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Analysis of Leptospira isolates from mainland Portugal and the Azores islands
FEMS Microbiology Letters 185 (2000) 181^187
www.fems-microbiology.org
Analysis of Leptospira isolates from mainland Portugal and the Azores islands M. Collares-Pereira a
a;
*, H. Korver b , B.V. Cao Thi c , M. Santos-Reis d , E. Bellenger c , G. Baranton c , W.J. Terpstra b
Instituto de Higiene e Medicina Tropical (UNL), R. da Junqueira 96, 1349-008 Lisboa, Portugal b Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands c Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France d Centro de Biologia Ambiental (UL), Campo Grande, Edif|¨cio C2, 1700 Lisboa, Portugal
Received 16 October 1999; received in revised form 22 February 2000; accepted 25 February 2000
Abstract From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n = 71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n = 45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n = 32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n = 3), Leptospira kirschneri (n = 8) and Leptospira borgpetersenii (n = 21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Portugal; Taxonomy ; Serotyping; Genotyping; Leptospira
1. Introduction Leptospirosis, a disease with great impact on both human and veterinary public health, was ¢rst described in Portugal in 1931 [1]. However, our understanding of the circulating serovars is still scarce being necessary to have a clear picture about the occurrence of at least the most important and most common strains. To obtain this information, we have isolated and examined Leptospira strains from mammals in various areas of the country [2,3]. There are various antigenic and genetic methods for the characterization and identi¢cation of leptospires. The few strains that had been isolated in the past in Portugal were originally classi¢ed on the basis of their antigenic traits following the conventional serotyping methods, i.e. the microscopic agglutination test and the cross-agglutinin absorption test (CAAT) with rabbit antisera [4]. More re-
* Corresponding author. Tel. : +351 (21) 365-2600; Fax: +351 (21) 363-2105; E-mail : [email protected]
cently, the Portuguese strains of hardjo [2] and mozdok [5] were identi¢ed by restriction endonuclease analysis (REA) and mouse monoclonal antibodies (MAbs), respectively. In fact, the serovar concept according to the elaborate CAAT is not fully satisfactory as it does not always concur with the genetic classi¢cation originally proposed on the basis of total DNA/DNA hybridization studies [6]. Strains within one serovar may belong to di¡erent genospecies, for instance types Hardjobovis and Hardjoprajitno within serovar hardjo are respectively assigned to Leptospira borgpetersenii and Leptospira interrogans [7]. MAbs are used routinely as a simple and quick method for typing common isolates, sometimes even on the (sub)serovar level, i.e. strain or type [8], once suitable hybridoma secreting antibodies of the desired speci¢city have been prepared and MAbs are available [9,10]. The CAAT is used mainly in case of new strains representing new serovars or in case of problematic isolates. In addition to antigenic analysis by MAbs, genetic typing methods have recently proved to be valuable. They may serve as supplementary or alternative typing systems, particularly in epidemiological studies. They can identify strain di¡erences either at the (sub)ser-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 1 0 0 - 2
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ovar level, namely with pulsed-¢eld agarose gel electrophoresis (PFGE) [11,12], or at both the species and subspecies levels with the arbitrarily primed polymerase chain reaction (AP-PCR) and mapped restriction site polymorphisms (MRSPs) in PCR-ampli¢ed rrs and rrl eubacterial ribosomal genes [13]. However, despite the good sensitivity and reproducibility of these genetic tools, further studies are required before their usefulness can be evaluated on a routine basis, considering they still fail to di¡erentiate between important serovars such as icterohaemorrhagiae and copenhageni that are genetically and epidemiologically very similar but antigenically diverse [12]. In conclusion, it is important to compare MAbs, as an accurate and simple serological method for antigenic analysis in epidemiological studies to these new genetic techniques which also give quick results but need special equipment, trying to assess their respective applicabilities. The present work characterizes recent Portuguese Leptospira isolates and compares the results obtained with MAbs analysis and with PFGE, MRSPs in PCR-ampli¢ed rRNA genes and AP-PCR for DNA analysis. 2. Materials and methods A total of 228 recent Leptospira strains were characterized : 175 were typed as pathogenic L. interrogans sensu lato strains and 53 as Leptospira bi£exa sensu lato, the saprophytic species based on PCR with speci¢c primers. The majority of the pathogenic strains (n = 149) were analyzed for serovar identi¢cation with MAbs and a total of 71 strains (including 13 saprophytes) were randomly selected for the genetic comparison through DNA restriction (NotI) analysis by PFGE. Moreover, 45 of these 71 strains, i.e. 32 pathogenic isolates and the 13 saprophytes, were also analyzed through MRSPs of PCR-ampli¢ed 16S rrs genes and AP-PCR. 2.1. Bacterial strains and agglutinations The Leptospira strains were isolated from the kidneys of 725 wild mammals captured between 1993 and 1995 in di¡erent geographical areas, in mainland Portugal, Coim-
bra (n = 212) and Pancas (n = 230), and the islands of Azores (n = 283), mostly, from Terceira and Sa¬o Miguel (Table 1). Isolates were sub-cultured once in 5 ml semisolid EMJH (Difco) prior to the EMJH liquid medium [14,15] to get a density suitable for use in agglutination reactions with 23 standard antisera (group sera) for the ¢rst typing according to serogroup a¤nities [4]. Agglutinations were carried out by mixing equal quantities of a well grown culture (2U108 cells ml31 ) and serial dilutions of reference antisera produced in rabbits. The agglutination was read by dark-¢eld microscopy. Those strains with no agglutination were submitted to other phenotypic tests, namely, the growth at 13³C [15] and the resistance to 8azaguanine [16], and ¢nally to PCR detection of signatures from both saprophytic and pathogenic leptospires on rrs (16rRNA) genes (data not shown) to con¢rm a saprophytic nature [17]. Pathogenic strains were characterized by MAbs at serovar level and in order to compare this antigenic information with the information from new genetic techniques, a limited number of isolates was further analyzed through PFGE, MRSPs in PCR-ampli¢ed rRNA 16S genes and AP-PCR ¢ngerprinting. 2.2. Production of MAbs BALB/c mice were immunized against Icterohaemorrhagiae, Pomona and Ballum serogroup strains : serovars copenhageni strain Wijnberg, mankarso strain Mankarso, copenhageni strain M20, ndambari strain Ndambari, pomona strain Pomona, proechimys strain 1161 U, tropica strain CZ 299, mozdok strain 5621 (two fusions) and castellonis strain Castellon 3. MAbs were produced according to standard techniques [9]. For the characterization of Icterohaemorrhagiae group serovars copenhageni and icterohaemorrhagiae, a panel of six MAbs was used [18]; regarding the antigenic analysis of Pomona and Ballum group strains, another seven [10] and four [3] MAbs were respectively selected. 2.3. DNA restriction analysis by PFGE DNA of in situ lysed cells was cleaved with NotI, as
Table 1 Leptospira ¢eld isolates characterized in the present study (A = Azores; C = Coimbra; P = Pancas) Leptospira speciesa (number studied)
Serogroup
Mammal species
Antigenic analysis (n = 228)
DNA analysis (n = 71)
L. interrogans (n = 175)
Ballum
Apodemus sylvaticus; Arvicola sapidus; Mus musculus ; Mus spretus; Rattus norvegicus ; Rattus rattus Erinaceus europaeus ; Rattus norvegicus; Rattus rattus Crocidura russula; Mus spretus; Rattus norvegicus; Rattus rattus Apodemus sylvaticus; Crocidura russula; Mus spretus; Mustela nivalis; Rattus norvegicus
A = 63, C = 13, P = 38 A = 16, C = 3 C = 27, P = 15
A = 12, C = 5, P = 18 A=3 C = 10, P = 10
C = 52, P = 1
C = 13
Icterohaemorrhagiae Pomona L. bi£exa (n = 53) a
Not determined
As de¢ned by Dikken and Kmety [4].
