Anna Gajda

Managed honey bee colony losses in Canada, China, Europe, Israel and Turkey, for the winters of 2008–9 and 2009–10

Summary In 2008 the COLOSS network was formed by honey bee experts from Europe and the USA. The primary objectives set by this scientific network were to explain and to prevent large scale losses of honey bee (Apis mellifera) colonies. In June 2008 COLOSS obtained four years support from the European Union from COST and was designated as COST Action FA0803—COLOSS (Prevention of honey bee Colony Losses). To enable the comparison of loss data between participating countries, a standardized COLOSS questionnaire was developed. Using this questionnaire information on honey bee losses has been collected over two years. Survey data presented in this study were gathered in 2009 from 12 countries and in 2010 from 24 countries. Mean honey bee losses in Europe varied widely, between 7–22% over the 2008–9 winter and between 7–30% over the 2009–10 winter. An important finding is that for all countries which participated in 2008–9, winter losses in 2009–10 were found to be substantially higher. In 2009–10, winter losses in South East Europe were at such a low level that the factors causing the losses in other parts of Europe were absent, or at a level which did not affect colony survival. The five provinces of China, which were included in 2009–10, showed very low mean (4%) A. mellifera winter losses. In six Canadian provinces, mean winter losses in 2010 varied between 16–25%, losses in Nova Scotia (40%) being exceptionally high. In most countries and in both monitoring years, hobbyist beekeepers (1–50 colonies) experienced higher losses than practitioners with intermediate beekeeping operations (51–500 colonies). This relationship between scale of beekeeping and extent of losses effect was also observed in 2009–10, but was less pronounced. In Belgium, Italy, the Netherlands and Poland, 2008–9 mean winter losses for beekeepers who reported ‘disappeared’ colonies were significantly higher compared to mean winter losses of beekeepers who did not report ‘disappeared’ colonies. Mean 2008–9 winter losses for those beekeepers in the Netherlands who reported symptoms similar to “Colony Collapse Disorder” (CCD), namely: 1. no dead bees in or surrounding the hive while; 2. capped brood was present, were significantly higher than mean winter losses for those beekeepers who reported ‘disappeared’ colonies without the presence of capped brood in the empty hives. In the winter of 2009–10 in the majority of participating countries, beekeepers who reported ‘disappeared’ colonies experienced higher winter losses compared with beekeepers, who experienced winter losses but did not report ‘disappeared’ colonies.

Occurrence of parasites and pathogens in honey bee colonies used in a European genotype-environment interactions experiment

Summary Diseases are known to be one of the major contributors to colony losses. Within a Europe-wide experiment on genotype—environment interactions, an initial 621 colonies were set up and maintained from 2009 to 2012. The colonies were monitored to investigate the occurrence and levels of key pathogens. These included the mite Varroa destructor (mites per 10 g bees), Nosema spp. (spore loads and species determination), and viruses (presence/absence of acute bee paralysis virus (ABPV) and deformed wing virus (DWV)). Data from 2010 to the spring of 2011 are analysed in relation to the parameters: genotype, environment, and origin (local vs. non-local) of the colonies in the experiment. The relative importance of different pathogens as indicators of colony death within the experiment is compared. In addition, pathogen occurrence rates across the geographic locations are described.

Winter colony losses in Poland

Grażyna Topolska, Anna Gajda, Krystyna Pohorecka, Andrzej Bober, Sylwia Kasprzak, Marta Skubida and Piotr Semkiw Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Ciszewskiego 8, 02-786 Warsaw, Poland. National Veterinary Research Institute, Department of Parasitology with Laboratory of Honey Bee Diseases, Pulawy, Poland. Research Institute of Pomology and Floriculture, Apiculture Division, Kazimierska 2 str, 24-100 Pulawy, Poland.

Effect of genotype and environment on parasite and pathogen levels in one apiary - a case study

As part of the COLOSS GEI experiment, one apiary in Chalkidi, Greece was continuously monitored for various pests and pathogens including V. destructor mites, Nosema spp. spores, and quantitative titers of five viruses from late summer 2009 until March 2012. The apiary was established with 39 colonies which included ten A. m. carnica (CarV) colonies from Germany, ten A. m. ligustica (LigI) colonies from Italy, ten A. m. macedonica (MacB) colonies from Bulgaria and nine local A. m. macedonica (MacG) colonies from Greece. At the end of the monitoring period, eight colonies survived: six local MacG, one MacB and one LigI. The local MacG colonies consistently showed comparatively lower V. destructor infestation levels and, consequently, also low DWV titres. In contrast, LigI colonies had the highest Nosema spp. and BQCV titres. It is, however, difficult to attribute the low survival rate of non-local colonies to any single factor. Since all colonies were located in close proximity in a single apiary, and the local bees were in better health than non-local bees, we assume that the local bees were better adapted to the local environmental conditions, and handled environmental stressors better to avoid disease outbreak.

