Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche
Webzine Sanità Pubblica Veterinaria: Numero 32, novembre 2005 [http://spvet.it]Documento reperibile all'indirizzo: http://spvet.it/indice.html#300
The use of molecular biology for the investigation of foodborne pathogens.
Francesca Leoni, *Jim McLauchli
Approximately 1-3 incidents of foodborne and infant botulism occurs in humans each decade, however wound botulism amongst illegal injecting drugs users has become much more common with almost 100 cases being diagnosed over the past 5 years. Diagnosis can be problematic and requires the use of bioassays for the detection of neurotoxin.
Recent work in the UK has lead to the successful development and evaluation of real- time PCR assays for the detection of Clostridium botulinum neurotoxin genes. The assays are quick and straightforward to perform and can be applied to both detecting this bacterium directly to clinical food and environmental material as well as to identify cultures growing in vitro. The use of these assays has resulted in faster diagnosis as well as a 75% reduction in the use of animals in the mouse bioassay.
The numbers of cases of human listeriosis in England and Wales has almost doubled over the past five years, however the reason for this increase is unclear. Surveillance of human listeriosis in the UK is performed by voluntary reporting of cases as well as by centralized collection of Listeria monocytogenes isolates for confirmation and subtyping. Subtyping strategies involve the use of phenotypic as well as genotypic characterization systems. Progress has been made with the provision of rapid molecular fingerprinting by the use of automated DNA extraction from bacterial growth followed by profiling using amplified fragment length polymorphism (AFLP) analysis. Possible clusters of L.monocytogenes strains are confirmed by pulsed field gel electrophoresis as well as to comparing isolates from those recovered from foods. Molecular analysis revealed several clusters as human cases (although this was insufficient to explain the upsurge in case) and outbreaks associated with butter, and sandwiches sold in hospitals were identified. These data lead to direct interventions involving the withdrawal of contaminated foods as well as surveys of both butter and foods sold in hospitals which will be completed in 2005.
Human diarrhoeal disease are of considerable importance in the UK, however conventional analysis of faecal samples reveal an aetiological agent in the minority of cases. A very large case control study was completed in England during the 1990s of which a potential pathogen or toxin was detected in less than 50% of the cases. Following this study over 5,000 faecal samples were archived which were stored at -70°C for more than 10 years. Considerable progress has been made over the past 10 years in the extraction of both RNA and DNA directly from faeces and the use of this to detect intestinal pathogens using PCR based assays.
Funding was secured in the HPA from the UK Food Standards Agency to generate nucleic acid from the frozen faecal archive. Initial testing established that target nucleic acid survived the archiving process over the past decade from both DNA and RNA viruses, gram-negative and gram-positive bacteria and protozoan parasites. Nucleic acids have now been extracted from over 3,000 samples from which no potential pathogen or toxin were previously detected and more than 45% yielded evidence for the presence of either Salmonella, Campylobacter, rotavirus, norovirus, Cryptosporidium or Giardia. Work is progressing to identify further targets in these previously target negative samples and these will be invaluable in more accurately calculating the burden of diarrhoeal diseases for a wider range of aetiological agents.
The seminar was closed with a lively discussion highlighting many common interests between the IZS Umbria e Marche and HPA in terms of surveillance systems, diagnostic methods and advances in nucleic acid detection technologies.