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described [10,11,19] and analyzed by PFGE using a Pulsaphor apparatus (Pharmacia), with a hexagonal electrode. DNA from approximately 1.5^20U108 cells was loaded per lane. A program combining di¡erent pulse times (25^100 s) was respectively used for the separation of the medium-sized fragments (70^400 kb) and the largest NotI fragments, on one gel each time. The sizes of the DNA fragments were estimated by comparison of band mobilities with concatemerized NM1149 bacteriophage DNA (48.5-kb genome size). 2.4. MRSPs of PCR-ampli¢ed rrs genes MRSPs of PCR-ampli¢ed rrs genes analysis was performed according to Ralph et al. [13]. Primers used for rrs gene were : 16S-11, 5P-GGC TGC AGT CGA CGT TTG ATC CTG GCT CAG-3P, 16S-1507, 5P-CCA GAT CTG AGC TCA AGG AGG TGA TCC AGC-3P. PCR was performed with approximately 5 ng genomic DNA in the presence of [32 P]dCTP. Restriction enzyme digestions were performed with DdeI, HhaI, NlaIV and TaqI. Three Wl of each restriction digest was electrophoresed on a nondenaturing acrylamide gel. As in the AP-PCR (next described), the gel was exposed for 24^72 h on Kodak XOmat X-ray ¢lm and reference strains of Leptospira spp. were included as internal controls. 2.5. AP-PCR AP-PCR ¢ngerprints were performed according to Ralph et al. [13]. Only the most informative primers
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for Leptospira as identi¢ed from previous studies [7,13] were used : KF (5P-CAC GCA CAC GCA CAG AGA3P), KG (5P-CAC ACG CAC ACG GAA GAA-3P), RSP (5P-GGA AAC AGC TAT GAC CAT GA-3P). 32 P-labeled [33 P]dCTP ampli¢cation products were loaded onto a 4% acrylamide^50% urea sequencing gel. 3. Results 3.1. MAbs After a ¢rst typing according to serogroup a¤nities (Icterohaemorrhagiae, Pomona and Ballum), a panel of 17 MAbs was able to assign all Portuguese isolates to the serovar level (Table 2). The agglutination pro¢les within Icterohaemorrhagiae serogroup showed serovar copenhageni among mainland isolates from Rattus norvegicus and among Azores isolates from the genera Erinaceus, Mus and Rattus, while serovar icterohaemorrhagiae was only identi¢ed among Azores isolates from the two Rattus species (not shown). Serovar mozdok in the Pomona group was only identi¢ed among mainland isolates and MAbs showed consistently three types of patterns which allowed the separation of isolated mozdok strains into type 1 (n = 27), type 2 (n = 10) and type 3 (n = 4) (Table 2). All isolates obtained from Crocidura russula were identi¢ed as type 3 according to `serovar' tsaratsovo reclassi¢cation as discussed later in this paper. The other mozdok types were only isolated from the genera Mus and Rattus (data not shown).
