Contagious Equine Metritis: Characterization of Small and ...
Transcript of Contagious Equine Metritis: Characterization of Small and ...
Bull. Equine Res. Inst. No. 24, 23-32 (1987)
Contagious Equine Metritis: Characterization of Small and
Large Colonial Variants of Taylorella equigenitalis
lsolated from a Laboratory Strain
MaSanobu KAMADA, 1) Tohru ANZAI, 1) Takuml KANEMARU,1)
Ryuichi WADA 2) and Takeshi KUMANOMIDO2)
An experiment was carried out to clarify morphological, serological and bacteriological
characteristics of small and large colonial variants of Taylorella equigenitalis isolated from a laboratory strain. During subculture small colonial variants occurred spontaneously
by a reversion from small to large colonies in vitro. They were convex, smooth, friable and colorless with entire edges and grew slower on EGA than the large colonial variants
to reach a diameter of 0.7 mm or less at 7 days of incubation. On the contrary, the large
colonial variants were convex, smooth, viscid and grayish-white to light brown with entire
edges and grew relatively rapid and 3 to 5 mm in diameter at 7 days. Both colonial variants were visible on EGA at 3 days of incubation. When observed by the stereoscopic micro-
scope, the small and large colonial variants were classified into the transparent and opaque
phenotypes in opacity. No considerable difference was in biochemical characteristics be-tween both colonial variants. Besides, these variants had a close antigenic relationship
in the cross complement fixation and indirect hemagglutination tests.
Key words : Taylorella equigenitalis, Haemophilus equigenitalis, colonial variation, colonial
morphology, GEM
Introduction
Contagious equine metritis (CEM) is a contagious venereal disease of horses characterized by acute endometritis, show-ing clinical signs, such as vaginal exudate, and congestion and edema of the cervix and vagina. 1) It is caused by a micro-aerophilic, Gram-negative and nonfer-mentative coccobacillus or bacillus, which was newly classified as Taylorella equi-
genitalis. 2, 3)In general, changes in the colonial
morphology of bacteria are often indica-
tive of changes in antigenicity4-6) and in
virulence. 4, 7) Up to date, there have
been some reports on colonial variations
of T. equigenitalis in vitro ,8_10) but few
reports on changes in its antigenicity or
its virulence. In their previous paper, 11)
the authors described that small colonial
variants isolated from a laboratory strain
were much weaker in pathogenicity for
mares than large colonial variants. As a
result, it was indicated that changes in
colonial morphology were closely assoc-
iated with changes in virulence of T.
equigenitalis, as well as a number of other
pathogens.
Received for publication May 31, 1987.1) Epizootic Research Station, Equine Research Institute, Japan
Racing Association. 1400-4, Shiba, Kokubunji-machi, Shimotsuga-
gun, Tochigi 32904 JAPAN2) Equine Research Institute. Japan Racing Association.
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Kamada, M. et al.
The present study was carried out to
determine whether small colonial variants
of T. equigenitalis showing changes in
virulence for mares were associated with
changes in antigenicity. Its aims were to
classify these variants morphologically
according to the description of Hitchcock
et al.lo) and to know their bacteriological
characteristics. This paper deals with
various characteristics obtained from cul-
tural, morphological, biochemical and
serological tests.
Materials and Methods
Isolation history of bacteria and strains used. The To-89 strain 12) of T. equigenitalis was isolated from a mare with metritis in the Hidaka district of Hokkaido during the 1980 breeding season. After the 3rd sub-culture on Eugon chocolate agar (ECA) in an atmosphere containing 10% CO2, it revealed the presence of numerous colonial phenotypes. Small and large colonial variants, called the To-895 and To-89L strains, respectively, were picked up and then subcultured to ensure a single colony type. Small colonial var-iants were initially isolated at a low frequency and grew slower on ECA than any other colonial variant. They reached 0.7 mm or less in diameter at 7 days of incubation. On the contrary, large colo-nial variants 3 to 5 mm in diameter were isolated at a high frequency at 7 days of incubation. Both colonial variants were visible on ECA within 3 days after cultivation. After the 8th subculture, they were used for cultural, morphological,
biochemical and serological examinations.Cultural and morphological examinations.
Growth conditions were tested with ECA
(BBL) supplemented with glucose or
without. Moreover, X and V factor
requirements for growth were tested with
X and V discs (Nissui) on ECA and
brain heart infusion agar (Nissui). These
cultures were incubated at 37•Ž in an
atmosphere containing 10% CO2.
Colonial morphology was examined
with cultures on ECA and clear typing
medium, which was a modification of
the medium described by James and
Swanson. 13) Namely, each liter of med-
ium contained 3.75 g of trypticase pep-
tone (BBL), 7.5 g of thiotone peptone
(BBL), 4 g of K2HPO4 (anhydrous) 1 g
of KH2PO4 (crystalline), 5 g of NaGI, 2
g of soluble starch (BBL), and 10 g of
Noble agar (Difco). The medium was
autoclaved at 121 •Ž for 15 min. Ten
milliliters of Iso Vital X (BBL) and 2
ml of factors X and V containing 1 mg
of hemin (Sigma) per ml, 1 mg of L-
histidine per ml, and 0.3 mg of NAD
(Sigma) per ml were thoroughly mixed
into a cooled (50•Ž) medium before
pouring on plates. Plates had been stored
at 4•Ž until use. Cultures were incubated
in an atmosphere containing 10% CO2
at 37•Ž for 96 to 120 h. Morphological
observation on colonies was carried out
by a stereoscopic microscope equipped
with a substage reflecting mirror with a
diffusing surface and with coaxial vertical
illumination (SZH-151; Olympus Opti-
cal Co., Ltd., Tokyo).
Biochemical examination. The presence
of oxidase was determined by Kovac's
method. 14) The production of catalase
was assessed with growth removed from
an agar culture and placed in a drop of
3% H2O2 on a glass slide. Motility was
tested with growth by the hanging drop method,. The porphyrin test was carried
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Characterization of Colony Variants of CEMO
out by the method of Killian. 15) Most
of the biochemical tests were generally
performed with media described by Edw-ards and Ewing 16) according to the
previous reports. 2)
Fig. 1. Reversion from small to large colonial variants of the To-89S strain of T.
equigenitalis on Eugon chocolate agar at 8 days of incubation. Large colonies
looking like white powder
Cellular enzyme test. The presence of
97 specific cellular enzymes was tested
by means of API ZYM and additional
eight experimental ZYM galleries con-
sisting of ZYM II and AP 1-7 (API
System, S. A. ). Each strain was cultured
on an agar plate at 35•Ž for two days,
and the growth suspended in distilled
water (turbidity: No. 6 on the McFarland
scale). Two drops of the suspension were
incubated into each of the cupules in
the test galleries, which were incubated
at 35•Ž for 4 h. After the incubation, one
drop of each of API reagents A and B
was added to each of the cupules. The
results were read according to the direc-
tions of the manufacturer. Color grades
3+, 4+, and 5+ were interpreted as
positive.
Streptomycin sensitivity test. Two strains
of T. equigenitalis were examined for
susceptibility to streptomycin by the disk
diffusion method 17) with commercial discs
(50 ƒÊg per disc) produced by the Showa
Yakuhin Kanko Co., Ltd., Tokyo. Before
incubation at 37•Ž for 48 to 72 h, discs
were placed on the agar upon which a
lawn of bacteria had been streaked by a
bending glass rod. Resistance was scored
on the basis of inhibition of colonial
growth within a 5-mm radius of the discs.
Serological tests. After incubated on
ECA for 4 days, the To-89S and To-89L
strains were suspended in PBS and
centrifuged at 2000 rpm for 10 min.
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Kamada, M. et al.
After washed three times with PBS, the
sediment was suspended in PBS contain-
ing 0.2% formalin solution and stored at
4•Ž until use. It was used as an inocula
for immunization or as an antigen for
the complement fixation (CF) and in-direct hemagglutination (IHA) tests.
Fig. 2. Transparent colony of the To-89S
strain grown on clear typing medium,
as observed under the stereoscopic
microscope with reflection light
Fig. 3. Transparent colonies of the To-89S
strain grown on Eugon chocolate agar,
as observed under the stereoscopic mi-
croscope with coaxial vertical illumina-
tion
Fig. 4. Opaque colony of the To-89L strain
grown on clear typing medium, as ob-
served under the stereoscopic micros-
cope with reflection light
Fig. 5. Opaque colonies of the To-89L strain
grown on Eugon chocolate agar, as
observed under the stereoscopic micros-
cope with coaxial vertical illumination
Rabbits were injected intramuscularly five times with the above-mentioned inocula ranging from 109 to 1010 colony
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Characterization of Colony Variants of CEMO
forming units per ml at 3- to 4-day
intervals. They bled 2 weeks after the
last injection. Before the CF and IHA
tests, they were inactivated at 56•Ž for
30 min.
Table 1. Reversion of the To-89 strain of
T. equigenitalis from small to large
colonies in vitro during subculture
Time in days of incubation
Remarks. * Small and large colonies.** Small colonies alone .
The antigen was adjusted and both
tests were performed in the same manner
as described by Kamada et al. 18) and
Sahu et al. 19) The CF titer was expressed
as the reciprocal of the highest serum
dilution that had reduced hemolysis to
50% or less. The IHA titer was expressed
as the reciprocal of the highest serum
dilution that had agglutinated sheep
erythrocytes to 50% or over.
Results
Cultural and morphological characteristics. During subculture the To-89S strain of T. equigenitalis showed a reversion from small to large colonies in vitro (Table 1). Namely, after the 5th subculture, large colonies grew spontaneously on ECA at a low frequency after 6 days of incubation
(Fig. 1). When growth of the organism on ECA without glucose was comparable
to that on ECA with glucose, it had a
tendency to show a reversion from small
to large colonies at a very low frequency
during the prolonged cultivation.
Table 2. Colony size of the To-89S and To-
89L strains of T. equigenitalis at the
8th passage level grown on Eugon
chocolate agar
Time in days of incubation
Remark. The figure indicates the colony size (mm in diameter).
Table 3. Characteristics of the To-89S and
To-89L strains of T. equigenitalis
Remarks.* Negative . ** Positive. *** Arabinose, fructose,
glucose, inulin, lactose, maltose, mannitol, mannose,
raffinose, rhamnose, saccharose, salicin, sorbitol,
starch, trehalose, and xylose.
The To-89S strain grew slower on
ECA than large colonial variants to
reach 0.7 mm or less in diameter at 7
days of incubation, whereas the To-89L
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Kamada, M, et al.
strain grew relatively rapid to reach 3 to 5 mm in diameter (Table 2). Both colonial variants were visible on ECA within 3 days after cultivation. When observed by the stereoscopic microscope, small colonial variants were transparent with only a few translucent foci (Figs. 2 and 3) and large colonial variants opaque
(Figs. 4 and 5). The To-89S strain was classified as a transparent phenotype in opacity and the To-89L strain as an opaque phenotype.
Table 4. Enzymes of the To-89S and To-89L
strains of T, equigenitalis detected
commonly in the cellular enzyme test.
Table 5. Antigenic relationship between the To-89S and To-89L strains of T. equigenitalis by cross complement fi-xation (CF) and indirect hemagglu-tination (IHA) tests
Remarks.* The CF titer is expressed as the reciprocal of
the highest serum dilution that had reduced hemo-lysis to 50% or less. ** The IHA titer is exp-ressed as the reciprocal of the highest serum dilu-tion that had agglutinated sheep erythrocytes to 50% or over.
Biological and biochemical characteristics.
The To-89S and To-89L strains were
Gram-negative, nonmotile and capsulated
coccobacilli or short rods. The To-89L strain was convex, smooth, viscid and
grayish-white to light brown with entire edges, whereas the To-89S strain was convex, smooth, friable and colorless with entire edges.
In the conventional biochemical tests, no significant difference was observed in biochemical characteristics between both strains. Namely, they were positive only in catalase, oxidase and phosphatase tests, being negative in most of the tests
(Table 3). They were also positive for the porphyrin test and resistant to strepto-mycin. When cellular enzymes were ex-amined, many enzymes, such as phos-
phoamidase, glutamate transpeptidase, and arylaminidases of amino acids and oligopeptidases, were detected (Table 7).
Antigenic characteristics. In the CF and IHA tests, there have been few significant differences in antigenicity between the To-89S and To-89L strains of T, equi-
genitalis (Table 5). Namely, they were found to have a close antigenic relation-ship in both tests.
