Veterinary Research Communications, 25 (2001) 565^575 # 2001 Kluwer Academic Publishers. Printed in the Netherlands

Demonstration of Heterogeneous Genotypes of Taylorella equigenitalis Isolated from Horses in Six European Countries by Pulsed-Field Gel Electrophoresis S. Kagawa1*, F. Klein2, L. Corboz3, J.E. Moore4, O. Murayama1 and M. Matsuda1 1 Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, 1-17-71 Fuchinobe, Sagamihara, 229-8501 Japan; 2Laboratoire Departemental de L'Orne, 19^21 rue Candie, BP7, 61001 Alencon Cedex, France; 3 Institut fur Veterinarbakteriologie der Universitat Zurich, 8057 Zurich 11, Switzerland; 4 Molecular Epidemiology Research Unit, N. Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, N. Ireland, UK *Correspondence: E-mail: [email protected] Kagawa, S., Klein, F., Corboz, L., Moore, J.E., Murayama, O. and Matsuda, M., 2001. Demonstration of heterogeneous genotypes of Taylorella equigenitalis isolated from horses in six European countries by pulsed-¢eld gel electrophoresis. Veterinary Research Communications, 25(7), 565^575 ABSTRACT Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-¢eld gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classi¢ed into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE pro¢les to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related di¡erence in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classi¢ed into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM. Keywords: contagious equine metritis, diagnosis, DNA, genotypes, horses, pulsed-¢eld electrophoresis, Taylorella equigenitalis Abbreviations: CEM, contagious equine metritis; MIC, minimal inhibitory concentration; STM, streptomycin; PFGE, pulsed-¢eld gel electrophoresis

INTRODUCTION Taylorella equigenitalis, a Gram-negative eubacterium, is an important pathogen responsible for contagious equine metritis (CEM) (Taylor et al., 1978; Timoney and Powell, 1982; Ter Laak et al., 1989). CEM was ¢rst reported in England (Crowhurst, 1977) but, since then, CEM and its causative agent have been detected in many countries, and in various breeds of horses (Timoney et al., 1977; Hughes et al., 1978; 565

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Simpson and Eaton-Evans, 1978; Swerczek, 1978; Ter Laak et al., 1989). It was isolated in Japan in 1980 (Sugimoto et al., 1980). Genomic restriction fragment length polymorphism analysis, based on the pulsed¢eld gel electrophoresis (PFGE) pro¢les of restricted genomic DNA fragments from isolates of T. equigenitalis has enabled us to genotype strains of this organism (Miyazawa et al., 1995; Matsuda et al., 1998). In the present study, we describe the detection of heterogeneous genotypes using PFGE after separate digestion with ApaI and with NotI of the genomic DNA from 46 isolates of T. equigenitalis obtained from six European countries, namely Belgium, England, Finland, France, Sweden and Switzerland. We also analysed 12 European isolates of T. equigenitalis obtained from male horses and 11 streptomycin (STM)susceptible European isolates by PFGE, for the ¢rst time. MATERIALS AND METHODS Two Belgian isolates (Bel266-5923 and Bel266-6959) obtained in 1986 and in 1988 were provided by Dr L. Deveriese, Universiteit Gent, Merelbeke. Two of the English isolates (N217-79 and N480-82) were provided by Dr A.M. van der Zeijst, University of Utrecht, Utrecht. Three English isolates (Eng1, Eng2 and Eng3) were provided by Dr N. Chanter, The Animal Health Trust, Newmarket. Nine English isolates (Eng4 to Eng12), obtained in Newmarket, were provided by Mr S. Neil, Department of Agriculture for Northern Ireland, Belfast. Two Finnish isolates (Fin583 and Fin1698) were provided by Dr E.-L. Hintikka, National Veterinary and Food Research Institute, Helsinki. Ten French isolates were obtained from nine horses, including two males, in France from 1993 to 1998. Five Swedish isolates, one from a female and four from male horses, were provided by Dr E. Olsson, The National Veterinary Institute, Uppsala. Twelve Swiss isolates were obtained from clinical cases in ¢ve male and six female horses in Switzerland from 1988 to 1990, since when they have been stored at the University of Zurich. All of these strains are listed in Table I, which shows that 3 of the 5 Swedish isolates and 8 of the 12 Swiss isolates were susceptible to STM. T. equigenitalis NCTC11184T, isolated in 1977 in England (Taylor et al., 1978), an American prototype strain, Kentucky 188, which was isolated in 1978 (Bryans and Hendricks, 1979) and a Japanese strain, EQ59, isolated in 1980 (Sugimoto et al., 1980; Miyazawa et al., 1995) were used as reference strains to facilitate a genotypic comparison based on PFGE pro¢les. The culture of the strains, the preparation of agarose blocks and the techniques for digesting the DNA separately with ApaI and with NotI have been described previously by Miyazawa and colleagues (1995), who also described the basis for choosing these two particular restriction enzymes.

