Påvisande av Helicobacter spp hos hund : en metodologisk studie

Abstract: The purpose of this study was to develop a reliable molecular-genetic method to determine the different species of helicobacter in dogs. The study is part of a larger project to map the prevalence of Helicobacter spp in healthy and sick dogs in Sweden, and to determine the possible connection of Helicobacter spp infection with gastrointestinal diseases in dogs. Several published studies have reported on the prevalence of Helicobacter spp in dogs. The problem is that three of the most common species are so alike that a 16SrRNA-PCR with sequencing is not able to differentiate between them. In this study, DNA has been purified from samples and then a Multiplex PCR has been performed. Multiplex PCRs use multiple primers in one single PCR. The primers have been attached with fluorescent markers, and capillary electrophoresis is used to illustrate the results. In this way, very similar species can be differentiated from each other. This paper also includes a short review of previous studies made in this field and the different methods used for diagnosis of infection with Helicobacter spp. Samples from 17 dogs were collected and analyzed. Multiplex PCR was used on 12 healthy dogs. For the Multiplex PCR non-invasivly collected samples were used, namely faeces and mouth swabs. The remaining 5 dogs were patients with different gastrointestinal problems. From these dogs, in addition to faeces and mouth swab samples, biopsies were taken from the stomach and duodenum. The study shows that the Multiplex PCR works well, but that the capillary electrophoresis is not able to illustrate the results. Also, all samples from all dogs were analyzed in a helicobacteria-specific 16SrRNA PCR and this showed Helicobacter spp in nearly all samples, the frequency of mixed helicobacter-infection being as common in mouth swabs as it was in faeces samples. This unexpected result further emphasises the need to develop the Multiplex PCR-technology, as normal sequencing cannot be used in severe mixed infections. Thus, the Multiplex PCR should prove to be a diagnostically reliable method, but further development and testing are needed.