CAMPYNET NEWS
NOVEMBER/DECEMBER 2001
Campynet final meeting – a brief summary
As all participants will be aware, the final meeting of the Campynet project took place at Duerdent Court in London, England, on November 17-18th 2001. At this meeting the status of the individual groups was presented. These are summarised below.
Strain collection: One hundred strains were selected and form "the Campynet collection". These were the basis for the standard methods developed during the project. They have been extensively characterised by "classical" Penner, and the new Colindale serotyping systems, PFGE, fla-PCR, ribotyping and AFLP analysis. A paper describing the strain characteristics and the implications for typing methods therof is being prepared and the data are also expected for publication on this website, pending acceptance of the aforementioned article and appropriate clearance of copyright issues.
The collection will be made available to researchers upon request to Jaap at ID-DLO for the cost of 910 Euro. A standard form giving the terms and conditions for the release of the strains must be signed prior to supply.
PFGE: The protocol described on this website was designed to account for the differences in equipment used in the European Union. This method has been used to examine all 100 strains (83 C. jejuni, 17 C. coli) and profiles produced on two electrophoresis models (Bio-Rad units DR-III and CHEF Mapper), and two different agarose grades (standard and high resolution). All duplicate profiles matched at 100% similarity when subjected to numerical analysis using appropriate parameters. This database was used as the basis of an interlaboratory trial to test the suitability of the system for comparitive purposes between European laboratories. At the time of the final meeting, two laboratories from the UK and Sweden each provided two gels that included a number of Campynet strain profiles for identification via the database established at the Danish Veterinary Laboratory. Gels from one laboratory were supplied in a non-standard format. One gel from each lab has for far been analysed, and 30/32 strains correctly assigned to type using default parameters. The remaining two strains (from the non-standard gel) were successfully identified using minor changes to the analysis parameters. The non-standard gels will be rescanned to conform to standard format and reanalysed.
Since this time, data has been supplied from two further laboratories for comparison. It is intended to complete all interlaboratory comparisons in January and subsequently assess the potential of a common PFGE typing system for C. jejuni for use in European laboratories. It was proposed that distinct profiles will be given SMAP (SMA1 Pfge profile) numbers and strains not digested by this enzyme will be designated SMAP RD (refractory to digestion). Although duplicate C. coli profiles were successfully clustered together at 100% similarity, the scheme is not wholly suited for definitive typing of this species since many of the component fragments fall outside the frame of reference used for C. jejuni. A paper describing the scheme will be prepared early in 2002.
Fla-PCR typing: to enable standardisation of nomenclature for fla-PCR types based on the Campynet recommended protocol, a coding system was presented that assigns fragments to band classes according to molecular weight. This approach does not therefore rely upon computerised data analysis and is intended to complement the rapidity and level of discrimination provided by the method. A paper describing this method will be prepared early in 2002.
AFLP: all strains had been characterised by each of the methods described by the groups in Holland (Birgitta Duim) and Denmark (Stephen On) respectively. A more limited study was conducted on a few strains using the method described by the group in Norway (B. Lindtstedt). The discriminatory potential of the methods appeared comparable, but profile matching between laboratories equipped with different separation and detection apparatus was not achieved. Thus, the existing methods are well suited for use in a single laboratory but data comparisons are limited where matching equipment is not used.
Information technology: the WWW site has been the principal means of informing both participants and the wider scientific community of the progress of the Campynet project. The Website will remain at its present URL to ensure that the protocols can be readily accessed, and to continue to act as a source of information about the project and the bacteria in general. It is expected that updates of the newspage will continue through 2002 at least, pending preparation of the final report to the EC.
The IT group have also evaluated three software packages for comparison of molecular type profiles. PFGE patterns (not the Campynet standard) were used as a model. The three programs evaluated were BioNumerics, Taxotron, and Phoretix 1-D. The following summarises our findings.
Phoretix 1-D
Advantages: The program works intuitively & most functions are easy to implement
Disadvantages:
Taxotron
Advantages: The manual is short and useful, but does not explain everything. Normalization works well and indistinguishable profiles can be matched.
