Milanka Jezdimirovic

The application of adsorbent bentonite in oxidative stress induced by paraquat

The protective effect of bentonite (natural and synthetic) in oxidative stress induced by paraquat was studied on 32 adult male Wistar rats. The animals were divided into four groups (n=8). The first group received only paraquat p.o. (via a gastric tube). An hour after administration of paraquat, the second and the third group were treated with natural and synthetic bentonite, respectively, while the fourth group was untreated (control). All three experimental groups were treated once a day, throughout 8 consecutive days. Blood samples were taken 0, 1, 4, 6 and 8 days after the beginning of the treatment. Oxidative stress induced by paraquat was estimated by means of catalase activity (CAT - mmol/H2O2/min/g Hb) and malondialdehyde quantity (MDA - nmol/g Hb). Oxidative stress proved to be significant an hour and 192 hours after the beginning of chronic intoxication by paraquat. Both natural and synthetic bentonite expressed a protective action from oxidative stress in the period from 24 h to 144 h post application, acting through the external capacity of cationic exchange, which is known to be approximately 10%. The absorption of paraquat depends on the size of the molecule and its polarisation and is performed by the mechanisms of ion exchange (ionic and electrostatic interaction). Nevertheless, both absorbents developed a significant protective effect (approximately 50 %) 1 h and 192 h after the first application of paraquat, indicating that these protectors act as molecular sieves and thus suppress the process of lipid peroxidation and the development of even more intensive oxidative stress.

Downregulation of nicotinic and muscarinic receptor function in rats after subchronic exposure to diazinon

Highlights • We studied the development of tolerance to subchronic p.o. administration of DZN in rats, under both in vivo and in vitro conditions• As a consequence of AChE inhibition, ACh neurotransmitters are being accumulated in over stimulated nicotinic and muscarinic receptors.• With isolated diaphragm and ileum, we examined the down regulation of nicotinic and muscarinic receptor function through EFS technique.• The results of our research could be useful in forensic diagnostics of organophosphate poisoning.

Investigation of the sensitivity of E. coli strains isolated from domestic animals to antibiotics and hemiotherapeutics in vitro.

Resistance to antibiotics is not a modern phenomenon. On the contrary, penicillin resistance in some bacterial strains developed quickly after its introduction into daily practice. At the same time some bacterial strains developed resistance to almost all known antibiotics, vancomycin included. Vancomycin was for a long time the only efficient antibiotic against staphylococcal infections. It is of special concern the fact that antibiotics are in everyday exploitation in agriculture and veterinary clinical practice which use them not only as a mean of therapeutic treatment, but as an additive in animal feedstuffs in order to promote growth and prevent bacterial infections. The same antibiotics are used in human medicine, which is a persistent problem. In such a way it is possible to develop resistance which can be transferred to human pathogenic bacteria via mobile genetic elements. The incidence of resistant bacterial strains increases year after year not only on a local level, but on a global scale, as well. Monitoring of the use of antibiotics and chemotherapeutics in the Republic of Serbia is not established as such, our intention was to study a number of bacteria isolated from cattle, pigs, poultry, dogs and cats. At this time we are presenting the results for pathogenic strains of E. coli in order to determine the use of antibiotics and chemotherapeutics of the old and new generations in domestic animals. E. coli sensitivity was investigated with the disc diffusion test for: ampicillin, amoxicillin with clavulanic acid, tetracycline, chloramphenicol, gentamicin, and ceftriaxon, sulphamethoxasole with trimethoprim, ciprofloxacin and florfenicol. E. coli strains resistant to three or more antibiotics were tested by means of agar dilution method for ciprofloxacin, tetracycline, chloramphenicol, gentamicin and amoxicillin with clavulanic acid by determination of the minimal inhibitory concentration (MIC). The tested E. coli strains resulted resistant to all antibiotics and chemotherapeutics with the exception of ceftriaxon and florfenicol. The highest resistance incidence (87.5%) was to tetracycline in E. coli strains isolated from pigs, 60% for E. coli strains isolated from cattle, 56% isolated from poultry and 20% originating from dogs. E. coli strains isolated from cats were sensitive to tetracycline. The highest incidence of ampicillin resistance was determined for E. coli strains originated from poultry (78%).