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RESULTS The PFGE pro¢les after separate digestion with ApaI or NotI of genomic DNA from the two Belgian isolates of T. equigenitalis, the reference strains NCTC11184T, Kentucky 188 and EQ59, and the two English isolates N217-79 and N480-82, are shown in Figure 1. As shown in Figure 1A, ApaI cleaved the genomic DNA from the two Belgian isolates into two subtypes. Figure 1B demonstrates that NotI also allowed the classi¢cation of these two isolates into two subtypes. Figure 1 also shows that the ApaI and NotI pro¢les of the Belgian isolate Bel1266-5923 were identical to those of the English isolate, N217-79. The PFGE pro¢les given by separate digestion with ApaI or NotI of the genomic DNA from the ¢ve English isolates (Eng1 to Eng3, N217-79 and N480-82) of T. equigenitalis, and the three reference isolates are shown in Figure 2. As shown in Figure 2A, ApaI cut the genomic DNA from the ¢ve English isolates into two subtypes. The pro¢le for one of these subtypes is seen in lanes 4^7 and the other in lane 8 of this ¢gure. Figure 2B demonstrates that NotI also divided the ¢ve isolates into two subtypes in a similar manner to ApaI. PFGE pro¢les after separate digestion with ApaI or NotI of the genomic DNA from the other nine English isolates (Eng4 to Eng12) and NCTC11184T classi¢ed the 10 isolates into seven genotypes (see Figure 7). Two English isolates (Eng6 and Eng9) had the identical PFGE pro¢les to those of Kentucky 188 (see Figure 7). As shown in Figure 3, ApaI (Figure 3A) and NotI (Figure 3B) cleaved the genomic DNA from the two Finnish isolates (Fin583 and Fin1698) into two subtypes. ApaI cleaved the genomic DNA from the 10 French isolates (Fr-1 to Fr-10) into two subtypes (Figure 4A). The pro¢le for one of these is shown in lanes 7, 9, 12 and 13 and the other in lanes 4 to 6, 8, 10 and 11 in Figure 4A. Figure 4A also shows that the ¢rst of these two subtypes had an identical ApaI pro¢le to that of Kentucky 188. Figure 4B demonstrates that NotI also classi¢ed the 10 isolates into two subtypes in a similar manner to ApaI. By the same methodology, the genomic DNA from the ¢ve Swedish isolates (Swe-1 to Swe-5) was shown to comprise three subtypes (Figures 5 and 7) and that from the 12 Swiss isolates (Swi-1 to Swi-12) fell into three subtypes (Figures 6 and 7). Consequently, after separate digestion with ApaI or NotI of the genomic DNA from 46 isolates of T. equigenitalis obtained from horses in six European countries, the PFGE pro¢les were heterogeneously classi¢ed into 18 genotypes. DISCUSSION To our knowledge, this is the ¢rst report di¡erentiating a number of isolates of T. equigenitalis obtained from male horses by PFGE. The analysis of genomic DNA from three French, four Swedish and ¢ve Swiss isolates of T. equigenitalis obtained from male horses gave no evidence of a sex-related di¡erence in the genomic DNA of isolates of T. equigenitalis.

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TABLE I The origin and characteristics of isolates of T. equigenitalis analysed in the present study

Strain no. Bel266-5923 Bel266-6959 NCTC11184Ta N217-79 N480-82 Eng1 Eng2 Eng3 Eng4 Eng5 Eng6 Eng7 Eng8 Eng9 Eng10 Eng11 Eng12 Fin583 Fin1698 Fr-1 Fr-2 Fr-3 Fr-4 Fr-5 Fr-6 Fr-7 Fr-8b Fr-9 Fr-10 Swe-1 Swe-2 Swe-3 Swe-4 Swe-5 Swi-1 Swi-2 Swi-3 Swi-4 Swi-5 Swi-6

Breed

Streptomycin susceptibility

Sex

Nonthoroughbred Nonthoroughbred Thoroughbred Thoroughbred Thoroughbred NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA French Trotter French Trotter French Trotter French Trotter French Trotter Trotter Cold-blood Trotter Fiord Horse Ardeness Fiord Horse Cold-blood Cold-blood Cold-blood Thoroughbred Thoroughbred Cold-blood

NA NA Resistantd Resistantd Resistantd NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Resistant Resistant Resistant Resistant Resistant Resistante Resistante Susceptiblee Susceptiblee Susceptiblee Resistant Resistant Resistant Susceptible Susceptible Resistant

NA NA Female NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Female Male Male Male Female Female Male Male Male Male Female Male Female Malef Female Female

Country of isolation Belgium Belgium England England England England England England England England England England England England England England England Finland Finland France France France France France France France France France France Sweden Sweden Sweden Sweden Sweden Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland

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TABLE I (Cont)

Strain no. Swi-7c Swi-8c Swi-9 Swi-10 Swi-11 Swi-12 Kentucky 188 EQ59

Breed

Streptomycin susceptibility

Sex

Thoroughbred Thoroughbred Thoroughbred Warm-blood Thoroughbred Thoroughbred NA Thoroughbred

Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible Resistantd Resistantd

Female Female Malef Female Malef Malef Female Female

Country of isolation Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland USA Japan

NA, not available a

Type strain

b c

An isolate from the same horse as Fr-7, but collected 1 week later

Swi-7 and Swi-8, isolates from di¡erent localization of the same horse

d

MICs for STM-resistant isolates ranged from 128 to 4512 mg/ml

e

Resistance to STM was judged by the ability to grow on agar plates containing STM (0.2 g in 1000 ml). No MICs for the resistant strains were estimated

f

Four di¡erent horses from a circus

In a previous study (Taylor et al., 1978), it was shown that the CEM organism was highly resistant only to STM, although some degree of resistance was found against lincomycin, clindamycin, sulphamethoxazole and trimethoprim. Sugimoto and colleagues (1983) reported that 10 Japanese isolates (MIC, 128 mg/ml), NCTC11184T and Kentucky 188 were all highly resistant to STM. Most strains of T. equigenitalis isolated internationally have been found to be resistant to STM. The assessed susceptibility to STM was not available for some strains of T. equigenitalis (Table I), but we were able to analyse the genomic DNA from 11 STM-susceptible isolates: 3 of 5 obtained in Sweden and 8 of 12 in Switzerland (Table I). Each of the six genotypes involved had a single phenotype of resistance or susceptibility to STM in both countries. It would be very interesting if genotypes by PFGE were found to contain both STM-susceptible and STM-resistant isolates, since this would suggest that they have a common origin. Previously, a common genotype was demonstrated for the Kentucky 188 and 18 isolates of T. equigenitalis among 27 obtained in Ireland and in the United States (Matsuda et al., 1998). In addition, this genotype occurred in 4 of 7 isolates obtained in South Australia (Matsuda et al., 2000). The present study showed that this genotype was found in six isolates obtained in England and in France. Overall, the genotype of Kentucky 188 has been detected in 28 isolates from ¢ve countries. Thus, these 28 isolates may have been derived from a common source.

