Nevenka Aleksic

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).

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.

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.

The first determination of Eustrongylides excisus Jägerskiöld, 1909 — larvae (Nematoda: Dioctophymatidae) in the pike-perch Sander lucioperca in Vojvodina (Serbia)

Twenty-one specimens of pike-perch (Sander lucioperca) were caught in the Danube-Tisa-Danube Canal in the city area of Novi Sad for parasitological examination. The presence of nematodes in the muscles was revealed in three fish. The parasites were identified to belong to the species Eustrongylides excisus, for which the pike-perch is a paratenic host. This finding represents the first determination of the larvae in the pike-perch in Serbia. The pike-perch is infected by ingestion of benthos- or plankton-eating fishes, the second intermediate hosts harbouring the fourth-stage nematode larvae. E. excisus is pathogenic to humans, who may be infected by consuming raw or undercooked fish.

The influence of pulverised sugar dusting on the degree of infestation of honey bee colonies with Varroa destructor.

The aim of this work was the investigation on the efficacy of pulverised sugar dusting on knocking-down Varroa destructor mites and the influence of the dynamics of the treatment on the degree of infestation in honey bee colonies. Two methods were deployed to measure the degree of infestation of bee colonies with V. destructor mites: the sugar shake method and the technique which involves mesh bottom boards equipped with sticky inserts. The research was carried out on 30 strong honey bee colonies in three successive years. The results proved that dusting with pulverised sugar (particle diameter below 40 μm) influenced the fall of V. destructor in comparison with both their fall off prior to the treatment and the negative control. The most discernible effects on the mite fall and the decline in their population in the hives was obtained with dustings repeated at threeday intervals. To conclude, the dynamics of the treatment affected the degree of infestation in bee colonies. However, the efficacy of sugar dusting was significantly lower in comparison with the one of flumethrin (positive control). Thus, in spite of considerable favourable effects on the decline in the degree of infestation with mites, by no means may dusting with pulverised sugar be advised to beekeepers as the one and only means of bee protection against V. destructor. The use of the technique which involves mesh bottom boards equipped with sticky inserts proved more efficacious and sensitive in the judgment of the degree of infestation of bee colonies in comparison with the sugar shake method, which includes dusting 300 bees with 40 g of pulverised sugar (particle diameter below 40 μm).

Influence of phytogenic feed additive on Lawsonia intracellularis infection in pigs

Lawsonia intracellularis is known to cause proliferative enteropathy (PE), one of the economically most important swine diseases with global distribution. Not unlike other enteric diseases, PE is a frequent indication for antibiotic therapy. However, their unjustified use leads to an emerging problem - antimicrobial resistance. Thus, the aim of this research was to assess if a phytogenic additive may replace antibiotics in the control of PE in 144 weaned piglets (72 treated and 72 controls) naturally infected with L. intracellularis. The quantity of L. intracellularis faecal shedding was monitored by real-time polymerase chain reaction (PCR) assay in faecal samples on day 0, 14 and 28, whilst the level of the ileum damage was determined by immunohistochemistry (IHC) assay performed on gut sections. Real-time PCR assay revealed that cycle-threshold (Ct) values in the treatment group increased significantly over time and were higher than in the control. These results indicate that the use of the phytogenic additive decreases the faecal excretion of L. intracellularis both throughout the experiment and in comparison to the control. The expression of the L. intracellularis antigen in IHC assay was lower in treated animals, implying that the additive leads to the decrease in the pathogen quantity in the ileum. Significantly higher feed conversion ratio was recorded in the treatment group. The results indicate that the phytogenic additive may be beneficial in the control of PE, but additional research is necessary to assess its use in various pig categories and define the optimum concentrations.

Quantitative PCR assessment of Lotmaria passim in Apis mellifera colonies co-infected naturally with Nosema ceranae

A recently described trypanosomatid species Lotmaria passim and the microsporidium Nosema ceranae infect the honey bee (Apis mellifera), but the interspecific dynamic of these two common gut parasites is unknown. In this study, a real-time qPCR assay was developed to enable the specific detection and quantification of L. passim. The annual dynamics of N. ceranae and L. passim infections were evaluated in ten A. mellifera colonies naturally infected with both parasites at one apiary in Serbia from March 2016 to March 2017. Ten samples (60 bees abdomens) were taken from each colony on 8 sampling occasions. L. passim infection level was evaluated with qPCR, while N. ceranae infection was measured by spore counts. N. ceranae infection level was significantly higher in comparison with that of L. passim (spore or cell equivalents/bee, respectively). Significant positive correlation between infection levels of the parasite species indicates their similar annual dynamics, whilst the differences in the levels of infection between particular months point to a seasonal pattern in the incidence of both parasites. The assay which has been developed and validated creates opportunity for detailed study of L. passim infection kinetics and the improvement in the management practices in beekeeping related to these two parasites.

Impact of antibiotics used as growth promoters on bacterial resistance

For decades intensive husbandry has more or less been based on the use of antibiotics in sub-minimum inhibitory concentrations (sub-MIC) aimed at growth promotion. Continuous exposure of animal intestinal microbiota, including opportunistic zoonotic pathogens, to sub-MIC poses a pressure to selection and spread of bacteria strains with developed mechanism of antibiotic resistance. These bacteria may be transferred to people either by direct contact with farm animals or indirectly, via the food chain. Although in the EU a ban on the use of antibiotics as growth promoters was imposed in 2006, in many countries, including the largest producers and consumers of antibiotics in the world, it has yet to be done. Given that we are faced with a global problem of the loss of the efficacy of several antibiotic classes which are available for the treatment of human bacterial infections, it is unacceptable that antibiotic use in husbandry is not under global control. Reduction in antibiotic use in clinical practice in human medicine remains in dispute, but non-therapeutic use in husbandry remains a field in which much can be done to contribute substantially to the extension of antibiotic effectiveness and health care of future generations.

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.

Pasteurlla multocida mastitis in cow

Pasteurella (P.) multocida is a heterogeneous species of Gram-negative bacteria which are common commensals of the upper respiratory system of various mammal and bird species, but are also opportunistic contagious zoonotic pathogens which cause a wide spectre of infections in domestic animals and humans. P. multocida is a rare cause of mastitis in dairy cows. The source of infection mainly remains unknown, mastitis usually is acute, and the therapy by intramammary administration of antibiotics does not lead to satisfactory results. Lethality is possible due to presence of endotoxins in blood. Literature data on P. multocida mastitis in dairy cows is particularly scarce, which is why such a case is described in the current work, with past medical history, clinical findings, laboratory diagnostics and therapeutic approach.