Table 2 Reciprocals of agglutination titers of MAbs from serogroups Icterohaemorrhagiae, Pomona and Ballum with their serovars/reference strains and 149 Portuguese ¢eld isolates (by origin and number) MAbs
Serogroup Icterohaemorrhagiae copenhageni/M20 [4] Azores (9); Coimbra (3) icterohaemorrhagiae/RGA [4] Azores (3)
Serogroup Pomona mozdok/5621 [4] Coimbra (20); Pancas (7) (type 1) Coimbra (5); Pancas (5) (type 2) Coimbra (1); Pancas (3) (type 3)
F12C3 [18]
F52C1 [18]
F70C7 [18]
F70C14 [18]
F70C24 [18]
F89C12 [18]
20 480 20 480^40 960 10 240 10 240^20 480
5 120 1 280^5 120 1 280 640^2 560
81 920 40 960^81 920 40 960 40 960^81 920
80 negative 40 960 40 960
40 960 20 480^40 960 160 40^320
2 560 640^5 120 negative negative
F43C9 [10]
F46C2 [10]
F46C4 [10]
F46C9 [10]
F48C6 [10]
F58C1 [10]
F61C7 [10]
20 80 negative to 640 80^2 560 40^1 280 negative 20^320 20 480^40 960
1 280 2 560^40 960 negative negative
1 280 2 560^20 480 1 280^40 960 10 240^40 960
2 560 640^5 120 640^5 120 320^1 280
40 960 20 480^81 920 1 280^40 960 10 240^40 960
5 120 5 120^40 960 2 560^40 960 20 480^81 920
F74C1 [3]
F74C4 [3]
F74C7 [3]
F74C12 [3]
negative negative
20 negative
20 negative
5 120 10 240
1 280 5 120
2 560 10 240
Serogroup Ballum arborea/Arborea [4] 5 120 Azores (48); Coimbra (13); Pancas 1 280^5 120 (31) ballum/Ballum [4] 10 240 Pancas (1) 2 560
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The agglutination Ballum pro¢les (Table 2) showed a clear predominance of serovar arborea among the mainland rodent isolates, with the only evidence of serovar ballum in just one Mus spretus isolate from Pancas. The Ballum pro¢les among the Azores isolates from Mus musculus and Rattus rattus were all characterized as serovar arborea. 3.2. PFGE By PFGE (Table 3), the three Icterohaemorrhagiae isolates from the Azores showed to belong to this serogroup since they shared common fragments with icterohaemorrhagiae (or copenhageni) serovars. Isolates A37 and A46 shared a common pattern close to icterohaemorrhagiae (Fig. 1) while A02 (Fig. 1) had three additional fragments and di¡ered from the patterns of every serovar in Icterohaemorrhagiae serogroup [11]. Most Pomona isolates presented a pro¢le similar to that of `serovar' tsaratsovo (Fig. 2 and data not shown). The remaining exhibited either a pro¢le quite similar to mozdok (e.g. Fig. 2) or a tsaratsovo-like pro¢le where only one or two small fragments di¡ered (e.g. Fig. 2 and data not shown). All but one of the studied Ballum isolates exhibited a characteristic common pattern similar to the arborea reference one (Fig. 1 and data not shown). They clearly differed from the castellonis pattern by a supplementary frag-
Fig. 2. PFGE of NotI restriction fragments from serogroup Pomona isolates. The digestion products were separated at 200 V for 24 h in 1% agarose^0.5UTris^borate^EDTA. The three pulse times were 25 s over 10 h, 50 s over 8 h and 100 s over 6 h. Lanes: 2^8, Pomona isolates ; 9, serovar tsaratsovo ; 10, Pomona isolate; 1 and 11, size markers (lambda, 48.5-kb genome size).
ment at 200 kb and from serovar ballum which exhibited a speci¢c fragment at 230 kb. 3.3. MRSPs of PCR-ampli¢ed rrs genes
Fig. 1. PFGE of NotI restriction fragments from serogroups Ballum and Icterohaemorrhagiae Azorean isolates. The digestion products were separated at 200 V for 24 h in 1% agarose^0.5UTris^borate^EDTA. The two pulse times were 25 s and 100 s over 24 h each. Lanes: 1, serovar castellonis ; 2^6, Ballum isolates ; 7, serovar arborea; 8^10, Icterohaemorrhagiae isolates (size markers, kb).
MRSP allowed a quick identi¢cation of the pathogenic and saprophytic Portuguese isolates at the species level. As demonstrated previously [7,13], the primers used in this study ampli¢ed a conserved portion of the rrs (from bases 11 to 1507). PCR ampli¢cation of rrs gene contained one product of the expected size (1.5 kb) for all tested strains (data not shown). An additional product of 260 bp was found for all strains from L. borgpetersenii genotype as described earlier [13]. Restriction digestions performed with DdeI, HhaI, NlaIV and TaqI allowed the assignment at the species level of the pathogenic isolates. Among the mainland strains, two genospecies were identi¢ed : L. borgpetersenii, including the nine isolates from serogroup Ballum, and Leptospira kirschneri, with all those from Pomona serogroup. The Azores isolates belonged also to either of two genospecies : L. interrogans, which included the three isolates from serogroup Icterohaemorrhagiae, and L. borgpetersenii, with the 12 Ballum group isolates.
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Table 3 Serovar identi¢cation results with MAbs, PFGE and AP-PCR of 32 pathogenic ¢eld isolates (A = Azores ; C = Coimbra ; P = Pancas) Serogroup of L. interrogans (genomic species) Icterohaemorrhagiae (L. interrogans) Pomona (L. kirschneri)
Ballum (L. borgpetersenii)
Isolate A02 A37 A46 C138 C149 P110 P112 P116 P122 P126 P159 A03 A07 A09 A11 A23 A30 A31 A32 A33 A36 A40 A44 P87 P90 P115 P124 P127 P135 P150 P167 P189
Mammal species Rattus norvegicus Rattus rattus Erinaceus europaeus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Crocidura russula Mus musculus Mus musculus Mus musculus Mus musculus Rattus rattus Mus musculus Mus musculus Mus musculus Mus musculus Rattus rattus Rattus rattus Rattus rattus Mus musculus Rattus norvegicus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Rattus norvegicus Mus spretus
MAbs a
ictero ictero copb moz 1c moz 1 moz 1 moz 1 moz 1 moz 1 moz 1 moz 3 arbe arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb
PFGE
AP-PCR
ictero/cop ictero/cop ictero/cop tsard tsar-like tsar tsar tsar tsar tsar moz arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb
ictero ictero ictero tsar tsar tsar tsar tsar tsar tsar moz arb/balf arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal
a
ictero = icterohaemorrhagiae. cop = copenhageni. c moz 1 = mozdok `type 1'. d tsar = tsaratsovo. e arb = arborea. f bal = ballum. b
3.4. AP-PCR AP-PCR leptospiral ¢ngerprints were obtained with the four primers tested. The level of complexity of the patterns was variable, depending on the primer used. The patterns were simple using primer SP and relatively complex with primers KF, KG and RSP. In each case, as previously reported [7,13], prominent species speci¢c products (not shared with the other leptospiral species) were observed and con¢rmed the genomic species assignments supported by MRSP data. AP-PCR ¢ngerprints of both mainland and Azores isolates were homogenous inside each identi¢ed genospecies (Table 3). The three L. interrogans isolates presented the characteristic AP-PCR ¢ngerprints from serovar icterohaemorrhagiae strains (data not shown). Fingerprints of L. borgpetersenii isolates were close to those of both reference strains from serovar arboreae and ballum which present similar AP-PCR ¢ngerprints whatever primer was used.
