Vol. 37, No. 3
INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
July 1987, p. 296297
0020-7713/87/030296-02$02.OO/O
Copyright 0 1987, International Union of Microbiological Societies
Classification of Seven Leptospira Water Strains by Classical
Methods and Identification of Three New Serovars
MARINA CINCO,l* ELENA BANF1,l ARNO SCHONBERG,2 AND CHRIS 0. R. EVERARD3
Istituto di Microbiologia, Universitd di Trieste, 34127 Trieste, Italy'; Institut fur Veterinarmedizin, Berlin West, Federal
Republic of Germany2;and Medical Research Council, Veterinary Diagnostic Laboratory, Barbados3
Six Leptospira strains isolated from surface water and one isolated from bovine urine were classified as
members of Leptospira bijlexa according to their antigenic and biological characteristics. Three new serovars
were identified, for which we propose the names barbados, krefeldi, and neville. Serum sensitivity was tested
for further biological characterization of the L . bijlexa species.
Saprophytic Leptospira bifEexa and parasitic Leptospira
interrogans can be differentiated on the basis of serological
and biological characteristics (8). These tests are important
to distinguish between the two species and especially to
classify leptospires of dubious identity, such as strains
isolated from biological fluids of animals, which do not react
with immune sera prepared against L. interrogans serovars.
In 1967, Johnson and Harris (10) demonstrated that pathogenic leptospires are serum resistant; later, we tested a
relevant number of standard pathogenic and saprophytic
Leptospira strains, confirming that nonpathogenic leptospires are susceptible to the killing activity of normal fresh
serum, while pathogenic strains survive at an 80 to 100% rate
(5).
Six strains of leptospires isolated from surface water and
one from the urine of a bovine having a history of abortion
and mastitis were sent to Trieste Reference Laboratory for
typing. We assayed the strains for their biological characteristics (growth at 13"C, resistance to 8-azaguanine, growth in
tryptic soy broth [TSB], and conversion to spherical forms
by 1 M NaCl) and performed serological typing. We also
examined their serum resistance to check whether it correlated with our previous results (3).
Strains and history. Strains HI, Ji, and P were isolated
from surface waters in Barbados. Strains 380, 379, and 383
were isolated in Germany from water resources of farms and
were sent to Trieste, with the specific immune sera, by A.S.
Strain K6196 came from Malaysia and was sent to us by
Neville Stallmann (Leptospira Reference Laboratory, Brisbane, Australia); this strain was isolated from a bovine in a
herd with a history of abortion and mastitis. Agglutination
tests performed at that laboratory revealed that the strain
was not L. interrogans, so it was thought to be a saprophytic
contaminant of the water.
Biological characteristics. The following biological characteristics of the seven strains were determined: ability to grow
in the presence of 225 pg of 8-azaguanineper ml (ll), at 13°C
(9), and in TSB (7); and conversion of cells to spherical
forms by 1 M NaCl after 2 h (8). In addition, serum
sensitivity was assayed as already described (3), using fresh
serum at the optimal dilution of 1:6. The serum sensitivity
was expressed as percent survival of colony-forming units
(CFU) recovered after 30 min of incubation compared with
* Corresponding author.
the number of CFU recovered in the presence of heatinactivated serum (3).
Serological tests. Immune sera were prepared as described
before (4, 5), and the new isolates were compared by a
microscopic agglutination test and cross-agglutinin absorption test (6) with serovars representative of L . biflexa.
Strains HI, P , 383, 380, Ji, and 379 all exhibited reactions
typical of the saprophytic leptospires (Table 1). These
strains did not grow in TSB and were serum sensitive, but to
various degrees. The highest level of resistance (strain 379)
approached the highest recorded for saprophytic strains (3).
However, strain K6196 was evidently a distinctive strain; it
was unable to grow in the presence of 8-azaguanine, at 13"C,
or in TBS, yet it was clearly serum sensitive. The serological
tests revealed that strains 383,379, and Ji belonged to known
serogroups and serovars. Strains HI and P belonged to
serogroup Basovizza, and strain 380 belonged to serogroup
Doberdo. The results of cross-agglutinin absorption (Table
2) show that strain 380 was not identical to any of the
members of Doberdo serogroup and represents a new
serovar, which we named krefeldi. Strains P and HI (both
antigenically identical) belonged to a new serovar within the
serogroup Basovizza, which we named barbados (Table 2).
