1. I – Exposure Assessment Charles Gerba - UA Chris Choi – UA Mark Nicas – UCB Paul Kiem – NAU Syed Hashsham - MSU

2. Development of decay constants Surrogate development and assessment Detection methodology development (for surrogates and assessment) Transport and Fate Studies U of A NAU U of A NAU MSU Aerosols U of C U of A NAU Water U of A NAU MSU Fomites U of A MSU NAU Soil NAU Development of Exposure Models U of A U of C Drexel MSU QMRA Group 1: Exposure

3. Overall Objectives Development of new BAC surrogates for application in methods assessment and transport and fate modeling efforts. Validation of detection methods for microbial risk assessment. Development of fate and transport models for BAC on fomites. Development of fate and transport models for BAC in water systems. Development and validation of a discrete-time Markov chain model for airborne BAC within a room. Measure the re-suspension of particle-associated BAC in a test room or chamber Determine the particle size distribution of respiratory aerosol

4. Year 1 Assessment of coliphage surrogates Characterization of PRD1/MS-2 as surrogates Assessment of fomite sampling protocols Development of k values (decay rates) Anthrax detection methods sensitivity Identification of anthrax surrogates Improvement to distribution system transport model Improvement to Markov Chain model

5. Assessment of Phage Surrogates PRD-1 and MS-2 being assessed (most extensively used phage models) PRD-1, which has been extensively used previously as a model appears to actually be P- 22 Primers developed and assess for PRD-1 application Sampling methods and survival on various fomites assessed

6. Variability and Associated Recovery Methods from Fomites

7. Comparison of Sampling Method and Survival of Coliphage MS-2 on Fomites SurfaceMethodT 90 (days)T 99 (days) CottonVortex10.621.2 LaminarVortex16.533.0 LaminarSwab12.023.9 LaminarFellowes11.523.0 SteelVortex5.611.1 SteelSwab5.110.1 SteelFellowes4.48.9

8. Decay Rate on Fomites (K) To be useful in model predictive models on the fate of agents of concern information on their decay on fomites needs to be normalized from various studies published in the literature. The existing literature was reviewed and data on agent decay was normalized against environmental conditions This data can now be used in models to predict survival of these agents on fomites. It was found that enteric viruses survive for days to months on surfaces compared to hours to days for respiratory viruses.

9. Respiratory virus inactivation rates (K)

10. Limit of Detection Methods for Anthrax in Water Real time polymerase chain reaction (PCR), conventional PCR, and selected antibody based and chemiluminiscence assays are among the most sensitive detection methods is one of the main accomplishments this year. A similar analysis for air and soil is also complete with similar conclusions.

11. Distribution of detection limit: various methods available to detect Anthrax in water

12. EviDot:CdSe/ZnS Core-shell Approx diameter 2-5 nm Non bio-available EviTag: Quantum dots with PEG lipid surface coating coupled to amine terminal groups Approx. hydrodynamic diameter 25nm Bio-available DNA strands can be attached Fluorescence varies with size of QD (2nm-5nm) EviTag with green fluorescence (diameter ~25nm, emission 520nm) was used EviFluor: antibodies or protein conjugated EviTag Approx. hydrodynamic diameter 25nm Bio-available DNA strands can be attached Quantum dots as surrogates* of bacterial pathogens (*Only selected characteristics)

13. Normal Human Lymphocytes Lymphocyte Showing DNA Damage DNA Break is taken as an indication of genotoxicity ALKALINE ELECTROPHORESIS STAINING (EtBr) Comet Assay

14. Effect of quantum dots on human lymphocytes: DNA damage by Comet assay * ** *** *p<0.05,**p<0.01, ***p<0.001 Values mean+ SD; Cell viability in all the treatment concentrations >90%; Positive control (EMS 2mM)- Tail moment >350

15. Distribution System – Fate and Transport Modeling Fate & Transport in Water System Investing water quality models at intersections of pressurized pipe systems Experimental verifications have been carried out at the Water Village. Creating several scenarios in order to generate training and testing data required for a back- propagation Artificial Neural Network (ANN) with six input (water demands) and six output (average concentration) units.

16. EPANET-based Simulation -HD Model - WQ Model Experimental Validation using Water-Distribution Networks at the Water Village ANN-based Prediction Models RISK ASSESSMENT Indicator Microorganisms Distribution System Modeling Distribution System Modeling

17. Key Components of a Typical Drinking Water System & Their Vulnerabilities

18. Water Systems & Microbiology Laboratory

19. Water Distribution Systems Laboratory

20. Mixing patterns along the interface

21. Corresponding Risk Microbial Risk Assessment & Consequences

22. Distribution System – Fate and Transport Modeling - Contributions Corrections in EPANET model and further calibration Construction of distribution system network and initial model testing

23. Modeling Airborne Fate and Transport of Agents of Concern Refinement of a simple Markov Chain Model Development method for incorporating information on air velocity and turbulent intensity values Validate Markov chain particle model via published data Construct test chamber

25. Year-1 Publications Boone, S. A. and C. P. Gerba 2006. The significance of fomites in the spread of respiratory and gastrointestinal disease. Applied and Environmental Microbiology, In Press. Romero-Gomez, P., C. Y. Choi, B. van Bloemen Waanders, and S. McKenna, Transport Phenomena at Intersections of Pressurized Pipe Systems, 2006, 8th Annual Water Distribution Systems Analysis Symposium, Cincinnati, OH. Alok K. Pandey, Amanda B. Herzog, Joan B. Rose, Syed A. Hashsham. Potential of Quantum Dots as Surrogates for Microbial Pathogens and Evaluation of Their Genotoxicity. 107 th General Meeting of the American Society for Microbiology, Toronto, Canada, May 21-25, 2007. Amanda B. Herzog, Alok Pandey, Tomoyuki Shibata, Joan B. Rose, and Syed A. Hashsham. Implications of Detection Limit of Various Methods of Bacillus anthracis in Computing Risk to Human Health. 107 th General Meeting of the American Society for Microbiology, Toronto, Canada, May 21-25, 2007.

26. Year 2 Determine attenuation rates of various Bacillus species under natural conditions. B. anthracis, B. thuringiensis, B. globigii compare qPCR and Cultivation for DL for P22 from fomites, from air and from water. Conduct aerosol release experiments to assess Markov chain particle model

27. Year 2 Criteria for Surrogates document Decay constants (K values) for BAC in water, fomites, aerosols Selection and assessment of a surrogate for B. anthracis Development of an algorithm for detection limit and risk Assessment of quantum dots as a surrogate

28. Year 2 Conduct dispersion studies of MS-2/PRD- 1 surrogates in water distribution system at the Water Village to aid model development Establish prediction models using artificial neural networks based on experimental data and EPAnet

29. Anticipated Products Improved standard operating procedure for sampling Set of methods criteria and algorithms for addressing the interpretation of a negative Models for air/surface/hand risks Models for microbial transport in water distribution systems