INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1992, p. 330-334 Vol. 42, No. 2 0020-7713/92/020330-05$02.00/0 Copyright 0 1992, International Union of Microbiological Societies

International Committee on Systematic Bacteriology Subcommittee on the of Minutes of the Meetings, 13 and 15 September 1990, Osaka, Japan

Minute 1. Call to order. The meeting was called to order reference laboratories maintain a full collection of serovars, by the Chairman, E. Kmety, at 1300 on 13 September 1990. and (v) production and distribution of monoclonal antibod- The opening consisted of a welcoming introduction. Follow- ies. ing this there were additional open sessions at 1800 on 13 Minute 9. General aspects of taxonomy. E. Kmety pre- September and at 0900 on 15 September. A closed session sented a paper on general aspects of taxonomy. He spoke of was held at 1300 on 15 September. early attempts at a phylogenetic classification and also of Minute 2. Record of attendance. The members present problems in the use of DNA base composition for compari- were E. Kmety (Chairman), R. Yanagawa (Vice-chairman), son purposes. Following this he discussed problems with N. Stallman (Secretary), M. Cinco, H. Dikken, W. Ellis, S. using DNA hybridization. It was stated that some microbi- Faine, R. Johnson, R. Marshall, and W. Terpstra. In addi- ologists have considered the possibility of having two clas- tion to members of the subcommittee, the following individ- sification systems, one phylogenetic and the other practical. uals attended one or more of the open sessions: B. Adler, A. Comments were then made on a report submitted by the Alexander, B. Cacciapuoti, W. Chang, S. Coghlan, T. Fu- International Committee on Systematic Bacteriology Ad jikura, Y. Kobayashi, H. Korver, T. Masuzawa, P. Perolat, Hoc Committee on Reconciliation of Approaches to Bacte- A. Schonberg, Y. Yanagihara, and S. Yamamoto. rial Systematics in 1987. In this report it was stated that at Minute 3. Apologies for absence. Apologies for absence present the species is the only taxonomic unit that can be were received from G. Baranton, Dai Bao-min, D. Brenner, defined in phylogenetic terms. According to the phylogenetic Nie Di-kai, C. Everard, K. Hovind-Hougen, A. Kaufmann, definition, a species generally includes strains that exhibit J. Mazzonelli, and A. Thiermann. approximately 70% or greater DNA-DNA relatedness. Minute 4. Mark of respect. The subcommittee recorded There is some evidence that the taxon subspecies is phylo- with deepest regret the death of a member, Y. G. Cher- genetically valid, and further guidelines are needed for nukha, and the death of H. Willinger, who was a collabora- definition of this taxonomic unit. It was also stated that tor. nomenclature should reflect genomic relationships to the Minute 5. Report on the activities of the subcommittee. A greatest extent possible. The overall concern of members of report of the subcommittee activities for the period from the Ad Hoc Committee on Reconciliation of Approaches to 1987 to 1990 was read and accepted. Bacterial Systematics was that any phylogenetically based Minute 6. Minutes of theprevious meeting. The minutes taxonomic schemes must show phenotypic consistency. of the meeting held in Manchester, England, on 5 and 6 Minute 10. Present status of Leptospira parva. W. Ellis September 1986, which were circulated on 10 November presented a paper on the known features of Leptospiraparva 1986, were accepted as a true record of the meeting. as described by Hovind-Hougen et al. in 1981, with subse- Minute 7. Matters arising from the minutes of the previ- quent additional observations by Yasuda et al. in 1987 and ous meeting. (i) Standardization of methods used for the by Saito et al. in 1987. A lengthy discussion followed on the identification of serovars. A discussion was held on heat- differences between Leptospira parva and the genera labile antigens, and it was agreed that the current method Leptospira and Leptonema. The members of the subcom- used for classification, in which whole antigen is used, mittee agreed that a number of differences exist and that a should continue to be used. proposal for a new genus, Tumeria, the type strain of which (ii) Proposed new classification based on DNA homology. is Tumeria parva H, should be published in the International An error in spelling was detected; Leptospira santarosan' Journal of Systematic Bacteriology. should be corrected to Leptospira santarosai. Minute 11. Use of Ambis as a taxonomic tool. W. Ellis Minute 8. WHO activities in international cooperation on presented a paper on the use of Ambis as a taxonomic tool. leptospirosis control and research. T. Fujikura presented a The Ambis system was described as a numerical classifica- paper on the activities being developed by the World Health tion system that is based on a comparison of polypeptide Organization (WHO) for the prevention and control of banding