Considerations for studying transmission of antimicrobial resistant enteric bacteria between wild birds and the environment on intensive dairy and beef cattle operations.

BACKGROUND: Wild birds using livestock facilities for food and shelter may contribute to dissemination of enteric pathogens or antimicrobial resistant bacteria. However, drivers of microbial exchange among wildlife and livestock are not well characterized. Predisposition for acquiring and retaining environmental bacteria may vary among species because of physiologic or behavioral differences, complicating selection of a bacterial model that can accurately characterize microbial connections among hosts of interest. This study compares the prevalence and antibiotic resistance phenotypes of two potential model bacterial organisms isolated from wild birds and their environments. METHODS: We compared prevalence and resistance profiles of Escherichia coli and Enterococcus species isolated from environmental swabs and bird feces on a residential control site, a confinement dairy, a pasture-based beef farm, and a confinement beef farm. RESULTS: Bird feces at all sites had low-to-moderate prevalence of Escherichia coli (range: 17-47%), despite potential for exposure on farms (range: 63-97%). Few Escherichia coli were isolated from the control environment. Enterococcus faecalis was dominant in birds at both beef farms (62% and 81% of Enterococcus isolates) and low-to-moderately prevalent at the dairy and control sites (29% and 23% of isolates, respectively). Antimicrobial resistance prevalence was higher in farm samples compared to those from the residential control, but distribution of resistant isolates varied between the bacterial genera. Birds on all farms carried resistant Enterococcus at similar rates to that of the environment, but resistance was less common in bird-associated Escherichia coli despite presence of resistant isolates in the farm environment. DISCUSSION: Bacterial species studied may affect how readily bacterial exchange among populations is detected. Selection of microbial models must carefully consider both the questions being posed and how findings might influence resulting management decisions.

Connectivity increases trophic subsidies in fragmented landscapes.

Landscape corridors mitigate the negative effects of habitat fragmentation by increasing dispersal. Corridors also increase biodiversity in connected habitat fragments, suggestive of metacommunity dynamics. What is unknown in this case is the mechanisms through which metacommunity dynamics act. Working in a large-scale fragmentation experiment, we tested the effect of corridors on the movement of prey species and subsequent effects on predator nutrition (which we call trophic subsidies). We enriched plants of central patches with 15 N, then measured δ15 N in green lynx spiders, the most abundant insect predator, in patches that were either connected to or isolated from the enriched patch. We found that corridors increased prey movement, as they increased spider δ15 N by 40% in connected patches. Corridors also improved spider body condition, increasing nitrogen relative to carbon. We suggest a novel mechanism, trophic subsidies, through which corridors may increase the stability or size of populations in connected landscapes.

Connectivity from a different perspective: comparing seed dispersal kernels in connected vs. unfragmented landscapes.

Habitat fragmentation can create significant impediments to dispersal. A technique to increase dispersal between otherwise isolated fragments is the use of corridors. Although previous studies have compared dispersal between connected fragments to dispersal between unconnected fragments, it remains unknown how dispersal between fragments connected by a corridor compares to dispersal in unfragmented landscapes. To assess the extent to which corridors can restore dispersal in fragmented landscapes to levels observed in unfragmented landscapes, we employed a stable-isotope marking technique to track seeds within four unfragmented landscapes and eight experimental landscapes with fragments connected by corridors. We studied two wind- and two bird-dispersed plant species, because previous community-based research showed that dispersal mode explains how connectivity effects vary among species. We constructed dispersal kernels for these species in unfragmented landscapes and connected fragments by marking seeds in the center of each landscape with 'IN and then recovering marked seeds in seed traps at distances up to 200 m. For the two wind-dispersed plants, seed dispersal kernels were similar in unfragmented landscapes and connected fragments. In contrast, dispersal kernels of bird-dispersed seeds were both affected by fragmentation and differed in the direction of the impact: Morella cerifera experienced more and Rhus copallina experienced less long-distance dispersal in unfragmented than in connected landscapes. These results show that corridors can facilitate dispersal probabilities comparable to those observed in unfragmented landscapes. Although dispersal mode may provide useful broad predictions, we acknowledge that similar species may respond uniquely due to factors such as seasonality and disperser behavior. Our results further indicate that prior work has likely underestimated dispersal distances of wind-dispersed plants and that factors altering long-distance dispersal may have a greater impact on the spread of species than previously thought.

