Phillip C. Williamson

Evidence of Borrelia lonestari DNA in Amblyomma americanum (Acari: Ixodidae) Removed from Humans

ABSTRACT We used a nested PCR with Borrelia flagellin gene (flaB) primers and DNA sequencing to determine if Borrelia lonestari was present in Amblyomma americanum ticks removed from military personnel and sent to the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine. In our preliminary investigation, we detected Borrelia sequences in 19 of 510 A. americanum adults and nymphs from Ft. A. P. Hill, Va. During the 2001 tick season, the flaB primers were used to test all A. americanum samples as they were received, and 29 of 2,358 A. americanum samples tested individually or in small pools were positive. PCRs with 2,146 A. americanum samples in 2002 yielded 26 more Borrelia-positive samples. The positive ticks in 2001 and 2002 were from Arkansas, Delaware, Kansas, Kentucky, Maryland, New Jersey, North Carolina, Tennessee, and Virginia. The last positive sample of the 2001 season was a pool of larvae. To further investigate larval infection, we collected and tested questing A. americanum larvae from Aberdeen Proving Ground, Md.; 4 of 33 pools (40 larvae per pool) were positive. Infection of unfed larvae provides evidence of the maintenance of B. lonestari by means of transovarial transmission. Sequence analysis revealed that the amplicons were identical to sequences of the B. lonestari flaB gene in GenBank. Despite the low prevalence of infection, the risk of B. lonestari transmission may be magnified because A. americanum is often abundant and aggressive, and many tick bite victims receive multiple bites.

Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States.

Serum from �%^4% of residents was positive for infection, compared with �%^9% for B. burgdorferi.

Borrelia, Ehrlichia, and Rickettsia spp. in Ticks Removed from Persons, Texas, USA

Some tick-borne agents may pose yet-unknown public health risks.

Preliminary Assessment of Microbiome Changes Following Blood-Feeding and Survivorship in the Amblyomma americanum Nymph-to-Adult Transition using Semiconductor Sequencing

The physiology of ticks supports a diverse community of non-pathogenic and pathogenic organisms. This study aims to initially characterize the microbial community present within colony-reared Amblyomma americanum using PCR of the variable region 5 of the 16S rRNA gene followed by semiconductor sequencing and classification of sequence data using the Ribosomal Database Project and MG-RAST analysis tools. Comparison of amplicon library datasets revealed changes in the microbiomes in newly engorged nymphs, newly-molted adults, and aged adults, as well as ticks exposed to different environmental conditions. These preliminary data support the concept that microbe survivorship and diversity are partially dependent upon environmental variables and the sequence of blood feeding, molting, and aging. The maintenance and/or emergence of pathogens in ticks may be dependent in part on temporal changes in the microbial community of the tick microbiome.

Development of shuttle vectors for transformation of diverse rickettsia species

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 – 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.

Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

BackgroundIn the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization.ResultsTo demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare false-positive SNPs were associated with variable nucleotide tandem repeats.ConclusionsThe high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution.

Geographic distribution and genetic diversity of the Ehrlichia sp. from Panola Mountain in Amblyomma americanum

BackgroundA novel Ehrlichia, closely related to Ehrlichia ruminantium, was recently discovered from Panola Mountain State Park, GA, USA. We conducted a study to determine if this agent was recently introduced into the United States.MethodsWe developed a sensitive PCR assay based on the conserved gltA (citrate synthase) gene and tested DNA samples extracted from 1964 field-collected and 1835 human-biting Amblyomma americanum from 23 eastern states of the USA.ResultsThe novel agent was detected in 36 ticks collected from 10 states between 1998 and 2006. Infected ticks were collected both from vegetation (n = 14, 0.7%) and from humans (n = 22, 1.2%). Fragments of the conserved gltA gene and the variable map1 gene were sequenced from positive samples. Two distinct clades, with 10.5% nucleic acid divergence over the 730 bp map1 sequence, were identified.ConclusionThese data suggest that the Panola Mountain Ehrlichia was not recently introduced to the United States; this agent has an extensive distribution throughout the range of its tick vector, has been present in some locations for several years, and displays genetic variability. Furthermore, people in several states were exposed to this agent through the bite of infected ticks, underscoring the potential public health risk of this emerging ehrlichiosis.

High Incidence of Chikungunya Virus and Frequency of Viremic Blood Donations during Epidemic, Puerto Rico, USA, 2014.

Deaths were rarely observed, but newborns and other vulnerable populations are at risk for severe complications.

Rickettsiae in Gulf Coast Ticks, Arkansas, USA

To determine the cause of spotted fever cases in the southern United States, we screened Gulf Coast ticks (Amblyomma maculatum) collected in Arkansas for rickettsiae. Of the screened ticks, 30% had PCR amplicons consistent with Rickettsia parkeri or Candidatus Rickettsia amblyommii.

Criteria for Validation of Methods in Microbial Forensics

A process for validation is essential in the development of methods that microbial forensics uses to generate reliable and defensible results. Law enforcement investigators need to respond quickly to the best leads to counter ever-increasing threats and will rely upon results generated from the analyses of any microbial forensic evidence to attempt to attribute any attack to a person(s) or group. Readily available technology and knowledge are making it easier for an individual or group to carry out biocrimes or bioterrorism using microorganisms and toxins as weapons. The potential that a biological weapon will be used is of serious concern for the safety and security of people and critical infrastructure. If a biocrime is committed, microbial forensic evidence will be sought, collected, and characterized to help investigators identify the perpetrator(s) and exclude innocent suspects. Analyses of collected material are often challenging because the identification of the signatures most useful for attribution often requires substantial effort (3). In addition, some microbial forensic specimens can be limited in quantity and/or quality. Despite these demands, accurate and credible results are needed because the interpretation of such results might seriously impact the course or focus of an investigation, thus affecting the liberties of individuals, or even be used as a justification for a government’s military response to an attack. Therefore, the methods for the collection, extraction, and analysis of microbial evidence that could generate key results need to be as scientifically robust as possible so that they are defensible to the legal community (12, 21) and, perhaps, to the international government, law enforcement, and scientific communities. Proper interpretation of the results of microbial forensic analysis relies substantially on understanding the performance and limitations of the methods of collection and the analytical processes, assays, and interpretation involved. Failing to properly validate a method or misinterpreting the results from a microbial forensic analysis or process may have severe consequences. DEFINING VALIDATION Validation is frequently used to connote confidence in a test or process. However, frequently, the process of validation is not well defined or properly described in context. Not being explicit about what is meant by validation can result in misinterpretation and misapplication of a properly performed test. It also can lead to a false sense of confidence in a poor method. In the nascent field of microbial forensics (5), there is a need to better describe what constitutes validation. A strict delineation of the steps needed to validate a method or process may be too restrictive; there are a myriad of methods, processes, targets, platforms, and applications. Yet some basic requirements transcend individual differences in methods, and these can be reinforced by contextual description and illustrated with examples. Failing to validate a method or misinterpreting the reliability of a method in a microbial forensic analysis can have dire consequences. This paper provides a framework for developing a validation plan that can be useful for microbial forensics and may have application to other scientific fields where “validation” may be used colloquially.