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PubMed2003

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.

Actinomycetales Infections↗
PubMed2001

Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination.

The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodococcus equi in impression smears from affected organs of foals on postmortem examination. The second aim was to demonstrate whether R. equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method. Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R. equi, and also from the caudal mesenteric lymph node of foal A. Impression smears were made from the tracheal exudates of all foals. An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R. equi. This antibody reacted with serotype 1 of R. equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp. equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M. paratuberculosis. The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R. equi could only be detected by this method. R. equi was demonstrated in smears of the tracheal exudates of all three foals. The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R. equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R. equi pneumonia in live foals by examining smears of tracheal aspirates.

Actinomycetales Infections↗
PubMed2002

Association of pneumonia in foals caused by Rhodococcus equi with farm soil geochemistry.

OBJECTIVE: To quantify and compare geochemical factors in surface soils from horse-breeding farms with horses with pneumonia caused by Rhodococcus equi (affected farms) and horse-breeding farms with no history of pneumonia caused by R equi (unaffected farms). SAMPLE POPULATION: Soil from 24 R equi-affected farms and 21 unaffected farms. PROCEDURE: Equine veterinary practitioners throughout Texas submitted surface soil samples from areas most frequented by foals, on R equi-affected and unaffected horse-breeding farms in their practice. Soil samples were assayed for the following factors: pH, salinity, nitrate, phosphorus, potassium, calcium, magnesium, sodium, sulfur, zinc, iron, manganese, and copper. Median values for all factors were recorded, and differences between affected and unaffected farms were compared. RESULTS: Significant differences in soil factors were not detected between affected and unaffected farms; hence, there was no association between those factors and R equi disease status of the farms. CONCLUSIONS AND CLINICAL RELEVANCE: The surface soil factors monitored in this study were not significant risk factors for pneumonia caused by R equi. As such, it is not possible to determine whether foals on a given farm are at increased risk of developing disease caused by R equi on the basis of these factors. Data do not support altering surface soil for factors examined, such as alkalinization by applying lime, as viable control strategies for R equi.

Actinomycetales Infections↗
PubMed2007

Evaluation of fecal samples from mares as a source of Rhodococcus equi for their foals by use of quantitative bacteriologic culture and colony immunoblot analyses.

OBJECTIVE: To determine whether mares are a clinically important source of Rhodococcus equi for their foals. SAMPLE POPULATION: 171 mares and 171 foals from a farm in Kentucky (evaluated during 2004 and 2005). PROCEDURES: At 4 time points (2 before and 2 after parturition), the total concentration of R equi and concentration of virulent R equi were determined in fecal specimens from mares by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively. These concentrations for mares of foals that developed R equi-associated pneumonia and for mares with unaffected foals were compared. Data for each year were analyzed separately. RESULTS: R equi-associated pneumonia developed in 53 of 171 (31%) foals. Fecal shedding of virulent R equi was detected in at least 1 time point for every mare; bacteriologic culture results were positive for 62 of 171 (36%) mares at all time points. However, compared with dams of unaffected foals, fecal concentrations of total or virulent R equi in dams of foals with R equi-associated pneumonia were not significantly different. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that dams of foals with R equi-associated pneumonia did not shed more R equi in feces than dams of unaffected foals; therefore, R equi infection in foals was not associated with comparatively greater fecal shedding by their dams. However, detection of virulent R equi in the feces of all mares during at least 1 time point suggests that mares can be an important source of R equi for the surrounding environment.

Actinomycetales Infections↗
PubMed1993

Rhodococcus equi infection in foals: a report of an outbreak on a thoroughbred stud in Zimbabwe.

Twenty-four foals were confirmed to be infected with Rhodococcus equi on a private stud in Zimbabwe over a two-year period. Six mares had foals which were affected in each of the two years. All the foals were febrile and early cases were detected by this pyrexia. Bronchopneumonia was only clinically detectable in advanced cases. In spite of energetic hygiene measures relating to pasture and housing management, the incidence was higher in the second year (23 per cent of foals born) than in the first (15 per cent of foals born). The mean age of the foals was significantly greater in the second year. The immunological status of some of the foals was obtained from zinc sulphate turbidity tests performed at 24 hours old and all the affected foals so tested were considered to have had effective colostral transfer. Clinically, affected foals showed pyrexia, tachypnoea and tachycardia and many had a scanty purulent nasal discharge. Only one animal had diarrhoea which was attributed to enteric infection with R equi. Diagnosis was confirmed by clinical examination, culture of the organism from tracheal aspirates and thoracic radiography and was supported by significant elevations of plasma fibrinogen and platelet and neutrophil counts in all cases. Anaemia was not a major finding in any case. Rifampicin and erythromycin were administered per os and weight-related doses were continued until plasma fibrinogen and the absolute neutrophil and platelet counts had been persistently normal for two weeks. Supportive radiography was obtained in some cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomycetales Infections↗
PubMed1987

Antibody response of horses to Rhodococcus equi antigens.