Barriers to, Efforts in, and Optimization of Integrated One Health Surveillance: A Review and Synthesis.

Insufficient data from existing surveillance systems underlie societal tolerance of acute and slow-onset health disasters that threaten, harm, and kill vast numbers of humans, animals, and plants. Here we describe barriers to integrated "One Health" surveillance, including those related to a lack of medical services, professional divisions, incompatible vocabularies, isolated data sets, and territorial borders. We draw from publications of experts who justify broader and more integrated surveillance, education, and stewardship focused on preventing and mitigating disease emergence and re-emergence. In addition, we highlight efforts from Illinois, the United States and the broader world, pointing to examples of relevant education; ways to acquire, compile, and analyze diagnostic and syndromic data; mapping of diseases of humans and animals; and rapid communication of findings and recommendations. For the future, we propose using needed outcomes for health and sustainability to set priorities for One Health programs of education, surveillance, and stewardship. Professionals and paraprofessionals should gather, interpret, and widely communicate the implications of data, not only on infectious diseases, but also on toxic agents, malnutrition, ecological damage, the grave impacts of warfare, societal drivers underlying these problems, and the effectiveness of specific countermeasures.

Temporal and Spatial Distribution of Tick-Borne Disease Cases among Humans and Canines in Illinois (2000-2009).

Four tick-borne diseases (TBDs), anaplasmosis, ehrlichiosis, Lyme disease (LD), and Rocky Mountain spotted fever (RMSF), are endemic in Illinois. The prevalence of human and canine cases of all four TBDs rose over the study period with significant differences in geographic distribution within the state. Among human cases, there were associations between cases of RMSF and LD and total forest cover, seasonal precipitation, average mean temperature, racial-ethnic groups, and gender. Estimated annual prevalence of three canine TBDs exceeded human TBD cases significantly in each region. There was concordance in the number of human and canine cases by county of residence, in annual prevalence trends, and in time of year at which they were diagnosed. To account for multiple environmental risk factors and to facilitate early diagnosis of cases, integrated surveillance systems must be developed and communication between veterinarians, physicians, and public health agencies must be improved.

Smartphone application for classification of motor impairment severity in Parkinson's disease.

Advanced hardware components embedded in modern smartphones have the potential to serve as widely available medical diagnostic devices, particularly when used in conjunction with custom software and tested algorithms. The goal of the present pilot study was to develop a smartphone application that could quantify the severity of Parkinson's disease (PD) motor symptoms, and in particular, bradykinesia. We developed an iPhone application that collected kinematic data from a small cohort of PD patients during guided movement tasks and extracted quantitative features using signal processing techniques. These features were used in a classification model trained to differentiate between overall motor impairment of greater and lesser severity using standard clinical scores provided by a trained neurologist. Using a support vector machine classifier, a classification accuracy of 0.945 was achieved under 6-fold cross validation, and several features were shown to be highly discriminatory between more severe and less severe motor impairment by area under the receiver operating characteristic curve (AUC > 0.85). Accurate classification for discriminating between more severe and less severe bradykinesia was not achieved with these methods. We discuss future directions of this work and suggest that this platform is a first step toward development of a smartphone application that has the potential to provide clinicians with a method for monitoring patients between clinical appointments.

Sublingual immunization with an engineered Bacillus subtilis strain expressing tetanus toxin fragment C induces systemic and mucosal immune responses in piglets.

UNLABELLED: Sublingual (SL) and intranasal (IN) administration of a Bacillus subtilis-based tetanus vaccine was tested in piglets, which more closely mimic the human immune system than mice. Piglets were immunized by the SL, IN or oral routes with vaccine expressing tetanus toxin fragment C, or commercial tetanus vaccine given by intramuscular injection as a control. Tetanus toxoid specific ELISA and passive neutralization tests were used to measure IgG and IgA levels in serum and mucosal secretions, and assess protective serum antibodies, respectively. The nature of the immune response was explored by MHC Class II, TGF-ß1 expression, and ELISA assays for multiple cytokines. SL or IN immunization of piglets induced neutralizing tetanus toxoid specific serum antibody and local salivary and vaginal IgA responses. Standard tetanus vaccine resulted in systemic antibodies, whereas oral administration of the Bacillus-based vaccine was ineffective. Further analyses indicated a balanced Th1/Th2 response to SL or IN immunization. CONCLUSION: This study demonstrates for the first time that SL or IN administration is effective for inducing both systemic and mucosal responses in a piglet model, indicating that SL or IN delivery of a B. subtilis-based tetanus vaccine can be a simple, non-invasive, low cost strategy to induce immunity to tetanus.

