Liesel B. Gende

Efficacy of natural propolis extract in the control of American Foulbrood

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease that affects larvae of the honeybees. Due to the serious effects associated with AFB and the problems related to the use of antibiotics, it is necessary to develop alternative strategies for the control of the disease. The aim of the present work was to evaluate the effect of a propolis ethanolic extract (PEE) against P. larvae and its potential for the control of AFB. In vitro activity of PEE against P. larvae isolates was evaluated by the disk diffusion method and the minimum inhibitory concentration (MIC) was determined. Toxicity for honeybees was evaluated by oral administration of PEE and its lethal concentration was assessed. Lastly, colonies from an apiary with episodes of AFB on previous years were divided into different groups and treated with sugar syrup supplemented with PEE by aspersion (group one), sugar syrup by aspersion (group two), fed with sugar syrup supplemented with PEE (group three) and fed with sugar syrup only (group four). All isolates were sensitive to PEE and the MIC median was 0.52% (range 0.32-0.64). PEE was not toxic for bees at least at 50%. Field assays showed that 21 and 42 days after the application of the treatments, the number of P. larvae spores/g of honey was significantly lower in colonies treated with PEE compared to the colonies that were not treated with PEE. To our knowledge, this is the first report about the use of propolis for the treatment of beehives affected with P. larvae spores.

An in vitro Evaluation of Tagetes minuta Essential Oil for the Control of the Honeybee Pathogens Paenibacillus larvae and Ascosphaera apis, and the Parasitic Mite Varroa destructor

Abstract Biological activity was evaluated of the Tagetes minuta essential oil in different in vitro laboratory experiments on the mite (Varroa destructor), honeybees (Apis mellifera), the bacterium that causes the American Foulbrood (Paenibacillus larvae), and the fungus that produces chalkbrood (Ascosphaera apis). Two methods of complete exposure were used for mite lethality test: by spraying in Burgerjons tower with 10 mg of active ingredient in solution with distilled water and emulsion, and in unmodified Petri dishes (60 x 20 mm) with oil (different concentrations) diluted in 1 mL of ethanol. Ratio selection was obtained as: LD50 of Apis mellifera/LD50 of V. destructor. Determination of Minimal Inhibitory Concentrations (MIC): to P. larvae was tested at concentrations of 25, 50, 100, 150, 200, 250, 350, 450, 500, 600, 650, 700, 800 and 1000 ppm. An A. apis strain was grown on agar MY20 supplemented with variable concentrations (between 0–800 ppm) of T. minuta oil was evaluated. Results obtained in tests of total exposure showed that the concentration was able to kill 50% of mites in 24 h (DL50) and was estimated to be 4.37 mg/cage. The efficacy after spray treatment reached 56%. The ratio selection was 3:11. Against P. larvae, the oil showed MIC values ranging from 700–800 μL/L depending on the tested bacterial strains. Tagetes minuta oil in agar MY20 inhibited mycelial growth of A. apis above concentrations of 200 ppm (p = 0.0001). Oil concentrations of 700 and 800 ppm achieved maximum growth inhibition of A. apis (67% of growth inhibition on average). Tagetes minuta oil demonstrated in vitro antibacterial, antifungical and miticide activity, although this oil shows a moderate inhibitor effect compared with other essential oils of native plants from Argentina. However, this oil presents a ratio selection that would allow it to be used in field conditions with a good safety margin. It is possible that this oil can be used in combination with others, in integrated pest management strategies in bee colonies.

Efficacy of thymol against Echinococcus granulosus protoscoleces.

The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 mug/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 microg/ml, viability reduced to 53.5+/-11.9% after 12 days of incubation. At day 42, viability was 11.5+/-15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 microg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.

Laboratory Evaluations of Syzygium aromaticum (L.) Merr. et Perry Essential Oil Against Varroa destructor

Abstract The oil obtained by hydrodistillation of the foral bottom of Syzygium aromaticum (L.) Merr. et Perry was analyzed by GC and GC/MS. Eugenol was the main constituent in the oil (86.7%). The biological activity of the oil applied to Varroa destructor and Apis mellifera was evaluated in two laboratory tests. Mite lethality was estimated using a complete exposure method test with the oil at different concentrations, and a systemic administration method of oil at different concentrations diluted in syrup was placed in feeders for bees. The LC50 for complete exposure method at 24 h was 0.59 μL/dish. The inferior and superior limits obtained were 0.47 x 10−6 μL/dish and 1.22 μL/dish, respectively. LC50 estimated at 48 h showed a slight decrease as compared to that recorded at 24 h. Ratio selection (LC50 of A. mellifera/LC50 of V. destructor) for complete exposure method was 26.46 and 13.35 for 24 h and 48 h, respectively. Regarding the systemic administration method, mites LC50 at 24 h was 12,300 ppm. The inferior and superior limits calculated were 9,214 ppm and 15,178 ppm, respectively. LC50 estimated at 48 h showed a slight decrease as compared to that recorded at 24 h. Ratio selection for systemic administration method was 3.05 and 2.22 for 24 h and 48 h, respectively. Syzygium aromaticum oil was found to be an attractant for V. destructor at 4.8% (w/w) concentration. The results showed that oil toxicity against V. destructor differed depending upon its administration. Nevertheless, the ratio selection calculated by this oil is expected to enable its application under field conditions with a good safety margin. This oil could also be used in combination with other oils in integrated pest management strategies in bee colonies.

