Progress 09/01/10 to 08/31/13
Outputs
Target Audience: 1. Entomologists with interest in control of vector-borne disease 2. Agriculturalists in international communities who are developing field-based strategies to enhance output 3. Biotechnologist with a focus on environmental applications 4. Global health officials with interst in global food security and agricultural output Changes/Problems: This grant was transferedfrom the University of New Mexico to the Biomedical Research Institute of New Mexico (BRINM) in July 2011. The new award number for this is 20-10-33120-19651, proposal number 2012-02379, accession number 0229441. This is the final report for this award from the University of New Mexico. All subsequent annual reports for this award will be submitted via BRINM. What opportunities for training and professional development has the project provided? This grant is currently supporting the stipend of agraduate student and a post-doctoral associate. How have the results been disseminated to communities of interest? Dr. Ravi Durvasula and Adam Forshaw were invited to present plenary seminars at the 1st Biodesert Consortium on Bacterial Symbiosis, Tunis, Tunisia. Molecular tools, environmental release strategies and the use of a microbial encapsulation strategy for risk mitigation of the paratransgenic vector control appraoch were presented to an international audience of scientists and students from US, Europe and North Africa. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) We have successfully developed an alginate-based calcium/barium hybrid microparticle with a Ca2+-ALG core and Ba2+-ALG shell that successfully contains "payload" microbes in liquid solution while maintaining microbe viability until the particles are dissolved 2) We have successfully demonstrated "tuning" the particle release profiles, where microbes can diffuse from the particle at variable rates depending on cross-linker composition (Ba2+:Cal2+) 3) We have successfully encapsulated P. agglomerans and B. subtilis and have demonstrated their rescue from the microcapsule as well as enhanced viability within the capsule 4) We have validated the premise that alginate microspheres would provide resistance to environmental insult such as ultraviolet radiation by demonstrating increased survival of encapsulated microbes when exposed to high energy UVC radiation 5) All attempts at forcing horizontal gene transfer (HGT) of our engineered DNA plasmid to Pseudomonas fluorescens and native P. agglomerans in liquid media, soil and other HGT-promoting environments have failed. This is encouraging since it suggests that theengineered DNA is unsuitable for bacterial uptake except under very, very specific laboratory conditions and thus lowers the likelihood for environmental contamination
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Durvasula R. Paratransgenic approaches to arthropod-borne disease - Act 1 (invited seminar)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Forshaw A and Durvasula R. Microencapsulation as a strategy for implementation and environmental safe-guarding of a paratransgenic approach to control of vector-borne diseases - Act 2, Risk assessment and mitigation (invited seminar)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Forshaw A, Arora A and Durvasula R. Microencapsulation of transgenic B. subtilis within chitosan-coated alginate microspheres
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Current outputs for USDA 2010-33120-21852 include: MICROENCAPSULATION OF TRANSGENIC BACILLUS SUBTILIS WITHIN CHITOSAN-COATED ALGINATE MICROSPHERES, American Society of Tropical Medicine and Hygiene, Philadelphia PA, 2011, (Poster presentation). Initial results regarding encapsulation of engineered bacteria were presented via oral poster session to interested parties at the ASTMH annual meeting in Philadelphia. PARATRANSGENIC APPROACHES TO ARTHROPOD-BORNE DISEASE -ACT 1, 1st Biodesert Consortium on Bacterial Symbiosis, Tunis Tunisia (Invited Seminar). In this plenary seminar, Dr. Durvasula (PI) provided a comprehensive overview of paratransgenic strategies directed at control of vector-borne disease. Molecular tools and environmental release strategies under development in the Durvasula Lab were presented to an international audience of scientists and students from US, Europe and North Africa (Morocco, Algeria, Tunisia and Libya). PARATRANSGENIC APPROACHES TO ARTHROPOD-BORNE DISEASE - ACT 2 (RISK ASSESSMENT AND MITIGATION), 1st Biodesert Consortium on Bacterial Symbiosis, Tunis Tunisia, 2011 (Invited Seminar. In this plenary seminar, Adam Forshaw (Research Fellow) introduced the idea of microbial encapsulation for risk mitigation of paratransgenic vector control strategies. Preliminary results of early encapsulation experiments were presented to an international consortium of scientists expert in the field of insect-microbial symbiosis. MICROENCAPSULATION AS A STRATEGY FOR IMPLEMENTATION AND ENVIRONMENTAL SAFE-GUARDING OF A PARATRANSGENIC APPROACH TO CONTROL OF VECTOR-BORNE DISEASES (Provisional Patent, # 2011-057-02) This provisional patent outlines microencapsulation strategies that can be used to deliver genetically engineered bacteria to disease-transmitting arthropods under field conditions. Composition of microspheres and methods of encapsulation and monitoring of microorganisms are described in detail. PARTICIPANTS: Dr. Ravi Durvasula, MD (Principal Investigator) Chief of Medicine and Acting ACOS for Research New Mexico VA Health Care System Vice Chairman for VA Affairs Professor of Medicine Director, Center for Global Health University of New Mexico School of Medicine Albuquerque, NM-87131. Dr. Thomas A. Miller, PhD (Collaborator) Professor of Entomology Entomology Department