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PubMed1990

Surgical management of Rhodococcus equi metaphysitis in a foal.

A chronic Rhodococcus equi metaphysitis involving the distal growth plate of the left third metatarsal bone had induced a longstanding lameness in a young foal. Abnormal hematologic values included mild anemia, hyperfibrinogemia, mild leukocytosis, and neutrophilia. Radiography of the distal portion of MT3 revealed a radiolucent zone on the medial aspect of the growth plate, and small pieces of bone suggestive of sequestra. Treatment with erythromycin estolate and rifampin, aggressive surgical debridement, and cancellous bone grafting helped resolve the bone infection.

Abscess↗
PubMed1991

[In vitro activity of twenty antibiotics against Rhodococcus equi].

The in vitro susceptibility of nine Rhodococcus equi strains (seven isolates from immunocompromised patients mainly HIV positive and two reference strains) to twenty various antibiotics were assessed for bacteriostatic effects by an agar dilution method. Imipenem and ceftriaxone were the most effective of the beta-lactams studied. The lowest MIC were noted with vancomycin, teicoplanin, erythromycin, clarithromycin, rifampicin, gentamicin and doxycycline. A longitudinal survey, including three strains isolated from the same patient, showed the emergence of rifampicin resistance and a marked increase of the MIC to imipenem.

Actinomycetales Infections↗
PubMed1997

Purulent meningitis due to Rhodococcus equi. A case of posttraumatic infection.

Opportunistic infections due to Rhodococcus equi have been increasingly reported in the immunocompromised population, especially in patients with AIDS. In this report, we present an unusual case of purulent meningitis that developed in an immunocompetent six-year-old child through direct inoculation of R. equi.

Animals↗
PubMed1987

Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages.

It has been suggested that R. equi causes pulmonary disease in foals by persisting within the lung as a facultative intracellular parasite of alveolar macrophages. This paper describes an ultrastructural study of the intracellular events after ingestion of R. equi by foal alveolar macrophages, in an attempt to determine the mechanism of intracellular survival of R. equi. Secondary lysosomes of alveolar macrophages recovered from foals by bronchoalveolar lavage were labelled with electron-dense ferritin, and the cells were challenged with either viable or formalin-killed R. equi. After 0-, 3-, 8- or 24-h incubation, the cells were fixed and processed for electron microscopy. There was no evidence of phagosome-lysosome fusion after ingestion of either viable or non-viable R. equi by foal alveolar macrophages. Rhodococcus equi persisted and multiplied within dilated phagosomes, which were often lined by elongate microvillous structures. After 24-h incubation, 75% of the ingested bacteria were still structurally intact. Macrophages with ingested viable R. equi were irreversibly damaged and released intracellular bacteria into the surrounding medium. These data confirm that R. equi is a facultative intracellular parasite of foal alveolar macrophages and is able to persist and multiply within the phagosome, apparently inhibiting phagosome-lysosome fusion by some as yet unknown mechanism.

Animals↗
PubMed1998

Cutaneous malakoplakia in pigs inoculated with Rhodococcus equi.

Cutaneous malakoplakia was observed in pigs inoculated intramuscularly with Rhodococcus equi strains of intermediate virulence. Macroscopically, the inoculation sites showed the indurated swelling of the skin. Histopathologically, abscess formation with histiocytic granulomatous reaction was observed. Many macrophages contained target or owl-eye shaped hematoxyphil intracytoplasmic inclusions or calcosherites (Michaelis-Gutmann bodies) of various sizes. The Michaelis-Gutmann bodies were also seen outside of the macrophages. Histochemically, most Michaelis-Gutmann bodies stained positively with the von Kossa silver method and periodic acid Schiff. Immunohistochemically, some of Michaelis-Gutmann bodies were stained by two rabbit polyclonal antibodies (rabbit anti-A5 serum and rabbit anti-ATCC 33701 serum) and a mouse monoclonal antibody (anti-20-kDa antigen monoclonal antibody). This is the first report of cutaneous malakoplakia in domestic animals, which also revealed the relationship between R. equi infection and malakoplakia immunohistochemically. This experimental swine model is useful to investigate the morphogenesis of Michaelis-Gutmann bodies in malakoplakia through chronological skin biopsies.

Actinomycetales Infections↗
PubMed1995

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.

Actinomycetales Infections↗
PubMed2005

VapB-positive Rhodococcus equi infection in an HIV-infected patient in Japan.

Rhodococcus equi, a bacterium present in soil, is a common cause of pneumonia in foals. This organism has been recognized as an opportunistic pathogen in humans, typically causing infection in immunocompromised hosts such as HIV-infected patients and organ transplant recipients. However, human infection with R. equi has not been reported in Japan except in a case involving a laboratory worker. We report the first human case of VapB-positive R. equi pneumonia, which involved an HIV-infected patient living in an urban area in Japan. The patient was treated successfully with 450 mg rifampicin and 600 mg tosufloxacin, even though his CD4+ lymphocyte count at the time of diagnosis was 10/microl. The patient's dogs were suspected in the epidemiology of this infection, but unfortunately we could not isolate the organism from canine-associated specimens in this case. R. equi infections in companion animals have been thought to be very rare, but they may be increasing in dogs. Therefore, further epidemiological research may clarify the prevalence of R. equi infection and the factors predisposing dogs to this infection.

AIDS-Related Opportunistic Infections↗
PubMed1996

Identification of intermediately virulent Rhodococcus equi isolates from pigs.

We recently reported the existence of Rhodococcus equi isolates with at least three virulence levels, isolated from AIDS patients: virulent R. equi having 15- to 17-kDa antigens that kills mice with 10(6) cells, intermediately virulent R. equi having a 20-kDa antigen that kills mice with 10(7) cells, and avirulent R. equi that does not kill mice with 10(8) cells or more (S. Takai, Y. Imai, N. Fukunaga, Y. Uchida, K. Kamisawa, Y. Sasaki, S. Tsubaki, and T. Sekizaki, J. Infect. Dis. 172:1306-1311, 1995). Virulent R. equi having the 15- to 17-kDa antigens has been isolated frequently from horses and their environment, but the source of intermediately virulent R. equi having the 20-kDa antigen is poorly understood. There are many reports of the isolation of R. equi from the lymph nodes of pigs with and without lesions resembling those of tuberculosis. Therefore, we analyzed antigens of R. equi isolates from the submaxillary lymph nodes of pigs by immunoblotting with monoclonal antibodies against these virulence-associated antigens. Immunoblots of whole-cell antigen preparations of R. equi pig isolates revealed the presence of the 20-kDa antigen in almost all the pig isolates studied, and these isolates were intermediately virulent for mice. We also demonstrated that the expression of the 20-kDa antigen and its pathogenicity in mice were associated strongly with the presence of five large, distinct plasmids of 70 to 95 kb; two of the five plasmids from pig isolates were the same sizes as those from human isolates. These results suggest that R. equi having the 20-kDa antigen exists in the submaxillary lymph nodes of pigs and that the source of infection in some human cases might be associated with pigs and their environment.

AIDS-Related Opportunistic Infections↗
PubMed1987

Experimental infection of piglets by aerosols of Rhodococcus equi.

The purpose of this study was to investigate experimental infection of the piglet as a model of Rhodococcus equi pneumonia in the foal. Three litters of eight piglets each were exposed to an aerosol of 3.4 X 10(7) R. equi per piglet per day for seven consecutive days. Over the next 23 days the piglets were observed for clinical signs of disease. Periodically after infection one piglet from each litter was killed, the lungs were cultured quantitatively for R. equi and the gross and microscopic pulmonary lesions were assessed. The only clinical evidence of disease was the occurrence of elevated temperatures in the infected piglets. Rhodococcus equi was slowly cleared from the piglets' lungs during the 23 days following aerosolization. Piglets sacrificed seven to ten days after aerosolization had the most extensive pulmonary lesions, consisting of severe consolidation of the cranioventral lobes. Microscopic examination revealed thickened interalveolar septa and alveoli containing many neutrophils and macrophages with intracytoplasmic Gram-positive coccobacilli. The pulmonary lesions in these piglets differed from those of naturally infected foals in that they were not characterized by macrophage-rich abscesses and the infection gradually resolved.

Actinomycetales Infections↗
PubMed2004

Innate immune responses to Rhodococcus equi.

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.

Adaptor Proteins, Signal Transducing↗
PubMed2002

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.

Actinomycetales Infections↗
PubMed1991

Rhodococcus equi plasmids: isolation and partial characterization.

Fifty-four strains of Rhodococcus equi from different clinical sources (mainly horses and pigs) were examined for their plasmid content by two screening methods. Plasmids were detected in 49 of 54 strains. A plasmid of approximately 80 kb was isolated from 21 of 22 isolates from horses and 20 of 28 isolates from pigs, and a 105-kb plasmid was isolated from 7 of 28 isolates from pigs. The 80-kb plasmid was significantly associated with strains of equine rather than porcine origin, and the 105-kb plasmid was significantly associated with strains of porcine origin. The type strain, ATCC 6939, consistently failed to yield a plasmid. Restriction enzyme analysis of purified plasmid DNA confirmed the relatedness of the 80-kb plasmids isolated from strains of equine and porcine origin. More differences between the restriction patterns of plasmids from strains isolated from horses and from pigs than among strains from either species were observed. Restriction enzyme analysis also showed relatedness of the 105-kb plasmid to the 80-kb plasmid. Three strains shown by others to be virulent in horses or mice possessed the 80-kb plasmid, whereas three other strains not virulent for horses or mice lacked the plasmid, although one had the 105-kb plasmid. There was a significant but not perfect association between the presence of the 80-kb plasmid and production of a diffuse 17.5-kDa thermoregulated, virulence-associated protein. Further study is needed to determine whether this plasmid is associated with virulence in R. equi.

