Isolation of canine and feline "gastrospirilla" is very demanding, yet possible, as was shown in the present study. We have described and successfully used an isolation method to detect two new Helicobacter spp., H. bizzozeronii and H. salomonis, from the gastric biopsies of dogs and cats. In addition, we have isolated two strains that resemble "F. rappini" in morphology, although the taxonomic position of these "Flexispira"-like organisms requires further investigation. Whilst the methods described here for isolation of these bacteria represent a considerable advance over those used previously, further improvements in culture conditions for these bacteria are still required, especially when examining cats.

The culture and characterisation of H. bizzozeronii and H. salomonis have extended knowledge of the ecology, prevalence, and culture characteristics of "gastrospirilla". The methods employed in this study can be applied to determine the clinical significance of gastric Helicobacter spp. in dogs and cats with gastrointestinal disease, as well as for animal model studies of H. pylori. Furthermore, potential virulence genes can be studied from the culturable gastric organisms.

Successfully cultured and taxonomically identified species are needed and can be used for the development of new selective culture media for the differentiation of H. felis, H. bizzozeronii and H. salomonis. Such studies may resolve the problem of mixed cultures of these species obtained from gastric biopsies. Similarly, organisms growing in culture can be used for the development of species-specific PCR-based methods of direct detection of these species in gastric biopsy samples.

Results of the 16S rRNA gene sequence comparisons between culturable and unculturable Helicobacter spp. within the "gastrospirilla" group reveal highly similar sequences for H. felis, H. bizzozeronii, H. salomonis, "G. hominis"1 and 2 and "G. suis". Therefore, the true relationship between these "strains" remains unknown. It is evident that this widely used gene cannot be used as a species identification tool for these bacteria. Its application to the taxonomy of these bacteria has been confusing and the true genetic relationship between cultured and uncultured animal and human "gastrospirilla" remains to be solved by more discriminative methods such as DNA-DNA hybridisation.

Similarly, the inertia of "gastrospirilla" in commonly used biochemical tests, and those comprising an extended (65 tests) phenotypic identification scheme, indicate that a polyphasic approach must be used for definition of these species. Certain biochemical features, detailed morphological determination by light- and electron microscopy, standardised SDS-PAGE protein profile analysis and DNA-DNA hybridisation should be used.

The widely accepted "gold standard" for delimiting species boundaries is DNA-DNA hybridisation. This technique must be applied in taxonomic studies of strains in this group of culturable Helicobacter spp. Dot-blot DNA-DNA hybridisation was found to be a good screening method that could be used for the selection of strains for quantitative DNA-DNA reassociation studies. For routine purposes we strongly recommend the use of highly standardised SDS-PAGE protein profile analysis for identification of culturable "gastrospirilla", since the results obtained were in excellent agreement with both the dot-blot, and quantitative DNA-DNA hybridisation analyses.

The genetic diversity of different H. felis strains isolated from cats and dogs and originating from three different continents was very high and comparable to that observed in its close taxonomic relative, H. pylori. The genome size of H. felis was determined to be 1.6 Mb.

Available data strongly indicate the need for further work concerning the specificity and application of PCR assays for detecting the various Helicobacter spp. known to inhabit the gastric mucosa of domestic pets and humans.