A Comparison of Different Matrices for the Laboratory Diagnosis of the Epizootic American Foulbrood of Honey Bees

American Foulbrood (AFB) of honey bees caused by the spore-forming bacterium Paenibacillus larvae is a notifiable epizootic in most countries. Authorities often consider a rigorous eradication policy the only sustainable control measure. However, early diagnosis of infected but not yet diseased colonies opens up the possibility of ridding these colonies of P. larvae spores by the shook swarm method, thus preventing colony destruction by AFB or official control orders. Therefore, surveillance of bee colonies for P. larvae infection followed by appropriate sanitary measures is a very important intervention to control AFB. For the detection of P. larvae spores in infected colonies, samples of brood comb honey, adult bees, or hive debris are commonly used. We here present our results from a comparative study on the suitability of these matrices in reliably and correctly detecting P. larvae spores contained in these matrices. Based on the sensitivity and limit of detection of P. larvae spores in samples from hive debris, adult bees, and brood comb honey, we conclude that the latter two are equally well-suited for AFB surveillance programs. Hive debris samples should only be used when it is not possible to collect honey or adult bee samples from brood combs

Bioassay for grooming effectiveness towards Varroa destructor mites in Africanized and Carniolan honey bees

Grooming behavior is considered a varroosis tolerance factor of Africanized honey bees, but this behavior is difficult to evaluate directly within the honey bee colony. A laboratory bioassay was developed to measure the intensity and effectiveness of grooming responses by worker bees artificially infested with one Varroa mite. At a study site in tropical Brazil, the sequence of seven well- defined grooming reactions towards mites of different colonial origin was compared. In a total of 226 assays, Africanized bees responded significantly faster and more intensively than Carniolan workers. But there were no statistical differences in the removal of mites according to the bee types. Even extensive grooming behavior never resulted in damage or death of the mites. The possible use of the bioassay as a screening for the extent of the grooming behavior is discussed

Scent or movement of Varroa destructor mites does not elicit hygienic behaviour by Africanized and Carniolan honey bees

Hygienic behaviour of mite-tolerant Africanized and susceptible Carniolan colonies was evaluated in Brazil by sham-manipulating or artificially inoculating 4175 capped worker brood cells with dead Varroa destructor mites or ants, or their odour extracts. Both bee types expressed the hygienic components `uncapping', `removal of introduced mite/ant' and `removal of brood' to the same extent and pattern. The similar response to dead mites of different origins and solvent-extracted mites indicates a minor role of scent or of movement of mites within sealed brood cells as releasers of hygienic behaviour. However, application of dichlormethane-extract of mites increased the hygienic response compared to pure solvent alone. Hygienic reactions to mite infested brood cells must, therefore, be elicited by other signals, possibly by the detection of specific reactions or odours of the infested larvae or pupae

Sterilization of beehive material with a double inductively coupled low pressure plasma

American Foulbrood is a severe, notifiable disease of the honey bee. It is caused by infection of bee larvae with spores of the gram-positive bacterium $\textit {Paenibacillus larvae}$. Spores of this organism are found in high numbers in an infected hive and are highly resistant to physical and chemical inactivation methods. The procedures to rehabilitate affected apiaries often result in the destruction of beehive material. In this study we assess the suitability of a double inductively coupled low pressure plasma as a non-destructive, yet effective alternative inactivation method for bacterial spores of the model organism $\textit {Bacillus subtilis}$ on beehive material. Plasma treatment was able to effectively remove spores from wax, which, under protocols currently established in veterinary practice, normally is destroyed by ignition or autoclaved for sterilization. Spores were removed from wooden surfaces with efficacies significantly higher than methods currently used in veterinary practice, such as scorching by flame treatment. In addition, we were able to non-destructively remove spores from the highly delicate honeycomb wax structures, potentially making treatment of beehive material with double inductively coupled low pressure plasma part of a fast and reliable method to rehabilitate infected bee colonies with the potential to re-use honeycombs

A comparison of the hygienic response of Africanized and European (Apis mellifera carnica) honey bees to Varroa-infested brood in tropical Brazil

In order to examine the significance of hygienic behavior for the tolerance to varroosis of Africanized honey bees, they were compared with non-tolerant Carniolans in tropical Brazil. Capped worker brood cells were artificially infested with living Varroa mites, and inspected some days later. Uncapping, disappearance of the introduced mite and removal of the pupa were recorded in a total of manipulated 3,096 cells during three summer seasons. The hygienic response varied between Africanized and Carniolan colonies, but this difference was significant only in one year, during which Africanized honey bees removed a significantly greater proportion of Varroa mites than European honey bees. A high proportion of the mites disappeared from artificially infested brood cells without damage to the pupae. The opening of the cell and the removal of the bee brood are independent traits of a graded response by adult workers towards mite-infested brood cells. We found a higher between-colony variation in the reaction towards Varroa-infested brood of Africanized honey bees compared to Carniolans. The overall similar response of the two bee types indicates that hygienic behavior is not a key factor in the tolerance to varroosis of Africanized bees in Brazil.<br>Com o intuito de examinar o significado do comportamento higiênico na tolerância à varroose de abelhas africanizadas, elas foram comparadas com as não tolerantes Cárnicas no Brasil tropical. Células de cria de operárias operculadas foram artificialmente infestadas com ácaros Varroa vivos e inspecionadas alguns dias depois. Desoperculação, desaparecimento dos ácaros introduzidos e remoção da pupa foram anotados em um total de 3096 células manipuladas durante três verões. A resposta higiênica variou entre as colônias africanizadas e de Cárnicas, mas esta diferença foi significante apenas em um ano, durante o qual as abelhas africanizadas removeram uma proporção significantemente maior de ácaros Varroa que as abelhas européias. Uma grande porporção de ácaros desapareceram das células das crias artificialmente infestadas, sem danos às pupas. A abertura da célula e remoção das crias das abelhas são características independentes de uma resposta graduada por parte de operárias adultas com relação a células de crias infestadas por ácaros. Nós encontramos uma variação maior entre colônias na reação de abelhas africanizadas à cria infestada por Varroa, quando comparadas a abelhas Cárnicas. A resposta geralmente similar dos dois tipos de abelhas indica que o comportamento higiênico não é um fator importante na tolerância à varroose de abelhas africanizadas no Brasil