85-kb Type Research Articles

Virulence plasmids in clinical isolates of Rhodococcus equi from sick foals in the Netherlands.

Clinical samples from 123 foals with suspected rhodococcosis submitted to the Veterinary Microbiological Diagnostic Centre of the Faculty of Veterinary Medicine between 1993 and 2006 were tested for the presence of the virulence gene vapA. Of the 123 samples, 120 were vapA-positive and 3 vapA-negative Rhodococcus equi were isolated. The 120 vapA-positive R. equi were isolated from 70 tracheal wash, 19 lung tissues, 7 lymph nodes, 6 synovial fluids, 13 abscesses or pus and single isolates from the uterus, gut, cerebrospinal fluid, abdomen fluid and faeces. Of the 120 isolates, 46 were from Dutch warmblood horses, 23 from Friesian horses, 14 from Trotters, 4 from Holsteiners, 3 from Arab breed, 2 from ponies, 1 from a Welsh pony and 27 from undefined breed horses. Using plasmid profile analysis of the 120 isolates, 117 isolates contained the 85-kb type I plasmid, 2 contained the 87-kb type I plasmid and 1 contained the novel 52-kb non-mobilizable virulence plasmid reported recently. These results showed that the virulent R. equi strains harbouring a virulence plasmid of 85-kb type I or 87-kb type I, which have been detected in clinical isolates from five European countries, are widespread in the Netherlands. This is the first report of plasmid types of clinical R. equi isolates in the Netherlands.

Molecular characterization of Rhodococcus equi isolates from horses in Poland: pVapA characteristics and plasmid new variant, 85-kb type V.

Background Rhodococcus equi is one of the most significant bacterial pathogens affecting foals up to 6 months of age worldwide. Rhodococcosis is present in Poland however information about molecular characterization of R. equi isolates is scarce.This study describes molecular characterization of Rhodococcus equi infection on 13 horse breeding farms in Poland between 2001 and 2012. Samples were collected by tracheobronchial aspiration from pneumonic foals or during necropsy. The R. equi isolates were genotyped by plasmid profiling and pulsed-field gel electrophoresis.ResultsTotally, 58 R. equi isolates were investigated. One isolate lost its plasmid. Among the 57 VapA-positive isolates, 48 contained 85-kb type I plasmid (82.8%), 8 contained 87-kb type I plasmid (13.8%). One isolate (1.7%) had a unique restriction cleavage pattern and the 2nd fragment of EcoRI digests of this plasmid DNA was about 2600 bases smaller than that of the 85 kb type I. This new plasmid variant was designated as the “85-kb type V”.Among the 58 isolates typeable with VspI-PFGE, ten PFGE clusters were detected. The majority of foals were infected mostly with isolates of low genetic diversity.ConclusionsMost of clinical isolates of R. equi from foals in Poland contain pVapA 85-kb type I and 87-kb type I similarly to the other European countries and the United States. However, the new variant of pVapA 85-kb type V was identified.The chromosomal variability was detected among some of the investigated isolates and the presence of farm-specific isolates might be possible.

Molecular epidemiology of Rhodococcus equi in slaughtered swine, cattle and horses in Poland.

BackgroundRhodococcus equi is an emerging zoonotic presumably foodborne pathogen. Since the data on the worldwide prevalence of R. equi in meat animals are scarce, the present study aimed to investigate the molecular epidemiology of R. equi in swine, cattle and horse carcasses intended for human consumption in Poland.ResultsTotally 1028 lymph node samples were examined. R. equi was isolated from 26.6 % (105/395) swine and 1.3 % (3/234) bovine healthy submaxillary lymph nodes. In horses, R. equi was isolated only from 0.5 % (1/198) samples of middle tracheo-branchiales lymph node while no lymphocentrum retropharyngeum sample was positive (0/198). The purulent lesions were observed only in 0.8 % swine submaxillary lymph nodes samples (3/398) and in two of them R. equi was detected.All bovine and most of swine isolates (98.1 %) were vapB-positive. 87.9 % of swine isolates carried 95-kb type 5 plasmid, 3.7 % type 1 and plasmid types: 4, 7, 10, 11, 21, 31 were carried by a single isolate (0.9 %). All bovine isolates carried VAPB type 26. Single horse isolate was vapA-positive and carried plasmid VAPA 85-kb type I.ConclusionsThe prevalence of vapB-positive R. equi in investigated healthy swine intended for human consumption was very high.Not only swine, but also even apparently healthy cattle or horse carcasses should be considered as a potential source of R. equi for humans, especially in countries where undercooked or raw beef or horsemeat is traditionally consumed.

