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Abstract: Nosema apis and Nosema ceranae are two microsporidian pathogens of the European honey bee, Apis mellifera. There is evidence that N. ceranae is more virulent than N. apis subject to environmental factors like climate. This makes N. ceranae one of the suspects in the increasing colony losses recently observed in many regions of the world. Correct differentiation between N. apis and N. ceranae is important and best accomplished by molecular methods. So far only protocols based on species–specific sequence differences in the 16S rRNA gene are available. However, recent studies indicated that these methods may lead to confusing results due to polymorphisms in and recombination between the multi-copy 16S rRNA genes. To solve this problem and to provide a reliable molecular tool for the differentiation between the two bee pathogenic microsporidia we here present and evaluate a duplex-PCR protocol based on species–specific sequence differences in the highly conserved gene coding for the DNA-dependent RNA polymerase II largest subunit. A total of 102 honey bee samples were analyzed by the novel PCR protocol and the results were compared with the results of the originally published PCR–RFLP analysis and two recently published differentiation protocols, based on 16S rRNA sequence differences. Although the novel PCR protocol proved to be as reliable as the 16S rRNA gene based PCR–RFLP it was superior to simple 16S rRNA based PCR protocols which tended to overestimate the rate of N. ceranae infections. Therefore, we propose that species–specific sequence differences of highly conserved protein coding genes should become the preferred molecular tool for differentiation of Nosema spp. [Copyright &y& Elsevier]

The population of managed honey bees has been dramatically declining in the recent past in many regions of the world. Consensus now seems to be that pathogens and parasites (e.g. the ectoparasitic mite Varroa destructor, the microsporidium Nosema ceranae and viruses) play a major role in this demise. However, little is known about host-pathogen interactions for bee pathogens and attempts to develop novel strategies to combat bee diseases have been hampered by this gap in our knowledge. One reason for this dire situation is the complete lack of cell cultures for the propagation and study of bee pathogens. Here we present a cell culture model for two honey bee-pathogenic microsporidian species, Nosema apis and N. ceranae. Our cell culture system is based on a lepidopteran cell line, which proved to be susceptible to infection by both N. ceranae and N. apis and enabled us to illustrate the entire life cycle of these microsporidia. We observed hitherto undescribed spindle-shaped meronts and confirmed our findings in infected bees. Our cell culture model provides a previously unavailable means to explore the nature of interactions between the honey bee and its pathogen complex at a mechanistic level and will allow the development of novel treatment strategies. [ABSTRACT FROM AUTHOR]

Nosema ceranae and Nosema apis are two fungal pathogens belonging to the phylum Microsporidia and infecting the European honeybee, Apis mellifera. Recent studies have suggested that N. ceranae is more virulent than N. apis both at the individual insect level and at the colony level. Severe colony losses could be attributed to N. ceranae infections, and an unusual form of nosemosis is caused by this pathogen. In the present study, data from a 5-year cohort study of the prevalence of Nosema spp. in Germany, involving about 220 honeybee colonies and a total of 1,997 samples collected from these colonies each spring and autumn and analyzed via species-specific PCR-restriction fragment length polymorphism (RFLP), are described. Statistical analysis of the data revealed no relation between colony mortality and detectable levels of infection with N. ceranae or N. apis. In addition, N. apis is still more prevalent than N. ceranae in the cohort of the German bee population that was analyzed. A possible explanation for these findings could be the marked decrease in spore germination that was observed after even a short exposure to low temperatures (+4°C) for N. ceranae only. Reduced or inhibited N. ceranae spore germination at low temperatures should hamper the infectivity and spread of this pathogen in climatic regions characterized by a rather cold winter season. [ABSTRACT FROM AUTHOR]

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae–infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis. [ABSTRACT FROM AUTHOR]

Abstract: The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93–95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30–37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR–RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping. [Copyright &y& Elsevier]