Enhancing the Department of Defense's Capability to Identify Environmental Exposures Into the 21st Century.

Military service members come in contact with a wide range of hazardous substances especially during deployment. The identification of service member's with potential exposures to infectious diseases and environmental toxic substances has been a problem for the U.S. military almost since the formation of the services and continues to be an issue today. In June and July of 2013, the Armed Forces Health Surveillance Center sponsored two meetings to address the need by the Department of Defense to perform retrospective exposure analysis that would support military force health protection efforts. The first meeting included medical professionals who were familiar with health problems that followed potential environmental or infectious disease exposures but that the military health system was unprepared to address. The second group was composed of technical experts who were asked to suggest potential material and nonmaterial solutions to address the needs of the military public health community. This supplement to Military Medicine includes the outcome of these two meetings, descriptions of some of the Department of Defense biorepositories including the large serum repository housed at Armed Forces Health Surveillance Center and discussion of additional topics related to the establishment and use of biorepositories that would support public health practice in the 21st century.

Building a DoD Biorepository for the Future: Potential Benefits and Way Forward.

Significant advances have been made in the molecular analyses of the human physiological state. In general, these techniques have been termed "omics" because of their requirements for sophisticated analyses of large datasets. Application of these new omics technologies has led to advances in medical practice related to public health as well as a new field termed personalized medicine. The Department of Defense (DoD) consistently needs the ability to identify people who have been exposed to environmental hazards during deployments and in their day-to-day jobs. The department currently has a biorepository of sera collected from military service members and has used that repository to study potential environmental exposures (toxins and infectious diseases) since 1987. The DoD Serum Repository is also linked to service member health records, making it a very powerful tool for studies related to force health protection and public health practice. However, this repository does not contain a reliable source of nucleic acid. Accordingly, to take advantage of modern molecular omics technologies, the DoD should establish an enhanced biospecimen repository that can support future questions related to force health protection. This article briefly discusses the various omics techniques, and how they can be used for analyses to support medical practice and public health.

Description and utilization of the United States department of defense serum repository: a review of published studies, 1985-2012.

Specimens in the United States Department of Defense (DoD) Serum Repository have accumulated in frozen storage since 1985 when the DoD began universal screening for human immunodeficiency virus. Use of the stored serum for health research has been carefully controlled, but the resulting publications have never been systematically identified or described. The Armed Forces Health Surveillance Center (AFHSC) information systems and open (online) sites were used as data sources. Through 2012, the repository contained 54,542,658 serum specimens, of which 228,610 (0.42%) have been accessed for any purpose. Between 2001 (the first year that comprehensive, digital records were available) and 2012, 65.2% of all approved requests for serum were for healthcare or public health investigations, but greater than 99% of all shipped samples were for research. Using two different methods - a structure search of PubMed and an exhaustive online search based on records from AFHSC - we identified 76 articles published between October 1988 and March 2013 that covered a multitude of infectious diseases, injuries, environmental exposures and mental health conditions through analysis of antibodies, biological metabolic, signaling and regulatory substances, Vitamin D, organochlorines, dioxin, omega-3-fatty acid, and portions of human deoxyribonucleic acid. Despite its operational and scientific value, it appears that the DoD Serum Repository has been underutilized. Changes to policy and increased capacity for specimen processing could increase use of the repository without risking privacy or the availability of specimens for the healthcare of individual service members in the future.

Serosurvey of bacterial and viral respiratory pathogens among deployed U.S. service members.

