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PubMed1993

Involvement of interferon-gamma and tumor necrosis factor-alpha in host defense against Rhodococcus equi.

Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in AIDS patients. In vitro, splenic cells of noninfected euthymic mice produced tumor necrosis factor-alpha (TNF alpha) in greater amounts when incubated with live R. equi rather than with killed bacteria. In vivo, interferon-gamma (IFN-gamma) and TNF alpha serum levels of infected euthymic mice remained below the level of detectability. Treatment of infected nude mice, which developed chronic infection, with discontinuous injections of IFN-gamma, TNF alpha, or both did not decrease bacterial colony-forming units in liver, spleen, or lungs. However, treatment of infected euthymic mice, which cured a R. equi inoculum within 3 weeks, with anti-IFN-gamma or anti-TNF alpha antibodies (or both) significantly increased tissue colony counts. These data argue that, in this murine model, endogenous IFN-gamma and TNF alpha are involved in the cell-mediated immunologic response against R. equi infection.

Actinomycetales Infections↗
PubMed1994

Haemolytic and phospholipase C (PLC) activities of Rhodococcus equi.

Rhodococcus equi, an intracellular organism causing pneumonia and lung abscesses in foals, is generally thought to be non-haemolytic. In the present study, however, 13 of 14 representative isolates were found to be haemolytic when tested on agar media containing washed red blood cells rather than whole blood. Red cells of rabbits, dogs, horses and man were more sensitive to lysis than were those of ruminants. Two new enzymatic activities of the species were defined: a lecithinase and a phosphatidylinositol-specific phospholipase C (PI-PLC). As judged from tests for trypsin, temperature and ethanol sensitivity, the haemolytic activity was primarily dependent on PI-PLC though the participation of lecithinase seemed probable. The haemolytic activity of growing strains, but not of cell-free preparations, was partially inhibited by lecithin but enhanced by cholesterol; however, cholesterol oxidase (CO) activity, known to mediate cooperative lysis of RBC sensitized with sphingomyelin-specific phospholipases C or D of some other species, did not contribute to the direct haemolysis caused by R. equi as demonstrated here.

Actinomycetales Infections↗
PubMed2000

Isolation of rare opportunistic pathogens in Hungary: case report and short review of the literature. Rhodococcus equi.

Rhodococcus equi is a well-established pathogen in foal pneumonia and is increasingly recognized as a pathogen in immunocompromised humans. We have isolated a Gram-positive coccobacillus from 8 blood samples and lung tissues of a renal transplant patient. Colony morphology, growth in Lowenstein-Jensen medium, 21 biochemical reactions, the characteristic morphological cycle (coccus-rod-coccus) and the CAMP test established the R. equi diagnosis. Histological studies of 2 lung biopsy specimens revealed numerous microabscesses with aggregates of polymorphonuclear leukocytes surrounded by abundant foamy macrophages. Our isolates proved to be sensitive to majority of antibacterial drugs. The appropriate therapy (amoxicillin-clavulanate) proved to be effective, however six months later a relapse was observed. Data show that in spite of its rare occurrence, R. equi infection represents a diagnostic and therapeutic challenge. The taxonomical, epidemiological, clinico-pathological, diagnostic and therapeutic data of R. equi are discussed.

Actinomycetales Infections↗
PubMedDate not supplied

Rhodococcus equi.

Rhodococcus equi is an important cause of subacute or chronic abscessating bronchopneumonia of foals up to 3-5 months of age. It shares the lipid-rich cell wall envelope characteristic of the mycolata, including Mycobacterium tuberculosis, as well as the ability of pathogenic members of this group to survive within macrophages. The possession of a large virulence plasmid in isolates recovered from pneumonic foals is crucial for virulence. The plasmid contains an 27 kb pathogenicity island (PI) that encodes seven related virulence-associated proteins (Vaps), including the immunodominant surface-expressed protein, VapA. Only PI genes are differentially expressed when the organism is grown in macrophages in vitro. Ten of the PI genes, including six Vap genes, have signal sequences, suggesting that they are exported from the cell to interact with the macrophage. Different PI genes are regulated by temperature, pH, iron, oxidative stress and probably also by magnesium, all environmental changes encountered after environmental R. equi are inhaled in dust and are ingested into macrophages in the lung. The basis of pathogenicity of R. equi is its ability to multiply in and eventually to destroy alveolar macrophages. Infectivity is largely or exclusively limited to cells of the monocyte-macrophage lineage. Current evidence suggests that infection of foals with virulent R. equi results in some foals in subversion of cell-mediated immunity and development of an ineffective and sometimes lethal Th2-based immune response. Significant progress has been made recently in the development of R. equi-E. coli shuttle vectors, transformation and random and site specific mutagenesis procedures, all of which will be important in molecular dissection of the mechanisms by which R. equi subverts normal macrophage killing mechanisms and cell-mediated immunity.

Actinomycetales Infections↗
PubMed2006

Two cases of equine abortion caused by Rhodococcus equi.

Rhodococcus equi was isolated from lung, liver, spleen, and stomach content of two aborted equine fetuses of 7 and 8 months gestation from two different farms. Lesions included diffuse pyogranulomatous pneumonia with numerous Gram-positive coccobacilli within the cytoplasm of macrophages, multinucleated Langhans giant cells and neutrophils, and enhanced extramedullary hematopoiesis with megakaryocytosis within the liver and spleen. Detection of R. equi was made by bacteriology and immunohistochemistry for R. equi and VapA, the virulence factor of R. equi. R. equi and VapA were identified within the lungs of both fetuses, and its distribution correlated with lesions. Fetal lesions were similar to those observed in foals. We speculate that the fetuses contracted infection from the placenta by normal breathing movements or by swallowing of the amniotic fluid contaminated with R. equi.

Aborted Fetus↗
PubMed1999

Tumour necrosis factor and interferon-gamma are required in host resistance against virulent Rhodococcus equi infection in mice: cytokine production depends on the virulence levels of R. equi.

Rhodococcus equi is a facultative intracellular bacterial pathogen that causes pneumonia in foals and immunosuppressed humans. There are at least three virulence levels of R. equi and these pathogenicities are associated, in mice, with the presence of virulence plasmids. This study focused on cytokine secretion, in mice, in the course of a primary infection with sublethal doses of R. equi strains of different virulence levels (virulent, intermediately virulent and avirulent). Tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma), but not interleukin-4 (IL-4) and interleukin-10 (IL-10), were induced endogenously in mice in relation to the multiplication and clearance of virulent and intermediately virulent strains of R. equi. These cytokines were not detected in mice infected with avirulent R. equi. Deaths occurred among mice treated with monoclonal antibodies (mAbs) against either TNF or IFN-gamma prior to sublethal dose infection with virulent and intermediately virulent strains of R. equi, but not with avirulent R. equi. These results suggested that cytokine production depended largely on the virulence levels of R. equi: TNF and IFN-gamma were required early during infection with virulent R. equi to limit replication and clearance of bacteria within the organs, but they were not necessary for limiting infection with avirulent R. equi.

Actinomycetales Infections↗
PubMed1991

Association between a large plasmid and 15- to 17-kilodalton antigens in virulent Rhodococcus equi.

Rhodococcus equi strains showing 15- to 17-kDa antigens in immunoblots were found to be virulent in mice. To study the genes specific to these antigens in virulent R. equi, we compared plasmid profiles, immunoblot profiles, and murine pathogenicity profiles of 10 strains of R. equi. All the strains showing 15- to 17-kDa antigens contained a large plasmid of approximately 85 kbp and were virulent in mice; however, the remaining strains lacked both the antigens and the large plasmid and were avirulent in mice. Mutants of virulent strains ATCC 33701 and L1, which were cured of the large plasmid by repeated passage at 38 degrees C, lacked the 15- to 17-kDa antigens and showed a dramatic decrease in lethality in mice. These results suggested that the presence of an 85-kbp plasmid may be essential for virulence and expression of 15- to 17-kDa antigens of R. equi and offered support for earlier observations that freshly isolated strains of R. equi killed mice, whereas laboratory-adapted strains did not.

Actinomycetales Infections↗
PubMed2005

Isocitrate lyase activity is required for virulence of the intracellular pathogen Rhodococcus equi.

Rhodococcus equi is an important pathogen of foals, causing severe pyogranulomatous pneumonia. Virulent R. equi strains grow within macrophages, a process which remains poorly characterized. A potential source of carbon for intramacrophage R. equi is membrane lipid-derived fatty acids, which following beta oxidation are assimilated via the glyoxylate bypass. To assess the importance of isocitrate lyase, the first enzyme of the glyoxylate bypass, in virulence of a foal isolate of R. equi, a mutant was constructed by a strategy of single homologous recombination using a suicide plasmid containing an internal fragment of the R. equi aceA gene encoding isocitrate lyase. Complementation of the resulting mutant with aceA showed that the mutant was specific for this gene. Assessment of virulence in a mouse macrophage cell line showed that the mutant was killed, in contrast to the parent strain. Studies in the liver of intravenously infected mice showed enhanced clearance of the mutant. When four 3-week-old foals were infected intrabronchially, the aceA mutant was completely attenuated, in contrast to the parent strain. In conclusion, the aceA gene was shown to be essential for virulence of R. equi, suggesting that membrane lipids may be an important source of carbon for phagocytosed R. equi.

Actinomycetales Infections↗
PubMed1999

Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Actinomycetales Infections↗
PubMed2002

Identification of pulmonary T-lymphocyte and serum antibody isotype responses associated with protection against Rhodococcus equi.

Rhodococcus equi infects and causes pneumonia in foals between 2 and 4 months of age but does not induce disease in immunocompetent adults, which are immune and remain clinically normal upon challenge. Understanding the protective response against R. equi in adult horses is important in the development of vaccine strategies, since those mechanisms likely reflect the protective phenotype that an effective vaccine would generate in the foal. Twelve adult horses were challenged with virulent R. equi and shown to be protected against clinical disease. Stimulation of cells obtained from bronchoalveolar lavage fluid with either R. equi or the vaccine candidate protein VapA resulted in significant proliferation and a significant increase in the level of gamma interferon (IFN-gamma) expression by day 7 postchallenge. The levels of interleukin-4 expression were also increased at day 7 postchallenge; however, this increase was not antigen specific. Anamnestic increases in the levels of binding to R. equi and VapA of all immunoglobulin G (IgG) antibody isotypes [IgGa, IgGb, IgG(T)] examined were detected postchallenge. The levels of R. equi- and VapA-specific IgGa and IgGb antibodies, the IgG isotypes that preferentially opsonize and fix complement in horses, were dramatically enhanced postchallenge. The antigen-specific proliferation of bronchoalveolar lavage fluid cells, the levels of IFN-gamma expression by these cells, and the anamnestic increases in the levels of opsonizing IgG isotypes are consistent with stimulation of a memory response in immune adult horses and represent correlates for vaccine development in foals.

Actinomycetales Infections↗
PubMed2003

Analysis of anamnestic immune responses in adult horses and priming in neonates induced by a DNA vaccine expressing the vapA gene of Rhodococcus equi.

Rhodococcus equi remains one of the most important pathogens of early life in horses, yet conventional vaccines to prevent rhodococcal pneumonia have not been successful. DNA vaccination offers an alternative to conventional vaccines with specific advantages for immunization of neonates. We developed a DNA vaccine expressing the vapA gene (pVR1055vapA) that induced an anamnestic response characterized by virulence associated protein A (VapA)-specific IgG antibodies in sera and bronchoalveolar lavage fluid (BALF) as well as VapA-specific proliferation of pulmonary lymphocytes when tested in adult ponies. In contrast, none of the adults receiving the control plasmid responded. To determine if pVR1055vapA induced VapA-specific responses in the foal, the targeted age group for vaccination against R. equi, 10 naïve foals were randomly assigned at birth to two groups of five. At 8-15 days of age (day 1), foals were vaccinated by intranasal and intradermal (i.d.) routes with either pVR1055vapA or the negative control pVR1055vapA_rev. All foals were DNA boosted at day 14 and protein boosted at day 30 with either recombinant VapA or recombinant CAT (control group). Prior to the protein boost, neither group developed VapA-specific immune responses. However, at day 45, two of the VR1055vapA-vaccinated foals had increased titers of VapA-specific IgGb, IgM and IgGa in the sera, and IgG in the BALF. The induction of the opsonizing isotypes IgGa and IgGb has been previously shown to be associated with protection against R. equi. No VapA-specific immune responses were detected in the control group. This study indicates that the DNA vaccine effectively stimulates anamnestic systemic and pulmonary immune responses in adult horses. The results in foals suggest that the DNA vaccine also primed a subset of immunized neonates. These data support further development and modification to produce a DNA vaccine to more effectively prime neonatal foals.

Actinomycetales Infections↗
PubMed1992

In-vitro antimicrobial susceptibility of Rhodococcus equi.

Rhodococcus equi is an intracellular facultative, Gram-positive cocco-bacillary organism of increasing importance as a pulmonary pathogen in HIV-positive patients. This study was carried out to evaluate the optimal antibiotic combinations for treating such infections. Four human R. equi isolates and one reference strain were tested for their susceptibilities to 36 antibiotics. In-vitro the most active antibiotics were amikacin, gentamicin, netilmicin, erythromycin, clarithromycin, roxithromycin, ciprofloxacin, sparfloxacin, rifampicin, vancomycin, teicoplanin, doxycycline, minocycline, imipenem, meropenem and trimethoprim/sulphamethoxazole. The only bactericidal antibiotics were the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin. As determined by FIC indices, four combinations were synergistic: rifampicin-erythromycin, rifampicin-minocycline, erythromycin-minocycline and imipenem-amikacin. However, no antibiotic combinations were synergistic with the time-kill kinetic method at achievable serum concentrations or at ten-fold and half-fold the MICs. Frequencies of selection of antibiotic-resistant mutants determined at concentrations of five- and ten-fold the MICs ranged from less than 1 x 10(-8) for erythromycin and trimethoprim/sulphamethoxazole to 5 x 10(-4) for amikacin. These results may be of help in selecting the antibiotics for treating infected HIV-positive patients.

Anti-Bacterial Agents↗
PubMed2000

[A diagnostic medium for Arcanobacterium haemolyticum and other bacterial species reacting with hemolytic synergism to the equi-factor of Rhodococcus equi].

Colonies of Arcanobacterium haemolyticum on common blood agar can be easily overlooked. Therefore a diagnostic medium was developed, on which A. haemolyticum colonies produce a conspicuous zone of complete hemolysis. The medium under question is blood agar prepared from the Columbia Blood Agar Base and 5% washed sheep erythrocytes sensitised with equi factor (EF) of Rhodococcus equi. Optimally, 10 activity units (AU) of EF per 1 ml were used. EF was titrated on a non-nutrient medium consisting of agar Purified (Difco) and 5% washed sheep erythrocytes sensitized with beta-haemolysin (BL) of Staphylococcus aureus, 10 AU/ml. On the same medium, staphylococcal BL was titrated on the basis of its direct haemolytic effect. Despite the very distinct haemolytic reaction of the control strains evident on the diagnostic medium with EF, A. haemolyticum failed to grow from 2.597 throat swabs examined especially for the particular microbe during the a period of two years. However, A. haemolyticum was isolated on this medium twice from 223 swabs from wounds and skin lesions. The proposed medium makes also the rapid diagnosis of species Corynebacterium ulcerans, Dermatophilus congolensis and Listeria monocytogenes on the basis of haemolytic synergism possible.

Actinomycetales↗
PubMed1993

Monoclonal antibody specific to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Virulent Rhodococcus equi produces 15- to 17-kDa surface protein antigens. These antigens are used as markers to identify virulent R. equi isolates from foals and their environment by Western blot (immunoblot) analysis with naturally infected foal serum. In the present study, a monoclonal antibody (MAb; 10G5) was generated against the 15- to 17-kDa antigens excised from sodium dodecyl sulfate-polyacrylamide gels to develop sensitive and specific immunoblot assays for the identification of virulent R. equi. MAb 10G5 strongly reacted with R. equi ATCC 33701 and L1, which expressed 15- to 17-kDa antigens by Western blot, colony blot, and dot immunobinding assays, but it did not react with strains ATCC 33701P- and L1P-, which lacked the antigens. For identification of virulent R. equi, clinical and environmental isolates were tested by these assays with the MAb, and all virulent strains were successfully identified; these strains possessed virulence plasmids. These results suggest that the MAb is a useful reagent for the identification of virulent R. equi.

Animals↗
PubMed1999

Restriction fragment length polymorphisms of virulence plasmids in Rhodococcus equi.

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries-Argentina, Australia, Canada, France, and Japan-were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738-740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world.

Plasmids↗
PubMed2003

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection.

Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.

Actinomycetales Infections↗
PubMed1996

Radiological findings in nine AIDS patients with Rhodococcus equi pneumonia.

Rhodococcus equi (R. equi) infections have been incidentally reported as a cause of pulmonary infection in severely immunocompromised hosts, including AIDS patients. Our purpose is to describe the radiological findings in nine AIDS patients with R. equi pneumonia assessed by bronchoalveolar lavage (BAL), biopsies, cultures of sputum, and hemocultures. All patients were examined by chest radiographs and contrast-medium-enhanced chest CT. Dense pulmonary consolidations with or without cavitations accounted for the most striking radiological patterns. Chest CT also revealed six mediastinal involvements, strongly mimicking a lymphoma. Two of them had multiple bilateral pulmonary nodular opacities. Pleural effusion was not identified. Although intensive therapies were administered, seven among nine patients died within few months. In an AIDS patient living in a rural area or exposed to horses and presenting these radiological patterns, the possibility of R. equi pneumonia should be considered in the differential diagnosis along with other infectious diseases or lymphomas.

AIDS-Related Opportunistic Infections↗
PubMed1995

Radiologic findings in two AIDS patients with Rhodococcus equi pneumonia.

Rhodococcus equi (R. equi) has been reported as an occasional cause of pulmonary infection in severely immunocompromised hosts, including AIDS patients. Our purpose is to describe the radiologic findings in two AIDS patients with R. equi pneumonia. Chest radiographs showed right-upper-lobe consolidation and cavitation in both patients. Chest CT confirmed upper mediastinal involvement and precarinal lymphadenopathy in both cases. Multiple lung nodules related to the bronchi were also identified in one patient. In an AIDS patient from a rural area or with exposure to horses, the possibility of R. equi infection should be considered when cavitary pneumonia is present, even if there is mediastinal involvement and/or lymphadenopathy, or if multiple lung nodules are also present.

AIDS-Related Opportunistic Infections↗