The antigens extracted from strains belonging to seven capsular serotypes of Rhodococcus equi, as well as from two wild strains isolated from pneumonic foals, were examined. Whole-cell antigens and soluble products present in broth culture supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose, and stained with serum from hyperimmunized rabbits or foals. Foal sera used included sera from pneumonic animals with known titer to equi factors; from animals bled monthly on a farm with enzootic pneumonia, and from animals bled monthly on a farm with no history of R. equi pneumonia. The humoral response of foals to somatic antigen preparations was negligible, with few differences noted between sera from healthy, subclinically affected, and sick foals. The humoral response to R. equi broth culture supernatant products appeared more marked and was related to equi factor antibody titer. These findings suggest that the humoral response to R. equi whole-cell antigens is unimportant in protection against disease, which is consistent with the behavior of the organism as a facultative intracellular pathogen.

Animals↗
PubMed2002

Isocitrate lyase of the facultative intracellular pathogen Rhodococcus equi.

Isocitrate lyase is the first enzyme of the glyoxylate shunt which is required for the assimilation of fatty acids and acetate. The intracellular pathogen Rhodococcus equi contains high activities of this enzyme following growth on acetate and lactate, indicating that it plays an important role in the metabolism of these substrates. The gene encoding isocitrate lyase (aceA) was cloned and sequenced. It specifies a 46846 Da protein, which was shown to be functional by expressing it in Escherichia coli. A gene similar to fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, was located 90 bp downstream from aceA. Northern hybridization and RT-PCR experiments showed that aceA and fadB are cotranscribed into a 2.8 kb transcript. A smaller 1.6 kb aceA transcript was also observed which was 2.5-fold more abundant than the aceA-fadB transcript. It is proposed that a stable hairpin structure with a free energy (DeltaG) of -28.5 kcal x mol(-1) and located in the 90 bp aceA-fadB intergenic region is involved in stabilizing the aceA transcript.

Acetates↗
PubMed2001

Epidemiology of Rhodococcus equi strains on Thoroughbred horse farms.

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.

Actinomycetales Infections↗
PubMed1990

Influence of Rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses.

Opsonized Rhodococcus equi activated the respiratory burst of resident alveolar macrophages (AM) from adult horses in a logarithmic-linear, mass-related manner. The effect of R equi was not significantly different from that of equal masses of opsonized zymosan A. Therefore, R equi does not appear to attenuate the respiratory burst of equine AM. The stimulatory effect of R equi was not reflected by increased production of superoxide anion (O2-), but increased activity of the hexose monophosphate shunt was observed. These results suggest a similarity between the respiratory burst of AM from horses and that of AM from rabbits. We concluded that resident AM from adult horses do not produce O2- concurrently with an increase in activity of the hexose monophosphate shunt when stimulated with either opsonized zymosan A or opsonized R equi. This suggests that O2- is not an important component of the antibacterial defenses of equine AM. Whether equine AM are incapable of producing O2- or require different stimuli to produce it was not determined.

Animals↗
PubMed2002

Rhodococcus equi pulmonary infection in a pancreas-alone transplant recipient: consequence of intense immunosuppression.

We report the case of a pancreas-alone transplant recipient who developed Rhodococcus equi pneumonia after receiving multiple courses of antilymphocyte therapy for the treatment of recurrent acute pancreas allograft rejection. We also review and discuss the diagnosis, clinical course, and treatment of 18 cases of R. equi infection reported in solid organ transplant recipients. The lung is the most common primary site of infection, but R. equi infection is difficult to diagnose because of the pleomorphic, gram-positive, and partially acid-fast nature of the organism. Treatment usually involves a combination of antibiotics including rifampin, macrolides, vancomycin, and ciprofloxacin. The optimal duration of therapy is unknown, but relapse is common if the duration of treatment is less than 14 days. The duration of therapy should be guided by clinical recovery, culture results, and radiographic findings. Monitoring levels of immunosuppressive agents-such as tacrolimus and cyclosporine-is needed in order to avoid clinically significant drug interactions with rifampin or the macrolides when these agents are used in order to treat R. equi infection in the transplant population.

