The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay.

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.

The prevalence of Acarapis woodi in Spanish honey bee (Apis mellifera) colonies.

Acarapis woodi is an internal obligate parasite of the respiratory system of honey bees which provokes significant economic losses in many geographical areas. The main aim of this study was assess the A. woodi role in the "higher honey bee colony losses phenomenon" in Spain. Therefore, a new polymerase chain reaction (PCR) was developed to amplify the mitochondrial cytochrome oxidase I gene (COI) and so the actual prevalence of A. woodi in Spanish honey bee colonies in 2006 and 2007 was determined as part of a wider survey. The results revealed a greater prevalence than expected in most of the geographical areas studied where has been generally underestimated One problem encountered in this study was to distinguish between A. woodi and other species (Acarapis dorsalis and Acarapis externus) at the molecular level. Furthermore, the patterns of genetic divergence across sequences raised serious doubts about the current classification of these organisms.

Further evidence of an oriental origin for Nosema ceranae (Microsporidia: Nosematidae).

Although Nosema ceranae was first isolated from the Asian honeybee (Apis cerana) in Asia and then subsequently recognized as a widespread gut parasite of the Western honeybee (Apis mellifera), its origins and primary host are yet to be accurately established. In this study we examined the possibility of an Asian origin for the parasite by looking for evidence of its ongoing spread out of Asia. To do this, we surveyed for the presence of N. ceranae in A. cerana and A. mellifera on isolated islands of the Solomon Islands (Pacific region), most of which were inhabited with A. mellifera that had been introduced from Australia and New Zealand at a time when N. ceranae was not present in either country, but on which some had also recently become inhabited with invasive A. cerana that originated from Asia with no prior history of contact with A. mellifera infected with N. ceranae. We also sought to verify previous findings that N. ceranae was widespread in Asian honeybees by surveying for its presence in isolated populations of the Asian honeybees, A. cerana, A. koschevnikovi, A. nigrocincta and A. florea. We obtained evidence that A. cerana introduced N. ceranae to A. mellifera in the Solomon Islands and also confirmed the widespread occurrence of the parasite in Asian honeybees, even reporting it for the first time in A. koschevnikovi from Borneo. Our findings provide further support for the hypothesis that N. ceranae has only recently emerged from Asia to become a parasite of A. mellifera.

Linking evolutionary lineage with parasite and pathogen prevalence in the Iberian honey bee.

The recent decline in honey bee colonies observed in both European countries and worldwide is of great interest and concern, although the underlying causes remain poorly understood. In recent years, growing evidence has implicated parasites and pathogens in this decline of both the vitality and number of honey bee colonies. The Iberian Peninsula provides an interesting environment in which to study the occurrence of pathogens and parasites in the host honey bee populations due to the presence of two evolutionary lineages in A. m. iberiensis (Western European [M] or African [A]). Here, we provide the first evidence linking the population structure of the Iberian honey bee with the prevalence of some of its most important parasites and pathogens: the Varroa destructor mite and the microsporidia Nosema apis and Nosema ceranae. Using data collected in two surveys conducted in 2006 and 2010 in 41 Spanish provinces, the evolutionary lineage and the presence of the three parasitic organisms cited above were analyzed in a total of 228 colonies. In 2006 N. apis was found in a significantly higher proportion of M lineage honey bees than in the A lineage. However, in 2010 this situation had changed significantly due to a higher prevalence of N. ceranae. We observed no significant relationships in either year between the distributions of V. destructor or N. ceranae and the evolutionary lineage present in A. m. iberiensis colonies, but the effects of these organisms on the genetic diversity of the honey bee populations need further research.

Nosema spp. parasitization decreases the effectiveness of acaricide strips (Apivar(®) ) in treating varroosis of honey bee (Apis mellifera iberiensis) colonies.

Given the key role played by honey bees in almost all terrestrial ecosystems, maintaining bee populations in adequate sanitary conditions is crucial for these essential pollinators to continue their work. From the beginning of the 21st century, beekeepers have reported a progressive increase in the overwintering mortality of honey bee colonies worldwide. Despite the failure to reach a consensus regarding the cause of this phenomenon, pathogens are thought to be strongly implicated. In the present work, we provide evidence of the negative effects of colony parasitization by Nosema spp. - primarily by N. ceranae- on the effectiveness of acaricide strips to treat Varroa destructor. The effectiveness of the Varroa mite strip treatment (Apivar(®) ) was greater in colonies in which Nosema spp. parasitization had been controlled. Several studies report that infection by Nosema spp. may affect the behaviour of worker bees. As the effectiveness of Varroa strip treatment depends on bees contacting the strips and their subsequent interaction within the colony, such behavioural and social alterations could interfere with the treatment and allow more severe effects to develop in the colonies infected by Nosema. These results should be considered when assessing acaricide treatments in field conditions due to the high prevalence of both pathogens worldwide.

The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade.

Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of samples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey.

A preliminary study of the epidemiological factors related to honey bee colony loss in Spain.

In recent years, a worldwide decline in the Apis mellifera populations has been detected in many regions, including Spain. This decline is thought to be related to the effects of pathogens or pesticides, although to what extent these factors are implicated is still not clear. In this study, we estimated the prevalence of honey bee colony depopulation symptoms in a random selected sample (n = 61) and we explored the implication of different pathogens, pesticides and the flora visited in the area under study. The prevalence of colony depopulation symptoms in the professional apiaries studied was 67.2% [95% confidence interval (CI) = 54.6-79.8; P < 0.0001]. The most prevalent pathogen found in the worker honey bee samples was Nosema ceranae[65.6%; 95% CI = 52.8-78.3; P < 0.0001], followed by Varroa destructor[32.7%; 95% CI = 20.2-45.4; P < 0.0001] and 97.5% of the colonies infected by N. ceranae were unhealthy (depopulated). Co-infection by V. destructor and N. ceranae was evident in 22.9% (95% CI = 11.6-34.3; P < 0.0001) of the samples and only in unhealthy colonies. Of the 40 pesticides studied, only nine were detected in 49% of the stored pollen samples analysed. Fipronil was detected in only three of 61 stored pollen samples and imidacloprid was not detected in any. Acaricides like fluvalinate, and chlorfenvinphos used to control Varroa mite were the most predominant residues in the stored pollen, probably as a result of their application in homemade formulae. None of the pesticides identified were statistically associated to colony depopulated. This preliminary study of epidemiological factors suggests that N. ceranae is a key factor in the colony losses detected over recent years in Spain. However, more detailed studies that permit subgroup analyses will be necessary to contrast these findings.

Effect of temperature on the biotic potential of honeybee microsporidia.

The biological cycle of Nosema spp. in honeybees depends on temperature. When expressed as total spore counts per day after infection, the biotic potentials of Nosema apis and N. ceranae at 33 degrees C were similar, but a higher proportion of immature stages of N. ceranae than of N. apis were seen. At 25 and 37 degrees C, the biotic potential of N. ceranae was higher than that of N. apis. The better adaptation of N. ceranae to complete its endogenous cycle at different temperatures clearly supports the observation of the different epidemiological patterns.

Honeybee colony collapse due to Nosema ceranae in professional apiaries.

Honeybee colony collapse is a sanitary and ecological worldwide problem. The features of this syndrome are an unexplained disappearance of adult bees, a lack of brood attention, reduced colony strength, and heavy winter mortality without any previous evident pathological disturbances. To date there has not been a consensus about its origins. This report describes the clinical features of two professional bee-keepers affecting by this syndrome. Anamnesis, clinical examination and analyses support that the depopulation in both cases was due to the infection by Nosema ceranae (Microsporidia), an emerging pathogen of Apis mellifera. No other significant pathogens or pesticides (neonicotinoids) were detected and the bees had not been foraging in corn or sunflower crops. The treatment with fumagillin avoided the loss of surviving weak colonies. This is the first case report of honeybee colony collapse due to N. ceranae in professional apiaries in field conditions reported worldwide.

Regurgitated pellets of Merops apiaster as fomites of infective Nosema ceranae (Microsporidia) spores.

The importance of transmission factor identification is of great epidemiological significance. The bee-eater (Merops apiaster) is a widely distributed insectivorous bird, locally abundant mainly in arid and semi-arid areas of southern Europe, northern Africa and western Asia but recently has been seen breeding in central Europe and Great Britain. Bee-eaters predominantly eat insects, especially bees, wasps and hornets. On the other hand, Nosema ceranae is a Microsporidia recently described as a parasite in Apis mellifera honeybees in Europe. Due to the short time since its description scarce epidemiological data are available. In this study we investigate the role of the regurgitated pellets of the European bee-eater as fomites of infective spores of N. ceranae. Spore detection in regurgitated pellets of M. apiaster is described [phase-contrast microscopy (PCM) and polymerase chain reaction (PCR) methods]. Eighteen days after collection N. ceranae spores still remain viable and their infectivity is shown after artificial infection of Nosema-free 8-day-old adult bees. The epidemiological consequences of the presence of Nosema spores in this fomites are discussed.

Detection of infective Nosema ceranae (Microsporidia) spores in corbicular pollen of forager honeybees.

Nosema ceranae is a Microsporidia recently described as a parasite in Apis mellifera honeybees in Europe. Due to the short time since its description, no epidemiological data are available. In this study, spore detection in both pollen baskets and pollen collected from commercial traps is described (PCM, TEM and PCR methods). Spore infectivity is shown after artificial infection of Nosema-free adult bees. The epidemiological consequences of the presence of Nosema spores in corbicular pollen require more study and must be considered in beekeeping practices.

Outcome of colonization of Apis mellifera by Nosema ceranae.

A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.