INTERNATIONAL
JOURNALOF SYSTEMATIC
BACTERIOLOGY,
Oct. 1987, p. 357-364
0020-7713/87/040357-08$02.OO/O
Copyright 0 1987, International Union of Microbiological Societies
Vol. 37, No. 4
Revised Group Classification of the Genus Spiroplasma (Class
Mollicutes), with Proposed New Groups XI1 to XXIII
JOSEPH G. TULLY,'" DAVID L. ROSE,' EDWARD CLARK,* PATRICIA CARLE,3 JOSEPH M. BOVE,3 ROBERTA
B. HENEGAR,2 ROBERT F. WHITCOMB,2 DAVID E. COLFLESH,4 AND DAVID L. WILLIAMSON4
Mycoplasma Section, Laboratory of Molecular Microbiology, Frederick Cancer Research Facility, National Institute of
Allergy and Infectious Diseases, Frederick, Maryland 21 701 Insect Pathology Laboratory, U . S . Department of
Agriculture, Beltsville, Maryland 2070j2; Laboratoire de Biologie Cellulaire et Moleculaire, Institut Nationale Recherche
Agronomique, Pont-de-la-Maye, France3; and Department of Anatomical Sciences, State University of N e w Y w k , Stony
Brook, New York 117944
';
Fourteen spiroplasma strains, primarily of insect origin, were analyzed according to criteria previously
proposed for description of new serogroups of the genus Spiroplasma. When tested by reciprocal metabolism
inhibition, growth inhibition, and deformation serological procedures, 12 of the strains were serologically
unrelated to each other and to representative strains previously assigned to groups I to XI and subgroups 1-1
to 1-8. Examination by dark-field and electron microscopy indicated that each of the 12 strains possessed
morphological features typical of spiroplasmas (helicity,motility, lack of a cell wall, and absence of periplasmic
fibrils). All strains were resistant to 500 U of penicillin per ml and catabolized glucose but were unable to
hydrolyze urea. Ability to hydrolyze arginine varied among strains. The guanine-plus-cytosine contents of the
deoxyribonucleic acid of the 12 strains varied from 24 to 29 mol%. Two other strains (MQ-6 and Ar-1357)
shared only a partial serological relationship to strain CC-1 (group XVI), suggesting that this group may consist
of an assemblage of heterogeneous serovars. On the basis of the unique serological distinctions and other
properties reported herein, we propose that the 12 representative strains be assigned new consecutive group
designations XI1 to XXIII.
(30). In this scheme, group I was subdivided into eight
subgroups (1-1 to I-8), including a recently described (21)
plant-pathogenic spiroplasma (strain P40 and allied spiroplasmas, subgroup 1-8). Three groups (111, IV, and V) have been
given species epithets (S.Jloricola [lo], S. apis [MI, and S .
mirum [24], respectively). In addition, following recommendations by the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes (15)
for elevation of group I subgroups to species levels, we
recently characterized representatives of subgroups I-2,1-3,
and 1-8 as Spiroplasma melliferum (9), Spiroplasma kunkelii
(29), and Spiroplasma phoeniceum (20), respectively.
The number of new spiroplasma isolates has continued to
increase as new insect hosts have been examined (6, 8, 11,
12: Clark et al., in press). Many of these new isolates from
insects have been shown in preliminary serological tests to
be unrelated to previously established groups or species. In
1984, the Subcommittee on the Taxonomy of Mollicutes
recognized (16) the advantages of a group classification
system for spiroplasma strains, particularly for strains representing putative species for which no formal taxonomic
description had been offered. Although authors were encouraged to provide a complete taxonomic description of new
Spiroplasma species based upon proposed minimum standards (14), the subcommittee recommended continued use of
the grouping scheme as an interim and informal means for
maintenance of an updated compilation of currently recognized species and putative species within the genus. However, the subcommittee also indicated (16) that it would be
advisable to provide guidelines for establishment of new
Spiroplasma groups within this interim classification. An ad
hoc committee appointed by the subcommittee recently
proposed (28) a set of procedures for determination of
morphological, serological, and genomic properties of spiroplasmas. This proposal constituted, in effect, minimal criteria for describing new Spiroplasma groups.
