BackgroundIn 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses.Methodology/Principal FindingsWe used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006–2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone.Conclusions/SignificanceThese findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.

Timely classification and identification of bacteria is of vital importance in many areas of public health. We present a mass spectrometry (MS)-based proteomics approach for bacterial classification. In this method, a bacterial proteome database is derived from all potential protein coding open reading frames (ORFs) found in 170 fully sequenced bacterial genomes. Amino acid sequences of tryptic peptides obtained by LC-ESI MS/MS analysis of the digest of bacterial cell extracts are assigned to individual bacterial proteomes in the database. Phylogenetic profiles of these peptides are used to create a matrix of sequence-to-bacterium assignments. These matrixes, viewed as specific assignment bitmaps, are analyzed using statistical tools to reveal the relatedness between a test bacterial sample and the microorganism database. It is shown that, if a sufficient amount of sequence information is obtained from the MS/MS experiments, a bacterial sample can be classified to a strain level by using this proteomics method, leading to its positive identification.

Public reporting burden for fthi collection of Informaiaon is estimated to, average I hour per response, including the time for reviewing Instructions. searchilng erristing data sources, gatheing and maintsteng the data needed, and completing and review"n this collection of Information. Send cofmment regarding this burden estimate or any othier aspect of this collection of Infomuation, Indluding suggestions for reducing fti burden to Departmrent PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORTDIR ECBC, ATTN: AMSRD-ECBC-RT-II, APG, MD 21010-5424 NUMBER GEO-CENTERS, Inc., Gunpowder Branch, APG, MD 21010-5424 ECBC-TR-415 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORIMONITOR'S ACRONYM(S) SPONSORIMONITOR'S REPORT NUMBER(S) DISTRIBUTION I AVAILABILITY STATEMENTApproved for public release; distribution is unlimited. SUPPLEMENTARY NOTES ABSTRACTThis work discusses the significance of the pyrolyzate peaks observed in the gas chromatography-ion mobility spectrometry (GC-IMS) dataspace of the pyrolysis-GC-IMS (Py-GC-IMS) briefcase system. This system has the ability to detect and classifyi deliberately released bioaerosols in outdoor field scenarios. The bioaerosols include Gram-positive spores and Gram-negative bacteria, the MS-2 Escherichia coliphage virus, and ovalbumnin (OV) protein species. The work suggests certain improvements that can be made to the IMS detection System. A pyrolysis-gas chromatography-ion mobility spectrometry (Py-GC-IMS) briefcase system can detect and classify deliberately released bioaerosols in outdoor field scenarios. The bioaerosols include Gram-positive spores and Gram-negative bacteria, the MS-2 Escherichia coliphage virus, and ovalbumin (OV) protein species. However, the origin and structural identities of the pyrolyzate peaks observed in the GC-IMS dataspace, their microbiological information content, and taxonomic importance with respect to biodetection have not been determined. SUBJECT TERMS PyrolysisThe present work interrogates the identities of the peaks by inserting a time-offlight (TOF) mass spectrometry (MS) system in parallel with the IMS detector through a Tee connection in the GC module. Biological substances, producing ion mobility peaks from the pyrolysis of microorganisms, have been identified by their GC retention times, by matching their electron ionization mass spectra with authentic standards, and by the National Institute of Standards and Technology (NIST) mass spectral database.Strong signals from 2-pyridinecarboxamide were identified in Bacillus samples, including Bacillus anthracis, and their origins were traced to the cell wall peptidoglycan macromolecule. 3-Hydroxymyristic acid is a component of lipopolysaccharides (LPS) in the cell walls of Gram-negative organisms. The Gram-negative E. coli organism showed significant amounts of 3-Hydroxymyristic acid derivatives and degradation products in Py-GC-MS analyses. Some of the fatty acid derivatives were observed in very low abundance in the ion mobi...