BACKGROUND: Respiratory illnesses can cause substantial morbidity during military deployments. Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, adenovirus, parainfluenza, and respiratory syncytial virus (RSV) are hypothesized causes. PURPOSE: To determine pathogen-specific seroprevalence prior to and after deployment in support of Operation Enduring Freedom (OEF). METHODS: A retrospective cohort study of 1000 service members deployed between June 30, 2004, and June 30, 2007, was conducted from 2008 through 2009. Pre- and post-deployment sera were tested for the presence of antibody to each pathogen. RESULTS: Pre-deployment IgG seropositivity was high for adenovirus, RSV, and parainfluenza (98.7%, 97.8%, and 81.6%, respectively), whereas seropositivity for B. pertussis, M. pneumoniae, and C. pneumoniae was 14.2%, 21.9%, and 65.1%, respectively. As defined by seroconversion in 1000 subjects, the following were identified: 43 new parainfluenza infections (24% of susceptibles); 37 new pertussis infections (4% of susceptibles); 33 new C. pneumoniae infections (10% of susceptibles); and 29 new M. pneumoniae infections (4% of susceptibles). B. pertussis seroconversion was two to four times higher than reports for the general U.S. population. Overall, 14.2% of the service members seroconverted to at least one of these six pathogens; this increased to 30.1% seroconversion when influenza was included. However, serologic testing was not clearly associated with clinical illness in this report. CONCLUSIONS: Serologic evidence for respiratory infections was common among the 2004-2007 OEF-deployed military, sometimes at a higher rate than the general U.S. population. Awareness of this risk and implementation of preventive measures should be emphasized by leadership prior to and during deployment.

Influenza and respiratory disease surveillance: the US military's global laboratory-based network.

The US Department of Defense influenza surveillance system now spans nearly 500 sites in 75 countries, including active duty US military and dependent populations as well as host-country civilian and military personnel. This system represents a major part of the US Government's contributions to the World Health Organization's Global Influenza Surveillance Network and addresses Presidential Directive NSTC-7 to expand global surveillance, training, research and response to emerging infectious disease threats. Since 2006, the system has expanded significantly in response to rising pandemic influenza concerns. The expanded system has played a critical role in the detection and monitoring of ongoing H5N1 outbreaks worldwide as well as in the initial detection of, and response to, the current (H1N1) 2009 influenza pandemic. This article describes the system, details its contributions and the critical gaps that it is filling, and discusses future plans.

Attenuation of defined Brucella melitensis wboA mutants.

Rough Brucella mutants have been sought as vaccine candidates because they do not induce seroconversion. In this study, two defined nonreverting rough mutants were derived from virulent Brucella melitensis strain 16M: a wboA deletion mutant designated WRR51 and a wboA purEK dual deletion mutant designated WRRP1. Strain WRRP1 exhibited reduced survival in human monocyte-derived macrophages (hMDMs) compared with parent strain WRR51 or with ΔpurEK strain WR201. Strain WRRP1 persisted for 1 week or less in BALB/c mice after intraperitoneal infection, while less severe attenuation was exhibited by the two single mutants in this model. Trans complementation of wboA restored the survival of WRR51 in hMDMs comparable to strain 16M and the survival of WRRP1 comparable to strain WR201.

Genome sequence of the deep-rooted Yersinia pestis strain Angola reveals new insights into the evolution and pangenome of the plague bacterium.

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.

A novel brain heart infusion broth supports the study of common Francisella tularensis serotypes.

Francisella tularensis Schu S4, LVS and U112 have become model organisms for the study of Francisella pathogenesis, and represent a cross section of the different F. tularensis subspecies. Both Schu S4 and LVS are fastidious organisms, requiring medium fortified with supplements and nutrients for enhanced growth. Chamberlains defined medium, Tryptone Soy Broth supplemented with cysteine (TSBc), and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2% IsoVitaleX are typically used in the cultivation of these bacteria. In this report, we describe a simple brain heart infusion broth formulation that can be used to obtain superior growth characteristics in all of these model organisms, and can support bacterial growth from low inoculum. Surprisingly, CAMHB, which is favored in the literature for culturing Schu S4 and LVS, induced the worst growth characteristics of the four formulations studied. To expand on these observations, an additional seven strains of F. tularensis, representing types A.I, A.II, and B were selected from the Department of Defense United Culture Collection (UCC) and a comparative analysis of their growth characteristics performed in the four broth formulations. Results demonstrate differences in the growth characteristics of Francisella species that are significantly influenced by both strain type and the choice of growth medium. Though four of the five additional Type A strains displayed superior growth characteristics in Chamberlain's defined medium, growth characteristics of all three model organisms, as well the Type B strains, were enhanced by the new BHI-based broth formulation. We conclude that this medium represents the optimal choice for cultivation of the three model organisms used for Francisella research.