Classification of helical, wall-less procaryotes (members
of the genus Spiroplasma [class Mollicutes]) has changed
dramatically in the 14 years since the genus was first
described. Spiroplasma citri, the causative agent of citrus
stubborn disease, was the first species of the genus to be
described (19). Since that time, but especially within the past
5 years, numerous new species (or putative species) have
been recovered from plant surfaces and the gut fluids or
hemolymph of a wide variety of insects and other arthropods
(2, 7 , 8, 25, 26; Clark et al., Isr. J. Med. Sci., in press).
Serological, biochemical, and biological characteristics
commonly employed in classification schemes for other
mollicutes (14) were used initially for spiroplasmas (19).
When genomic and other molecular techniques (e.g., deoxyribonucleic acid [DNA] base analysis, genome size determination, DNA restriction endonuclease patterns, DNA-DNA
hybridization, and cell protein analysis by one- and twodimensional gel electrophoresis) were applied to spiroplasmas, they confirmed other indications that the genus
Spiroplasma consisted of a number of distinct species.
Molecular techniques were also instrumental in confirming
serological results, indicating that certain plant- and arthropod-derived strains were partially related to S. citri (3).
An approach to spiroplasma classification based upon
both serological and molecular genetic data was proposed in
1980 (17). In this scheme, five groups (I to V) and four
subgroups (1-1 to 1-4) were defined. The designation of
subgroups was made necessary by discovery of partial
DNA-DNA homologies that closely paralleled the early
serological results. Although this proposed scheme gained
general acceptance, it was later revised to accommodate
isolation and characterization of new spiroplasmas (30, 31).
Eleven groups were recognized in the most recent revision
* Corresponding author.
357
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358
INT. J. SYST.BACTERIOL.
TULLY E T AL.
TABLE 2. Growth inhibition
TABLE 1. Metabolism inhibition
~
Group
Strain
Homologous
reactionU
Heterologous reaction
Direction"
Group
Strain
Positive"
Homologous
reaction'
Heterologous reaction
Directionb
Positive'
XI1
DU-1
13,000
Antigen
Antiserum
None
None
XI1
DU-1
5
Antigen
Antiserum
None
None
XI11
Ar-1343
13,000
Antigen
Antiserum
None
XIX (162)
XI11
Ar-1343
8
Antigen
1-4 (2), VII (3),
XIV
EC-1
13,000
Antigen
Antiserum
None
None
xv
1-25
>117,000
Antigen
Antiserum
None
None
XVI
cc-1
39,000
Antigen
Antiserum
None
None
XVII
DF-1
39,000
Antigen
Antiserum
None
VIII (162)
XVIII
TN-1
>117,000
Antigen
Antiserum
None
None
XIX
PUP-1
Antigen
Antiserum
1-3 (162), X (486),
XI (486), XI11
(162), XXI (162)
None
13,000
xx
LD-1
ND
Antigen
Antiserum
ND
None
XXI
W115
4,374
Antigen
Antiserum
None
VIII (162), XIX
(162)
XXII
CT-1
13,000
Antigen
Antiserum
None
None
XXIII
TG-1
13.000
Antigen
Antiserum
None
None
Reciprocal of endpoint metabolism inhibition titer when antigen is tested
against homologous antiserum.
Responses given in the antigen line represent reactions between the
designated spiroplasma strain (antigen) versus antisera to 31 other
spiroplasmas. Responses given in the antiserum line represent reactions
between antiserum to the designated group spiroplasma versus 31 other
spiroplasmas as antigens.
Heterologous cross-reactions are recorded by group number and reciprocal titer (in parentheses) of cross-reactions. None indicates an inhibition titer
of 1 5 4 or less. ND, Not done.
Antiserum
XIV
EC-1
5
Antigen
Antiserum
xv
1-25
8
Antigen
Antiserum
None
XIX (2)
XVI
cc-1
5
Antigen
Antiserum
None
XVII (2)
XVII
DF-1
10
Antigen
VIII (4), XXII
(2)
XIX (2)
Antiserum
XVIII
TN-1
XIX
PUP-1
In this report, we provide a further revision of the group
classification of spiroplasmas, including characterization of
12 new groups that we propose be given numerical designations from XI1 to XXIII. The description of each of these
new groups follows aforementioned published recommendations of the ad hoc committee of the subcommittee (28).
MATERIALS AND METHODS
Spiroplusrnu strains. The origin and history of spiroplasma
isolates representing group I (subgroups 1-1 through 1-7) and
groups I1 through XI have been given in detail earlier (17, 30,
31). The isolation (21) and characterization (20) of the P40
strain (subgroup 1-8) as Spiroplasma phoeniceum have been
reported recently. Details of the isolation of the following
spiroplasma strains from insects or flowers were recently
6
14
Antigen
Antiserum
Antigen
Antiserum
xx
LD-1
ND
Antigen
Antiserum
ND
None
XXI
W115
15
Antigen
Antiserum
None
XI11 (2)
XXII
CT-1
8
Antigen
Antiserum
None
None
XXIII
TG-1
7
Antigen
Antiserum
None
None
LI
"
XVI (2), XXI
(2)
None
~~
~
Zone of growth inhibition (in millimeters) when the designated spiroplasma is used as an antigen versus homologous antiserum.
See Table 1, footnote b.
Heterologous cross-reactions in the growth inhibition test are recorded by
group number and inhibition zones of cross-reactions. None indicates an
inhibition titer of 1 mm or less. ND, Not done.
"
summarized by Clark and associates (in press): DU-I from
Diabrotica undecimpunctata (beetle), EC-1 from Ellychnia
corrusca (beetle), 1-25 from Cicadulina bipunctella bipunctella (leafhopper), PUP-1 from Photuris pennsylvanicus
(firefly), W115 from the flower of an ornamental Prunus
plant, CC-1 from Cantharis carolinus (beetle), TG-1 from
Tabanus gladiator (horsefly), and CT-1 from Culex tritaeniorhynchus (mosquito). The Ar-1343 and Ar-1357 strains
were recovered from Aedes sticticuslvexans and Aedes
cantanslannulipes mosquitoes, respectively, in the French
Alps by Chaste1 and colleagues (6). The MQ-6 spiroplasma
was isolated from the gut fluid of a Monobia quadridens
wasp by T. B. Clark (unpublished data). The LD-1 isolate
from Leptinotarsa decemlineata (Colorado potato beetle)
and the DW-1 strain of the sex ratio spiroplasma from
Drosophila willistoni (fruit flies) were recently cultivated by
Hackett and associates (11, 12). The DF-1 strain from
Chrysops sp. (deerfly) and the TN-1 strain from a Tabanus
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VOL. 37, 1987
SPIROPLASMA GROUPS
TABLE 3. Deformation
Group
Strain
Homologous
reaction"
Heterologous reaction
Directionh
Positive"
XI1
DU-1
5,120
Antigen
Antiserum
111 (80)
111 (40)
XI11
Ar-1343
2,560
Antigen
Antiserum
None
XIX (160)
XIV
EC-1
2,560
Antigen
Antiserum
XXIII (40)
1-5 (40), IV (40), VII
(40), IX (40), XI (401,
XX (40), XXII (160)
XV
1-25
10,240
Antigen
Antiserum
None
XX (40), XXIII (40)
XVI
CC-1
2,560
Antigen
Antiserum
None
None
XVII
DF-1
10,240
Antigen
Antiserum
None
VIII (go), XIX (40)
XVIII
TN-1
2,560
Antigen
Antiserum
None
IX (40), XIX (go), XX
(160). XXI (40)
XIX
PUP-1
10,240
Antigen
Antiserum
1-1 (go), 1-2 (go), 1-3
(40), 1-5 (40), 1-6 (40),
1-8 (40), X (go), XI
(1601, XI11 (160),
XVII (40), XVIII (go),
XX (40), XXI (160),
XXII (40), XXIII (40)
XXI (160)
Antigen
1-8 (40), XIV (40), XV
xx
XXI
LD-1
Wll5
20,480
40,960
Antiserum
(401, XVIII (160)
1-2 (320), XI (40), XIX
(40)
Antigen
1-5 (40), 1-6 (40), XVIII
Antiserum
(40), XIX (160), XXII
(40)
1-6 (40), XIX (160)
XXII
CT-1
640
Antigen
Antiserum
1-4 (40), XIV (160)
XI (go), XIX (40),
XXIII (40)
XXIII
TG-1
20,480
Antigen
Antiserum
XV (40), XXII (40)
XIV (401, XIX (40)
a Reciprocal of endpoint in deformation test in which the designated antigen
is tested versus homologous antiserum.
