INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Jan. 1987, p. 82-84
0020-7713/87/010082-03$02.OO/O
Copyright 0 1987, International Union of Microbiological Societies
Vol. 37, No. 1
Proposed Criteria for an Interim Serogroup Classification for
Members of the Genus Spiroplasma (Class Mollicutes)?
ROBERT F. WHITCOMB,l* JOSEPH M. BOVE,2 T S E H AN CHEN,3 JOSEPH G. TULLY,4
AND DAVID L. WILLIAMSON'
Insect Pathology Laboratory, U . S . Department of Agriculture, Beltsville, Maryland 20705l; Laboratoire de Biologie
Cellulaire et Moltculaire, Institut National de Recherche Agronomique et Universitt de Bordeaux 11, Pont de la Maye,
France2; Department of Plant Pathology, Cook College, Rutgers University, New Brunswick, New Jersey 089033;
Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,
Frederick, Maryland 21 7014; and Department of Anatomical Sciences, State University of New York, Stony Brook,
New York 1I 7945
Criteria for description of new groups of the serogroup classification scheme for the Spiroplasma genus are
proposed. New groups, if shown to be referable to the MoZZicutes class and Spiroplasmataceae family, should be
shown to differ from established groups by (i) reciprocal deformation tests and (ii) one of three other serologic
tests (growth inhibition, metabolism inhibition, or enzyme-linked immunosorbent assay). Also, the ability or
inability to utilize glucose, arginine, and urea should be determined. Determination of guanine-plus-cytosine
content is strongly recommended but optional.
The genus Spiroplasma (class Mollicutes) was established
in 1973 when the causal agent of citrus stubborn disease
(Spiroplasma citri) was characterized (18) as the first genus
and species of a new family (21) of helical, wall-less
procaryotes. In the following decade, many new Spiroplasma species were recovered from flower surfaces and the
hemolymph or gut fluids of a wide variety of insects and
other arthropods (5). As with other members of the class
Mollicutes, serologic techniques were first used to classify
the Spiroplasma genus (29, 30). However, other molecular
and genetic techniques were soon applied to this group of
microorganisms, including analysis of guanine-plus-cytosine
content, genome size of the deoxyribonucleic acid (DNA),
DNA-DNA hybridization, DNA restriction endonuclease
patterns, and polyacrylamide gel electrophoretic patterns of
cell proteins (3). Results from molecular, genetic, and
serologic analyses were complementary and consistent.
Each technique indicated the existence of groups that differed in guanine-plus-cytosine content, had negligible
intergroup DNA-DNA homology, and exhibited unique
polyacrylamide gel electrophoretic patterns (14, 15). It thus
appeared that the genus contained species other than the
type, Spiroplasma citri. On the other hand, a certain number
of strains isolated from plant or arthropod sources showed
partial serologic or molecular interrelationships with S. citri
(2)*
These observations led to a concept of Spiroplasma classification based upon both serologic and molecular genetic
data. In the first scheme, proposed in 1980 (14),five groups
(I to V ) and four subgroups (1-1 to 1-4) were defined. This
proposal gained general acceptance and has been subsequently revised (26, 27) to accommodate discovery of new
strains. The current classification recognizes 11 groups;
group I now contains eight subgroups, including the recently
added subgroup 1-8 (20). Three of the groups (111, IV, and V)
have been given species designations (Spiroplasmajforicola,
Spiroplasma apis, and Spiroplasma mirum, respectively).
Initially, the subcommittee recommended that elevation of
the group I subgroups (other than the type species, S. citri)
to species status should be postponed until more information
on subgroup interrelationships became available (10). In
1982, however, the subcommittee proposed (11) that subgroups could be elevated to species under certain conditions.
The most important requirement was that the level of
DNA-DNA homology between the candidate subgroup and
all other subgroups was less than 70%. Strains of subgroups
1-2 and 1-3 met the recommended conditions and have been
characterized recently as Spiroplasma melliferum (6) and
Spiroplasma kunkelii (25), respectively.
Within the past few years, Spiroplasma strains that represent 13 additional groups have been isolated, primarily
from insects (C. Chastel, D. Devaux, F. Le Goff, A. M.
Simitzis-Le Flohic, R. Ruffaz, G. Kerdraon, and B. Gilot,
Isr. J. Med. Sci, in press; T. B. Clark, R. B. Henegar, L.
Rosen, K. J. Hackett, R. F. Whitcomb, J. E. Lowry, C.
Saillard, J. M. Bove, J. G. Tully, and D. L. Williamson, Isr.
J. Med. Sci., in press). At its interim meeting in Jerusalem,
Israel, in June 1984, the subcommittee acknowledged (12)
that the currently informal serogroup system of classification
had a number of obvious advantages for the genus Spiroplasma. Although workers were encouraged to assign Latin
binary combinations to properly characterized Spiroplasma
species, it was recognized that the large number of emerging
(and putative) species might delay or indefinitely postpone
development of a useful classification system for these
organisms. An ad hoc committee was therefore appointed by
the subcommittee to develop a series of recommendations
for standard criteria for a serogroup designation for these
organisms. The subcommittee emphasized that this scheme
would serve only as an interim system in lieu of established
specific epithets.
The ad hoc committee convened on 11 June 1985 and
developed the following recommendations.
1. Cloning of the organism, by standard techniques (9, 23),
should constitute the first step in designation of a new
Spiroplasma serogroup.
2. It must be demonstrated that the organism is a member of
the class Mollicutes (8, 17). This requires (i) demonstration (9) that the organism has an absolute insusceptibility
* Corresponding author.
t Submitted for, and with the approval
of, the International
Subcommittee on the Taxonomy of Mullicutes.
82
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VOL. 37, 1987
3.
4.
5.
6.
7.
1
PROPOSAL
to penicillin, and (ii) demonstration, by thin-section electron microscopic techniques (7), that the helical cells are
bounded by a single limiting cytoplasmic membrane and
are devoid of a cell wall and periplasmic fibrils. These
latter morphologic features would be observed with spirochetes (13) that might be cultivated on artificial media
and confused with a spiroplasma.
It must be demonstrated that the organism is referable to
the genus Spiroplasma by (i) examination of a broth
culture of the organism by dark-field microscopy (22) to
confirm that helicity and motility are present in at least
some stage of the growth cycle or cultural history and (ii)
demonstration of the ability of the organism to utilize
glucose (16) and its ability (or inability) to utilize (a)
arginine (1) and (b) urea (9). In conjunction with experiments demonstrating the status as a member of the
Mollicutes this morphologic and biochemical evidence
establishes that the organism is a member of the genus
Spiroplasma and the family Spiroplasmataceae.
Establishment of a new Spiroplasma serogroup requires
fulfillment of the following additional criteria. (i) The
organism must be shown to be serologically distinct from
all other previously established serogroups or species in
the genus Spiroplasma by a reciprocal deformation (DF)
test (3&32) and by at least one of three other serologic
tests: growth inhibition (GI) (28), metabolism inhibition
(MI) (30), or enzyme-linked immunosorbent assay
(ELISA) (19) tests. If the candidate organism shows, in
any of the serologic tests selected, evidence of moderate
sharing of serologic properties with a member of any
other established serogroup or subgroup (for example, a
DF titer of 1:40, an MI titer of 1:160, or a GI zone of 2
mm), the organism should not be described as a new
serogroup or subgroup. To properly classify such an
organism, antiserum must be prepared to the candidate
strain, and reciprocal DF, MI, and GI tests must be
performed between the candidate organism and the strain
with which it shows partial crossing. Such tests could
show (i) that the candidate organism is either a previously
unrecognized subgroup (such as those in group I [2, 14,
271) or a closely related serovar of the existing serogroup
that does not merit recognition at the subgroup level
(such as group IV strains [24]) or (ii) that the appearance
of partial relationship seen in a given serologic test is
exhibited only as one-way crosses or is not supported by
results from other serologic tests. In the latter case,
designation of a new serogroup may be justified.
The determination of guanine-plus-cytosine content of
the DNA of the organism (4) is optional and may be
postponed if the authors eventually wish to publish a
taxonomic description and give a species designation to
the organism. If there is no intention to provide a more
extensive taxonomic description, the committee strongly
recommends that this optional procedure be completed.
The candidate spiroplasma should be deposited in a
recognized national culture collection and made available
for circulation to other workers.
Characterization of Spiroplasma species will continue to
be determined by the minimal standards for description of
new Mollicutes species previously recommended by the
subcommittee (9).
1.
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