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Veterinary Microbiology 97 (2003) 111–122

Recent advances in molecular epidemiology and

detection of Taylorella equigenitalis associated

with contagious equine metritis (CEM)

Motoo Matsuda a , ∗ , John E. Moore b

a Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University,

Fuchinobe 1-17-71, Sagamihara 229-8501, Japan

b Northern Ireland Public Health Laboratory, Department of Bacteriology,

Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK

Received 18 February 2003; received in revised form 29 July 2003; accepted 15 August 2003

Abstract

In the present review article, recent molecular advances relating to studies with Taylorella equigen-

italis , as well as the recently described second species of the genus Taylorella , namely Taylorella

asinigenitalis , have been described. Molecular genotyping of T. equigenitalis strains by pulsed-ﬁeld

gel electrophoresis (PFGE) after digestion with the suitable restriction enzyme(s) enabled the ef-

fective discrimination of strains, thus allowing the examination of the scientiﬁc mechanism(s) for

its occurrence and transmission of contagious equine metritis (CEM). Alternatively, polymerase

chain reaction (PCR) ampliﬁcation and nucleotide sequencing of the 16S ribosomal DNA sequence

and/or the other species speciﬁc sequence(s) as targets were conﬁrmed to be effective for

identiﬁcation of T. equigenitalis . These new analytical methods at the genomic DNA level also en-

abled the discrimination of the newly discovered donkey-related T. asinigenitalis from T. equigen-

italis , and moreover, the performance of phylogenetic analysis of genus Taylorella organisms

with other closely related genera. Furthermore, detailed analysis of the genes responsible for CEM

within the T. equigenitalis genome would be useful to help elucidate the pathogenic virulence

and transmission mechanisms associated with the important equine pathogen associated with

CEM.

Keywords: Taylorella equigenitalis ; Pulsed-ﬁeld gel electrophoresis; Molecular genotyping; Phylogenetic

analysis; Molecular identiﬁcation; Taylorella asinigenitalis

∗ Corresponding author. Tel.: + 81-42-769-1942; fax: + 81-42-754-7661.

E-mail address: matsuda@azabu-u.ac.jp (M. Matsuda).

doi:10.1016/j.vetmic.2003.08.001

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M. Matsuda, J.E. Moore / Veterinary Microbiology 97 (2003) 111–122

1. Introduction

Contagious equine metritis (CEM) is a bacterial infectious disease of horses caused by

Taylorella equigenitalis , a Gram-negative eubacterium. CEM is characterized by discharge

of large amounts of mucopurulent ﬂuid from the vagina, infertility, or early abortion, and is

a sexually transmitted disease. An inﬂammation of the endometrium of mares often results

in temporary infertility followed by uneventful recovery. However, a proportion of mares

may become long-term symptomless carriers and these together with infected stallions, may

spread infection during mating.

CEM was ﬁrst reported in the mares of thoroughbred horses during the 1977 breeding sea-

son in UK ( Crowhurst, 1977 ) and the causative organism was ﬁrst described as Haemophilus

equigenitalis by Taylor et al. (1978) . Thereafter, the disease spread rapidly among horses

in Ireland ( Timoney et al., 1977 ), France ( Powell et al., 1978 ), Australia ( Hughes et al.,

1978 ), Belgium ( ter Laak et al., 1989 ), the US ( Swerczek, 1978 ) and Germany ( Mumme

and Ahlswede, 1979 ) during the period from 1977 to 1979, as shown in Fig. 1 . CEM is trans-

mitted by mating and is so highly contagious that efforts have been made in every country

to control and prevent this infectious disease. In the UK, the Horserace Betting Levy Board

(HBLB) has published the Board’s Code of Practice to eliminate the transmission of the

disease in horses ( Anonymous, 2002b ).

