HALYNA TKACHENKO1, JOANNA GRUDNIEWSKA2, ANASTASIIA ANDRIICHUK3, NATALIA KURHALUK1 1Department of Zoology and Animal Physiology Institute of Biology and Environmental Protection, Pomeranian University, Slupsk, Poland 2Department of Salmonid Research, Inland Fisheries Institute Rutki, 83-330 Zukowo, Poland 3Institute of Animal Breeding of National Academy of Agricultural Sciences Kharkiv, Ukraine

(Поступила в редакцию 28.01.2014)

Introduction. Salmonids are an important species for pond aquaculture and extensive open water fisheries in several European countries. Rapid growth and disease resistance are the most important concerns in the present aquaculture industry [1]. Salmonids are vulnerable to furunculosis, a disease caused by the Gram-negative bacterium Aeromonas salmonicida and Aero-monas hydrophila [2, 3]. Infections with A. salmonicida and A. hydrophila are probably the most important disease problems in European aquaculture as they are widespread and cause disease both in fresh water and sea water [4]. The term furunculosis is derived from the characteristic furuncles in muscles, which are common during a chronic course of the disease. Otherwise, the dominant pathological findings are a swollen, dark spleen, and pe-techial hemorrhages in internal organs [4].

Increased incidence of infectious diseases connected with of Aeromonas infection has traditionally been treated with antibiotics, chemotherapeutics and vaccines [5]. Vaccination is a very effective way of protecting animals against infectious disease. Where properly applied in aquaculture, it has significantly reduced the need for antibiotic use as a compensation method for the immunosuppression associated with the intensification of farming fish [6]. However, fish respond differently from avians and mammals to vaccination. Major differences between fish and other vertebrates are that their metabolism and immune response are temperature-dependent. Moreover, fish produce antibodies with lower affinity for antigens [7]. It is generally accepted that successful fish farming depends on the use of vaccination particularly when pathogen eradication is unlikely to be successful [6].

Different kinds of vaccines have been investigated against A. hydrophila including whole cell, outer membrane proteins, extra-cellular proteins, lipopolysaccharides, biofilms attenuated vaccines [4]. While each medicine probably are effective in the treatment of a particular disease, problems arise with the development of possible pathological side effects of immunization in fishes, as well as the emergence of antibiotic resistant pathogenic strains. For optimal protection of salmonids in sea-water, vaccination should be carried out some time before sea transfer, in order to give immunity sufficient time to develop, and to avoid handling stress during smoltifi-cation. On the other hand however, vaccination should not be carried out too early, as the degree of immunity declines with time [4].

Despite the importance and success of vaccination, little is known about the mechanisms of oxidative stress and antioxidant defense in fish during vaccination. In the present study, we determined the influence of vaccination against Aeromonas spp. on responses of oxidative stress and antioxidant defense biomarkers in brain tissue of rainbow trout (Oncorhynchus mykis).

Materials and methods. Clinically healthy rainbow trout with a mean body mass of 135.5±1.5 g were used in the experiments. The study was carried out in a Department of Salmonid Research, Inland Fisheries Institute near the village of Zukowo (Poland). All enzymatic assays were carried out at Department of Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University (Slupsk, Poland).

The fish were divided into two groups and held in 250-l square tanks (70-75 fish per tank) supplied with the same water as during the acclimation period (2 days). Before vaccination, the fish were anaesthetized by Propis-cin solution. Fish were grouped as follows: I) unhandled controls, II) vaccinated by vaccine against furunculosis. The vaccine against furunculosis is a vaccine containing an inactivated strain of A. salmonicida and A. hydrofila in concentration 1x1010 colony forming units (CFU). The vaccine was produce in Department of Epizootology, Faculty of Veterinary Medicine, University of Warmia and Mazury (Olsztyn, Poland). Immersion solution contained 1 liter of vaccine per 10 liters of water. It was prepared immediately prior to vaccination. Immersion lasted from 60 to 120 seconds. The fish were kept for 30 days at 14.5°C after vaccination at a water temperature of 14.5±0.5°C and the pH 7.5.

The animals were quickly captured and killed on 31 days post vaccination (n=15 in each group). Brain tissue were removed in situ. Tissue samples were homogenized in ice-cold buffer (100 mM Tris-HCl, pH 7.2) using a glass homogenizer immersed in an ice water bath to a yield a 10 % homo-genate. Homogenates were centrifuged at 3,000g for 15 min at 4°C. After centrifugation, the supernatant was collected and frozen at -20 °C until analyzed. Protein contents were determined using the method of Bradford (1976) with bovine serum albumin as a standard. All enzymatic assays were carried out at 22±0.5 °C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany) in duplicate.

The enzymatic reactions were started by the addition of the tissue supernatant. An aliquot of the homogenate was used to determine the lipid perox-idation status of the sample by measuring the concentration of thiobarbitur-ic-acid-reacting substances (TBARS), carbonyl groups as an indication of oxidative damage to proteins, as well as superoxide dismutase (SOD), cata-lase (CAT), glutathione reductase (GR) glutathione peroxidase (GPx), and total antioxidant activity (TAC).

Data were checked for assumptions of normality using the Kolmogorov-Smirnov one-sample test and Lilliefors tests (p>0.05). Homogeneity of variance was checked using the Levene test. Significance of differences in the lipid peroxidation level, level of carbonyl derivatives of amino acids reaction, antioxidant enzymes activities was examined using Mann-Whitney U test according to Zar (1999) [8]. Differences were considered significant at p<0.05. All statistical analysis was performed by STATISTICA 8.0 software (StatSoft, Poland).

Results and discussion. Vaccination caused a significant decrease the TBARS level in the brain tissue by 24 % (p=0.017). Aldehyde and ketonic derivates of carbonyl content in the trout vaccinated against Aeromonas spp. were significantly reduced (by 27 %, p=0.008 and by 24 %, p=0.006, respectively) compared to the level in the controls (Fig. 1).


Fig. 1. Aldehyde and ketonic derivates of carbonyl content of oxidatively modified protein (OMP) in the brain tissue of the trout vaccinated against Aeromonas spp. Data are represented as mean±S.E.M. (n=15).

* the significant difference was shown as p<0.05 when compared vaccinated group and unhandled group values.

Brain SOD activity was non-significantly higher than that in the control (by 14 %, p>0.05). CAT, GR, and GPx activities in the brain were significantly inhibited in vaccinated group (by 33.8 %, p=0.033, by 6.5 %, p=0.021, by 62.5 %, p=0.000, respectively). The total antioxidant capacity was significantly decreased by 43 % (p=0.002) in vaccinated group compared to those in the control (Table 1).

T a b l e 1. Enzymatic antioxidant defenses in the brain tissue of the rainbow trout vaccinated against Aeromonas spp.


The study show a post-treatment changes in oxidative stress profile in brain tissue of rainbow trout treated by vaccine against Aeromonas spp. The decrease of aldehyde and ketonic derivates of carbonyl content was observed. However, the post-treatment levels of antioxidant defenses as well as total antioxidant capacity showed decrease after vaccination. Impairment in the synthesis of enzymatic and nonenzymatic antioxidant of vaccinated fish may be the most important factor in reducing levels of cellular total an-tioxidant.

Certain conditions (such as disease, exposure to toxins, immunization, aging, exercise etc.) can increase the rate of oxidative damage, a condition called oxidative stress [9, 10]. Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, which may lead to a series of biochemical and physiological changes, thus, altering normal body homeostasis and tissue injury [9]. Despite the potential danger of the ROS, cells have a variety of defence mechanisms to neutralize the harmful effects of free radicals [10].

The first line of defence against oxidative stress consists of the antioxidant enzymes SOD, CAT and GPx, which convert superoxide radicals into hydrogen peroxide and then into water and molecular oxygen [10]. Induction of antioxidant enzymes is an important line of defense against oxidative stress in fish [11]. SOD is a group of metalloenzymes that catalyzes the dismutation of superoxide to hydrogen peroxide, plays a crucial antioxidant role and constitutes the primary defense against the toxic effects of superoxide radicals in aerobic organisms [12]. In our study, nonsignificant increase of SOD activity was observed in brain of vaccinated trout. It could be adaptive response to the immunization which neutralizes the impact of ROS and may be of importance in preventing membrane lipid peroxidation when the latter is initiated by a combination of Fe3+ and O2--generating system [13]. A similar result of increased SOD activity has been reported in carp tissues following xenobiotics exposure [10, 14].

Skugor et al. (2009) used multiple gene expression profiling to outline the mechanisms that determine success of vaccine protection against Aeromonas in Atlantic salmon and to search for the correlates of protection [15]. Several genes with known immune functions showed higher expression levels in liver of salmon, including the phosphotyrosine independent ligand for lymphocyte-specific protein tyrosine kinase Lck SH2 or nucleoporin p62 that regulates activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) by tumor necrosis factor a (TNFa) [15]. Up-regulation of NF-kB and activator protein AP-1 by pathogens and cytokines induces mass production of immune mediators and effector proteins. NF-kB and Jun proteins respond to various cell damaging factors, including free radicals and other genotoxic agents that can cause apoptosis, growth arrest, altered DNA repair or altered differentiation. NF-kB can also activate protection against oxidative and cellular stress by providing anti-apoptotic and proliferation-promoting signals. A suite of chaperones and protein adaptors of different types (heat shock proteins, 14-3-3 proteins, glucose regulated proteins, DnaJ, cyclophilins) were expressed at higher level in fish with low resistance to furunculosis and this could be evidence of cellular stress [15]. Genes for proteins involved in regulation of redox status and protection against ROS had higher expression levels in vaccinated fish with high resistance to furunculosis [15].

In our study, the activities of CAT, GPx, as well as GR were significantly decreased in the brain tissue of vaccinated trout. The decreased CAT activities indicate the reduced capacity to scavenge hydrogen peroxide produced in brain tissue of vaccinated trout in response to oxidative stress. Similarly, the inhibition of the CAT activity by pesticides has been reported in various studies in fish species [10]. GPx is dependent on access to gluta-thione disulfide by the NADPH-dependent GR. Decrease of glutathione-mediated antioxidant defense system results in oxidative stress and increased cytotoxicity, whereas elevation of intracellular GSH levels is recognized as an adaptive response to oxidative stress [9].

Conclusion. Oxidative stress biomarkers analyses revealed significant differences between vaccinated fish against furunculosis. We noted strong association between oxidative stress and brain tissue responses. Both the glutathione-mediated antioxidant defense system and endogenous CAT play a critical role in intracellular antioxidant defense in vaccinated fishes. Glu-tathione-dependent enzymes activity decreased in vaccinated trout. In contrast, SOD activity showed increase, which indicate a different response of antioxidant enzymes to vaccination. Furthermore, brain of vaccinated trout had lower level of aldehyde and ketonic derivates of oxidatively modified protein, as well as lipid peroxidation biomarkers, while antioxidant defenses became more susceptible to oxidative damage induced by vaccination. Acknowledgments.

This work was supported by grant of the Pomeranian University for Young Scientists. This study was also carried out during Scholarship Program (N51200912) of Anastasiia Andriichuk supported by The International Visegrad Fund in the Department of Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University (Slupsk, Poland). We thank to The International Visegrad Fund for the support of our study.


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