Temporal and spatial distribution of blastomycosis cases among humans and dogs in Illinois (2001-2007).

OBJECTIVE: To compare the temporal and spatial distribution of cases of blastomycosis among humans and dogs in Illinois. DESIGN: Retrospective cross-sectional survey. SAMPLE: Human and canine populations in Illinois from 2001 through 2007. PROCEDURES: For each year, human population data were obtained from the US Census Bureau, and the total number of dogs was estimated by use of a human population-based formula. Data regarding infections with Blastomyces dermatitidis in humans were accessed from the Illinois Department of Public Health. Data regarding B dermatitidis infections in dogs were acquired through a survey of a random sample of the 747 veterinary medical practices in Illinois. Statistical analyses of human and canine data were performed by use of t tests, ANOVA, odds ratio assessment, and regression modeling. RESULTS: Estimated annual incidence of human cases of blastomycosis in Illinois increased from 3.8 to 10.7 cases/1 million persons/y from 2001 through 2007. Analysis of data from 221 veterinary practices revealed that the mean estimated annual incidence of canine cases of blastomycosis was 8.3 times the mean estimated annual incidence of human cases, with a similar pattern of change and regional distributions. Thirty-eight counties reported either human or canine cases but not both. CONCLUSIONS AND CLINICAL RELEVANCE: The estimated annual incidence of blastomycosis in humans and dogs in Illinois increased during the period of interest. Veterinarians, physicians, and public health agencies should be encouraged to communicate with each other regarding diagnoses of blastomycosis in either species to facilitate early diagnosis and treatment.

Sublingually administered Bacillus subtilis cells expressing tetanus toxin C fragment induce protective systemic and mucosal antibodies against tetanus toxin in mice.

Sublingual (SL) immunization against infectious agents or bacterial toxins is not a common route for antigen delivery. However, in our continued search for a needle-free platform for vaccine administration, we evaluated the efficacy of SL immunization with Bacillus subtilis engineered to express tetanus toxin fragment C (TTFC). We compared the results obtained with those for intranasal (IN) immunization with the same vaccine, which we recently reported to induce complete protection in mice against a 2×LD100 challenge of tetanus toxin (Lee et al., Vaccine 28:6658-65). Groups of animals received 3-4 immunizations of 10(9)B. subtilis vegetative cells expressing TTFC given IN or SL. Other SL immunized groups received either purified recombinant TTFC (rTTFC) or B. subtilis placebo. A non-toxic mutant of Escherichia coli heat labile enterotoxin (mLT) was included as adjuvant in some of the studies. Mice inoculated by either IN or SL administration developed protective IgG antibodies against tetanus toxin challenge. Similar of higher IgA levels in saliva, vaginal wash and feces were detected in animals immunized SL with B. subtilis cells expressing TTFC compared with IN-immunized mice or mice immunized SL with rTTFC. SL immunization promoted a mixed Th1/Th2 response, based on cytokine analysis (IL-2, IL-4, IL-10 and INFγ). Antigen-stimulated tissues (lung, intestine, spleen and lymph nodes) revealed a dramatic increase in the density of MHC class II+ expressing cells compared to all other groups. The antibody response to TTFC was superior when the adjuvant mLT was excluded from IN and SL immunizations. However, SL administration of mLT induced strong systemic and mucosal antibody responses, indicating that successful use of this route of immunization is not specific to tetanus toxin. We conclude that SL immunization is a promising, effective, safe, non-invasive and convenient method for mucosal delivery of B. subtilis cells expressing tetanus vaccine and, potentially, other immunogens. SL immunization appears to induce both systemic and mucosal immune responses.

Differential effects of habitat isolation and landscape composition on wasps, bees, and their enemies.

Habitat loss and fragmentation are major threats to biodiversity and ecosystem functioning. Effects of these usually intercorrelated processes on biodiversity have rarely been separated at a landscape scale. We studied the independent effects of amount of woody habitat in the landscape and three levels of isolation from the next woody habitat (patch isolation) on trap nesting bees, wasps, and their enemies at 30 farmland sites in the Swiss plateau. Species richness of wasps was negatively affected by patch isolation and positively affected by the amount of woody habitat in the landscape. In contrast, species richness of bees was neither influenced by patch isolation nor by landscape composition. Isolation from woody habitats reduced species richness and abundance of natural enemies more strongly than of their hosts, so that parasitism rate was lowered by half in isolated sites compared to forest edges. Thus, population regulation of the hosts may be weakened by habitat fragmentation. We conclude that habitat amount at the landscape scale and local patch connectivity are simultaneously important for biodiversity conservation.