Draft genome sequences of Yersinia pestis isolates from natural foci of endemic plague in China.

To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague.

Typing methods for the plague pathogen, Yersinia pestis.

Phenotypic and genotypic methodologies have been used to differentiate the etiological agent of plague, Yersinia pestis. Historically, phenotypic methods were used to place isolates into one of three biovars based on nitrate reduction and glycerol fermentation. Classification of Y. pestis into genetic subtypes is problematic due to the relative monomorphic nature of the pathogen. Resolution into groups is dependent on the number and types of loci used in the analysis. The last 5-10 years of research and analysis in the field of Y. pestis genotyping have resulted in a recognition by Western scientists that two basic types of Y. pestis exist. One type, considered to be classic strains that are able to cause human plague transmitted by the normal flea vector, is termed epidemic strains. The other type does not typically cause human infections by normal routes of infection, but is virulent for rodents and is termed endemic strains. Previous classification schemes used outside the Western hemisphere referred to these latter strains as Pestoides varieties of Y. pestis. Recent molecular analysis has definitely shown that both endemic and epidemic strains arose independently from a common Yersinia pseudotuberculosis ancestor. Currently, 11 major groups of Y. pestis are defined globally.

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells.

BACKGROUND: Effective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs. METHODS: To detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays. RESULTS: We found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin. CONCLUSION: Host gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.

The complete genome sequence of Yersinia pseudotuberculosis IP31758, the causative agent of Far East scarlet-like fever.

The first reported Far East scarlet-like fever (FESLF) epidemic swept the Pacific coastal region of Russia in the late 1950s. Symptoms of the severe infection included erythematous skin rash and desquamation, exanthema, hyperhemic tongue, and a toxic shock syndrome. The term FESLF was coined for the infection because it shares clinical presentations with scarlet fever caused by group A streptococci. The causative agent was later identified as Yersinia pseudotuberculosis, although the range of morbidities was vastly different from classical pseudotuberculosis symptoms. To understand the origin and emergence of the peculiar clinical features of FESLF, we have sequenced the genome of the FESLF-causing strain Y. pseudotuberculosis IP31758 and compared it with that of another Y. pseudotuberculosis strain, IP32953, which causes classical gastrointestinal symptoms. The unique gene pool of Y pseudotuberculosis IP31758 accounts for more than 260 strain-specific genes and introduces individual physiological capabilities and virulence determinants, with a significant proportion horizontally acquired that likely originated from Enterobacteriaceae and other soil-dwelling bacteria that persist in the same ecological niche. The mobile genome pool includes two novel plasmids phylogenetically unrelated to all currently reported Yersinia plasmids. An icm/dot type IVB secretion system, shared only with the intracellular persisting pathogens of the order Legionellales, was found on the larger plasmid and could contribute to scarlatinoid fever symptoms in patients due to the introduction of immunomodulatory and immunosuppressive capabilities. We determined the common and unique traits resulting from genome evolution and speciation within the genus Yersinia and drew a more accurate species border between Y. pseudotuberculosis and Y. pestis. In contrast to the lack of genetic diversity observed in the evolutionary young descending Y. pestis lineage, the population genetics of Y. pseudotuberculosis is more heterogenous. Both Y. pseudotuberculosis strains IP31758 and the previously sequenced Y. pseudotuberculosis strain IP32953 have evolved by the acquisition of specific plasmids and by the horizontal acquisition and incorporation of different genetic information into the chromosome, which all together or independently seems to potentially impact the phenotypic adaptation of these two strains.

Multiple antimicrobial resistance in plague: an emerging public health risk.

Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.

Multiplex real-time SYBR Green I PCR assay for detection of tetracycline efflux genes of Gram-negative bacteria.

