Several negative factors contribute to a decline in the number of insect pollinators. As a novel approach in therapy, we hypothesize that the EM^® for bees could potentially have an important therapeutic and immunomodulatory effect on honey bee colonies. The aim of our study was to evaluate its impact on honey bees at the individual and colony level. This is the first appliance of the commercial probiotic mix EM^® PROBIOTIC FOR BEES in honey bees as economically important social insects. The sugar syrup with 10% of probiotic was administered by spraying or feeding the honey bee colonies in the field conditions, in order to evaluate the infection levels with spores of Nosema spp. and colonies’ strength. Moreover, in laboratory-controlled conditions, in the hoarding cages, adult workers have been fed with sugar syrup supplemented with 2.5, 5, and 10% of EM^® for bees for biochemical and immunological analyses of hemolymph, and with 5 and 10% for measuring the size of hypopharyngeal glands. It was found that following the EM^® for bees administration the Nosema spp. spore counts in colonies were significantly reduced, and colonies’ strength was increased. The results at the individual level showed significant positive physiological changes in treated groups of adult bees, revealing at the same time a higher mortality rate when feeding sugar syrup supplemented with the probiotic.

Various factors contribute to a decline in diversity and number of bees. Here, an integrated approach in experimental BPC 157 therapy was implemented, combining laboratory-controlled and field study results. The aim of a study was to assess the effects of BPC 157 additional feeding of newly emerged worker honeybees on few biochemical and immunological parameters in hemolymph (glucose, trehalose, lipids, proteins, vitellogenin, glucose-oxidase (GOX)), and hypopharyngeal gland (HPG), in laboratory-controlled conditions. Additionally, to examine the physiological status of protein digestion, the enzymatic activity of leucine aminopeptidase (LAP) in the mid-guts of worker honeybees was analyzed. It was found that individual honeybees, in hoarding cages, following BPC 157 administration through carbohydrate food, showed positive physiological changes when compared to the control groups. Those results were complemented by strong and visible LAP activity, particularly noticeable in the apical parts of the epithelial cells in the mid-guts of young worker honeybees originated from treated hives, suggesting a link between alternative oral therapy with BPC 157 and honeybees’ immunity.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.

Recent animal experiments suggested that centrally transported botulinum toxin type A (BoNT-A) might reduce an abnormal muscle tone, though with an unknown contribution to the dominant peripheral muscular effect observed clinically. Herein, we examined if late BoNT-A antispastic actions persist due to possible central toxin actions in rats. The early effect of intramuscular (i.m.) BoNT-A (5, 2 and 1 U/kg) on a reversible tetanus toxin (TeNT)-induced calf muscle spasm was examined 7 d post-TeNT and later during recovery from flaccid paralysis (TeNT reinjected on day 49 post-BoNT-A). Lumbar intrathecal (i.t.) BoNT-A–neutralizing antiserum was used to discriminate the transcytosis-dependent central toxin action of 5 U/kg BoNT-A. BoNT-A-truncated synaptosomal-associated protein 25 immunoreactivity was examined in the muscles and spinal cord at day 71 post-BoNT-A. All doses (5, 2 and 1 U/kg) induced similar antispastic actions in the early period (days 1–14) post-BoNT-A. After repeated TeNT, only the higher two doses prevented the muscle spasm and associated locomotor deficit. Central trans-synaptic activity contributed to the late antispastic effect of 5 U/kg BoNT-A. Ongoing BoNT-A enzymatic activity was present in both injected muscle and the spinal cord. These observations suggest that the treatment duration in sustained or intermittent muscular hyperactivity might be maintained by higher doses and combined peripheral and central BoNT-A action.

Botulinum toxin type A (BoNT-A) induces a bilateral analgesic effect following unilateral injection in rodent bilateral or mirror pain models. This occurs either by indirect plasticity-related actions, or by the toxin’s direct central action in bilateral spinal circuits. Herein, we aimed to resolve this question by assessing the role of trans-synaptic toxin traffic in a bilateral inflammatory pain model. The analgesic effect of the toxin was examined in rats pre-treated with unilateral intraplantar BoNT-A (7 U/kg) and subsequently challenged with bilateral carrageenan-evoked hind-paw inflammation (2%, 50 µL/paw, 6 days post BoNT-A). Specific neutralizing antitoxin injected into the lumbar intrathecal space (2 IU, 24 h post BoNT-A), aimed at preventing the spinal trans-synaptic traffic of BoNT-A, abolished its bilateral analgesic effect. The toxin trans-synaptic effect was associated with reduced c-Fos neuronal activation and BoNT-A-mediated cleavage of synaptosomal-associated protein 25 (SNAP-25) in the bilateral dorsal horn. Here, we showed that, in bilaterally occurring pain, BoNT-A exerts a direct contralateral analgesic action extending beyond the level of the dorsal root ganglion sensory neuron that directly links the hindlimb injection site to the primary sensory region. This points to the crucial role of the toxin’s central trans-synaptic traffic, and its direct action at propriospinal nociceptive circuits in its pain-relieving efficacy.