The biological characteristics of the isolates indicated that
they can be ascribed to the L. bifexa species; these data
were confirmed by serum sensitivity, which was in the range
of 22.2 to 79.8% survival. The range of survival found in
pathogenic strains is reported to be 80 to 100% (3). Only
strain K6196 gave contradictory responses, because its
growth was inhibited by 8-azaguanine and temperatures of
TABLE 1. Biological Characteristics of seven newly isolated
Leptospira strains
Reaction
Strain
HI
P
383
380
379
Ji
K6196
a
Resistance to
8-azaguanine
(225 kg/ml)
Growth
+a
+*
+
+
+
+
+-
at
l3OC
+
+
+
+
+
-
Growth
in TSB
(% survival)
-b
-a
45.2
62.7
29.9
60.8
79.8
22.2
28.3
-
-
-
-
-
Growth in the first subculture (+) or no growth (-).
Conversion of 75% of cells in 1 M NaCl (within 2 h).
296
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Serum
sensitivity
Conversion
to spherical
forms
VOL.37, 1987
NOTES
TABLE 2. Cross-agglutinin absorption tests
Immune
serum
P
HI
HI
P
P
380
380
380
Khera 11
a
Absorbed
with strain
S. Giusto
Vf2
RPE
Khera 11
380
% Agglutination"
titer with strain
P
0
0
HI
Ob
0
S. Giusto P
0
50
Vl2
380
0
50
RPE
380
0
100
Khera 11 380
0
12.5
6.5
0
Taxonomic status
Serogroup
serovar
Basovizza
Barbados
297
A better definition of these isolates should require additional biological probes. We have tested the serum sensitivity, which has some correlation with the classical biological
tests, but it is time-consuming and cannot give a clear-cut
distinction when survival values are close to 80% A genetic
or immunological species-specific probe should be preferable, such as the monoclonal antibody that was reported by
Adler and Faine (1)to react only with L . interroguns strains.
This work was performed with a grant by Minister0 della Pubblica
Instruzione.
Doberdo
Krefeldi
Expressed as a percentage of the homologous titer before absorption.
1:lOO titer in postabsorption microagglutination test.
Therrnolabile (5) antigen on Khoshamian.
13"C,like parasitic leptospires; on the other hand, it was not
converted to the spherical form by 1M NaCl. It was strongly
serum sensitive, having only 28% survival. On the basis of
these results we are inclined to consider this strain a
nonparasitic leptospire and include it in the species L.
biflexu. This finding may suggest that strain K6196 is a
contaminant of bovine urine, or of specimens or media
employed for cultivation. There are other examples of
nonparasitic Leptospiru strains isolated from body fluids of
mammals but whose true origins are uncertain, e.g., the
strain illini, also described as Leptonemu illini (2), a strain
derived from the urine of a bull (7), and the strain andamana
CH11, which was isolated from human cerebrospinal fluid
(12). Strain K6196 was distinguished from L . illini by its
inability to grow in TSB.
Serological results indicated that strains P and HI, 380,
and K6196 represent three new serovars, one of which
belongs to a new serogroup. Serovars each representing
single serogroups are quite common in L. bifEexu serology;
this peculiarity probably reflects the role of varied and
inconstant environmental conditions acting as selecting
agents on the antigenic pattern of free-living leptospires.
This taxonomic study has revealed that biological tests are
still able to distinguish saprophytic from parasitic strains,
but there exist also leptospires which have an intermediate
behaviour (like strain K6196).
LITERATURE CITED
1. Adler, B., and S. Faine. 1983. Species- and genus-specific
antigens in Leptospira, revealed by monoclonal antibodies and
enzyme immunoassay. Zentralbl. Bakteriol. Parasitenkd.
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1983. Leptospira sp. strain Dimbovitza, first isolate in Europe
with characteristics of the proposed genus Leptonema. Int. J.
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11. Johnson, R. C., and P. Rogers. 1964. Differentiation of pathogenic and saprophytic leptospires with 8-azaguanine. J. Bacte1501. 88:1618-1623.
12. Taylor, D., and A. N. Goyle. 1931. Leptospirosis in the
Andamans. Indian Med. Res. Mem. 2O:l-190.
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