Actinomycetales Infections↗
PubMed2003

The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties.

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.

Animals↗
PubMed2004

The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.

An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.

Actinomycetales Infections↗
PubMed2001

Characterization of mutations in the rpoB gene associated with rifampin resistance in Rhodococcus equi isolated from foals.

Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rif(r))-determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 microg/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 microg/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in R. equi, and the resistance levels are dependent on both the location and the nature of the substitution.

Actinomycetales Infections↗
PubMed2006

Effect of hyperimmune plasma on the severity of pneumonia caused by Rhodococcus equi in experimentally infected foals.

This study evaluated the prophylactic effectiveness of hyperimmune plasma (HIP) as an aid in the prevention of pneumonia caused by experimental infection with Rhodococcus equi. Thirty neonatal foals were administered R. equi HIP or saline at 2 days of age and were infected with virulent R. equi at 7 days. All foals developed signs or symptoms of respiratory disease. Radiographic scores on day 28 and neutrophil concentrations on day 49 were significantly greater in control foals, and time to respiratory effort score of 2 or higher was significantly shorter for control foals. Three foals, all in the principal group, died or were euthanized before the end of the study, but there was no significant difference in mortality between groups. VapA titers were significantly greater in principal foals. Administration of R. equi HIP decreased the severity of radiographic lesions and prolonged time to increased respiratory effort due to R. equi-induced pneumonia.

Actinomycetales Infections↗
PubMed1997

Restriction enzyme analysis of the virulence plasmids of VapA-positive Rhodococcus equi strains isolated from humans and horses.

Restriction enzyme digestion patterns of the large virulence plasmids of 8 human and 37 foal isolates of virulence-associated protein (VapA)-positive Rhodococcus equi strains from different sources were compared. Foal isolates came from five continents. Digestion with EcoRI divided these plasmids into three closely related types, and digestion with BamHI divided them into three major types which corresponded to the EcoRI types. The only EcoRI and BamHI type 3 plasmid was from a single foal isolate obtained from Japan. There are thus two major but related virulence plasmids in isolates from foals. Geographic differences were noted, since foal isolates with the EcoRI type 1 plasmid digestion pattern tended to come mostly from the United States, Canada, European countries, India or Zimbabwe and foal isolates with EcoRI type 2 pattern tended to come mostly from Latin American countries. Only 8 of 38 different human isolates, mostly from AIDS patients, were VapA positive, in contrast to 37 of 42 foal isolates. VapA-positive isolates from humans possessed virulence plasmids of either EcoRI type 1 or EcoRI type 2. These results confirm that only a small proportion of human patients with R. equi infections acquire foal virulent R. equi.

AIDS-Related Opportunistic Infections↗
PubMed1998

Report of invasive Rhodococcus equi infections in Taiwan, with an emphasis on the emergence of multidrug-resistant strains.

From November 1995 to October 1997, seven patients with invasive infections due to Rhodococcus equi were treated in Taiwan. Four patients had pulmonary lesions, and one each of the remaining three patients had a recurrent Port-A-Cath (Kabi-Pharmacia, North Ryde, New South Wales, Australia)-related bacteremia, a primary bacteremia, and a brain abscess. Three patients had underlying hematologic malignancies, and one each of the remaining four patients had diabetes mellitus, Waldenström's macroglobulinemia, long-term use of steroids, and AIDS. The 13 isolates of R. equi recovered from these patients were identified by using API Coryne System (bioMérieux, Marcy l'Etoile, France), VITEK GPI card (bioMérieux Vitek, Hazelwood, MO), supplemental biochemical tests, and cellular fatty acid chromatograms. Susceptibilities of these isolates to 16 antimicrobial agents, with use of the agar dilution method, varied; among them, amikacin and trimethoprim-sulfamethoxazole were the most active agents. Different random amplified polymorphic DNA (RAPD) patterns of isolates from different patients documented the lack of epidemiological relatedness of the causative organisms of these infections. This study confirms the emergence of multidrug-resistant R. equi infection in Taiwan and documents the relapsing or reactivating nature of this infection.

Actinomycetales Infections↗
PubMed2005

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.

Actinomycetales Infections↗