Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research

Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera

<p>Abstract</p> <p>Background</p> <p>For years, the understanding of the pathogenetic mechanisms that underlie honey bee viral diseases has been severely hindered because of the lack of a cell culture system for virus propagation. As a result, it is very imperative to develop new methods that would permit the <it>in vitro </it>pathogenesis study of honey bee viruses. The identification of virus replication is an important step towards the understanding of the pathogenesis process of viruses in their respective hosts. In the present study, we developed a strand-specific RT-PCR-based method for analysis of Deformed Wing Virus (DWV) replication in honey bees and in honey bee parasitic mites, <it>Varroa Destructor</it>.</p> <p>Results</p> <p>The results shows that the method developed in our study allows reliable identification of the virus replication and solves the problem of falsely-primed cDNA amplifications that commonly exists in the current system. Using TaqMan real-time quantitative RT-PCR incorporated with biotinylated primers and magnetic beads purification step, we characterized the replication and tissue tropism of DWV infection in honey bees. We provide evidence for DWV replication in the tissues of wings, head, thorax, legs, hemolymph, and gut of honey bees and also in Varroa mites.</p> <p>Conclusion</p> <p>The strategy reported in the present study forms a model system for studying bee virus replication, pathogenesis and immunity. This study should be a significant contribution to the goal of achieving a better understanding of virus pathogenesis in honey bees and to the design of appropriate control measures for bee populations at risk to virus infections.</p

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer

<p>Abstract</p> <p>Background</p> <p>Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk.</p> <p>Methods</p> <p>A case-control study design was used to test the association between prostate cancer risk and the polymorphisms <it>TNF-A</it>-308 A/G (rs 1800629), <it>RANTES</it>-403 G/A (rs 2107538), <it>IL1-A</it>-889 C/T (rs 1800587) and <it>MCP-1 </it>2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area.</p> <p>Results</p> <p>Diagnosis of prostate cancer was significantly associated with <it>TNF-A </it>GA + AA genotype (OR, 1.61; 95% CI, 1.09–2.64) and <it>RANTES </it>GA + AA genotype (OR, 1.44; 95% CI, 1.09–2.38). A alleles in <it>TNF-A </it>and <it>RANTES </it>influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and <it>IL1-A </it>or <it>MCP-1 </it>polymorphisms.</p> <p>Conclusion</p> <p>Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.</p

Priming with a Recombinant Pantothenate Auxotroph of Mycobacterium bovis BCG and Boosting with MVA Elicits HIV-1 Gag Specific CD8+ T Cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8+ T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4+ T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge

Predictive Markers of Honey Bee Colony Collapse

Across the Northern hemisphere, managed honey bee colonies, Apis mellifera, are currently affected by abrupt depopulation during winter and many factors are suspected to be involved, either alone or in combination. Parasites and pathogens are considered as principal actors, in particular the ectoparasitic mite Varroa destructor, associated viruses and the microsporidian Nosema ceranae. Here we used long term monitoring of colonies and screening for eleven disease agents and genes involved in bee immunity and physiology to identify predictive markers of honeybee colony losses during winter. The data show that DWV, Nosema ceranae, Varroa destructor and Vitellogenin can be predictive markers for winter colony losses, but their predictive power strongly depends on the season. In particular, the data support that V. destructor is a key player for losses, arguably in line with its specific impact on the health of individual bees and colonies

Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia

Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (∼1011 viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January

The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10 7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10 6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge