MS

thesisThe quantitative extent of resistance induced by specific and nonspecific immunization to systemically induced C. albicans infection was investigated in mice. Animals were specifically immunized by i.p. injections of formalinized suspension of C. albicans. Nonspecific resistance was induced by temporally spaced subcutaneous injections of endotoxin. The mice were hallenged by i.v. injections of known numbers of C. albicans. Animals were sacrificed at 24 hours intervals; cultures of spleen, liver, and kidney for viable C. albicans were done on antibiotic blood agar. In vitro studies were evaluated by comparing kinetic curves of inactinations of c. albicans by macrophages from various systems, that is, from animals that wer immunized, endotoxin treated or treated with saline. Lymphocytes plus macarophasges from the various systems were also challenged to determine if the lymphocyte was functionally involved with the destruction of C. albicans. The most significant in vivo result was that fewer yeast were found in the kidneys of immunized animals. The kidneys of the endotoxin treated animasl and the saline treated animals were heavily infected. No significant differences were observed in the livers and spleens of the different groups throughout the experiement. In vitro results indicate that macarophages from immunized animals have slightly but sutatistically significant enhance cytopeptic capacities when compared to macrophages from endotoxin treated and saline treated animals. Lylmphocytes appeard to enhace the cytopeptic response after a temporal delay which was not observed in cultures containin gonoy macrophages. The data suggest that macrophages from immunized animals, endotoxin treated animals or saline treated animals yieled a steeper inactivation curve. Reflecting death of the challenge organism, than the systems containing lymphocytes form the various systems acting in conjunction with macrophages from a homologous or heterologous system. Lylmphocytes from immunized animals and lymphocytes from endotoxin treated animals did enhance the cytopeptic capacities of macrophages from saline treated animals when comparisons to the lymphocyte-macrophage saline control system were made. This enhanced cytopepsis that occurred n the presence of the lymphocyte was not as pronounced n systems containing lymphocytes and macrophages form immunized animals and/or endotoxin treated animals. The fact that endotoxin treated lymphocytes stimulate the cytopeptic capacities of saline treated macrophages indicates a mechanism for the induction of nonspecific resistance. This implies that the mechanism of nonspecific resistance included cellular components and that both the lymphocyte and macrophage participate

Electron monochromator mass spectrometry for the analysis of whole bacteria and bacterial spores. Anal. Chem

Spores from a variety of Bacillus species were analyzed with direct probe mass spectrometry using an electron monochromator to select electrons of distinct energies for ionization. Electron energies were chosen to match the electron capture energies of taxonomically important compounds such as dipicolinic acid and fatty acids. Previous negative ion interferences were not observed when the monochromator was used, and the signal-tonoise ratio of targeted compounds was significantly enhanced using this approach. To demonstrate the selectivity of the technique, the monochromator was swept over a range of electron energies while monitoring the masses of compounds with known electron capture energies. Scanning the monochromator while the mass spectrometer was operated in single-ion mode enabled dipicolinic acid to be detected in 10 5 spores. The results presented here demonstrate the utility of the electron monochromator for selectively ionizing compounds directly in bacteria and bacterial spores

Longitudinal Metagenomic Analysis of the Water and Soil from Gulf of Mexico Beaches Affected by the Deep Water Horizon Oil Spill

Estimates of 7x105 cubic meters of crude oil were released into the Gulf of Mexico as a consequence of the April 20th, 2010 Deep Water Horizon drilling rig explosion, leaving thousands of square miles of earth's surface covered in crude oil. Dispersants were used on large slicks and injected at the well head, resulting in oil being suspended throughout the water column. Starting in June 2010, oil reached hundreds of miles of Louisiana, Alabama, Mississippi, and Florida shoreline disturbing the ecological balance and economic stability of the region. While visible damages are evident in the wildlife populations and marine estuaries, the most significant affect may be on the most basic level of the ecosystems: the bacterial and plankton populations.We present results from high throughput DNA sequencing of close-to-shore water and beach soil samples before and during the appearance of oil in Louisiana and Mississippi. Sixteen samples were taken over a two month period at approximately two week intervals from Grand Isle, LA and Gulfport, MS and were sequenced using the Illumina GAIIx platform. Significant genomic-based population fluctuations were observed in the soil and water samples. These included large spikes in the human pathogen Vibrio cholera, a sharp increase in Rickettsiales sp., and decrease of Synechococus sp. in water samples. Analysis of the contiguous de-novo assembled DNAs (contigs) from the samples also suggested the loss of biodiversity in water samples by the time oil appeared at the shores in both locations. Our observations lead us to the conclusion that oil strongly influenced microbial population dynamics, had a striking impact on the phytoplankton and other flora present prior to the appearance of oil, and that the microbial community had not recovered to pre-spill conditions by the end of our observational period

Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning

Historical Distribution and Molecular Diversity of Bacillus anthracis, Kazakhstan

This study provides useful baseline data for guiding future disease control programs

High-throughput sequencing of SARS-CoV-2 in wastewater provides insights into circulating variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) likely emerged from a zoonotic spill-over event and has led to a global pandemic. The public health response has been predominantly informed by surveillance of symptomatic individuals and contact tracing, with quarantine, and other preventive measures have then been applied to mitigate further spread. Non-traditional methods of surveillance such as genomic epidemiology and wastewater-based epidemiology (WBE) have also been leveraged during this pandemic. Genomic epidemiology uses high-throughput sequencing of SARS-CoV-2 genomes to inform local and international transmission events, as well as the diversity of circulating variants. WBE uses wastewater to analyse community spread, as it is known that SARS-CoV-2 is shed through bodily excretions. Since both symptomatic and asymptomatic individuals contribute to wastewater inputs, we hypothesized that the resultant pooled sample of population-wide excreta can provide a more comprehensive picture of SARS-CoV-2 genomic diversity circulating in a community than clinical testing and sequencing alone. In this study, we analysed 91 wastewater samples from 11 states in the USA, where the majority of samples represent Maricopa County, Arizona (USA). With the objective of assessing the viral diversity at a population scale, we undertook a single-nucleotide variant (SNV) analysis on data from 52 samples with \u3e90% SARS-CoV-2 genome coverage of sequence reads, and compared these SNVs with those detected in genomes sequenced from clinical patients. We identified 7973 SNVs, of which 548 were “novel” SNVs that had not yet been identified in the global clinical-derived data as of 17th June 2020 (the day after our last wastewater sampling date). However, between 17th of June 2020 and 20th November 2020, almost half of the novel SNVs have since been detected in clinical-derived data. Using the combination of SNVs present in each sample, we identified the more probable lineages present in that sample and compared them to lineages observed in North America prior to our sampling dates. The wastewater-derived SARS-CoV-2 sequence data indicates there were more lineages circulating across the sampled communities than represented in the clinical-derived data. Principal coordinate analyses identified patterns in population structure based on genetic variation within the sequenced samples, with clear trends associated with increased diversity likely due to a higher number of infected individuals relative to the sampling dates. We demonstrate that genetic correlation analysis combined with SNVs analysis using wastewater sampling can provide a comprehensive snapshot of the SARS-CoV-2 genetic population structure circulating within a community, which might not be observed if relying solely on clinical cases

Rapid - PCR ( LightCycler ) in diagnosis of biological agents

Although the historic use of biological weapons has been infrequent, a belief that state sponsored armies or terrorist organizations will use this type of weapon has never been greater which demands a capability for rapid medical response and early intervention. Molecular diagnostic methods, based on DNA amplification known as PCR (Polymerase Chain Reaction) are promising tools in fast and specific detection and identification of biological agent(s). The R.A.P.I.D.TM - PCR ( Ruggedized Advanced Pathogen Identification Device ) (fig.1), is a 32 sample capacity, automated instrument integrating Idaho Technology's LightCycler® technology into a portable, impact resistant package. This allows field identification of pathogens quickly. Monitoring the fluorescence from the double-stranded DNA dye SYBR® Green (fig.2), followed by differentiation of products by melting curves or from TaqMan® probes (6-FAM-oligo-TAMRA,), allows inexpensive quantification of low initial template copy number. Cycle sequencing reactions done in the thermocycling module of the R.A.P.I.D. system are faster, cleaner and more readable than parallel reactions done in a conventional heat block cycler. The use of air as the cycling medium ensures temperature uniformity and rapid heat exchange with the sample loaded in thin micro-capillary tubes which are ideally suited to temperature cycling, because of their extremely high surface area to volume ratio. A conventional PCR protocol takes up-to 3 hours to do 30 three temperature cycles. The R.A.P.I.D. system can complete a 40 cycle reaction in less than 20 minutes ( 6 to 30 min.). This makes R.A.P.I.D. system the fastest thermal cycler in the world. The United States Air Force has developed over 50 assays for infectious agents on the R.A.P.I.D. system. Assays for infectious agents typically consist of two temperature cycling for 40 cycles. Protocols for isolation of bacteria and viral DNA (or RNA) have been developed for clinical specimens, air samples and water samples. Protocols for food samples are being developed now. Assays in use for : Bacillus anthracis, Yersinia pestis, botulinum toxin, staphylococcal enterotoxins, Francisella tularensis, Salmonella, Shigella, Vibrio cholerae, E. coli, Campylobacter, VEE, West Nile, Yellow Fever, Brucella spp., and many others. The continuing effort and advances in testing design make this capability an asset for Army Commanders and Medical facilities alike

Oral Vaccination with Brucella melitensis WR201 Protects Mice against Intranasal Challenge with Virulent Brucella melitensis 16M

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis