J. Med. Microbiol. - Vol. 46 (1997), 173-181 0 1997 The Pathological Society of Great Britain and Ireland MOLECULAR DlAG NOS IS Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR P. D. BROWN and P. N. LEVETT" School of Clinical Medicine and Research, University of the West Indies, and * Leptospira laboratory, Barbados, West Indies Reference strains from 30 serovars representing seven species of Leptospira and 48 recent isolates from human patients, dogs and rats, were characterised by polymerase chain reaction-restriction endonuclease analysis (PCR-REA), arbitrarily primed PCR (AP-PCR) and low stringency PCR (LS-PCR). PCR-REA analysis yielded seven groups among 29 serovars of pathogenic Leptospira; the non-pathogenic L. bzjiexa serovar patoc was not amplified with the primer pairs studied. AP-PCR and LS-PCR fingerprinting resulted in 25 and 21 distinct profiles, respectively, among the 30 reference strains. The results of the three PCR-based techniques were highly concordant and were in general agreement with those from previous DNA studies, confirming the high level of polymorphism among Leptospira species and serovars, and supported the concept of the serovar as the basic taxonomic unit of leptospiral classification. Results of the PCR- based typing methods for 11 randomised leptospiral strains, 36 clinical isolates from human patients and dogs and 12 survey isolates from trapped rats agreed with those from serological identification. With one exception, isolates of the same serovar gave identical profiles irrespective of the source. AP-PCR and LS-PCR are simple to perform and interpret, and appear to be useful for characterising isolates of Leptospira spp. for diagnostic and epidemiological purposes. 3 Introduction health surveillance, as different serovars can exhibit different host specificities and may not be associated Leptospirosis is an ubiquitous zoonotic infection, the with a particular clinical form of infection [5, 61. spectrum of which ranges from subclinical infection to Serological detection of antibodies to individual the severe, multi-organ syndrome characterised by serogroups requires the use of the microscopic jaundice and renal failure and known as Weil's disease. agglutination test (MAT), the interpretation of which Pathogenic leptospires of the species Leptospira is complex [7]. Although the MAT detects antibodies interrogans sensu Zato were traditionally classified into against specific serovars, cross-reactions occur between 23 serogroups, each comprising several serologically related serovars and hence the results of the MAT can related serovars [ 1,2]. Currently, over 200 pathogenic be interpreted only as serogroup-specific (e.g., the serovars have been described [2]. Non-pathogenic serogroup Tcterohaemorrhagiae rather than the serovar serovars were similarly classified into a single species, copenhageni). In addition, the use of the MAT to L. bzjiexa. However, the phenotypic classification has diagnose infection requires paired sera to allow the been replaced by a genomic one in which 11 species detection of rising titres. Paradoxical and anamnestic are defined, based upon DNA relatedness [3,4]. responses complicate interpretation further. However, Pathogenic serovars are found within eight of these the test remains important because of the epidemio- genomic species of Leptospira. logical value of knowing presumptively which ser- ogroup is involved in an infection. The precise identification and classification of Leptos- pira spp. is important for epidemiological and public On the island of Barbados, about 97% of clinical leptospiral infections are caused by three serogroups of leptospires. Serogroups Autumnalis, Icterohaemor- Received 18 Jan 1996; revised version accepted 17 July rhagiae and Ballum account for 60%, 23% and 14% 1996. of infections, respectively [8]. Only four serovars have Corresponding author: Dr. P. N. Levett. been isolated in Barbados despite intensive investiga- 174 P. D. BROWN AND P. N. LEVETT tion for over 20 years: him (serogroup Autumnalis), an arbitrary primer at low stringency with the copenhageni (serogroup Icterohaemorrhagiae), arborea specificity of ‘diagnostic’ primers [23]. The potential (serogroup Ballum) and bajan (serogroup Australis) of the methods to identify pathogenic Leptospira [9]. The clinical syndrome caused by each of these isolates was also assessed with several randomised four serovars in Barbados is identical [9]. leptospiral strains. Several molecular techniques have been applied successfully to the subtyping of leptospiral strains Materials and methods [lo-161 but these are not ideally suited to diagnostic Leptospiral strains and culture conditions laboratories, being complex and time-consuming. Recently, a new procedure for rapid differentiation The 30 reference leptospiral serovars used in this study of micro-organisms known as random amplified (Table 1) included those which are detected in the polymorphic DNA (RAPD) or arbitrarily primed Caribbean region, together with serovars that are of polymerase chain reaction (AP-PCR), was described ubiquitous distribution [2]. Cultures were derived from [17, 181. This technique is based on the use of a the collection of the Barbados Leptospira Laboratory; single arbitrary primer in a PCR of low stringency to 11 of these were randomised, coded and investigated amplify segments of the genome. Highly diverse DNA blind by each method. Twenty-seven isolates from banding patterns can be generated, which may allow patients with acute leptospirosis in Barbados between discrimination between and within species [ 19-22]. July 1990 and October 1993, nine isolates from four The development of a PCR-based typing system for sick dogs that were euthanised between December 1992 Leptospira spp. would be a considerable advance and September 1993, and 12 from rats trapped between because of its requirement for very small quantities of September 1994 and April 1995 (Table 2) were also DNA and its simplicity. studied. Leptospires were maintained in semi-solid or liquid EMJH medium (Difco, USA), supplemented In this study, PCR followed by restriction endonu- with 5-fluorouracil [24, 251. clease analysis of the amplified target (PCR-REA), AP-PCR, and low stringency PCR (LS-PCR) with a DNA isolation and purijication pair of specific primers, were used to characterise Leptospira reference serovars and then to type Genomic DNA was isolated and purified from 7-day leptospires isolated from recent specimens. LS-PCR cultures of all Leptospira isolates by methods described was studied because it combined the principle of using previously [26], followed by treatment with RNAase A Table 1. Leptospira reference serovars used in this study Species Serogroup Serovar Strain L. interroguns Australis bratislava Jez Bratislava L. interroguns Bataviae butaviae Swart L. interroguns Canicola canicolu Hond Utrecht IV L. interrogans Canicola portlandvere MY 1039 L. interroguns Hebdomadis kremustos Kremastos L. interrogans Icterohaemorrhagiae copenhugeni M20 L. interroguns Icterohaemorrhagiae icterohaemorrhagiae RGA L. interroguns Louisiana lanka R 740 L. interrogans Pomona pomonu Pomona L. interroguns Pyrogenes pyrogenes Salinem L. interroguns Sejroe hardjo Hardjoprajitno L. borgpetersenii Ballum arborea Arborea L. borgpetersenii Ballum ballum Mus 127 L. borgpetersenii Sejroe sejroe M 84 L. borgpetersenii Tarassovi tarassovi Perepelitsin L. noguchii Autumnalis fortbrugg Fort Bragg L. noguchii Louisiana louisiana LSU 1945 L. noguchii Louisiana orleans LSU 2580 L. noguchii Panama panama CZ 214 L. sunturosui Bataviae brasiliensis An 776 L. santurosui Mini georgia LT 117 L. santurosui Sejroe trinidad TRVL 34056 L. sunturosai Sejroe caribe TRVL 61866 L. kirschneri Autumnalis bim 1051 L. kirschneri Cynopteri cynopteri 3522 C L. kirschneri Grippotyphosa grippotyphosa Moskva V L. bifexu Semaranga patoc Patoc I *L. inudai Panama mangus TRVL 137774 *L. noguchii Australis bajan Toad 60 *L. noguchii Australis barbadensis Toad 67 *New species designations (A.Kaufmann, personal communication). PCR-BASED DIFFERENTIATION OF LEPTUSPIRA 175 Table2. Summary of the Leptospira isolates used in this study Strain Source Serovar Strain Source Serovar P692 blood, human arboreaa D752b kidney, dog copenhageni RE06 kidney, rat arborea D756 kidney, dog copenhageni RE07 kidney, rat arborea P678 blood, human bimb RE09 kidney, rat arborea P687 blood, human bim RK02 kidney, rat arborea P710 blood, human bim RM02 kidney, rat arborea P714 blood, human bim P703 blood, human bajunc P715 blood, human bim P786 blood, human copenhagenid P72 1 urine, human bim P796 blood, human copen h agen i P739 blood, human bim P808 urine, human copenhageni P746 blood, human bim P824 blood, human copen hageni P747 blood, human him P895a aqueous humour, human copenhageni P788 urine, human bim P895b CSF, human copen hagen i P822 blood, human bim RE1 1 kidney, rat copen hageni P823 blood, human bim RF03 kidney, rat copenhageni P826 blood, human birn RGO 1 blood, rat copenhagen i P842 blood, human bim RHO6 kidney, rat copenhageni P843 urine, human bim RJO 1 kidney, rat copen h agen i P85 1 blood, human bim RJ08 kidney, rat copen hageni P877 urine, human bim RK04 kidney, rat copen hageni P883 blood, human bim D744 blood, dog copen hagen i P937 blood, human birn D745a blood, dog copen hagen i D762a cerebellum, dog birn D745b kidney, dog copen hagen i D762b medulla oblongata, dog bim D752a aqueous humour, dog copen