 11 days, and during this period, fish fed 3% of body weight, twice a day. Experimental diet preparation: In this study, commercial food, Biomar (BioMar SAS, Nersac, France) (Table 1) was used as the basal diet and different doses (10, 30 and 50 mg/kg) of FF were added. Skin mucus immune parameters: Skin mucus samples were obtained based on Ross et al. (2000). Accordingly, three fish from each tank were randomly selected and anaesthetized. The mucus samples were kept in -80°C before determining the lysozyme activity and the total Ig level. Enzymatic assay of lysozyme was performed using turbidimetric method (Subramanian et al., 2007). In this procedure, Micrococcus Luteus (ATCC4698) was used as substrate. The mucus total Ig levels was measured based on the method suggested by Siwicki and Anderson (1993). Determination of immune genes expression: The intestinal tissue samples were obtained from samples and immediately transferred to liquid nitrogen tank. To extract total DNA, 50-100 milligrams of the tissue was homogenized in 1 ml of RNX-Plus for 15 minutes in room temperature according to company guidelines. After drying the product, it was dissolved in 50 ml of distilled water and then kept in the refrigerator at - 80°C. Prior transferring samples to refrigerator, the concentration enzyme activity of RNA were checked using spectrophotometer in 260/280 nanometers. Table 1. The proximate composition of the basal diet. Proximate analysis (%) 93.2 Dry matter 38.6 Crude protein 15.1 Crude lipid 11.0 Ash 213 Int. J. Aquat. Biol. (2019) 7(4): 211-217 Also, the quality of RNA was assessed using agarose gel electrophoresis as well as dyeing with ethidium bromide (Miandare et al., 2013). One ml of total RNA was used to build cDNA using bio rad kit. Copying process was conducted according to the manufacturer's instructions. The resulted cDNA was kept in -20°C (Gioacchini et al., 2010). The primers of qPCR were designed for TNF-∝, IL-1 and Lys genes in gene bank based on protected areas of DNA sequences (Table 2). Then, the primers were added to the samples for analyzing gene expressions. Copying process was conducted according to a specific protocol. Cyber green method was used to investigate gene expression and the samples were put in Real Time PCR instrument to measure gene expression. Statistical Analysis: SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The statistically significant differences (P<0.05) were determined using one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. Results Skin mucus immune parameters: The skin mucus lysozyme activity of fish in control group's was significantly lower than that of the experimental groups treated by various doses of antibiotic (P<0.05) (Fig. 1). The results showed the total immunoglobulin in fish mucus was not significantly different between treatments (P>0.05) (Fig. 2). Immune related genes expression: gene expression studies revealed the highest expression of TNF-α gene in 10 mg of FF treatment (Fig. 3), whereas the others did not have significant differences compared to control. In addition, in 10 mg of FF treatment the expression of IL-1β gene was the highest (Fig. 4), and those of 30 and 50 mg had the lowest level, and the control one showed an intermediate levels (P<0.05). The expression of Lysozyme gene in 10 mg/g FF was Table 2. Sequences and accession numbers of primers used. Primer name Primer Sequence Application Accession number i1b q-PCRF CGTCTCCACATCTCGTACTCA Immune AY340959.1 il1b q-PCRR GTGTCTTTCCTGTCCATCTCC tnf-alpha q-PCRF CTGCTTCACGCTCCATAAGA Immune AY427649.1 tnf-alpha q-PCRR CTGGTCCTGGTCATCTCTCC lyz q-PCRF GGCAGTGGTGTTTTTGTGTC Immune NM_139180.1 lyz q-PCRR CGTAGTCCTTCCCCGTATCA β-actin q-PCRF AGCAGATGTGGATCAGCAAG Housekeeping gene NM_131031.1 β-actin q-PCRR TACCTCCCTTTGCCAGTTTC Figure 1. The effect of different doses of florfenicol on mucus lysozyme activity in zebrafish. Values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. Different letters above the bars denote a significant difference between treatments (P<0.05). Figure 2. The effect of different doses of florfenicol on mucus total Ig in zebrafish. Values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. Different letters above the bars denote a significant difference between treatments (P<0.05). 