Reactions listed in antigen line represent positive results of test in which
indicated spiroplasma strain was used as an antigen in deformation test versus
antisera to each of the thirty-one other spiroplasma groups and subgroups.
Other results were negative. Reactions summarized in the antiserum line
represent positive results of tests in which antiserum against the indicated
spiroplasma was tested in deformation tests against antigens of each of the
other 31 spiroplasma groups and subgroups. Other results were negative.
Heterologous cross-reactions in deformation test are listed by grc.
number and titer of cross-reaction. None indicates a deformation titer of 1:LO
or less. ND, Not done.
Culture medium. Most spiroplasmas were cultured on
SP-4 or M1D broth medium (27). Strain LD-1 was grown
initially in DCCM medium containing an insect cell line (12),
but was subsequently cultured in cell-free DCCM medium.
Most culture media contained 500 U of penicillin per ml, and
broth and agar cultures were generally incubated at 30 or
32°C.
Antisera. Antisera to all strains were prepared in rabbits,
using previously described techniques (23). Serum was inactivated at 56°C for 30 min and stored at -20°C.
Serological tests. Disk growth inhibition tests were carried
out as previously noted (30, 32). Procedures for the metabolism inhibition and deformation tests have also been described earlier (29, 31, 33, 34).
Morphological tests. Cells of all strains from cultures in
logarithmic growth phase were examined by dark-field microscopy, using a magnification of 1 , 2 5 0 ~ For
.
electron
microscopic examination, cells were grown in approximately
20 ml of liquid medium and pelleted by centrifugation. The
cells were then fixed for 2 h in 3% glutaraldehyde, postfixed
in 1% osmium tetroxide for 1 h, dehydrated in acetone,
embedded in Epon, sectioned, and stained with 1% aqueous
uranyl acetate and Reynold's lead citrate.
Biochemical and physiological properties. Tests for the
ability of spiroplasmas to catabolize glucose and to hydrolyze arginine or urea were performed as described earlier (9,
28).
Genomic analysis. Methods used for DNA extraction and
determination of guanine-plus-cytosine (G+ C) content of the
DNA (by both buoyant density and melting temperature [T,]
procedures) were reported earlier (4, 5, 17).
RESULTS
Serological tests. The results of serological comparisons of
the 12 representative spiroplasma strains are summarized in
Tables 1 to 3. Although the LD-1 strain was compared in
reciprocal metabolism inhibition and deformation tests, difficulties in obtaining colonies of the organism on agar medium prevented its evaluation by the growth inhibition test.
In metabolism inhibition tests (Table l ) , most of the new
isolates showed few significant cross-reactions. When such
heterologous affinities were observed, they usually occurred
only as one-way crosses. For example, the PUP-1 antigen
was inhibited to high levels by the homologous antiserum
(metabolism inhibition titer of 1:13,000), but only to moderate levels (titers of 1:162 to 1:486) by antisera to five other
spiroplasma groups (1-3, X, XI, XIII, and XXI). In contrast,
when reciprocal metabolism inhibition tests, using antigens
to these five groups, were performed with PUP-1 antiserum,
none of the five antigens showed titers greater than 154. A
lesser number of one-way cross-reactions was observed with
TABLE 4. Reciprocal metabolism inhibition and growth
inhibition tests with three spiroplasmas of group XVI
Antigen
nigrovittatus (horsefly) were reported earlier by Clark and
colleagues (8). All spiroplasmas in this collection were triply
cloned (14), and representative cultures were deposited in
the American Type Culture Collection (see Table 5 for
accession numbers).
359
Ar-1357 (mosquito)
CC-1 (beetle)
MQ-6 (wasp)
Result" with antiserum prepared to strain:
Ar-1357
cc-1
MQ-6
39,00016"
48615
1,45815
16215
39,00015
4,37415
16214
48615
39,00015
(' Reciprocal of metabolism inhibition titedzones of growth inhibition (in
millimeters).
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360
INT. J .
TULLY ET AL.
SYST.
BACTERIOL.
FIG. 1. Electron micrograph of sectioned and stained spiroplasma cell pellets of strains representative of proposed new groups: (A) UU-1,
(B) EC-1, (C) 1-25, (D) CC-1, (E) DF-1, (F) TN-1, (G) PUP-1, (H) LD-1, (I) W115, (J) TG-1. Sections stained with 2% aqueous uranyl acetate
and Reynold’s lead citrate. Arrows indicate unit membrane. Bar, 100 nm.
other spiroplasma strains in both growth inhibition (Table 2)
and deformation (Table 3) tests.
Reciprocal serological cross-reactions in at least two serological test procedures (metabolism and growth inhibition)
were observed between the Ar-1357 and MQ-6 strains and
the CC-1 spiroplasma, designated herein as the representative of group XVI (Table 4). These results suggest the
existence of considerable serological heterogeneity in the
CC-1 group.
Morphological features. When broth cultures of the strains
in this collection were examined by conventional dark-field
microscopic techniques, usually after incubation at 30 to
32°C for 2 to 4 days, all showed the helical filaments of
spiroplasmas. Most organisms in the group also exhibited
some motility and the flexing movement observed with other
helical mollicutes. Thin-section electron microscopic examination of each of the representative organisms confirmed
the absence of a cell wall and periplasmic fibrils (Fig. 1).
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VOL. 37, 1987
SPIROPLASMA GROUPS
361
TABLE 5. Revised group classification of the genus Spiroplasma
Binomial andlor
common name
Group”
or
subgroup
Strainb
G+C
Glucose/
content arginine
(mal%) utilization
Principal host
Disease incited
S. cirri; citrus stubborn
spiroplasma
1-1
Mar0c-R8A2~(27556)
C189 (27665)
Israel
26
+/+
Dicots, leafhoppers
Citrus stubborn
S . melliferum; honey bee
spiroplasma
1-2
BC-3T (33219)
AS 576 (29416)
26
+/+
Bees
Honeybee spiroplasmosis
S . kunkelii; corn stunt spiroplasma
1-3
E275T (29320)
1-747 (29051)
B655 (33289)
26
+/+
Maize, leafhoppers
Corn stunt
277F spiroplasma
1-4
277F (29761)
26
+/+
Rabbit tick
None known
Green leaf bug spiroplasma
1-5
LB-12 (33649)
26
+/+
Green-leaf bug
None known
Maryland flower spiroplasma
1-6
M55 (33502)
ET- 1
28
+/+
Flowers, Eristalis fly
None known
Cocos spiroplasma
1-7
N525 (33287)
N628
26
+/+
Coconut palm
None known
S . phoeniceum; Vinca spiroplasma
1-8
P40T (43115)
26
+/+
Catharanthus roseus
Periwinkle disease
Sex ratio spiroplasmas
I1
DW-1 (43153)
26
ND
Drosophila
Sex ratio trait
S . fEoricola
I11
23-6T (29989)
BNRl (33220)
OBMG (33221)
26
+/-
Insects. flowers
None known
S . apis
IV
B31T (33834)
SR3 (33095)
PPSl (33450)
30
+/+
Bees, flowers
“May disease”
S . mirum
V
SMCAT (29335)
30
+I+
Rabbit ticks
Suckling mouse
cataract
GT-48 (29334)
TP-2 (33503)
Ixodes spiroplasma
VI
Y32 (33835)
25
+/-
Ixpdes pacifcus ticks
None known
Monobia spiroplasma
VII
MQ-1 (33825)
28
+ /-
Monobia wasp
None known
Syrphid spiroplasma
VIII
EA-1 (33826)
30
+I+
Eristalis arbustorum fly
None known
Cotinus spiroplasma
IX
CN-5 (33827)
