Milena Radakovic

Dominance of Nosema ceranae in honey bees in the Balkan countries in the absence of symptoms of colony collapse disorder

Nosema species were determined in honey bees from Balkan countries. A total of 273 Nosema-positive samples were analysed. Duplex PCR and PCR-RFLP with newly designed primers, nos-16S-fw/rv, were used to differentiate between N. apis and N. ceranae. N. apis was detected in only one sample (collected in 2008 in Serbia) and N. ceranae in all the others (N = 272) including 35 older samples from Serbia collected between 2000 and 2005. No co-infection was detected. The results suggest (1) the dominance of N. ceranae infection in all Balkan countries monitored throughout the last three years; (2) the presence of N. ceranae in Serbia at least since 2000, which means that N. ceranae has not recently displaced N. apis; (3) the higher efficacy of PCR-RFLP with newly designed primers, nos-16S-fw/rv, in comparison with duplex PCR (100%:82%, respectively). The prevalence of N. ceranae in Balkan countries was not associated with an increase in nosemosis or colony losses resembling Colony Collapse Disorder (CCD).

In vitro evaluation of the clastogenicity of fumagillin.

Fumagillin, an antibiotic compound produced by Aspergillus fumigatus, is effective against microsporidia and various Amoeba species, but is also toxic when administered systemically to mammals. Furthermore, a recent in vivo study by Stanimirovic Z et al. 2007 : (Mutat Res 628:1–10) indicated genotoxic effects of fumagillin. The aim of the present study was to investigate and explain the clastogenic effects of fumagillin (in the form of fumagillin dicyclohexylamine salt) on human peripheral blood lymphocytes in vitro by sister‐chromatid exchanges (SCE), chromosome aberrations (CA), and micronucleus (MN) tests. The mitotic index (MI), proliferation index (PI), and nuclear division index (NDI) were calculated to evaluate the cytotoxic potential of fumagillin. Five concentrations of fumagillin (0.34, 0.68, 1.02, 3.07, and 9.20 μg/ml) were applied to lymphocyte cultures. All the tested concentrations of fumagillin increased the frequency of SCE per cell significantly (P < 0.001 or P < 0.01) compared with the negative control. A significant (P < 0.001) increase in frequency of structural CA was observed at the three highest concentrations in comparison with the negative control. In addition, the three highest test concentrations increased MN formation and decreased MI, PI, and NDI significantly compared with the negative control. The present results indicate that fumagillin is clastogenic and cytotoxic to cultured human lymphocytes. Environ. Mol. Mutagen., 2008.

Biogeographic study of the honey bee (Apis mellifera L.) from Serbia, Bosnia and Herzegovina and Republic of Macedonia Based on mitochondrial DNA analyses

In this work, Apis mellifera carnica and A. m. macedonica honey bees from Serbia, Bosnia and Herzegovina and Republic of Macedonia were analysed using molecular techniques in order to improve our knowledge about biogeography of A. mellifera on the Balkan peninsula. This is the first time that the indigenous honey bees from Bosnia and Herzegovina and Republic of Macedonia have been analyzed using a molecular approach. Sampling was carried out from 560 stationary apiaries where bees were kept in traditional hives (woven skeps). The COI–COII regions of 1680 samples were PCR-amplified and sequenced. To reveal the haplotype of studied bees, the obtained sequences were aligned with published sequence data of haplotypes that belong to A. mellifera C phylogenetic lineage. The C2D mtDNA haplotype was found in all honey bees sampled from Serbia, Bosnia and Herzegovina and Republic of Macedonia, These results show that A. m. carnica and A. m. macedonica share the same C2D mtDNA haplotype. COI gene segments of 1680 samples were PCR-amplified and digested with restriction enzymes NcoI and StyI in order to discriminate A. m. macedonica from A. m. carnica. Amplified fragment patterns produced by both restriction enzymes matched with diagnostic pattern characteristic for A. m. macedonica in case of samples from east, south and south-west parts of Serbia, and Republic of Macedonia, fragments of samples from northern part of Serbia and Bosnia and Herzegovina did not include NcoI, and StyI restriction sites. These results indicate that honey bees from east, south and south-west parts of Serbia, and Republic of Macedonia belong to the A. m. macedonica, and honey bees from northern part of Serbia and Bosnia and Herzegovina belong to another subspecies, probably to the A. m. carnica. Therefore A. m. macedonica has much wider area of distribution than it was previously considered.