Disadvantages:
Bionumerics
Advantages:
Disadvantages
Conclusions in brief:
Phoretix 1-D performs poorly and cannot be recommended for comparing and identifying types r
Taxotron is somewhat laborious to use, but is inexpensive and effective Ö
BioNumerics is expensive but is flexible, reasonably easy to use (but difficult to master!) and performs well Ö
New and complementary typing methods: much of the groups activities had focused upon the use of the Riboprinter (automated ribotyping) for standardised typing of campylobacters. Three groups in Holland (Jan Van der Plas), Northern Ireland (Bob Madden/Clare Harrington) and Denmark (Vivian Fussing) had collaborated in evaluating three enzymes (PvuII, PstI, HaeIII) for use with this system. HaeII proved to be the most discriminatory and is thus the enzyme of choice for riboprinting campylobacters. An article describing the results in full will be prepared in 2002.
The group had also hosted several lectures at Campynet meetings from outstanding speakers. At the November meeting, Dr. Jerry Wells (Institute of Food Research, UK) gave a lecture on the use of microarrays for genomotyping and expression analysis of campylobacter, and of their complementary research on genomics and proteomics. Previously, Martin Maiden and Trudy Wassenaar had spoken on Multi-Locus Sequence Typing (MLST) and genomic instability respectively, at the September 2000 meeting. Clearly new advances in typing methods continue to be made and the developments in post-genomics research promise to herald the dawn of a new era in campylobacter research.
The future: Campynet 2? Trudy Wassenaar led a discussion at the November meeting on the future of Campynet. Various scenarios were put forward and Jaap Wagenaar agreed to initiate further developments. It is now my understanding that Jenny Frost at CPHL, London, has assumed the role of Coordinator for the new project. Further developments will be posted as and when I receive information.
2001, A Campynet Odyssey: December 31st marks the official end of the Campynet project. For many of us the project has been extremely demanding but we can be extremely proud of our achievements. We have accomplished what we set out to achieve: standardised and harmonised methods for subtyping Campylobacter. Thanks to everybody who participated for making this possible.
Let us all wish each other a Merry Christmas and a Happy and Successful 2002 - we certainly deserve it!
March 21st, 2001
STANDARD FLAGELLIN TYPING METHOD ONLINE!
The Flagellin typing group (Bob, Diane and Clare) have completed their investigations on the different fla-typing methods and a standard protocol is now available. Click here to access. A full report of the groups investigations will appear shortly.
December 18th, 2000
PFGE TRIAL READY TO GO!
As described in the "News" of Nov. 7, the plans for an interlaboratory trial of the Campynet and CDC PFGE plug preparation methods, combined with an assessment of the potential for international comparisons, are now made. A subset of 32 strains from the Campynet strain collection has been selected for this purpose and will be examined by the following laboratories:
Danish Veterinary Laboratory, Denmark
Veterinary Laboratories Agency, UK
Central Public Health Laboratory, UK
Preston Public Health Laboratory, UK
Public Health Laboratory, Norway
Swedish Veterinary Laboratory, Sweden
The participants will compare the two PFGE-DNA plug preparation methods to see what advantages and disadvantages the methods hold, particularly with respect to European laboratories. TIFF files of the two gels (conforming to the detailed description on the methods page, please!) will be sent to Stephen at the Danish Vet Lab who will then try to identify the strains by pattern analysis. These participants should take special consideration of the amendments to the standard gel array noted below.This is an exciting moment in the project´s history and as the PFGE Subgroup leader I would like to thank the above laboratories for agreeing to participate.
AMENDMENTS TO THE STANDARD PFGE METHOD
As mentioned previously (see October news), the concensus to focus efforts for typing C. jejuni led to a reconsideration of the normalisation standard. As a consequence, A NEW PROTOTYPE NORMALISATION STANDARD IS SUGGESTED. This is a 1:1 mixture of CNET 068 and CNET 112 and covers a much wider MW range than the former single standard, CNET 068. A new gel set-up is also suggested and these amended conditions will form the basis of the interlaboratory comparison, as described above. The standard PFGE protocol has been amended accordingly (click here to access.
CAMPYNET TO CONTINUE?
Four of the six members of the Management Team - Diane, Stephen, Jan and Jaap - were able to discuss the project during the recent WHO Consultation on the Increasing incidence of human campylobacteriosis, held in Copenhagen from November 21-25. It was agreed that efforts to continue the project should be made. Plans for this are at a very early stage and all comments and suggestions are welcome.
It simply remains at this stage to wish everyone:
Thank you all for your participation and continued support!!!
November 7th, 2000
Special message for all PFGE users: As discussed at the September meeting, I am looking for a few laboratories to evaluate the Rapid DNA plug preparation method developed by CDC and compare it with the present Campynet method. Would all interested parties please mail me by clicking here for further details.