Lastly, eight of the nine L. kirschneri strains were identi¢ed as `serovar' tsaratsovo except strain P159. This isolate looked closer to mozdok, with primers KF and KG (data not shown). 4. Discussion Present results emphasize a perfect agreement for species classi¢cation with MRSP rrs genes and AP-PCR, but a correlation was not always evident at the serovar level for MAbs on the one hand and PFGE and AP-PCR on the other hand. This is due to the great intra-species heterogeneity of the strains and to the di¡erent discrimination power of the techniques used. The practical value of MRSP rrs genes and AP-PCR as quick identi¢cation methods at the species level is stressed. On the other hand, the use of MAbs con¢rmed to be generally a powerful method to identify Leptospira organ-
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isms at the serovar level. Comparing MAbs, PFGE and AP-PCR ¢ndings, a good correlation at the serovar level within the Ballum serogroup was only observed between the ¢rst two methods, where the same common arborea pattern was seen in all the isolates; AP-PCR did not discriminate between arborea and ballum (Table 3). The PFGE of NotI restriction fragments showed a supplementary 200-kb fragment speci¢c for serovar castellonis and the absence of a speci¢c 230-kb fragment for serovar ballum, which con¢rmed serovar arborea as the most likely identi¢cation. However, there was no complete agreement between the Pomona and the Icterohaemorrhagiae agglutination pro¢les at the serovar level and the PFGE digestion ¢ngerprints of their speci¢c isolates. Three types of mozdok within the Pomona group were identi¢ed by MAbs while PFGE suggested the presence of three di¡erent ¢ngerprint patterns. Those that were typed as mozdok type 1 by MAbs were generally typed as tsaratsovo by genetic methods while the reference strain B81/7 of tsaratsovo was antigenically typed as mozdok type 3 (data not shown). Tsaratsovo actually belongs to serovar mozdok according to antigenic (CAAT) and genetic (REA) analysis [13] and the designation of tsaratsovo as a separate serovar should be abandoned. Interestingly, although there was not an equal designation of the di¡erent types and patterns within the Pomona ¢eld isolates, all the tests used were at least in agreement considering the mozdok identity of the isolates grown from C. russula. This result con¢rms the already observed antigenic and genetic di¡erentiation according to the host speci¢city of certain Pomona ¢eld strains [20]. In addition, PFGE was not able to discriminate between serovars copenhageni and icterohaemorrhagiae as previously reported [12]. However, PFGE showed in this case, through repeated observations, a pattern diversity within the serovar icterohaemorrhagiae, unless A02 represents a new serovar or genotype. This diversity has not been reported so far. The saprophytic strains which were analyzed by MRSP rrs genes, AP-PCR and PFGE were not related with any pathogenic species and shared a common pro¢le, identical to the one from L. bi£exa reference strain. Considering these strains were unusually isolated from the kidneys of trapped animals, once disregarded the possibility of contamination due to non-sterile isolation procedures, their particular existence is probably the result of a prolonged exposure to fresh water in £ooded areas. In conclusion, this study shows that phenotypic and genotypic methods mostly lead to similar results albeit varying degrees of detail and applicability. Due to this agreement, the use of a selected panel of MAbs of the already known serovars or types is advisable in cost^bene¢t terms, for situations of a limited diversity of local isolates, such as in Portugal. However, to solve the possible existence of non-reactive isolates corresponding to new strains as well as the problem of mixed lepto populations
(isolates are not usually cloned by limited dilution), the use of AP-PCR ¢ngerprints would be important in heterogenic populations: using much less DNA than PFGE, this method is a de¢nite improvement for species identi¢cation, also providing a simple and rapid tool to intra-species comparison. So, for epidemiological studies in leptospirosis, the use of di¡erent but complementary methods is always preferable. Acknowledgements Field work was possible thanks to the contribution of the other researchers of the team: M.L. Mathias, M.G. Ramalhinho, M.M. Oom, F. Petrucci-Fonseca, R. Fons and R. Libois, with the assistance of J.P. Clara. We are also grateful to both L. Floªr (Laborato¨rio de Sanidade Animal) and J.E. Matos (Universidade dos Ac°ores) for the cooperation and logistic facilities in the islands, and to M.J. Gama (IHMT, Lisboa) for her laboratorial assistance. Funding was provided through the national projects PBIC/C/SAU/1535/92 and STRDA/C/CEN/447/92, and complemented by Secretaria Regional da Sau¨de e Seguranc°a Social and Secretaria Regional da Agricultura e Pescas (Azores). We also thank Centro de Mala¨ria e Outras Doenc°as Tropicais (IHMT, UNL) for providing technical support.
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spira interrogans with monoclonal antibodies. Zent.bl. Bakteriol. Mikrobiol. Hyg. A 259, 498^506. Terpstra, W.J., Korver, H., Schoone, G.J., Leeuwen, J.V., Schonemann, C.E., De Jonge-Aglibut, S. and Kolk, A.H.J. (1987) Comparative classi¢cation of Leptospira serovars of the Pomona group by monoclonal antibodies and restriction-endonuclease analysis. Zent.bl. Bakteriol. Mikrobiol. Hyg. A 266, 412^421. Herrmann, J.L., Baril, C., Bellenger, E., Pe¨rolat, P., Baranton, G. and Saint-Girons, I. (1991) Genome conservation in isolates of Leptospira interrogans. J. Bacteriol. 173, 7582^7588. Herrmann, J.L., Bellenger, E., Pe¨rolat, P., Baranton, G. and SaintGirons, I. (1992) Pulsed-¢eld gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identi¢cation. J. Clin. Microbiol. 30, 1696^1702. Ralph, D., McClelland, M., Welsh, J., Baranton, G. and Pe¨rolat, P. (1993) Pathogenic Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-ampli¢ed rRNA genes. J. Bacteriol. 175, 973^981. Ellinghausen, H.C. and McCullough, W.G. (1965) Nutrition of Leptospira pomona and growth of 13 other serotypes : fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am. J. Vet. Res. 26, 45^51.
