Jevrosima Stevanovic

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).

Variability in germination and in temperature and storage resistance among Paenibacillus larvae genotypes

There are several methods for cultivation of Paenibacillus larvae, the causative agent of American foulbrood (AFB) in honey bees. Protocols for detection of sub-clinical levels of the bacterium from honey and bee samples include heat treatment of samples. The main objective of this study was to investigate if there is variability in temperature resistance among P. larvae genotypes, potentially leading to biased diagnose and disease monitoring. The variation in germination and proliferation ability among type collection (N=4) and field isolates (N=4) of P. larvae representing four different genotypes was investigated. Results demonstrate a significant variability between P. larvae genotypes in germination rate on solid media as well as in endospore resistance to heat treatment and storage. It is concluded that strains of different genotypes should be included in evaluation of standard laboratory protocols for cultivation of P. larvae to avoid bias in disease monitoring and quantification of the pathogen.

Sex Determination in 58 Bird Species and Evaluation of CHD Gene as a Universal Molecular Marker in Bird Sexing

The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim.

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.

Heritability of grooming behaviour in grey honey bees (Apis mellifera Carnica)

Grooming behaviour is considered an important defensive mechanism of honey bees against Varroa mites. The aim of this study was to reveal whether grooming behaviour is a useful criterion in breeding of Varroa-tolerant bees. To obtain a reliable evaluation the environmental influences were excluded. The degree of grooming potential was estimated by the percentage of damaged mites in the total number of fallen mites. The heritability of grooming behaviour throughout the three consecutive generations of queens was assessed by mother-daughter regression method. Among unselected queens, expressed grooming behaviour was recorded only in colonies with F1 queens (36.27%), but not in colonies with P queens and F2 queens (33.69%, 31.66%, respectively). Significant differences in grooming behaviour were found between colonies of P and F1 queens (p 0.05) difference among them. Nevertheless, the relatively low heritability of grooming behaviour in the three generations of queens examined (h2yx=0.49±0.02; h2zx=0.18±0.01; h2zy=0.16±0.01) indicate that breeding colonies for grooming behaviour only cannot be advised to beekeepers whose aim is to breed bees highly tolerant to Varroa mites.

Genetic variation of Apis mellifera from Serbia inferred from mitochondrial analysis

Genetic variation of Apis mellifera from Serbia inferred from mitochondrial analysis Two honeybee subspecies inhabit Serbia; Apis mellifera carnica and A. m. macedonica. Both belong to eastern Mediterranean (C) evolutionary lineage. Furthermore three Serbian honeybee ecotypes restricted to particular regions, were defined through morphometry and cytogenetic analyses. In this study, mitochondrial data have been used to analyze the molecular diversity of the honeybee population from Serbia. Seven haplotypes of the C evolutionary lineage have been found, two of them are newly described (C2o and C2p) and restricted to two regions, which ultimately increased the number of haplotypes found in this lineage. Comparisons with surrounding honeybee populations suggest a hybrid situation between A. m. carnica and A. m. macedonica and also introgression from A. m. ligustica. The results should be considered when dealing with future conservation strategies, and for pathogen-parasite-tolerant breeding programs. Różnorodność Genetyczn a Apis Mellifera w Serbii na Podstawie Badań Mitochondrialnych Na terenie Serbii występują dwa podgatunki pszczoły miodenj: Apis mellifera carnica i A. m. macedonica. Oba te podgatunki należą do wschodnio-śródziemnomorskiej linii ewolucyjnej (C). Na podstawie analiz morfometrycznych oraz cytogenetycznych zidentyfikowano trzy ekotypy pszczoły miodnej występujące w określonych rejonach Serbii. W przedstawionych badaniach, do określenia różnorodności molekularnej serbskiej populacji pszczół wykorzystano wyniki analiz mitochondrialnych. Stwierdzono siedem haplotypów linii ewolucyjnej C, z których dwa to nieznane wcześniej haplotypy C2o i C2p o występowaniu ograniczonym do dwóch regionów, co w efekcie zwiększa liczbę haplotypów tej linii. Porównania z okolicznymi populacjami pszczół sugerują mieszanie się pszczół A. m. carnica i A. m. macedonica oraz introgresję A. m. ligustica. Uzyskane wyniki należy uwzględniać podczas ustalania programów hodowli zachowawczych oraz w programach hodowlanych zorientowanych na selekcję pszczół o zwiększonej tolerancji na patogeny i pasożyty.