Similarly, we discussed the need to evaluate the method for interlaboratory comparisons. I want volunteers to run 2-3 gels of selected Campynet strains using the standard conditions, and to supply me with photos or TIFF files so that I can try to identify them. Please mail me by clicking here if you would like to participate.
October 2000
Report of the First meeting of the Campynet second-phase participants
The meeting on 23-24 September between all participants has been held and can be considered very successful indeed, thanks to some great organisation from Diane. Feedback from the attendees was very positive and I was personally very pleased with the very positive responses to both the PFGE- and Information Technology (IT) subgroups: it´s nice to know the WWW page is appreciated!
For those who did not attend: Diane introduced the project and the meeting schedule. A summary of the progress made in each of the subgroups (strain collection/distribution, PFGE, fla-typing, AFLP, IT and novel techniques) was given by the subgroup leaders. The most important points are as follows.
The matters raised in the open letter were discussed and it was agreed unanimously to focus on standardised PFGE typing for C. jejuni strains. As a consequence, work is presently underway to identify a better normalisation standard. The Subgroup leader is maintaining frequent contact with workers at the Centers for Disease Control, USA, in the hope that the "Pulsenet" and "Campynet" systems can be harmonised.
In addition to the Subgroup leaders, presentations were made by a number of guests at the meeting. Collette Fitzgerald from CDC gave a report on the Pulsenet system used in the United States, Trudy Wassenaar from Johannes Gutenberg University discussed genetic instability and the implications for genotyping, and Martin Maiden of the Wellcome Trust Centre for the Epidemiology of Infectious Disease presented his work on Multi-Locus Sequence Typing (MLST) of Campylobacter. Many thanks to the above speakers for their valuable and interesting contributions.
At the end of the meeting, the prospects of continuing Campynet were briefly mentioned. There seemed to be a general concensus that the project should continue and this prospect was discussed briefly at the Management Committee meeting in April. We would appreciate a brief comment from you on the subject: would you like the project to continue? If so, how would you like the project to progress? Please mail your brief comments (click here), I will collate them and they will be discussed at the next Management meeting.
September 14, 2000
IMPORTANT MESSAGE FOR ALL PFGE USERS! I have posted an open letter to raise important issues for consideration and discussion at the forthcoming meeting of participants on 23-24 September. Access the letter by clicking here.
August 2000, Newsflash for PFGE users: please note, the standard reference strain is R-38, the correct CNET number for which is CNET 068, not CNET 063 as posted originally. This typographic error has been corrected in the on-line PFGE protocol.
August 2000
IMPORTANT MESSAGE TO ALL PARTICIPANTS: plans for the inaugral Phase 2 meeting which will involve ALL PARTICIPANTS are now in an advanced stage. Diane has prepared a draft agenda (view by clicking here: this also contains useful information concerning travel and reimbursement of expenses) and it is hoped that all aspects of the project - PFGE, fla-typing, AFLP, information/communications technology etc. will be covered. The meeting is scheduled for SEPTEMBER 23-24 at DURDENT COURT, Tilehouse Lane, Denham, Uxbridge, Middlesex UB9 5DU, UK (Tel +44 1895 833338: Fax +44 1895 832156). Uxbridge is essentially a suberb of London and is serviced by the London Underground, as well as bus services of course. The closest airport is London Heathrow. PLEASE MARK THIS DATE IN YOUR DIARY AND MAIL DIANE TO LET HER KNOW YOU ARE COMING.
VERY FEW RESPONSES ABOUT RECEIVING THE STRAIN SET HAVE BEEN RECEIVED. The strains will NOT be distributed without your feedback concerning when you would like to receive them, so please contact Jaap or Birgitte and let them have some idea of a convenient time to receive the strains. PLEASE REMEMBER - THE SUCCESS OF THIS PROJECT DEPENDS ON YOUR CO-OPERATION.
May I remind you that the prototype standard PFGE protocol is now available (click here!) and I hope to discuss any and all aspects of this with you. Please, if you have tried it and would like to share your experiences - good or bad - then mail me - Stephen.
Additional news: the AFLP Subgroup members (Birgitte, Clare, Stephen) will convene in Copenhagen during the week starting August 21st to work closely on comparing results of the two available AFLP typing methods. Hopefully, preliminary results can be discussed at the Phase 2 meeting the following month.
We look forward to seeing you in September.
June/July 2000
PROTOTYPE STANDARD PROTOCOL FOR PFGE TYPING OF CAMPYLOBACTER NOW AVAILABLE
The PFGE Subgroup convened in Copenhagen, Denmark on June 15th to discuss the standard protocol for PFGE typing campylobacters. After extensive discussions lasting the whole day. As a result, a prototype standardised method for PFGE typing is now available. Click here to access the protocol.