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[15] Johnson, R.C. and Harris, V.G. (1967) Di¡erentiation of pathogenic and saprophytic leptospires. Growth at low temperatures. J. Bacteriol. 94, 27^31. [16] Johnson, R.C. and Rogers, P. (1964) Di¡erentiation of pathogenic and saprophytic leptospires with 8-azaguanine. J. Bacteriol. 88, 1618^ 1623. [17] Murgia, R., Riquelme, N., Baranton, G. and Cinco, M. (1997) Oligonucleotides speci¢c for saprophytic and pathogenic Leptospira occuring in water. FEMS Microbiol. Lett. 148, 27^34. [18] Korver, H., Kolk, A.H.J., Vingerhoed, J., Leeuwen, J.V. and Terpstra, W.J. (1988) Classi¢cation of serovars of the Icterohaemorrhagiae serogroup by monoclonal antibodies. Isr. J. Vet. Med. 44, 15^ 18. [19] Herrmann, J.L., Bakoss, P., Korver, H., Bulu, A.A., Bellenger, E., Terpstra, W., Saint-Girons, I. and Baranton, G. (1994) A new serovar in the Grippotyphosa serogroup comprising leptospiral isolates from di¡erent regions. Int. J. Syst. Bacteriol. 44, 362^364. [20] Hathaway, S.C., Marshall, R.B., Little, T.W.A., Headlam, S.A. and Winter, P.J. (1985) Di¡erentiation of reference strains of leptospires of the Pomona serogroup by cross-agglutination absorption and restriction endonuclease analysis. Res. Vet. Sci. 39, 145^150.
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Analysis of Leptospira isolates from mainland Portugal and the Azores islands M. Collares-Pereira a
a;
*, H. Korver b , B.V. Cao Thi c , M. Santos-Reis d , E. Bellenger c , G. Baranton c , W.J. Terpstra b
Instituto de Higiene e Medicina Tropical (UNL), R. da Junqueira 96, 1349-008 Lisboa, Portugal b Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands c Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France d Centro de Biologia Ambiental (UL), Campo Grande, Edif|¨cio C2, 1700 Lisboa, Portugal
Received 16 October 1999; received in revised form 22 February 2000; accepted 25 February 2000
Abstract From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n = 71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n = 45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n = 32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n = 3), Leptospira kirschneri (n = 8) and Leptospira borgpetersenii (n = 21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Portugal; Taxonomy ; Serotyping; Genotyping; Leptospira
1. Introduction Leptospirosis, a disease with great impact on both human and veterinary public health, was ¢rst described in Portugal in 1931 [1]. However, our understanding of the circulating serovars is still scarce being necessary to have a clear picture about the occurrence of at least the most important and most common strains. To obtain this information, we have isolated and examined Leptospira strains from mammals in various areas of the country [2,3]. There are various antigenic and genetic methods for the characterization and identi¢cation of leptospires. The few strains that had been isolated in the past in Portugal were originally classi¢ed on the basis of their antigenic traits following the conventional serotyping methods, i.e. the microscopic agglutination test and the cross-agglutinin absorption test (CAAT) with rabbit antisera [4]. More re-
* Corresponding author. Tel. : +351 (21) 365-2600; Fax: +351 (21) 363-2105; E-mail : [email protected]
cently, the Portuguese strains of hardjo [2] and mozdok [5] were identi¢ed by restriction endonuclease analysis (REA) and mouse monoclonal antibodies (MAbs), respectively. In fact, the serovar concept according to the elaborate CAAT is not fully satisfactory as it does not always concur with the genetic classi¢cation originally proposed on the basis of total DNA/DNA hybridization studies [6]. Strains within one serovar may belong to di¡erent genospecies, for instance types Hardjobovis and Hardjoprajitno within serovar hardjo are respectively assigned to Leptospira borgpetersenii and Leptospira interrogans [7]. MAbs are used routinely as a simple and quick method for typing common isolates, sometimes even on the (sub)serovar level, i.e. strain or type [8], once suitable hybridoma secreting antibodies of the desired speci¢city have been prepared and MAbs are available [9,10]. The CAAT is used mainly in case of new strains representing new serovars or in case of problematic isolates. In addition to antigenic analysis by MAbs, genetic typing methods have recently proved to be valuable. They may serve as supplementary or alternative typing systems, particularly in epidemiological studies. They can identify strain di¡erences either at the (sub)ser-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 1 0 0 - 2
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ovar level, namely with pulsed-¢eld agarose gel electrophoresis (PFGE) [11,12], or at both the species and subspecies levels with the arbitrarily primed polymerase chain reaction (AP-PCR) and mapped restriction site polymorphisms (MRSPs) in PCR-ampli¢ed rrs and rrl eubacterial ribosomal genes [13]. However, despite the good sensitivity and reproducibility of these genetic tools, further studies are required before their usefulness can be evaluated on a routine basis, considering they still fail to di¡erentiate between important serovars such as icterohaemorrhagiae and copenhageni that are genetically and epidemiologically very similar but antigenically diverse [12]. In conclusion, it is important to compare MAbs, as an accurate and simple serological method for antigenic analysis in epidemiological studies to these new genetic techniques which also give quick results but need special equipment, trying to assess their respective applicabilities. The present work characterizes recent Portuguese Leptospira isolates and compares the results obtained with MAbs analysis and with PFGE, MRSPs in PCR-ampli¢ed rRNA genes and AP-PCR for DNA analysis. 2. Materials and methods A total of 228 recent Leptospira strains were characterized : 175 were typed as pathogenic L. interrogans sensu lato strains and 53 as Leptospira bi£exa sensu lato, the saprophytic species based on PCR with speci¢c primers. The majority of the pathogenic strains (n = 149) were analyzed for serovar identi¢cation with MAbs and a total of 71 strains (including 13 saprophytes) were randomly selected for the genetic comparison through DNA restriction (NotI) analysis by PFGE. Moreover, 45 of these 71 strains, i.e. 32 pathogenic isolates and the 13 saprophytes, were also analyzed through MRSPs of PCR-ampli¢ed 16S rrs genes and AP-PCR. 2.1. Bacterial strains and agglutinations The Leptospira strains were isolated from the kidneys of 725 wild mammals captured between 1993 and 1995 in di¡erent geographical areas, in mainland Portugal, Coim-
bra (n = 212) and Pancas (n = 230), and the islands of Azores (n = 283), mostly, from Terceira and Sa¬o Miguel (Table 1). Isolates were sub-cultured once in 5 ml semisolid EMJH (Difco) prior to the EMJH liquid medium [14,15] to get a density suitable for use in agglutination reactions with 23 standard antisera (group sera) for the ¢rst typing according to serogroup a¤nities [4]. Agglutinations were carried out by mixing equal quantities of a well grown culture (2U108 cells ml31 ) and serial dilutions of reference antisera produced in rabbits. The agglutination was read by dark-¢eld microscopy. Those strains with no agglutination were submitted to other phenotypic tests, namely, the growth at 13³C [15] and the resistance to 8azaguanine [16], and ¢nally to PCR detection of signatures from both saprophytic and pathogenic leptospires on rrs (16rRNA) genes (data not shown) to con¢rm a saprophytic nature [17]. Pathogenic strains were characterized by MAbs at serovar level and in order to compare this antigenic information with the information from new genetic techniques, a limited number of isolates was further analyzed through PFGE, MRSPs in PCR-ampli¢ed rRNA 16S genes and AP-PCR ¢ngerprinting. 2.2. Production of MAbs BALB/c mice were immunized against Icterohaemorrhagiae, Pomona and Ballum serogroup strains : serovars copenhageni strain Wijnberg, mankarso strain Mankarso, copenhageni strain M20, ndambari strain Ndambari, pomona strain Pomona, proechimys strain 1161 U, tropica strain CZ 299, mozdok strain 5621 (two fusions) and castellonis strain Castellon 3. MAbs were produced according to standard techniques [9]. For the characterization of Icterohaemorrhagiae group serovars copenhageni and icterohaemorrhagiae, a panel of six MAbs was used [18]; regarding the antigenic analysis of Pomona and Ballum group strains, another seven [10] and four [3] MAbs were respectively selected. 2.3. DNA restriction analysis by PFGE DNA of in situ lysed cells was cleaved with NotI, as
Table 1 Leptospira ¢eld isolates characterized in the present study (A = Azores; C = Coimbra; P = Pancas) Leptospira speciesa (number studied)
Serogroup
Mammal species
Antigenic analysis (n = 228)
DNA analysis (n = 71)
L. interrogans (n = 175)
Ballum
Apodemus sylvaticus; Arvicola sapidus; Mus musculus ; Mus spretus; Rattus norvegicus ; Rattus rattus Erinaceus europaeus ; Rattus norvegicus; Rattus rattus Crocidura russula; Mus spretus; Rattus norvegicus; Rattus rattus Apodemus sylvaticus; Crocidura russula; Mus spretus; Mustela nivalis; Rattus norvegicus
A = 63, C = 13, P = 38 A = 16, C = 3 C = 27, P = 15
A = 12, C = 5, P = 18 A=3 C = 10, P = 10
C = 52, P = 1
C = 13
Icterohaemorrhagiae Pomona L. bi£exa (n = 53) a
Not determined
As de¢ned by Dikken and Kmety [4].
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described [10,11,19] and analyzed by PFGE using a Pulsaphor apparatus (Pharmacia), with a hexagonal electrode. DNA from approximately 1.5^20U108 cells was loaded per lane. A program combining di¡erent pulse times (25^100 s) was respectively used for the separation of the medium-sized fragments (70^400 kb) and the largest NotI fragments, on one gel each time. The sizes of the DNA fragments were estimated by comparison of band mobilities with concatemerized NM1149 bacteriophage DNA (48.5-kb genome size). 2.4. MRSPs of PCR-ampli¢ed rrs genes MRSPs of PCR-ampli¢ed rrs genes analysis was performed according to Ralph et al. [13]. Primers used for rrs gene were : 16S-11, 5P-GGC TGC AGT CGA CGT TTG ATC CTG GCT CAG-3P, 16S-1507, 5P-CCA GAT CTG AGC TCA AGG AGG TGA TCC AGC-3P. PCR was performed with approximately 5 ng genomic DNA in the presence of [32 P]dCTP. Restriction enzyme digestions were performed with DdeI, HhaI, NlaIV and TaqI. Three Wl of each restriction digest was electrophoresed on a nondenaturing acrylamide gel. As in the AP-PCR (next described), the gel was exposed for 24^72 h on Kodak XOmat X-ray ¢lm and reference strains of Leptospira spp. were included as internal controls. 2.5. AP-PCR AP-PCR ¢ngerprints were performed according to Ralph et al. [13]. Only the most informative primers
183
for Leptospira as identi¢ed from previous studies [7,13] were used : KF (5P-CAC GCA CAC GCA CAG AGA3P), KG (5P-CAC ACG CAC ACG GAA GAA-3P), RSP (5P-GGA AAC AGC TAT GAC CAT GA-3P). 32 P-labeled [33 P]dCTP ampli¢cation products were loaded onto a 4% acrylamide^50% urea sequencing gel. 3. Results 3.1. MAbs After a ¢rst typing according to serogroup a¤nities (Icterohaemorrhagiae, Pomona and Ballum), a panel of 17 MAbs was able to assign all Portuguese isolates to the serovar level (Table 2). The agglutination pro¢les within Icterohaemorrhagiae serogroup showed serovar copenhageni among mainland isolates from Rattus norvegicus and among Azores isolates from the genera Erinaceus, Mus and Rattus, while serovar icterohaemorrhagiae was only identi¢ed among Azores isolates from the two Rattus species (not shown). Serovar mozdok in the Pomona group was only identi¢ed among mainland isolates and MAbs showed consistently three types of patterns which allowed the separation of isolated mozdok strains into type 1 (n = 27), type 2 (n = 10) and type 3 (n = 4) (Table 2). All isolates obtained from Crocidura russula were identi¢ed as type 3 according to `serovar' tsaratsovo reclassi¢cation as discussed later in this paper. The other mozdok types were only isolated from the genera Mus and Rattus (data not shown).