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Figure 1. Fractionation by PFGE after separate digestion with ApaI (A) and NotI (B) of restricted genomic DNA from isolates of T. equigenitalis obtained in Belgium, as well as some isolates obtained in England. Lane Y, chromosomal genomic DNA from Saccharromyces cerevisiae YNN295. Lane L, lambda concatemeric DNA. Lane H, HindIII digest of lambda DNA. Lane 1, NCTC11184T; lane 2, Kentucky 188; lane 3, EQ59; lane 4, N480-82; lane 5, N217-79; lane 6, Bel266-5923; lane 7, Bel266-6959

Figure 2. Fractionation by PFGE after separate digestion with ApaI (A) and NotI (B) of restricted genomic DNA from isolates of T. equigenitalis obtained in England. For lanes Y, L and H, refer to the legend for Figure 1. Lane 1, EQ59; lane 2, Kentucky 188; lane 3, NCTC11184T; lane 4, Eng1; lane 5, Eng2; lane 6, Eng3; lane 7, N217-79; lane 8, N480-82

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Figure 3. Fractionation by PFGE after separate digestion with ApaI (A) and NotI (B) of restricted genomic DNA from isolates of T. equigenitalis obtained in Finland. For lanes Y, L and H, refer to the legend for Figure 1. Lane 1, NCTC11184T; lane 2, Kentucky 188; lane 3, EQ59; lane 4, Fin583 (A) or Fin1698 (B); lane 5, Fin1698 (A) or Fin583 (B)

Figure 4. Fractionation by PFGE after separate digestion with ApaI (A) and NotI (B) of restricted genomic DNA from isolates of T. equigenitalis obtained in France. For lanes Y, L and H, refer to the legend for Figure 1. Lane 1, EQ59; lane 2, NCTC11184T; lane 3, Kentucky 188; lane 4, Fr-1; lane 5, Fr-2; lane 6, Fr-3; lane 7, Fr-4; lane 8, Fr-5; lane 9, Fr-6; lane 10, Fr-7; lane 11, Fr-8; lane 12, Fr-9; lane 13, Fr-10

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Figure 5. Fractionation by PFGE after separate digestion with ApaI (A) and NotI (B) of restricted genomic DNA from isolates of T. equigenitalis obtained in Sweden. For lanes Y, L and H, refer to the legend for Figure 1. Lane 1, EQ50; lane 2, NCTC11184T; lane 3, Kentucky 188; lane 4, Swe-1; lane 5, Swe-2; lane 6, Swe-3; lane 7, Swe-4; lane 8, Swe-5

Figure 6. Fractionation by PFGE after separate digestion with ApaI (A) and NotI (B) of restricted genomic DNA from isolates of T. equigenitalis obtained in Switzerland. For lanes Y, L and H, refer to the legend for Figure 1. Lane 1, EQ59; lane 2, NCTC11184T; lane 3, Kentucky 188; lane 4, Swi-1; lane 5, Swi-2; lane 6, Swi-3; lane 7, Swi-4; lane 8, Swi-5; lane 9, Swi-6

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Figure 7. Summary of the results of genotyping by PFGE analysis after separate digestion with ApaI and with NotI of genomic DNA from isolates of T. equigenitalis. Boxes indicate identical genotypes by PFGE analysis