It is important for the success of the project that any problems are identified early. Please, would all those who intend to use PFGE use the method described and tell me your experiences - good and bad - so that we can work towards an effective method for PFGE typing across Europe. E-mail your comments to Stephen. We look forward to hearing your experiences at the September meeting.
April/May 2000
Progress of Campynet was reviewed at a Management Meeting held in London on April 27th. The most important points are summarised below.
We welcome Clare Harrington as a member of the AFLP Subgroup! During her tenure at the Danish Veterinary Laboratory, Clare gained a tremendous amout of experience with this method and it was felt such experiene would be invaluable to the group as a whole. Clare will continue her work on AFLP in her new post at Queen´s University of Belfast, working with Bob Madden!
Sending the Campynet strain set to the evaluating laboratories (i.e. second-phase participants) was discussed. This collection will contain 100 strains, which involves a lot of work for both sending and receiving laboratories. The distribution will be phased over a period of time, but we need to know WHEN IT IS CONVENIENT FOR YOU TO RECEIVE THE STRAINS!!! Jaap and Birgitte are beginning the culture of the strains now, so please E-mail them (just click on their names!) and let them know when you could receive the strains for testing.
Each of the subtyping groups have been working hard on developing/evaluating suitable protocols for standardised typing of C. jejuni/coli. It is hoped that standard SOPs for performing flagellin PCR-RFLP, and PFGE typing will be available by the late June/early July. Methods for standard PFGE typing will be discussed in detail by the members of the PFGE subgroup (Stephen, Fiona, Marja-Liise) at a one-day meeting in Copenhagen tentatively scheduled for THURSDAY, JUNE 15TH.
The first major meeting for ALL participants is tentatively scheduled for THE WEEKEND OF 23-24 SEPTEMBER 2000, probably in the UK and hosted in student-type accomodation (ie. no frills!). It is hoped that all participants will be able to come to the meeting with at least one photograph/scan of a selection of Campynet strains typed with the method(s) of their choice. AGAIN, CAN WE STRESS THE IMPORTANCE OF CONTACTING JAAP/BIRGITTE TO ENSURE THE STRAIN SET IS DELIVERED TO YOU, SO YOU TEST CAMPYNET STRAINS!!!
We also need to know if you are still interested in participating in the project, which methods you are interested in using, and what your level of expertise is (from 1-5, 1=inexperienced, 5=very experienced). Please send a mail to Diane, detailing the above information.
Finally, we welcome Sigrun Gudmundsdottir at the Icelandic Fisheries Agency, Iceland, as a subsidiary member! Although Sigrun learned of the project too late to be formally included in the project, she is very keen to use the recommended standard methods for typing campylobacters to examine Icelandic strains from diverse sources and will be kept fully informed of all developments.
March 2000: The major review of new developments in subtyping of campylobacters has now been published in the ASM book "Campylobacter, 2nd edition". The chapter was written by several participants in the Campynet project and methods included in the chapter include serotyping, phage typing, PFGE, flagellin PCR-RFLP, RAPD, AFLP, and Riboprinting. The full reference is: Newell, D. G., Frost, J. A., Duim, B., Wagenaar, J. A., Madden, R. H., Van der Plas, J. and On, S. L. W. 2000. New Developments in the subtyping of campylobacters. In: Campylobacter, second edition (Nachamkin, I. and Blaser, M. J., eds.), pp. 27-44. ASM Press.
The book also contains two other chapters co-written by Campynet contributors on genomic instability and the consequences for genotyping (with useful step-by-step suggestions for detecting genetic instability and interpreting genomic profiles for epidemiological purposes) and poultry infections, as well as chapters encompassing various aspects of taxonomy, pathogenesis, epidemiology, molecular genetics, and food safety by other distinguished authors in the field.
For ordering and other details on the book, click here to access the ASM website.February 2000: The flagellin subtyping group have completed their week-long combined investigation of available methods at the University of Belfast. The results will be used to help decide which assay will be chosen as a standard method for further evaluation.
January 2000: Happy New Year!