In relation to H. equigenitalis , on the basis of the characterization in terms of a numerical

analysis of phenotypic characteristics, determination of genome DNA G

Fig. 1. Chronological detection of CEM in several countries and geographical distribution of CFGE genotypes of

T. equigenitalis as described previously ( Kagawa et al., 2001a; Matsuda et al., 1997, 1998, 1999, 2000; Miyazawa

et al., 1995 ).

C content and

DNA–DNA hybridization to determine its taxonomic position, Sugimoto and colleagues

proposed the transfer of H. equigenitalis Taylor et al. (1978) to the new genus Taylorella gen.

M. Matsuda, J.E. Moore / Veterinary Microbiology 97 (2003) 111–122

113

nov. in 1983 ( Sugimoto et al., 1983 ), which was subsequently approved by the International

Committee on Systematic Bacteriology ( Anonymous, 1984 ).

In Japan, the ﬁrst occurrence of CEM in horses and the isolation of its causal agent

from cervical swabs obtained from mares with endometritis, occurred in the spring of 1980

( Sugimoto et al., 1980 ). Moreover, CEM and its causal agent, T. equigenitalis , has since

been detected in many countries and in various breeds of horses ( ter Laak et al., 1989 ). More

recently, the presence of CEM and the ﬁrst isolation of T. equigenitalis from thoroughbred

mares in Turkey have been described ( Ozgur et al., 2001 ). CEM still occurs sporadically, as

was observed recently in the UK, with 14 cases in 1996, 2 cases in 1997, and 3 cases in 2002

( Anonymous, 2002a,c, 2003 ; Jackson et al., 2002; Moore et al., 2003 ) having been described.

These cases indicate that outbreaks of CEM, caused by T. equigenitalis may occur in new

countries as well as in countries where the disease was previously described. In these coun-

tries, CEM continues to be an economic problem, although effective control measures and

restrictions of CEM have been introduced to prevent the transmission of the disease locally,

nationally and internationally. Moreover, these recent reports on the isolation of T. equigen-

italis raise the issue of the importance of molecular genotyping, phylogenetic analysis and

molecular identiﬁcation of the causal bacterium of CEM. As described later, a phylogenetic

analysis of the 16S ribosomal DNA sequence of T. equigenitalis revealed a phylogenetic

position of the organism distinctly separate from Haemophilus inﬂuenza , Alcaligenes xy-

losoxidans , Bordetella bronchiseptica , Spirillum volutans and Chromobacterium ﬂuviatile

( Bleumink-Pluym et al., 1993 ). Recently, three T. equigenitalis -like organisms that were

phenotypically indistinguishable from T. equigenitalis have been isolated from three male

donkeys in USA ( Katz et al., 2000; Jang et al., 2001 ). Based on the results of the 16S rDNA

sequence analysis, genomic DNA–DNA hybridization studies, and the G

2. Molecular genotyping of T. equigenitalis

No serological differences have been observed among the isolates of T. equigenitalis

examined throughout the world ( Croxton-Smith et al., 1978; Taylor et al., 1978; Benson

et al., 1978; Fernie et al., 1979 ), hence no attempts have thus been made to serologically

type T. equigenitalis isolates to date.

Since CEM was reported for the ﬁrst time by Crowhurst in UK in 1977 ( Crowhurst,

1977 ), no serological and biological typing methods were applied during the ﬁrst 10 years

of the disease in order to clarify the scientiﬁc mechanism(s) for its occurrence and transmis-

sion. In the 1990s, Bleumink-Pluym and her colleagues at the University of Utrecht, The

C composition

analysis of the genomic DNAs between the T. equigenitalis -like organisms and T. equigen-

italis , a new species, Taylorella asinigenitalis , within the genus Taylorella was proposed

for the T. equigenitalis -like organisms from the male donkeys ( Jang et al., 2001 ).

Although some review articles of CEM and T. equigenitalis have already been published

( Eaglesome and Garcia, 1979; Brewer, 1983; ter Laak et al., 1989; Timoney, 1996 ), recent

advances in the application of molecular methods to the study of T. equigenitalis has not

been reviewed. Therefore, in the present paper, we describe recent molecular advances

in the study of T. equigenitalis , as well as the second species of the genus Taylorella , T.

asinigenitalis , which has recently been added to this genus.