In an effort to find a rapid, efficient, and reliable method for screening and classifying large numbers of tetracycline-resistant bacterial isolates, we developed a multiplex, real-time PCR assay using SYBR Green I and the Roche LightCycler. The assay can rapidly identify eight genes encoding tetracycline resistance efflux pumps including tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H) and tet(J). Primers were selected for PCR amplification of these eight tetracycline resistance determinant (tet) genes commonly found in Gram-negative organisms. We combined primer pairs together to make a single-tube multiplex PCR reaction followed by melting curve analysis. Amplification of the expected tet gene products was confirmed by both agarose gel electrophoresis and DNA sequence analysis. Based on melting temperature differences, we could identify the different classes of tet genes. To test the multiplex PCR, the assay was used on 107 tetracycline-resistant clinical isolates of various Gram-negative organisms isolated in several locations around the world. About 49.5% of those strains carried a tet(A) gene, 35.5% carried a tet(B), 7.5% carried a tet(J), 5.6% carried a tet(C) and 1.9% carried a tet(D) gene. DNA sequence analysis of the amplicons confirmed that the specificity of the test was 100%. The sensitivity of the multiplex test varied from 10 to 1000 CFU per PCR reaction. Our real time PCR assay utilizing SYBR Green I and melting point analysis on the Lightcycler system showed not only a high confidence level in differentiation of the classes of tet genes but also precise reproducibility. Our multiplex PCR tet gene class identification assay offers a significant savings of time and labor in the analysis of large numbers of clinical strains compared with assays using individual gene PCR or traditional phenotype methods.

Current trends in plague research: from genomics to virulence.

Yersinia pestis is the causative agent of plague, which diverged from Yersinia pseudotuberculosis within the past 20,000 years. Although these two species share a high degree of homology at the DNA level (>90%), they differ radically in their pathogenicity and transmission. In this review, we briefly outline the known virulence factors that differentiate these two species and emphasize genetic studies that have been conducted comparing Y. pestis and Y. pseudotuberculosis. These comparisons have led to a better understanding of the genetic contributions to the differences in the virulence and pathogenicity between these two organisms and have generated information that can be applied in future diagnostic and vaccine development. Comparison of the genetic differences between Y. pestis and Y. pseudotuberculosis has also lent insight into the emergence of acute pathogens from organisms causing milder diseases.

Comparison of Acinetobacter baumannii isolates from the United Kingdom and the United States that were associated with repatriated casualties of the Iraq conflict.

Acinetobacter isolates associated with casualties from the Iraq conflict from the United States were compared with those from the United Kingdom by pulsed-field gel electrophoresis and integron analysis. Representatives of the main outbreak strain associated with casualties from both countries were indistinguishable in DNA profile. Two further outbreak strains were common to both sets of isolates.

Real-time PCR assays targeting a unique chromosomal sequence of Yersinia pestis.

BACKGROUND: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. METHODS: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis. RESULTS: The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference. CONCLUSIONS: Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.

The complete genome sequence of Francisella tularensis, the causative agent of tularemia.

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

Microevolution and history of the plague bacillus, Yersinia pestis.

The association of historical plague pandemics with Yersinia pestis remains controversial, partly because the evolutionary history of this largely monomorphic bacterium was unknown. The microevolution of Y. pestis was therefore investigated by three different multilocus molecular methods, targeting genomewide synonymous SNPs, variation in number of tandem repeats, and insertion of IS100 insertion elements. Eight populations were recognized by the three methods, and we propose an evolutionary tree for these populations, rooted on Yersinia pseudotuberculosis. The tree invokes microevolution over millennia, during which enzootic pestoides isolates evolved. This initial phase was followed by a binary split 6,500 years ago, which led to populations that are more frequently associated with human disease. These populations do not correspond directly to classical biovars that are based on phenotypic properties. Thus, we recommend that henceforth groupings should be based on molecular signatures. The age of Y. pestis inferred here is compatible with the dates of historical pandemic plague. However, it is premature to infer an association between any modern molecular grouping and a particular pandemic wave that occurred before the 20th century.