The Campynet project continues onward. The flagellin gene typing group have arranged to directly compare the various fla-PCR typing systems available during a week-long sojurn at the University of Belfast. Bob Madden (subgroup leader and host!), Clare Harrington (Danish Veterinary Laboratory) and Anne Ridley (Veterinary Laboratory Agencies) will perform the evaluation. Stephen distributed TIFF files of the digitized gels to be used for the software evaluation. A minireview of genotyping methods published under the auspices of the Campynet project and co-authored by Diane was published in the journal Applied and Environmental Microbiology (Wassenaar, TM and Newell, DG. Genotyping of Campylobacter spp. Appl. Environ. Microbiol. 66:1-9).
December 1999:
Campynet Management meeting, hosted during the final COST 97 ("Pathogenic microorganisms in poultry and eggs") meeting in Lelystad, Holland. Issues discussed included the key meetings in 2000, at which model typing systems for fla-PCR and PFGE would be selected and the major meeting at which standard methods would be distributed. The first of these, a subgroup meeting, was likely to be hosted at VLA in New Haw towards the end of March 2000: the second was likely to be hosted by ID-DLO in Holland sometime in May 2000. More details will be made available in due time.
September 1999:
Campynet in America! A meeting for participants and interested parties was held during the 10th International Workshop for Campylobacter, Helicobacter and related organisms, in Baltimore this month. A brief summary was given of the project and each Subgroup member summarised the progress made to date in their respective groups. A poster was also presented to inform others of the project.
August 11th, 1999: Campynet goes to America!
June 4th, 1999: Second Management meeting of Campynet at the Danish Veterinary Laboratory in Copenhagen. The most important points are summarised below.
Strain distribution. A few strains have still not been received from the UK, Sweden and Belgium. It was decided that the standard set of strains would be distributed in two phases. 36 selected strains to be used for the development of standard methods would be sent to first-stage partners in late June/early July. The remaining strains will be sent during September/October.
Flagellin, PFGE and AFLP typing subgroups. Various means of facilitating the comparison of available techniques were discussed, especially for the fla- and AFLP methods. It was suggested that interlaboratory visits from subgroup members would be effective and one such visit (for AFLP comparison) is presently in an advanced stage of preparation.
Information technology. The structure and content of the Campynet WWW site has been warmly approved (comments and suggestions remain welcome!). All selected software for pattern analysis had now been ordered and was awaiting delivery.
Visits. Applications for interlaboratory visits during the second Campynet year (ie. from October 1999) will shortly be considered. If you are a participant and would like to visit another participating laboratory, please send a brief note to Diane. We hope to make the official application form available from this site shortly.
Future use of standard strain set. There was some debate over the use of the strains collected via Campynet for other national- and international (eg. Fifth Framework) projects. It was agreed that approval from all contributing laboratories be sought to allow the Management Committee to make executive decisions on the use of strains for non-Campynet related projects. It is intended that any partner wishing to use the strain collection for projects that are not directly related to Campynet should ask permission from the Management Committee first. However, grant applications to obtain funding to cover characterisation of the strain set does NOT need to be approved in this way. The intention here is simply to provide proper and ethical control over use of the strains collected by the Campynet project, and to ensure that appropriate recognition to the funding body is given. More news will follow on this subject shortly.
Future meetings. Stephen was given the task of trying to arrange a satellite symposium for Campynet participants attending the Tenth International Workshop on Campylobacter, Helicobacter and related organisms scheduled for Baltimore in September 1999. More details will follow in due course. Preliminary discussions were made regarding the major technology transfer workshop for all participants next year. This is likely to take place in March or April 2000: the UK or Holland are possible host countries.
Publications. A book chapter concerning new developments in subtyping methods, written by several Campynet participants, has just been accepted by ASM. Publication of the book is scheduled for 2000. A poster for describing the Campynet project has also been submitted to the Baltimore congress.
April 28th, 1999: URGENT!!! WE ARE STILL WAITING FOR STRAINS FOR THE CAMPYNET STANDARD STRAIN SET - PLEASE, WILL THOSE PARTICIPATING LABORATORIES IN THE UNITED KINGDOM, BELGIUM AND SWEDEN WHO HAVE PROMISED ISOLATES CONTACT JAAP WAGENAAR OR BIRGITTA DUIM NOW AND CONFIRM THE STATUS OF THOSE STRAINS. THIS IS A PRIORITY ISSUE SINCE WE ARE NOW BEHIND SCHEDULE. THE SUCCESS OF THIS PROJECT DEPENDS ON YOUR COOPERATION.
April 28th, 1999: Campynet goes online!
October 16th, 1999: First Management meeting of Campynet. This was featured in our first report to members, which was produced as a two-sheet newsletter. You can access this little piece of history by clicking here.
October 1st, 1999: Campynet officially starts!
