Yukako Sasaki

Characterization of virulence plasmid types in Rhodococcus equi isolates from foals, pigs, humans and soil in Hungary

Rhodococcus equi isolates (204) obtained from foals (lung abscesses, lymph nodes, nasal discharge, rectal swabs) bred in 15 studs located throughout Hungary, isolates from soil samples, lymph nodes of pigs and from lesions of human patients were examined to determine genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17 kDa virulence-associated protein antigen (VapA) and 20k Da (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisms. Of 146 clinical isolates from foals in 15 studs, 129 (88.3%) gave positive results for the VapA gene, showing a 564 bp product of the expected size in the PCR amplification. Of the 129 clinical isolates from foals, 123 contained an 85 kb type I plasmid and the remaining six contained an 87 kb type I plasmid. Of 48 soil isolates from two horse studs, 26 (54.2%) were positive for VapA gene and contained an 85 kb type I plasmid. Of three pig isolates, one was positive for VapA gene and contained an 85 kb type I plasmid, and the remaining two were positive for the VapB gene, showing a 827 bp product of the expected size in the PCR amplification and were R. equi of intermediate virulence which contained a 95 kb type S5 plasmid. Of the seven human isolates, five were positive for VapB gene by PCR, these were R. equi of intermediate virulence, which contained a 95 kb type S5 plasmid. These results revealed that virulent R. equi strains harbouring a virulence plasmid of 85 kb type I or 87 kb type I, which have been found in clinical isolates from Europe and North and South America, are widespread in Hungary. Furthermore, same intermediately virulence plasmid type was found in both human and pig isolates.

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence‐associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 106 cells expresses 15‐ to 17‐kDa antigens and intermediately virulent R. equi that kills mice with 107 cells expresses a 20‐kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence‐associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence‐associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20‐kDa antigen was found to be located on the cell surface, as were the 15‐ to 17‐kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence‐associated antigens of R. equi is dependent on the environmental conditions.

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.

Characterization of Virulence Plasmids and Serotyping of Rhodococcus equi Isolates from Submaxillary Lymph Nodes of Pigs in Hungary

ABSTRACT The plasmid types and serotypes of 164 Rhodococcus equi strains obtained from submaxillary lymph nodes of swine from different piggeries in 28 villages and towns located throughout the country were examined. The strains were tested by PCR for the presence of 15- to 17-kDa virulence-associated protein antigen (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphism characteristics. None of the 164 isolates contained the vapA gene, and 44 (26.8%) isolates were positive for the vapB gene, showing a product of the expected 827-bp size in the PCR amplification. The 44 isolates of intermediate virulence contained virulence plasmids that were identified as types 1 (3 isolates), 4 (1 isolate), 5 (36 isolates), 6 (1 isolate), and 7 (2 isolates) and as a new variant (1 isolate). On the basis of restriction digestion patterns of plasmid DNAs, we tentatively designated the variant as type 17. Use of the serotyping method of Prescott showed that 110 (67.1%) out of the 164 isolates were typeable and that serotype 2 predominated (83 isolates [50.6%]), followed by serotype 1 (26 strains [15.9%]). Only one isolate belonged to serotype 3. A total of 54 (32.9%) isolates were untypeable in Prescotts system. The prevalence of R. equi strains of intermediate virulence among the isolates that came from the submaxillary lymph nodes of swine in Hungary was lower than that seen with isolates obtained elsewhere.

Virulence of Rhodococcus equi Isolated from Cats and Dogs

ABSTRACT 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.

Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan.