Molecular Characterization of Poxviruses Associated with Tattoo Skin Lesions in UK Cetaceans

There is increasing concern for the well-being of cetacean populations around the UK. Tattoo skin disease (characterised by irregular, grey, black or yellowish, stippled cutaneous lesions) caused by poxvirus infection is a potential health indicatora potential health indicator for cetaceans. Limited sequence data indicates that cetacean poxviruses (CPVs) belong to an unassigned genus of the Chordopoxvirinae. To obtain further insight into the phylogenetic relationships between CPV and other Chordopoxvirinae members we partially characterized viral DNA originating from tattoo lesions collected in Delphinidae and Phocoenidae stranded along the UK coastline in 1998–2008. We also evaluated the presence of CPV in skin lesions other than tattoos to examine specificity and sensitivity of visual diagnosis. After DNA extraction, regions of the DNA polymerase and DNA topoisomerase I genes were amplified by PCR, sequenced and compared with other isolates. The presence of CPV DNA was demonstrated in tattoos from one striped dolphin (Stenella coeruleoalba), eight harbour porpoises (Phocoena phocoena) and one short-beaked common dolphin (Delphinus delphis) and in one ‘dubious tattoo’ lesion detected in one other porpoise. Seventeen of the 18 PCR positive skin lesions had been visually identified as tattoos and one as a dubious tattoo. None of the other skin lesions were PCR positive. Thus, visual identification had a 94.4% sensitivity and 100% specificity. The DNA polymerase PCR was most effective in detecting CPV DNA. Limited sequence phylogeny grouped the UK samples within the odontocete poxviruses (CPV group 1) and indicated that two different poxvirus lineages infect the Phocoenidae and the Delphinidae. The phylogenetic tree had three major branches: one with the UK Phocoenidae viruses, one with the Delphinidae isolates and one for the mysticete poxvirus (CPV group 2). This implies a radiation of poxviruses according to the host suborder and the families within these suborders.

Oxytetracycline Residues in Honey Analyzed by Liquid Chromatography with UV Detection

Summary A liquid chromatography method with UV detection for determination of oxytetracycline (OTC) in honey has been developed. The samples were extracted with the solution of oxalic acid. The clean-up procedure was performed by solid phase extraction (SPE) using polymeric Strata X and carboxylic acid cartridges. Chromatographic separation was carried out on the Luna C8 analytical column with mobile phase consisting of acetonitrile-0.02 M oxalic acid. The method has been successfully validated according to the requirements of the European Decision 2002/657/EC and this method is used in routine control of oxytetracycline in honey samples. The limit of detection (LOD) and limit of quantification (LOQ) of the presented method were 10 and 12.5 μg/kg, respectively. The developed method has also been verified in quantitative determination of oxytetracycline residues in honey after experimental treatment with this product in bee colonies. Streszczenie Opracowano metodę chromatografii cieczowej z detektorem UV do oznaczania oksytetracykliny (OTC) w miodzie. Do ekstrakcji próbek wykorzystano roztwór kwasu szczawiowego. Do oczyszczania zastosowano zestaw ekstrakcji do fazy stałej (SPE) z użyciem kolumienek polimerycznych Strata X oraz kolumienek karboksylowych. Rozdziału chromatograficznego dokonano przy użyciu kolumny analitycznej Luna C8 z faza ruchomą składającą się z acetonitrylu i 0.02 M roztworu kwasu szczawiowego. Metoda została zwalidowana zgodnie z wymogami Decyzji Komisji Europejskiej 2002/657/EC i jest stosowana w rutynowych badaniach kontrolnych oksytetracykliny w próbkach miodu. Opracowana metoda została zweryfikowana w ilościowym oznaczaniu pozostałości oksytetracykliny w miodzie po eksperymentalnym podaniu pszczołom tego antybiotyku. Celem przeprowadzonego doświadczenia było określenie, jak długo oksytetracyklina może pozostawać w miodzie. Badano także stabilność oksytetracykliny w warunkach laboratoryjnych. Wyniki badania stabilności wskazują, że oksytetracyklina w miodzie przechowywanym w warunkach laboratoryjnych ulega powolnemu zmniejszaniu. W pobranych do badań próbkach miodu stwierdzono zawartość OTC w stężeniach od 17 300 μg/kg do 34 000 μg/kg. Przeprowadzone badania wskazują, że OTC w znacznych ilościach przedostaje się do miodu po jej zastosowaniu u pszczół. Po 12 miesiącach od podania antybiotyku pszczołom, stężenie w miodzie było poniżej wyznaczonej granicy wykrywalności metody (10 μg/kg).

