Ispitivanje mogućnosti korišćenja izoenzimskih oblika superoksid dizmutaze 1 kao biomarkera intenziteta parazitskih infekcija kod ovaca / Investigating the possibilities of using superoxide dismutase 1 isoenzyme forms as biomarkersfor intensities of paras

In clinical pathology of sheep, parasitic infections occupy a significant place and besides health, pose a great economic problem in sheep breeding. Sheep are highly susceptible to parasitic infections, even under conditions of very low pasture infections. In this way, sheep support the sustainability and amplification of parasitic fauna intensity in an epizootiological region and may at the same time serve as “indicators” of animal species with the aim of evaluating the presence of parasitic fauna in a specific region. The evaluation of parasitic infection intensity in the host is of special significance, primarily for the therapeutic measures necessary to be taken, but also, represents an essential criterion employed in selecting more resistant individuals. The presence of parasites and their developing forms during migration, leads to health disturbances in the animals, when the degree of damage is in direct correlation with the number of parasites present and/or their larval forms, ie. with the intensity of parasitic infection. Free radical species originating during parasitic infections, directly inhibit some of the antioxidative enzymes. Changes on these enzymes and their inhibition have an influence on the protective capacity from oxidative damages and a relative participation of certain components in the total antioxidative potential. For this reason, the hypothesis has been established that the analysis of these changes may serve as a diagnostic tool with the aim of determining the intensity of parasitic infection. SOD (EC 1.15.1.1) presents the first line of defense under conditions of disturbed redox balance, as they prevent the formation of a series of very harmful reactive oxygen and nitrogen species (hydroxyl radicals and peroxynitrites) and the initiation of oxidative chain reactions, which as an outcome are significant damages to biomolecules (lipids, proteins and nucleic acids). A known “paradox” is that SOD 1 is irreversably inactivated at high concentrations of hydrogen peroxide, which is a product of the reaction catalyzed by this enzyme. Considering the previously elaborated fact that parasitic infections lead the organism of the host into oxidative stress, the aim of this doctoral thesis was to more precisely investigate changes in the isoenzymatic profile of SOD 1 in sheep and examine if these may serve as biomarkers for the intensity of parasitic infection. The study was performed on a total of 300 sheep (aged 12 to 18 months), on strains of autochtonous Zackel sheep breed (Sjeničko-pešterska, Lipska and Vlaška vitoroga) which are included in the programme for conserving animal genetic resources in the Republic of Serbia as well as on imported breeds of sheep (Würtemberg, Ile de France and Charollais) kept under semi intensive conditions in four regions. Within each investigated breed/strain, a control group of sheep was established (n=10), negative to parasitic infection. Sampling of feces for parasitological and blood for biochemical assaying was performed during the course of three days , in the morning and in the evening.The distribution of parasites in sheep was evaluated using the native smear corpological technique, by sedimentation and flotation methods. The degree of infection intensity per sheep was quantitatively established by the method of McMaster (modified by Zajiček). The oxidative stress parameters were measured for catalase activity (CAT), the red cell membrane damage by level of malondialdehyde (MDA), while carbonyl and thiol plasma protein group concentrations were used as indicators of the degree of protein oxidative modification. The activity of Cu,Zn - superoxide dismutase (SOD 1) and relative distribution of lactate dehydrogenase (LDH1-LDH5) activity were determined electrophoretically. In the studied regions, in the strains of autochtonous Zackel sheep breed and imported breeds of sheep, parasitic infections were detected as follows: Trichostrongylidae, Nematodirinae, Protostrongylidae, Trichuris spp. Strongyloides spp. Anoplocephalidae, Dicrocoelium dendriticum and Eimeria spp. Coprological examinations revealed various extensities and intensities of parasitic infections in the strains of autochtonous Zackel sheep breed as well in the imported breeds in the regions studied. In the strains of autochtonous Zackel sheep breed, lower extensities and intensities of parasitic infections were determined (p<0.05). The level of oxidative stress in autochtonous strains of Zackel and imported sheep breeds is different depending on the extensity and intensity of parasitic infection. It has been determined that the oxidative modifications of proteins from imported breeds are more expressed under same extensity and intensity of parasitic infection when compared with the autochtonous Zackel sheep strains (p<0.01). It has been determined that the basal level of cell membrane lipid peroxidation is higher in the control group of imported sheep breeds when compared with the autochtonous Zackel strains (p<0.001). In the presence of parasitic infection, both in the autochtonous Zackel strains and imported sheep breeds, results in oxidative modification of lipids, this being more expressed in the imported sheep breeds (p<0.05). The total SOD 1 activity is higher in strains of the autochtonous Zackel breed when compared to the imported sheep breeds (p<0.05), this confirming the stronger response of Zackel strains to oxidative stress provoked by parasitic infection. With the increase of parasite infection intensity, the total activity of SOD 1 enzyme increases in the strains of the autochtonous Zackel breed as well as in the imported breeds. Catalase activity is reduced with the increase in parasite infection intensity, in all studied breeds, strains of the autochtonous Zackel breed and imported sheep breeds (p<0.05). On the basis of the relative isoenzyme distribution of the lactate-dehydrogenase (LDH) enzyme, damages to cells from various organs were determined, this being dependent on the presence of the species and intensity of parasitic infection. In the studied strains of the autochtonous Zackel and imported sheep breeds infected with Strongyloides spp, Dicrocoelium dendriticum and Protostrongylidae, damages to the cells of the miocard (LDH2 ) and lung (LDH3) were determined. In the presence of parasitic infections with Strongyloides spp, Trichuris spp and Dicrocoelium dendriticum damage to liver cells has been detected (LDH5). Depending on the intensity of parasitic infection, production of RNS increases in strains of the autochtonous Zackel breed as well as in the imported sheep breeds. In the imported sheep breeds, the level of nitrosative stress is more intense when compared to the Zackel strains (p<0.001). Applying SDS-PAG electrophoresis and HPLC/MS, the molecular mass of the sheep SOD 1 was determined more precise, 31.84 kDa and 29.5 kDa. By isoelectrofocusing, the isoelectric point of sheep SOD 1 was determined to be 6.42. In in vitro experiments, during exposure of SOD 1 to the action of hydrogen peroxide, a 50% activity inhibition of this enzyme was proven. The degree of activity inhibition of this enzyme depended on the exposure time and applied concentration of hydrogen peroxide. With electrophoretic analyses, it has been determined that exposure of SOD 1 to hydrogen peroxide leads to the dimerization and fragmentation of this enzyme and formation of 6 protein bands with molecular weights of: 63.5 kDa, 31.84 kDa, 26.91 kDa, 21.97 kDa, 15 kDa and 13 kDa. By GC/MS analysis, it was determined that hydrogen peroxide leads to the carbonylation of SOD 1 (m/z 881.91 and m/z 909.42) and irreversible oxidation of the enzyme thiol groups (m/z 938.65 and m/z 969.96). By isoelectrofocusing, also the presence of SOD 1 with an isoelectric point of 5.0 has been determined, which was the consequence of the oxidative modifications of this enzyme. With the aid of spectral electron paramagnetic resonance, in in vitro experiments, for the first time, the formation of the nitrogen dioxide radical was proven as a consequence of SOD 1 oxidative modification. It has been determined that isoenzymatic forms of SOD 1 may be used as biomarkers for evaluating the intensities of parasitic infections in sheep. Depending on the intensity of the parasitic infection and the consequences of oxidative modifications, changes in isoenzymatic forms of SOD 1 appear.