Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Martin Hugh-Jones

Abstract

Two primer sets were manufactured from the cytadhesin P1 gene of M. Pneumoniae in an attempt to amplify homologous sequences in M. mycoides subsp. mycoides "SC" Type in a search for a P1-like gene in the latter organism. The first primer set MP-2 amplifies a 488 bp fragment in M. pneumoniae. The second primer set MP-3 amplifies a 303 bp fragment bracketing the 13-amino acid binding domain in the same organism. Both primer sets amplified the expected fragments in all the virulent strains of M. mycoides subsp. mycoides "SC" type. The primer sets did not however amplify the expected fragments in the vaccine T1 strain of the organism. A third primer set (MP-V) was then selected from the sequence of the CAP-21 gene insert described by Taylor et al, 1992. This primer set amplifies a 458 bp fragment in all members of the M. mycoides cluster including the T1 strain. The primer set amplified the expected fragment in all the virulent strains as well as the T1 vaccine strain of M. mycoides subsp. mycoides "SC" type but did not amplify it in M. pneumoniae and M. genitalium. A comparison of the protein pattern of the virulent strain PG1 and the vaccine T1 strain of M. mycoides subsp. mycoides "SC" type revealed considerable protein differences. A multiplex PCR assay was then developed to differentiate the virulent strains from the T1 vaccine strain of M. mycoides subsp. mycoides "SC" type using the primer sets MP-3 and MP-V. The assay amplified two fragments in all the virulent strains of the organism (the 303 bp and the 458 bp) and only one fragment (the 458 bp) in the T1 strain thereby differentiating the strains in one amplification procedure. The assay was then employed to test crudely prepared field samples from cattle suspected of CBPP infection but previously vaccinated with the T1 strain.

ISBN

9780591289213

Pages

97

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