A protein multiplex microarray substrate with high sensitivity and specificity.

The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three-dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen-specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specific signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laboratory environment.

Development of a Bacillus subtilis-based rotavirus vaccine.

Bacillus subtilis vaccine strains engineered to express either group A bovine or murine rotavirus VP6 were tested in adult mice for their ability to induce immune responses and provide protection against rotavirus challenge. Mice were inoculated intranasally with spores or vegetative cells of the recombinant strains of B. subtilis. To enhance mucosal immunity, whole cholera toxin (CT) or a mutant form (R192G) of Escherichia coli heat-labile toxin (mLT) were included as adjuvants. To evaluate vaccine efficacy, the immunized mice were challenged orally with EDIM EW murine rotavirus and monitored daily for 7 days for virus shedding in feces. Mice immunized with either VP6 spore or VP6 vegetative cell vaccines raised serum anti-VP6 IgG enzyme-linked immunosorbent assay (ELISA) titers, whereas only the VP6 spore vaccines generated fecal anti-VP6 IgA ELISA titers. Mice in groups that were immunized with VP6 spore vaccines plus CT or mLT showed significant reductions in virus shedding, whereas the groups of mice immunized with VP6 vegetative cell vaccines showed no difference in virus shedding compared with mice immunized with control spores or cells. These results demonstrate that intranasal inoculation with B. subtilis spore-based rotavirus vaccines is effective in generating protective immunity against rotavirus challenge in mice.

Efficacy, heat stability and safety of intranasally administered Bacillus subtilis spore or vegetative cell vaccines expressing tetanus toxin fragment C.

Bacillus subtilis strains expressing tetanus toxin fragment C (TTFC) were tested as vaccine candidates against tetanus in adult mice. Mice received three intranasal (IN) exposures to 10(9) spores or 10(8) vegetative cells of B. subtilis expressing recombinant TTFC. Immunized mice generated protective systemic and mucosal antibodies and survived challenge with 2× LD(100) of tetanus toxin. Isotype analysis of serum antibody indicated a balanced Th1/Th2 response. Lyophilized vaccines stored at 45° C for ≥ 12 months, remained effective. Immunized conventional and SCID mice remained well, and no histological changes in brain or respiratory tract were detected. Lyophilized/reconstituted B. subtilis tetanus vaccines administered IN to mice appear safe, heat-stable, and protective against lethal tetanus challenge.

Separation of suspended particles in microfluidic systems by directional locking in periodic fields.

We investigate the transport and separation of overdamped particles under the action of a uniform external force in a two-dimensional periodic energy landscape. Exact results are obtained for the deterministic transport in a square lattice of parabolic, repulsive centers that correspond to a piecewise-continuous linear-force model. The trajectories are periodic and commensurate with the obstacle lattice and exhibit phase-locking behavior in that the particle moves at the same average migration angle for a range of orientation of the external force. The migration angle as a function of the orientation of the external force has a Devil's staircase structure. The first transition in the migration angle was analyzed in terms of a Poincare map, showing that it corresponds to a tangent bifurcation. Numerical results show that the limiting behavior for impenetrable obstacles is equivalent to the high Peclet number limit in the case of transport of particles in a periodic pattern of solid obstacles. Finally, we show how separation occurs in these systems depending on the properties of the particles.

A public-policy practicum to address current issues in human, animal, and ecosystem health.

There are recognized needs for cross-training health professionals in human, animal, and ecosystem health and for public health policy to be informed by experts from medical, science, and social science disciplines. Faculty members of the Community Health and Preventive Medicine Section at the University of Illinois at Urbana-Champaign, College of Veterinary Medicine, and the Institute of Government and Public Affairs, University of Illinois at Urbana-Champaign, have offered a public-policy course designed to meet those needs. The course was designed as a practicum to teach students the policy-making process through the development of policy proposals and to instruct students on how to effectively present accurate scientific, demographic, and statistical information to policy makers and to the public. All students substantially met the learning objectives of the course. This course represents another model that can be implemented to help students learn about complex, multifactorial issues that affect the health of humans, animals, and ecosystems, while promoting participation in public health policy development.

