This is a R21 proposal in response to PA-01-113 on Therapeutics on AIDS-Associated Opportunistic infections. Given the technical difficulties associated with clinical, diagnostic, laboratory and epidemiological investigations on E. bieneusi, sequencing the genome of this microsporidia will enhance progress on this significant pathogen.
The specific aims are as follows: ? ? 1. Identify patients with chronic microsporidiosis, collect stool and purify spores in sufficient quantities to generate monoclonal and polyclonal antibodies against E. bieneusi spores. The collection of stools will be done as part of an ongoing clinical and laboratory investigations on the epidemiology of cryptosporidiosis and microsporidiosis at the Mulago Hospital in Kampala, Uganda. Patients with chronic microsporidiosis will be recruited as part of the study. Spores will be identified by microscopy and confirmed by PCR, and stools will be collected for 8-10 weeks. They will be concentrated, sent to Tufts University, and purified for monoclonal antibody (mAb) production. We will require ~5x109 spores to immunize mice and screen by indirect immunofluorescence using spores fixed on slides as a source of antigen for clones and subclones. This will improve considerably the efficiency of collection and purification of spores required to sequence the entire genome of EB. The production of specific antibodies will also greatly enhance ow ability to produce rapid and simplereagents for diagnostic, epidemiologic, laboratory and clinical investigations. ? ? 2. Develop immunomagnetic separation (IMS) procedure to concentrate, and purify large quantities of spores from stools of patients chronically infected with E. bieneusi. The IMS procedure will allow a more efficient concentration and purification of E. bieneusi spores from stools of infected individuals required to sequence the EB genome. ? ? 3. (Optional): Construct a genomic library and perform chromosome analysis by PFGE. (Time, material and funds permitting). ? ? At the conclusion of this project, we will have collected, concentrated and purified sufficient quantities of EB spores (10 x 1010) to sequence the entire genome of this medically important and enigmatic parasite. The outcome of this proposal will lead to a submission of a full proposal to sequence the EB genome.