Oxidative stress and DNA damage in horses naturally infected with Theileria equi

The aim of this study was to determine the concentrations of oxidative stress parameters and DNA damage in horses infected by Theileria equi. Initial screening of 110 horses with duplex PCR enabled the selection of 30 infected horses with T. equi and 30 free of infection (control). Specimens from the 60 horses were further analysed by determining the following oxidative stress parameters: extent of haemolysis (EH), plasma free haemoglobin (PHb), catalase (CAT), Cu,Zn superoxide dismutase (SOD1), paraoxonase (PON1), nitrite (NO2-), total nitrate and nitrite (NOx), malondialdehyde (MDA) and free thiol groups (-SH). In addition, relative distribution of lactate dehydrogenase (LDH1-LDH5) activity and the DNA-damaging effects of T. equi infection were evaluated. Compared to control horses, horses infected with T. equi had significantly higher SOD1 activities (P <0.05) and PHb (P <0.01), NO2- (P <0.001), NOx (P <0.05) and MDA concentrations (P <0.001), and significantly lower EH (P <0.001), CAT (P <0.01) and PON1 (P <0.001) activities, and thiol group concentrations (P <0.05). The comet assay demonstrated significantly increased DNA damage in T. equi infected cells compared to non-infected cells (P <0.001). Infected horses had significantly increased LDH5 isoenzyme activities (P <0.05). There was higher production of ROS/RNS in T. equi-infected horses, which resulted in changes in osmotic fragility, damage to lipids, proteins and DNA, haemolysis and hepatocellular damage. Oxidative stress in horses naturally infected with T. equi could contribute to the pathogenesis of the infection.

Evaluation of cytogenetic and DNA damage in human lymphocytes treated with adrenaline in vitro.

Catechol groups can be involved in redox cycling accompanied by generation of reactive oxygen species (ROS) which may lead to oxidative damage of cellular macromolecules including DNA. The objective of this investigation was to evaluate possible genotoxic effects of a natural catecholamine adrenaline in cultured human lymphocytes using cytogenetic (sister chromatid exchange and micronuclei) and the single cell gel electrophoresis (Comet) assay. In cytogenetic tests, six experimental concentrations of adrenaline were used in a range from 0.01-500 μM. There were no indications of genotoxic effects of adrenaline in sister chromatid exchange and micronucleus tests. However, at four highest concentrations of adrenaline (5 μM, 50 μM, 150 μM and 300 μM) we observed a decreased mitotic index and cell-cycle delay. In addition, in the Comet assay we used adrenaline in a range from 0.0005-500 μM, at two treatment times: 15 min or 60 min. In contrast to cytogenetic analysis, there was a dose-dependent increase of DNA damage detected in the Comet assay. These effects were significantly reduced by concomitant treatment with quercetin or catalase. Therefore, the obtained results indicate that adrenaline may exhibit genotoxic effects in cultured human lymphocytes, most likely due to production of reactive oxygen species.

Evaluation of the effects of ephedrine on human lymphocytes in the comet assay.

Ephedrine, a natural alkaloid from plants of the genus Ephedra, has a chemical structure similar to catecholamines. It is well established that catecholamines (adraneline, noradrenaline and dopamine) cause genotoxic and mutagenic effects. Therefore, the objectives of this investigation were to examine weather ephedrine can exhibit genotoxic effects on isolated human lymphocytes in the Comet assay. Dose-response of human lymphocytes was determined at the concentration range of ephedrine from 0.0005 μM to 500 μM. Three concentrations of ephedrine (1, 50 and 300 μM) which had acceptable cell viability (over 90%) were used for further experiments with inhibitors of DNA reparation (cytosine arabinoside and hydroxyurea). The obtained results showed that ephedrine did not induce DNA damage in isolated human lymphocytes. However, co-treatment of the negative control with DNA repair inhibitors caused a slight but significant increase of DNA damage, due to an endogenous DNA damage. Interestingly, cells treated with ephedrine and DNA repair inhibitors did not express increased DNA damage. On the basis of the obtained results it can be concluded that ephedrine did not exhibit genotoxic effects on isolated human lymphocytes. This result is in accordance with previous investigations showing negative genotoxicological results for ephedrine using bacterial gene mutation test-systems and in vitro cytogenetic analysis.

A molecular and haematological study of Theileria equi in Balkan donkeys

Equine piroplasmosis in donkeys has been recognised as a serious problem of major economic importance. The present molecular study is the first investigation of the presence of Theileria equi and Babesia caballi in Balkan donkeys and of the possible haematological alterations related to it. A total of 70 apparently healthy donkeys from Serbia were included in this study. The overall prevalence of T. equi infection in donkeys tested with multiplex PCR was 50%. There was no B. caballi-positive sample. Infections in donkeys included in this study seem to be associated with decreased red blood cell count, haemoglobin concentration, haematocrit and platelet count, and with increased white blood cell count, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Altered haematological parameters in donkeys can lead to a decrease in working capacity and production performance. Further molecular research and long-term monitoring of equine piroplasmosis is needed in Serbia and throughout Europe.