patterns and levels of DNA homology. It provides leptospirosis and the promotion of research in collaboration methods for recording, comparing, and manipulating sodium with the scientific community, member states, research dodecyl sulfate-polyacrylamide gel electrophoresis separa- institutions, and other international organizations. The ac- tions. The scan patterns can be compared with patterns of tivities were discussed under the following headings: (i) other known and unknown organisms which are held in a monitoring and surveillance of leptospirosis; (ii) develop- data bank. The short scan time allows rapid identification. ment of a world network of WHO collaborating centers and The system is very costly and not cost effective at this stage. related international reference institutions; (iii) international It was stated that it is too early to comment on the use of this collaborative research on leptospirosis; (iv) WHO meetings system as an epidemiological tool. A comparison of serovar and training courses and elaboration of guidelines for diag- hardjo bovis strains obtained from various areas of the world nosis and control of leptospirosis; and (v) perspectives of was described. international cooperation. Following the presentation a dis- Minute 12. Genetic characterization of pathogenic cussion was held on (i) vaccine production, (ii) assistance Leptospira species by DNA hybridization. R. Marshall pre- from the WHO in translating Chinese publications into sented a paper on genetic characterization of pathogenic English, (iii) designation of a reference laboratory in Africa, Leptospira species by DNA hybridization. He described a (iv) problems in diagnostic procedures because only a few quantitative slot blot hybridization method that he used to

330 VOL.42, 1992 MINUTES 331

determine the levels of DNA relatedness among 66 patho- alternative classification is considered, a system based on genic Leptospira serovars; in this study 16 strains were used phylogenetic relationships would be the most logical. Also, as sources of labeled reference DNA, and 57 of the serovars restriction endonuclease analysis is not very suitable for were placed into six DNA homology groups. The results classification. In the long term, investigation into the com- were in general agreement with the results of other workers position of DNA as a basis for determining phylogenetic who used different DNA homology methods. As a result of relationships might lead to a natural classification system. It differences, the new species Leptospira kirschneri was pro- was concluded that pending the possible development of a posed and into it were placed serovars cynopteri, grippoty- new classification system based on phylogenetic relation- phosa, kabura, tsaratsovo, butembo, kambale, ramisi, dania, ships, it is practical to maintain the system based on cross- and bulgarica. It was suggested that after serogroup Hard- agglutinin absorption results. However, data for new strains joprajitno strains are examined, the terms Hardjoprajitno should be complemented by restriction endonuclease analy- and Bovis should be used. A discussion was held, and it sis patterns. was agreed that confusion may occur if this change were Minute 16. Thermolabile antigens. E. Kmety commented made. on thermolabile antigens. He stated that sometimes these Minute 13. Leptospira biflexa: remarks on a very hetero- antigens are present and sometimes they are not. He also geneous species. M. Cinco presented a paper on Leptospira stated that the Vi antigen is present in the pathogenic biflexa, a very heterogeneous species. Using various meth- leptospires and is related to virulence. Thermolabile antigens ods, he described the genetic diversity of strains in the were discovered in 1972 and have been shown to be present Leptospira biflaa group and stated that the diversity in this in serogroups Icterohaemorrhagiae and Pomona. The fact species is greater than that in Leptospira interrogans. Of 65 that this finding has been neglected in the past has led to known saprophytic strains, 24 do not fit into groups. These discrepant results. It was stated that thermolabile antigens 24 strains are individual strains, and each represents one could be lost over the years. The presence of thermolabile serovar and one serogroup. It was stated that on the basis of antigens is not important taxonomically. At present we use a observations Leptospira biflexa is a very heterogeneous two-antigen system based on thermolabile antigens. Antisera taxon which contains strains that do not exhibit sufficient are prepared from whole antigen, which is a mixture of DNA relatedness to accept them as members of one species. thermolabile and thermostable antigens. It is possible that It would be more appropriate to use the term nonpathogenic the recommendation relating to thermostable antigens may leptospires instead of the binomial Leptospira biflaa. If we have to be