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Goals of this project are: <OL> <LI> To develop a new approach for management of stable flies based on better understanding of stable fly-bacterial symbiosis; <LI> To better understand the significance of house flies in transmission of Escherichia coli O157:H7 and antibiotic resistant enterococci. <LI> Identify sand fly midgut molecules that can be used in vector-based strategies against transmission of Leishmania </ol>
Objectives of this project are: <OL> <LI>To investigate the temporal changes in the microbial community structure of cattle/horse manure and resulting changes in oviposition attractants/stimulants for stable flies; <LI>To study the role of house flies in the ecology of 1) Escherichia coli O157:H7 in the cattle environment and 2) antibiotic resistant bacteria (enterococci) in around municipal waste water treatment facilities. 3) Identify midgut proteins that are involved with Leishmania development within the sand fly midgut via RNA interference.
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The specific sub-objectives: <OL><LI> Assess the effect of silencing specific midgut molecules thought to directly or indirectly influence L. major development within P. papatasi midgut. <LI> Biochemically and molecularly characterize P. papatasi midgut molecules that exhibit an effect on Leishmania development when knocked down by RNAi. <LI> Investigate the role of midgut chitinase PpChit1 and the peritrophins PpPER1, PpPER2 and PpPER3 in PM formation, and PpChym2, PpKaz1 and PpKaz2 in blood meal digestion within P. papatasi midgut
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Expected outcomes: Stable flies (SF) are the most important pest of cattle in the US and worldwide. This study will lead to better understanding of SF- bacterial associations and the role of bacteria in SF oviposition behavior. Newly identified bacterial isolates and volatile/contact compounds stimulating SF oviposition can be used for the development of novel SF management strategies based on disruption of SF oviposition and larval development. House flies (HF) are an important mechanical and biological vector of human food-borne pathogens and antibiotic resistant strains. This study will directly assess the role of HF in transmission of E. coli O157:H7 and antibiotic resistant enterococci under field conditions. This will lead to greater emphasis for management of house flies as a part of pre- and post-harvest food safety programs. Leishmaniases are serious vector-borne diseases with an important socio-economic impact in many countries. As effective therapies are often un-affordable to most patients and no vaccines are available, vector-based strategies may lead to novel approaches to fight disease transmission. However, many aspects of the interaction between sand fly and Leishmania are still unknown. Our studies assessing the effect of silencing transcripts thought to be involved in Leishmania development within the sand fly will provide data on the role of midgut molecules in the physiology of sand flies, with potential application as transmission blocking vaccines (TBV). We expect a significant reduction in protein expression and activity of those messages targeted by RNAi and an effect on Leishmania development .
NON-TECHNICAL SUMMARY: Stable fly (SF) is a blood-sucking fly that is considered as the most important insect pest of cattle in the USA. SF larval development strictly depends on a live microbial community in the natural habitat, and SF females are capable of selecting an oviposition site based on the microbially-derived stimuli that indicate the suitability of the substrate for larval development. The aim of this project is to identify changes in the microbial community structure of aging cattle and horse manure, identify which bacteria are critical for SF oviposition and which volatile and contact compounds these bacteria produce that stimulate SF oviposition. This information can be then used for development and formulation of novel SF management approaches based on disruption of SF oviposition and larval development. Improvement of food safety and security is one of the research priorities in developed as well as developing world. a) House flies (HF), due to their larval developmental sites (e.g animal feces) and behavior of adult flies including unrestricted movement and attraction to human food are an important vector of bacteria originating from feces and therefore they represent a great public health risk. In this project, we will assess the role of HF in transmission of a) Escherichia coli O157:H7 between two cattle farms; b) antibiotic resistant enterococci from waste water treatment plants, to the surrounding urban/residential settings. Phlebotomine sand flies are the main vectors of leishmaniasis, a multi-spectrum disease that causes substantial morbidity and mortality in the developing world. As effective therapies are un-affordable to most patients and no vaccines are available, vector-based strategies, specifically transmission-blocking vaccines are becoming a choice strategy to control many vector transmitted diseases. Our studies aim at assessing the effect of silencing a selected number of sand fly proteins thought to be involved in Leishmania development within the sand fly; elucidating how these proteins interact with Leishmania parasites undoubtedly will lead to the development of novel strategies to fight parasite transmission. <P>
APPROACH:<BR> Project 1. Microbial community structure and semiochemicals of aging manure and stable fly oviposition: a) Culture-based approach: MacConkey, mENT, mFC, TSB agars will be used following the manufacturer protocols. b) Culture-independent approach: 16S rDNA tag-encoded FLX amplicon parallel pyrosequencing will be used. The partial 16S rDNA will be amplified by the universal eubacterial primers. 454 sequence run will be performed using GS FLX Titanium plate and Genome Sequencer FLX System. The FASTA file for each sample will be evaluated by BLASTn against the NCBI GenBank database. c) Volatile compounds collected by Tennex and SPME will be analyzed by GC-MS as described before (conducted USDA-ARS, Lincoln, NE). d) Stable fly oviposition assays with individual and mixture of compounds will be followed as described previously (Romero et al., 2006). <BR> <BR>
Project 2. House flies and ecology of E. coli O157:H7 and enterococci a) Transmission of E. coli O157:H7 by house flies (HF) between two confined cattle facilities. Two cattle confined facilities about 2 km apart have been selected. The isolation and characterization of E. coli O157 from cattle and associate house flies will be conducted as described previously (Alam and Zurek, 2006). b) Prevalence and characterization of antibiotic resistant enterococci at waste water treatment plants (WWTP) and associated HF. HF and influx water/sludge at the 3 WWTP will be collected and enterococci will be characterized for antibiotic resistance and virulence factors by a polyphasic approach described previously (Macovei and Zurek, 2006). <BR> <BR>
Project 3. Aim1: We have prepared dsRNAs using primers spanning the mature sequence of each cDNA in this study. Each dsRNAs will be injected into adult female P. papatasi. Two to four days following dsRNA injection in the thorax, flies will be fed on blood and individual midguts dissected 24-72h following a blood meal. To test success of RNAi, mRNA abundance and protein expression and/or activity will be evaluated by comparing to control flies. Aim 2: Among the molecules involved in this study, we will characterize, molecular and biochemically, those that decrease parasite survival/load within the sand fly midgut when knocked-down by RNAi. For each selected midgut molecule, recombinant proteins will be produced by transfecting HEK293-F cells with their respective cDNAs. Expression and purification of recombinant proteins will be carried out at KSU and at the NIH. Aim 3: Evaluate the effect of each dsRNA on blood meal digestion (PpChym2, PpKaz1 and PpKaz2) and PM formation (PpChit1, PpPER1, PpPER2 and PpPER3). Following dsRNA injection, flies will be maintained on 30% sucrose between 48 to 96h before blood feeding. Injected flies will be blood fed, either artificially on defibrinated blood through membranes feeders or directly on BALB/c mice. Sand fly midguts will be dissected at various time points following the blood meal, depending on the targeted silenced gene normal expression profile. Also, we will evaluate microscopically whether RNAi targeting PM-related molecules, digestive proteases or thrombin inhibitors can influence the synthesis of the PM and the digestion of the blood.