Table 2 Reciprocals of agglutination titers of MAbs from serogroups Icterohaemorrhagiae, Pomona and Ballum with their serovars/reference strains and 149 Portuguese ¢eld isolates (by origin and number) MAbs
Serogroup Icterohaemorrhagiae copenhageni/M20 [4] Azores (9); Coimbra (3) icterohaemorrhagiae/RGA [4] Azores (3)
Serogroup Pomona mozdok/5621 [4] Coimbra (20); Pancas (7) (type 1) Coimbra (5); Pancas (5) (type 2) Coimbra (1); Pancas (3) (type 3)
F12C3 [18]
F52C1 [18]
F70C7 [18]
F70C14 [18]
F70C24 [18]
F89C12 [18]
20 480 20 480^40 960 10 240 10 240^20 480
5 120 1 280^5 120 1 280 640^2 560
81 920 40 960^81 920 40 960 40 960^81 920
80 negative 40 960 40 960
40 960 20 480^40 960 160 40^320
2 560 640^5 120 negative negative
F43C9 [10]
F46C2 [10]
F46C4 [10]
F46C9 [10]
F48C6 [10]
F58C1 [10]
F61C7 [10]
20 80 negative to 640 80^2 560 40^1 280 negative 20^320 20 480^40 960
1 280 2 560^40 960 negative negative
1 280 2 560^20 480 1 280^40 960 10 240^40 960
2 560 640^5 120 640^5 120 320^1 280
40 960 20 480^81 920 1 280^40 960 10 240^40 960
5 120 5 120^40 960 2 560^40 960 20 480^81 920
F74C1 [3]
F74C4 [3]
F74C7 [3]
F74C12 [3]
negative negative
20 negative
20 negative
5 120 10 240
1 280 5 120
2 560 10 240
Serogroup Ballum arborea/Arborea [4] 5 120 Azores (48); Coimbra (13); Pancas 1 280^5 120 (31) ballum/Ballum [4] 10 240 Pancas (1) 2 560
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The agglutination Ballum pro¢les (Table 2) showed a clear predominance of serovar arborea among the mainland rodent isolates, with the only evidence of serovar ballum in just one Mus spretus isolate from Pancas. The Ballum pro¢les among the Azores isolates from Mus musculus and Rattus rattus were all characterized as serovar arborea. 3.2. PFGE By PFGE (Table 3), the three Icterohaemorrhagiae isolates from the Azores showed to belong to this serogroup since they shared common fragments with icterohaemorrhagiae (or copenhageni) serovars. Isolates A37 and A46 shared a common pattern close to icterohaemorrhagiae (Fig. 1) while A02 (Fig. 1) had three additional fragments and di¡ered from the patterns of every serovar in Icterohaemorrhagiae serogroup [11]. Most Pomona isolates presented a pro¢le similar to that of `serovar' tsaratsovo (Fig. 2 and data not shown). The remaining exhibited either a pro¢le quite similar to mozdok (e.g. Fig. 2) or a tsaratsovo-like pro¢le where only one or two small fragments di¡ered (e.g. Fig. 2 and data not shown). All but one of the studied Ballum isolates exhibited a characteristic common pattern similar to the arborea reference one (Fig. 1 and data not shown). They clearly differed from the castellonis pattern by a supplementary frag-
Fig. 2. PFGE of NotI restriction fragments from serogroup Pomona isolates. The digestion products were separated at 200 V for 24 h in 1% agarose^0.5UTris^borate^EDTA. The three pulse times were 25 s over 10 h, 50 s over 8 h and 100 s over 6 h. Lanes: 2^8, Pomona isolates ; 9, serovar tsaratsovo ; 10, Pomona isolate; 1 and 11, size markers (lambda, 48.5-kb genome size).
ment at 200 kb and from serovar ballum which exhibited a speci¢c fragment at 230 kb. 3.3. MRSPs of PCR-ampli¢ed rrs genes
Fig. 1. PFGE of NotI restriction fragments from serogroups Ballum and Icterohaemorrhagiae Azorean isolates. The digestion products were separated at 200 V for 24 h in 1% agarose^0.5UTris^borate^EDTA. The two pulse times were 25 s and 100 s over 24 h each. Lanes: 1, serovar castellonis ; 2^6, Ballum isolates ; 7, serovar arborea; 8^10, Icterohaemorrhagiae isolates (size markers, kb).
MRSP allowed a quick identi¢cation of the pathogenic and saprophytic Portuguese isolates at the species level. As demonstrated previously [7,13], the primers used in this study ampli¢ed a conserved portion of the rrs (from bases 11 to 1507). PCR ampli¢cation of rrs gene contained one product of the expected size (1.5 kb) for all tested strains (data not shown). An additional product of 260 bp was found for all strains from L. borgpetersenii genotype as described earlier [13]. Restriction digestions performed with DdeI, HhaI, NlaIV and TaqI allowed the assignment at the species level of the pathogenic isolates. Among the mainland strains, two genospecies were identi¢ed : L. borgpetersenii, including the nine isolates from serogroup Ballum, and Leptospira kirschneri, with all those from Pomona serogroup. The Azores isolates belonged also to either of two genospecies : L. interrogans, which included the three isolates from serogroup Icterohaemorrhagiae, and L. borgpetersenii, with the 12 Ballum group isolates.