29
+/+
Cotinus beetle
None known
S. cdicicola; mosquito
spiroplasma
X
AES-lT (35112)
26
+/-
Aedes mosquito
None knawn
Monobia spiroplasma
XI
MQ-4 (35262)
26
+/+
Monobia wasp
None known
Cucumber beetle spiroplasma
XI1
DU-1 (43210)
25
+/-
Diabrotica undecimpunctata None known
beetle
S . sabaudiense; mosquito spiroplasma
XI11
Ar-1343= (43303)
30
+/+
Aedes mosquito
None known
Ellychnia spiroplasma
XIV
EC-1 (43212)
26
+I-
Ellychnia corrusca beetle
None known
Leafhopper spiroplasma
xv
1-25 (43262)
26
+ /-
Cicadulina leafhopper
None known
Cantharis spiroplasma
XVI
CC-1 (43207)
Ar-1357
MQ-6
26
+/-
Cantharis beetle
None known
Continued on next page
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362
TULLY ET AL.
INT. J. SYST.BACTERIOL.
TABLE 5-Continued
Binomial and/or
common name
Group"
or
subgroup
Strainh
G+C
content
(mol%)
Glucose/
arginine
utilization
+I+
Disease incited
Chrysops fly
None known
Tabanus nigrovittatus
None known
Photuris pennsylvanicus
beetle
None known
+I+
Leptinotarsa decemlineata
None known
24
+ 1-
Prunus flower
None known
CT-lT (43302)
25
+I-
Culex tritaeniorhynchus
None known
TG-1 (43525)
26
+ 1-
Tabanus gladiator
None known
Deer fly spiroplasma
XVII
DF-1 (43209)
29
Tabanid spiroplasma
XVIII
TN-1 (43211)
25
Firefly spiroplasma
XIX
PUP-1 (43206)
26
+ 1+ 1-
Colorado potato beetle
spiroplasma
xx
LD-1 (43213)
25
Flower spiroplasma
XXI
W115 (43260)
S . taiwanense; mosquito
spiroplasma
XXII
Tabanid spiroplasma
XXIII
a
Principal host
Groups assigned on the basis of failure to cross-react in growth inhibition, metabolism inhibition, and deformation tests.
Accession numbers (in parentheses) are those of the American Type Culture Collection (ATCC). ND, Not done.
Biochemical features. Substrate utilization tests were confined to definition of the ability of the 12 new strains to
catabolize glucose and to hydrolyze arginine or urea. All
representatives in the group catabolized glucose, and the
majority of strains were able to hydrolyze arginine (Table 5 ) .
However, urea hydrolysis was not demonstrated with any
isolate.
Genome features. The DNA base composition (guanine
plus cytosine [G+C] content) varied from as low as 25 k 1
mol% to as high as 29 5 1 mol% (Table 5 ) . Most spiroplasmas in the new groups had a base content of 26 2 1 mol%.
DISCUSSION
Historical perspective. The classification scheme for
spiroplasmas proposed by Junca and colleagues in 1980 (17)
offered several important features and advantages. Perhaps
the most important of these was the demonstration that
classification concepts derived from genomic techniques
(DNA-DNA hybridization and base composition) were
closely analogous with concepts derived from serological
tests. Prior to that report (17), it was not yet clear that the
genus Spiroplasrna would eventually consist of a wide
assemblage of genomically (and therefore also serologically)
distinct clusters. During the 1970s, emphasis on the assemblage of group I subgroups had given the impression that
many new spiroplasmas would turn out to be closely or
partially related to known strains. For example, whereas
four strain groups (organisms assigned to subgroups 1-1 to
1-4) discovered during this period shared serological crossreactions, only three new strain groups (organisms assigned
to groups I1 to IV) were unrelated to group I organisms.
