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Crossed Immunoelectrophoresis analysis of bee venom. Enzymatic identification of antigens.

Bee venom obtained by electrical stimulation has been analysed by crossed immunoelectrophoresis against high-titer rabbit antibodies. The antigenic analysis of bee venom revealed that the extract contained 17 antigens, which were detected in the crossed immunoelectrophoresis pattern after staining with Coomassie Brilliant Blue. The crossed immunoelectrophoresis pattern indicated heterogeneity in several of the antigens, as most of the precipitates (exhibiting enzyme and hemolytic activities) represented multiple forms of phospholipase A, hyaluronidase, acid phosphatase and melittin.

Acid Phosphatase↗

Death caused by wasp and bee stings in Denmark 1960-1980.

During a 21-year period in Denmark a total of 26 deaths were caused by wasp or bee stings (according to the National Health Service). The deaths might be classified, with some overlapping, as caused by either anaphylactic/anaphylactoid shocks (between 65% and 80%), suffocation after stings in the airways (about 15%) or preexisting diseases, especially arteriosclerotic heart disease (approx. 20%). Characteristically, in most persons with shock reactions unconsciousness and death occurred very shortly after the sting (within 45 min), while the interval between sting and death was longer (30 min to a couple of hours) when death was caused by suffocation. In more than 21 of the 26 cases it seemed reasonable to assume that insect allergy might have contributed to the fatal outcome. Six of these cases had a previous history of abnormal reactions to insect venom, thus only a small group would have benefited from the prophylactic effect of hyposensitisation. There was no known previous history of reactions to insect stings in the other cases, but it is likely that more than six persons had had severe reactions to insect stings on other occasions. Presumably many deaths where insect stings have been involved--through not verified as causal--are classified as inexplicable or accidental, thus the real number of deaths caused by wasp or bee stings could be substantially greater. Consequently hyposensitisation after severe insect sting reactions of verified allergic genesis can still be advised.

Adolescent↗

A preliminary study of immunotherapy with a monomethoxy polyethylene glycol modified honey bee venom preparation.

Monomethoxy polyethylene glycol (mPEG) modified honey bee venom (HBV) immunotherapy (IT) has been studied in 14 patients allergic to honey bee venom. Doses could be increased more rapidly and higher doses were reached compared to regular venom immunotherapy. No general side effects were seen, although large local swellings were found somewhat more often than with regular HBV. Most patients could easily be switched from the modified to the unmodified venom. Eight patients experienced and tolerated field stings. Skin testing showed a decreased allergenicity of the mPEG-HBV. The mean HBV-specific IgE level was below pre-treatment level already after only 6 weeks of IT. The HBV-specific IgG response was very good.

Adult↗

Systemic T-cell unresponsiveness during rush bee-venom immunotherapy.

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-gamma do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO+ vs CD45RO- T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-gamma-expressing cells among the CD45RO- subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO- T cells. In most allergic patients, a considerable percentage of Th cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself.

Adult↗

The purification and characterisation of hyaluronidase from the venom of the honey bee, Apis mellifera.

Hyaluronidase has been purified from the venom of the honey bee, Apis mellifera. The purification proved remarkably difficult, requiring a large number of chromatographic steps culminating in the removal of traces of phospholipase A2 with an affinity purified rabbit anti-phospholipase A2 immunosorbent column. The purified enzyme showed a 1143-fold increase in specific activity and was homogeneous. Electrophoresis in polyacrylamide gels (12%) containing sodium dodecyl sulphate (pH 8.9) or urea (pH 2.8) and electrofocusing in polyacrylamide (5%) gave a single band. The final product contained less than 0.1% phospholipase A2 and less than 1.5% acid phosphatase and gave a single line of precipitation against rabbit anti-hyaluronidase but was not precipitated by rabbit anti-phospholipase A2. Previous reports of instability were not confirmed, and we found the enzyme to be highly stable over a wide range of temperature and pH, and to denaturing agents. Purified hyaluronidase was found to be 'sticky' when highly pure and at low concentration, and adhered strongly to Sephadex G-75. The relative molecular mass was estimated at 35 000-37 000 by gel filtration, and at 41 000 by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. A value of 50 000 was obtained by ultracentrifugation assuming a partial specific volume of 0.73 cm3/g. Hyaluronidase was found to be a minor allergen in bee venom allergic patients.

Amino Acids↗

Experimental examinations concerning the problem of deformed emerging bees after infestation with Varroa jacobsoni.

The deformations of bees occurring in connection with varroatosis was examined both in case of natural and artificial infestation. Under both conditions, the number of bees showing wing deformations increased with the degree of mite infestation. Shortened abdomina, however, only appeared in the case of natural infestation. Inadequate brood care in colonies severely infested by Varroa mites is supposed to be responsible. Wing deformations could be produced by experimental haemolymph extraction.

Animals↗

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level.

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.

Animals↗

Biodemographic analysis of male honey bee mortality.

Biodemographic studies of insects have significantly enhanced our understanding of the biology of aging. Eusocial insects have evolved to form different groups of colony members that are specialized for particular tasks and highly dependent on each other. These different groups (castes and sexes) also differ strongly in their life expectancy but relatively little is known about their mortality dynamics. In this study we present data on the age-specific flight activity and mortality of male honey bees from two different genetic lines that are exclusively dedicated to reproduction. We show that males initiating flight at a young age experience more flight events during their lifetime. No (negative) relation between the age at flight initiation and lifespan exists, as might be predicted on the basis of the antagonistic pleiotropy theory of aging. Furthermore, we fit our data to different aging models and conclude that overall a slight deceleration of the age-dependent mortality increase at advanced ages occurs. However, mortality risk increases according to the Gompertz-Makeham model when only days with flight activity (active days) are taken into account. Our interpretation of the latter is that two mortality components act on honey bee males during flight: increasing, age-dependent deaths (possibly from wear-and-tear), and age-independent deaths (possibly due to predation). The overall mortality curve is caused by the interaction of the distribution of age at foraging initiation and the mortality function during the active (flight) lifespan.

Aging↗

Imaging diagnosis: acute lung injury following massive bee envenomation in a dog.

A 5-year-old neutered male mixed breed dog presented for increased respiratory effort after being stung by over 100 bees and developing anaphylactic shock. Given the history, clinical signs and thoracic radiographic findings of a mild bilateral interstitial pattern, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) was suspected. Further testing was performed to support this diagnosis. On computed tomographic images, there was a diffuse bilateral opacification of the lungs, with preservation of bronchial and vascular margins. Pulmonary function testing indicated decreased pulmonary compliance, decreased diffusion capacity and decreased functional residual capacity. These results supported the diagnosis of ALI/ARDS secondary to bee sting envenomation and development of anaphylactic shock. After 8 days of treatment with oxygen, steroids, antibiotics, and bronchodilators the dog improved. This case demonstrates the usefulness of computed tomography and pulmonary function testing in the diagnosis of ALI/ARDS.

Anaphylaxis↗

Unwarping confocal microscopy images of bee brains by nonrigid registration to a magnetic resonance microscopy image.

Confocal microscopy (CM) is a powerful image acquisition technique that is well established in many biological applications. It provides 3-D acquisition with high spatial resolution and can acquire several different channels of complementary image information. Due to the specimen extraction and preparation process, however, the shapes of imaged objects may differ considerably from their in vivo appearance. Magnetic resonance microscopy (MRM) is an evolving variant of magnetic resonance imaging, which achieves microscopic resolutions using a high magnetic field and strong magnetic gradients. Compared to CM imaging, MRM allows for in situ imaging and is virtually free of geometrical distortions. We propose to combine the advantages of both methods by unwarping CM images using a MRM reference image. Our method incorporates a sequence of image processing operators applied to the MRM image, followed by a two-stage intensity-based registration to compute a nonrigid coordinate transformation between the CM images and the MRM image. We present results obtained using CM images from the brains of 20 honey bees and a MRM image of an in situ bee brain.