changed. assume that a genetic criterion for taxonomy should be used, Minute 17. Hybridization. R. Marshall presented a paper further studies on the serovars will be needed, and individual on hybridization and its impact on classification. He outlined species names may be given to each genetic group. a method in which single-stranded DNA from one organism Minute 14. Present concept of serogroups and serovars. is used and stated that this method is only one method that H. Dikken presented a paper on the present concept of can be used for classification. This method is useful for serovars and serogroups. He stated that the concept of looking at taxonomic relationships and phylogenetic rela- serovars and serogroups has changed little in the last 30 tionships. Marshall also discussed the 70% threshold value years and that this concept has fulfilled a useful role. for species definition and stated that it has the best discrim- However, there are weaknesses in some procedures, and the inating power; however, a great deal of work will still be procedures should be standardized. These problems have required. The method is useful at the research level. been discussed at subcommittee meetings over many years. Minute 18. New technologies: interest in identification As a result of these discussions, the following recommenda- and taxonomy. P. Perolat presented a paper on rRNA gene tions have been introduced: (i) amendment of the definition restriction patterns that can be used for molecular typing of of serovar; (ii) standardization of typing methods (standard- Leptospira strains. He studied 74 strains, including 67 sero- ized preparation of rabbit immune serum, standardization of var reference strains and 7 isolates, and characterized them the microscopic agglutination test, and standardization of by their rRNA gene restriction patterns. In this study 50 the agglutinin absorption test), and (iii) rules for serovar patterns were observed. Perolat found that strains that recognition. A number of new techniques and their impact belonged to different genomic species always produced on classification were discussed. These items included different patterns. However, genomic species could be sub- monoclonal antibodies, restriction endonuclease analysis, divided on the basis of several different patterns. A total of and thermolabile antigens. Additional studies will be re- 43 serovars produced specific patterns, and it was found that quired before any possible change can be made in the some serovars could not be separated on the basis of their classification of leptospires. It was decided that the serolog- rRNA gene restriction patterns. Strains belonging to sero- ical methods which have been the basis of classification for vars icterohaemorrhagiae, copenhageni, lai, pyrogenes, and so long should continue to be used. However, more detailed jalna produced pattern 1; serovar birkini, mankarso, and characteristics within a serovar might have a practical im- wolffi strains produced pattern 4; strains belonging to sero- pact. var canicola, gem, hebdomadis, pomona, and hardjo (strain Minute 15. Typing from the perspective of a reference Hardjoprajitno) produced pattern 12; serovar valbuzzi and Zaboratoly. W. Terpstra presented a paper on typing from zanoni strains produced pattern 14; serovar jonsis, malaya, the perspective of a reference laboratory. In this paper, he and sumneri strains produced pattern 16; serovar arborea, defined classification as placing strains in a logical and ballum, castellonis, and kenya strains produced pattern 35; coherent order so that strains are grouped on the basis of and serovar borincana and shermani strains produced pat- common traits and so that identification characterizes a tern 43. Perolat stated that the data obtained provided the strain on the basis of traits which set it apart from other basis for a molecular typing system for the genus Leptospira. strains. He then discussed the advantages and disadvantages The method used for pulsed-field electrophoresis was also of the cross-agglutinin absorption test, factor analysis, described. In this method an electric field is applied alter- monoclonal antibodies, restriction endonuclease analysis, nately in two perpendicular directions. This method results and Southern blotting. Terpstra then suggested that when an in high resolution when restriction enzymes that are charac- 332 MINUTES Im. J. SYST.BACTERIOL. terized by rare cutting sites are used, and variations in the the Japanese isolation, but there are no records as far as pulse time allow optimal separation of all of the restricted Babudieri and Smith were aware of agglutinin absorption fragments. tests. The early assumption that these strains are identical Minute 19, Fatty acidprofiles: a chemotaxonomic key for appears to have been based solely on the results of cross- the classif cation of the . B. Cacciapuoti agglutination reactions. In 1966 at the subcommittee