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.