214 Ezatrahimi et al./ Florfenicol effect on zebrafish immunity significantly higher than others (Fig. 5). However, no significant difference was noticed when compared with control group. Discussions Improvement and protection of fish health is one of the main factor in the aquaculture industry (Mahghani et al., 2014). Increasing the productivity of fish farming is associated with stressful conditions, which provides a sensitive environment for infectious diseases (Dehghan et al., 2016). Lysozyme is one of the most important parameters of the non-specific humoral immune system. It is a polypeptide with a molecular weight of 14 to 18 kDa. It exists in a wide range of vertebrates, including freshwater and marine species, and is known as an antibacterial agent (Itami et al., 1992). This enzyme is secreted from white blood cell granules (mostly by neutrophils, monocytes and less by macrophages) as well as leukocyte-rich tissues such as kidneys, mucus membranes, spleen, gills and digestive tract since these organs are highly susceptible to invasion by pathogenic bacteria (Holloway et al., 1993). The enzyme can break down the glycosidic bond of peptidoglycan layer in the bacterial cell wall. Lysozyme has direct effects on gram-positive bacteria; and can eliminate the gram- negative bacteria with the help of complement system (Yano, 1996). In the present study, evaluation of skin mucus lysozyme activity revealed significant increases in all experimental groups compared to the control group. However, the treatment containing 30 mg FF showed more activity, which was not statistically significant. Reda et al. (2013) reported that lysozyme activity is significantly increased in tilapia after FF administration, but had no significant effect on IgM, phagocytosis and ALT as seen in the present study. In contrast, administration of FF in hybrid tilapia diet at rate of 0.02 g kg-1 had no effect on serum lysozyme. It may be due to different conditions used in previous studies, fish species, temperature, antibiotic dose and Figure 2. The effect of different doses of florfenicol on relative expression of TNF-α gene in zebrafish. Values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. Different letters above the bars denote a significant difference between treatments (P<0.05). Figure 4. The effect of different doses of florfenicol on relative expression of IL-1β gene in zebrafish. Values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. Different letters above the bars denote a significant difference between treatments (P<0.05). Figure 5. The effect of different doses of florfenicol on relative expression of lysozyme gene in zebrafish. Values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. Different letters above the bars denote a significant difference between treatments (P<0.05). 215 Int. J. Aquat. Biol. (2019) 7(4): 211-217 route of administration which can affects drug absorption (Bjorklund and Bylund, 1990). According to Ren et al. (2014), juveniles of Litopenaeus vannamei revealed no significant differences in antibacterial activities in plasma following administration of 100 mg kg-1 FF, whereas 200 mg kg- 1 FF treatment suppressed them, the total hemocyte count and phagocytic activity decreased significantly in both treatments. In the present study, the total Ig level in the skin mucus of fish fed diet containing different antibiotic doses showed no significant difference compared to control. Since the function of a gene at the mRNA level varies from that of the protein after translation, the evaluation of the expression of lysozyme gene can be reliable in evaluation of the effect of immunostimulants on this gene. The level of lysozyme or its activity is viewed as an important indicator in the primary defense of fish. There are changes in levels of lysozyme activity or mRNA expression in response to infectious diseases or stress. Based on the results, the highest expression of lysozyme was in fish treated with 10 mg/g FF. However, there was no significant differences when compared with the control. IL1β and TNF-α expression in the 10 mg/kg FF were highest; however, 30 and 50 mg/kg of FF had the lowest expression level, and the fish in control group showed an intermediate level. The tumor necrosis factor (TNF) is a signaling protein (cytokine) participating in systemic inflammation as one of the cytokines forming the reaction of the acute phase. TNF-α serves as intermediate proteins in immune cells and puts effects on them (Gruss, 1996). TNF-α is the main mediator of acute inflammatory response to Gram-negative bacteria and other bacterial microbes and accounts for most of the systemic disorders of acute infections. The results showed that the expression of this gene in fish treated with 10 mg/kg was highest. In contrast, suppressing effects of FF on rat immune responses showed that FF could suppress humoral and cellular immune responses in mice (Guan et al., 2011). Similarly, some antibiotics have a suppressive effect on increased levels of TNF-α (Er et al., 2010; Er and Yazar, 2012). In this study, the expression of this gene decreased in a dose-dependent manner. In brown trout, serum levels of TNF-α in fish treated with FF were not suppressed following LPS injection. This may be due to the dose of FF or differences in the species type (Ayse and Burak, 2014). According to Caipang et al. (2009), oral administration of FF and oxolinic acid significantly increase the anti-inflammatory cytokines, including IL-1β and IL-8 in Atlantic cod (Gadus morhua). Changes in the immune response and antioxidant defense of the Atlantic cod have also been documented in relation to the oral administration of oxolinic acid and FF. 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