Discussion
Hitchcock et al. 10) described that all the colonial variants of T. equigenitalis
grown on clear typing medium could be classified into one of three groups based on opacity phenotype: transparent, o-
paque, and intermediate. In their report, transparent variants with a few opaque foci were classified as a transparent
phenotype. The small colonial variants used in the present experiment were transparent with only a few translucent foci on ECA and clear typing medium when observed by the stereoscopic micro-scope equipped with a substage reflecting
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Characterization of Colony Variants of CEMO
mirror with a diffusing surface and
equipped with coaxial vertical illumina-
tion. So the To-895 strain of T. equigenitalis
should be classified into the transparent
phenotype described by those authors. 10) On the other hand, the large colonial
variants used in this study were opaque
under the stereoscopic microscope with
reflection light. When observed by the
stereoscopic microscope with coaxial ver-
tical illumination, they were opaque with
a few translucent foci. According to
Hitchcock et al., 10) the opaque pheno-
type was expressed as opaque colonies in
opacity under the stereoscopic microscope
with reflection light or transmitted light.
Therefore, the To-89L strain should be
classified into the opaque phenotype de-
scribed by them. 10)
In general, variation of bacteria show-
ing the opaque or transparent appearance
of the colony can be induced by the
presence or absence of a capsule, 10) outer membrane protein 5, 20, 21) and other sur-
face appendages.7, 22, 23) These changes in
morphological properties often reflect
changes in virulence, 7, 21) and antigenic
properties. 20) On the other hand, morph-ological observations of T. equigenitalis
showed the presence of a fibrillar layer
on the outer membrane of various variants
or the presence of intracellular strands
comprised in a bleb-forming outer mem-
brane like pill of Neisseria gonorrhoeae. 10)
In another previous paper, 11) the authors
indicated that changes in colonial mor-
phology were closely associated with changes in virulence in T. equigenitalis, as
well as in a number of pathoens.
Besides, it was suggested that small
colonial variants might probably be close-
ly associated with changes in such adhesive
properties of bacteria as pili or outer membrane proteins because of rapid
elimination of them from the uterus and
cervix. The results in this study, however,
indicated that small and large colonial
variants showing an apparent difference
in virulence for mares had a close anti-
genic relationship, since no considerable difference was in serological and bio-
chemical characteristics between them.
Therefore, it is thought that changes in
the colonial morphology may probably
reflect changes in antigenic quantity, as
well as in antigenic quality. In future, it
will be necessary to carry out a molecular
microbiological investigation of colonial
variations of T. equigenitalis. Such inves-
tigation has been performed in the
studies of N, gonorrhoeae. 5, 6, 20, 21) It will
bring about success to the antigenic
analysis of colonial variations of T. equi-
genitalis.Sahu and Weber 8) and Sahu et al. 9)
described six coloniall variants of T.
equigenitalis: five phenotypes isolated from
clinical specimens and observed at 14
days of incubation or later, and a sixth
slow-growing phenotype grown in ECA
plates at 5 days of incubation or later. The tiny colonies of the six phenotypes
were 0.25 mm or less in diameter after 10
days of incubation, showing a reversion
from tiny to large colonies in vivo. At
present, it is unknown whether their tiny colonies exhibit opacity variation when
grown on ECA or clear typing medium or whether large colonies occur when
the tiny ones are grown on ECA during
a prolonged cultivation.
On the other hand, the To-895 strain
of T. equigenitalis, transparent phenotype
in opacity, presented the following char-
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Kamada, M. et al.
acteristics. Namely, small colonial var-
iants were convex, smooth, friable and
colorless with entire edges, grew slower
on ECA than the large colonial variants
to reach 0.7 mm or less in diameter at 7
days of incubation, and were visible on
ECA within 3 days after cultivation.
The bacteria exhibited a reversion spon-
taneously from small to large colonies in
vitro, but not in vivo. It is possible, how-
ever, that the small colonial variants
used in this study may give rise to large
colonies in vivo when inoculated at a
rather large inoculum size into experi-
mental mares because of the occurrence
of the above-mentioned reversion in vitro.