Virulent Rhodococcus equi isolates from foals in Poland – pVapA characteristics and plasmid new variant, 85-kb type V

Virulent Rhodococcus equi isolates from foals in Poland – pVapA characteristics and plasmid new variant, 85-kb type V

Enteric Pathogens and Coinfections in Foals with and without Diarrhea.

Diarrhea is a major clinical problem affecting foals up to 3 months of age. The aim of this study was to identify enteric microorganisms involved in monoinfections and coinfections and the associated virulence factors in healthy and diarrheic foals. Diarrheic (D) (n = 56) and nondiarrheic (ND) foals (n = 60) up to three months of age were studied. Fecal samples were analyzed for identification of infectious agents (microbiological culturing, molecular techniques, and microscopic analyses). Escherichia coli fimH (30% versus 25%), Salmonella spp. (25% versus 7%), Strongyloides westeri (25% versus 25%), Clostridium perfringens type A (21% versus 10%), E. coli ag43 (20% versus 35%), Strongylus (11% versus 18%), and vapA-positive Rhodococcus equi (5% versus 2%) were the most frequent enteric pathogens detected in D and ND foals, respectively. The frequency of toxin A-positive C. perfringens was significantly increased in the D (p = 0.033) compared with the ND animals. R. equi strains harboring virulent plasmids were also identified (VapA 85-kb type I and VapA 87-kb type I) in D and ND foals. Coinfections were observed in 46% of the D and 33% of the ND foals. Our results demonstrate the great diversity of enteric pathogens, virulence factors, and coinfections involved in enteric infections of foals.

Disseminated Rhodococcus equi infection in dromedary camels (Camelus dromedarius)

Rhodococcus (R). equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized people. It affects also New World camelids, but there are no reports of R. equi infection in Old World camelids yet.Four cases of disseminated R. equi infection in adult breeding dromedaries occurred at one camel farm near Dubai within 16 months of each other. At necropsy the lungs were diffusely consolidated with large caseous areas. Histology revealed severe suppurative to necrotising pneumonia with multiple encapsulated abscesses. Immunohistochemistry enabled the detection of 15- to 17-kDa antigens (VapA) of R. equi in the lung sections. High numbers of R. equi were isolated from the lung lesions as well as from liver, spleen and mediastinal lymph nodes, indicative of septicaemia. The isolated strains were PCR-positive for the specific virulence plasmid (VapA-Gen) of R. equi, indicating virulent strains and containing an 85-kb type I plasmid.This is the first report of disseminated R. equi infection in Old World camelids. Since adult camels in general do not suffer from bacterial caused pneumonia (except tuberculosis), this is a new emerging disease for camels.

Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid

To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene.

Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) ( P < 0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive. R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive. The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.