Also, group IV organisms proved to be serologically heterogeneous (22), although less so than group I organisms. The
initial perception of increasing numbers of clusters of partially related spiroplasmas was reinforced between 1980 and
1982 when three additional spiroplasma subgroups (1-5
[strain LB-121, 1-6 [M55and allied strains], and 1-7 [strain
N525 and allies]) were discovered and added (31) to the
classification scheme.
However, largely because of the discoveries of Clark (7),
by the time the second (31) and third (30) revisions of the
classification scheme had appeared, it was evident that
spiroplasma classification would probably reflect an increasing number of new, genomically distinct, insect-derived
organisms. With expansion of the search to holometabolous
insects, few, if any, of these new strains could now be
expected to show serological cross-reactions to S. citri or
other group I organisms.
The results reported herein, in which 12 clearly distinct
spiroplasma strain groups are added to the previously recognized 11groups, leave no doubt that other additions to the
list of Spiroplasma species (or groups) will occur in the near
future.
Revision of serological classification. In Table 5 we present
a revised group classification for the genus Spiroplasma and
propose group designations (XI1 to XXIII) for 12 new
strains. The recognition of 23 groups and 8 subgroups within
the genus considerably modifies and complicates existing
requirements for the classification of new isolates or strains.
Serological methods continue to be the most rapid and
reliable means for identification of spiroplasma strains. The
advantages of the deformation test as a procedure for
preliminary serological screening of candidates and the use
of the metabolism inhibition test for refined analysis of the
serological position of a new strain has been noted earlier
(31). However, each of these tests requires an extensive
inventory of group-specific antisera. As additional groups
are proposed in the future, the ability to meet the recommended serological criteria for description of new spiroplasma strains will be further complicated.
The experiences reported here with strain PUP-1 (group
XIX) and the two strains assigned to CC-1 (group XVI)
illustrate several important concepts in the classification of
spiroplasmas. An ad hoc committee appointed by the Subcommittee on Taxonomy of Mollicutes has recently recommended (28), in the event that heterologous cross-reactions
are observed in tests of a new spiroplasma strain with any
established group or subgroup, that the candidate organism
should be compared with existing groups by reciprocal
deformation, metabolism inhibition, and growth inhibition
tests. Such tests would determine whether the observed
reactions were consistent among serological tests and
whether they occurred only as one-way responses. As noted
above (Tables 1 to 3), moderate one-way serological crossreactions were often observed when strain PUP-1 was tested
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VOL. 37, 1987
SPIROPLASMA GROUPS
against sera directed against the other established and candidate groups. However, these results were only rarely
observed in the other direction and were never observed in
even two of the three serological tests. Thus, strain PUP-1
appears to represent a distinct group with no established link
to any other group or subgroup.
In contrast, consistent partial cross-reactions were observed in reciprocal serological tests between the group XVI
strain CC-1, MQ-6, and Ar-1357. These results suggest that
it may eventually be appropriate to designate serovars, or
possibly subgroups, within group XVI.
Spiroplasmas assigned to three of the new groups within
this revised classification (XII, XIII, and XXII) have recently been characterized, and new species epithets have
been proposed (I, 13; Abalain-Colloc et al., Int. J. Syst.
Bacteriol., in press). It is anticipated that at least three
additional groups (XIV, XVI, and XX) of the putative new
Spiroplasma species in this cluster of 12 new strains, and
perhaps all representative strains in groups I1 to XI, will
eventually be assigned species epithets, following completion of appropriate tests recommended for taxonomic descriptions of new species by the subcommittee (14). Whether
the remaining spiroplasmas described herein or new isolates
reported in the future should be given taxonomic designations will depend, to some extent at least, upon their
perceived basic or economic importance.
ACKNOWLEDGMENT
We thank Colis Blood for excellent technical assistance in the
preparation of numerous antisera employed in this study.
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