Doxycycline depletion and residues in eggs after oral administration to laying hens

The depletion of doxycycline (DC) residues in eggs was determined after oral drug administration by drinking water to laying hens. The antibiotic was supplied to birds for 5 consecutive days and the eggs were collected during medication and 18 days after withdrawal. DC residues were determined by LC-MS/MS. DC was isolated from eggs with a solution of 0.02 M of oxalic acid (pH 4), 0.1 M Na2EDTA and acetonitrile. The limit of detection (LOD) and limit of quantification (LOQ) of the method were 2 and 5 µg kg–1, respectively. Analyses were performed on whole egg, egg white and yolk separately. DC was detectable 24 h after the beginning of administration. The concentration of antibiotic increased daily, resulting in the highest DC concentration in whole eggs at the first day of the withdrawal period. Thirteen days after withdrawal, the content of DC in whole eggs was below the LOQ of the method. However, some differences were found in the depletion curve of DC between egg white and yolk. Residues of DC in egg white were much higher during treatment and 1 day after withdrawal, but later the concentration in egg white decreased fairly rapidly and a higher DC content in egg yolk was observed. The depletion period was shorter for egg white than for yolk, and DC was detected in the egg white until 12 days after withdrawal and 2 days more in egg yolk than in white. DC reached a peak faster in egg white, but the residues were detectable for longer period in the yolk. Graphical Abstract

Occurrence of Veterinary Antibiotics and Chemotherapeutics in Fresh Water, Sediment, and Fish of the Rivers and Lakes in Poland

Abstract The occurrence of commonly used veterinary antimicrobial agents was investigated in 159 fresh water, 443 fish, and 150 sediment samples from Polish rivers and lakes. The agents included aminoglycosides, ß-lactams, diaminopyrimidines, fluoroquinolones, lincosamides, macrolides, pleuromutilins, sulfonamides, and tetracyclines. The analysis was performed by three different sample preparation procedures for each matrix and it was performed by liquid chromatography-tandem mass spectrometry with electrospray ionisation source in positive mode, under the same conditions. All analytical methods used were validated and showed good sensitivity, accuracy, and precision. The LOQ was in the range from 5 μg/kg to 125 μg/kg for fish samples, from 0.02 μg/L to 10 μg/L for fresh water samples, and from 1 μg/kg to 8 μg/kg for sediment samples.

LC-MS/MS analysis of doxycycline residues in chicken tissues after oral administration

Abstract For the purpose of quantitative determination of doxycycline (DC) residues in tissues, a sensitive liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed. The method was used to determine DC residues in chicken tissues (breast and thigh muscle, liver and kidney) after oral administration with drinking water to five-weak-old broiler chickens. The DC was administered for five consecutive days at a therapeutic dose of 10 mg/kg b.w. once a day. The tissues were collected after 6 h, 24 h, 7 d, and 8 d. The method was validated and the decision limit was established for muscle - 109.2 μg/kg, for liver - 326.1 μg/kg, and for kidney - 634.0 μg/kg. The detection limit was 2 μg/kg and the limit of quantification was 5 μg/kg. In a short period after ceasing the treatment, the detected concentrations of DC were much higher than the established maximum residue limit values. The highest residue concentrations of DC were observed in the kidney, followed by the liver and muscle. The lowest concentration of DC was determined in tight muscle.

Determinaton of enrofloxacin and ciprofloxacin in albumin and freeze-dried-eggs by liquid chromatography with fluorescence detection

Abstract The study presents a method for determination of enrofloxacin and ciprofloxacin in albumin and freeze-dried-eggs with the use of liquid chromatography with fluorescence detection. The procedure enables a simple isolation of fluoroquinolones from albumin based on extraction with acetonitrile, and from freeze-dried-eggs with acetonitrile under alkaline conditions. The samples have been analysed on ultracarb C8 liquid chromatography column. The gradient elution programmes consisted of the mixture of 0.03 M phosphoric acid with 0.002 M sodium 1-heptanesulfonate monohydrate and acetonitrile. The method has been validated according to requirements of the European Decision 2002/657/EC. Recoveries ranged from 78% to 83% for spiked freeze-dried-eggs and from 89% to 91% for albumin. The developed method can be applied for determination and confirmation of enrofloxacin and ciprofloxacin in albumin and freeze-dried-eggs.