Endonuclease Fragments

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p /90/ $02.00/0 Copyright (O 1990, American Society for Microbiology Vol. 28, No. 9 Epidemiologic Study of Taylorella equigenitalis Strains by Field Inversion Gel Electrophoresis of Genomic Restriction Endonuclease Fragments NANCY BLEUMINK-PLUYM,' ED A. TER LAAK,2 AND BERNARD A. M. VAN DER ZEIJST1* Department of Bacteriology, Institute of Infectious Diseases and Immunology, School of Veterinary Medicine, University of Utrecht, P.O. Box , 3508 TD Utrecht,' and Department of Bacteriology, Central Veterinary Institute, 8200 AB Lelystad,2 The Netherlands Received 11 December 1989/Accepted 18 June 1990 Contagious equine metritis (CEM), a sexually transmitted bacterial disease, was first described in thoroughbred horses. It also occurs in nonthoroughbred horses, in which it produces isolated, apparently unrelated outbreaks. Thirty-two strains of Taylorella equigenitalis, the causative agent of CEM, from all over the world were characterized by field inversion gel electrophoresis of fragments of genomic DNA obtained by digestion with low-cleavage-frequency restriction enzymes. This resulted in a division into five clearly distinct groups. Strains from thoroughbred horses from all continents belonged to one group. Strains from nonthoroughbred horses from various countries were different from strains from thoroughbred horses; four groups could be determined. Two groups contained both streptomycin-resistant and streptomycin-susceptible strains. The data indicate that CEM in nonthoroughbreds did not originate from the thoroughbred population; also, the reverse was not demonstrated. Thus, extensive international transportation directives regarding the testing of nonthoroughbred horses for CEM may need reconsideration. Contagious equine metritis (CEM) is a sexually transmitted bacterial disease of horses caused by Taylorella equigenitalis (formerly Haemophilus equigenitalis [9]). CEM was reported for the first time in the United Kingdom by Crowhurst in 1977 (3). Originally, the disease spread among thoroughbred horses to France and the United States and occurred in Australia. Many outbreaks in thoroughbreds could be traced to the first outbreak of Since then, the disease has also been detected in other breeds of horses in many other countries (13). In nonthoroughbred horses, CEM produces apparently unrelated outbreaks for which a common source cannot be found. In The Netherlands, CEM was first recognized in 1987 in five mares and in the fetus and placenta of another mare of the Dutch saddle horse breed in three separate outbreaks. The origin of these infections was unknown. In 1988, an outbreak occurred among trotters; T. equigenitalis was isolated from 14 mares. In 1988, another outbreak occurred in the Haflinger breed: a mare and a stallion were found to be infected with T. equigenitalis (13). The origin of these outbreaks also remained unclear. Retrospectively, two strains isolated in 1985 and 1986 from Dutch saddle horses were identified as T. equigenitalis. These strains were not recognized initially because of a strong autoagglutination (E. A. ter Laak and C. M. F. Wagenaars, Res. Vet. Sci., in press). In this paper, we describe the use of field inversion gel electrophoresis (FIGE) for the separation of large DNA fragments obtained from T. equigenitalis strains to study the epidemiology of CEM. A total of 20 Dutch isolates and 12 isolates from other countries and continents were compared. * Corresponding author MATERIALS AND METHODS Bacterial strains. The history and properties of the T. equigenitalis strains are summarized in Table 1. The bacteria were grown on Columbia blood agar base (Oxoid Ltd.) chocolate agar supplemented with sodium sulfite and L- cysteine (1) in 7% C02 in air. The identity of the Dutch isolates was confirmed by cultural, biochemical, and serologic properties: cream-colored colonies after growth on enriched chocolate agar media for at least 48 h in an atmosphere of 5 to 10% carbon dioxide in air; gram-negative, nonmotile, coccobacillary forms; negative results in tests for glucose, nitrate, indole, urea, and hydrogen sulfide; positive results in oxidase, catalase, and phosphatase tests; positive result in a slide agglutination test or indirect immunofluorescence test, with the aid of hyperimmune serum of a goat immunized with type strain NCTC This strain was obtained in 1978 from J. E. Shreeve, Central Veterinary Laboratory, Weybridge, United Kingdom. The Irish strain was obtained in 1978 from P. J. Timoney, Veterinary Research Laboratory, Dublin. The other strains were obtained from M. E. Mackintosh, Equine Research Station, Newmarket, United Kingdom. Haemophilus influenza and Haemophilus aphrophilus A860032/A were obtained from S. M. van Ham, Department of Medical Microbiology, Faculty of Medicine, University of Amsterdam. Preparation of DNA in agarose blocks. Cells were incorporated into agarose blocks (LMP agarose GIBCO/BRL), and their DNA was purified in situ as described by McClelland et al. (5). The blocks were stored in TE buffer (10 mm Tris hydrochloride [Boehringer Mannheim] [ph mm disodium EDTA) at 4 C. Restriction endonuclease digestion of DNA in agarose blocks. Blocks were placed in 0.5 ml of buffer appropriate for the restriction enzyme and incubated at 37 C. After 1 h, 400,ul of buffer was removed and 20 U of restriction endonuclease (GIBCO/BRL or New England BioLabs, Inc.) was added. Restriction digestion was performed for 6 to 12 h.

2 VOL. 28, 1990 EPIDEMIOLOGIC STUDY OF T. EQUIGENITALIS 2013 TABLE 1. History and properties of T. equigenitalis strains Horse Country Yr Breed Streptomycin FIGE group Strain no. Remarks 24 Ireland 1977 Thoroughbred R A L England 1977 Thoroughbred R A Type strain, NCTC England 1979 Thoroughbred R A N217/79 33 United States 1979 Thoroughbred R A N203/79 37 Australia 1979 Thoroughbred R A N206/79 32 England 1982 Thoroughbred R A N480/82 36 Belgium 1978 Nonthoroughbred S B N202/79 NCTC United States 1979 Thoroughbred(?) S B N210/79 22 Netherlands 1985 Dutch saddle horse R B L10783 Mare 38 Austria 1982 Nonthoroughbred S C N415/82 39 Austria 1982 Nonthoroughbred R C N412/82 40 Switzerland 1988 Nonthoroughbred R C N610/88 35 Germany 1979 Nonthoroughbred R D N211/79 23 Netherlands 1986 Dutch saddle horse R D L24902 Stallion 1 Netherlands 1987 Dutch saddle horse R D L46960 Mare 1, outbreak 1 2 Netherlands 1987 Dutch saddle horse R D L50354 Mare 2, outbreak 1 3 Netherlands 1987 Dutch saddle horse R D L48987 Mare 1, outbreak 2 4 Netherlands 1987 Dutch saddle horse R D L50353 Mare 2, outbreak 2 5 Netherlands 1988 Dutch saddle horse R D L60219 Fetus of mare 3, outbreak 2 16 Netherlands 1988 Haflinger R D L Stallion 25 Netherlands 1988 Haflinger R D L68138 Mare 6 Netherlands 1987 Dutch saddle horse R E L52721 Mare 1, outbreak 3 7 Netherlands 1988 Trotter R E L67215 Mare 1 9 Netherlands 1988 Trotter R E L Mare 2 10 Netherlands 1988 Trotter R E L Mare 3 il Netherlands 1988 Trotter R E L Mare 4 12 Netherlands 1988 Trotter R E L Mare 5 14 Netherlands 1988 Trotter R E L Mare 6 17 Netherlands 1988 Trotter R E L Mare 7 18 Netherlands 1988 Trotter R E L Mare 8 19 Netherlands 1988 Trotter R E L Mare 9 20 Netherlands 1988 Trotter R E L71205 Mare 10 a R, Resistant; S, susceptible. Strain number refers to the strain collection of the Equine Research Station (N) or the Central Veterinary Institute (L). After 3 h, an additional 20 U of restriction enzyme was added. FIGE separation of restriction endonuclease fragments. Blocks were inserted into the gel slots of a 1% agarose gel (15 [width] by 20 [length] cm) and electrophoresed by the FIGE technique of Carle et al. (2) by using a GENE-TIC (Biocent b.v., Lisse, The Netherlands) power supply. Electrophoresis was performed for 24 to 48 h in Tris borate buffer (44.5 mm Tris hydrochloride [ph 8.3]-44.5 mm boric acid-1.25 mm disodium EDTA) at 90 V. The FIGE program consisted of 6 to 12 identical cycles of 4 h. An exponentially increasing forward switch time (0.05 to 55 s) was used; 50% of the switch time was reached at 40% of the cycle time. The reverse time phase and the pause time were 33 and 2%, respectively, of the forward time phase. Agarose gels were stained with ethidium bromide (Sigma Chemical Co., St. Louis, Mo.), and DNA fragments were visualized with long-wave UV light. Lambda concatamers were used as molecular weight markers. RESULTS The G+C content of the genome of T. equigenitalis is only about 36.5% (9). As pointed out by McClelland et al. (5), such genomes can be cleaved in only a few large fragments by restriction enzymes with G+C-rich recognition sites. We have digested the DNA of two T. equigenitalis strains (belonging to group E; see below) with ApaI (GGGCCC), BssHI (GCGCGC), NaeI (GCCGGC), NarI (GGCGCC), and NciI (CCG/CGG). BssHI, NarI, and NciI did not cut the genome, but digestion with ApaI and NaeI resulted in 9 to 12 large fragments, which were separated by FIGE. ApaI digestion of the type strain gave 11 bands (410, 340, 240, 160 doublett), 120, 80, 50, 20, 15, and 12 kilobase pairs). For the FIGE group E (see below), the sizes of the fragments were 390, 310, 220, 140 (doublet), 90, 60, 50, 35, 20, 15, and 12 kilobase pairs. NaeI digestion of the latter strain resulted in 9 fragments (410, 360, 340, 170, 150, 60, 50, 30, and 15 kilobase pairs). The sum of the molecular weights of these fragments predicts a genome size of 1.5 x 106 to 1.6 x 106 base pairs. No intense bands characteristic for multicopy plasmids were observed. For the characterization of the other T. equigenitalis strains, ApaI digestion was chosen because it resulted in well-separated bands after electrophoretic separation. All 32 strains could be designated to one of five different restriction patterns, which were designated A, B, C, D, and E) (Fig. 1 and 2). The digestion patterns for H. influenza and H. aphrophilus were different from each other and from those of all T. equigenitalis strains.