Plasmid profiles of virulent Rhodococcus equiisolates from soil environment on horse-breeding farms in Hungary

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

Molecular epidemiology of virulent Rhodococcus equi from foals in Brazil: virulence plasmids of 85-kb type I, 87-kb type I, and a new variant, 87-kb type III

We investigated the prevalence of virulent Rhodococcus equi in clinical isolates from 41 foals (19 sporadic and seven endemic cases) in Brazil between 1991 and 2003. Of the 41 virulent isolates, six contained an 85-kb type I plasmid, 33 contained an 87-kb type I plasmid, both of which have been found in isolates from the Americas, and the remaining two contained a new variant, which did not display the Eco RI, Eco T22I and Bam HI digestion patterns of the 11 representative plasmids already reported (85-kb types I–IV; 87-kb types I and II; 90-kb types I–V). We tentatively designated the new variant as the ‘87-kb type III’ plasmid, because its Bam HI digestion pattern is similar to that of the 87-kb type I plasmid. This is the first report of the molecular epidemiology surveillance of virulent R. equi in clinical isolates from Brazilian foals. Nous avons étudié la prévalence de Rhodococcus equi dans des isolats cliniques de 41 poulains (19 cas sporadiques et 7 endémiques) au Brésil entre 1991 et 2003. Parmi les 41 isolats virulents, 6 contenaient un plasmide de 85-kb de type I, 33 contenaient un plasmide de 87-kb de type I, ces deux types ayant déjà été retrouvés dans des isolats originaires des Etats-Unis. Les deux isolats restants contenaient un nouveau variant qui, après digestion par Eco RI, Eco T22I et Bam HI, ne présentait pas le même profil que les onze types de plasmide reportés jusqu'à présent (85-kb types I, II, III et IV; 87-kb types I et II; 90-kb types I, II, III, IV et V). Nous avons suggéré de dénommer le nouveau variant ‘87-kb type III’, car son profil après digestion par Bam HI est identique à celui des plasmides de 87-kb de type I. Il s'agit du premier rapport d'une étude épidémiologique au niveau moléculaire de R. equi sur des isolats cliniques de poulains au Brésil.

Genotypic Characterization of Virulent Rhodococcus equi Isolated from the Environment of Hokkaido Native Horses in Hakodate, Hokkaido

Rhodococcus equi was isolated from soil samples obtained from seven Hokkaido native horse farms in Hakodate, Hokkaido. R. equi was prevalent in all 59 soil samples collected from the seven farms at concentrations of between 7.5 × 102 and 7.5 × 104 colony-forming units per gram of soil. The isolates were examined for presence of the gene that encodes the virulence-associated 15-17 kDa antigen protein, VapA, using PCR. VapA-positive R. equi was isolated from 1-6% of isolates from three of the farms. This represents 1.4% of isolates from all farms examined (8 out of 590). The eight virulent isolates were characterized by plasmid profiling, restriction fragment length polymorphism analysis, and pulsed-field gel electrophoresis. All of the virulent isolates contained an 87-kb type II plasmid, but there were eight distinct VspI profiles. These results suggest that, although the Hokkaido horses of Hakodate are exposed to a variety of distinct types of virulent R. equi, the incidence of exposure is low or zero. An 87-kb type II plasmid was strongly associated with Hokkaido native horses as has already been observed in virulent isolates from Hokkaido native horses at Bekkai, Hokkaido.

Molecular epidemiology of virulent Rhodococcus equi from foals in Brazil: virulence plasmids of 85-kb type I, 87-kb type I, and a new variant, 87-kb type III

We investigated the prevalence of virulent Rhodococcus equi in clinical isolates from 41 foals (19 sporadic and seven endemic cases) in Brazil between 1991 and 2003. Of the 41 virulent isolates, six contained an 85-kb type I plasmid, 33 contained an 87-kb type I plasmid, both of which have been found in isolates from the Americas, and the remaining two contained a new variant, which did not display the EcoRI, EcoT22I and BamHI digestion patterns of the 11 representative plasmids already reported (85-kb types I–IV; 87-kb types I and II; 90-kb types I–V). We tentatively designated the new variant as the ‘87-kb type III’ plasmid, because its BamHI digestion pattern is similar to that of the 87-kb type I plasmid. This is the first report of the molecular epidemiology surveillance of virulent R. equi in clinical isolates from Brazilian foals.

