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Development of molecular techniques for the detection of C.Psittaci infection in sheep and the typing of chlamydial isolates

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BaxterSIF_1991redux.pdf (44.54Mb)
Date
1991
Author
Baxter, Stuart Ian Forgan
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Abstract
 
 
Enzootic abortion of ewes (EAE) is an economically important disease of sheep caused by a specific type of Chlamydia psittaci. Despite the considerable losses caused by the disease, the pathogenesis, immunology and epidemiology of EAE are poorly understood, as are the immune mechanisms evoked by the current vaccine used to control the disease. Moreover, the efficacy of the vaccine has been found to vary. In contrast to Chlamydia trachomatis, for which 15 serovars have been defined, and the other recently defined species Chlamydia pneumoniae, a suitable comprehensive systematic classification of C.psittaci has not been developed. This has presented a central problem to the study of EAE since more than one type of C.psittaci infect sheep. In addition to this, the unsuitability of cell culture based tests for the detection of the organism in biological specimens has limited both pathological and epidemiological studies.
 
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.
 
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.
 
URI
http://hdl.handle.net/1842/29882
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  • Biological Sciences thesis and dissertation collection

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