Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

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.     

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 virulent Rhodococcus equi in isolates from soil collected from two horse farms in South Africa and restriction fragment length polymorphisms of virulence plasmids in the isolates from infected foals, a dog and a monkey

The prevalence of virulent Rhodococcus equi in soil isolates from two horse farms in South Africa and nine clinical isolates from six foals, a foal foetus, a dog, and a monkey was investigated. The isolates were tested for the presence of virulence plasmid DNA and 15- to 17-kDa antigens by immunoblotting. Rhodococcus equi was isolated from almost all of the soil samples obtained from the two farms with 5.0 x 10(1) to 3.3 x 10(4) colony forming units per gram of soil. Virulent R. equi was isolated from three soil samples from one of the farms and appeared in 3.8% (three of 80 isolates), but not in any of the 182 isolates from the other farm. Of the three virulent R. equi isolates, one contained an 85-kb type I plasmid and two an 87-kb type I plasmid. Of nine clinical isolates from the foals, foal foetus, dog and monkey, five from the foals were virulent R. equi which expressed the virulence-associated antigens and contained a virulence plasmid 85-kb type I, and were all isolated from cases of pneumonia typical of that induced by R. equi in young foals living in widely separated areas in South Africa. The isolates from the other four foals, the dog and the monkey were avirulent R. equi.     

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.     

Plasmid profiles of virulent Rhodococcus equi isolates 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 VapA-positive Rhodococcus equi in thoroughbred horses in Kagoshima,Japan

The prevalence of virulent R. equi having 15- to 17-kDa antigens (VapA) in fecal isolates from 13 thoroughbred foals and their dams on 5 farms in Kagoshima, Japan, and the plasmid profiles of VapA-positive isolates by restriction fragment digestion patterns were investigated to compare the genotypic variation among virulence plasmids of R. equi isolates from Japan. In total, 218 (24.6%) of 886 isolates from the feces of the 13 foals and 13 (12.5%) of 104 isolates from the feces of their dams demonstrated VapA-positive R. equi. Plasmid DNA preparations of 231 virulent isolates from foals and dams were analyzed by restriction enzyme digestion with endonucleases EcoRI, EcoT22I and HindIII and were divided into 3 types: 172 isolates contained a 90-kb type I plasmid, 57 contained a 90-kb type III plasmid and 2 contained a 90-kb type IV plasmid. This study demonstrates a geographic character in the distribution of virulence plasmids found in VapA-positive isolates from thoroughbred foals in Kagoshima.     

Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.     

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.     

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

Rhodococcus equi was isolated from fecal and soil samples from four native Jeju horse farms and six Thoroughbred farms in Jeju, Korea. The isolates were examined for the presence of virulence-associated 15-17-kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and for the gene encoding VapA by PCR. R. equi was isolated from all 36 soil samples collected from the 10 farms with between 5.0 x 10(2) and 7.5 x 10(4) colony-forming units (cfu) per gram of soil, and from 37 of 40 fecal samples with between 5.0 x 10(1) and 1.1 x 10 (5) cfu per gram of feces. Virulent R. equi was isolated from seven farms and appeared in 2.0% of isolates (10 of 508). Of the 10 virulent isolates, four contained a 90-kb type II plasmid, which has been found in isolates from the Kiso native horses of Japan, and the other six contained a new variant, which did not display the EcoRI and EcoT22I digestion patterns of the 10 representative plasmids already reported (85-kb types I, II, III, and IV; 87-kb types I and II; 90-kb types I, II, III, and IV). We designated the new variant as the "90-kb type V" plasmid, because its EcoRI digestion pattern is similar to that of the 90-kb type II plasmid. This is the first report of the prevalence of virulent R. equi in Jeju, Korea. The same virulence plasmid type is found in both Korean and Japanese isolates, providing insight into the origin, ancestry, and dispersal of native horses in Korea and Japan.     

Typing of Rhodococcus equi isolated from submaxillary lymph nodes of pigs in Japan

Isolation of Rhodococcus equi from the submaxillary lymph nodes of pigs, with or without caseous lymphadenitis, and typing of the isolates by two serological methods were carried out. The rate of isolation of the organisms from the lymph nodes of pigs was 5 times higher in the lymph nodes with caseous lymphadenitis than in those without the lesion. Of 219 isolates, 146 (66.7%) were typable by the method of Prescott, while all the 219 isolates (100%) were typable by the method of Nakazawa et al. The most frequently isolated were serotype 2 of Prescott (identical to serogroup 16 of Nakazawa et al.), and serogroup 3 of Nakazawa et al., which did not correspond with any serotypes of Prescott. Serotypes/serogroups of R. equi from pigs were thus first clarified in Japan.     