Animals↗
PubMed1996

Isolation of virulent and intermediately virulent Rhodococcus equi from soil and sand on parks and yards in Japan.

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. However, little is known about the distribution of virulent and intermediately virulent R. equi in human environment. In the present study, R. equi was isolated from 173 of 234 (73.9%) samples collected from soil and sand on 115 parks and 49 yards in Japan. The numbers of R. equi from soil and sand ranged from 2.5 x 10(1) to 1.2 x 10(5) per gram of sample. None of 1,294 isolates from those samples showed virulence-associated 15- to 17-kDa antigens and a 20-kDa antigen. These results suggest that avirulent R. equi is widespread in parks and yards, but the human environment has not been contaminated with virulent and intermediately virulent R. equi strains yet.

Antigens, Bacterial↗
PubMed2006

Comparison of two selective media for the recovery, isolation, enumeration and differentiation of Rhodococcus equi.

The use of selective media to facilitate the isolation of Rhodococcus equi from environmental and clinical samples has aided studies of the ecology of R. equi and the epidemiology of disease caused by R. equi. Here, we compared the efficacy of two selective media (NANAT and modified CAZ-NB) for the recovery of six defined strains of R. equi and for the isolation and enumeration of both avirulent and virulent R. equi from 60 paired soil samples from horse farms using colony blotting and DNA hybridisation. No difference was found between the two media in the recoverability of defined strains of R. equi or the proportion of soil cultures positive for R. equi or virulent R. equi. NANAT medium was significantly less inhibitory of bacterial growth from soil culture compared to mCAZ-NB (P = 0.001), but there was no difference between the media in the number of R. equi colonies recovered. Soil cultured on mCAZ-NB medium yielded a significantly greater number of virulent R. equi colonies than NANAT (P = 0.03). The proportion of R. equi that were virulent in soil cultures on mCAZ-NB (32%) was more than three times that seen in cultures on NANAT (9%). Thus modified CAZ-NB appeared to be a better selective media for studies where the optimal recovery of virulent R. equi is required, such as in studies of the gastrointestinal carriage of virulent R. equi and of subclinically infected foals.

Actinomycetales Infections↗
PubMed1994

Biochemical and serological characteristics of Rhodococcus equi isolates from animals and humans.

In the present study, 17 Rhodococcus equi isolates from animals and 13 R. equi isolates from humans were correctly identified by the use of the API Coryne-test system. The biochemical characteristics revealed no significant differences between isolates from animals and humans. Heat extractions at acid pH or autoclave extractions of the bacteria and the use of monospecific antisera against type antigens 1-7 allowed the serotyping of the bacteria. The majority of the R. equi isolates reacted with serotype 1 and serotype 2-specific antisera, extracts of two human isolates reacted with serotype 5 and serotype 6-specific antisera, respectively. The determination of biochemical and serological properties of R. equi might help to characterize individual cultures of this species. This might be useful in epidemiological studies.

Actinomycetales Infections↗
PubMed2001

B-Cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals.

Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with R. equi disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for R. equi infection in foals.

Actinomycetales Infections↗
PubMed2001

Induction of vap genes encoded by the virulence plasmid of Rhodococcus equi during acid tolerance response.

The response of the intracellular pathogen Rhodococcus equi to acid shock, a stress potentially encountered after phagocytosis by macrophages, was analyzed. The wild-type and its avirulent plasmid-cured strain acquired increased acid tolerance during the exponential growth phase upon exposure to sublethal acid stress, a response referred to as the acid tolerance response. Maximal adaptation was observed when cells were pretreated for 90 min at pH 5.0 before exposure to the pH challenge. Search for plasmid-encoded proteins regulated by an acidic pH was performed using two-dimensional gel electrophoresis, and enabled us to detect several membrane and cytoplasmic proteins with altered expression during the adaptation phase, but none of them were plasmid-encoded. However, using a strategy based on plasmid-encoded gene expression, we showed that two operons located on the virulence plasmid of strain 85F were upregulated by acid pHs with a maximal induction at pH 5.0. One operon, containing vapA, was monocistronic whereas the other was polycistronic composed of vapD and an unknown open reading frame. Our combined results suggest that these genes may play an important role in the pathogenicity of R. equi.

Actinomycetales Infections↗