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We had previously detected nine distinctly di¡erent genotypes among 42 isolates of T. equigenitalis obtained from horses in Norway, Ireland, the United States, and Australia (Matsuda et al., 1997, 1998, 2000). The 18 genotypes that were demonstrated in the present study among the 46 isolates obtained in Belgium, England, Finland, France, Sweden and Switzerland appear to be distinctly di¡erent from the other eight genotypes, except for Kentucky 188 genotype. These results strongly suggest that such heterogeneous genotypes of T. equigenitalis have a global distribution. Finally, in the present study, we did not ¢nd any isolates whose genomic DNA could not be cleaved by the two restriction enzymes in order to di¡erentiate isolates of T. equigenitalis. ACKNOWLEDGEMENTS The authors thank Dr L. Deveriese, Faculty of Veterinary Medicine, Universiteit Gent, Merelbeke; Dr N. Chanter, Animal Health Trust, Newmarket; Dr E.-L. Hintikka, National Veterinary and Food Research Institute, Helsinki; Mr S. Neil, Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Belfast; Dr E. Olsson, Laboratory of Bacteriology, National Veterinary Institute, Uppsala; and Dr A.M. van der Zeijst, School of Veterinary Medicine, University of Utrecht, Utrecht. REFERENCES Bryans, J.T. and Hendricks, J.B., 1979. Epidemiological observations on contagious equine metritis in Kentucky, 1978. Journals of Reproduction and Fertility Supplements, 27, 343^349 Crowhurst, R.C., 1977. Genital infection in mares. The Veterinary Record, 100, 476 Hughes, K.L., Bryden, J.D. and Macdonald, F., 1978. Equine contagious metritis. Australian Veterinary Journal, 54, 101 Matsuda, M., Miyazawa, T., Ishida, Y. and Moore, J.E., 1997. Pro¢les of fragments after pulsed-¢eld gel electrophoresis of cleaved genomic DNA from strains of Taylorella equigenitalis isolated from horses in Norway. Microbiological Research, 152, 217^220 Matsuda, M., Miyazawa, T., Moore, J.E., Buckley, T.C. and Thomas, L.A., 1998. Molecular genotyping by pulsed-¢eld gel electrophoresis of restricted genomic DNA of strains of Taylorella equigenitalis isolated in Ireland and in the United States. Veterinary Research Communications, 22, 217^224 Matsuda, M., Kagawa, S., Sakamoto, Y., Miyajima, M., Barton, M. and Moore, J.E., 2000. Detection of heterogeneous genotypes among Australian strains of Taylorella equigenitalis. Australilan Veterinary Journal, 78, 56^57 Miyazawa, T., Matsuda, M., Isayama, Y., Samata, T., Ishida, Y., Ogawa, S., Takei, K., Honda, M. and Kamada, M., 1995. Genotyping of isolates of Taylorella equigenitalis from thoroughbred brood mares in Japan. Veterinary Research Communications, 19, 265^271 Simpson, D.J. and Eaton-Evans, W., 1978. Sites of CEM infection. The Veterinary Record, 102, 488 Sugimoto, C., Isayama, Y., Kashiwazaki, M., Fujikura, T. and Mitani, K., 1980. Detection of Haemophilus equigenitalis, the causal agent of contagious equine metritis, in Japan. National Institute of Animal Health Quarterly (Japan), 20, 118^119 Sugimoto, C., Isayama, Y., Sakazaki, R. and Kuramochi, S., 1983. Transfer of Haemophilus equigenitalis Taylor et al., 1978 to the genus Taylorella gen. nov. as Taylorella equigenitalis comb. nov. Current Microbiology, 9, 155^162 Swerczek, T.W., 1978. Contagious equine metritis in the USA. The Veterinary Record, 102, 512^513

575 Taylor, C.E.D., Rosenthal, R.O., Brown, D.F.J., Lapage, S.P., Hill, L.R. and Legros, R.M., 1978. The causative organism of contagious equine metritis 1977: proposal for a new species to be known as Haemophilus equigenitalis. Equine Veterinary Journal, 10, 136^144 Ter Laak, E.A., Fennema, G. and Jaartsveld, F.H.J., 1989. Contagious equine metritis in the Netherlands. Tijdschrift voor Diergeneeskunde, 114, 189^201 Timoney, P.J. and Powell, D.G., 1982. Isolation of the contagious equine metritis organism from colts and ¢llies in the United Kingdom and Ireland. The Veterinary Record, 111, 478^482 Timoney, P.J., Ward, J. and Kelly, P., 1977. A contagious genital infection of mares. The Veterinary Record, 101, 103 (Accepted: 12 January 2001)