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M. Matsuda, J.E. Moore / Veterinary Microbiology 97 (2003) 111–122

Netherlands, employed ﬁeld inversion gel electrophoresis (FIGE; Southern and Elder, 1995 )

in order to perform an epidemiologic study of T. equigenitalis strains ( Bleumink-Pluym

et al., 1990 ). FIGE is a variation of pulsed-ﬁeld gel electrophoresis (PFGE), capable of

separating DNA fragments up to 2000 kb (2 Mb) long ( Schwartz and Cantor, 1984 ). In

their study, strains of T. equigenitalis from throughout the world were characterized by

FIGE through examination of fragments of genomic DNA obtained by digestion with the

low-cleavage-frequency restriction enzyme, Apa I, resulting in a division into ﬁve distinct

FIGE groups, A–E ( Bleumink-Pluym et al., 1990 ). Strains from thoroughbred horses from

all continents belonged to one group, A. The other strains from non-thoroughbred horses

from various countries were different and could be divided into four groups (groups B–E).

Intact and high molecular genomic DNA was prepared from cells embedded in agarose

blocks ( Schwartz and Cantor, 1984 ) and was used for restriction enzyme digestion as a

substrate in the study.

In addition, Thoresen et al. (1995) demonstrated the genomic homogeneity among strains

of T. equigenitalis from Norwegian trotting horses, as well as strains from various Swedish

breeding horses by chromosomal DNA ﬁngerprinting after polyacrylamide slab gel elec-

trophoresis of restriction enzyme Bam HI-digested genomic DNA. The other strains of T.

equigenitalis isolated in Sweden, The Netherlands and elsewhere were divided into 10

groups.

In 1995 and 1999, Miyazawa and colleagues ( Miyazawa et al., 1995; Matsuda et al., 1999 )

reported genotyping by crossed-ﬁeld gel electrophoresis (CFGE) of restricted genomic

DNAs after separate digestion with Apa I and Not I of 130 isolates, of T. equigenitalis from

thoroughbred mares with CEM and from thoroughbred stallions, isolated in Japan from

1980 to 1996 and concluded that a single genotype, designated genotype J was present

among all of the isolates. CFGE is a variation of PFGE in which the circular gel is turned

at each pulse interval under the ﬁxed-electric ﬁeld ( Southern and Elder, 1995 ). A genotypic

difference between the Japanese isolates and an American prototype strain, Kentucky 188

( Bryans and Hendricks, 1979 )of T. equigenitalis and also a slight genotypic difference

between NCTC11184 T ( Taylor et al., 1978 ) and Kentucky 188 were demonstrated in their

CFGE proﬁles after separate digestion with Apa I and Not I. Consequently, no strains of T.

equigenitalis with any genotype other than genotype J may have reached Japan from 1980 to

1996 and strains with the genotype J have survived in Japan since the ﬁrst dissemination of

CEM into Japan. Therefore, the authors have suggested that it is important to take rigorous

countermeasures against carrier horses of T. equigenitalis since import inspection for CEM

from the abroad could be successful in this country.

The genomic DNA of isolates of T. equigenitalis , isolated from seven Norwegian trotters

and a Norwegian pony with CEM in Norway, was examined by the same methodology as

that used for the Japanese strains described above ( Matsuda et al., 1997 ). The respective

electrophoretic proﬁles of the fragments were essentially identical among the eight isolates

but differed from those of T. equigenitalis NCTC11184 T and Kentucky 188. They also

exhibited slight differences from proﬁles obtained from Japanese isolates. Conclusively,

these results may possibly suggest a common genotype, designated genotype N and a

common source of infection for all these eight isolates in Norway. Also the strains examined

by Thoresen et al. (1995) and described above were known to belong to the same genotype

N( Matsuda et al., 1997 ).

M. Matsuda, J.E. Moore / Veterinary Microbiology 97 (2003) 111–122

115

Matsuda et al. (1998) , then described the molecular genotyping of isolates of T. equigen-

italis isolated in Ireland and the US. The CFGE proﬁles after separate digestion with Apa I

and Not I of genomic DNAs from isolates of T. equigenitalis isolated in Ireland in 1978–1982

were of three distinct genotypes. The isolates in one of these three genotypes gave CFGE

proﬁles identical to that of an American prototype strain, Kentucky 188, but were different

from T. equigenitalis strains NCTC11184 T and an isolate, EQ59, in Japan. Additional strains

isolated in the US (Maryland, Kentucky, and Virginia) gave four distinct genomic CFGE

proﬁles. All four genotypes in the US were distinctly different from that of T. equigeni-

talis NCTC11184 T or that of the Japanese isolate. The proﬁle of one genotype in the US

was identical to that of Kentucky 188. Thus, one genotype, that of the Kentucky 188, was

common to certain of the Irish and American isolates, suggesting a common source. It is of

considerable interest that the subtyping into four distinct genotypes clearly correlated with

the geographical differences in the origin of the eight isolates of T. equigenitalis , including

Kentucky 188. Two genotypes were demonstrated among strains isolated in Maryland and

it was suggested that this may be due to the presence of a quarantine station in that state for

horses entering the US, though the number of strains examined was limited.

The CFGE proﬁles of ﬁeld isolates of T. equigenitalis isolated between 1979 and 1980

from thoroughbred horses in South Australia could be classiﬁed into three distinctly different

genotypes, one of which was identical to the CFGE proﬁles of Kentucky 188 ( Matsuda

et al., 2000 ). Overall, the Kentucky 188 genotype has been detected for 22 isolates among

34 isolates of T. equigenitalis in the three countries.

In six European countries, namely Belgium, UK, Finland, France, Sweden and Switzer-

land, 18 genotypes were demonstrated using CFGE proﬁles after separate digestion with

Apa I and Not I of the genomic DNA from 46 isolates of T. equigenitalis ( Kagawa et al.,

2001a ). Between two and nine genotypes were detected in each country. Two English iso-

lates and four French isolates gave identical CFGE proﬁles to those of Kentucky 188 from

the US. Overall, the genotype of Kentucky 188 has been detected in 28 isolates from the ﬁve

countries. Thus, these isolates may have been derived from a common source. A common

genotype was found in isolates from Belgium and UK and also in isolates from France and

Switzerland. The 18 genotypes that were demonstrated among the 46 isolates obtained in

Belgium, UK, Finland, France, Sweden, and Switzerland appear to be distinctly different

from the other eight genotypes, obtained from horses in Norway, Ireland, US, and Australia,

as described above ( Matsuda et al., 1997, 1998, 2000 ), except for the Kentucky 188 genotype

( Kagawa et al., 2001a ). These results strongly suggest that such heterogeneous genotypes

of T. equigenitalis have a global distribution ( Fig. 1 ). Moreover, no isolates whose genomic

DNA could be cleaved by the two restriction enzymes, Apa I and Not I, and fractionated by

CFGE were found ( Kagawa et al., 2001a ).

The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from stallions

in France, Sweden and Switzerland gave no evidence of a sex-related difference in the ge-

nomic DNA. In the pioneering study ( Taylor et al., 1978 ), it was shown that the CEM organ-

ism was highly resistant only to streptomycin, although some degree of resistance was found

against lincomycin, clindamycin, sulphamathoxazole and trimethoprim. Sugimoto et al.

(1983) reported that 10 Japanese isolates, NCTC11184 T , and Kentucky 188 were all highly

resistant to streptomycin (MIC, 128

g/ml). Most, but not all, isolates of T. equigenitalis

isolated internationally have been found to be resistant to this antibiotic. The genomic DNA

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