Therefore, it is of great interest in a
future study to determine whether the
two small colonial variants described
above are classified into the same pheno-
type, because they resemble in various
characteristics, except a slow-growing
phenotype.
Observation on the colonial morpho-logy of T. equigenitalis grown on ECA revealed the presence of small colonial variants in 15 of 33 primary isolations from mares with GEM (unpublished data). To date, pure isolates of small colonial variants have been obtained directly from 3 cases in the field. When these isolates belonging to the transparent
phenotype were subcultured on ECA, large colonial variants grew within 8 days after cultivation. These results indicate that colonial variations of T. equigenitalis exhibiting changes in virulence occur in vitro and in vivo and that they have been spread among mares in the field. Since GEM broke out in Japan for the first
time during the 1980 breeding season, 12)
it is still difficult to detect carrier mares
of the disease clinically, because there is
an increase in number of naturally in-
fected mares presenting mild or no clinical
signs of the disease. The occurrence of
colonial variations exhibiting changes in
virulence might have been responsible
for the gradual decrease in number of
naturally infected horses showing typical
clinical signs of GEM in the field.
It depends upon the development of a
diagnostic method available for the de-
tection of carrier mares that any measure
taken for the prevention of GEM finally
leads to a success in the control or erad-
ication of the disease in Japan and other
counties. Usually, the final diagnosis of
the disease is carried out by the isolation
of etiological bacteria from the genital
tract of mares and stallions. The small
colonial variants have no characteristics
in colonial morphology, except that they
are transparent and of small phenotype.
It is very difficult to differentiate them
from other bacteria showing almost the
same colonial appearance at the time of
primary isolation. In addition, the pre-sence of such slow-growing strains of T.
equigenitalis as described in the previous
reports17, 24) makes bacterial differentiation
rather difficult. Therefore, the serological
reactions, including the CF and IHA
tests and enzyme linked immunosorbent
assay, may be available as additional
means for the diagnosis of the disease.
The results of the present cross CF and
IHA tests indicated a close antigenic
relationship between small and large
colonial variants of T. equigenitalis.
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Characterization of Colony Variants of CEMO
Acknowledgments
The authors are grateful to Drs. T. Hirasawa, A. Amada and Y. Fukunaga, of their institute, for val-uable advice given to the present work, and to Miss Inoue for technical assistance. Thanks are also due to the members of the Hidaka Agricultural Mutual Aid Association who extended a helping hand in collect-ing specimens, and to Dr. K. Hirose and the staff members of the Hidaka Livestock Hygiene Service Center, Prefecture of Hokkaido, for cooperation for this work.
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馬伝染性 子宮炎:保 存株か ら分離 されたTaylorella equigenitalisの 小型 および大型集落 変異株
の性 状:鎌 田正信1)・安斉 了1)・兼丸卓 美1)・和 田隆一・2)・熊埜 御堂 毅2)(1)日本 中央 競馬会競走馬総
合研究所栃 木支所, 2)日本中央競馬会競走馬総合研究所)一 保存株 か ら分離 されたT. equigenitalzs
の小型お よび大型集落変異株について形態学的,血 清学的,細 菌学的性状を調べた.小 型集落変異株 は
継代 中にご大 型集落を散 発的に産 生 した.こ の現象は ブ ドウ糖無添加培地 よ り添加培地で高頻 度で認め
られ る傾 向を示 した.小 型集落変異株は隆起 した平滑 な粘稠性 の少 ない無 色透 明な円形 集落 を形成 し,
発育速度は大型集落変異株にご比べて遅 く,培 養7日 目で直径0.7mm以 下 であった.一 方,大 型集落
変異 株は隆起 した平滑な粘稠性の強い灰 白色か ら淡褐色不透 明な円形集落 を形成 し,発 育速度が早 く,
培 養7日 目で直径3か ら5mmで あ った.実 体顕微鏡に よる集落形態 の観 察で,小 型 お よび大 型集落
変異株は透明度変異においてそれぞれ透 明,不 透 明変異株 と分類 された.な お,今 回の実験 において
両株間での生化学性状 の差は認め られず,交 叉補体結合 お よび間接血球凝集 試験 において両株は類似
した抗原性を有 す ることが明 らか とな った.
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