meeting presented a paper on fatty acid profiles, a chemotaxonomic in Moscow, it was agreed that strain RGA should be ac- characteristic that is used for classification of the family cepted as the neotype strain of the genus Leptospira since it Leptospiraceae. He examined the fatty acid profiles of 59 was believed that it was the oldest strain. A number of strains, representing 28 serovars of Leptospira interrogans, groups of workers have now investigated strains RGA and 29 serovars of Leptospira biJlexa, the single serovar of Ictero I No 1, and it has been shown by using a variety of Leptospira pawa, and the single serovar of the monospecific methods that these strains are different. It was proposed that genus Leptonema (Leptonema illini). The investigations a new serovar, serovar icterogenes, should be created, with were conducted by using gas-liquid chromatography of fatty strain RGA as the reference strain. The members of the acid methyl ester derivatives. The interstrain differences in subcommittee agreed that consideration of this matter the gas-liquid chromatography fatty acid methyl ester pro- should be postponed; it will be considered at a future files were quantified by performing a linear regression anal- meeting. ysis. This analysis allowed Cacciapuoti to compare the fatty Minute 24. Species designation. R. Yanagawa presented acid methyl ester profiles for pairs of strains by using a single a paper on species designation. He noted that International digital value, the correlation coefficient. From the results Code of Nomenclature of Rule 15 states that the obtained, it was shown that strains could be classified on a type of species is the designated strain; “in special cases, the chemotaxonomic phenotypic basis. place of the type strain may be taken by a description, Minute 20. Summary of diflerentiation within the species. preserved specimen, or an illustration.” In 1907 Stimson E. Kmety summarized the papers presented in the section on saw in a kidney section of a patient an organism which he differentiation within the species. He stated that consider- named Spirochaeta interrogans. It was later thought that the able progress has been made in the differentiation of lepto- patient had had Weil’s disease and that the correct name for spires. Knowledge has increased because of studies of the organism was Leptospira interrogans. In 1958 Alston genetic properties, monoclonal antibodies, and biochemical and Broom did not accept this and stated that the name properties. It was stated that the present serovar concept has Leptospira icterohaemorrhagiae should be retained. It was limitations and that the concept is attached to the rabbit stated that since Stimson did not isolate a Leptospira immune system. Within some serovars, there are groups of strain, the name Leptospira interrogans is not in accordance strains that differ in serological, genetic, and biochemical with Rule 15. For this reason, it was proposed that the properties. All of these markers have been shown to have species name Leptospira icterohaemorrhagiae should be limitations. It was also stated that all of these differentiating restored. markers are at the subserovar level. The practical impor- Minute 25. Replacement of strain RGA by strain Ictero tance of this was recognized; however, additional studies No 1 as the type strain of Leptospira interrogans. Y. will be needed. Kobayashi presented a paper on the replacement of strain Minute 21. Proposal for the replacement of strain RGA RGA by strain Ictero No 1 as the type strain of Leptospira Ljy strain Ictero No 1 as the reference strain of serovar interrogans. He stated that the members of the subcommit- icterohaemorrhagiae. R. Yanagawa presented a paper on tee discussed this change at the meeting held in Manchester the replacement of strain RGA by strain Ictero No 1 as the in 1986 and that it was agreed that there would be no change reference strain of serovar icterohaemorrhagiae. He stated because of insufficient documentation. In order to solve this that the members of the subcommittee discussed this change problem, 17 monoclonal antibodies were produced against at the meeting held in Manchester in 1986 and that it was strain Ictero No 1 and compared with strains belonging to agreed that there would be no change because of insufficient serogroup Icterohaemorrhagiae, including strain RGA, for documentation. This was followed by a description of the antigenic properties. The comparison tests were performed history of maintenance of the strain Ictero No 1culture since by using the microscopic agglutination test and leptospires it was isolated in May 1915. It was concluded that strain which had been treated with heat, formalin, or sodium azide. Ictero No 1 has been maintained without change and that The results of the tests showed that strain Ictero No 1 was since it is the oldest strain, it should be the reference strain clearly distinguished from