This report describes the discovery of two new virulence plasmid types from a crossbred foal with Rhodococcus equi pneumonia in Kumamoto died with severe R. equi pneumonia and ulcerative enteritis. R. equi was isolated in large numbers and isolates from the foal were investigated for the presence of virulence-associated 15-17 kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and by gene coding for VapA by PCR. Plasmid DNAs extracted from the isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII. The digestion patterns that resulted divided the plasmids of these isolates into two closely related types. The digestion patterns were then compared with eight representative virulence plasmid types (85 kb types I, II, III and IV, 87 kb types I and II, 90 kb types I and II), which have already been reported. None of the EcoRI and EcoT22I digestion patterns of the eight representative plasmids matched those of the two plasmid types. We tentatively designated these new plasmid types as 90 kb type III and type IV, since HindIII and BamHI digestion patterns of the two plasmid types were identical with those of a 90 kb type I plasmid. This study, demonstrated that there are at least 10 distinct but closely related plasmids present in isolates from horses in the world.

Detection of virulent Rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of R. equi pneumonia in foals

Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Humoral immune responses in 16 foals to virulence-associated 15- to 17-kDa antigens of Rhodococcus equi were studied during the first fourteen weeks of life on two horse-breeding farms with a persistent incidence of R. equi infection. Serum antibody levels specific for 15- to 17-kDa antigens were measured by enzyme-linked immunosorbent assay and Western immunoblotting. Immunoglobulin G (IgG) antibodies specific to 15- to 17-kDa antigens were detected by all the foals. R. equi was found in the feces of foals during week 1 of life, and the number of fecal R. equi rapidly increased to the highest level. Virulent R. equi were isolated from the feces of the foals at a high frequency and from their environmental soil on the farms. Evidence that serum antibody response to 15- to 17-kDa antigens of virulent R. equi occurred naturally in every foal in correlation with the quantitative changes of fecal R. equi during the first 1 to 3 months of life suggests that intestinal virulent R. equi might be the most important source of antigenic stimulation in foals from contaminated farms.

Isolation and characterisation of Rhodococcus equi from submaxillary lymph nodes of wild boars (Sus scrofa)

Rhodococcus equi has been isolated from the submaxillary lymph nodes of domesticated pigs, but little is known about the presence of R. equi in wild boars. The aim of the study was the evaluation of the incidence of R. equi in wild boars and the characterisation of them. Of 482 submaxillary lymph nodes of wild boars shot in 39 settlements throughout Hungary, R. equi was isolated from 60 specimens, and plasmid types of 82 isolates were examined. The isolates were tested for the presence of 15-17-kDa (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. None of the 82 isolates contained vapA gene but 21 isolates (25.6%) were positive for vapB gene showing 827bp product of the expected size in the PCR amplification. Sixty-one strains (74.4%) did not contain plasmid. The 21 isolates of intermediate virulence contained virulence plasmids that were identified as types 1 (1 isolate), 5 (16 isolates), 21 (1 isolate), and three new distinct plasmid variants (1-1-1 isolate), respectively. On the basis of restriction digestion patterns of plasmid DNAs, we tentatively designated the new variants as types 25-27, respectively. The prevalence of R. equi strains of intermediate virulence among the isolates originated from the submaxillary lymph nodes of wild boars (25.6%) is very similar to those of domestic pigs (26.8%) in Hungary, and plasmid type 5 is the predominating one in both groups. This is the first report of isolation of VapB-positive R. equi from wild boars in the world.

Effect of growth temperature on maintenance of virulent Rhodococcus equi

Repeated passage of virulent Rhodococcus equi ATCC 33701 and L1 at 38 degrees C resulted in attenuation of the strains as a result of curing the virulence plasmid; at 30 degrees C, repeated passage had no such effect. At a temperature of 38 degrees C the plasmid-bearing cells replicated more slowly than their plasmid-cured derivatives and so were gradually replaced by cells lacking plasmids. In contrast, at a temperature of 30 degrees C the growth rate of either strain was not affected by the presence or absence of the plasmid. No plasmid-cured derivative was recovered from mouse organs at 48 h after inoculation of a mixture of equal numbers of bacteria with and without plasmids. It is concluded that under nonselective conditions growth temperature is an important factor in maintaining the virulence of R. equi.