One medicine, one university: the DVM/MPH program at the University of Illinois.

The University of Illinois at Urbana-Champaign College of Veterinary Medicine (UIUC-CVM) and the University of Illinois-Chicago School of Public Health (UIC-SPH) are in the fourth year of a collaborative Doctor of Veterinary Medicine and Master of Public Health dual-degree program. The two campuses, one urban and one rural, are 150 miles apart but are sister schools within the University of Illinois system. This article describes the origin of the program, how the program functions across two campuses, its academic focus, required coursework, and research projects designed to fulfill the program's capstone requirements. The article shows how two campuses can be linked through a combination of online and on-site didactic coursework, briefly describes innovative proposals for projects within the United States and abroad, and highlights faculty committed to educating cross-trained public-health professionals while addressing the national need for veterinarians trained in public health. The authors also discuss how the dual-degree program has led to the formation of the Illinois Center for One Medicine, One Health (ICOMOH), an intra-university collaboration focusing on the interface of human, animal, and ecosystem health.

A DNA oligonucleotide microarray for detecting human astrovirus serotypes.

Human astroviruses have been shown in numerous studies to be an important cause of gastroenteritis in young children worldwide. The present communication addresses their characterization by use of oligonucleotide microarray hybridization. The system developed consists of an RT-PCR using primers of low degeneracy capable of detecting all eight serotypes of human astroviruses. RT-PCR products are then hybridized against a microarray consisting of short oligonucleotide probes 17-18 nucleotides in length. Cy3-labeled ssDNA targets are generated using a Cy3-labeled primer in the RT-PCR. The non-labeled strand is enzymatically digested, and the labeled target is rescued by column purification. This method of generating labeled target uses equimolar concentrations of the amplifying primers and does not compromise assay sensitivity for initial detection of the virus. Hybridization can be performed without the need for additional amplification. Although the amplicon spans a relatively conserved region of the astrovirus genome, the use of short probes enables type distinction despite such limited diversity. Probes differing by as little as a single nucleotide can be used to distinguish isolates. The microarray developed was capable of distinguishing representatives of the eight known serotypes of human astroviruses.

Passive immunotherapy of Bacillus anthracis pulmonary infection in mice with antisera produced by DNA immunization.

Because of the high failure rate of antibiotic treatment in patients with anthrax there is a need for additional therapies such as passive immunization with therapeutic antibodies. In this study, we used codon-optimized plasmid DNAs (DNA vaccines) encoding Bacillus anthracis protective antigen (PA) to immunize rabbits for producing anti-anthrax antibodies for use in passive immunotherapy. The antisera generated with these DNA vaccines were of high titer as measured by ELISA. The antisera were also able to protect J774 macrophage cells by neutralizing the cytotoxic effect of exogenously added anthrax lethal toxin, and of the toxin released by B. anthracis (Sterne strain) spores following infection. In addition, the antisera passively protected mice against pulmonary challenge with an approximate 50 LD50 dose of B. anthracis (Sterne strain) spores. The protection in mice was obtained when the antiserum was given 1h before or 1h after challenge. We further demonstrated that IgG and F(ab')2 components purified from anti-PA rabbit hyperimmune sera retained similar levels of neutralizing activities against both exogenously added B. anthracis lethal toxin and toxin produced by B. anthracis (Sterne strain) spores. The high titer antisera we produced will enable an immunization strategy to supplement antibiotic therapy for improving the survival of patients with anthrax.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

DNA vaccines against enteric infections.

The first DNA vaccines for prevention of infectious diseases were described in 1993 and have since been shown to generate protective humoral and cellular immune responses to numerous infectious agents. For enteric infections, protective immunity has been obtained with DNA vaccines against several enteric viral, bacterial, and parasitic agents. Inoculation of DNA vaccines has generally been by intramuscular injection or by gene gun delivery of vaccine DNA-coated gold microparticles into the skin. Administration of DNA vaccines by the oral route would target the vaccines to enteric mucosal tissues, as well as providing a convenient means for vaccine delivery. Orally administered plasmid DNAs encapsulated in polymeric microparticles or inserted in live bacterial vectors have been effective in animal models for rotavirus DNA vaccines and Listeria monocytogenes DNA vaccines, respectively. Human trials of enteric DNA vaccines have not been initiated, but trials of veterinary vaccines have shown promise.