strain RGA when an anti-Ictero of serovar icterohaemorrhagiae. S. Yamamoto supported No 1 monoclonal antibody (IMA 1) was used. It was also this proposal and discussed the early Japanese history of the found that the antigen for IMA 1was inactivated by incuba- strain and the early German work. He also pointed out tion at 56°C for 30 min. The antigens for 16 other monoclonal difficulties in translating early Japanese publications. antibodies (IMA 2 to IMA 17) were thermostable and were Minute 22. Expression of appreciation. R. Marshall ex- common to strains Ictero No 1and RGA. As a result of these pressed his appreciation for the excellent presentation by R. findings, it was proposed that strain Ictero No 1 (= ATCC Yanagawa and S. Yamamoto. 43782) should be designated the type strain of Leptospira Minute 23. Proposal to create a new serovar, serovar interrogans in place of strain RGA. Following a lengthy icterogenes, with strain RGA as the reference strain. R. discussion, the members of the subcommittee agreed by a Yanagawa presented a paper on the proposal to create a new majority vote that strain Ictero No 1 should replace strain serovar, serovar icterogenes, with strain RGA as the refer- RGA as the type strain of Leptospira interrogans. ence strain. He stated that in 1968 Babudieri and Smith Minute 26. Proposal of two reference strains (strains indicated that the exact relationship of the original strain No RGA and Ictero No 1)for serovar icterohaemorrhagiae. The 1 of Inada and Ido to any existing well-documented serovar subcommittee did not accept a proposal that there should be icterohaemorrhagiae serogroup strain is unknown. The first two reference strains (strains RGA and Ictero No 1) for European isolation of a pathogenic leptospire (strain RGA, serovar icterohaemorrhagiae. isolated by Uhlenhuth and Fromme) occurred shortly after Minute 27. Catalog of serovars. H. Dikken discussed VOL.42, 1992 MINUTES 333 additions and corrections to the catalog of serovars which that a working party should be set up to resolve problems. In was published in 1988. The subcommittee accepted the his review Faine (i) considered the substantial evidence that proposed variations. there are genetic groups in the genus Leptospira and related Minute 28. Appreciation expressed. A. Alexander ex- bacteria and recognized that studies of DNA and DNA pressed appreciation on behalf of the subcommittee for the relatedness have shown that there are a number of distinct extensive time and effort expended by H. Dikken and E. groups; (ii) recognized that according to the available evi- Kmety in the preparation of the catalog of serovars. dence, if currently used criteria are used, the genetic groups Minute 29. Serovars isolated in the People’s Republic of are sometimes less closely related to one another than is China. On behalf of Gao Ji-yuan et al. E. Kmety read a paper often accepted for separate genera in other groups of bacte- entitled The Survey on ClassiJicationof Leptospira interro- ria; and (iii) recognized that the genetic evidence has been gans in China. The following suggestions were made: (i) to generally supported by the results of studies of chemotax- maintain the accuracy and consistency of international ref- onomy, fatty acid profile data, lipopolysaccharide composi- erence strains, reference strains should not be replaced by tion data, serological data, and data on other phenotypic strains which are considered similar, and the Subcommittee characteristics and that there are potential conflicts with on the Taxonomy of Leptospira should regularly check some current phenotypic groupings. The members of the reference strains; (ii) monoclonal antibodies should be ex- subcommittee agreed to set up a working party to recom- changed between reference laboratories; (iii) collaboration mend a taxonomic grouping of leptospires and related bac- between the reference laboratory in the People’s Republic of teria based on genetic criteria, supported by phenotypic data China and other reference laboratories should be strength- when possible. The members of the subcommittee also ened; and (iv) other reference laboratories should be in- agreed to direct the working party to (i) consider the possi- volved in checking more than 30 new serovars which have bility that new genera may need to be created; (ii) conserve been isolated in the People’s Republic of China. current serovar names as specific names within genera Minute 30. Collections of reference strains. H. Dikken where possible and appropriate, in an attempt to reconcile presented a paper on the collections of reference strains in the current serological classification with genetic taxonomy; reference laboratories. He stated that it has been proposed (iii) recommend the status of strains within groups when that reference strains held in reference laboratories should possible, but relegate temporarily to incertae sedis status be investigated and