Prevalence of Virulent Rhodococcus equi in Soil Environment on a Horse-Breeding Farm in Tennessee, U.S.A.

Virulent Rhodococcus equi is an important pathogen in foals aged 1 to 3 months, but its incidence in Tennessee, U. S. A., is poorly understood. Ten soil samples were collected from a Tennessee walking horse farm in Tennessee. The presence of R. equi was investigated and 107 isolates were tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. R. equi was isolated (103-104 colony forming units/g) from 8 of the 10 soil samples. Twenty-four VapA-positive isolates were detected from 7 of the 8 culture-positive samples with a prevalences ranging from 6.7 to 50%. Of the 24 virulent isolates, 21 contained an 85-kb type I plasmid, and 3 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in Texas. There was no evidence of R. equi infection on the farm, but the virulent form was widespread in the soil environment of the farm.

Virulence of Rhodococcus equi isolated from cats and dogs.

Nine cat isolates and nine dog isolates of Rhodococcus equi from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent R. equi strains and contained no virulence plasmids.

Prevalence of virulent Rhodococcus equi in soil from five R. equi-endemic horse-breeding farms and restriction fragment length polymorphisms of virulence plasmids in isolates from soil and infected foals in Texas.

Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R. equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims. Rhodococcus equi were isolated from soil of all 5 farms; however, virulent R. equi were only isolated from 3 of the 5 farms and represented 18.8% (87 of 462) of total isolates. Of the 87 virulent soil isolates, 56 (64.5%) contained an 85-kb type I plasmid, 23 (26.4%) an 87-kb type I plasmid, 7 (8%) a newly defined 85-kb type III plasmid (Tx 43), and 1 (1.1%) a newly defined 85-kb type IV plasmid (Tx 47). Of the 100 isolates from infected foals, 96 were virulent. Of the 96 virulent isolates, 51 (53.1%) contained an 85-kb type I plasmid, 39 (40.6%) an 87-kb type I plasmid, 4 (4.2%) an 85-kb type III plasmid (Tx 43), and 2 (2.1%) an 85-kb type IV plasmid (Tx 47). There are at least 4 different R. equi virulence-associated plasmids in Texas, 2 of which have not previously been described. Based upon virulence plasmid typing, there is geographic diversity among isolates of R. equi from clinical and environmental samples on horse-breeding farms in Texas. There is not a strong correlation between the presence of virulent R. equi in farm soils and the R. equi disease status of those farms.

Isolation of virulent Rhodococcus equi from native Japanese horses

R. equi was isolated from soil samples obtained from the environment of seven native Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, Miyako and Yonaguni) and from fecal samples collected from three native horse breeds (Hokkaido, Kiso and Misaki). Virulent R. equi at various levels (ranging from 0.5 to 12.9%) was isolated from the feces or soil environment of Hokkaido, Kiso and Misaki horses. Isolates were investigated both for the presence of 15- to 17-kDa antigens (virulence-associated protein antigens; VapA) by colony blotting, using the monoclonal antibody 10G5, and the gene of VapA by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases, and the digestion patterns of the plasmids of virulent isolates were divided into three types. Two of the three types (87-kb type II and 90-kb type I) had already been reported in Japanese isolates, and a new type (tentatively designated as 90-kb type II) had been found in isolates from Kiso horses. Six virulent R. equi isolates from the Hokkaido horses contained an 87-kb type II plasmid. Eight of 24 isolates from the Kiso horses contained an 87-kb type II plasmid, and the remaining 16 contained a 90-kb type II (a new type) plasmid. Two isolates from the Misaki horses contained a 90-kb type I plasmid. These results demonstrate the geographic difference in the distribution of virulence plasmids in R. equi isolates among native Japanese horses.

Prevalence of Virulence Plasmids in Soil Isolates of Rhodococcus equi from 5 Horse-Breeding Farms in Argentina.

Prevalence of virulent Rhodococcus equi in isolates from 5 horse-breeding farms with endemic infection in Argentina was investigated: isolates were tested for the presence of virulence plasmid DNA and virulence-associated 15-to 17-kDa protein antigens (VapA) by colony blotting with the monoclonal antibody 10G5. R. equi was isolated from all of the soil samples obtained from the 5 farms with 2.0 × 103 to 3.7 × 10 4 colony forming units per gram of soil. Virulent R. equi at various levels (ranging from 4.0 to 24.3%) was isolated from the 5 farms and appeared in 11.4% (74 of 650 isolates). Of the 74 virulent R. equi, 8 contained an 85-kb type I plasmid and 66 contained an 87-kb type I. These results revealed that environments of the horse-breeding farms with endemic infection in Argentina were contaminated with virulent R. equi harboring a virulence plasmid of 85-kb type I or 87-kb type I, which were found in clinical isolates previously reported from Argentina.

Two Cases of Dead Foals Associated with Rhodococcus equi Pneumonia in Turkey.

Rhodococcus equi has been recognized as an equine pathogen since 1923 and is a worldwide pathogen in foals. Up to now, there has been one report on R. equi infection in an Arabian foal in Turkey. We report here two cases of R. equi pneumonia diagnosed in Turkish foals. R. equi was isolated from pure cultures obtained from lung abscesses of two dead foals which were sent to the laboratory from distinct stud-farms in the Izmit district, Turkey, due to the increasing mortality of foals with pneumonia. These isolates were analyzed by plasmid and protein profiles: one isolate had an 85-kb type I virulence plasmid and the other was considered avirulent. Virulent R. equi strains possessing an 85-kb type I or an 87-kb type I were isolated from one of the stud-farms with the problem. These results indicate that the stud-farm was contaminated with virulent R. equi.

Some Epidemiological Aspects of Rhodococcus equi Infection in Foals in Japan: A Review of 108 Cases in 1992-1998.

One hundred and eight foals were necropsied and examined for Rhodococcus equi infection from 1992 to 1998 in Hokkaido and Aomori, Japan. Fifty-four of 108 cases had been clinically diagnosed as having R. equi pneumonia, whereas the remaining 54 were diagnosed with non-R. equi pneumonia (23 foals), arthritis (6 foals) and other diseases (25 foals). Pathologically, 104 foals were shown to have suppurative pneumonia with abscesses, and the remaining 3 had intestinal involvement without pulmonary lesions and one had osteomyelitis. Based on the pathology and bacteriology, 108 were classified into 5 disease types: pneumonia (44 cases; suppurative bronchopneumonia), enteritis (3 cases; ulcerative enteritis associated with suppurative lymphadenitis of the intestines), mixed (25 cases; pneumonia, enteritis, and intestinal lymphadenitis), transitional (35 cases; pneumonia and intestinal lymphadenitis), and osteomyelitis (1 case). Eighty-three of the 108 foals (25 foals died due to non-infectious causes) were analyzed for age distributions at the first clinical examination and death. About 75% (62 foals) of the foals showed clinical signs within 2 months of age, peaking at 40 days, and the mean age at the clinical examination was 53.6 days. About 81% (67 foals) died within 3 months of age, peaking at 60 days, and the mean age at death was 77.2 days. About 57% (47 foals) died within 2 weeks after the first clinical examination, and mean days for treatment were 21.7. Mean ages at the clinical signs and death of the 83 foals were compared statistically according to breed, sex, birth month, and disease types, but the difference was not significant. Bacteriologically, virulent R. equi was isolated from lesions of all the 108 foals. Virulent isolates from the foals bred at Hokkaido and Aomori contained an 87-kb type II or a 90-kb plasmid with a different ratio. From this surveillance, it is clear that R. equi infection in foals is still one of the most important diseases in horse-breeding farms in Japan.

Restriction fragment length polymorphisms of virulence plasmids in Rhodococcus equi.

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries-Argentina, Australia, Canada, France, and Japan-were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738-740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world.