3 2014 BLEUMINK-PLUYM ET AL. J. CLIN. MICROBIOL. Go m,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ r B D E D r- A -<r- r--- A\ B A C D B D EB CI n -240 c c: -160.Sc --19o llv Cr, FIG. 1. Comparison of the genomic fragments, after ApaI digestion, of Dutch T. equigenitalis strains and control strains. The numbers of the strains and the designations of the groups correspond to those in Table 1. kbp, Kilobase pairs. Group A contained strains isolated from thoroughbred horses only. Group B contained the Belgian strain, the streptomycin-susceptible strain isolated in the United States from a horse thought to be thoroughbred, and the Dutch saddle horse strain isolated in Group C contained the strains from Austria and Switzerland. Group D contained the German strain, strains from the outbreak in Dutch Haflinger horses, and strains from Dutch saddle horses (the 1986 strain and all strains from the first two outbreaks of 1987). Group E contained all Dutch strains from trotters and the only strain of the third outbreak in the Dutch saddle horse in A summary of the data is given in Table 1. DISCUSSION This study shows the usefulness of restriction endonuclease analysis of T. equigenitalis genomic DNA for epidemiologic studies. In particular, the DNA digestion with lowcleavage-frequency enzymes in conjunction with FIGE has allowed a clear distinction of T. equigenitalis strains. Among the limited number of strains investigated, clusters of strains, by breed of horse, can be distinguished: group A strains were found only in thoroughbreds and group E strains were found in all Dutch trotters and in only one Dutch saddle horse. Clusters can also be distinguished by geography: group C strains from Austria and Switzerland were not found in horses from other regions. In The Netherlands, at least three different origins of infection exist, because strains belonging to three groups (B, D, and E) were isolated. All strains from each outbreak, x rc V ,>- 15.`12 FIG. 2. Comparison of the genomic fragments, after ApaI digestion, of a collection of international T. equigenitalis strains. The numbers of the strains and the designations of the groups correspond to those in Table 1. kbp, Kilobase pairs. however, belonged to only one group. This also applies to the great outbreak in trotters; from 14 infected mares in this outbreak, 10 strains have been stored. The methods used in this study are applicable for studying the epidemiology of bacteria in general. For instance, other methods require (monoclonal) antisera or (radioactive) probes. The only information needed for this method, however, is the G+C content of the genome. On the basis of this information, restriction enzymes can be chosen to produce large fragments; enzymes recognizing a sequence containing the tetranucleotide CTAG, which is extremely rare in many bacterial genomes (5), can also be used. Because the G+C content of T. equigenitalis is 36.5% and the restriction recognition sites used consist of six G or C bases, a frequency of = 1/27065 may be expected for such a restriction site. In reality, the average fragment is much larger. This can be explained by the nonrandom distribution of nucleotides among the genome; e.g., the trinucleotides CCG and CGG are rare (5). In principle, the method we have used only proves that two strains are different. Several methods have been described to derive the percentage of nucleotide substitutions per site (d) from the fraction of shared DNA restriction fragments (F) (6). These methods assume a random distribution of nucleotides among the genome; as mentioned, this condition is not satisfied. Nevertheless, according to the method of Nei and Li (7) it can be calculated that for 10 ApaI fragments, the minimal difference that can be observed (F = 0.9) corresponds to d = 0.59%. Sharing of just two bands (F = 0.2) predicts a value for d of 9.94%. Thus, the method we have used would be particularly useful for comparing genomes that are 90 to 99.4% identical.

