Deformed wing virus type a and b in managed honeybee colonies of Argentina.

Apis mellifera is infected by more than 24 virus species worldwide, mainly positive-sense, single-stranded RNA viruses of the Dicistroviridae and Iflaviridae families. Among the viruses that infect honey bees, Deformed wing virus is the most prevalent and is present as three master variants DWV-A, B, and C. Given that the ectoparasitic mite Varroa destructor vectors these virus variants, recombination events between them are expected, and variants and their recombinants can co-exist in mites and honeybees at the same time. In this study, we detect, through RT-qPCR, the presence of DWV-A and B in the same samples of adult bees from colonies of Argentina. Total RNA was extracted from pools of ten adult bees from 45 apiaries distributed across the main beekeeping Provinces of Argentina (Buenos Aires, Santa Fe, Córdoba, Santiago del Estero, Río Negro, and Mendoza); then RT-qPCR reactions were performed to detect DWV-A and B, with specific primer pairs. After the amplifications, PCR products (204 and 660 bp amplicons for DWV-B, and ~250 bp for DWV-A) were purified and sequenced to verify that they corresponded to reported sequences, analyzing them using the Blast software. Of the 45 samples analyzed by RT-qPCR, over 90% were infected with DWV-A and 47% were also positive for DWV-B, where it was found in high prevalence specifically in colonies of A. mellifera of the Buenos Aires Province. Future studies will determine the impact of this type of the virus and its ability to recombine with the other DWV types in the apiaries of our country.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America / Primeira detecção molecular de co-infecção de vírus de abelhas em Bombus atratus assintomática na América do Sul

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

A polinização é essencial para a produção de alimentos e tem como particularidade a conexão entre os ecossistemas naturais com sistemas de produção agrícola. Recentemente, as perdas de espécies de bumblebee em todo o mundo têm sido relatadas. Neste trabalho, amostras de uma exploração comercial de bumblebee da Argentina, com recente história de mortes foram estudadas utilizando uma Multiplex PCR para a detecção de vírus de abelha mais frequentemente detectados na América do Sul. Todas as amostras analisadas foram positivas para as co-infecções com Deformed wing virus, Black queen cell viruses e Sacbrood virus. Este trabalho descreve o primeiro relato de infecção de Bombus atratus com vírus de abelhas. Uma melhor compreensão das infecções virais em bumblebee e da epidemiologia dos vírus poderia ser de grande importância, uma vez que tais abelhas podem servir como reservatório viral, com possível repercussão tanto na produtividade de abelhas melíferas como afetando-as diretamente.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America.

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

Pharmacokinetics of ceftazidime administered to lactating and non-lactating goats.

The aim of this work was to determine the pharmacokinetics of intravenous (i.v.) and intramuscular (i.m.) ceftazidime administered to lactating (LTG; n=6) and non-lactating (NLTG; n=6) healthy Creole goats in 2 trials (T1 and T2). During T1 and T2, goats randomly received a single dose of i.m. or i.v. ceftazidime (10 mg/kg). Serum concentration of iv ceftazidime in NLTG and LTG goats is best described by 2 and 3 compartment models, respectively. The pharmacokinetic parameters of iv and im ceftazidime administered to LTG and NLTG showed statistically significant differences (P < 0.05) in the constants (lamda(z), T1 vs. T2 [i.v.] 0.5 +/- 0.1 vs. 0.3 +/- 0.1/h; T1 vs. T2 [i.m.] 0.5 +/- 0.2 vs. 0.3 +/- 0.1/h) and in the mean times (t(1/2), T1 vs. T2 [i.v.] 1.6 +/- 0.3 vs. 2.3 +/- 0.6 h; T1 vs. T2 [i.m.] 1.6 +/- 0.7 vs. 2.6 +/- 0.9 h) of elimination. The bioavailability of ceftazidime in LTG and NLTG was 113.0 +/- 17.8 and 96.0 +/- 18.0%, respectively. Ceftazidime concentration in milk at 2 h was: i.v. = 1.9 +/- 0.2 and i.m. = 2.4 +/- 0.5 microg/ml; the penetration in milk was i.v. = 18.3 +/- 13.5 and im = 14.3 +/- 10.6%. Ninety-six hours after i.v. and i.m. administration, residues of the drug were not found in milk. In conclusion, ceftazidime, when administered to goats, showed high concentration times in serum, good penetration into milk and a bioavailability that makes it suitable to be used by the i.m. route.

Unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a proteínas presentes en abejas, larvas y productos de la colmena de Apis mellifera L / Binding of tylosin, tilmicosin and oxytetracycline to proteins from honeybees, larvae and beehive products

Las abejas melíferas son afectadas por gran cantidad de enfermedades infecciosas principalmente producidas por bacterias, hongos, virus y parásitos eucariotas. Dentro de las ocasionadas por procariotas, la loque americana es una enfermedad extremadamente grave que afecta a larvas y pupas de abejas; su agente causal es la bacteria esporulada Paenibacillus larvae. La administración de antibióticos es la principal alternativa para el control de esta enfermedad en colmenares con altos niveles de infección. El objetivo del presente trabajo fue determinar, mediante un método biológico, la unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a las proteínas presentes en abejas adultas, larvas menores de 72 horas, larvas mayores de 72 horas, jalea de obreras, miel y polen, con la finalidad de diseñar un modelo de ruta cinética de los antibióticos. Los límites de sensibilidad de la técnica de valoración de estos antibióticos fueron 0,05 μg/ml para tilosina y tilmicosina, y 0,01 μg/ml para oxitetraciclina. Los coeficientes de correlación fueron superiores a 0,90 y los coeficientes de variación intra e inter-ensayo inferiores al 5%. Tanto tilosina como oxitetraciclina presentaron un porcentaje de unión a proteínas de un 15% en promedio en tejidos y subproductos de la colmena, lo cual resultó inferior a lo observado con tilmicosina (29% en promedio). En conclusión, por sus características químicas, su actividad antimicrobiana y su baja tasa de unión a las abejas, larvas y subproductos de la colmena, la tilosina presenta propiedades farmacocinéticas que podrían representar una ventaja terapéutica para el tratamiento de la loque americana en colmenas.

American Foulbrood (AFB) caused by the spore-forming bacterium Paenibacillus larvae is the most serious disease of bacterial origin affecting larvae and pupae of honeybees. Antibiotics are used in many countries for the control of AFB in high incidence areas, but their misuse may lead to antibiotic resistance of bacterial strains and honey contamination. The objective of the present work was to determine, through a biological method, the protein binding of tylosin, tilmicosin and oxytetracycline to worker jelly; honey; pollen; adult bees and larvae in order to propose their kinetic routes. The sensitivity limit of the technique used was 0.05 μg/ml for tylosin and tilmicosin and 0.01 μg/ml for oxytetracycline, respectively. The method had intra and inter-assay correlation coefficients over 0.90, respectively and a coefficient variation of intra-and inter-assay for all antibiotics and processed samples under 5%. Tylosin and oxytetracycline presented lower percentages of protein binding in tissues and hive products (average 15%) in relation to those observed for tilmicosin (29%). In conclusion, tylosin is useful for AFB control in honey bee colonies due to its chemical characteristics, antimicrobial activity and levels of protein binding in bees, larvae, and beehive products.

[Binding of tylosin, tilmicosin and oxytetracycline to proteins from honeybees, larvae and beehive products]. / Unión de los antibióticos tilosina, tilmicosina y oxitetraciclina a proteínas presentes en abejas, larvas y productos de la colmena de Apis mellifera L.

American Foulbrood (AFB) caused by the spore-forming bacterium Paenibacillus larvae is the most serious disease of bacterial origin affecting larvae and pupae of honeybees. Antibiotics are used in many countries for the control of AFB in high incidence areas, but their misuse may lead to antibiotic resistance of bacterial strains and honey contamination. The objective of the present work was to determine, through a biological method, the protein binding of tylosin, tilmicosin and oxytetracycline to worker jelly; honey; pollen; adult bees and larvae in order to propose their kinetic routes. The sensitivity limit of the technique used was 0.05 µg/ml for tylosin and tilmicosin and 0.01 µg/ml for oxytetracycline, respectively. The method had intra and inter-assay correlation coefficients over 0.90, respectively and a coefficient variation of intra-and inter-assay for all antibiotics and processed samples under 5%. Tylosin and oxytetracycline presented lower percentages of protein binding in tissues and hive products (average 15%) in relation to those observed for tilmicosin (29%). In conclusion, tylosin is useful for AFB control in honey bee colonies due to its chemical characteristics, antimicrobial activity and levels of protein binding in bees, larvae, and beehive products.

[Optimization of the growth of Paenibacillus larvae in semi-selective media]. / Optimización del desarrollo de Paenibacillus larvae en medios semiselectivos.

The sensitivity of media MYPGP, MYPGP(NALPIA) A (6 microg/ml nalidixic acid and 10 microg/ml pipemidic acid) and MYPGP(NALPIA) B (9 microg/ml nalidixic acid and 20 microg/ml pipemidic acid) for the recovery of viable spores of Paenibacillus larvae from honey, was evaluated by using different incubation times and different spore concentrations. No significant differences between incubation times, spore concentration or culture media were found. In the case of the recovery of vegetative cells from PBS at different incubation times and different dilutions no significant differences were found between the incubation times or the dilutions tested, while significant differences were found in the three media when compared with one another, MYPGP(NALPIA)B providing the lowest recovery of vegetative cells. Considering these results, we propose the use of MYPGP(NALPIA)B to recover spores of P. larvae from honey, specially for honeys with heterogeneous populations of bacterial spores; when culturing vegetative cells, MYPGP or MYPGP(NALPIA)A must be used to obtain good growth.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey.

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist/A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey / Inhibición del crecimiento de Ascosphaera apis mediante cepas de Bacillus y Paenibacillus aisladas de miel

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist / A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

La cría yesificada es una micosis invasiva ocasionada por el hongo heterotálico Ascosphaera apis que afecta exclusivamente a las larvas de las abejas. La enfermedad tiene difusión mundial y en la Argentina se halla diseminada en todas las áreas donde se realiza apicultura. Se estudió la potencialidad de 249 cepas de bacterias esporuladas aeróbicas aisladas de miel como agentes biocontroladores del hongo mediante un ensayo en disco central en condiciones de laboratorio. Se seleccionaron como mejores antagonistas 10 cepas bacterianas identificadas como Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), y Paenibacillus alvei (m321). Para probar la eficiencia de las cepas seleccionadas, se empleó la técnica de cultivo dual con 5 repeticiones de cada combinación antagonista bacteriano / cepa de A. apis, 5 para cada control y 4 medios de cultivo diferentes empleando 10 cepas del hongo de distintos orígenes geográficos. El análisis de la variancia y posterior comparación de medias LSD (Least Square Dfferences) mostró que los mejores antagonistas fueron B. subtilis (m329) y B. megaterium (m435) para el caso del medio YGPSA, mientras que para MYPGP las más eficientes fueron las cepas de B. circulans Fr 231 y m448b.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey.

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist/A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.