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Table 3 Serovar identi¢cation results with MAbs, PFGE and AP-PCR of 32 pathogenic ¢eld isolates (A = Azores ; C = Coimbra ; P = Pancas) Serogroup of L. interrogans (genomic species) Icterohaemorrhagiae (L. interrogans) Pomona (L. kirschneri)
Ballum (L. borgpetersenii)
Isolate A02 A37 A46 C138 C149 P110 P112 P116 P122 P126 P159 A03 A07 A09 A11 A23 A30 A31 A32 A33 A36 A40 A44 P87 P90 P115 P124 P127 P135 P150 P167 P189
Mammal species Rattus norvegicus Rattus rattus Erinaceus europaeus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Crocidura russula Mus musculus Mus musculus Mus musculus Mus musculus Rattus rattus Mus musculus Mus musculus Mus musculus Mus musculus Rattus rattus Rattus rattus Rattus rattus Mus musculus Rattus norvegicus Mus spretus Mus spretus Mus spretus Mus spretus Mus spretus Rattus norvegicus Mus spretus
MAbs a
ictero ictero copb moz 1c moz 1 moz 1 moz 1 moz 1 moz 1 moz 1 moz 3 arbe arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb
PFGE
AP-PCR
ictero/cop ictero/cop ictero/cop tsard tsar-like tsar tsar tsar tsar tsar moz arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb arb
ictero ictero ictero tsar tsar tsar tsar tsar tsar tsar moz arb/balf arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal arb/bal
a
ictero = icterohaemorrhagiae. cop = copenhageni. c moz 1 = mozdok `type 1'. d tsar = tsaratsovo. e arb = arborea. f bal = ballum. b
3.4. AP-PCR AP-PCR leptospiral ¢ngerprints were obtained with the four primers tested. The level of complexity of the patterns was variable, depending on the primer used. The patterns were simple using primer SP and relatively complex with primers KF, KG and RSP. In each case, as previously reported [7,13], prominent species speci¢c products (not shared with the other leptospiral species) were observed and con¢rmed the genomic species assignments supported by MRSP data. AP-PCR ¢ngerprints of both mainland and Azores isolates were homogenous inside each identi¢ed genospecies (Table 3). The three L. interrogans isolates presented the characteristic AP-PCR ¢ngerprints from serovar icterohaemorrhagiae strains (data not shown). Fingerprints of L. borgpetersenii isolates were close to those of both reference strains from serovar arboreae and ballum which present similar AP-PCR ¢ngerprints whatever primer was used.
Lastly, eight of the nine L. kirschneri strains were identi¢ed as `serovar' tsaratsovo except strain P159. This isolate looked closer to mozdok, with primers KF and KG (data not shown). 4. Discussion Present results emphasize a perfect agreement for species classi¢cation with MRSP rrs genes and AP-PCR, but a correlation was not always evident at the serovar level for MAbs on the one hand and PFGE and AP-PCR on the other hand. This is due to the great intra-species heterogeneity of the strains and to the di¡erent discrimination power of the techniques used. The practical value of MRSP rrs genes and AP-PCR as quick identi¢cation methods at the species level is stressed. On the other hand, the use of MAbs con¢rmed to be generally a powerful method to identify Leptospira organ-
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isms at the serovar level. Comparing MAbs, PFGE and AP-PCR ¢ndings, a good correlation at the serovar level within the Ballum serogroup was only observed between the ¢rst two methods, where the same common arborea pattern was seen in all the isolates; AP-PCR did not discriminate between arborea and ballum (Table 3). The PFGE of NotI restriction fragments showed a supplementary 200-kb fragment speci¢c for serovar castellonis and the absence of a speci¢c 230-kb fragment for serovar ballum, which con¢rmed serovar arborea as the most likely identi¢cation. However, there was no complete agreement between the Pomona and the Icterohaemorrhagiae agglutination pro¢les at the serovar level and the PFGE digestion ¢ngerprints of their speci¢c isolates. Three types of mozdok within the Pomona group were identi¢ed by MAbs while PFGE suggested the presence of three di¡erent ¢ngerprint patterns. Those that were typed as mozdok type 1 by MAbs were generally typed as tsaratsovo by genetic methods while the reference strain B81/7 of tsaratsovo was antigenically typed as mozdok type 3 (data not shown). Tsaratsovo actually belongs to serovar mozdok according to antigenic (CAAT) and genetic (REA) analysis [13] and the designation of tsaratsovo as a separate serovar should be abandoned. Interestingly, although there was not an equal designation of the di¡erent types and patterns within the Pomona ¢eld isolates, all the tests used were at least in agreement considering the mozdok identity of the isolates grown from C. russula. This result con¢rms the already observed antigenic and genetic di¡erentiation according to the host speci¢city of certain Pomona ¢eld strains [20]. In addition, PFGE was not able to discriminate between serovars copenhageni and icterohaemorrhagiae as previously reported [12]. However, PFGE showed in this case, through repeated observations, a pattern diversity within the serovar icterohaemorrhagiae, unless A02 represents a new serovar or genotype. This diversity has not been reported so far. The saprophytic strains which were analyzed by MRSP rrs genes, AP-PCR and PFGE were not related with any pathogenic species and shared a common pro¢le, identical to the one from L. bi£exa reference strain. Considering these strains were unusually isolated from the kidneys of trapped animals, once disregarded the possibility of contamination due to non-sterile isolation procedures, their particular existence is probably the result of a prolonged exposure to fresh water in £ooded areas. In conclusion, this study shows that phenotypic and genotypic methods mostly lead to similar results albeit varying degrees of detail and applicability. Due to this agreement, the use of a selected panel of MAbs of the already known serovars or types is advisable in cost^bene¢t terms, for situations of a limited diversity of local isolates, such as in Portugal. However, to solve the possible existence of non-reactive isolates corresponding to new strains as well as the problem of mixed lepto populations
(isolates are not usually cloned by limited dilution), the use of AP-PCR ¢ngerprints would be important in heterogenic populations: using much less DNA than PFGE, this method is a de¢nite improvement for species identi¢cation, also providing a simple and rapid tool to intra-species comparison. So, for epidemiological studies in leptospirosis, the use of di¡erent but complementary methods is always preferable. Acknowledgements Field work was possible thanks to the contribution of the other researchers of the team: M.L. Mathias, M.G. Ramalhinho, M.M. Oom, F. Petrucci-Fonseca, R. Fons and R. Libois, with the assistance of J.P. Clara. We are also grateful to both L. Floªr (Laborato¨rio de Sanidade Animal) and J.E. Matos (Universidade dos Ac°ores) for the cooperation and logistic facilities in the islands, and to M.J. Gama (IHMT, Lisboa) for her laboratorial assistance. Funding was provided through the national projects PBIC/C/SAU/1535/92 and STRDA/C/CEN/447/92, and complemented by Secretaria Regional da Sau¨de e Seguranc°a Social and Secretaria Regional da Agricultura e Pescas (Azores). We also thank Centro de Mala¨ria e Outras Doenc°as Tropicais (IHMT, UNL) for providing technical support.
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