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.

Morphological, biochemical and hematological characterization of endangered Balkan donkey breed.

Abstract The aim of the study was to establish morphometric, biochemical and hematological values for the endangered Balkan donkey breed (Serbia) and to explore the possible age dependence of the parameters tested. Inter-breed similarity of morphometric parameters was assessed by comparing the data obtained for the Balkan donkey with morphometric measurements of several previously characterized domestic donkey breeds. The study population included 74 donkeys, divided in two age groups (group A ≤ 3 years; group B > 3 years). In total, 18 morphometric, 13 hematological and 14 biochemical parameters were assessed. Significant morphometric differences (p<0.05) in body length, head length, chest circumference and body weight were found between the two age groups. Significant differences in morphological parameters were revealed among the Balkan donkey and other donkey breeds (Catalonian, Croatian and Albanian), but results of cluster analysis demonstrated the smallest distance between the Balkan donkey and Albanian donkeys. The results of morphometric analyses showed consistency of the obtained values within the breed, and diversity as compared to other donkey breeds, and, thus, could be taken as referent for the Balkan donkey. Hematological and biochemical profiles obtained for the Balkan donkey were consistent with previous reports and within the recommended reference ranges. White blood cell, mid cell and granulocyte counts, showed significantly higher (p<0.05) values in donkeys under 3 years of age, while the only biochemical parameter affected by age was alkaline phosphatase. The information gained through characterization of the Balkan donkey breed provides a basis for conservation and development of the breed standard.

Stimulating effect of sugar dusting on honey bee grooming behaviour

The aim of this research was to investigate whether or not sugar dusting can stimulate the grooming behaviour in Apis mellifera L. (Hymenoptera: Apidae), an important defensive mechanism against Varroa destructor Anderson & Trueman (Acari: Varroidae), and to assess the most effective dose and frequency of treatment. The criterion for evaluation of grooming potential was the percentage of damaged mites (PDM) among the total number collected on the bottom boards of the hives. In each sugar‐treated group PDM was significantly higher in comparison both with the negative control (no treatment) and with the values preceding the treatment. The results point to a stimulating effect of sugar on the grooming behaviour at all doses and frequencies tested. Treatment frequency influenced the stimulating effect of sugar: treatments at 3‐ and 7‐day intervals with 30 and 40 g resulted in significantly higher PDMs than the least frequent treatment (every 14 days); dusting with 20 g influenced PDM only when repeated at 3‐day intervals. Because treatments at 3‐day intervals are time‐consuming, those with 40 or 30 g repeated every 7 days may be recommended. In the positive control (hives treated with amitraz), average PDM was significantly lower than in the negative control and all sugar‐treated groups. Possible causes of the stimulating effect of sugar dusting on bee grooming behaviour are discussed.

Clinical babesiosis and molecular identification of Babesia canis and Babesia gibsoni infections in dogs from Serbia

Canine babesiosis is a frequent and clinically significant tick-borne disease. Sixty symptomatic dogs with clinical findings compatible with babesiosis were included in this study conducted in Serbia. After clinical examination, blood samples were taken for microscopic examination, complete blood count (CBC), Canine SNAP 4Dx Test, DNA analyses and sequencing. The main clinical signs included apathy, anorexia, fever, brown/red discoloration of urine, pale mucous membranes, icterus, splenomegaly, and vomiting. The main clinicopathological findings in Babesia infections were a slight to severe thrombocytopenia and a mild to very severe normocytic normochromic anaemia. Microscopic evaluation revealed 58 positive samples with the presence of large and small intraerythrocytic piroplasms in 57 and 1 sample(s), respectively. No co-infections were found using SNAP test. Two Babesia species, B. canis (58/60) and B. gibsoni (2/60), were differentiated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Species identification was further confirmed by sequencing PCR products of B. gibsoni samples and six randomly selected B. canis samples. All dogs were treated with imidocarb dipropionate (6.6 mg/kg of body weight), given intramuscularly twice at an interval of 14 days. This report presents the first molecular evidence of the occurrence of B. gibsoni and B. canis, confirmed by DNA sequencing, in sick dogs from Serbia.