Animals↗

A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor.

We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.

Amino Acid Sequence↗

Gene expression profiles in the brain predict behavior in individual honey bees.

We show that the age-related transition by adult honey bees from hive work to foraging is associated with changes in messenger RNA abundance in the brain for 39% of approximately 5500 genes tested. This result, discovered using a highly replicated experimental design involving 72 microarrays, demonstrates more extensive genomic plasticity in the adult brain than has yet been shown. Experimental manipulations that uncouple behavior and age revealed that messenger RNA changes were primarily associated with behavior. Individual brain messenger RNA profiles correctly predicted the behavior of 57 out of 60 bees, indicating a robust association between brain gene expression in the individual and naturally occurring behavioral plasticity.

Aging↗

Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera.

We characterized Apis mellifera in both native and introduced ranges using 1136 single-nucleotide polymorphisms genotyped in 341 individuals. Our results indicate that A. mellifera originated in Africa and expanded into Eurasia at least twice, resulting in populations in eastern and western Europe that are geographically close but genetically distant. A third expansion in the New World has involved the near-replacement of previously introduced "European" honey bees by descendants of more recently introduced A. m. scutellata ("African" or "killer" bees). Our analyses of spatial transects and temporal series in the New World revealed differential replacement of alleles derived from eastern versus western Europe, with admixture evident in all individuals.

Africa↗

Enzymatic basis of mannose toxicity in honey bees.

Honey bees have a negligible amount of phosphomannoseisomerase, together with a high content of a hexokinase which phosphorylates mannose more efficiently than fructose or glucose. Competition at the phosphorylation level plus accumulation of mannose-6-phosphate can fully account for the toxicity of mannose in honey bees.

Animals↗

Brood care in halictine bees.

Four species of the halictine bee genus Evylaeus keep their brood cells open during most or part of the development of the larvae. In the colonial summer phase, house bees care for the young and keep brood cells clean from feces and exuviae. Progressive feeding of nector is present at least in Evylaeus malachurus, whose fully fed larvae are, on an average, 60 percent heavier than the egg-and-pollen stage. Interactions between the two generations of social Halictinae are of general occurrence, and their intensity corresponds to the level of social behavior attained.

Animals↗

Detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase pcr.

A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3' end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.

Animals↗

Case report. Bee sting brachial block.

A case of brachial plexus block is presented, following a bee sting in the posterior triangle of the neck. The onset of neurological symptoms was rapid as was their subsequent resolution. Delayed peripheral neurological symptoms believed to have an immunological basis have been reported in response to stings from bees and other Hymenoptera both in the central and peripheral nervous systems (Goldstein et al., 1960; Means et al., 1973; Bachman et al., 1982; Weatherall et al., 1987; Van Antwerpen et al., 1988), but to the authors' knowledge no similar case of immediate peripheral block has been reported.

Adult↗

DNA fingerprinting analysis in the solitary bee Megachile rotundata: variability and nest mate genetic relationships.

The most conventional approach for evaluating genetic variability in an insect population involves assessing the degree of enzyme polymorphism. Hymenoptera display a relatively low level of genetic variability compared with most insect species. DNA probes consisting of tandemly repeated sequences are powerful tools for detecting polymorphisms when employed to develop DNA fingerprinting (DNAfp) profiles in a wide range of organisms. This report describes genetic variability in the solitary bee species Megachile rotundata as assessed by DNAfp using the M13 sequence and a synthetic oligonucleotide sequence homologous to a hypervariable region of the alpha-globin gene. DNAfp comparisons among offspring were used to analyze genealogical structure in M. rotundata nests. The results indicate that polyandry, by a large number of males, is not a common phenomenon in M. rotundata bee species. In the present analysis, it is likely that the broods raised in single nests are mostly the offspring of one singly mated female. However, the data does not preclude that for certain nests two males could have been involved in the mating process.

Animals↗