Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Johannes Storz

Abstract

Sensitive detection and differentiation of the bacterial genus Chlamydia (C.) was accomplished using the polymerase chain reaction (PCR), and phylogenetic relationships among chlamydiae were inferred from DNA sequences of amplified major outer membrane protein (MOMP) genes. DNA of strains of C. psittaci was efficiently amplified using primers homologous to nontranslated regions of the MOMP genes (ompA). Portions of these reactions were reamplified using one different primer specific for an internal ompA sequence. Primary amplification products were differentiated by restriction analysis. Degenerate, inosine containing oligonucleotide primers homologous to terminal translated regions amplified DNA fragments of all chlamydial MOMP genes. Partial ompA DNA sequences of 25 chlamydial strains representing all species, C. psittaci, C. pneumoniae, and C. trachomatis, were obtained by dideoxy-DNA sequencing using genus-specific primers. MOMP gene sequences were generally identical in related chlamydial strains. Chlamydial phylogenetic relationships were inferred by computer-assisted maximum parsimony (cladisitic) analysis of 23 aligned MOMP genotypes. The phylogram supported monophyly of Chlamydia in the evolution of the MOMP gene. C. psittaci strain MN (meningopneumonitis) was closest to an hypothetical chlamydial ancestor. Avian types of C. psittaci with wide host range and broad disease potential represented ancestral chlamydiae. Highly evolved groups of C. trachomatis, C. pneumoniae with the MOMP genotype KOALA of chlamydial isolates from Phascolarctus cinereus (Koala), and ruminant and porcine isolates of C. psittaci with disease propensity for polyarthritis were uniquely host-adapted. All examined MOMP genotypes of Chlamydia were detected in another two-step PCR. Primers homologous to translated terminal ompA regions were used in primary PCR. A portion was amplified in secondary genus-specific reactions with primers internal to the first step primers. Three chlamydial genomes were detected in a background of 1 $\mu$g unrelated DNA. Group-specific secondary PCR used one genus-specific primer and primers derived from fingerprint regions of major chlamydial groups. Four groups were differentiated as amplification products. Simultaneous presence of DNA from different chlamydial groups was detected. Restriction analysis differentiated MOMP genotypes of the groups. DNA sequences corresponding to B577/LW508 MOMP genotypes of C. psittaci were detected in two out of seven milk samples from cases of bovine mastitis.

Pages

139

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