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

Abstract

Abstract. 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 virulenceassociated 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.Rhodococcus equi is one of the most important bacterial pathogens of young foals. Infections caused by this organism are characterized by chronic, suppurative bronchopneumonia and enteritis associated with a high mortality rate. 1,7,18 It was previously reported that the 15-17-kDa virulence-associated protein antigens (VapA) of R. equi are associated with virulence in mice 15,19 and that the presence of an 85-or a 90-kb plasmid is essential for virulence and the expression of VapA. 10,14,[22][23][24] These virulence-associated antigens and virulence plasmids have been used as epidemiologic markers to identify virulent R. equi isolates from horses and their environment by Western blot (immunoblot) assay using a monoclonal antibody and plasmid profiles. 4,11,12,16,17,21 More recently, restriction enzyme digestion patterns of virulence plasmids in human and foal isolates from several countries were examined. 6,20 The digestion patterns divided the plasmids of virulent isolates into 5 closely related types. Three of the 5 types had already been reported in Canadian and Japanese isolates, 6 and 2 new types were identified in French and Japanese isolates. 9,20 These 5 types of plasmids were designated as 85-kb type I (p-REAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2), 6 87-kb type II (a new type), and 90-kb (pREL1). The 85-kb type I plasmid was found in isolat...

Rhodococcus equi Virulence Plasmids Recovered from Horses and Their Environment in Jeju, Korea: 90-kb Type II and a New Variant, 90-kb Type V

“…Most clinical isolates from the Americas, Australia, and Europe contain 85-kb type I or 87-kb type I plasmids. The 85-kb type II plasmid is found only in French isolates and the 85-kb type III and type IV plasmids only in isolates from Texas [12,16,27]. These five types of virulence plasmids have never been isolated from Japanese samples.…”

“…1B). The digestion patterns were then compared with 10 representative virulence plasmid types (85-kb types I, II, III, and IV; 87-kb types I and II; and 90-kb types I, II, III, and IV), which have already been reported [16,22,27,31]. As shown in Fig.…”

“…The bacterial strains used in this study were the reference strains of R. equi ATCC 33701 (85-kb type I), 96E35 (85-kb type II), T47-2 (85-kb type III), T43 (85-kb type IV), 222 (87-kb type I), 96B6 (87-kb type II), L1 (90-kb type I), S11-8 (90-kb type II), Kuma83-3 (90-kb type III), and Kuma83-10 (90-kb type IV) [16,22,23,27].…”

Molecular Typing of VapA-Positive Rhodococcus equi Isolates from Jeju Native Horses, Korea

“…However, avirulent R. equi, which have no virulence-associated antigens or plasmids and do not cause disease in foals, are distributed widely in horses and their environments [10,23]. Epidemiological studies have revealed that horse-breeding farms with endemic infections are heavily contaminated with virulent R. equi [9,17].The restriction-enzyme digestion patterns of virulent virulence plasmids from foals have allowed the plasmids of virulent isolates to be categorized into 12 closely related types and have revealed geographic differences in the distributions of these virulence plasmids, not only throughout the world, but also within Japan [7,11,15,16,19,20,22]. In a recent study, we isolated virulent R. equi containing the 90-kb type II plasmid that has been found in isolates from the native Kiso horses of Japan, from fecal and environmental samples of the native Jeju horse and thoroughbred horse farms on Jeju Island, Korea, and a new variant, the 90-kb type V plasmid [20].…”

“…The restriction-enzyme digestion patterns of virulent virulence plasmids from foals have allowed the plasmids of virulent isolates to be categorized into 12 closely related types and have revealed geographic differences in the distributions of these virulence plasmids, not only throughout the world, but also within Japan [7,11,15,16,19,20,22]. In a recent study, we isolated virulent R. equi containing the 90-kb type II plasmid that has been found in isolates from the native Kiso horses of Japan, from fecal and environmental samples of the native Jeju horse and thoroughbred horse farms on Jeju Island, Korea, and a new variant, the 90-kb type V plasmid [20].…”