that many laboratories had already sent strains for which evidence is inadequate or not available; (iv) information. This information will be tabulated at a later write a draft formal description of any new groups, including date. Many laboratories have mislabeled reference strains, relevant genetic criteria; and (v) recommend methods and and there is a problem as to how to arrange that all reference procedures for effective rapid genetic characterization of strains in all reference laboratories are the same. The project isolates that are useful for general Leptospira laboratories as is too large for one laboratory to check the strains held by well as for reference laboratories. In addition, the members other laboratories. All new strains should be sent to those of the subcommittee agreed that (i) the working party should laboratories which have a complete reference collection. It be composed of S. Faine (Convenor), the Chairman of the was suggested that if a reference laboratory is in doubt about Subcommittee on the Taxonomy of Leptospira (ex officio), a strain, it should ask for a new strain. All new serovars W. Ellis, P. Perolat, R. Marshall, M. Cinco, and Y. Yanagi- should have their restriction endonuclease patterns deter- hara and should have the power to co-opt; (ii) the working mined. A discussion was held on whether there is a need to party should confer by using correspondence and should have three major reference laboratories to service Europe, produce an interim report for the Subcommittee on the North America, Australia, and New Zealand or whether it Taxonomy of Leptospira within 12 months so that a draft may be necessary to have only one reference laboratory in resolution can be prepared for consideration by correspon- the world. dence or at any future meeting within 2 years; and (iii) the Minute 31. Use of monoclonal antibodies for the identifi- working party should recommend a research program and cation of Leptospira strains. H. Korver presented a paper on funding requirements for the most time- and cost-effective the use of monoclonal antibodies for the identification of resolution of outstanding problems. Leptospira strains. He discussed the monoclonal antibodies Minute 33. Changes in membership and oficers. R. which are available, how reaction patterns can be shown in Yanagawa was elected Chairman, and R. Marshall was different ways, and the practical aspect of the use of mono- elected Secretary. Y. V. Ananjina, B. Cacciapuoti, and Y. clonal antibodies. Not all strains can be identified by using Yanagihara were elected members. The lists of observers monoclonal antibodies. Of the 205 reference strains in the and collaborators were reviewed. G. Baranton, C. Bolin, P. collection examined, 122 could be identified to the serovar Perolat, T. Vinh, E. Volina, and M. Wiemers were elected level, 75 could be identified to the subgroup level, and eight observers. J. Hookey and D. Miller were elected collabora- could not be identified. Monoclonal antibodies can be sero- tors. S. Yamamoto was elected an honorary member. group, subserogroup, and serovar specific. Isolates can be Minute 34. Expression of appreciation. On behalf of the identified in peripheral laboratories. Panels of antibodies subcommittee, R. Yanagawa thanked the retiring Chairman, have been sent to 21 laboratories. New strains can be E. Kmety, for his work during the past 4 years and N. D. recognized by monoclonal antibodies because patterns can Stallman, who had served as Secretary for the preceding 12 be different. It was stated that there is a definite advantage in years. using monoclonal antibodies. It was suggested that the Minute 35. Current membership. The current member- following points should be discussed: (i) designation of ship of the subcommittee is as follows: R. Yanagawa standard monoclonal antibodies for rapid typing and for (Chairman), Hokkaido, Japan; R. Marshall (Secretary), checking the identification of strains; (ii) central administra- Palmerston North, New Zealand; Y. V. Ananjina, MOSCOW, tion; (iii) distribution of data; and (iv) distribution of mono- CIS; B. Cacciapuoti, Rome, Italy; M. Cinco, Trieste, Italy; clonal antibodies. H. Dikken, Amsterdam, The Netherlands; W. A. Ellis, Minute 32. Draft resolution. S. Faine gave the review Belfast, Northern Ireland; S. Faine, Melbourne, Victoria, described below at the subcommittee meeting and suggested Australia; K. Hovind-Hougen, Copenhangen, Denmark; 334 MINUTES INT. J. SYST.BACTERIOL.

R. C. Johnson, Minneapolis, Minn.; E. Kmety, Bratislava, Minute 36. Adjournment. The final session was closed by Czechoslovakia; N. D. Stallman, Brisbane, Queensland, the Chairman at 1700 on 15 September 1990. It was an- Australia; W. Terpstra, Amsterdam, The Netherlands; M. nounced that the next meeting would be held in Prague, Torten, Ness-Ziona, Israel; and Y. Yanagihara, Shizuoka- Czechoslovakia, in 1994. ken, Japan. R. Marshall, Secretury