Review of the Food-borne Zoonoses Research ... - ARCHIVE: Defra

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To test the suitability of the AFLP method for sensitive characterisation of Salmonella species, over 400 isolates were selected for investigation, from the collection of the Scottish Salmonella Reference Laboratory at Stobhill Hospital in Glasgow. A catalogue of representative strains was produced, including 243 isolates of human origin, 83 animal isolates, 37 from a variety of foods and 26 strains acquired from environmental sources. The isolates had already been characterised by classical typing methods by the Scottish Reference Laboratory. In addition, the majority of the strains had been typed using PFGE, and their antibiotic resistance profile determined. DNA extracts from the resuscitated Salmonella strains were provided by the Scottish Salmonella Reference Laboratory for AFLP analysis at LGC. The first stage of the project focused on development of a robust protocol for the generation of AFLP profiles. To facilitate inter-laboratory comparison it was decided to utilise commercially available AFLP reagents provided as a Microbial Fingerprinting Kit. However, in the last year of the project, the highly variable quality of commercially available reagents necessitated a change to independently-sourced materials. The effect of a variety of parameters on the robustness and quality of AFLP profiles was assessed, and an optimised protocol was developed. To promote reproducible data interpretation and comparability of results between analysts and laboratories, methods for normalisation of data and criteria for AFLP profile acceptance were developed. However, the inherent variability in profile intensity was a significant challenge to the reproducibility of the method, with subjective calling of signals on the borderline of automated detection. The variation in fragment intensity is one of the main problems with robust isolate characterisation by AFLP analysis. An extensive range of commercially available PCR primer and restriction enzyme combinations were evaluated for sensitivity against a set of closely related isolates, and novel combinations targeting hyper-variable regions of the Salmonella genome were also developed and tested. Genomic digestion with EcoRI and Taq I, followed by amplification using EcoRI-T and TaqI-0 selective primers was identified as the most discriminatory approach, and this was used to profile the extensive catalogue of isolates. The AFLP profiles produced were then compared with known phenotypic characteristics of the isolates to identify markers correlating with particular traits. To facilitate identification of unique markers for particular characteristics, a searchable database was developed at LGC using Microsoft Access. The database allowed both storage of isolate profiles and comparison of molecular and phenotypic characteristics of strains, enabling AFLP profiles of unknown samples to be matched with related profiles already entered into the database. The effectiveness of the database system was demonstrated in a blind trial, although in contrast to previous studies characterising smaller numbers of isolates, robust correlation between molecular markers and phage type or other phenotypic characteristics could not be established. In conclusion, the AFLP typing approach offers several advantages over currently used typing methods, including accuracy and precision of fragment sizing, and the suitability of the profile information for database storage. However, the complex, multi-step nature of the method can lead to high failure rates, and the relatively high cost of commercial reagents and equipment are further drawbacks to widespread uptake of the AFLP approach. Overall, the discriminatory power of the technique was not found to be sufficiently high to warrant extensive further effort in overcoming the very real drawbacks of the method, particularly with the advent of newer techniques including proteomic and microarray methods, and the widespread adoption of PFGE analysis for international surveillance initiatives. . 40
Review Summary This project aimed to improve 1) the speed of salmonella typing and 2) the ability to type some of the epidemic serotypes and subtypes (for example, Salmonella Enteritidis and Salmonella Typhimurium DT 104) to improve outbreak tracing and epidemiology in general. The use of AFLPs (amplified fragment length polymorphisms) was assessed as a typing method with these aims in view. At the time of funding, this was important methodology to try to develop. The work was appropriately carried out and, although it was reasonably successful, AFLP was not sufficiently robust to be taken further when other typing methods showed more promise. 41
Page 2 and 3: Contents Section A - Introduction..
Page 4 and 5: Review of the Food-borne Zoonoses (
Page 6 and 7: Plans for taking forward these prio
Page 8 and 9: Policy Objectives The key policy ob
Page 10 and 11: Figure 2. Laboratory reports of Sal
Page 12 and 13: Figure 4. Laboratory reports of Cam
Page 14 and 15: 6. Aims of the FBZ research review
Page 16 and 17: Section B - Review Summaries 16
Page 18 and 19: epidemiology of Salmonella and anti
Page 20 and 21: There is a need to objectively asse
Page 22 and 23: 2. Campylobacter review summary 2.1
Page 24 and 25: for this organism. Research within
Page 26 and 27: 3. E. coli review summary 3.1 Succe
Page 28 and 29: 4. Other zoonotic pathogens of inte
Page 30 and 31: Annex 1 - Projects Reviewed 30
Page 32 and 33: OZ0311 Conventional versus capillar
Page 34 and 35: OZ0402 OZ0404 OZ0405 OZ0407 OZ0604
Page 36 and 37: OZ0704 OZ0705 OZ0706 OZ0707 Quantif
Page 38 and 39: Annex 2 - Project Abstracts and Pro
Page 42 and 43: Project code: OZ0324 Project title:
Page 46 and 47: Review Summary The knowledge that t
Page 48 and 49: Review Summary The project was felt
Page 52 and 53: It is clear that the EC legislative
Page 54 and 55: if the vaccines currently available
Page 56 and 57: Four published papers have come out
Page 58 and 59: Review Summary This epidemiological
Page 60 and 61: Project code: OZO316 Project title:
Page 64 and 65: agricultural industry/public health
Page 66 and 67: testing laboratories. In addition,
Page 68 and 69: animal pathogen but is a human path
Page 70 and 71: esearch has also substantially impr
Page 72 and 73: to elucidate the antigenic repertoi
Page 74 and 75: and have contributed to our underst
Page 76 and 77: Fundamental issues concerning the f
Page 78 and 79: Review summary Project OZ0606 focus
Page 80 and 81: Abstract of research OZ0608 The ado
Page 82 and 83: thinning process for molecular trac
Page 84 and 85: Project code: LK0665 Project title:
Page 86 and 87: Review summary While poultry meat i
Page 88 and 89: Review summary A key aim of this pr
Page 90 and 91:
Review summary This project aimed t
Page 92 and 93:
Project code: OZ0709 Project title:
Page 94 and 95:
Page 96 and 97:
Project code: OZ0138C Project title
Page 98 and 99:
Page 100 and 101:
Page 102 and 103:
Abstract of research Projects OZ070
Page 104 and 105:
produced were both relevant and imp
Page 106 and 107:
List of External Reviewers. Mr. Pau
Page 108 and 109:
Review of Defra’s FBZ Research Pr
Page 110 and 111:
Review of Defra’s FBZ Research Pr
Page 112 and 113:
Annex 5 - Review Timetable 112
Page 114 and 115:
11.45 - 12.00 Bovine and Porcine Fo
Page 116:
Campylobacter Other 11.50 - 12.05 P
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