Title

The identification of novel virulence-associated determinants in Mycoplasma gallisepticum through in vivo screening of transposon mutants

Date of Completion

January 2006

Keywords

Biology, Molecular|Biology, Microbiology|Agriculture, Animal Pathology

Degree

Ph.D.

Abstract

Mycoplasma gallisepticum is one of several infectious agents contributing to chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys. It is highly contagious in industry flocks spreading via airborne dust, droplets or feathers. Infection causes lowered food consumption by the host, leading to loss of weight and lowered egg production, which causes significant economic losses to the industry. Due to its large economic impact in the poultry industry, numerous vaccines to control M. gallisepticum infection in chicken flocks have been produced, however they have had only limited success. The ineffectiveness of these vaccines has been attributed to a general lack of understanding of the molecular basis of pathogenesis. Currently the only consistent method for molecular manipulation in M gallisepticum is transposon mutagenesis. Transformation with a plasmid carrying the transposon Tn4001 mod leads to its random insertion into the M. gallisepticum genome. ^ Signature sequence mutagenesis (SSM) is a global screening technique that allows for the identification of attenuated virulence mutants. Chickens are challenged with a pool of Tn4001 mod mutants and later these transposon mutants are recovered from various tissues and screened for the presence or absence of various mutants in the output populations. This is a negative selection screening technique whereby those mutants possessing transposon insertions in genes essential for virulence will not be recovered from the host animal. Mutants that are not recovered from the chicken are used in a single infection study to confirm their virulence deficiency. The technique allows comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants. Using this technique, we have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion into the middle of the gene resulted in diminished metabolic function and reduced virulence of the mutant designated as Mg 7. There is also early work on a second mutant, P2H12, to indicate that an insertion into a six-gene operon is responsible for its attenuated virulence phenotype. These attenuated virulence mutants may have potential as efficacious vaccines.^