title: DNA fingerprinting of Haemophilus somnus, Histophilus ovis and Actinobacillus seminis
Creators: name: Appuhamy, Sarath
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Microbiology.

description: H. somnus, H. ovis and A. seminis are closely-related Gram-negative pleomorphic bacteria. They are very similar in cultural and biochemical characteristics and these phenotypic characteristics are highly variable between strains of each species. Thus their identification is often uncertain. H. somnus causes thromboembolic meningoencephalitis, pneumonia and reproductive failure including abortion in cattle. H. ovis causes similar disease syndromes in sheep. A. seminis is a common cause of epididymitis and infertility in rams. Genotypic characterisation of bacteria, in many cases, has been shown to provide a clearer, more discriminating and more reliable means of differentiating species and strains within species. Polymerase chain reaction (PCR) fingerprinting methods, using different primer sets targeted against specific chromosomal regions are being applied successfully for the typing of a wide range of human and veterinary pathogens. This work has involved the characterisation of a large number of isolates from bovine and ovine sources, including strains provided by Scottish Veterinary Investigation Centres or isolated as part of this study from slaughterhouse materials. This slaughterhouse survey showed that 18% of randomly selected reproductive tracts of cows contained H. somnus and this was much higher than earlier reports from elsewhere. The identity of the strains was confirmed by conventional cultural and biochemical tests (including API ZYM) and the strains were also characterised in terms of their plasmid profiles and antibiotic resistance. A high proportion of H. ovis isolates contained plasmids (63%) when compared to H. somnus (13%) and A. seminis (8%). Nine H. ovis and two A. seminis isolates contained two plasmids while the rest contained single plasmids. The size range of these plasmids was 1.7 kb to 5 kb. The sensitivity of these strains to a number of commonly used antibiotics was determined but there was no apparent relationship between resistance and the presence of plasmids. Although A. seminis is generally regarded as an ovine pathogen, one isolate was obtained from a bovine source. The main thrust of the work was to develop PCR techniques for identification and typing. Three primer sets, namely REP, ERIC and Ribosomal, have been applied in order to generate reproducible profiles of PCR amplimers. Of the 29 H. somnus strains studied, 11, 16 and eight groups were recognised by the use of REP, ERIC, and Ribosomal primers respectively. For the 19 H. ovis isolates 11, seven and five groups were identified by the three primer sets respectively. For the 24 isolates of A. seminis, REP- and ERIC- PCR yielded five and nine types respectively, but PCR-ribotyping gave a similar- pattern for all isolates except one. The use of a combination of the primer sets provided a high resolution fingerprinting method for these strains which could be useful for epidemiological studies of these related bacteria. PCR-ribotyping produced a relatively simple pattern which was useful for rapid identification of these species after primary isolation and for differentiation of these species from each other and from other related bacteria. These PCR fingerprinting methods were simple to perform and reproducible. Clearly distinguishable profiles were obtained between respiratory and genital isolates of H. somnus by all three typing methods. The discrimination of H. ovis isolates was better in REP-PCR than in ERIC-PCR. In general, PCR-ribotyping produced a simple pattern but REP- and ERIC- PCR produced complex patterns. The genetic heterogeneity of A. seminis was revealed. The two major amplimers of PCR-ribotyping of A. seminis were cloned and sequenced. The sequences showed that A. seminis contains at least two ribosomal operons, termed rrnA and rmB and that these encode one and two tRNAs, respectively, in the spacer region between the 16S and 23S rRNA genes. Species-specific primers for A. seminis were developed from the sequence of the spacer region of rrnB for the specific identification and detection of A. seminis by PCR. The PCR assay was able to detect approximately 300 colony forming units of A. seminis per ml of 10-fold diluted raw semen sample. Storage solution added to semen for long-term storage was found to inhibit the PCR and the assay would best be performed for diagnostic purposes on fresh semen prior to storage.