UTICAJ PRODUŽENE PERORALNE PRIMENE EUGENOLA NA HEMATOLOŠKE I NEKE BIOHEMIJSKE PARAMETRE KRVI KOD PACOVA

The possible haematotoxic, hepatotoxic and nephrotoxic effects of eugenol in rats after two-week and the four-week continuous use were tested. The experiment was conducted on 72 male Wistar rats divided into six equal groups. Four were treated with different doses of eugenol (10 mg/kg bw/day, 50 mg/kg/d, 200 mg/kg bw/d and 400 mg/kg bw/d), the control group received the vehikulum (0.5% methyl cellulose, 20% propylene glycol and water), and the sixth group was the absolute, untreated control. Eugenol and the vehikulum were administered with a gastric probe, on a daily basis, for four weeks at a rate of 1 ml/100 g body weight of rats. Blood was collected by cardiac puncture of the anesthetised rats on days 14 and 28 in order to assess the haematological and biochemical blood parameters: haematocrit, red blood cell count, white blood cell and platelet counts, leukocyte formula, hemoglobin, MCV, MCH, total protein concentration, albumin, urea, creatinine, and the activity of the alanine aminotransferase (ALT), alkaline phosphatase (ALP) and creatine kinase (CK). The results showed that, having been applied for two and four weeks at doses of 10, 50, 200 and 400 mg/kg bw/day, eugenol did not show any haematotoxic, hepatotoxic or nephrotoxic effects. Eugenol administered for four weeks does not significantly affect the number of erythrocytes, haemoglobin concentration, haematocrit, red blood Ветеринарски журнал Републике Српске Veterinary Journal of Republic of Srpska (Бања Лука-Banja Luka), Вол/Vol.XIII, бр/No.2, 131–142, 2013 Milanka Jezdimirović и сар.: Uticaj produžene peroralne primene eugenola na hematološke i neke biohemijske parametre krvi kod pacova 133 cell volume, leukocyte count and leukocyte formula. Applied for two weeks it caused a significant increase in the mass of haemoglobin in erythrocytes. This effect is not specific and does not depend on the dose or the treatment duration. Eugenol in doses of 10 and 200 mg/kg/day applied for four weeks caused a significant reduction in the number of platelets, whilst the highest dose tested (400 mg/kg/day) caused a significant increase in comparison to the number of platelets in rats treated with 10 and 200 mg/kg/day. Changes in the number of platelets induced by eugenol qualitatively different and are not related to dose or length of treatment. Eugenol applied for two and four weeks does not significantly affect proteinaemia, albuminaemia, serum urea and creatinine concentrations, and serum ALP activity in rats. Three assessed doses of eugenol (10, 50 and 200 mg/kg/d) significantly decreased, whereas the highest dose increased the ALT activity. However, eugenol applied for four weeks at a dose of 200 and 400 mg/kg/d caused a statistically significant decrease in CK activity.

