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Sponsor: US-Israel Binational Agricultural Research Development Fund (BARD)
Grant Number: IS-4281-10 R
Title: Identification of Genes Involved in Virulence of Escerichia coli Mastitis by Signature Tagged Mutagenesis
Annual Direct Cost: $26,000
Project Period: 10/01/10-09/30/13
DESCRIPTION (provided by applicant): Escherichia coli is an important cause of mammary gland infection and inflammation (mastitis) in dairy cows leading to considerable economic losses worldwide. Transient infection is the most frequent clinical presentation, however, some animals might be severely affected and develop sepsis or conversely progress into a mild or subclinical persistent infection. Although host factors play an important role in the progression of the disease, transient, persistent and severe E. coli strains have been identified among field strains and confirmed in bovine and murine mastitis models. LPS is undoubtedly an important virulence factor in E. coli mastitis, however, using the murine mastitis model we have shown that other virulence factors probably exists. To identify additional virulence determinants and genes affecting LPS virulence, we propose to use a Signature-Tagged Mutagenesis (STM) screen.
We propose to construct a library of thousands of random mutants of E. coli strain P4. This strain causes severe bovine mastitis that may include systemic infection. Furthermore, this infection process can be mimicked using the P4 strain in a mouse model. Next, the mutant library will be screened and genes involved in colonization of the mammary gland in the mouse mastitis model will be identified. The most promising mutants will be explored in dairy cows by experimental intramammary challenge. We also plan to perform a molecular epidemiological screening of field isolates of mastitis E. coli strains for the presence of the identified virulence genes. In the longer term (beyond the scope of this proposal), the role of the gene products in the disease process will be investigated.
The PIs include Dr. Schukken (Cornell University), who has access to field data, and epidemiologically well defined field strains and has experience with and facilities for mastitis challenge models in cows and Dr. Shpigel (The Hebrew University), who has established in the last four years mouse models to study host-pathogen interactions in the mammary gland.
The Collaborator, Dr. Rosenshine (The Hebrew University), has extensive experience in construction of genomic libraries of transposon-mutants of pathogenic E. coli and in library screening. We believe that this research will help to elucidate the molecular basis of coliform mastitis in dairy cows and will identify genetic differences between strains that cause mild and severe mastitis. The identified genes may provide new targets for diagnostic, preventive and therapeutic intervention.