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Title: Development of molecular techniques for the detection of C. psittaci infection in sheep and the typing of chlamydial isolates
Author: Baxter, Stuart Ian Forgan
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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The aim of this project was to use nucleic acid based techniques for the detection and typing of C.psittaci strains. A representative genomic library of C.psittaci, S26/3, was constructed and used to isolate the 16S rRNA and major outer membrane protein (MOMP) genes. The 16S rRNA gene was subcloned into a transcription vector to allow the generation of single stranded RNA probes complementary to native C.psittaci ovine abortion (OA), 16S rRNA. This anti-sense probe was used in an RNase protection test (Hybrid Duplex RNA Analysis-HYDRA). HYDRA was shown to be a sensitive and highly specific detection test for C.psittaci, OA strain infection in biological samples including ovine faeces, ovine and human placenta, ovine ileal and tonsilar tissues and ovine vaginal swabs. The same anti-sense probe was also used to detect C.psittaci OA strains by in situ hybridisation. Chlamydia-specific oligonucleotide primers were designed and polymerase chain reaction (PCR) amplification of both the 16S rRNA and MOMP genes was developed, at a preliminary level, as a detection test for infection by C.psittaci. The application of both Southern blot analysis and restriction endonuclease (RE) profiling of PCR amplified fragments (PCR-RE profiling), using the MOMP and 16S rRNA genes, allowed the identification of C.psittaci subspecies types and the easy discrimination of the three chlamydial species. A combination of Southern blot analysis, PCR-RE profiling and direct DNA sequence determination of the MOMP genes from a large group of C.psittaci,OA isolates was used to investigate possible strain variation within this C.psittaci type. The comparison demonstrated a high degree of identity within the OA group and a contrasting high level of variation within the other group of ovine C.psittaci, non-abortion ruminant strains. This study has established simple and convenient techniques for both detection and identification of the 2 different types of C.psittaci which infect sheep, independent of culture and extensive purification of the causative organism.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available