Haemorrhagic-necrotic enteritis in heavy breeds broilers

The aim of the investigation was to determine the influence of Clostridium perfringens type A on the development of pathomorphological substrate, its intensity and distribution in fifteen weeks old heavy breeds broilers. The investigation was carried out on corpses of 8 hens and 7 roosters of heavy breeds of provenance COBB 500. After the completion of the autopsy, samples of altered parts of jejunum and liver were taken for histopathological examination, and jejunum intestinal contents for bacteriological examination. In all the corpses, in open pleuroperitoneal cavity, even in situ, an altered part of jejunum can be noticed. It was extremely dilated the entire length, and its wall was bluish-gray with disseminated subserous punctiform blood extravasates. When opened, semi-liquid content with blood coagulums and patches of necrotic mucosa went out of it. By microscopic examination of small intestine tissue cuttings, colored by HE method, there was observed a diffuse necrosis of intestinal villi. They were desroyed and replaced by eosinophilic structureless mass. Furthermore, there could be noticed submucose oedema, capillary congestion and blood extravasates in mucosa, as well as infiltration of neutrophilic granulocytes in lamina propria. These microscopic alterations reflect hemorrhagic necrotic enteritis. By microscopic examining of small intestine cuttings colored according to Brown & Brenn method, colonies of bacteria in distal parts of the submucosa were found out. Using bacteriological tests in anaerobic conditions, there was isolated a culture identified as Clostridium perfringens. After applying of multiplex PCR, the obtained isolate was genotyped as Clostridium perfringens type A. On the basis of pathomorphological, bacteriological and molecular examinations, it can be concluded that the infection of heavy breeds with Clostridium perfringens type A is manifested by appearance of haemorrhagic-necrotic jejunitis, that the causer penetrates deeply into jejunum tissue and that wheat and wheat bran were a favoring factor for proliferation of the etiological agent. [Projekat Ministarstva nauke Republike Srbije, br. III 46009 i br. TR 31062]

The determination of the concentrations of metamizol sodium in inflamed joints of pigs after intravenous and iontophoretic application

Concentrations of the NSAID metamizol sodium (MmNa) in the synovial fluid and hyaline cartilage of inflamed knee and elbow joints of pigs after i.v. application and iontophoresis (IPh) were investigated. The research was conducted on 14 male pigs divided equally into two experimental groups and exposed to artificial inflammation of knees and elbows prior to the application of MmNa. The first group (A) was administered 2.5 g (twice the maximum therapeutic dose) MmNa intravenously, whilst the second group (B) was exposed to the same dose, but applied by IPh into contralateral knee and elbow joints. Four hours after the application of MmNa biopsies of the affected knee and elbow joints were performed. The average concentration of MmNa in the synovial fluid of inflamed joints in group A was 9.81±1.96μg/g, while in group B was 170.66±2.07 μg/g, being 17 times higher. The average concentration of MmNa in the hyaline cartilage was 2.29±1.16μg/g following i.v. administration and 98.36±21.58μg/g after IPh, i.e. approximatelly 43 times higher. This led to the conclusion that IPh of MmNa, resulting in incomparably higher concentrations in inflamed joints without any adverse systemic effects, has an advantage over i.v. application.

The evaluation of the concentrations of methylprednisolone applied intravenously and by iontophoresis in the pig.

The current clinical research on pharmacokinetics of methylprednisolone was performed on male pigs to whom was administered either intravenously or locally, via iontophoresis. Equal doses of methylprednisolone sodium succinate (MPSS) were applied, i.e. 40 mg it total per animal. In all pigs artificial inflammation of knee and elbow joints was provoked four days prior to the treatment. Four hours after the application of methylprednisolone tissue samples (both synovial fluid and hyaline cartilage) were obtained from the inflamed joints and subjected to analysis. The quantification of the drug was performed by HPLC technique. The results indicated high quantities of methylprednisolone both in the synovial fluid and hyaline cartilage, the concentrations being significantly higher in animals after iontophoretic application (17.15±3.11 and 12.70±2.19 μg/g, respectively) in comparison with the animals treated intravenously (0.33±0.11 and 0.21±0.06 μg/g, respectively). Thus, iontophoresis was proved a highly advisable clinical means of application of methylprednisolone, especially having in mind the possibility of avoiding systemic adverse effects which are present after parenteral drug administration. In addition, it enables higher therapeutic concentrations of MPSS to be obtained both in the synovial fluid and in the hyaline cartilage of the treated inflamed joints.

Investigations of eugenol efficacy in treatment of mange in swine

The acaricide efficacy, tolerability and safety of the active ingredient of the etheric oil of cloves eugenol was investigated in the treatment of mange in swine, and the obtained results were compared with the results of acaricide efficacy of the synthetic acaricide permethrin, which has been in use for quite a some time. A single application of permethrin in the form of a 1% solution showed maximum efficacy of 62.5%, and after three applications of 75.0% in the treatment of sarcoptes in swine mange. A single application of eugenol in the form of a 10% solution had maximum efficacy of 75.0%, and applied three times an efficacy of 100% in curbing Sarcoptes scabiei var. suis. A single administration of 20% eugenol solution showed maximum efficacy of 87.5%, and applied three times it was 100% efficient in curbing Sarcoptes scabeiei var. suis. The best efficacy in the treatment of sarcoptes mange in swine was achieved with three applications of eugenol in a concentration of 20%. This maximum effect (100%) was obtained already after the second treatment. Eugenol in a concentration of 10% was safe for local application on skin because it does not cause any undesired reactions, while a 20% concentration caused irritation followed by a passing redness and disquiet in a smaller number of treated animals. The results of comparative investigations of acaricide efficacy of permethrin and eugenol demonstrate that there is resistence in Sarcoptes scabiei var. suis to permethrin. The biocide eugenol can safely be recommended for the treatment of sarcoptes mange in swine.

Pharmacokinetics of diclofenac in pigs after intramuscular administration of a single dose.

The pharmacokinetics of diclofenac was studied in 10 clinically normal male Yorkshire pigs, following intramuscular (i.m) administration of a single dose of diclofenac-sodium (2.5 mg/kg body weight). Diclofenac serum concentrations were determined by high pressure- liquid-chromatography (HPLC), with UV detection (226 nm). Following i.m. administration all individual diclofenac serum levels best fitted the one-compartment open model for extravascular administration. The maximal diclofenac serum concentration of 5.88 ± 0.934 mg/L was reached after 0.80 ± 0.35 h. The absorption half-life was 0.36 ± 0.25 h, and the area under the concentration vs. time curve (AUC0→∞) was 20.32 ± 4.521 mgh/L. A monoexponential concentration decline and small volume of distribution (Vd) of 0.29 ± 0.100 L/kg indicated a rapid, but not extensive distribution of diclofenac between central and peripheral compartment(s). Total clearance was 0.13 ± 0.034 L/h/kg, and elimination half-life was short (1.67 ± 0.743 h), as a result of a rapid distribution and extensive metabolism of diclofenac in the pigs body. When administered i.m. to pigs, diclofenac is absorbed and distributed rapidly. Distribution is not extensive, suggesting that diclofenac is predominantly retained in the central compartment. The elimination of the drug from the pigs circulation is also rapid, most of it probably being a result of extensive metabolism in the liver.

Determination of diclofenac residue in swine tissue following intramuscular administration

Diclofenac residue (DF) in swine tissue (liver, kidney, muscle, injection site) were examined using the method of High Performance Liquid Chromatography (HPLC) 15h, 72h,and 120h after i.m. administration of an individual therapeutic dose of diclofenac-sodium. At 15h after administration, the highest concentration of DF was found in the kidney (0.614 mg/kg), while a concentration about two times lower was found in the liver (0.316 mg/kg). At the injection site, the DF concentration was 0.432 mg/kg, while DF remained in a very low concentration (0.052 mg/kg) in the muscle outside the injection site, which was the lowest concentration in comparison with all the other examined tissues. At 72h after administration, DF was present in all examined tissues, but its concentrations were lower than the level that could be determined using the analytic procedure. Traces of the administered medicine disappeared after the waiting period of 120h in all tissues, except for the injection site. The longer presence of the medicine at the injection site could be a consequence of local inflammation, or irritation, that could cause a reduction in pH values of the area around the injection site, thus leading to slower dissolving and a longer presence of the DF at the injection site.