Repellent and acaricidal effects of botanical extracts on Varroa destructor

Extracts of indigenous plants from South America have shown a broad spectrum of bioactivities. No-contaminant and natural substances have recently resurged as control treatment options for varroosis in honey bee colonies from Argentina. The aim of this work was to evaluate the biological activity of botanical extracts from Baccharis flabellata and Minthostachys verticillata on Varroa destructor and Apis mellifera. The acaricidal and insecticidal activities were assessed by the spraying application method. Both ethanolic extracts showed high levels of toxicity against the mites and were harmless to their host, A. mellifera. During the attractive-repellent test, the olfactory stimulus evoked for the extract from B. flabellata resulted as a repellent for mites. The aromatic stimulus of these extracts would be strong enough to cause disturbance on the behavior of V. destructor. Thus, the repellent effect of these substances plus the toxicity on mites postulate these botanical extracts like promising natural compound to be incorporated for the control of varroosis.

In vitro and in vivo efficacy of carvacrol against Echinococcus granulosus

Currently, benzimidazoles are used as chemotherapeutic agents and as a complement to surgery and PAIR in the treatment of cystic echinococcosis (CE). They are generally applied at high doses causing side effects and, 50% of cases do not respond favorably to such chemotherapy. The use of essential oils obtained by distillation from aromatic plants would be an effective alternative or complementary to the synthetic compounds, because would not bring the appearance of side effects. Carvacrol and his isomer thymol are the main phenolic components from essential oils of Origanum vulgare (oregano) and Thymus vulgaris (thyme). The aim of the present work was to evaluate the in vitro and in vivo efficacy of carvacrol against Echinococcus granulosus metacestodes. For the in vitro assay, protoscoleces and cysts of E. granulosus were incubated with carvacrol at the following final concentrations: 10, 5 and 1μg/ml of carvacrol. The maximum protoscolicidal effect was found with 10μg/ml of carvacrol. Results of viability tests were consistent with the structural and ultrastructural damage observed in protoscoleces. Ultrastructural studies revealed that the germinal layer of cysts treated with carvacrol lost the multicellular structure feature. In the clinical efficacy study, a reduction in cyst weight was observed after the administration of 40mg/kg of carvacrol during 20days in mice with cysts developed during 4 months, compared to that of those collected from control mice. Given that the in vivo effect of carvacrol was comparable with the treatment of reference with ABZ and the fact that is a safe compound, we postulated that carvacrol may be an alternative option for treatment of human CE.

Oral administration of essential oils and main components: Study on honey bee survival and Nosema ceranae development

Diverse parasites and pathogens affect productivity and survival of honey bees. Plant secondary metabolites are potential alternative treatments, however, their effect has been little studied on microsporidian diseases. Furthermore, there is poor information about the toxicity resulting from prolonged oral administration of these substances to bees. In this research, we evaluated in vivo effects of different essential oils and main components (MCs) on bee survival and Nosema ceranae development under an ad libitum non-choice regimen. Substances administered on sucrose syrup to newly emerged bees at different concentrations were avidly consumed and caused different survival performances. Nevertheless, sublethal doses of substances did not control the parasite.

A scientific note on the first report of honeybee venom inhibiting Paenibacillus larvae growth

In the eusocial honeybee, Apis mellifera, worker bees use a stinging apparatus for defense. The sting is supplied with venom by glands localized in the abdomen. Honeybee venom (BV) is composed of at least 18 bioactive molecules, ranging from biogenic amines to proteins whose structure and function have been largely determined. These include peptides such as melittin, apamin, adolapin, and mast cell degranulating peptide; biologically active amines; enzymes as phospholipase A2 (PLA2); and a few nonpeptide components (Peiren et al. 2005; Matysiak et al. 2011). Melittin and PLA2 are the most abundant proteins, representing 50 and 12 % of BV dry weight, respectively. Furthermore, the venom gland has been recently reported as an important source of antimicrobial substances with proven antibacterial and antifungal action (Yu et al. 2012; Han et al. 2013). Nevertheless, data about the effects of BV on infectious pathogens of honeybees are previously absent from the literature. In this study, we proved that BV is able to inhibit the growth of a pathogenic bacterium affecting honeybee colonies. Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most destructive brood diseases of A. mellifera. Traditional control by chemotherapy has caused residues in hive products, and, more disturbingly, resistant pathogens (Evans 2003). These facts have driven studies on natural alternatives for AFB control. Thus, the aim was to analyze the effects of honeybee venom on P. larvae growth as well as its effect on adult bee survival after chronic exposure to BV. P. larvae strains had been isolated from larvae with AFB from four apiaries in Argentina and PL33 (UB-CIDEFI) like reference strain. All strains were genotypically identified using PL2-Fw and PL2-Rv primers, and characterized like ERIC I with ERIC1R -and ERIC2 primers (Genersch et al. 2006). For BV extraction, A. mellifera forager bees were obtained by covering the hive entrance and collecting them in their back flight. Venom sacs were dissected by pulling the sting apparatus from 30 bees. The resultant droplet of BV from the tip of the sting was placed on sterile slides, allowed to dry in dark for 24 h, were collected by scraping and then weighed. P. larvae isolates were exposed to serial dilutions of BV. One hundred microliters of Mueller–Hinton broth, yeast extract, and thiamine broth (Gende et al. 2008) were placed per well. BV stock solution and 50 μL of the bacterial suspension (0.5 McFarland scale) were added to each well (serial dilutions from 200 to 1.56 μg/mL). Incubation took place at 37oC for 48 h. The inhibition of bacterial growth was confirmed by resazurin method (Damiani et al. 2014). Serial dilutions from 100 to 0.04 μg/mL of oxytetracycline (Fluka Analytical) were included as a positive control (Alippi et al. 2007). The lowest concentration of each substance that showed inhibition was considered as the minimal inhibitory concentration (MIC). The minimal bactericidal concentration (MBC) was estimated transferring 100 μL honeybee venom/ Paenibacillus larvae / antimicrobial activity / Apismellifera