Virulence-associated protein characterisation of Rhodococcus equi isolated from bovine lymph nodes

Rhodococcus equi has a low pathogenicity in cattle, but it occasionally causes lymph node granulomas, which are detected at abattoir post mortem inspection, and must be distinguished from tuberculous granulomas. Lymph node lesions were detected in 6719 cattle, from a total of 3,263,622 cattle examined post mortem in abattoirs, in the Republic of Ireland, during 1997 and 1998. Histological examination was performed on all lesions, principally for the purpose of identifying animals with tuberculosis. A total of 1122 of the lesions were cultured on blood agar and on Stonebrinks and Lowenstein-Jensen medium containing pyruvate, because the histological findings were difficult to interpret or were suggestive of R. equi infection. R. equi was isolated from 264 lesions. Almost all of the R. equi granulomas were confined to a single lymph node, and were present predominantly in the retropharyngeal, bronchial and mediastinal lymph nodes. R. equi granulomas were present in a significantly higher proportion of the lesions detected in steers and heifers compared to cows. The prevalence in the total population of 3.3 million cattle examined post mortem was 0.008%. The 15-17kDa antigens, associated with virulence in this organism, and the 20kDa antigen, associated with intermediate virulence, were not detected in isolates from 146 cattle, analysed by immunoblot assays. A PCR assay to detect the plasmid gene encoding the 15-17kDa antigens was also negative for isolates from these 146 animals. Plasmids were not detected in 30 isolates which were examined.     

Molecular typing of VapA-positive Rhodococcus equi isolates from Jeju native horses, Korea

We recently demonstrated the presence of virulence-associated protein antigen (VapA)-positive Rhodococcus equi in Jeju Island, Korea. These bacteria contained one of two distinct plasmid types, a 90-kb type II plasmid, which has been found in isolates from the native Kiso horses of Japan, and a new variant, a 90-kb type V plasmid. However, the genotypic characters of the VapA-positive R. equi from Jeju native horses and their environments are poorly understood. Ninety-eight isolates from soil samples and 89 isolates from fecal samples were collected from five farms that breed or have bred Jeju native horses, and were tested for the presence of VapA by immunoblotting and PCR. Of the 98 soil isolates and 89 fecal isolates, seven and 13 were VapA-positive R. equi, respectively. In 2003, two Jeju foals died suddenly and were brought to the Faculty of Veterinary Medicine, Cheju National University, for postmortem examination. Pure cultures of R. equi were isolated from the lung lesions of both foals. Of the 16 clinical isolates, 14 were VapA-positive R. equi. Of the 34 VapA-positive clinical and environmental isolates, 16 contained the 90-kb type II plasmid and 18 contained a 90-kb type V plasmid. Pulsed-field gel electrophoresis (PFGE) patterns of the VapA-positive isolates from Jeju horses and Kiso horses, containing the 90-kb type II plasmid, were compared and formed two distinct groups. Furthermore, 18 virulent isolates containing the 90-kb type V plasmid formed two distinct PFGE groups (of 16 and two isolates). These results demonstrate that two virulence plasmid types are widespread in R. equi in Jeju native horses. However, there is little diversity in the PFGE patterns of virulent isolates, suggesting the clonal spread of virulent R. equi. The PFGE pattern of the virulent R. equi isolates from Jeju native horses in Korea is not identical to those of Kiso native horses in Japan.     

Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen.     

Attempts to find phenotypic markers of the virulence plasmid of Rhodococcus equi.