University of California Riverside, CA-92521. Adam Forshaw Howard Hughes Medical Inst. Research Fellow University of New Mexico School of Medicine Albuquerque, NM-87131. Arinder K. Arora Graduate Student, Biology Department University of New Mexico, Albuquerque NM-87131. Sudeep Kumar Post Doctoral Fellow Department of Internal Medicine University of New Mexico, Albuquerque NM-87131. John A. Shelnutt (Consultant) Distinguished Member of Technical Staff Sandia National Laboratories Albuquerque, NM 87106. TARGET AUDIENCES: Whereas the proposal defines a narrow audience of agriculturalists working on Pierce's Disease and regulatory authorities chartered with oversight of transgenic technologies, the impact of this project, when fully realized, extends the target audience to: 1) Entomologists with interest in control of vector-borne disease 2) Agriculturalists in several international communities (Europe, Asia, Africa and North America) who are developing filed-based strategies to enhance output 3) Public health officials tasked with development of novel methods for control of vector-borne human diseases 4) Biotechnologists with a focus on environmental applications 5) Medical personnel, such as infectious disease specialists, with particular focus on vector-borne disease. 6)Global health officials with interest in global food security and agricultural output (i.e. the Gates Foundation). PROJECT MODIFICATIONS: Whereas the initial proposal describes a paratransgenic method involving Alcaligenes species, concerns remained about the potential adverse impact on human health, especially in immunocompromised individuals. Recent finding form the Miller Lab suggest that P. agglomerans plays a symbiotic role in H. vitripennis. Since the Pantoea strain E-325 has been approved for environmental dissemination and poses no threat to human and animal populations, we elected to move forward with this organism instead of the Alcaligenes species described in the original proposal. We do not believe that this constitutes a major change or scientific departure from our original proposal or intended project. Furthermore, we have developed robust molecular tools for the transformation of Pantoea strains, and have been able to make significant progress with the stated aims of this proposal.
Impacts
Our current work in microencapsulation of genetically modified bacteria for use in paratransgenic insect control has yielded several very promising results: 1) We have successfully developed an alginate-based calcium/barium hybrid microparticle with a Ca2+-ALG core and Ba2+-ALG shell that successfully contains "payload" microbes in liquid solution while maintaining microbe viability until the particles are dissolved. 2) We have successfully demonstrated "tuning" the particle release profiles, wherein microbes can diffuse from the particle at variable rates depending on cross-linker composition (Ba2+:Cal2+). 3) We have successfully encapsulated P. agglomerans and B. subtilis and have demonstrated their rescue from the microcapsule as well as enhanced viability within the capsule. 4) We have validated the premise that alginate microspheres would provide resistance to environmental insult such as ultraviolet radiation by demonstrating increased survival of encapsulated microbes when exposed to high energy UVC radiation. 5) We are currently developing a novel encapsulation formula incorporating a high-carbon dye which should further increase this UVC resistance. 6) The initial proposal aims to develop microencapsulation technology for paratransgenic control of Pierce's disease. The progress to date with encapsulated P. agglomerans advances the goal and we anticipate contained filed trials in 2012 to evaluate efficacy, both in terms of payload delivery and risk mitigation, i.e., decreased non-target spread of payload. 7) Other applications of this approach greatly increase the global impact of this project. Microencapsulation in being developed for use in desert communities through the collaboration with the BioDesert Program (European Union collaboration). Furthermore, microencapsulation is being developed to drive paratransgenic strategies directed at arthropod vectors of human disease, such as sand flies (visceral leishmaniasis) and kissing bugs (Chagas disease). Current work on the prevention of horizontal gene transfer (HGT) within the rhizosphere from our engineered microbes has yielded similarly promising results: 1) All attempts at forcing HGT (a very low probability event in nature) of our engineered DNA plasmid to Pseudomonas fluorescens and native P. agglomerans in liquid media, soil and other HGT-promoting environments have failed. This is encouraging since it suggests that the engineered DNA is unsuitable for bacterial uptake except under very, very specific laboratory conditions and thus lowers the likelihood for environmental contamination. We are currently investigating several other microbes to confirm this hypothesis. 2) Preliminary results demonstrate that P. agglomerans is viable in the earthworm gut as well as the soil. We are currently investigating whether any possible HGT occurs within these systems. 3) We are preparing studies on HGT prevention utilizing alginate microspheres, which act as physical barriers containing engineered DNA from entering the rhizosphere, thereby even further decreasing the likelihood of environmental contamination.
Publications
- Arora, A.K., Durvasula, R., and Miller T.A. 2012. Distinguishing between two closely related strains of Xylella fastidiosa. (in prep)
- Forshaw, A.P., Miller, T.A., Arora A.K. and Durvasula, R., 2010. Microencapsulation of engineered microbes in paratransgenic control strategies. (in prep)
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