4 VOL. 28, 1990 Studies of enzyme activities and cellular fatty acid composition have not resulted in methods to distinguish strains of T. equigenitalis. Sugimoto et al. (9) investigated 12 strains of T. equigenitalis for the presence of 97 enzymes. For 20 enzymes, activity varies within the species. Because information for singular strains was not reported, it could not be determined whether these enzyme activities might serve as an epidemiologic marker. Tainturier et al. (11) investigated 17 strains of T. equigenitalis for the presence of 85 enzymes. For 18 enzymes, activity varied within the species. Although information for singular strains was not reported, the authors concluded that some enzyme activities could be used for strains of various geographical origins in epidemiologic studies. Such studies, however, have not been published. Sugimoto et al. (10) investigated eight T. equigenitalis strains, and Neill et al. (8) investigated 36 T. equigenitalis strains for cellular fatty acid composition. All strains showed a grossly similar pattern, so that the cellular fatty acid composition could not serve as an epidemiologic marker. Strains of T. equigenitalis can be divided into two groups on the basis of their resistance or susceptibility to streptomycin. This single difference between strains did not permit epidemiologic studies. MICs for resistant strains vary from 128 to >512 mg/liter and the MIC for susceptible strains is 1 mg/liter (4, 12). Strains for which MICs are intermediate have not been reported. Because no plasmids were found in the T. equigenitalis strains used in this study, resistance to streptomycin is not expected to be easily transferred to susceptible strains. The majority of T. equigenitalis strains isolated internationally are resistant to streptomycin (13). Streptomycin-susceptible strains have been isolated sporadically in Belgium, the United States, France, and England. In Japan, streptomycin-susceptible strains have been isolated only since In the following countries, streptomycin-susceptible strains are more numerous: Austria (40% of the strains), the Federal Republic of Germany (83% of the strains), and Denmark (nearly all strains) (13). In our study, groups B and C contained both streptomycin-resistant and streptomycin-susceptible strains. Thus, since susceptibility to streptomycin was not correlated to a particular group, it remains an additional epidemiologic marker. CEM was discovered in the United Kingdom in thoroughbred horses as a new disease caused by an unrecognized bacterium. Although the origin of infection has never been detected, it is clear from its epidemic onset in 1977 and its rapid spread within thoroughbreds, even to those in other countries, that it was a new infection for the thoroughbred. In view of the great economic losses caused by CEM, it has been controlled vigorously, resulting in a nearly complete eradication of the infection in thoroughbreds (13). At the same time, many countries enforced examination of horses to be imported or exported to ensure the absence of CEM. In this way, CEM has been detected in various other breeds in many countries. As opposed to the situation with the thoroughbred, it was hardly possible to trace outbreaks or cases. Signs in nonthoroughbred breeds are generally milder than those observed in the thoroughbred, although signs in the latter have become milder in the years since it was discovered. This also suggests that CEM was a new disease for the thoroughbred and that the infection already existed for some time in nonthoroughbreds. Where did the outbreak among thoroughbreds