Four isolates of Rhodococcus equi, from pneumonic foals, and containing the 85 kb virulence plasmid, a porcine isolate containing an 80 kb plasmid, and their plasmid cured derivatives, were examined for 239 phenotypic properties in an attempt to find characters other than the virulence-associated protein (VapA) which might be encoded by the virulence plasmid in organisms grown at 37 degrees C. Tests chosen included those which have previously given variable results for R. equi isolates, since such variability might be attributed to plasmid curing, and characteristics which have been described as properties of plasmids of Rhodococcus species other than R. equi. Tests included cadmium resistance, Congo red binding, resistance to 26 antibiotics, conventional clinical microbiological tests, utilization of 95 different carbon sources, enzymatic activities in API ZYM, fluorogenic assays for exo- and endopeptidase, glycosidase activities, and testosterone degradation. Apart from production of VapA by foal isolates, no phenotypic property was identified in the plasmid-positive isolates. Phenotypic characteristics of R. equi that have not been described before, and might be useful in identification were: metabolism of N-acetyl-beta D-glucopyranoside, alpha- and beta-hydroxybutyric, alpha-ketobutyric and N-acetyl-glutamic acids, of methylpyruvate, heptanoate, nonanoate and stearate esters; exopeptidase activity against alanine-alanine-tyrosine, alanine-phenylalanine-lysine, glycine-arginine, lysine-alanine, and valine-glycine-alanine; endopeptidase activity against arginine and methionine; and hydrolysis of bis-phosphate ester.     

Isolation and characterisation of Mycobacterium avium and Rhodococcus equi from granulomatous lesions of swine lymph nodes in Slovenia

Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.     

Experimental infection of European wild boars and domestic pigs with pseudorabies viruses with differing virulence

OBJECTIVE: To determine susceptibility of European wild boars (Sus scrofa) to infection with pseudorabies virus (PrV) and to characterize the virulence of a wildboar PrV isolate for wild and domestic pigs. ANIMALS: 18 wild boars and 16 domestic pigs. PROCEDURE: Three groups of 4 wild boars were inoculated with PrV Bartha, Kaplan, and a wild-boar isolate (BFW1) and housed with uninfected pigs. Two groups of domestic pigs (4 and 8 pigs/group, respectively) were inoculated with various doses of BFW1. Animals were observed daily for clinical signs, and samples were tested for PrV excretion and homologous antibodies. After reactivation of latent infection by induced immunosuppression, PrV was detected in tissues of necropsied animals, using cell culture and a polymerase chain reaction (PCR). RESULTS: Clinical signs depended on virulence of the PrV strain and dose of inoculum. Only infection with PrV Kaplan resulted in severe disease and death. Virus was isolated from nasal and genital swab specimens. Antibodies were first detected on day 7 after inoculation; a specific humoral immune response was delayed in BFW1-infected animals. Virus was isolated from various tissues of Kaplan-infected wild boars, whereas mainly viral DNA was detected in a few tissues of Bartha- and BFW1-infected animals, using PCR after immunosuppression. CONCLUSIONS AND CLINICAL RELEVANCE: European wild boars are susceptible to transmission of PrV infection from domestic pigs and vice-versa. The PrV isolate BFW1 is of low virulence and seems to be adapted to the wild boar population from which it was isolated.     

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.     

Isolation of Rhodococcus equi from the feces of indigenous animals and soil from the Lower Zambezi National Park and Lochinvar National Park, Zambia

Rhodococcus equi is an important pathogen in foals; however, its incidence in African indigenous animals is poorly understood. Fecal samples (92 from nine indigenous species) and 43 soil samples were collected from two Zambian National Parks. The presence of R. equi was investigated and 533 isolates were tested for the presence of 15- to 17-kDa antigens (VapA) and a 20-kDa antigen (VapB) by immunoblotting and PCR. R. equi was isolated (10(2)-10(4) colony forming units/g) from 75% of fecal and 74% of soil samples. Neither antigen was detected; however, about 20% of the isolates contained cryptic plasmids of various sizes. There was no evidence of virulent R. equi, but the avirulent form was widespread in the animals and the soil.     

A physical map of the 85 kb virulence plasmid of Rhodococcus equi 103.

A physical map of the 85 kb virulence plasmid pOTS from Rhodococcus equi 103 was constructed. The restriction map contains 2 AsnI, 5 BglII, 9 EcoRI, 4 HindIII, and 3 XbaI sites. The positions of the EcoRI and HindIII of pOTS are identical to that of the 85 kb virulence plasmid of R. equi ATCC 33701 reported recently by others. EcoRI restriction fragment sizes were similar in the 85 kb plasmids isolated from 4 horse derived R. equi but, except apparently for the 28.3 and possibly 2.0 and 1.5 kb fragments, were different in an 80.1 kb plasmid isolated from a pig source R. equi.