originate? Our data show that the six strains isolated from thoroughbreds in England, Ireland, Australia, and the United States belong to group A, to which no strains from other breeds EPIDEMIOLOGIC STUDY OF T. EQUIGENITALIS 2015 belong. One possible exception is strain 34, for which the horse breed is thought to be thoroughbred. If it was isolated from a thoroughbred, it had no epidemiologic connection to other cases of CEM in the thoroughbred, because only one outbreak of CEM caused by a streptomycin-susceptible organism has been reported internationally for the thoroughbred. Because strains isolated from thoroughbreds in England in 1977, 1979, and 1982 were investigated, it may be concluded that the restriction pattern did not change in those 5 years and that patterns found in the other breeds belong to different strains. Our limited data indicate that T. equigenitalis naturally occurring in the nonthoroughbred breeds was not transmitted from the United Kingdom thoroughbred population and vice versa. This absence of transfer is probably explained by the closed system of thoroughbred breeding, which separates thoroughbreds from other breeds. These data suggest that the sometimes extremely severe rules for importing nonthoroughbred horses from countries where T. equigenitalis occurs could be reconsidered. If indeed there is no transfer of T. equigenitalis from nonthoroughbreds to thoroughbreds, there seems to be no reason to take more severe measures against T. equigenitalis than against Streptococcus zooepidemicus, Klebsiella pneumoniae, or Pseudomonas aeruginosa, which also can cause severe infections of the genital tract of the horse. ACKNOWLEDGMENTS We thank M. E. Mackintosh, Equine Research Station, Animal Health Trust, Newmarket, United Kingdom, for supplying 10 T. equigenitalis strains from various countries; S. M. van Ham, Department of Medical Microbiology, Faculty of Medicine, University of Amsterdam, for supplying two Haemophilus strains; and Chrysantha M. F. Wagenaars for technical assistance. LITERATURE CITED 1. Atherton, J. G Inhibition of CEM organism in mixed cultures. Vet. Rec. 103: Care, G. F., M. Frank, and M. V. Olson Electrophoretic separation of large DNA molecules by periodic inversion of the electric field. Science 232: Crowhurst, R. C Genital infections in mares. Vet. Rec. 100: Dabernat, H. J., C. F. Delmas, D. J. Tainturier, and M. B. Lareng In vitro susceptibility of Haemophilus equigenitalis, the causative organism of contagious equine metritis 1977, to antimicrobial agents. Antimicrob. Agents Chemother. 18: McClelland, M. R. C., R. Jones, Y. Patel, and M. Nelson Restriction endonucleases for pulsed field mapping of bacterial genomes. Nucleic Acids Res. 15: Nei, M. (ed.) Molecular evolutionary genetics, p Columbia University Press, New York. 7. Nei, M., and W.-H. Li Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76: Neill, S. D., J. J. O'Brien, C. H. McMurray, and W. J. Blanchflower Contagious equine metritis. Use of gas liquid chromatography in identifying the causal agent. Equine Vet. J. 16: Sugimoto, C., Y. Isayama, R. Sakazaki, and S. Kuramochi to the Transfer of Haemophilus equigenitalis Taylor et al. genus Taylorella gen. nov. as Taylorella equigenitalis comb. nov. Curr. Microbiol. 9: Sugimoto, C., E. Miyagawa, K. Mitani, M. Nakazawa, and Y. Isayama Cellular fatty acid composition of Haemophilus equigenitalis. J. Clin. Microbiol. 15:

5 2016 BLEUMINK-PLUYM ET AL. 11. Tainturier D. J., C. F. Delmas, and H. J. Dabernat Bacteriological and serological studies of Haemophilus equigenitalis, agent of contagious equine metritis. J. Clin. Microbiol. 14: Taylor, C. E. D., R. O. Rosenthal, D. F. J. Brown, S. P. Lapage, L. R. Hill, and R. M. Legros The causative organism of J. CLIN. MICROBIOL. contagious equine metritis 1977: proposal for a new species to be known as Haemophilus equigenitalis. Equine Vet. J. 10: Ter Laak, E. A., G. Fennema, and F. H. J. Jaartsveld Contagious equine metritis in the Netherlands. Tijdschr. Diergeneeskd. 114: